Identification of Conserved Chromatin-Regulatory Complexes 
among
the Class B Synthetic Multivulva Proteins
by
Melissa M. Harrison
A.B., Biological Sciences
Harvard University, 1998
Submitted to the Department of Biology
in partial fulfillment of the requirements for the degree of
DOCTOR OF PHILOSOPHY
dt La IW
MASSACUSETTS INSTITUTE OF TECHNOLOGY
July 
2006
@ 2006 Melissa M. Harrison. All rights reserved.
MASSACHUSETTS
OF TECHNO
AUG 16
LIBRAR
The author hereby grants MIT permission to reproduce and to distribute 
publicly paper
and electronic copies of this thesis document in whole or in part.
I 'I
4 1 /
Signature of Author: ,, ., ,, , ; ,, ; .
Department of Biology
Certified by:
Accepted by
H. Robert Horvitz
Professor of Biology
Thesis Supervisor
Stephen P. Bell
Chairman of the Graduate Committee
Department of Biology
ARCHIVES
INST E
LOGY
2006
IES
I I / I
Identification of Conserved Chromatin-Regulatory Complexes among
the Class B Synthetic Multivulva Proteins
Submitted to the Department of Biology in July 2006 in partial fulfillment of the
requirements for the Degree of Doctor of Philosophy in Biology
Abstract
The class A, B, and C synthetic Multivulva (synMuv) genes act redundantly to
antagonize Ras-mediated vulval induction in C. elegans. Many of these genes encode
proteins that are likely to function in transcriptional repression and chromatin
remodeling. The class B synMuv protein LIN-35 is similar to the mammalian tumor
suppressor Rb. Studies of mammalian systems have identified many chromatin-
remodeling factors that are recruited to promoters through association with Rb or other
pocket proteins. We have identified a complex of at least seven class B synMuv
proteins, including LIN-35 Rb. While this complex contains proteins such as DPL-1 DP
and LIN-53 RbAp48 that were thought to interact with LIN-35 Rb based on similarities to
mammalian systems, it also includes four additional proteins that were not previously
known to be associated with pocket proteins- LIN-9, LIN-37, LIN-52, and LIN-54. We
have named this complex the DRM complex for DP, Rb, and MuvB, complex. As similar
protein complexes were simultaneously identified in flies, and these proteins all have
mammalian homologs, it is likely that a similar complex exists in humans to regulate
gene expression. We have further shown that although pocket proteins are known to
interact with histone deacetylases, in C. elegans the synMuv protein HDA-1 HDAC1 is
not a component of the DRM complex although it is component of a NuRD-like complex.
Furthermore, the NuRD-like complex has functions distinct from the DRM complex in
vulval development. We have also characterized 
the class B synMuv protein LIN-61,
which is not a core member of either the DRM or NuRD-like complexes. LIN-61 contains
four malignant brain tumor (MBT) repeats, which are in multiple transcriptional
repressors. Thus like other class B synMuv proteins, LIN-61 likely functions to repress
transcription of genes that induce vulval development. Our biochemical and genetic
characterizations of the synMuv genes have identified a new pocket protein-containing
complex and have demonstrated the existence of multiple complexes among the class
B synMuv proteins. Thus the many proteins that have previously been classified as
class B synMuv proteins are likely to be functioning in independent complexes to
regulate vulval development through transcriptional repression.
Thesis Supervisor: H. Robert Horvitz
Title: David H. Koch Professor of Biology
Acknowledgments
First, I would like to thank my adviser, Bob Horvitz, who gave me the freedom to pursue
a more biochemical than genetic project. My science (and my grammar) has improved
immensely under his supervision. I would also like thank my thesis committee members
Steve Bell, Tyler Jacks, Ilaria Rebay, Chris Kaiser, and Dennis Kim. I would especially
like to thank Steve Bell who shared his biochemical expertise with me when I needed
assistance.
Much of my education over the last six years in the Horvitz laboratory was provided by
the graduate students and postdoctoral fellows with whom I have worked. I would like to
thank Craig Ceol who patiently taught me how to work with C. elegans during my
rotation project and who continued to mentor me in my first years in the laboratory. I
have also worked closely with Ewa Davison, Erik Andersen, and Adam Saffer in our
studies of vulval development. Ezequiel Alvarez-Saavedra has shared a bay with me for
most of my time in the lab and while my timer and pipettemen are always disappearing
on his bench I could not think of a better person with whom to have shared such a close
work environment. I would also like to thank Megan Higginbotham for being a friend and
helping me to survive being a teaching assistant for Graduate Genetics. Lunchtime
would not have been the same without Adam Saffer and Dave Harris who have kept me
company on our ritual lunch trips to Anna's. Finally, I would like to thank all the
members of the Horvitz laboratory for providing both intellectual and entertaining
discussions.
Through the years I have gotten invaluable advice from my father, Rick Harrison, and
my uncle, Stephen Harrison. As scientists they have advised me on many major
decisions and have understood the frustrations that are inherent in the career path I
have chosen. My mother, Ellen Harrison, might constantly wonder how my father and I
deal with these frustrations but she has been supportive and caring throughout. I must
also thank my sister, Rebekah Harrison, who, as a medical doctor, reaffirms my
decision to pursue basic research every time we talk as she drives home from work
ridiculously early in the morning. I want to acknowledge my grandmothers, Helen
Harrison and Marjorie Zucker, for the inspiration they have provided me through their
career choices. Both received PhDs at a time when this was incredibly uncommon for
women and went on to pursue successful research careers.
I must also thank Andy Mehle for being a wonderful, supportive and fun companion as
well as a smart and creative colleague. I am so lucky to have found some one who can
sees value in both work and play and recognizes the importance of a good hug after a
hard day.
Table of Contents
A bstract ....................................... ........... .. ............ .............. 2
Acknowledgments ................ ................ .................................................. 3
Table of Contents ................................. ...................................... 4
CHAPTER 1: Pocket proteins interact with a diversity of chromatin-
rem odeling proteins............................................. .................. 10
Introduction ................................................................................. ......... ......... 11
Figure 1. The C. elegans vulva develops from three of six ectodermal blast
cells. .................................. .. ....... .......... ................. 12
Control of transcription levels can be achieved by changes in chromatin
structure ...... ................... ..................... 15
Pocket proteins and their E2F binding partners are conserved across species. 17
The role of Rb in cell cycle control .................. ................. 20
Figure 2. Cell-cycle progression is regulated by the sequential activation of
multiple proteins. ................................................... 21
Pocket proteins repress E2F-responsive genes by two distinct mechanisms..... 24
Pocket proteins interact with histone deacetylases .................................... .26
Pocket proteins interact with ATP-dependent chromatin remodeling enzymes.. 29
Pocket proteins interact with histone methyltransferases ................................ 31
Pocket proteins interact with additional potential chromatin modifying enzymes 34
Pocket proteins likely control different sets of genes by different mechanisms.. 35
Figure 3: Pocket proteins regulate transcription of target genes by association
with different transcription factors ....................... ......... ........ 36
Pocket proteins have roles beyond cell-cycl control ................. ............... 38
Conclusions ..... .................. ......... ........... 40
Acknowledgments. ......... ........... ...... ................ 41
Literature Cited ................................................................................... 42
Table 1. synMuv genes encode proteins with homologs in other species.......... 56
CHAPTER 2: A subset of C. elegans class B synMuv proteins encodes a
conserved LIN-35 Rb-containing complex distinct from a NuRD-like
com plex ........................................................ ......... 57
Abstract .................................... ........ ................ 58
Introduction ......................................................................................................... 59
R e s u lts .................................................................................. . . .. ........... ....... 6 1
Iin-37 and lin-54 are class B synMuv genes.......... . ............................. 61
LIN-37 encodes a small hydrophilic protein with weakly conserved homologs
in other species.................................. ...... .... ......... 61
LIN-54 encodes a protein with two copies of a conserved cysteine-rich motif 62
LIN-37 and LIN-54 are broadly expressed in nuclei throughout development. 63
LIN-37, LIN-54 and other class B synMuv proteins form a large-molecular-
weight- protein complex ..... ........... ... . .. ............. . . ................... 64
The DRM complex is distinct from a C. elegans NuRD-like complex .............. 66
Vulval development of animals with mutations in NuRD-like complex
components is phenotypically distinguishable from that of animals mutant for
DRM complex components ......................... .................. 67
Some DRM complex components are absent in mutants lacking certain other complex
components ............... ......... ... ................. 68
D iscussion .............. ...... ..... ...... . ........... ......................... 71
lin-37 and lin-54 likely act in the DRM complex................................. 71
The DRM complex might act in vulval development ......................... .71
The DRM complex probably functions in transcriptional repression............. 72
The DRM complex and a NuRD-like complex have separable functions
during vulval developm ent ................................... ................................. 75
Does Rb function within the context of a DRM-like complex to control
development in mammals? ...........
. .
..
. . . . . .. . .. ..
.. ................
....... . . . . 
76
M aterials and M ethods ....................... ........................... ........ .... ............. 77
Culture conditions and strains.......................................... ...................... 77
T ransgenic S trains ................................................... ................................. 77
RNAi Analyses ................. ........ .............. ................ 77
Antibody Preparation, Immunocytochemistry and Western Blots................. 77
Em bryo lysates ............................................................................ ... 77
Immunoprecipitation experiments ............................... ........ . .......... 78
Gel filtration chromatography .................................. 78
Acknow ledgm ents ........................................ 80
Literature Cited ................................. .............................. ........ 81
Tables.............. ..................................... ................. 87
Table 1. lin-37and lin-54 Mutations Cause a Class B synMuv Phenotype..... 87
Figures ................................................ ....... ................ ......... 88
Figure 1. Cloning and expression of lin-37.............................. .88
Figure 2. Cloning and expression of lin-54 ........................................... 90
Figure 3. A subset of class B synMuv proteins form a complex in vivo ........ 92
Figure 4. NuRD-like complex components do not precipitate with the DRM
complex .......................................... ......... .. ......... 94
Figure 5. Analysis of the DRM complex and its components in mutant
backg ro und s. ...................................................... .. ........................ 96
Figure 6. Schematic indicating the components of the DRM and NuRD-like
complexes and a model for how these complexes act in vulval development. 98
Supplemental Information................................................ 100
Supplem ental Results................................................. 101
C loning of lin-37.............................. ............. ................ 101
Cloning of lin-54..... ...................................... 101
Supplemental Material and Methods ...... ................................ 102
Culture conditions and strains ............................................. 102
Transgenic Strains ............... .... ......... ... ................ 102
Determination of lin-54 cDNA sequence .................................. 102
Antibody Preparation ........................................ ............... ......... 103
Supplemental Literature Cited ................. ....................... 104
Supplem ental Figures............................................................ 106
Supplemental Figure 1. Molecular cloning of lin-37.............................. 106
Supplemental Figure 2. Molecular cloning of lin-54................... 108
Supplemental Figure 3. Antibodies specifically recognize LIN-9, 
LIN-35,
LIN-52 and LIN-53. .......................... .................. 110
Supplemental Figure 4. LIN-37 does not coimmunoprecipitate with
LIN-36::GFP ..................................................................... .......... ........ .. ......... 112
CHAPTER 3: LIN-61, one of two C. elegans MBT-repeat-containing
proteins, acts separately from the DRM and NuRD-like complexes in
developmental regulation ........... ............................. ................ .114
A b s tra c t ........................................................................................... 1 1 5
Introduction... ...................................................... ...... .............. 116
Materials and Methods ......... .... .. ......... ... ......... 119
S tra ins................................................. 1 19
Transgenic strains ....................................... 119
R N A i analysis..................... ................ 119
Antibody preparation, immunocytochemistry and western blots .................. 119
Phenotypic characterization ............. .................. ........... 120
Embyro lysates ................... ................... .................... 121
Immunoprecipitation experiments ........... .................... 121
Results ....................................................... 122
lin-61 is a class B synMuv gene................ .......................... 122
lin-61 encodes an MBT-repeat-containing protein .................... ......... 122
Characterization of lin-61 alleles ............................ ........ 123
The C. elegans genome encodes one additional MBT-repeat-containing
protein ................................... .... . .............................. 124
LIN-61 is broadly expressed in nuclei throughout development................. 126
Missense mutations in LIN-61 might disrupt protein stability ........... 127
Analysis of pleiotropies associated loss of function in lin-61 or mbtr- I ......... 128
LIN-61 is not a core member of the DRM or NuRD-like complexes ............ 130
D iscussion ................................................................................ ... ......... 132
MBT-repeat-containing proteins are not required for C. elegans viability...... 132
LIN-61 is likely involved in transcriptional repression ............................... 133
lin-61 functions separately from the DRM and NuRD-like complexes ........... 133
Acknow ledgm ents ........................................ .......... ................................. 136
Literature Cited .............. ................................... ........... ....... 137
Tables ................. ... ... ... .... .... ... .... ............... 144
Table 1. lin-61 mutations cause a class B synMuv phenotype................... 144
Table 2. Sequences of lin-61 alleles and allele strengths .......................... 145
Table 3. Phenotypic characterization of lin-61(n3809) and mbtr- 1(n4775)... 146
Figures ...................................... ................ 147
Figure 1. Molecular cloning of lin-61 ................. .................. 147
Figure 2. Identification of mbtr- 1 .................................. 149
Figure 3. Expression of LIN-61 ........................................ 151
Figure 4. LIN-61 levels are reduced in many lin-61 mutant animals ............. 153
Figure 5. LIN-61 is not a core component of the DRM complex................. 155
CHAPTER 4: lin-8, which antagonizes C. elegans Ras-mediated vulval
induction, encodes a novel nuclear protein that interacts with the
LIN-35 Rb protein.................................................. 157
Abstract ......... . . ...... .................................................... 158
Introduction ............................................ 159
Materials and Methods .............. ........................... 162
Strains and general techniques.......... ...................... 162
Deletion and Polymorphism Mapping .................................. 162
Transgenic Animals ................................ .. . ............. 164
Antibody Preparation, Immunoblotting, and Immunocytochemistry ....... . 164
Two-Hybrid and In Vitro Binding Experiments ................. .......... ........ 165
Results ......................................... ..................... 166
LIN-8 defines a family of novel C. elegans proteins ....... ......................... 166
Characterization of lin-8 alleles ........ ..... ................... 167
LIN-8 is a nuclear protein expressed in many cells ................................... 169
LIN-8 interacts with LIN-35 Rb in vitro................................... 170
D iscussion .................... .. ......... ........ ........... ..................... 172
Class A synMuv genes may regulate transcription ............................ ... 172
Physical interaction between synMuv A and B proteins................................ 173
Partial redundancy in the LIN-8 family .................................. 174
Interactions within the class A synMuv pathway ...................... .................. 175
Implications for human cancer ....................................... 176
Acknowledgments ........ ............................... 177
Literature Cited ............ ......... .......... ........ 178
Tables............................... ........ ................ ......... 186
Table 1. Sequences of lin-8 mutations ......... .................. 186
Table 2. lin-8 allele strengths ..... ............... ...................... ................ 187
Figures ...................................... ................. 189
Figure 1: Cloning of lin-8 ..... ................ ..................... 189
Figure 2. LIN-8 defines a family of C. elegans proteins ............... 192
Figure 3. LIN-8 protein is expressed broadly and localized in nuclei.......... 196
Figure 4. LIN-8 interacts with LIN-35 Rb in vitro ......................... 199
CHAPTER 5: Identification and classification of genes that act
antagonistically to let-60 Ras signaling in Caenorhabditis elegans
vulval development ........ ........................ ................... 202
Abstract .................... .............................. 203
Introduction ........... ................................................ 204
Materials and Methods ........ ...................... ................. 208
Strains and general techniques ............... .. ....... ............ ................ 208
Isolation of new alleles ............ . .. ..................................... ........ 209
Linkage group assignm ent............................................... 209
Complementation tests ........................ ............. 209
Construction of deficiency heterozygotes ........................... 210
Construction of single mutant and unlinked double mutant strains ............ 210
Construction of linked double mutant strains .................................. 211
Assay for P(3-8).p vulval cell fates................................ 212
Construction of linked double mutant strains .................................. 212
RNA-mediated interference................................ 212
lin-65 rescue .............. ............................................................. 212
Allele sequence...................................................... 212
Results ............................................................. 213
Isolation of new synM uv m utants ........................................ ......... ....... 213
Phenotypes of new mutants ....... ............ ......... . ............... 214
New synM uv genes ...... .............................................. ....................... 214
Interactions with other synMuv mutations ........................ 215
Suppression of let-23 mutations ......... .................... 215
Interactions with ark-1, gap-1 and sli-1 mutations ................. .... 217
Molecular identification of iin-65 ................. ......... ........ 218
Sequences of synMuv mutations ...................................... 219
Discussion ................... ....... ................ 221
Frequency of m utant isolation ...................................................... ............... 221
Different synMuv gene classes likely act in parallel to antagonize let-60
Ras pathw ay activity ...................................................................................... 222
Different synMuv gene classes control distinct biochemical activities ........... 223
Acknowledgments ........................................ 226
Literature Cited ........................................ ............................. 227
Tables ................... ............................... 235
Table 1. Phenotypes of synMuv mutant strains ............... ........... ..... 235
Table 2. Three- and four-factor crosses................................. 240
Table 3. Deficiency heterozygote mapping ....................................... 243
Table 4. Interactions of new mutations with class A and B synMuv
m utations .......................................... ................. 244
Table 5. Interactions of new mutations with the class C synMuv mutation
trr- 1(n3712) ................. ................... .......... 247
Table 6. Suppression of the let-23 vulvaless phenotype ................................... 249
Table 7. Interactions of new mutations with gap-1 and sli- I mutations......... 251
Table 8. Sequences of new mutations of class B and class C synMuv
proteins .................................... ....... .. .......... ...... . ................ 252
Table 9. Summary of synMuv genetic interactions.................. 258
Figures .................................... ......... ................ 261
Figure 1. Molecular cloning of lin-65 .... .............................. .... 261
Figure 2. lin-65 cDNA sequence .................... . .............. 263
CHAPTER 6: Future Directions ........................................ 265
synMuv proteins form multiple complexes that likely regualte transcription...... 266
synMuv proteins could regulate vulval development through transcriptional
repression of lin-3....................................... 267
Class B synMuv genes do not all function similarly to regulate vulval
developm ent ............................ ... ........ ........................... 269
synMuv proteins regulate many processes in addition to vulval development.. 270
Different target genes are likely to be regulated by different sets of synMuv
proteins ...................... ........................................... 271
Multiple proteins likely act to recruit synMuv complexes to DNA................... 272
How do the coordinated function of different transcription factors
cooperate to regulate development? ......
... ... . . . .. ...
....
... .. .. . . . .
.. 273
Conclusions ........................................... 274
Acknowledgments ........................................ 275
Literature Cited ............... ....... ....... ........ . ....... 276
Figure 1. Schematic indicating two possible models for how complexes
among the class B synMuv proteins could regulate transcription during vulval
developm ent ......................................... ........ ................................... 278
CHAPTER 1
INTRODUCTION:
Pocket proteins interact with a diversity of chromatin-remodeling
enzymes
Introduction
To understand how an entire organism develops 
requires first an understanding
of the coordinated control of cell division and differentiation. These complex processes
are in turn regulated by changes in gene expression that can be mediated by
modifications to the chromatin state. Biochemical studies have identified many factors
that function to regulate chromatin and to control transcription levels, and genetic
studies have implicated many of these factors in developmental regulation. Future
studies must be directed towards understanding how these changes in gene expression
result in cellular differentiation and ultimately in the development of the organism as a
whole.
Proper development of the vulva of the nematode Caenorhabiditis elegans
results from the combined activities of a number of signaling pathways, including a
receptor tyrosine kinase (RTK)/ Ras pathway, a Notch-signaling pathway, and a Wnt
pathway (YOCHEM et al. 1988; BEITEL et al. 1990; HAN et al. 1990; GLEASON et al. 2002).
The hermaphrodite vulva is specified to develop during the second larval stage from
three of six ectodermal blast cells (P3.p-P8.p) (Figure 1). Although all six blast cells are
capable of adopting vulval cell fates, in wild-type development only the descendants of
P5.p, P6.p, and P7.p contribute to the vulva. P3.p, P4.p, and P8.p generally divide just
once and fuse with the surrounding hypodermis.
Mutations in genes required for vulval specification result in either vulvaless (Vul)
mutant animals or multivulva (Muv) mutant animals. Analysis of mutants with these
phenotypes demonstrated the importance of an inductive RTK/Ras pathway in properly
specifying P5.p, P6.p, and P7.p. Gain-of-function mutations in the Ras homolog let-60
result in extra cell divisions and a Muv phenotype. By contrast, loss of function in Ras
pathway components results in Vul animals (STERNBERG and HAN 1998). Studies of
other systems have shown that the components of these pathways are highly
conserved in other organisms, as are the roles these proteins play in development. For
example, an RTK/Ras pathway is required to properly specify the photoreceptors in the
development of the eye in Drosophila melanogaster (WASSARMAN et al 1995). In
addition to its roles in wild-type development, Ras also has been shown to be an
Figure 1. The C. elegans vulva develops from three of six ectodermal blast cells.
During the L2 and early L3 stages, an EGF-like signal from the gonadal anchor cell
induces P5.p, P6.p, and P7.p to divide and 
generate the vulva. Although P3.p, P4.p,
and P8.p are competent to adopt vulval cell fates, in wild-type development they divide
once and fuse with the surrounding hypodermis. The lineages for the descendants of
P3.p-P8.p are shown.
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oncogene in mammals, and gain-of-function mutations have been found in many human
cancers (BARBACID 1987).
The activity of the Ras-pathway in C. elegans vulval cell-fate specification is
antagonized by the activities of the synthetic multivulva (synMuv) genes. Based on
genetic interactions, the synMuv genes fall into three classes, A, B, and C. Loss of
function in any single class does not result in a Muv animal. However, animals that have
loss-of-function mutations in members of any two classes are Muv.
Four class A synMuv genes have been identified, and all encode novel proteins.
Two of the class A synMuv proteins have THAP domains (CLARK et al. 1994; HUANG et
al. 1994), which have been shown to bind DNA (CLOUAIRE et a. 2005). The presence of
THAP domains in LIN-15A and LIN-56 suggests that these proteins might directly
interact with DNA. Among the class B and class C synMuv proteins are many proteins
that likely are involved in transcriptional repression and chromatin remodeling (Table 1).
These include HDA-1, a homolog of a histone deacetylase, and LET-418, a homolog of
an ATP-dependent chromatin-remodeling 
enzyme Mi2 (voN ZELEWSKY et al 2000;
DUFOURCo etal. 2002). These proteins along with LIN-53, a homolog of RbAp48, are
similar to components of the mammalian Nucleosome Remodeling and Deacetylase
(NuRD) complex (Lu and HORVITZ 1998; XUE et al. 1998; ZHANG et al. 1998a; ZHANG et
al. 1999). The class B synMuv proteins MET-2 and HPL-2 are homolgous to proteins
with histone methyltransferase activity and histone methyl binding activity, respectively
(COUTEAU et al. 2002; POULIN et al. 2005). The class C proteins likely form a histone
acetyltransferase complex (CEOL and HORVITZ 2004)
Among the class B synMuv proteins also is a protein similar to the mammalian
tumor suppressor Rb (Lu and HORVITZ 1998). In addition, the class B synMuv proteins
EFL-1 and DPL-1 are homologous to the mammalian heterodimeric transcription factor
E2F, which is known to recruit Rb to DNA (CEOL and HORVITZ 2001). Extensive studies
have been performed in mammals to better understand the function of Rb, and it has
been demonstrated that Rb interacts with many proteins to control proper gene
expression and ultimately to regulate cell cycle and development. Understanding the
important role of Rb and related proteins will require unifying our knowledge of the
function of these proteins in multiple organisms.
Control of transcription levels can be achieved by changes in chromatin
structure
In the nucleus, eukaryotic DNA is packaged with proteins to form chromatin. The
basic unit of chromatin is the nucleosome, which consists of 147 base pairs of DNA
wrapped around a protein octamer consisting of two molecules each of the histone
proteins H2A, H2B, H3, and H4. Each histone protein is composed of a globular core
region and an N-terminal tail of unstructured amino acids that protrude from the
octamer. These histone tails are subject to post-translational modifications including
phosphorylation, ubiquitination, acetylation, methylation, and ADP-ribosylation (SIMS et
al. 2003). These histone modifications and other changes in the chromatin structure can
modulate the transcription levels of nearby genes. The two best studied modifications
are acetylation and methylation.
Acetylation occurs on lysines on the N-terminal tails of all four histones and has
been linked to transcriptional activation. Histone acetyltransferases or proteins that can
recruit histone acetyltransferases through protein/protein interactions often act as
transcriptional activators. Acetylation of lysines decreases the interactions between
histones and the negatively-charged DNA by blocking the positive charge on the lysine
residue. This is proposed to open the chromatin structure and thereby allow proteins
involved in transcription to access the DNA. Conversely, hypoacetylation of histones
has been linked with transcriptional repression, and many transcriptional repressors are
associated with histone deacetylase activity (KOUZARIDES 1999).
Methylation, another post-translational modification of histones, occurs on
arginine and lysine moieties in the tails of histone H3 and histone H4. Methylation has
been linked with both transcriptional repression and transcriptional activation depending
on the methylated residue. For example, methylation of lysines 9 and 27 on histone H3
and methylation of lysine 20 on histone H4 are associated with transcriptional
repression. By contrast, methylation of lysine 4 of histone H3 has been associated with
transcriptional activation. Amino acids can also be mono-, di-, or tri-methylated, adding
a further level of complexity. Until recently, no enzymes capable of removing methyl
groups from amino acids had been discovered, leading to the suggestion that
methylation could result in a more permanent change in transcriptional activity than
changes in the acetylation state. This proposal is supported by the fact that methylation
of lysine 9 of histone H3 (H3K9) is highly associated with heterochromatin, a region of
the genome that is transcriptionally repressed (SIMS et al. 2003). The recent discovery
of enzymes that remove methyl groups suggests that, like acetylation, histone
methylation is also reversible (SHI et al. 2004; TSUKADA et al. 2006; WHETSTINE et al.
2006). However, the association of H3K9 methylation with heterochromatin implicates
this modification in generating or maintaining long-term transcriptional repression.
Chromatin structure also can be modulated through the activities of ATP-
dependent chromatin-remodeling enzymes. These enzymes utilize ATP to change the
contacts between histones and the DNA and to move nucleosomes along the DNA.
Proteins with these activities are involved with depositing nucleosomes on newly
replicated DNA as well as in transcriptional control. In transcriptional control, the
activities of ATP-dependent chromatin-remodeling enzymes are context dependent and
can result in either transcriptional activation or repression (SUDARSANAM and WINSTON
2000).
Chromatin-modifying enzymes do not necessarily act individually. Sometimes
these enzymes function in a step-wise fashion. For example, methylation on H3K9 is
blocked by prior acetylation of the same residue. Thus H3K9 methyltransferases often
require the prior activity of histone deacetylases. In other instances, histone
deacetylases depend on the actions of chromatin-remodeling enzymes, which alter the
chromatin structure to allow for deacetylation.
Enzymes that can modify chromatin structure are recruited to specific areas of
the genome through their interactions with transcription factors that bind sequence-
specifically to DNA. Regulation of these protein-protein interactions allow transcription
levels of individual genes to be modulated during certain times in development, in
specific cell types or through the cell cycle. One well-studied example of such a highly
regulated transcription factor is the E2F family of proteins. They are regulated in large
part through their interactions with a family of proteins that are related to the tumor
suppressor Rb.
Pocket proteins and their E2F binding partners are conserved across species
The retinoblastoma gene (Rb) was the first identified tumor suppressor. The
original mutation was discovered because of its causative role in a hereditary form of
pediatric retinoblastoma (FRIEND et al. 1986; FUNG et al. 1987; LEE et al 1987).
Mutations in Rb have subsequently been 
found in a large number of human cancers,
and the Rb-pathway is probably inactivated in most human cancers. The gene encodes
a 928-amino acid protein that has several functional domains. Together, two domains in
the C-terminus, the Walker A and B domains, form the central pocket, and it is to this
region that many of the proteins that interact with Rb bind. Many proteins bind Rb
through an LXCXE motif (where X is any amino acid), which has been shown to
associate with domain B (WHYTE et al. 1988; DYSON et al. 1989; LUDLOW et al. 1989; LEE
et al. 1998). However, proper folding of domain B can only be achieved through
interactions with domain A (LEE etal. 1998). Additional Rb-binding partners, such as
E2F proteins, interact with the Rb pocket region, but do not utilize an LXCXE motif for
the interaction (HUANG et al. 1992; LEE et al. 1998), suggesting these proteins bind at
distinct sites, and that Rb could interact with multiple binding partners simultaneously.
Mammals contain two additional pocket-domain-containing proteins, p107 and
p130 (EWEN etal. 1991; HANNON etal. 1993; LI etal. 1993; MAYOL etal. 1993). The
sequences of these two proteins are more similar to each other than to Rb, but all three
proteins contain the pocket domain and have been shown to have many of the same
binding partners. p107 and p130, but not Rb, both have a spacer region in domain B
that contains binding sites for cell-cycle regulated kinases, allowing p107 and p130 to
specifically interact with these cyclin-dependent kinases (EWEN et al. 1992; FAHA et a.
1992; LEES etal. 1992). In addition, the N-termini of p107 and p130 differ significantly
from Rb. Despite the conserved pocket 
domains and the many shared binding partners,
p107 and pl30 are functionally distinct from Rb, as demonstrated by the fact that
mutations in p 107and p130 have not been found in a large number of cancers.
However, studies of knockout mice demonstrated that functional redundancies exist
among these three proteins and that p107 and p130 might have roles as tumor
suppressors (COBRINIK et al. 1996; LEE et al 1996).
Rb-/- embryos die between day 13 and day 15 (CLARKE et al. 1992; JACKS et al.
1992; LEE etal. 1992). By contrast, p107and p130 null mice in the same genetic
background, 129/sv, develop normally (COBRINIK etal. 1996; MULLIGAN and JACKS
1998). Mice mutant for both p107and p130 die shortly after birth, suggesting that p107
and p130 have redundant functions important for development (COBRINIK et al. 1996).
Characterization of mice mutant for Rb and either p107or p130 also show that
functional overlap exists between these proteins, as doubly mutant embryos die earlier
than Rb-/- embryos alone (LEE et a. 1996). As highlighted by the fact that in a Balb/c
genetic background rather than in a 129/sv background p130-/- causes embryonic
lethality and p 107-/- causes severe developmental defects (LECOUTER et al. 1998a;
LECOUTER et al. 1998b), it is important only to compare phenotypes for mutations made
in the same genetic backgrounds.
While there are published reports of Rb interacting with over 100 different
proteins (MORRIS and DYSON 2001), the function of Rb is understood best in relation to
the E2F family of sequence-specific DNA-binding transcription factors. E2F DNA-
binding activity is generally attributed to heterodimeric complexes that contain one
member of the E2F family and one member of the DP family. There are eight identified
mammalian E2F proteins, five of which (E2F1-5) have been shown to interact with
pocket proteins. Six of the E2F proteins have been shown to form heterodimers with
either one of the two mammalian DP-family members (TRIMARCHI and LEES 2002). The
recently identified E2F7 and E2F8 have two DNA-binding domains and do not require
dimerization with a DP-family member to bind DNA (DE BRUIN et al. 2003; DI STEFANO et
al. 2003; LOGAN etal. 2004; CHRISTENSEN etal 2005; LOGAN etal. 2005; MAITI et al.
2005).
E2F-family members bind to DNA and, when not bound by pocket proteins, can
activate transcription of nearby genes. By contrast, interactions of pocket proteins with
their E2F-binding partners generally result in transcriptional repression of E2F-
responsive genes. Characterization of the different E2F-family members suggests that
some are generally found bound to DNA without an associated pocket protein and are
predominantly involved in transcriptional activation, E2F1-3, while others are more often
associated with transcriptional repression and are likely found bound to DNA and a
pocket protein simultaneously, E2F4-5. Overexpression of E2F1, E2F2, and E2F3
results in transcriptional activation (HELIN et al. 1992; JOHNSON et al. 1993; LEES et al.
1993; QIN etal. 1994; LUKAS et al. 1996), while E2F4, E2F5, and E2F6 have been
associated with transcriptional repression 
(LUKAS etal. 1996; MANN and JONES 
1996;
TRIMARCHI etal. 1998).
The different results that have been observed in the overexpression studies
might result from distinct subcellular localizations of the two classes of E2F family
members. E2F1, E2F2, and E2F3, which have been associated with transcriptional
activation, have a nuclear localization signal and are consistently localized to the
nucleus. By contrast, the repressor E2Fs, E2F4 and E2F5, each have a nuclear export
signal and are found in the nucleus only during GO and G1. Binding to their partner
pocket proteins can induce nuclear localization of E2F4 and E2F5 (TRIMARCHI and LEES
2002).
Individual pocket proteins associate with subsets of the E2F-family members. Rb
has been found to specifically associate with E2F1, E2F2, and E2F3, and to a lesser
extent E2F4. Conversely, p107 and p130 interact with the repressor E2F proteins, E24
and E2F5 (TRIMARCHI and LEES 2002).
Homologs of these mammalian pocket proteins can be found in a large number
of species. Drosophila melanogaster contains two Rb homologs, RBF1 and RBF2, two
E2F family members, dE2F1 and dE2F2, and a single DP family member, dDP
(DYNLACHT etal. 1994; OHTANI and NEVINS 1994; HAO et al. 1995; Du et al. 1996;
SAWADO et al. 1998; FROLOV et al 2001; STEVAUX et al. 2002). The amino acid
sequences of RBF1 and RBF2 are both more similar to the p107 and p130 than they
are to mammalian Rb. Similar to the characterization of "activator" E2F proteins and
"repressor" E2F proteins found in mammals, 
dE2F1 has been linked with transcriptional
activation and dE2F2 has been associated with transcriptional repression (FROLOV et al.
2001). As discussed above, Caenorhabditis elegans contains 
a single pocket protein,
LIN-35 (Lu and HORVITZ 1998). However, LIN-35 is more similar in sequence to the
mammalian pocket proteins p107 and p130. C. elegans also contain two E2F-like
proteins, EFL-1 and EFL-2 and a single DP family member, DPL-1 (CEOL and HORVITZ
2001; PAGE et al. 2001). The reduced complexity of these organisms makes studies of
the functions of these proteins simpler than in mammalian systems; however there are
obvious functional differences among the homologs. For example, animals that are null
for the only worm pocket protein are viable, whereas mice lacking multiple pocket
proteins die as fetuses (CLARKE et al. 1992; JACKS et al. 1992; LEE et al. 1992; COBRINIK
etal. 1996; LEE etal. 1996; Lu and HORVITZ 1998).
The role of Rb in cell cycle control
The initial identification of a potential role for Rb in controlling cell-cycle
progression was through the observation that DNA tumor viruses transform cells by the
ability of viral proteins, such as adenovirus E1A, SV40 large tumor antigen, and human
papillomavirus (HPV) E7, to bind and inactivate Rb (DECAPRIO et al. 1988; WHYTE et al.
1988; DYSON et al 1989). It was shown that these proteins bind to Rb through their
LXCXE motifs and induce inappropriate entry into the cell cycle. Subsequent studies
have resulted in a detailed understanding of how Rb, through its interactions with E2F,
might control cell cycle progression.
The cell cycle is divided up into four distinct phases: G1, S, G2, and M. G1 and
G2 are gap phases and allow time for the cell to grow and monitor its metabolic state.
During S phase, the cell replicates its DNA, and during M phase, the cell divides. GO
refers to the state of cells that have exited the cell cycle. When stimulated, cells in GO
reenter the cell cycle in G1. The progression of the cell through the cell cycle is
regulated by cyclins and cyclin-dependent kinases (cdks) (Figure 2A). Cyclin-dependent
kinases are a set of proteins whose ability to phosphorylate their substrates varies
through the cell cycle and whose activities are controlled by binding with specific cyclins.
Figure 2: Cell-cycle progression is regulated by the sequential activation of multiple
proteins.
A) Different cyclin-dependent kinases are sequentially activated by expression of their
cyclin binding partners.
B) Rb is phosphorylated as cells progress through the cell-cycle. Phosphorylation state
is indicated by the circled P.
C) The chromatin factors associated with pocket proteins are controlled by the level of
phosphorylation. In GO, pocket proteins are hypophosphorylated and can interact with
both HDAC and BRG1. This complex can repress expression from both the cyclinE and
cyclinA promoters. As the cell progresses through G1, cyclinD/cdk4/6 can
phosphorylate the pocket proteins and inhibit interaction with HDAC. cyc/inE expression
is activated. By contrast, the association of pocket proteins with BRG1 can maintain
repression of cyclinA. cdk2 can now associate with cyclinE and further phosphorylate
the pocket proteins, releasing BRG1 and allowing for cyclinA expression. Activation of
cyclinA/cdk2 permits the cell to progress through S phase.
0)
1.
Figure 2
G00
)
V
G2
P P p
od.A
/ cyclinE
cyclinA
cyclinE
cyclinA
cyclinE
cyclinA
1
While the levels of cdks are relatively constant throughout the cell cycle, cyclin levels
vary substantially.
Rb contains 16 potential cyclin dependent kinase (cdk) phosphorylation sites. As
a consequence of cdk regulation, Rb alternates between hypophosphorylated and
hyperphosphorylated states as cells progress through the cell cycle. In GO, Rb is
hypophosphorylated, and this form of the protein binds tightly to E2F proteins. Cyclin D-
cdk4/6 phosphorylates Rb during early G1, cyclin E/cdk2 phophorylates Rb near the
end of G1, and cyclin A/cdk2 might maintain phosphorylation of Rb during S phase. As
Rb becomes increasingly phosphorylated contacts with different binding partners are
interrupted and, in the simplest model, hyperphosphorylated Rb can no longer bind to
E2F and repress its function as a transcriptional activator, allowing expression of E2F-
regulated cell cycle genes (TRIMARCHI and LEES 2002).
The details above clearly are simplified. Rb is not the only pocket protein that can
regulate transcription of genes necessary for progression through the cell cycle. In
addition, levels of each pocket protein change as the cell progresses through the cell
cycle. p130 levels are highest in non-cycling quiescent and differentiated cells.
Conversely, p107 levels rise as the cell is stimulated to proliferate (CLASSON and DYSON
2001). In addition, studies have shown that the spectrum of interactions between pocket
proteins and the E2F family of proteins are not static throughout the cell cycle. During
GO, the most predominant E2F/pocket-protein 
complexes are repressor complexes,
either E2F4 or E2F5 bound by p130. As the cell progresses into G1 these complexes
are replaced by pocket-protein complexes containing either E2F1, E2F2, E2F3, or
E2F4. Finally, during S phase many of the activator E2F proteins are free and able to
activate transcription (TAKAHASHI et al 2000; RAYMAN et al. 2002).
Pocket proteins repress E2F-responsive genes by two distinct mechanisms
E2F proteins positively regulate transcription of many genes required for cell
cycle progression. While E2F1-5 all contain a potential transactivation domain,
overexpression of only E2F1, E2F2, and E2F3 can drive cells into the cell cycle
(JOHNSON et al. 1993; QIN et al. 1994; LUKAS et al. 1996), suggesting that these three
proteins, but not E2F4 and E2F5, cause potent transcriptional activation of genes
required for cell cycle entry. In addition, recruitment of E2F to promoters can lead to
activation of transgenes containing upstream E2F binding sites (HELIN et al 1992; SHAN
et al. 1992).
E2F might activate transcription by a number of different mechanisms. E2F binds
directly to the TATA-binding protein, a key component of the transcriptional machinery
(HAGEMEIER et al 1993; EMILI and INGLES 1995). This interaction could allow E2F to
recruit the transcriptional machinery to the promoters of specific genes, and this, in turn,
can lead to transcriptional activation. Alternatively, E2F recruits transcriptional
coactivators, like CREB-binding protein (CBP), to promoter regions. CBP is able to
interact with known transcriptional activators, such as histone acetyltransferase
complexes, and can bridge an interaction between these activators and transcription
factors, like E2F, that are bound to DNA (TROUCHE et at. 1996). Finally, E2F binding can
alter the DNA structure at the promoter, and this bending of the DNA can cause
transcriptional activation even in the absence of a functional E2F activation domain
(CRESS and NEVINS 1996).
Experiments in which Rb is overexpressed suggest that the binding of pocket
proteins to E2F causes transcriptional repression (HAMEL etal. 1992). Pocket proteins
bind close to the transactivation domain of E2F, allowing them to repress transcription
by blocking the ability of E2F to interact with proteins required for activation
(FLEMINGTON et al. 1993; HELIN et al. 1993).
Rb also has an active role in transcriptional 
repression (BREMNER et al. 1995;
SELLERS et al. 1995; WEINTRAUB et al. 1995). Artificial recruitment of Rb to promoters in
the absence of E2F can result in transcriptional repression, suggesting that some
function of Rb itself is capable of mediating transcriptional repression (BREMNER et al.
1995; SELLERS et al. 1995). The ability to actively repress transcription is shared by the
other pocket proteins and this function is mediated by the ability of the pocket proteins
to interact with a large number of enzymes that modify chromatin structure.
Pocket proteins interact with histone deacetylases
Pocket proteins can actively repress transcription through the recruitment of
histone deacetylase activity to the promoters of E2F-repsonsive genes (BREHM et al.
1998; FERREIRA etal. 1998; Luo etal. 1998; MAGNAGHI-JAULIN etal. 1998). The pocket
domains of Rb, p107, and p130 are essential for the interaction of these proteins with
histone deacetylase 1 (HDAC1) (BREHM et al. 1998; FERREIRA et al 1998; Luo et al.
1998; MAGNAGHI-JAULIN et al. 1998; RAYMAN et al. 2002). Despite the fact that both E2F
proteins and HDAC1 interact with pocket proteins through the pocket domain, ternary
complexes can be formed containing an E2F, a pocket protein, and HDAC1 (BREHM et
al. 1998; MAGNAGHI-JAULIN et al. 1998; FERREIRA et al. 2001). The existence of such
ternary complexes suggests that pocket proteins can actively repress transcription by
interacting with E2F and recruiting histone deacetylase activity to promoters.
HDAC1 contains an IACEE motif, which is similar to the LXCXE motif that
mediates the interactions of viral proteins with Rb. This IACEE motif might be important
for the interaction between pocket proteins and HDAC1 (BREHM et al. 1998; FERREIRA et
al 1998; MAGNAGHI-JAULIN etal. 1998), since HDAC1 lacking the IACEE motif no longer
strongly associates with pocket proteins. Because viral proteins interrupt the interaction
between HDAC1 and pocket proteins, it is likely that both viral proteins and HDAC1
interact with pocket proteins through the same domain (BREHM et al 1998; FERREIRA et
al. 1998; MAGNAGHI-JAULIN et al 1998). However, it remains unclear whether the
interactions between HDAC1 and pocket proteins are directly mediated by the IACEE
motif or whether other proteins are involved in the interaction (KENNEDY et al. 2001; LAI
et al. 2001).
The interaction of Rb with histone deacetylases is not limited to HDAC1. Rb can
also interact with HDAC2 and with HDAC3. HDAC2, but not HDAC3 contains an IACEE
motif that might be important for its interactions with pocket proteins (DAHIYA et al.
2000). Histone deacetylases have been categorized into three distinct classes based on
sequence similarity. HDAC1, HDAC2, and HDAC3 are all class I histone deacetylases
that are most similar to the yeast histone deacetylase Rpd3. Class II histone
deacetylases are more similar to yeast Hdal but share similarity to class I histone
deacetylases in their active sites. Class III histone deacetylases require an NAD
cofactor and are more similar to yeast Sir2 (EKWALL 2005). No interaction was detected
between Rb and the class II histone deacetylases HDAC4-6 (DAHIYA et al. 2000).
The interaction between Rb and histone deacetylases controls the acetylation
state of histones at certain promoters, and the acetylation state is correlated with
changes in transcription levels (FERREIRA et al. 2001; MORRISON et al. 2002; SIDDIQUI et
al. 2003). At the cyclin Epromoter, which is specifically regulated by Rb through its
interaction with E2F, Rb recruitment of HDAC activity can result in the deacetylation of
histones H3 and H4 of a single nucleosome located at the transcriptional start site. It is
to this nucleosome that Rb and HDAC1 are localized (MORRISON et al. 2002). Thus, the
recruitment of Rb to the cyclin Epromoter by E2F proteins might result in transcriptional
repression, at least in part, through the deacetylation of this nucleosome. Rb also
mediates H4 deacetylation at the cyclin A, cdc2, topoisomerase Ila, and thymidylate
synthase promoters. This deacetylation likely causes transcriptional repression as
Trichostatin A (TSA), an HDAC inhibitor, prevented repression of many of these genes
(SIDDIOUI et al. 2003). Additionally, DHFR is an E2F target gene that is silent in early G1
and is expressed at the G1- S transition. HDAC1 is specifically associated with the
DHFR promoter during GO and G1, during which time the promoter is hypoacetylated
and transcriptionally inactive. During the transition into S phase, HDAC1 is no longer
associated with the promoter, and the promoter becomes acetylated at lysines 5 and 12
of histone H4 (FERREIRA et al. 2001). These data suggest that the association of pocket
proteins with histone deacetylase activity is likely to be functionally relevant in
controlling the expression levels of at least some E2F-responsive genes.
Studies of histone deacetylases from a number of organisms have demonstrated
that they are often found in large, multimeric complexes. HDAC1 and HDAC2 have been
found to be components of both the mammalian Nucleosome Remodeling and
Deacetylase (NuRD) complex and the Sin3 complex (XUE et al. 1998; ZHANG et al.
1998a; ZHANG etal. 1998b). In addition to HDAC1 and HDAC2, these complexes both
contain RbAp48 and RbAp46 but differ in a number of other components (QIAN et al.
1993; XUE et al. 1998; ZHANG et al. 1998a; ZHANG et al. 1998b. RbAp48 and RbAp46
were initially identified as proteins that could bind to Rb in vitro {Qian, 1995 #22). They
were later shown to be components of a number of chromatin-remodeling complexes
and are capable of binding histone H4 (VERREAULT et al. 1996; XUE et al. 1998; ZHANG
et al. 1998a; ZHANG et al 1999). Pocket proteins specifically interact with the Sin3
complex, but not with the NuRD complex (LAI et al. 2001; RAYMAN et al. 2002).
The Sin3 complex is recruited by the LXCXE-containing protein RBP1 to pocket
proteins rather than through direct interaction of HDACs with pocket proteins (LAI et al.
2001). Despite the requirement of RBP1 for Sin3 recruitment, some Rb-associated
HDAC activity remains in the absence of RBP1 (LAI etal. 2001). This activity could
come from a number of different sources. The HDAC activity could result from
interactions between Rb and HDAC3, which is not a member of the Sin3 complex.
Alternatively, the residual activity could be accounted for by direct interaction between
Rb and HDAC1 or HDAC2 that is not associated with the Sin3 complex as has been
proposed (BREHM et al 1998; MAGNAGHI-JAULIN et al. 1998). Finally, HDAC activity could
be recruited to Rb by a protein distinct from RBP1. For example, RbAp48 is a protein
that can mediate interactions between Rb and HDAC1, HDAC2, and HDAC3 (NICOLAS
etal. 2000; NICOLAS etal. 2001).
Studies of murine cells have demonstrated that Sin3 and HDAC1 are associated
with the promoters of some endogenous genes in primary cells during quiescence but
that these proteins dissociate during G, and S phase when gene expression is initiated
(RAYMAN et al. 2002). The recruitment of HDAC1 to specific promoters in quiescent cells
depended on the presence of p107 and p130 but not Rb (RAYMAN et al 2002). Thus, at
least in quiescent cells, HDAC-mediated repression of E2F-responsive genes might
depend primarily on the pocket proteins p107 and p130. This is in agreement with data
showing that during quiescence specific E2F promoters are predominantly occupied by
a repressor E2F, E2F4, bound by p130 and are hypoacetylated. By late G
1
, these
promoters are bound by the activator E2F proteins, E2F1 and E2F3, and are acetylated
(TAKAHASHI et al. 2000).
The recruitment of histone deacetylase activity to promoters of E2F-responsive
genes is an important means for pocket proteins to repress transcription whether
HDACs are recruited individually or as components of the larger Sin3 complex.
However, histone deacetylation is not the only means of Rb-mediated transcriptional
repression. All pocket proteins retain the ability to partially repress a reporter construct
with upstream E2F binding sites even in the presence of the HDAC inhibitor Trichostatin
A, indicating that these proteins can repress these transgenes through an HDAC-
independent pathway (FERREIRA et al. 1998). In addition, the repression of the SV40
enhancer or the herpes virus thymidine kinase promoter by artificial recruitment of Rb
cannot be alleviated by the addition of TSA, further demonstrating that this repression is
not dependent on HDAC activity (Luo et al. 1998). The expression of a constitutively
active form of Rb results in cell cycle arrest, and this arrest cannot be alleviated by the
addition of TSA, showing that even without HDAC function the constitutively active form
of Rb is likely to be repressing genes necessary for progression through the cell cycle
(SIDDIQUI et al. 2003). These data demonstrate that pocket proteins do not repress
transcription only through the recruitment of histone deacetylase activity. Consistent
with these data, pocket proteins have been found to interact with a number of other
enzymes that modify chromatin structure.
Pocket proteins interact with ATP-dependent chromatin-remodeling enzymes
BRG1 (brahma/SWI2-related gene 1) is in a class of proteins related to the yeast
SWI2/SNF2 ATP-dependent chromatin-remodeling enzyme (KHAVARI et al. 1993).
Murine BRG1 was initially shown to bind to Rb in a yeast two-hybrid assay (DUNAIEF et
al. 1994). Given the yeast two-hybrid interaction, 
that BRG1 contains an LXCXE motif,
that BRG1 interacts with the pocket region of Rb and that the interaction between BRG1
and Rb can be disrupted by viral oncoproteins, it is likely that the interaction between
BRG1 and Rb is direct (DUNAIEF et al. 1994). Experiments using SW13 cells that lack
BRG1 demonstrated a functional role for BRG1 in Rb-mediated cell-cycle arrest
(DUNAIEF et al. 1994; STROBECK et al 2000; ZHANG et al. 2000). Overexpression of
BRG1 in SW13 cells can cause growth arrest and flattened cell morphology, indicating
the cells can no longer properly progress through the cell cycle. This morphology and
growth arrest is dependent on interaction with pocket proteins (DUNAIEF et al. 1994).
BRM (brahma), another mammalian ATP-dependent chromatin-remodeling
enzyme similar to yeast SWl2/SNF2, is also capable of binding to pocket proteins in the
yeast two-hybrid system (STROBER et al. 1996). Expression of BRM, like BRG1, in
SW13 cells induces flattened, growth-arrested cells, suggesting BRM might also
function with pocket proteins in mediating cell-cycle arrest (STROBECK etal. 2000). BRM
and BRG1 are functionally redundant in cooperating 
with Rb to mediate cell-cycle arrest,
since only cell lines lacking both proteins are resistant to the cell-cycle arrest caused by
constitutive activation of Rb, while cell lines lacking only BRG1 are not resistant
(STROBECK et al. 2002). These data show that BRM and BRG1 both bind Rb and
mediate the ability of Rb to block cell-cycle progression.
SWI2/SNF2 was initially identified in yeast as a transcriptional activator (LAURENT
et al. 1991; LAURENT et al 1993). However, BRG1 binds to the hypophosphorylated
form of Rb (DUNAIEF et al. 1994), suggesting that BRG1 might cooperate with Rb to
mediate transcriptional repression. hBRM forms a complex with E2F1 and Rb, and this
interaction represses E2F1-driven transcription in transient transfection assays
(TROUCHE et al. 1997). By contrast to this role in transcriptional repression, there are
also data to suggest that BRM cooperates with Rb to activate E2F-independent
transcription by the glucocorticoid receptor (SINGH et at 1995). Together these results
demonstrate that the functional consequence of the interaction between Rb and BRM
likely depends on the promoter to which these proteins are recruited.
An in vivo role for SWI/SNF-like proteins in regulating E2F-responsive genes also
was shown in Drosophila. Loss-of-function mutations 
in the fly homologs of SWI1,
SWI2, and SWI3, brahma, moira, and osa, respectively, enhanced the rough eye
phenotype caused by overexpression of dE2F1 and dDP. A similar phenotype was
caused by loss-of-function in a Drosophila Rb homolog RBF1, suggesting that
Drosophila SWI/SNF proteins function together with Rb in the fly eye (STAEHLING-
HAMPTON et al. 1999).
The role of SWI/SNF proteins in Rb-mediated transcriptional repression was
demonstrated for the cyclin A promoter. Neither the HDAC inhibitor TSA nor the DNA-
methylation inhibitor 5-aza-2-deoxycytidine were able to block the transcriptional
repression of the cyclin A promoter caused by constitutive activation 
of Rb (ZHANG et al.
2000; SIDDIOUI et al. 2003). By contrast, overexpression of a dominant negative form of
BRG1 could alleviate some of the repression (SIDDIouI et al. 2003). These data suggest
that HDAC activity and ATP-dependent chromatin remodeling might separately mediate
repression by pocket proteins by acting at different promoters. By contrast, data for
repression of Plkl, another E2F-regulated gene, demonstrate that in some cases HDAC
activity could be dependent on prior chromatin-remodeling (GUNAWARDENA et al. 2004).
Repression of Plkl expression by pocket proteins requires functional ATP-dependent
chromatin-remodeling enzymes, since activation of pocket proteins only repressed Plkl
in cells that expressed BRG1 and BRM1. E2F proteins and pocket proteins still
associated with the Plkl promoter in the 
absence of these two proteins. However,
deacetylation of the Plkl promoter required functional BRG1 and BRM1 (GUNAWARDENA
et al. 2004). Thus for the Plkl promoter prior chromatin remodeling by BRG1 or BRM1
might be required for histone deacetylation.
Pocket proteins interact with histone methyltransferases
In addition to histone deacetylases and ATP-dependent chromatin-remodeling
proteins, pocket proteins have also been shown to interact with the histone
methyltransferase SUV39H1, possibly through its LXCXE motif (NIELSEN et al. 2001;
VANDEL et al. 2001; NICOLAS et al. 2003). SUV39H1 is the human homolog of the
Drosophila protein Su(var)3-9 that was initially identified in screens for mutations that
could suppress the silencing of genes located in heterochromatic regions. It was later
shown that Su(var)3-9 is a methyltansferase that methylates lysine 9 of histone H3
(H3K9), a mark often associated with heterochromatin. Further research demonstrated
that the H3K9 methyl mark can be bound by the chromodomain protein HP1 (BANNISTER
et al. 2001), and that HP1 is also associated with silenced chromatin (JAMES et al.
1989).
A domain shared between Su(var)3-9 and other methyltransferases, the SET
domain, is known to be necessary for the methyltransferase activity. Expression of
SUV39H1 can repress the cyclin Epromoter when coexpressed with Rb only when the
SET domain is intact, indicating that the histone methyltransferase activity of SUV39H1
is necessary to mediate this repression. In agreement with these data, cyclin E
expression is upregulated in cells lacking SUV39H1 and the closely related histone
methyltransferase SUV39H2 (NIELSEN et al. 2001). The interaction between Rb and
SUV39H1 might be cell-cycle regulated, as phosphorylation of Rb by cyclin E/cdk2
interrupts the interaction between the two proteins (VANDEL et a. 2001). Rb is capable of
interacting with both SUV39H1 and E2F, so it is possible that Rb is recruiting SUV39H1
to E2F-responisve promoters (VANDEL et al 2001).
Rb also binds HP1 with the yeast two-hybrid assay (WILLIAMS and GRAFI 2000),
and HP1 can coimmunoprecipitate Rb from nuclear extract (NIELSEN et al. 2001).
Furthermore, HP1, Rb, and SUV39H1 can simultaneously bind to a methylated H3
peptide. Thus these three proteins might form a complex when bound to modified H3
that is incorporated into chromatin (NIELSEN et al. 2001).
The ability of Rb to recruit a histone methyltransferase to promoters was
demonstrated to be important for the cyclin Epromoter. A single nuceleosome is
required for the regulation of cyclin Etranscription (MORRISON et al. 2002). This
nucleosome is methylated at lysine 9 of histone H3 and is bound by HP1 only in Rb+/+
and not Rb-/- cells, demonstrating that methylation and binding of HP1 might be a
mechanism to ensure repression of the cyclin Egene (NIELSEN et al. 2001).
Studies of the endogenous DHFR promoter indicate that methylation also is likely
to be important in regulating expression of this gene. During GO, histones in the
promoter are methylated at lysine 9 of histone H3, but as the cells progress to the G
1
-S
transition the methyl mark disappears and the H3 tails are primarily acetylated (NICOLAS
etal. 2003). These data combined with the fact that HDAC activity is associated with the
DHFR promoter during GO (FERREIRA et al 2001) suggest that there might be a
cooperative interaction between HDAC activity and histone methyltransferase activity to
properly control the expression of some E2F-responsive genes. Because histone H3
acetylation of lysine 9 can block methylation of the same residue, such cooperation
between histone deacetylases and methyltransferases has been proposed. In some
cases methyltransferases might require HDAC activity to first remove the acetyl group
from the lysine. This model is supported by the fact that SUV39H1 has been shown to
interact with histone deacetylases as well as with RbAP48 and RbAp46, components of
a number of histone deacetylase-containing complexes (ZHANG et al 1997; XUE et al.
1998; ZHANG et al. 1998a; VAUTE et al. 2002).
Given the association between the 
H3K9 methyl mark and heterochromatin,
methylation on lysine 9 of histone H3 is correlated with long term repression of gene
expression. Pocket proteins are known to have roles both in cell cycle control and in
differentiation. The transcriptional repression of cell cycle-regulated genes must be
alleviated as the cell progresses through the cell cycle. By contrast, as cells differentiate
and exit the cell cycle these genes often become stably repressed. It has been
proposed that the more transient repression of cell-cycle genes in cycling cells might be
regulated by histone deacetylation that is easily reversed. In differentiating cells, the
more permanent repression of the cell-cycle genes might be achieved through
methylation and the subsequent binding of HP1.
For the DHFR, B-Myb, cyclin E, and cyclin DI promoters, histone acetylation
increases in cells as they progress through the cell cycle. Acetylation levels are low in
both GO of cycling cells and in differentiated cells (AIT-SI-ALI et al. 2004). Levels of
H3K9 methylation at the DHFR promoter do not change as cells progress through the
cell cycle. Conversely, in differentiating cells H3K9 methylation levels at the DHFR, B-
Myb, cyclin E, and cyclin D1 promoters increase dramatically as the cells differentiate
(AIT-SI-ALI etal. 2004). The transcriptional repression 
of cyclin D1 in differentiating cells,
but not in cycling cells is dependent on the histone methyltransferase SUV39H1 (AIT-SI-
ALl et al. 2004). These data suggest that in differentiating cells Rb might utilize
interactions with SUV39H1 to repress transcription, while in cycling cells Rb recruits
histone deacetylases.
Rb also has a role in the formation of heterochromatic bodies during senescence
and is found at the promoters of many E2F responsive genes in senescent, but not
quiescent cells where p107 and p130 appear to be the predominant pocket proteins
(RAYMAN et a. 2002; NARITA etal 2003). The interaction between Rb and SUV39H1
might be important for the role of Rb in the formation of this heterochromatic region and
for long-term repression of E2F-responsive genes.
Pocket proteins interact with additional potential chromatin modifying enzymes
The best characterized chromatin modifying proteins associated with pocket
proteins are those discussed above, histone deacetylases, ATP-dependent chromatin-
remodeling proteins, and histone methyltransferases. However, pocket proteins are
known to associate with over 100 different proteins, and these include additional
proteins that might be involved in chromatin remodeling.
Rb also interacts with the Jumonji domain 2-containing protein JMJD2A (GRAY et
al. 2005), which has recently been shown to be a demethylase for trimethylated lysine 9
and trimethylated lysine 36 of histone H3 (WHETSTINE et al. 2006). JMJD2A
demethylation results in dimethylated lysines, which are likely to have functions distinct
from the trimethylated form of H3K9 (WHETSTINE et al. 2006). In addition to interacting
with Rb, JMJD2A can interact with class I histone deacetylases and the coreceptor N-
CoR and when recruited to promoters can repress transcription (YOON et al. 2003; GRAY
et al. 2005; ZHANG et al. 2005).
Rb is associated also with the DNA methyltransferase DNMT1 (ROBERTSON et al.
2000; PRADHAN and KIM 2002), which is the major methyltransferase acting to maintain
DNA methylation through the cell cycle (LEONHARDT et al. 1992). DNA methylation has
been linked with many biological processes, including gene silencing (BIRD and WOLFFE
1999), suggesting that recruitment of DNMT1 could function in transcriptional
repression. DNMT1 interacts with Rb, E2F, and HDAC1 and acts as a transcriptional
repressor when recruited to promoters (ROBERTSON et al. 2000). However, other data
demonstrate that Rb instead might act by binding to DNMT1 and inhibiting the
interaction between DNMT1 and its substrate DNA. This interaction interferes with the
ability of DNMT1 to methylate DNA and could possibly lead to transcriptional activation
(PRADHAN and KIM 2002).
Pocket proteins likely control different sets of genes by different mechanisms
Given that pocket proteins interact with a large number of proteins involved in
gene regulation and the fact that a large number of genes are regulated by these
proteins, it is likely that various protein combinations will control transcription at different
gene targets (Figure 3A). Studies of expression of the cyclin A and cyclin Egenes have
demonstrated that transcriptional repression at these promoters, while both mediated by
Rb and its interaction with E2F, is controlled by different chromatin modifying enzymes.
Furthermore, this difference in transcriptional control can 
help to explain how Rb,
through its interactions with E2F proteins and chromatin-remodeling enzymes, can
control cell-cycle progression.
Overexpression of Rb and BRG1 in SW13 cells arrested them in S phase and not
in G1. Thus Rb and BRG1 likely function together to control the transition between S
and M phase rather than in the earlier G1-S transition (ZHANG et al. 2000). Rb and
BRG1 might retain the ability to repress transcription during G1. Indeed, the cyclin A
gene, which has been shown to require BRG1 but not HDAC for Rb-mediated
repression, is repressed through the G1 phase, showing that a functional Rb-BRG1
repressor complex likely exists during GI. By contrast, the cyclin Egene, which is
normally activated during G1, is repressed by a histone deacetylase activity (ZHANG et
al. 2000; MORRISON et al. 2002), suggesting that the Rb-HDAC repressor complex is
inactivated during GI. Phosphorylation of Rb by cyclin D/cdk4 during G1 is capable of
interrupting the interaction between Rb and HDAC1 
(HARBOUR et al. 1999). However,
Rb remains associated with BRG1, even when cdk4 is activated by cyclin D (ZHANG et
al. 2000). Activation of cdk2 by cyclin E can disrupt the association between Rb and
BRG1 (ZHANG et al 2000). Along with data showing that Rb, HDAC1, and BRG1 can
form a single complex, these data are most easily explained by a model for the
sequential role of Rb in controlling the expression of cyclin genes. Initially a single
complex including Rb, HDAC1, and BRG1 can repress transcription. The expression of
cyclin D in G1 activates cdk4, which can phosphorylate Rb and disrupt the association
of Rb and HDAC, but leaves a functional Rb-BRG1 complex. Due to the disruption of
Figure 3: Pocket proteins regulate transcription of target genes by association 
with
different transcription factors.
A) Pocket proteins repress transcription of different target genes by association with
different chromatin-remodeling factors. Pocket protein association with HDACs, but not
BRG1, is required to repress cyclin Eexpression. By contrast, repression of cyclinA
requires pocket proteins to recruit BRG1, but not HDACs, to the promoter.
B) Pocket-protein repression of some genes utilizes multiple chromatin-remodeling
activities in a sequential fashion. A single nucleosome in the cyclinEpromoter is
regulated by both histone deaceytlase activity and histone methyltransferase activity.
Histone methyltransferase activity provided by SUV39H1 might require prior
deacetylation of the histone by HDAC.
C) Pocket proteins might utilize association with different chromatin factors to regulate
the same gene in different cellular contexts. The DHFR promoter is methylated in
differentiated cells, presumably from an association of pocket proteins with histone
methyltransferases like SUV39H1. By contrast, in cycling cells the DHFR promoter is
regulated by histone acetylation and is repressed by pocket proteins associating with
HDAC.
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the Rb-HDAC complex, cyclin E is now expressed. Cyclin E/l cdk2 can now
phosphorylate Rb resulting in the interruption of the Rb-BRG1 complex permitting
expression of cyclin A and allowing the cells to progress through S phase (ZHANG et al.
2000).
Chromatin-remodeling enzymes do not only work alone at different promoters but
can cooperate to regulate the transcriptional profile of a gene through the cell cycle or
development (Figure 3B). For example, a single nucleosome in the cyclin Epromoter
can be modified by both methylation and acetylation, suggesting a cooperation between
pocket protein-associated histone deacetylase activity and histone methyltransferase
activity (NIELSEN et al. 2001; MORRISON et al. 2002). Perhaps the histone
methyltransferase requires deacetylation of the histone before the histone can be
methylated. In another example, histone deacetylation of the Plkl promoter required
activity of ATP-dependent-chromatin-remodeling factors (GUNAWARDENA et al 2004).
Finally, the same promoters might be regulated by different transcriptional
regulators at different stages of development or in different cell types (Figure 3C).
Histone methylation of a number of promoters is correlated with differentiation and
permanent exit from the cell cycle (AIT-SI-ALI et al. 2004). HDAC activity or chromatin-
remodeling activity might be more important for regulation of these genes in cycling
cells. Because different pocket proteins can be found associated with the same
promoter under varying circumstances, chromatin-modifying enzymes could be
recruited to the promoter by one specific pocket protein in cycling cells and a different
pocket protein in differentiating cells. For example, p107 was not found associated with
the E2Fand cyclin A promoters during quiescence. However once the cells progressed
through a round of the cell cycle, p107 could be detected readily on the same promoters
(TAKAHASHI et at 2000).
Pocket proteins have roles beyond cell-cycle control
Studies of E2F-responsive genes in Drosophila suggest that, as discussed
above, not all genes regulated by E2F are controlled in the same manner. Microarray
experiments using RNAi to reduce levels of either the activator E2F, dE2F1, the
repressor E2F, dE2F2, the single DP homolog, dDP, or the two pocket proteins, RBF1
and RBF2, either singly or together, in Drosophila SL2 cells demonstrated that the
different E2F proteins did not regulate the same sets of genes (DIMOVA et al. 2003).
Genes activated by dE2F1 included many homologs of genes involved in cell-cycle
regulation that are known to be regulated by E2F in mammalian cells. Loss of dE2F2
did not greatly affect transcription of these genes. Conversely a set of genes that are
repressed by dE2F2 and that were not affected by loss of dE2F1 included many genes
that are sex or cell-type specific and might be involved in developmental regulation
(DIMOVA et al 2003). The microarray data also show significant redundancy among the
two pocket proteins in Drosophila (DIMOVA et al. 2003), as has been suggested for
mammals from the phenotypes of knockout mice (COBRINIK et al. 1996; LEE et al. 1996).
Recently, two complexes have been identified in Drosophila that contain the fly
pocket proteins, RBF1 and RBF2, the Myb-MuvB and dREAM complexes (KORENJAK et
al. 2004; LEWIS et al. 2004), and Chapter 2 of this thesis contains the characterization of
a similar complex from C. elegans, the DRM complex. These 
three complexes contain,
in addition to pocket proteins, a number of other proteins that have been shown
previously to interact with Rb, including dE2F2/ EFL-1, dDP/DPL-1, and p55/LIN-53, a
homolog of RpAp48. In addition to these previously 
identified Rb-associated proteins,
these complexes contain many proteins that have not been previously implicated in
pocket protein function such as Mip40/LIN-37, Mip130/LIN-9, Mipl20/LIN-54, and
dLin52/LIN-52. In Drosophila, these complexes repress genes that might be important
for differentiation, but do not seem to be required for repression of cell-cycle regulated
genes (KORENJAK et al. 2004; LEWIS et al. 2004). In C. elegans, all members of this
complex are all class B synMuv genes, suggesting that this complex is likely involved in
the repression of genes that induce vulval specification.
The data from Drosophila suggest that a large number of genes regulated by
pocket proteins might be involved in differentiation and development (DIMOVA et al.
2003). There is also evidence in mammalian systems that pocket proteins have roles in
developmental regulation (LIPINSKI and JACKS 1999). For example, Rb is required for
neuronal and muscle differentiaton. Some of these genes might be regulated by
interaction of pocket proteins with E2F family members. However it is important to
recognize that E2F proteins are only a subset of the transcription factors bound by Rb
(MORRIS and DYSON 2001). For example, the role of Rb in muscle differentiation is best
understood in its regulation of transcription factors such as MyoD and not E2F.
Interestingly, Rb might function to increase transcriptional activity from promoters
regulated by these transcription factors (LIPINSKI and JACKS 1999).
Conclusions
Pocket proteins interact with a diverse number of transcription factors and
chromatin-remodeling enzymes, and while they have been most well studied as
transcriptional corepressors, they can also lead to gene activation (SINGH et al 1995).
Through these many protein-protein interactions, pocket proteins can regulate
transcription of a large number of genes. Further complexity is found in mammals, which
contain three pocket proteins, eight E2F family members, and two DP proteins. Genetic
background can further complicate studies of these proteins at the organismal level.
This is exemplified by the embryonic lethality caused by a p 130 null allele in a Balb/c
background, but the similar null allele is viable in a 129/sv genetic background
(COBRINIK et al. 1996; LECOUTER et al. 1998a; LECOUTER etal. 1998b; MULLIGAN and
JACKS 1998). Studies of protein function in cell culture systems should be performed
with the knowledge that different cell types might give different results.
Most studies of the role of pocket proteins in transcriptional control have focused
on E2F-regulated genes that are involved in cell-cycle control. More recently it is has
become clear that pocket proteins also have central roles in developmental regulation.
Studies of how these developmentally-controlled genes are regulated by the association
of pocket proteins with different chromatin modifying enzymes are important to get a
better understanding for the role these associations play in vivo.
Studies of non-mammalian systems, such as flies and nematodes, are important
for understanding how pocket proteins and the many proteins with which they interact
function in an organism as a whole. Given both the reduced complexity of these
systems and their amenability to genetic manipulation, studies of flies and nematodes
will aid in resolving the ever-broadening role of pocket proteins.
Acknowledgments
I thank Andrew Mehle, Michael Hurwitz, and Megan Higginbotham for helpful comments
about this chapter.
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Table 1
synMuv genes encode proteins with homologs in other species
Gene name Homolog or domains synMuv class
lin-8 Class A
lin- 15A THAP domain Class A
lin-38 Zinc finger Class A
lin-56 THAP domain Class A
mcd- 1 Zinc finger Class A
lin-9 Mipl30 Class B
lin- 13 Zinc fingers Class B
lin- 15B THAP domain Class B
lin-35 p107, p130 Class B
lin-36 THAP domain Class B
lin-37 Mip40 Class B
lin-52 dLin-52 Class B
lin-53 RbAp48, p55 Class B
lin-54 Mipl20 Class B
lin-61 h 1(3)mbt-like2 Class B
lin-65 Class B
dpl- 1 DP Class B
efl- 1 E2F4, 5 Class B
hpl-2 HP1 Class B
met-2 SETDB1 Class B
hda- HDAC1 Class B
let-418 Mi2 Class B
mep- 1 Zinc fingers Class B
tam-1 RING finger/B-box Class B
sfi-1 cCbl Class B
gap-1 GTPase activating protein Class B
ark- 1 Ack-related tyrosine kinase Class B
epc- 1 Enhancer of Polycomb Class C
mys- 1 Tip60/ Esal p Class C
trr- I TRRAP Class C
ss/-1 SWI/SNF Class C
CHAPTER 2
A subset of C. elegans class B synMuv proteins encodes a conserved
LIN-35 Rb-containing complex distinct from a NuRD-like complex
Melissa M. Harrison, Craig J. Ceol', Xiaowei Lu
2
, H. Robert Horvitz
3
Craig Ceol cloned Iin-54, generated and purified the anti-LIN-54 antiserum and
characterized the LIN-54 expression pattern. Xiaowei Lu cloned lin-37, generated the
anti-LIN-37 antiserum and generated and purified the LIN-53 antiserum.
1Present address: Howard Hughes Medical Institute, Department of
Hematology/Oncology, Children's Hospital, Boston, MA 02115
2
Present address: Department 
of Cell Biology, University 
of Virginia School of Medicine,
Charlottesville, VA 22908
3
Corresponding author: Department of Biology, Howard 
Hughes Medical Institute, Room
68-425, Massachusetts Institute of Technology, 
77 Massachusetts Ave., Cambridge,
MA 02139. E-mail: horvitz@mit.edu
ABSTRACT
The synthetic multivulva (synMuv) genes act redundantly to antagonize the
specification of Caenorhabditis elegans vulval cell fates, which are promoted by an
RTK/Ras pathway. At least 26 synMuv genes have been genetically identified, several
of which encode proteins with homologs that act in chromatin remodeling or
transcriptional repression. Here we report the molecular characterization of two synMuv
genes, lin-37 and lin-54. We show that lin-37 and lin-54 encode proteins in a complex
with at least seven synMuv proteins, including LIN-35, the only C. elegans homolog of
the mammalian tumor suppressor Rb. Three components of this complex are likely to be
required for complex stability. This complex is distinct from a second complex of synMuv
proteins with a composition similar to that of the mammalian Nucleosome Remodeling
and Deacetylase (NuRD) complex. The class B synMuv complex we identified is
evolutionarily conserved and is likely to function in transcriptional repression and
developmental regulation.
INTRODUCTION
Cell differentiation requires coordinated changes in gene regulation. Such
changes in gene expression levels are controlled by transcription factors and often are
mediated through the modification of chromatin states. Biochemical techniques have
identified many protein complexes that function in transcriptional repression and
chromatin remodeling (AYER 1999; BECKER and HORZ 2002). However, the in vivo
functions of these complexes in organismal development have been unclear.
Conversely, genetic studies of the nematode Caenorhabditis elegans have implicated
genes likely to be involved in chromatin-mediated transcriptional repression as
important in the development of the hermaphrodite vulva (see below), but the
biochemical properties of the products of these genes have been largely unknown.
The C. elegans vulva arises from three of six ectodermal blast cells that form the
vulval equivalence group (SULSTON and HORVITZ 1977; SULSTON and WHITE 1980).
These six cells, P3.p-P8.p, are generated during the first of four larval stages (L1). In
the second larval stage (L2) these cells are specified to adopt one of three distinct cell
fates, called primary, secondary, or tertiary, which are distinguished by their patterns of
cell divisions and the descendant cell types they generate. P6.p adopts a primary fate,
dividing to form eight nuclei, while P5.p and P7.p adopt secondary fates, dividing to form
seven nuclei each. These 22 descendants subsequently generate the vulva, through
which sperm can enter and eggs can exit. Although P3.p, P4.p, and P8.p are competent
to adopt vulval fates, instead they adopt a tertiary non-vulval fate, usually dividing once
and fusing with the adjacent syncytial hypodermis. The specification of the primary and
secondary cell fates (as opposed to the tertiary non-vulval cell fate) requires the
activities of several conserved pathways, including receptor tyrosine kinase (RTK)-Ras,
Notch, and Wnt signaling cascades (YOCHEM et al. 1988; BEITEL et al. 1990; HAN et al.
1990; GLEASON et al 2002). Mutations affecting these signaling pathways can cause
animals to lack a vulva and express a vulvaless or Vul phenotype, or to have ectopic
vulval protrusions and express a multivulva or Muv phenotype (YOCHEM et al. 1988;
STERNBERG and HORVITZ 1989; HAN et al. 1990; BEITEL et al. 1995; GLEASON et al.
2002).
59
The specification of the primary and secondary vulval cell fates promoted by
these pathways is antagonized by the synthetic multivulva (synMuv) genes (HORVITZ
and SULSTON 1980; FERGUSON and HORVITZ 1989). The synMuv genes have been
grouped into classes A, B, and C on the basis of genetic interactions (FERGUSON and
HORVITZ 1989; CEOL and HORVITZ 2004). Animals homozygous for loss-of-function
mutations in any single synMuv gene class are not Muv, whereas animals homozygous
for mutations in any two classes are Muv. Some class B synMuv genes encode proteins
with homologs in other species that function in chromatin remodeling and transcriptional
repression. These genes include lin-35, which encodes the only C. elegans homolog of
the mammalian Rb tumor-suppressor protein, and efl- 1 and dpl-1, which encode
proteins homologous to the E2F and DP subunits of the heterodimeric transcription
factor E2F (Lu and HORVITZ 1998; CEOL and HORVITZ 2001). The class B synMuv
proteins LIN-53 RbAp48, HDA-1 HDAC1 and LET-418 Mi2 are homologous to
components of the mammalian Nucleosome Remodeling and Deacetylase (NuRD)
complex (Lu and HORVITZ 1998; XUE et al. 1998; ZHANG et al. 1998a; ZHANG et al. 1999;
SOLARI and AHRINGER 2000; VON ZELEWSKY et al. 2000; CEOL and HORVITZ 2001;
DUFOURCQ et al. 2002; UNHAVAITHAYA et al. 2002).
At least 12 additional class B synMuv genes have products that might act with
LIN-35 Rb or components of a C. elegans NuRD-like complex in determining vulval cell
fates (HORVITZ and SULSTON 1980; FERGUSON and HORVITZ 1989; UNHAVAITHAYA et a.
2002; OWEN et al. 2003; THOMAS et al. 2003; POULIN et al. 2005). However, genetic
techniques are insufficient to determine how the protein products of the synMuv genes
interact. In this study, we report the molecular identification of two class B synMuv
genes, lin-37 and lin-54, and identify a new complex of class B synMuv proteins that
likely controls cell-fate specification through transcriptional repression.
RESULTS
lin-37 and lin-54 are class B synMuv genes: Two alleles 
of lin-37, n758 and n2234,
were previously isolated in independent screens for synMuv mutants (FERGUSON and
HORVITZ 1989; THOMAS et al. 2003). Two lin-54 alleles, n2231 and n2990, were isolated
in a screen for synMuv mutants in the lin-8(n 111) and lin- 15A(n433) class A mutant
backgrounds, respectively (THOMAS et al. 2003). lin-37 and lin-54 are class B synMuv
genes, as alleles in each gene cause a Muv phenotype with mutations in any of the four
identified class A synMuv genes or with mutations in the class C synMuv gene trr- 1
(Table 1; FERGUSON and HORVITZ 1989; THOMAS et al. 2003; data not shown). Like the
synMuv phenotype caused by loss-of-function mutations in other synMuv genes, the
synMuv phenotype caused by lin-37or lin-54 loss of function in a synMuv class A
mutant background requires let-60 Ras pathway activity, but not the upstream lin-3 EGF
signal (FERGUSON et al. 1987; HUANG et al. 1994; Lu and HORVITZ 1998; THOMAS and
HORVITz 1999; CEOL and HORVITZ 2001; CEOL and HORVITZ 2004; POULIN et al. 2005;
data not shown).
LIN-37 encodes a small hydrophilic protein with weakly conserved
homologs in other species: We mapped lin-37to a region of LGIII and performed
transformation rescue of the synMuv phenotype of lin-8(nl 11); lin-37(n758) with
cosmids and subcloned portions of cosmids in the region (Supplemental Results and
Supplemental Figure 1). Frameshift mutations of the predicted genes in the minimal
rescuing fragment demonstrated that lin-37 rescuing activity was encoded by ZK418.4
(Supplemental Results and Supplemental Figure 1). In addition, RNAi targeted against
ZK418.4 caused a highly penetrant synMuv phenotype in a lin- 15A(n767) background
but not in a wild-type or a lin- 15B(n744) background. 
To confirm that ZK418.4 is lin-37,
we determined the sequence of ZK418.4 in the two lin-37 mutants, n758 and n2234.
n758 contains a G-to-A transition at the splice donor site after the first exon probably
generating a truncated LIN-37 protein. n758 therefore is likely to be a null allele. n2234
contains an A-to-T transversion resulting in an amber mutation that should eliminate
more than half of the predicted protein product (Figure 1A).
We screened a cDNA library to obtain clones for the mRNA that encodes the
LIN-37 protein. The intron-exon structure of the lin-37 locus was determined by
comparing the complete sequences of the longest lin-37 cDNA clones with genomic
sequence (Figure 1A and Supplemental Results). We constructed a full-length lin-37
cDNA and showed that it was able to provide rescuing activity. Conceptual translation of
the reconstructed lin-37cDNA resulted in a hydrophilic protein of 275 amino acids with
weak similarity to the Drosophila melanogaster protein Mip40 and related vertebrate
proteins (BEALL et al. 2002; KORENJAK et al. 2004).
LIN-54 encodes a protein with two copies of a conserved cysteine-rich
motif: lin-54 had previously been mapped to an interval of LGIV between unc-30 and
lev- I (THOMAS et al. 2003). We performed germline-transformation experiments using
cosmids and subcloned portions of cosmids from this region and found the predicted
gene JC8.6 could rescue the synMuv phenotype of lin-54(n2231); lin-15A(n433)
mutants (Supplemental Results and Supplemental Figure 2). DNA sequence analysis of
JC8.6 in strains containing lin-54(n2231) or lin-54(n2990) identified missense mutations
in each (Figure 2A). RNAi targeted against JC8.6 caused a synMuv phenotype in either
a lin-8(n 111) or a lin- 15A(n767) background (OWEN et al. 2003; KORENJAK et al. 2004;
POULIN et al. 2005; data not shown), consistent with our identification of JC8.6 as lin-54.
JC8.6(RNAi) did not cause a Muv phenotype in wild-type or lin- 15B mutant
backgrounds, thus showing the same genetic interactions as lin-54 mutations and other
class B synMuv mutations. The recessive nature of the effects of lin-54(n2231) and
lin-54(n2990) and the similarity of their phenotypes to that caused by lin-54(RNAi)
suggest that these alleles result in a reduction of lin-54 function.
To establish the null phenotype of lin-54 we isolated two deletions affecting
JC8.6, n3423 and n3424 (Supplemental Results). In addition to causing a class B
synMuv phenotype (Table 1), both deletions also caused a fully penetrant sterile
phenotype, as do null mutations in some other class B synMuv genes (FERGUSON and
HORVITZ 1989; THOMAS and HORVITZ 1999; BEITEL et al. 2000; MELENDEZ and
GREENWALD 2000; VON ZELEWSKY et al. 2000; CEOL and HORVITZ 2001; THOMAS et al.
2003). Because of this sterility the synMuv phenotypes of lin-54(n3423) and
lin-54(n3424) mutants can be scored only in lin-54 homozygous 
progeny of
heterozygous mothers. This experimental design likely explains why the penetrances 
of
the synMuv phenotypes of the lin-54 deletion mutants we observed are weaker than
those of lin-54 missense mutants, as the lin-54 animals probably displayed a maternal
rescue (Table 1). Similar maternal effects have been noted for other synMuv
phenotypes (THOMAS et al. 2003).
The DNA sequences of lin-54 cDNAs revealed alternatively spliced transcripts
that would generate predicted proteins of 435 (LIN-54L) and 429 (LIN-54S) amino acids
in length (for details see Supplemental Materials and Methods). These proteins are
identical except for six additional amino acids found in LIN-54L. The missense allele
n2231 is predicted to cause an alanine-to-threonine substitution of amino acid 422 in
LIN-54L or amino acid 416 of LIN-54S. n2990 is predicted to cause a glycine-to-
glutamic acid substitution at amino acid 252 in LIN-54L and amino acid 246 in LIN-54S
(Figure 2B). Both predicted LIN-54 proteins are rich in cysteines, which are clustered in
two domains that share a nearly identical pattern and spacing. Domains with this
signature CXCX
4
CX
4
CXCX
6
CX2-3CXCX
2
C sequence are found in proteins from plants
and animals, including insects and mammals (CVITANICH et al. 2000). Some of these
proteins have similarity to LIN-54 outside the cysteine-rich domains. Notable among
these LIN-54 homologs are the fruit fly Drosophila melanogaster protein Mipl 20 and the
soybean Glycine max protein CPP1, which have sequence-specific DNA-binding
activities (CVITANICH et al. 2000; BEALL et al. 2002). The DNA-binding activity of CPP-1
was mapped to the cysteine-rich domains. Recently, yeast-one hybrid and chomatin
immunoprecipitation (ChlIP) studies have verified that LIN-54 can bind to the promoters
of multiple genes expressed in the digestive 
tract (DEPLANCKE et al. 2006). In addition,
Drosophila Mipl20 interacts with homologs of other class B synMuv proteins (BEALL et
al. 2002; BEALL et al. 2004; KORENJAK et al. 2004; LEWIS et al. 2004). Both human and
mouse Tesmin (SUGIHARA etal. 1999) share with LIN-54 and Mip120 an extended
region of similarity from the cysteine-rich motifs through their C-termini. This region
includes the A425T mutation found in n2231, but this residue is not conserved among
the other proteins. The missense mutation in n2990 converts a conserved glutamine
that immediately precedes the second cysteine motif to a glutamic acid, possibly
interrupting the function of this cysteine-rich motif (Figure 2B).
LIN-37 and LIN-54 are broadly expressed in nuclei throughout
development: We generated rabbit and guinea pig polyclonal antibodies that
specifically recognize LIN-37 and LIN-54, respectively, in western blots and
immunostaining. Antibodies raised against LIN-37 recognized a protein of
approximately 35 kD on western blots of wild-type protein extracts but not of protein
extracts from lin-37(n758) mutant animals (Figure 18B). In immunostainings, LIN-37 was
detected in most if not all nuclei of wild-type animals from the one-cell embryo through
the adult (Figure 1C- D, 1G and data not shown) and was absent in lin-37(n758) mutant
animals (Figure 1 E-F).
Antibodies raised against LIN-54 recognized a protein of 50 kDa on western blots
of wild-type protein extracts; this protein was absent in lin-54(n3423) mutant extracts
(Figure 2C). Immunostainings of wild-type animals demonstrated that, like LIN-37, LIN-
54 was present in the nuclei of all or almost all cells from the embryo through the adult
(Figure 2D-I and data not shown). In the hermaphrodite germline, LIN-54 was localized
to condensed chromosomes during the diakinesis phase of meiosis (Figure 2F-G).
LIN-54 was not detected in lin-54(n3423) mutant animals (Figure 2J-K).
LIN-37, LIN-54 and other class B synMuv proteins form a large-molecular-
weight- protein complex: Previous studies have demonstrated that some class B
synMuv proteins can interact in vitro and in vivo (Lu and HORVITZ 1998; WALHOUT et al.
2000; CEOL and HORVITZ 2001; UNHAVAITHAYA et al 2002). We performed gel-filtration
experiments and detected LIN-37 and LIN-54 in fractions corresponding to an apparent
molecular weight of between 440 and 669 kDa. Because both LIN-37 and LIN-54
coelute well above their monomeric molecular weight, it is likely that these proteins are
members of a multisubunit complex or complexes (Figure 3A). To identify components
of this complex we performed coimmunoprecipitation experiments.
We generated antibodies against 
the class B synMuv proteins LIN-9, 
LIN-35,
LIN-52, and LIN-53 (for details see Supplemental Materials and Methods). These
antibodies specifically recognize proteins of approximately 70 kD, 110 kD, 22 kD, and
50 kD, respectively, in wild-type but not in corresponding mutant protein extracts
(Supplemental Figure 3). Antibodies 
that recognize the synMuv proteins DPL-1, 
HDA-1,
HPL-2, LET-418, and LIN-56 were previously generated (CEOL and HORVITZ 2001;
COUTEAU et al. 2002; DUFOURCQ et al. 2002; UNHAVAITHAYA et al. 2002; E. Davison and
H.R.H. unpublished results).
We used anti-LIN-37 antibodies to immunoprecipitate proteins from both wild-
type and lin-37(n758) mutant embryo extracts. We determined the presence of synMuv
proteins that coimmunoprecipitated with LIN-37 using western blots. The class B
synMuv proteins LIN-9, LIN-35 Rb, LIN-52, LIN-53 RbAp48, LIN-54, and DPL-1 DP
were coimmunoprecipitated by anti-LIN-37 antiserum specifically from wild-type but not
from lin-37(n758) extracts (Figure 3B). The class B synMuv protein HPL-2, the class A
synMuv protein LIN-56 and tubulin did not coimmunoprecipitate with LIN-37 from either
wild-type or mutant protein extracts, demonstrating that the identified interactions are
specific (Figure 3B). The same set of proteins also coimmunoprecipitated with LIN-37 in
the presence of 50 [tg/ml ethidium bromide (EtBr). EtBr can disrupt complexes
dependent on DNA structure and DNA binding (LAI and HERR 1992), thereby suggesting
that these interactions are not DNA-dependent (Figure 3C). These class B synMuv
proteins were also precipitated by antibodies that recognize LIN-9, LIN-52, or LIN-54
(Figure 3D) but not by other rabbit polyclonal antibodies that recognize LIN-61 or LIN-56
(data not shown). We failed to detect DPL-1 in some of the coimmunoprecipitations, but
because we also failed to detect DPL-1 in the input we believe this result reflects poor
recognition by the antibody and not the absence of DPL-1. When DPL-1 was detected in
the input, DPL-1 was also seen in the immunoprecipitated proteins. Tubulin did not
coimmunoprecipitate with LIN-9, LIN-52, or LIN-54. Our analyses of immunoprecipitates
from extracts from synchronized cultures showed that these proteins remain associated
throughout larval development, including the late L2 and early L3 stages when the fates
of the Pn.p cells are specified (Figure 3E). The gel filtration chromatography and
coimmunoprecipitation experiments demonstrate that at least seven class B synMuv
proteins form a complex in vivo. Since this complex contains homologs of the known
transcriptional regulators Rb and DP and mutations in members of the complex cause a
65
Muv phenotype we refer to this complex as the DRM (DP, Rb, and MuvB) complex. This
complex includes only a subset of class B synMuv proteins as LIN-37 fails to
coimmunoprecipitate with LIN-36::GFP (Supplemental Figure 4) and HPL-2 does not
coimmunoprecipitate with LIN-37 or LIN-9.
Antibodies that recognize the class B synMuv protein EFL-1 recognize EFL-1 in
immunostaining (PAGE et al. 2001) but did not work in western blots, so we could not
assess precipitates for the presence of EFL-1. All DP-like proteins characterized to date
exist as heterodimers with E2F proteins (DYSON 1998). For this reason, we suggest that
at least one of the two C. elegans members of the E2F family, EFL-1 or EFL-2 (CEOL
and HORVITZ 2001; PAGE et al. 2001), associates with the precipitated proteins in vivo.
EFL-1 seems a likely candidate, since efl- I is a class B synMuv gene (CEOL and
HORVITZ 2001).
The DRM complex is distinct from a C. elegans NuRD-like complex: The
mammalian homologs of the synMuv proteins HDA-1 HDAC1 (DUFOURCQ et al. 2002),
LET-418 Mi2 (VON ZELEWSKY et al. 2000), and LIN-53 RbAp48 (Lu and HORVITZ 1998)
are components of the mammalian NuRD (Nucleosome Remodeling and Deacetylase)
complex (TONG et al. 1998; XUE et al. 1998; ZHANG et al. 1999). It has been proposed
that, by analogy to mammalian systems, these proteins form a NuRD-like complex in C.
elegans and are recruited to promoters of vulval genes by the class B synMuv proteins
LIN-35 Rb, EFL-1 E2F, and DPL-1 DP (SOLARI and AHRINGER 2000; VON ZELEWSKY et al.
2000; CEOL and HORVITZ 2001; DUFOURCQ et al. 2002; UNHAVAITHAYA et al. 2002). Since
we identified LIN-35 Rb and DPL-1 DP as components of the DRM complex, we tested
whether this complex also included the chromatin-modifying enzymes HDA-1 HDAC1 or
LET-418 Mi2 by testing if these proteins coimmunoprecipitate with LIN-37.
LET-418 did not coimmunoprecipitate with LIN-37 from wild-type protein extracts
(Figure 4A). A small amount of HDA-1 was present in the precipitate generated from
extracts from wild-type embryos after coimmunoprecipitation 
with anti-LIN-37 antisera,
but a similar low amount was detected in precipitates from lin-37 null mutant embryos.
Low levels of nonspecific precipitation of HDA-1 were also noted by Unhavaithaya etal.
(2002). We conclude that HDA-1 did not specifically coimmunoprecipitate with LIN-37.
HDA-1 was not observed in coimmunoprecipitates with LIN-37 in extracts from L3 larvae
(Figure 4B). In addition, HDA-1 did not precipitate with LIN-9, LIN-52, or LIN-54 (Figure
4B-C). Reciprocally, LIN-37 did not coimmunoprecipitate with HDA-1 (Figure 4A). By
contrast, we found that LIN-53 did coimmunoprecipitate with HDA-1 (Figure 4A). LET-
418 had previously been shown to coimmunoprecipitate with HDA-1 (UNHAVAITHAYA et
al. 2002), and we observed low levels of LET-418 in HDA-1 immunoprecipitates (Figure
4A).
These results suggest that HDA-1, LET-418, and LIN-53 are found in a complex
distinct from the DRM complex. We propose that LIN-53 is present in both the DRM
complex and a NuRD-like complex. RbAp48, the mammalian homolog of LIN-53, has
similarly been found in multiple chromatin-modifying complexes, including the chromatin
assembly complex Cafl, the NuRD complex, and the Sin3 complex (VERREAULT et al.
1996; XUE et al. 1998; ZHANG et al. 1998a; ZHANG et al. 1998b).
Vulval development of animals with mutations in NuRD-like complex
components is phenotypically distinguishable from that of animals mutant for
DRM complex components: All of the proteins identified in the DRM complex, as well
as HDA-1 and LET-418, have previously been considered to be class B synMuv
proteins (Lu and HORVITZ 1998; BEITEL et al. 2000; VON ZELEWSKY et al. 2000; CEOL and
HORVITZ 2001; DUFOURCO et al. 2002; THOMAS et al. 2003). Our data suggest that these
proteins can be found in two distinct complexes. Interestingly, mutations that disrupt
these two complexes seem to have distinct phenotypic consequences. Whereas most
class B synMuv mutants are completely non-Muv in the absence of mutations in class A
or class C synMuv genes, hda- 1 and let-418 single mutants display a weak Muv
phenotype. Specifically, hda-1 mutants are 20-31% Muv (DUFOURCQ et al. 2002; data
not shown), and when cultured at 22.50C about 1.5% of let-418(n3536) mutants are Muv
(our unpublished results). In addition, in 7% of let-418(arl 14) animals P8.p is induced
(VON ZELEWSKY et al. 2000); P8.p induction is a Muv phenotype (Ceol and Horvitz 2004).
Furthermore, let-418 is unlike most class B synMuv genes in that the level of P8.p
induction of let-418(arl 14) animals is enhanced to 13-20% by a typical class B mutation
(VON ZELEWSKY et al. 2000), whereas a second class B mutation does not alter the
vulval phenotype of most class B mutants.
In concert with our biochemical data, these genetic data support the hypothesis
that the members of the NuRD-like complex have functions distinct from those of the
DRM complex in the regulation of vulval development.
Some DRM complex components are absent in mutants lacking certain
other complex components: We investigated whether a DRM complex can form
properly when specific components are either absent or mutated. We analyzed extracts
from presumptive lin-35 and lin-37 null mutants, as these mutants can be maintained as
homozygotes and therefore can be grown in sufficient quantities for biochemical
analyses. For the genes lin-9, lin-52, lin-53, and lin-54, which have sterile null
phenotypes (BEITEL et at 2000; CEOL and HORVITZ 2001; THOMAS et al. 2003), we
isolated protein extracts from embryos with viable missense mutations that are known to
confer synMuv phenotypes. In general, we used anti-LIN-37 anitbodies to isolate the
DRM complex. To analyze extracts from lin-37(n758) embryos, which lack detectable
LIN-37, we used anti-LIN-9 or anti-LIN-52 antibodies to perform
coimmunoprecipitations.
In all but one of the immunoprecipitates 
from extracts of DRM complex mutants,
the presence of the unmutated complex members was unaffected (Figure 5A). The
absence of neither LIN-35 Rb nor LIN-37 significantly affected the ability of the
remaining complex components to coprecipitate. The missense mutations in
lin-52(n771), lin-53(n833), and lin-54(n223 1) also did not strongly affect the associations
among complex members (data not 
shown). Only a missense mutation 
in lin-9, n112,
which is predicted to change glycine 341 to glutamic acid (BEITEL et al 2000), altered
the composition of the complex by reducing the presence of another protein: LIN-53
RbAp48 failed to strongly associate with the complex when coimmunoprecipitated with
either anti-LIN-37 antiserum or anti-LIN-9 antiserum (Figure 5A). The n 112 mutation
affects a conserved residue in a region of LIN-9 that is highly conserved in the
homologous human protein hLin9 and the homologous Drosophila proteins ALY and
Mipl30/TWIT (WHITE-COOPER etal. 2000; BEALL etal. 2002; GAGRICA etal. 2004). The
only other identified missense mutation of lin-9 known to cause a homozygous viable
class B synMuv phenotype is n3675 (C.J.C., 
F. Stegmeier, M.M.H, and H.R.H.,
unpublished results), which changes the poorly conserved aspartic acid 305 to
asparagine. lin-9(n3675) did not reduce the levels of LIN-53 RbAp48 in the complex
(Figure 5A). The lin-9(n3675) mutation is in the same highly conserved region as the
lin-9(n 112) G341E mutation, but the altered residue is not conserved in Drosophila ALY
or human Lin-9.
These data suggest that an interaction between LIN-9 and LIN-53 might be
important for the incorporation of LIN-53 into the DRM complex, but that complex
assembly is not the sole function of LIN-9 in vulval development. Specifically another
function of LIN-9 might be modified in the lin-9(n3675) mutant. Alternatively, both of the
lin-9 mutations as well as the other missense mutations we tested might cause defects
in the formation of the complex too subtle to identify by assessing the presence of
known members by immunoprecipitation.
Because we could obtain quantities of lysate sufficient to analyze the association
of complex components by coimmunoprecipitation only from fertile mutants, we could
not directly ask whether sterile null mutants might affect the composition of the complex.
Instead, we analyzed the total protein levels of DRM complex components when each
member was removed by mutation. Since a null allele of lin-53 had not previously been
isolated, we screened a chemical-mutagenesis 
library and identified a deletion allele,
n3368, which starts after codon 52, removes 
more than half of the coding sequence,
and, is likely to be a null allele. Animals homozygous for lin-53(n3368) are sterile and
are synMuv in combination with the class A synMuv mutant lin- 15A(n767). To compare
sterile mutants to the wild type, we examined L4 larvae since adult wild-type
hermaphrodites contain developing embryos that are absent in sterile mutants.
Null mutants in lin-9, lin-53, and lin-54 decreased the protein levels of many other
complex components, including LIN-9, LIN-37, LIN-52, and LIN-54 (Figure 5B). In lin-9
null mutants, LIN-37, LIN-52, and LIN-54 levels were decreased as compared to wild-
type levels. Null mutants in lin-53 showed decreased levels of LIN-9, LIN-37, LIN-52,
and LIN-54. In lin-54 null mutants levels of LIN-9 and LIN-52 were decreased. These
decreases could reflect decreases 
in either protein synthesis or stability. 
Loss of LIN-35,
LIN-37, or DPL-1 proteins did not significantly affect the protein levels of any other DRM
complex components. LIN-35 and LIN-53 levels remained unchanged in all genotypes
examined, except as expected lin-35 or lin-53 mutants, respectively (Figure 5B). The
mammalian homologs of these two proteins- Rb and RbAp48 - are found in multiple
complexes, and thus their overall levels may not be dependent on the presence of any
one protein complex. Our data suggest that a complete loss of LIN-9, LIN-53, or LIN-54
can disrupt DRM complex formation and result in the degradation of other complex
members. Alternatively, it is possible that the loss of any of these three proteins causes
a decrease in transcription of some but not all of the genes that encode DRM complex
members.
DISCUSSION
lin-37 and Iin-54 likely act in the DRM complex: The synMuv genes act to
antagonize vulval cell-fate specification. At least 26 synMuv genes define at least three
distinguishable classes, A, B, and C (HORVITZ and SULSTON 1980; FERGUSON and
HORVITZ 1989; HSIEH et al. 1999; BEITEL et al. 2000; MELENDEZ and GREENWALD 2000;
THOMAS et al. 2003; CEOL and HORVITZ 2004), and it has been suggested that the
synMuv proteins form a transcriptional repressor complex or complexes (Lu and
HORVITZ 1998; SOLARI and AHRINGER 2000; VON ZELEWSKY et aL 2000; CEOL and
HORVITZ 2001; UNHAVAITHAYA et al. 2002; POULIN et al. 2005). The synMuv proteins
DPL-1 DP and EFL-1 E2F have been proposed to act through LIN-35 Rb to recruit a
NuRD-like complex to the promoters of genes that control vulval cell-fate specification
(SOLARI and AHRINGER 2000; CEOL and HORVITZ 2001).
In this paper we describe the molecular identification and characterization of two
class B synMuv genes, lin-37and lin-54. We show that LIN-37 and LIN-54 form a
multisubunit protein complex together with at least five other class B synMuv proteins:
LIN-9, LIN-35 Rb, LIN-52, LIN-53 RbAp48, and DPL-1 DP. This DRM complex is
biochemically and genetically distinct from a NuRD-like complex that includes HDA-1
HDAC1, LET-418 Mi2, and LIN-53 RbAp48. Based on these observations we propose
that LIN-35 Rb and DPL-1 DP likely have a function in vulval development distinct from
recruitment of the NuRD complex. However, mammalian Rb has been shown to bind
HDAC1 (BREHM et al. 1998; Luo et al. 1998; MAGNAGHI-JAULIN et al. 1998), and our data
do not preclude the possibility that LIN-35 Rb and DPL-1 DP function both in the context
of the DRM complex and in recruiting the NuRD complex.
The DRM complex might act in vulval development: Although the DRM
complex is composed of synMuv proteins, the complex was identified in embryos and
larvae, not specifically in the cells involved in vulval development. Therefore it is
possible that lin-37and lin-54 do not regulate vulval cell fates as components of the
DRM complex. Three observations lead us to suggest otherwise. First, that seven class
B synMuv proteins with similar roles in vulval development form a complex in vivo is
most simply explained if these proteins act together in the context of the DRM complex
to control vulval development; furthermore, as described below, these 
genes each act in
a variety of biological processes, but the only 
known normal biological process they all
share is a role in vulval development. Second, certain mutations that cause a synMuv
phenotype disrupt complex formation, suggesting that the synMuv phenotype
associated with these alleles could be caused by lack of DRM complex activity. Third,
the DRM complex exists during all developmental stages, including the stage when the
Pn.p cells are specified. The simplest 
hypothesis is that LIN-9, LIN-35, LIN-37, 
LIN-52,
LIN-53, LIN-54, and DPL-1 act as components of the DRM complex to antagonize the
specification of vulval cell fates. Given that homologs of DPL-1 are found as
heterodimers with E2F family members (DYsoN 1998), it is likely that one of the two C.
elegans E2F family members is also in the DRM complex. We suggest that this E2F
family member is EFL-1, since loss of function of efl-1 but not efl-2cause a class B
synMuv phenotype (CEOL and HORVITz 2001).
Vulval specification is unlikely to be the only function for the DRM complex or at
least of the components of the DRM complex, as these proteins are ubiquitously
expressed and mutations in genes that encode complex members are pleiotropic. A
subset of DRM complex members act in G, regulation (BOXEM and VAN DEN HEUVEL
2002), are needed for fertility (Beitel et al 2000; Ceol and Horvitz 2001; Thomas et al
2003) and cause hypersensitivity to RNAi (WANG et al. 2005). All complex members
modify expression of transgene arrays (HSIEH etal. 1999; E. Andersen and H.R.H.
unpublished results).
The DRM complex probably functions in transcriptional repression: The
DRM complex we have characterized is similar to two recently described and highly
similar complexes that contain Drosophila homologs of a number of class B synMuv
proteins (KORENJAK et al. 2004; LEWIS et al. 2004). First, the dREAM complex was
identified by biochemical purification of Drosophila Rb-containing complexes from
embryo extracts followed by mass spectrometry and western blot analysis. This
complex contains homologs of LIN-9, LIN-35, LIN-37, LIN-54, LIN-53, DPL-1, and
EFL-1; these homologs are called Mipl30, 
RBF, Mip40, Mipl20, p55, dDP, and dE2F2,
respectively. All of these proteins except the LIN-54 homolog, Mipl20, were identified
by mass spectrometry following biochemical purification. Mipl20 was found to copurify
with this complex based upon western blots, but was not identified using mass
spectrometry (KORENJAK et al. 2004).
Second, the Myb-MuvB complex was purified by immunoprecipitation of the LIN-
54 homolog Mip120 or the LIN-9 homolog Mipl30 from Drosophila tissue-culture cells
and the identification of coimmunoprecipitating proteins by mass spectrometry. The
Myb-MuvB complex contains all of the components in the dREAM complex as well as
stoichiometric amounts of dLin-52, the fly homolog of LIN-52, and substoichiometric
amounts of Rpd3, the fly homolog of HDA-1, and L(3)MBT, a protein similar to the class
B synMuv protein LIN-61 (LEWIS etal. 2004; M.M.H., X. Lu. and H.R.H. unpublished
results). The differences between the dREAM and Myb-MuvB complexes might be a
consequence of the different methods used for purification or might reflect the existence
in different tissues or during different developmental stages of multiple subcomplexes
with overlapping constituencies. Both the dREAM and Myb-MuvB complexes can
mediate transcriptional repression of many E2F-target genes (KORENJAK et al. 2004;
LEWIS et al. 2004). The high degree of similarity between the DRM complex and the
dREAM and Myb-MuvB complexes indicates that the DRM complex might, like the two
Drosophila complexes, act in transcriptional repression.
The DRM complex we have identified differs slightly from both the dREAM and
the Myb-MuvB complexes. Unlike the dREAM complex (KORENJAK et al. 2004), the DRM
complex contains a LIN-52 dLin52-like protein. Unlike the Myb-MuvB complex (LEWIS et
al. 2004), the DRM complex does not contain HDA-1 Rpd3 or LIN-61 L(3)MBT (M.M.H.
and H.R.H., unpublished results). In addition to LIN-61, one other L(3)MBT-like protein
exists in C. elegans (M.M.H. and H.R.H., unpublished results), and it is possible that this
protein associates with the DRM complex. It is also possible that like the LIN-54
homolog Mipl20, the LIN-52 homolog dLin-52 is present in the dREAM complex but
failed to be detected by mass spectroscopy. Given the similarities of the DRM, dREAM
and Myb-MuvB complexes, we propose that there is a core complex that consists of
LIN-35 RBF, EFL-1 E2F2, DPL-1 DP, LIN-9 Mipl30, LIN-37 Mip40, LIN-52 dLin-52, LIN-
53 p55, and LIN-54 Mipl20 and that this complex might associate with other proteins
during specific stages of development or in certain cell types (Figure 6B).
The dREAM and Myb-MuvB complexes both contain the DNA-binding protein
Myb. In these complexes, Myb acts in site-specific DNA replication but apparently not in
transcriptional repression (KORENJAK et al. 2004; LEWIS et al. 2004). There is no clear
Myb homolog in C. elegans (KORENJAK et al. 2004; LIPSICK 2004). Two predicted C.
elegans proteins F32H2.1 and D1081.8 have weak similarity to Myb restricted to their
SANT domains, which mediate the binding of Myb to DNA (PETERS et al. 1987). We and
others have found that RNAi targeting either of these genes did not cause a synMuv
phenotype in the background of the class A synMuv mutant lin-15A(n767) (KORENJAK et
al. 2004; data not shown). It is possible that F32H2.1 or D1081.8 associate with the
DRM complex in vivo but are not required for the function the DRM complex might have
in vulval development. Alternatively, the C. elegans functional ortholog of the
Drosophila Myb protein found in the dREAM and Myb-MuvB complexes might not be
readily identifiable by comparisons of primary sequence. It is also possible that the C.
elegans DRM complex does not contain a Myb ortholog.
The dREAM complex is required for the repression of dE2F-regulated genes with
sex-specific or tissue-specific expression patterns, but not for the repression of dE2F-
regulated genes involved in cell-cycle regulation (DIMOVA et al. 2003; KORENJAK et al.
2004). synMuv proteins are known to control transgene expression, cell-cycle
regulation, viability, and RNAi sensitivity in addition to vulval development (HSIEH etal.
1999; BEITEL et al. 2000; MELENDEZ and GREENWALD 2000; VON ZELEWSKY et al 2000;
CEOL and HORVITZ 2001; BOXEM and VAN DEN HEUVEL 2002; UNHAVAITHAYA et al. 2002;
CEOL and HORVITZ 2004; WANG et al. 2005). Given the broad expression patterns of the
synMuv genes and the numerous different phenotypic abnormalities caused by the loss
of individual synMuv proteins, including members of the DRM complex, we propose
that, similar to their Drosophila counterparts, the DRM complex proteins are involved in
the repression of many targets important for diverse biological functions, including the
specification of the vulval-cell fate.
The DRM complex likely contains at least two sequence-specific DNA-binding
activities. Both the EFL-1 E2F and DPL-1 DP heterodimer and LIN-54 Mipl20 are likely
to bind DNA in a sequence-specific manner, since their homologs in other species
function as DNA-binding proteins. These proteins might target the DRM complex
specifically to loci containing sequence motifs recognized by both proteins or to loci
containing sequence motifs recognized by either protein.
We propose the following two alternative models for how the DRM complex might
act in transcriptional repression (Figure 6C, D). First (Figure 6C), the DRM complex
could be recruited to DNA by multiple DNA-binding factors including LIN-54 and the
heterodimeric transcription factor formed by EFL-1 and DPL-1. The DRM complex could
then act with the NuRD complex to repress transcription. Alternatively (Figure 6D), the
DRM complex could bind DNA and subsequently bind unmodified histone tails and
prevent their acetylation; similar activities have been proposed for the dREAM complex
by Korenjak etal. (2004), who demonstrated that the dREAM complex can bind
unmodified histone H4 tails. This binding might be mediated by LIN-53, since the
mammalian homolog RbAp48 can bind histone H4 (VERREAULT et al. 1996). At some
target genes the histone deacetylase activity might be provided by a NuRD-like
complex, containing HDA-1 HDAC1, LET-418 Mi2 and LIN-53 RbAp48; alternatively the
NuRD-like complex might act on other target genes.
The DRM complex and a NuRD-like complex have separable functions
during vulval development: Although neither the DRM nor the dREAM complexes
contains any known chromatin-modifying or chromatin-remodeling enzymes, these
complexes might require the activity of a histone deacetylase to mediate transcriptional
repression, as noted above. Mutations in genes encoding components of either the
DRM or the NuRD-like complex require an additional class A or class C synMuv
mutation to produce a highly penetrant Muv phenotype. However, we found that
mutations in at least two of the NuRD-like complex components, 
hda-1 and let-418,
alone can cause low penetrance Muv phenotypes, suggesting that the chromatin-
remodeling and chromatin-modifying activities of this complex might be required more
broadly for the transcriptional repression of genes necessary for proper vulval
development than is the activity of the DRM complex. Perhaps other class B synMuv
proteins not associated with the DRM complex, for example LIN-61, act redundantly
with the DRM complex to maintain the repressed state formed by the activity of the
NuRD-like complex.
Does Rb function within the context of a DRM-like complex to control
development in mammals? The high degree of conservation shared by the
DRM/dREAM/Myb-MuvB complexes in C. elegans and Drosophila and the important
roles that the components of DRM complex play in C. elegans development suggest
that a similar complex exists in other organisms, including humans. Indeed the human
homolog of LIN-9, hLin-9 can interact with Rb and might act with Rb to specifically
promote differentiation (but not to inhibit cell-cycle progression) (GAGRICA et al. 2004).
Perhaps Rb or other Rb-family proteins act within the context of a DRM-like complex to
control development. Rb could act as a tumor suppressor through such a role in
developmental regulation in addition to its role in cell-cycle regulation. In C. elegans
vulval development a mutation in lin-35 Rb can, in combination with an additional
mutation in a class A or C synMuv gene, cause extra cell divisions. Further biochemical
and genetic studies of nematodes, insects, and mammals should elucidate the role that
this complex plays in development and in carcinogenesis.
MATERIALS AND METHODS
Culture conditions and strains: All strains were cultured at 200C, unless
otherwise specified, on NGM agar seeded with E colistrain OP50, as described by
Brenner (1974). N2 (Bristol) was the wild-type strain. For a list of the mutant alleles used
see Supplemental Material and Methods.
Transgenic Strains: Germline transformations were performed as described by
Mello et al. (1991). For details see Supplementary Materials and Methods.
RNAi Analyses: Templates for in vitro transcription reactions were made by
PCR amplification of either cDNAs including flanking T3 and T7 promoter regions or
coding exons from genomic DNA using T3-and T7-tagged oligonucleotides. RNA was
transcribed in vitro using T3 and T7 polymerases. In vitro transcribed RNA was
denatured for 10 min and annealed prior to injection.
Antibody Preparation, Immunocytochemistry and Western Blots: Animals
were injected with recombinant protein corresponding to all or part of the protein of
interest. For details see Supplemental Material and Methods. All rabbits and guinea pigs
were maintained by Covance (Denver, PA), except the guinea pigs used to generate the
LIN-54 antibodies were maintained at MIT. All antibodies were used at a 1:1000 dilution
for western blots, except for anti-LIN-53, anti-LIN-54, and anti-DPL-1, which were used
at 1:500. Affinity purified antibodies were used in all cases except for LIN-35 antibodies.
For LIN-35 western blots we used unpurified serum from the third production bleed. For
details on affinity purification see Supplemental Material and Methods. Larvae and
adults for immunostaining were fixed in 1% paraformaldehyde for 30 min, as described
by Finney and Ruvkun (1990). Embryos were fixed for 20 minutes in 0.8%
paraformaldehyde, as described by Guenther and Garriga (GUENTHER and GARRIGA
1996). Affinity-purified anti-LIN-37 and anti-LIN-54 antibodies were used at a 1:100
dilution for immunocytochemistry.
Embryo lysates: Embryos were harvested from liquid cultures and bleached to
kill larvae and adults in a solution containing 5N NaOH and 20% hypochlorite. The
embryos were resuspended in 1 ml of lysis buffer (25 mM HEPES pH 7.6, 150 mM
NaCI, 1 mM DTT, 1 mM EDTA, 0.5 mM EGTA, 0.1% NP-40, 10% glycerol with
Complete EDTA-free protease inhibitors (Roche Diagnostics)) for each gram of embryos
and frozen in liquid nitrogen. The embryos were thawed at room temperature and
sonicated 15 times for 10 seconds using a Branson sonifier 450 at setting 5. The
homogenate was then centrifuged at top speed in a microcentrifuge for 15 min at 40C.
The supernatant was removed to a new tube and was spun for 15 min at top speed in a
microcentrifuge. The remaining supernatant was pooled and the protein concentration
was determined using the Pierce Coomassie Plus Protein Assay Reagent (Pierce
Biotechnology). The lysate was diluted to 5-10 mg/ml and was used immediately or
stored at -80
0
C and thawed no more than once before use.
Immunoprecipitation experiments: Antibodies were crosslinked to Protein A
Dynabeads (Invitrogen) using dimethyl pimelimidate (Pierce) essentially as described by
Harlow and Lane (1999), with the following exceptions: reactions were stopped with
0.1 M Tris pH 8.0, and beads were washed three times for 1 min in 100 mM glycine pH
2.5 and then in lysis buffer. The beads were resuspended in PBS. 500 [l of lysate,
corresponding to 2.5-5 mg of total protein, were incubated with 25 tl of affinity-purified
antibody bound to 25 tl of beads for each immunoprecipitation reaction. Prior to
incubation with antibody-bound beads, lysates were initially precleared with 25 .l of
beads for between 1 hr and overnight at 40C. The precleared lysates were recirculated
over the antibody-bound beads at 40C for 1-2 hr and were then washed three times for 5
min each at 40C in lysis buffer. Following the final wash, the beads were resuspended in
20 tl of 2X protein sample buffer, boiled for 5 min, and loaded on an SDS-
polyacrylamide gel. HRP conjugated protein A (Bio-Rad) was used for detection of
protein by western blot following coimmunoprecipitation experiments, except for
detection of tubulin in which HRP conjugated goat anti-mouse (Bio-Rad) was used.
Gel filtration chromatography: Embryo 
extract in buffer (15 mM HEPES pH7.6,
0.1 M KCI, 3 mM MgCI
2
, 0.1 mM EDTA, 10% glycerol with Complete EDTA-free
protease inhibitors (Roche Diagnostic)) was precipitated with solid ammonium sulfate
added to a final concentration of 20%. The supernatant from this precipitation was then
precipitated with a concentration of 30% ammonium sulfate. The pellets following
protein precipitation were resuspended in elution buffer (EB) 
(25 mM HEPES pH 7.6,
150 mM KCI, 50 mM MgCI
2
, 1 mM DTT, 0.1 mM EDTA, 0.1% NP-40, 10% glycerol),
combined, and 50 pl were loaded onto a pre-equlibrated Superose 6 PC 3.2/30 column
(Amersham). The sample was eluted using EB at a flow rate of 0.02 ml/min. 75 01
fractions were collected. Proteins of known molecular weights (158 kD aldolase, 232 kD
catalase, 440 kD ferritin, 669 kD thyroglobulin; Amersham) were used as standards and
were detected by measuring the absorbance of each collected fraction at 280 nm.
ACKNOWLEDGMENTS
We thank Erik Andersen, Daniel Denning, Richard Harrison, Stephen Harrison, Andrew
Mehle, Niels Ringstad, Adam Saffer, and Hillel Schwartz for comments about the
manuscript; Beth Castor for DNA sequence determination; Na An for strain
management; members of the Horvitz laboratory for construction of the deletion library;
Jayson Bowers and Stephen Bell for assistance with gel filtration chromatography; Alan
Coulson for cosmid clones; Yuji Kohara for cDNA clones; the C. elegans Genome
Sequencing Consortium for genomic sequence; and the C. elegans Genetics Center for
some of the strains used in this work. This work was supported by NIH grant GM24663.
M.M.H. was a Howard Hughes Predoctoral Fellow. C.J.C. was a David H. Koch Fellow.
H.R.H. is the David H. Koch Professor of Biology at M.I.T. and an Investigator of the
Howard Hughes Medical Institute.
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I
Table 1. lin-37 and lin-54 Mutations Cause a Clas
Genotype
Single mutants lin- 15A(n433)
lin- 15A(n767)
lin- 15B(n744)
lin-35(n745)
lin-37(n758)
lin-37(n2234)
lin-54(n223 1)
lin-54(n2990)
lin-54(n3423)
lin-54(n3424)
trr- 1(n3712)
a
Double mutants lin-37(n758); lin- 15A(n433)
with class A lin-37(n758); lin- 15A(n767)
mutations lin-37(n2234); lin- 15A(n433)
lin-37(n2234); lin- 15A(n767)
lin-54(n2231); lin- 15A(n433)
lin-54(n223 1); lin- 15A(n 767)
lin-54(n2990); lin- 15A(n433)
lin-54(n3423); lin- 15A(n433)
lin-54(n3423); lin- 15A(n767)
lin-54(n3424); lin- 15A(n767)
Double mutants lin-37(n758); lin- 15B(n744)
with class B lin-54(n3423); lin- 15B(n744)
mutations lin-35(n745); lin-37(n758)
lin-35(n745); lin-54(n3423)
lin-37(n758); lin-54(n3423)
Double mutants trr- 1(n3712); lin-37(n758)a
with class C trr- 1(n3712); lin-54(n223 1) a
mutations
All animals were raised at 200C. The Muv phenotype was scored 
using a dissecting
microscope except in the cases noted. trr- 1 mutant homozygotes were recognized as
the non-GFP progeny of ttr- 1/mln 1[dpy- 10 mls 14] heterozygous parents. lin-54(n3423)
and lin-54(n3424) homozygotes were recognized as the non-GFP progeny of
lin-54/nT1[qls51]; +/nTI[qls51] heterozygous 
parents. lin-54(n3423); lin-15A(n433),
lin-54(n3423); lin-15A(n767), lin-54(n3424); lin- 15A(n767), and lin-54(n3423);
lin- 15B(n744) were also homozygous for unc-30(e 191).
a Muv, greater 
than three 
Pn.p cells 
induced.
b From 
Ceol and 
Horvitz 
(2004).
s B synMuv Phenotype
Percent Muv (n)
0 (302)
0 (350)
0 (272)
0 (104)
0 (318)
0 (170)
0 (368)
0 (327)
0 (541)
0 (242)
13 (89)b
100 (111)
100 (102)
100 (104)
100 (124)
99 (227)
100 (221)
100 (291)
92 (96)
100 (314)
99 (87)
0 (94)
0 (88)
0 (115)
0 (108)
0 (329)
55 (20) 
b
42 (33)
----
Figure 1. Cloning and expression of iin-37
(A) lin-37maps between sma-3 and mec- 14 on LGIII. lin-37gene structure as
determined from cDNA and genomic sequences is indicated. Shaded boxes indicate
coding sequence, and open boxes indicate 5' and 3' untranslated regions. Predicted
translation initiation and termination codons are shown along with the poly(A) tail and
the SL2 leader sequence. Mutations found in the two lin-37 alleles are indicated above
the gene structure. The cosmid ZK418 is shown below as a gray bar. (B) Affinity-purified
antibodies raised against recombinant LIN-37 were used to blot extracts from both wild-
type and lin-37(n758) mutant animals. The asterisk denotes non-specific
immunoreactivity. (C, E, G) Whole-mount staining with anti-LIN-37 antisera. (C) LIN-37
is expressed in all nuclei of the developing embryo. (D) 4,6-Diamidino-2-phenylindole
(DAPI) staining of the embryo shown in (C). (E) LIN-37 is absent from lin-37(n758)
embryos. (F) DAPI staining of the embryo shown in (E). (G) LIN-37 expression in the
mid body of an L1 larvae. (H) DAPI staining of the larvae shown in (G). WT, wild type.
Scale bars, 10 pm.
Figure 1
lin-37
III
100 -
75"
50-
37-
LGIII
0.05 m.u.
aLIN-37
DAPI
aLIN-37
lb
i
4
aII
I
I
,p
B \
4 LIN-37
Figure 2. Cloning and expression of lin-54
(A) lin-54 maps between unc-30 and lev- on LGIV. lin-54 
gene structure as derived
from cDNA and genomic sequences is indicated as in Figure 1. The positions of the
mutations in the four lin-54 alleles are indicated. Positions of the alternative splice
acceptors for exon 2 or 3 are shown by asterisks. In both cases, the use of alternative
splice acceptors creates three amino acid differences in the lin-54 coding sequence
(see Supplemental Materials and Methods). As described in Supplemental Materials
and Methods, the SL1 leader is also alternatively spliced. The cosmid JC8 is shown
below as a gray bar. (B) Alignment of the cysteine-rich motifs of LIN-54 with proteins
with sequences from Drosophila melanogaster, Glycine max, Homo sapiens, and Mus
musculus. Solid boxes indicate identity with LIN-54. The conserved cysteine residues of
the two cysteine-rich domains are shown by black dots. The arrowhead indicates the
site of the n2990 missense mutation. (C) Affinity-purified antibodies raised against
recombinant LIN-54 were used to blot extracts from both wild-type and lin-54(n3423)
mutant animals, which were derived from heterozygous mothers. (D, F, H, J) Whole-
mount staining with anti-LIN-54 antisera. (D) LIN-54 is broadly expressed in the adult
hermaphrodite germline. (E) DAPI staining of the animal shown in (D). (F) Enlargement
of the boxed portion of (D). LIN-54 is localized to condensed chromosomes (arrow). (G)
DAPI staining of the enlarged portion shown in (F). (H) LIN-54 is broadly expressed in
the embryo. (I) DAPI staining of the embryo shown in (F). (J) LIN-54 is absent in
lin-54(n3423) hermaphrodites. (K) DAPI staining of the normal germline shown in (J).
VVT, wild type. Scale bars, 10 pm.
Figure 2
lin-54
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Figure 3. A subset of class B synMuv proteins form a complex in vivo.
(A) Gel filtration chromatography fractions immunoblotted with antibodies specific to the
antigen indicated on the left. LIN-37 and LIN-54 coeluted in early fractions. Numbers
beneath correspond to the fraction in which a protein of the size indicated (in
kilodaltons) eluted from the column. (B) Embryo extracts from both wild-type and
lin-37(n758) mutant animals were precipitated with anti-LIN-37 antibodies. Proteins
were separated by SDS-PAGE and immunoblotted with antibodies specific to the
antigen indicated on the left. (C) Immunoprecipitations were performed using wild-type
embryonic extracts in the presence of 50 pg/ml ethidium bromide (EtBr). (D)
Immunoprecipitations were performed using embryonic extracts from the wild type and
antibodies that specifically recognize LIN-9, LIN-52, or LIN-54. Coimmunoprecipitation
of LIN-52 with anti-LIN-54 antibodies was from L1 larval extracts. (E) Extracts from wild-
type L1 or late L2/ early L3 larvae were precipitated as in (B). WT, wild type. IN, 2% of
input for immunoprecipitations. 10% of input for gel filtration chromatography. Vo void
volume. Vt total volume.
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Figure 4. NuRD-like complex components do not precipitate with the DRM complex (A)
Extracts from either wild-type or lin-37(n758) mutant animals (as indicated above the
lanes) were precipitated with antibodies directed against either HDA-1 or LIN-37 or with
non-specific anti-rabbit IgG. Immunoprecipitations were analyzed using antibodies
specific to the antigens indicated to the left. (B) Extracts from either wild-type or
lin-37(n758) mutant L3 larvae were 
precipitated with antibodies directed 
against LIN-37,
LIN-9, or LIN-52. Immunoprecipitations were analyzed using antibodies specific to the
antigens indicated to the left. (B) Wild-type embryo extracts were precipitated with
antibodies that recognize LIN-9, LIN-52, or LIN-54 and were analyzed by western blots
with antibodies that recognize HDA-1. WT, wild type. IN, 2% of the input. IP, 100% of
the immunoprecipitate.
A
LET-418 Mi2
IPaHDA-1 IgG
WT
IN IP IP
Figure 4
IPaLIN-37
lin-37 (n758)
II I
IP IN IP
4 ::
IDAC1
LIN-37
40000
I I
LIN-53 RbAp48
B
HDA-1 HDAC1
LIN-37
C
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IP IP
IPaLIN-37 aLIN-9 aLIN-52
WT lin-37(n7582 WT WT
IN IP IN IP IN IP IN IP
IN IP IN IP IN IP IN IP
]
IP IP IP
aLIN-9 aLIN-52 aLIN-54
WT WT WT
I I I II I
IN IP IN IP IN IP
HDA-1 i
I
I -
L
I
Figure 5. Analysis of the DRM complex and its components in mutant backgrounds.
(A) Anti-LIN-37 antibodies were used for immunoprecipitation of proteins from embryo
extracts from animals of the indicated genotype, except in the case of extracts from
lin-37(n758) animals, when anti-LIN-52 antibodies were used. For
immunuoprecipitations from lin-9(n 112) embryo extract, both anti-LIN-9 and anti-LIN-37
antibodies were used. Following immunoprecipitation, proteins were separated by SDS-
PAGE and immunoblotted using the antibodies specific to the antigens listed to the left.
IN, 2% of the input. IP, 100% of the immunoprecipitate. (B) Protein from 100 L4 larvae
of the genotype noted above each column was loaded per lane. The antibodies used on
each blot are indicated on the left. Anti-tubulin antibodies were used to assess protein
loading and transfer. Asterisks denote non-specific immunoreactivity.
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Figure 6. Schematic indicating the components of the DRM and NuRD-like complexes
and a model for how these complexes act in vulval development. (A) A list of DRM
complex members and the homologs found in Drosophila melanogaster and Homo
sapiens. (B) The DRM complex contains at 
least eight proteins: LIN-9, LIN-35 Rb,
LIN-37, LIN-52, LIN-53 RbAp48, LIN-54, DPL-1 DP, and EFL-1 E2F. Model: This
complex binds chromatin to repress the transcription of genes that promote the
expression of vulval cell fates. red box, a gene that promotes the expression of vulval
cell fates. (C) Model for joint action of the DRM and NuRD-like complexes. The DRM
complex could act with the NuRD-like complex to repress transcription. (D) Model for
sequential roles of the NuRD-like and DRM complexes. Genes that promote the
expression of vulval cell fates are transcriptionally active when the histones in the region
of their promoters are acetylated. The NuRD-like complex (which contains at least HDA-
1 HDAC1, LET-418 Mi2 and LIN-53 RbAp48) deacetylates these histones, causing
transcriptional repression. This transcriptional repression is maintained by the binding of
the DRM complex to the deacetylated histones. Note that is not known if the NuRD-like
complex and the DRM complex act on the same set of target genes. LIN-53 RbAp48 is
a component of both the NuRD-like and the DRM complexes. See text for references
cREAM corr
$
Figure 6
icleosome
Iu
DNA
NuRD-like complex
cREAM complex
tI N 7
C. elegans D. melanogaster 
H. sapiens
LIN-9 Mipl30 hLin-9
LIN-35 RBF Rb, p107, p130
LIN-37 Mip40 F25965
LIN-52 dLin-52 LOC91750
LIN-53 p55 RbAp48
LIN-54 Mipl 20 tesmin
DPL-1 dDP DP-1
EFL-1 dE2F2 E2F-4, E2F-5
il;
Supplemental Information
100
SUPPLEMENTAL RESULTS
Cloning of Iin-37: We mapped lin-37between sma-3 and mec- 14 on LGIII and
performed transformation rescue of the synMuv phenotype of lin-8(nl 11); lin-37(n758)
with cosmids in this region. A 13.8 kb fragment containing five predicted open reading
frames (ORFs) present in both of the rescuing cosmids F31H1 and C49B6, was able to
rescue the synMuv phenotype of lin-8(n 111); lin-37(n758) animals (Supplemental Figure
1). We introduced frameshift mutations into the minimal rescuing fragment to disrupt
each of the five complete open reading frames and tested each construct for rescue.
Only disruption of ZK418.4 eliminated the 
rescuing activity of the 13.8 kb fragment,
suggesting that ZK418.4 encodes lin-37 (Supplemental Figure 1).
By screening a cDNA library we obtained clones for mRNAs that encode lin-37.
Multiple cDNAs had the last four nucleotides of the C. elegans trans-spliced leader SL2
immediately preceding the first ATG; this SL2 sequence is not found in the genomic
sequence. That the immediately upstream gene ZK418.5 is transcribed in the same
direction as lin-37, the two genes are separated by only 170 base pairs and lin-37 is
SL2 trans-spliced suggest that ZK418.5 and lin-37 are co-transcribed as an operon
(BLUMENTHAL 1995).
Cloning of lin-54: Germline-transformation experiments using genomic DNA
clones from the interval of LGIV to which lin-54 was mapped identified two overlapping
cosmids, MMMC1 and JC8, each of which could rescue the synMuv phenotype 
of
lin-54(n223 1); lin-15A(n433) mutants (Supplemental Figure 2). DNA subclones of the
overlapping region that contained the predicted ORF JC8.6, including a subclone that
contained JC8.6 as the only complete predicted ORF, rescued the lin-54(n223 1); lin-
15A(n433) synMuv phenotype, whereas subclones that lacked a complete JC8.6
sequence failed to rescue (Supplemental Figure 2).
The deletion allele lin-54(n3423) removes 2338 base pairs beginning 280 base
pairs upstream of the predicted lin-54 initiator codon and leaves only the final ten lin-54
codons intact (Figure 2A). The deletion 
allele lin-54(n3424) removes 1140 
base pairs,
including the last exon and half of the preceding exon (Figure 2A).
101
SUPPLEMENTAL MATERIAL AND METHODS
Culture conditions and strains: Mutant alleles used are listed below and are
described by Riddle et al. (1997) unless otherwise 
noted: LGI: lin-35(n745), lin-53(n833,
n3368) (Lu and Horvitz, 1998; this study) ; LGII: 
dpl-1(n3316) (CEOL and HORVITZ 2001),
lin-8(n111), trr-1(n3712) (CEOL and HORVITZ 2004); LGIII: lin-9(n 112, n942, n3675)
(C.J.C., F. Stegmeier, M.M.H. and H.R.H unpublished results), lin-37(n758, n2234)
(FERGUSON and HORVITZ 1989; THOMAS et al. 2003), lin-52(n771, n3718) (THOMAS et al.
2003); LG IV: lin-54(n2231, n2990, n3423, n3424) (THOMAS et al., 2003; this study),
unc-30(e 191); LGV: hda- l(e 1795) (DUFOURCQ et al. 2002), let-418(arl 14, n3536)
(SEYDOUX et al. 1993; VON ZELEWSKY 
et al. 2000); LGX: lin-15A(n433, n767),
lin-15B(n744) (FERGUSON and HORVITZ 1989). The translocations nT1[qls51](IV;V) and
hT2[qls48] (I;111) and the chromosomal inversion mln [dpy- 10(e 128) mis 14] were also
used. qls48, qls51 and mls14 are integrated transgenes that express GFP and are
linked to the translocation or balancer (EDGLEY and RIDDLE 2001; MATHIES etal. 2003).
nls93 is an integrated transgene that expresses LIN-36::GFP and is capable of rescuing
the synMuv phenotype of lin-36(n766); lin-15A(n767) (THOMAS and HORVITZ 1999).
Transgenic Strains: For rescue of the 
lin-8(n 111); lin-37(n758) Muv phenotype,
cosmid or subclone DNA (20 ng/pl) was coinjected with a dominant rol-6 marker plasmid
(pRF4) (100 ng/pl). For rescue of the lin-54(n2231); lin-15A(n767) synMuv phenotype
cosmid (5-10 ng/pl) or plasmid (50-80 ng/pl) was injected along with the pRF4 plasmid
(50-80 ng/pl).
Determination of lin-54 cDNA sequence: lin-54 cDNA sequence was
determined from the EST clones yk414a8 and yk454f3 (kindly provided by Y. Kohara).
The 5' ends of lin-54 transcripts were determined using the 5' RACE system v2.0
(Invitrogen) according to the manufacturer's recommendations. We found SL1 splice-
leader sequence on 5' RACE isolates and detected two alternative SL1 splice sites:
gtttaattacccaagtttgagtattttcagtgttgacaATG and gtttaattacccaagtttgagtgttgacaATG, where
SL1 sequence is underlined and the predicted translational start site is in uppercase.
Two alternative splice acceptors exist for the first (ag/AACTTACAGGC versus
agaacttacag/GC) and second (ag/CTTTTTCAGCC versus agctttttcag/CC) introns. For
102
each intron, the use of the alternative splice acceptors only results in a nine nucleotide
variance in the length of the cDNA. The intronic sequence is shown in lowercase, and
the exonic sequence in uppercase.
Antibody Preparation: Anti-LIN-9 antiserum was generated by immunizing
rabbits with purified MBP-LIN-9 (full-length) fusion protein. The antiserum was affinity
purified against GST-LIN-9 (full-length) as described by Koelle and Horvitz (1996). Anti-
LIN-35 antiserum was generated by immunizing guinea pigs with purified MBP-LIN-35
(amino acids 270-961) fusion protein. Serum from the third production bleed was used
for western blot experiments. (Other anti-LIN-35 antiserum was previously reported (Lu
and HORVITZ 1998), but we were unable to establish specificity of this antiserum for
LIN-35 in the studies reported in this manuscript.) Anti-LIN-37 antiserum was generated
by immunizing rabbits with LIN-37 (full-length):His fusion protein. Anti-LIN-37 antiserum
was affinity purified against GST-LIN-37 (full-length). Anti-LIN-52 antiserum was
generated by immunizing rabbits with purified MBP-LIN-52 (full-length) fusion protein
and was affinity purified against GST-LIN-52 (full-length). Anti-LIN-53 rabbit antiserum
was generated by immunization with the peptide CNEVDEETPADVVERQQ (amino
acids 402-417) and was affinity purified using this peptide. This peptide is unique to
LIN-53 and is not contained in the related C. elegans protein RBA-1. Anti-LIN-54
antiserum was generated by immunizing guinea pigs with GST-LIN-54 (full-length)
fusion protein and was affinity purified against MBP-LIN-54 (full-length).
103
SUPPLEMENTAL LITERATURE CITED
BLUMENTHAL, T., 1995 Trans-splicing and polycistronic transcription in Caenorhabditis
elegans. Trends Genet 11: 132-136.
CEOL, C. J., and H. R. HORVITZ, 2001 dpl-1 DP and efl-1 E2F act with lin-35 Rb to
antagonize Ras signaling in C elegans vulval development. Mol Cell 7: 461-473.
CEOL, C. J., and H. R. HORVITZ, 2004 A new class of C. elegans synMuv genes
implicates a Tip60/NuA4-like HAT complex as a negative regulator of Ras
signaling. Dev Cell 6: 563-576.
DUFOURCO, P., M. VICTOR, F. GAY, D. CALVO, J. HODGKIN et al., 2002 Functional
requirement for histone deacetylase 1 in Caenorhabditis elegans gonadogenesis.
Mol Cell Biol 22: 3024-3034.
EDGLEY, M. L., and D. L. RIDDLE, 2001 LG II balancer chromosomes in Caenorhabditis
elegans: mTI(ll;Ill) and the mini set of dominantly and recessively marked
inversions. Mol Genet Genomics 266: 385-395.
FERGUSON, E. L., and H. R. HORVITZ, 1989 The multivulva phenotype of certain
Caenorhabditis elegans mutants results from defects in two functionally
redundant pathways. Genetics 123: 109-121.
KOELLE, M. R., and H. R. HORVITZ, 1996 EGL-10 regulates G protein signaling in the C.
elegans nervous system and shares a conserved domain with many mammalian
proteins. Cell 84: 115-125.
Lu, X., and H. R. HORVITZ, 1998 lin-35 and lin-53, two genes that antagonize a C.
elegans Ras pathway, encode proteins similar to Rb and its binding protein
RbAp48. Cell 95: 981-991.
MATHIES, L. D., S. T. HENDERSON and J. KIMBLE, 2003 The C. elegans Hand gene
controls embryogenesis and early gonadogenesis. Development 130: 2881-
2892.
RIDDLE, D. L., BLUMENTHAL, T., MEYER, B.J., PRIESS, J.R., 1997 C. elegans /I Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
104
SEYDOUX, G., C. SAVAGE and I. GREENWALD, 1993 Isolation and characterization of
mutations causing abnormal eversion of the vulva in Caenorhabditis elegans.
Dev Biol 157: 423-436.
THOMAS, J. H., C. J. CEOL, H. T. SCHWARTZ and H. R. HORVITZ, 2003 New genes that
interact with lin-35 Rb to negatively regulate the let-60 ras pathway in
Caenorhabditis elegans. Genetics 164: 135-151.
THOMAS, J. H., and H. R. HORVITZ, 1999 The C. elegans gene lin-36 acts cell
autonomously in the lin-35 Rb pathway. Development 126: 3449-3459.
VON ZELEWSKY, T., F. PALLADINO, K. BRUNSCHWIG, H. TOBLER, A. HAJNAL etal., 2000 The
C. elegans Mi-2 chromatin-remodelling proteins function in vulval cell fate
determination. Development 127: 5277-5284.
105
Supplemental Figure 1. Molecular cloning of lin-37.
Rescuing cosmids C49B6 and F31 H1 are shown as gray bars. Dotted bars indicate that
the cosmid continues in the direction shown. The subcloned portions of the cosmids
injected for rescue are shown below. The five predicted genes within the 13.8 kb
rescuing fragment are indicated on the bottom. The arrows indicate the direction of
transcription. The fraction of transgenic lines rescued for the lin-8(n 111); lin-37(n758)
Muv phenotype is shown on the right. +, rescue; -, no rescue.
106
00 C\ ( 00 C ZL
+o1 (0r N
7-C O L C)
a.
ELI
LIL
8
LU
-5
0,
C
a)
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0)
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r
.0
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0)
E
a)
a.
0n.
C')
I I
I I
Supplemental Figure 2. Molecular cloning of lin-54.
Rescuing cosmids MMMC1 and JC8.6 are shown as gray bars. Dotted bars indicate
that the cosmid continues in the direction shown. The subcloned portions of the cosmids
injected for rescue are shown below. The fraction of transgenic lines rescued for the
lin-54(n223 1); lin- 15A(n233) Muv phenotype is shown on the right. The lin-54 gene is
indicated on the minimal rescuing fragment. The arrow indicates the direction of
transcription.
108
a)
(I,
a4)
LU.
C
a)
E
a)
Q.
Q.
U,)
cO
r-
T
B
(L
'5
0
0)
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o
x
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i
cn
Supplemental Figure 3. Antibodies specifically recognize LIN-9, LIN-35, LIN-52 and
LIN-53
(A) Affinity-purified antibodies raised against recombinant LIN-9 were used to blot
extracts from 50 young adult wild-type or lin-9(n942) mutant animals. (B) Affinity-purified
antibodies raised against recombinant LIN-35 were used to blot extracts from a mixed
stage population of wild-type or lin-35(n745) mutant animals. (C) Affinity-purified
antibodies raised against recombinant LIN-52 were used to blot extracts from 50 young
adult wild-type or lin-52(n3718) mutant animals. (D) Affinity-purified antibodies raised
against a peptide specific to LIN-53 were used to blot extracts from 50 young adult wild-
type or lin-53(n3368) mutant animals.
110
Supplemental Figure 3
*-LIN-35
*LIN-53
B
250-
150-
100-
75-
150-
100-
75-
50-
C
50-
37-
25-
20-
D
75-
52
50-
A \:\
-: :a~4
~j~i(~
:~
-:: : -
:;?
,:;::
--
;-;
:-: ::
--
i_;,ri ?:-:-i: :~;: ::j:: :: :
Supplemental Figure 4. LIN-37 does not coimmunoprecipitate with LIN-36::GFP.
Extracts from both wild-type embryos and embryos carrying n1s93, a rescuing lin-36::.gfp
transgene, were immunoprecipitated with anti-GFP antibodies (mAb3E6, Qbiogen).
Proteins were separated by SDS-PAGE and immunoblotted with antibodies specific to
the antigen indicated on the left.
112
Supplemental 
Figure 4
IPaGFP
lin-36::
gfp 
WT
i IN IP' IN 
IP
I
LIN-36::GFP
LIN-37
CHAPTER 3
LIN-61, one of two C. elegans MBT-repeat-containing proteins, acts
separately from the DRM and NuRD-like complexes in developmental
regulation
Melissa M. Harrison, Xiaowei Lu', and H. Robert Horvitz
This manuscript is being prepared for publication.
Xiaowei Lu mapped and cloned lin-61. The original Iin-61 allele, sy223, was isolated in
Paul Sternberg's laboratory.
1
Present address: Department of Cell Biology, University of Virginia School of Medicine,
Charlottesville, VA 22908
114
ABSTRACT
Vulval development in Caenorhabiditis elegans is inhibited by the redundant functions of
the class A, B, and C synthetic multivulva (synMuv) genes. At least 31 synMuv genes
have been identified, many of which appear to act via transcriptional repression. Here
we report the identification of the class B synMuv gene lin-61, which encodes a protein
composed of four malignant brain tumor (MBT) repeats. MBT repeats are domains of
approximately 100 amino acids, and have been found in multiple copies in a number of
transcriptional repressors, including Polycomb group proteins. MBT repeats are
important for the transcriptional repression mediated by these proteins and in some
proteins act by binding to modified histones. C. elegans contains one other MBT-repeat-
containing protein, MBTR-1. We demonstrated that a deletion allele of mbtr- 1 does not
cause a class B synMuv phenotype nor does it enhance or suppress the abnormalities
caused by loss of function in lin-61. We further showed that lin-61 is biochemically and
genetically separable from other class B synMuv genes in a number of biological
processes. These data demonstrate that while the class B synMuv genes cooperate to
regulate vulval development, they have distinct functions in other processes.
115
INTRODUCTION
Development requires cells that are initially equivalent to differentiate into organs
and tissues. These changes in cell fate often are mediated by modulating gene
expression, which can be controlled through modifications to the surrounding chromatin.
Mutations in factors that control chromatin structure can lead to developmental defects
in numerous organisms reviewed in (reviewed in MARGUERON et al 2005). In
Caenorhabiditis elegans, the regulation of vulval development have involves
evolutionarily conserved factors important for signal transduction, chromatin remodeling
and transcriptional repression. Genetic and biochemical studies of these factors should
help to elucidate how proteins that modify chromatin structure can cooperate during
development.
The C. elegans hermphrodite vulva is formed from three of six equipotent blast
cells, P3.p-P8.p (SULSTON and HORVITZ 1977; SULSTON and WHITE 1980). Although all
six cells are competent to adopt a vuval cell fate, in wild-type development 
only P5.p,
P6.p and P7.p divide to generate the vulva. P3.p, P4.p, and P8.p normally divide once
and fuse with the surrounding syncytial hypodermis. Many signaling pathways are
required to properly specify vulval development, including a receptor tyrosine
kinase/Ras pathway, a Wnt signaling pathway, and a Notch signaling pathway (YOCHEM
etal. 1988; BEITEL etal. 1990; HAN etal. 1990; GLEASON etal. 2002). Mutations
affecting these pathways either can result in P3.p, P4.p, and P8.p aberrantly adopting
vulval cell fates, a multivulva (Muv) phenotype, or none of the Pn.p cells adopting a
vulval cell fate, a vulvaless (Vul) phenotype (YOCHEM et al. 1988; STERNBERG and
HORVITZ 1989; BEITEL et a. 1990; HAN etal. 1990; GLEASON etal. 2002).
Vulval induction is antagonized by the synthetic multivulva (synMuv) genes,
which have been placed into three classes, A, B, and C, based on their genetic
interactions (FERGUSON and HORVITZ 1989; CEOL and HORVITZ 2004). Because of
redundancy among the three classes, only animals with loss-of-function mutations in
two synMuv classes have a multivulva phenotype, whereas animals with a loss-of-
function mutation in a single class are not Muv. Many of the synMuv genes encode
proteins that have been implicated in chromatin remodeling and transcriptional
116
repression. These include proteins such as EFL-1 E2F, DPL-1 DP, and LIN-54, which
likely bind directly to DNA and repress transcription as components of the evolutionarily-
conserved DP, Rb and MuvB (DRM) complex (CEOL and HORVITZ 2001; M.M.H, C.J.
Ceol and H.R.H unpublished results.) The synMuv proteins LET-418, LIN-53, and HDA-
1 are homologous to components of the mammalian Nucleosome Remodeling and
Deacetylase (NuRD) complex (Lu and HORVITZ 1998; voN ZELEWSKY et al. 2000;
DUFOURCQ et al. 2002). The synMuv proteins HPL-2 and MET-2 are homolgous to HP1
and SETDB1, respectively (COUTEAU et al. 2002; POULIN et a. 2005). HP1 binds to
methylated lysine 9 of histone H3, and SETDB1 is a methylatransferase that can
methylate this lysine residue (BANNISTER et al. 2001; SCHULTZ et al. 2002). In other
organisms all of these proteins function in transcriptional repression, and recently in C.
elegans, a number of class B synMuv genes have been implicated in transcriptional
repression of lin-3 EGF (XUE eta!. 1998; ZHANG etal. 1998; ZHANG etal. 1999;
BANNISTER et al 2001; Cui et al. 2006a). The class C synMuv genes encode homologs
of a Tip60/NuA4-like histone acetyltransferase complex, which might act in
transcriptional repression or activation (CEOL and HORVITZ 2004).
Polycomb-group (PcG) proteins were initially identified by their ability to
transcriptionally repress Hox genes but have since been shown to repress additional
targets including genes regulated by E2F transcription 
factors (DAHIYA et al. 2001;
OGAWA et al. 2002). PcG proteins include enzymes with histone methyltransferase
activity and proteins that bind to these histone modifications as well as many other
proteins, including Sex Comb on Midleg (SCM). SCM contains two malignant brain
tumor (MBT) repeats, which are motifs of approximately 100 amino acids. MBT repeats
have been found in many transcriptional repressors, including 1(3)mbt, which is in a
complex with multiple other PcG group proteins and with E2F6 in human cells (OGAWA
et al. 2002).
Here we report the characterization of the class B synMuv gene, lin-61. lin-61
encodes a protein composed of four MBT repeats that is localized to chromatin. LIN-61
is not a core component of the pocket-protein-containing DRM complex or the NuRD-
like complex and acts separately from members of these complexes to regulate some
117
aspects of development. Thus MBT-repeat-containing proteins, such as Polycomb-
group proteins, might cooperate with Rb-containing complexes and histone deacetylase
complexes to repress a subset of genes, but likely act independently of these
complexes to regulate expression of other genes.
118
MATERIALS AND METHODS
Strains: Mutant alleles used are listed below and are described by Riddle et al.
(1997) unless otherwise noted: LGI: unc-14(e57), unc-15(e73), lin-61(sy223, 
n3442,
3446, n3447, n3624, n3687, n3736, n3807, n3809, n3922 (this study), mbtr- 1(n4775)
(this study), nls133 (H. Schwartz and H.R.H unpublished results); LGII: lin-8(n2731)
(THOMAS et al. 2003), lin-38(n751) (FERGUSON and HORVITZ 1989), lin-56(n2728)
(THOMAS et al. 2003), trr-1(n3712) (CEOL and HORVITZ 2004); LGIII: hpl-2(n4274) (E.
Andersen and H.R.H. unpublished results), lin-13(n770) (FERGUSON 
and HORVITZ 1989),
lin-37(n758) (FERGUSON and HORVITZ 1989), mat-3(ku233) (GARBE et al. 2004); LGV:
hda-1(e 1795) (Dufourcq et al. 2002); LGX: lin-15A(n433, n767), lin-15B(n744)
(Ferguson and Horvitz 1989), mys-1(n3681) (CEOL and HORVITZ 2004), qls56
[Iag-2.::gfp; unc- 119(+)] (SIEGFRIED and KIMBLE 2002), pkls 1605 [rol-6(su1006)
hsp 16/2::gfp/IacZ(out of frame)] (POTHOF et al. 2003), ccEx6188 [myo-3:.-Ngfp-lacZ
rol-6(su1006)] (HSIEH et al. 1999). The translocations nTl[qls51] (IV;V) and hT2[qls48]
(I;111) and the chromosomal inversion mIn [dpy-10(e128) mls I4]were also used. 
qls48,
qls51 and mls14 are integrated transgenes that express GFP and are linked to the
translocation or balancer (EDGLEY and RIDDLE 2001; MATHIES et al. 2003).
Transgenic strains: For rescue of the lin-61(sy223); lin- 15A(n767) or
lin-61(n3624); lin-15A(n767) Muv phenotype, cosmid or subclone DNA (5 or 10 ng/pl)
was coinjected with a dominant rol-6 marker plasmid (pRF4) (80 ng/pl).
RNAi analysis: Templates for in vitro transcription reactions were made by PCR
amplification of cDNAs including flanking T3 and T7 promoter regions. RNA was
transcribed in vitro using T3 and T7 polymerases. In vitro transcribed RNA was
denatured for 10 min and annealed prior to injection.
Antibody preparation, immunocytochemistry and western blots: Anti-LIN-61
antiserum was generated by immunizing rabbits and guinea pigs with purified GST-
LIN-61(amino acids 159-491). The antiserum was affinity purified against full-length
MBP-LIN-61. The rabbits and guinea pigs were maintained by Covance (Denver, PA).
All antibodies were used at a 1:1000 dilution for western blots, except for 
anti-LIN-8,
anti-LIN-53, anti-LIN-54, and anti-DPL-1, which were used at 1:500. Affinity purified
119
antibodies were used in all cases except for anti-LIN-35 antibodies, for which we used
unpurified serum from the third production bleed. Larvae and adults for immunostaining
were fixed in 1% paraformaldehyde for 30 min, as described by Finney and Ruvkun
(1990). Embryos were fixed for 20 min in 0.8% paraformaldehyde, as described by
Guenther and Garriga (1996). Affinity-purified anti-LIN-61 antisera were used at a 
1:100
dilution for immunocytochemistry.
Phenotypic characterization: To score RNAi hypersensitivity, L4 larvae were
placed on E. coli expressing either hmr- I or cel- 1 dsRNA. 24 hours later the young 
adult
hermphrodites were transferred to a fresh plate with E coliexpressing dsRNA. The
hermphrodites were allowed to lay eggs for 24 hr and were then removed. The progeny
of hermphrodites grown on E coli expressing cel- 1 dsRNA were scored for arrest at the
L2 larval stage. The progeny of hermphrodites grown on E coliexpressing hmr- 1
dsRNA were scored for embryonic lethality. For scoring ectopic PGL-1 expression, L1
larvae were permeabilized using a freeze-crack method followed by a methanol-acetone
fixation as described in Wang et al. (2005). Permeabilized larvae were incubated with
OIC1 D4 monoclonal anti-PGL-1 antibodies (Developmental Studies Hybridoma 
Bank,
University of Iowa) at a dilution of 1:20 overnight followed by a one hour incubation with
Alexa Fluor 594 goat anti-mouse IgM (Invitrogen) at a dilution of 1:25. To score for the
Tam phenotype, animals carrying the extrachromosomal array ccEx6188 [myo-3::Ngfp-
lacZ], hermphrodites were grown for at least two generations at 25oC. Relative GFP
expression was then scored under the fluorescence dissecting microscope.
Hermaphrodites carrying the transgene pkls 1605 [rol-6(su1006) hsp16/2::gfp/IacZ(out of
frame)] were scored for somatic repeat instability after being grown at 200C for five
days, heat-shocked at 310C two hr, and allowed to recover at 200C for one hour. The
hermaphrodites were then fixed and stained for the presence of p-galactosidase with X-
gal. Vulval defects in mat-3(ku233) mutants were scored by Nomarski differential
interference contrast (DIC) microscopy of mid-L4 hermaphrodites. Vulval development
was scored as abnormal if the invagination was asymmetric or contained fewer than the
22 cells found in wild-type animals.
120
Embyro lysates: Embryos were harvested from liquid cultures and bleached to
kill larvae and adults in a solution containing 5N NaOH and 20% hypochlorite. The
embryos were resuspended in 1 ml of lysis buffer (25 mM HEPES pH 7.6, 150 mM
NaCI, 1 mM DTT, 1 mM EDTA, 0.5 mM EGTA, 0.1% NP-40, 10% glycerol with
Complete EDTA-free protease inhibitors (Roche Diagnostics)) for each gram of embryos
and frozen in liquid nitrogen. The embryos were thawed at room temperature and
sonicated 15 times for 10 sec using a Branson sonifier 450 at setting 5. The
homogenate was then centrifuged at top speed in a microcentrifuge for 15 min at 40C.
The supernatant was removed to a new tube and was spun for 15 min at top speed in a
microcentrifuge. The remaining supernatant was pooled and the protein concentration
was determined using the Pierce Coomassie Plus Protein Assay Reagent (Pierce
Biotechnology). The lysate was diluted to 5-10 mg/ml and was used immediately or
stored at -80oC and thawed no more than once before use.
Immunoprecipitation experiments: Antibodies were crosslinked to Protein A
Dynabeads (Invitrogen) using dimethyl pimelimidate (Pierce) essentially as described by
Harlow and Lane (1999), with the two following exceptions: reactions were stopped with
0.1 M Tris pH 8.0 and beads were washed three times for 1 min in 100 mM glycine pH
2.5 followed by lysis buffer. The beads 
were then resuspended in PBS. 500 tl of 
lysate,
corresponding to 2.5-5 mg of total protein, were incubated with 25 ~l of affinity-purified
antibody bound to 25 !pl of beads for each immunoprecipitation reaction. Prior to
incubation with antibody-bound beads, lysates were initially precleared with 25 tl of
beads for between 1 hr and overnight at 40C. The precleared lysates were recirculated
over the antibody-bound beads at 40C for 1-2 hr and were then washed three times for 5
min each at 40C in lysis buffer. Following the final wash, the beads were resuspended in
20 ?l of 2X protein sample buffer, boiled for 5 min, and loaded on an SDS-
polyacrylamide gel. HRP-conjugated protein A (Bio-Rad) was used for detection 
of
protein by western blot following coimmunoprecipitation experiments, except 
for
detection of tubulin in which HRP-conjugated goat anti-mouse (Bio-Rad) 
was used.
121
RESULTS
lin-61 is a class B synMuv gene: Ten lin-61 alleles have been isolated in three
different screens. The original lin-61 allele, sy223, was isolated in the laboratory of Paul
Sternberg based on its synMuv phenotype in combination with a loss-of-function
mutation in lin-15A. Four alleles, n3687, n3807, n3809, and n3922, were isolated in a
screen for transgene misexpression (H. Schwartz, D. Wendell, and H.R.H. unpublished
results). Five alleles, n3442, n3446, n3447, n3624, and n3736 were isolated in a screen
for mutations that cause a synMuv phenotype with lin- 15A(n767) (CEOL et al. 2006).
As single mutants none of the ten lin-61 alleles caused a Muv phenotype
(Tablel). All ten alleles caused a synMuv phenotype in combination with loss of function
of the class A synMuv gene lin-56 (Table 2). n3809 caused a synMuv phenotype in
combination with loss of function in all four class A synMuv genes but not in combination
with mutations in the class B synMuv genes lin-37 and lin- 15B (Table 1; data not
shown). Loss of function of lin-61 also failed to enhance the Muv phenotype associated
with hda- 1(e 1795) (Table 1). Similar to 
previous reports for reduction of lin-9, 
lin-15B,
lin-35 and lin-37by RNAi (COUTEAU et al 2002), loss of function of lin-61 caused a Muv
phenotype in combination with a mutation in hpl-2 (Table 1). These data suggest that
lin-61 has a function in vulval development that is redundant with that of hpl-2 but not
with that of hda-1. The lin-61 allele n3809 did not enhance the weak Muv phenotype
associated with mutations in either of the class C genes trr- I1 or mys-1 (Table 1).
Because lin-61 caused a synMuv phenotype in combination with mutations in class A
but not class B synMuv genes and because it did not cause the P8.p induction 
observed
in animals mutant for class C synMuv genes (CEOL and HORVITZ 2004), we consider lin-
61 to be a class B synMuv gene.
lin-61 encodes an MBT-repeat-containing protein: We mapped sy223 to an
interval between unc- 14 and unc- 15 on LGI. A pool of cosmids covering 
the central
portion of this region rescued the synMuv phenotype of lin-61(sy223); 
lin- 15A(n767)
animals, and a single cosmid from this pool, R06C7, rescued the synMuv 
phenotype of
lin-61(n3624); lin-15A(n767) animals. A subcloned Stul-Sacll fragment containing
R06C7.7as the only complete predicted open-reading frame was 
capable of rescuing
122
the lin-61(n3624); lin- 15A(n767) synMuv phenotype (Figure 1A). In addition, RNAi
directed against R06C7. 7 caused a synMuv phenotype in combination with
lin- 15A(n767) (POULIN et at 2005). To confirm that R06C7 7is lin-61, we determined the
sequence of R06C7 7from lin-61(sy223) animals. sy223 contains a G-to-A transition at
the splice acceptor site before the last predicted exon (Figure 1B and Table 2).
Mutations affecting the coding region of lin-61 were also found in the nine other lin-61
alleles, including three nonsense mutations, five missense mutations and one mutation
in a splice acceptor (Table 2).
We determined the sequence of lin-61 cDNAs and determined that the lin-61
transcript is SL1-spliced and comprises six exons. lin-61 transcripts contain no 5' UTR
as the SL1 leader sequence is spliced directly to the ATG start codon. lin-61 encodes a
predicted protein of 491 amino acids that is composed almost exclusively of four
malignant brain tumor (MBT) repeats. MBT repeats are domains of approximately 100
amino acids and have been found in up to four copies per protein. MBT repeats were
initially identified in the D. melanagaster protein lethal (3) malignant brain tumor
(l(3)mbt) (WISMAR et al 1995) and 
are present in many other metazoan proteins,
including the Polycomb group protein Sex Comb on Midleg (SCM) (BORNEMANN et al.
1996; TOMOTSUNE et al. 1999; Usui et al. 2000; BoccUNI et al 2003; MARKUS et al.
2003; ARAI and MIYAZAKI 2005). In addition to their MBT repeats, 1(3)mbt and SCM both
contain atypical zinc fingers and a single sterile alpha motif (SAM) domain (WISMAR et
al. 1995; BORNEMANN et at 1996). The SAM domain of SCM mediates homodimerization
and interaction with the Polycomb protein Polyhomeotic (PETERSON et a. 1997; KIM et
al. 2005). Given that LIN-61 lacks both of these additional domains and that it is
composed almost exclusively of the four MBT repeats, the functionality of LIN-61 is
likely provided by the MBT repeats.
Characterization of lin-61 alleles: The mutation in lin-61(n3809) results in an
ochre stop codon at amino acid 159 that is predicted to result in a truncated LIN-61
protein and is likely to be a null allele. lin-61(n3809); lin-56(n2728) animals have a
highly penetrant synMuv phenotype (Table 2). Similarly penetrant synMuv phenotypes
are caused by an additional nonsense mutation and both of the splice acceptor
123
mutations (Table 2). Although n3687 
causes an ochre mutation at amino acid 
322,
animals carrying this Iin-61 allele have a weak synMuv phenotype. The low penetrance
of the synMuv phenotype suggests that a functional protein product might be made in
lin-61(n3687) animals. Alternatively, the strain containing n3687could contain an
additional mutation that suppresses the synMuv phenotype. This strain contains a
closely linked integrated transgene that overexpresses both lin- 15A and lin- 15B.
However, it is unlikely that this linked transgene is causing a reduction in the penetrance
of the synMuv phenotype, as overexpression of lin-15A is incapable of rescuing synMuv
defects caused by mutation in lin-56 (E. Davison and H.R.H. unpublished results).
Four of the five missense mutations in Iin-61, n3447, n3736, n3807, and n3922,
are in one of the four MBT repeats. The fifth missense mutation, n3624, causes a
proline-to-serine change in a residue between the first and second MBT repeats. The
five missense mutations cause weaker synMuv phenotypes than most of the putative
null alleles and form an allelic series. n3624 and n3807, the two strongest missense
mutants, cause a less penetrant synMuv phenotype than the nonsense and splice-
acceptor mutations, suggesting that some LIN-61 activity might remain in these mutants
(Table 2). Animals homozygous for lin-61(n3736) and a class A synMuv mutation have
an intermediate phenotype, as evident from the fact that lin-61(n3736) in combination
with lin-15A(n433) causes a lower penetrance synMuv phenotype than do lin-61(n3807)
or lin-61(n3624). However, when these strains are raised at 23?C or when the lin-61
alleles are combined with a mutation in the class A synMuv gene lin-56, lin-61(n3676)
causes a synMuv phenotype with a penetrance similar to the penetrance of the synMuv
phenotypes caused by lin-61(n3624) and lin-61(n3807) (Table 2 and data not shown).
lin-61(n3447) and lin-61(n3922) mutant animals have the least penetrant 
synMuv
phenotypes (Table 2).
The C. elegans genome encodes one additional MBT-repeat-containing
protein: Given the molecular identification of LIN-61 as containing four MBT repeats 
we
searched the C. elegans genome for additional MBT-repeat-containing proteins. 
Using
BLAST (ALTSCHUL etal. 1997), Pfam (BATEMAN et al 2002), and SMART (SCHULTZ 
et al.
2000) algorithms we identified a single additional MBT-repeat-containing protein
124
encoded by the predicted gene Y48GIA.6 (Figure 2A). We determined the sequence of
a full-length cDNA. The GENEFINDER prediction for the cDNA is predominantly 
correct,
except that the predicted fourth intron is not removed in this cDNA. The incorporation of
this predicted intron into the open-reading frame results in a larger fourth exon than
predicted but does not alter the frame of the predicted protein (Figure 2B). Given that
the protein encoded by Y48GIA.6 contains MBT-repeats, we named this gene mbtr-1
for malignant brain tumor repeats.
The structure of MBTR-1 is similar to that of LIN-61 
(Figure 2A). Like LIN-61,
MBTR-1 is composed almost exclusively of four MBT repeats and lacks the SAM
domain and zinc fingers found in other MBT-repeat-containing proteins (Figure 2).
MBTR-1 is 36% identical to LIN-61 and is more similar to LIN-61 than to proteins in any
other organisms. LIN-61 and MBTR-1 share an insertion of approximately 15 amino
acids in their second MBT repeat not found in their human homolog h-1(3)mbt-like II or in
other MBT-repeat-containing proteins (Figure 2A, Figure 4A, and data not shown). It is
unclear how these additional amino acids might alter the structure of the MBT repeat or
contribute to the function of the protein.
To determine the function of mbtr-1 we identified a deletion allele, n4775, which
removes exons 4 and 5 of mbtr- 1 and is predicted to result in a frame shift after amino
acid 165. GENEFINDER predicts and cDNAs confirm the existence of an open-reading
frame Y48G1A.2 in the first intron of mbtr- 1 (Figure 2B). Y48GIA.2 and mbtr- 1 are
transcribed from the opposite strands. While the n4775 deletion begins 1 kb upstream of
the Y48G1A.2 translational start site and does not remove coding sequences, it remains
possible that n4775 could remove sequences necessary for proper expression of
Y48GIA.2.
We have not identified a mutant phenotype associated with mbtr- 1(n4775).
mbtr- 1(n4775) does not cause a synMuv phenotype in combination with either lin-
15A(n767) or lin- 15B(n744). Animals mutant for both mbtr- 1 and lin-61 are not Muv.
mbtr- and Iin-61 are not redundantly required for a class A synMuv activity as
mbtr- 1(n4775) lin-61(n3809); lin- 15B(n744) animals are not Muv. Additionally, loss of
function in mbtr-1 does not enhance or suppress the synMuv phenotype of
125
lin-61(n3809), lin- 15A(n767) animals. 97% of lin-61(n3809), lin- 15A(n767) are Muv at
200C and 24% are Muv at 150C. Similarly, 94% of mbtr- 1(n4775) lin-61(n3809); lin-
15A(n767) are Muv at 200C and 27% are Muv at 150C.
LIN-61 is broadly expressed in nuclei throughout development: To
determine the expression pattern and localization of LIN-61, we generated guinea pig
and rabbit polyclonal antibodies against the C-terminal 332 amino acids of LIN-61 fused
with maltose-binding protein. The affinity-purified antibodies recognize a band
corresponding to a protein of approximately 60 kD on western blots of wild-type but not
lin-61(n3809) protein extracts (Figure 3A). This molecular weight is similar to the
predicted size of LIN-61 of 57 kD.
We used both the guinea pig and rabbit polyclonal antibodies to analyze the
localization of LIN-61 by immunostaining of embryos, larvae, and adult hermaphrodites.
Similar to all synMuv proteins studied to date, LIN-61 is localized to all or almost all
nuclei throughout development from the one-cell embryo to the adult (Figure 3B, F). In
the embryo, LIN-61 appears to localize to discrete foci in the nucleus (Figure 3B). Both
HPL-2 and LIN-13 similarly localize to foci in the nucleus (MELENDEZ and GREENWALD
2000; COUSTHAM et al. 2006). In addition, the human MBT-repeat-containing protein
1(3)mbt and Polycomb group proteins localize to foci in the nucleus (BUCHENAU et al.
1998; SAURIN etal. 1998; KOGA et al. 1999). In the adult hermaphrodite germline, LIN-61
is localized, at least in part, to condensed chromosomes during the diakinesis phase of
meiosis, suggesting that some LIN-61 might be localized to chromatin (Figure 3F). No
LIN-61 staining was seen in lin-61(n3809) 
mutant embryos, larvae, or adults (Figure 
3C,
G).
To better understand how LIN-61 might be acting with other synMuv proteins to
regulate vulval development we analyzed the localization of LIN-61 in animals mutant
for any of 28 genes that regulate vulval development, including four class A synMuv
genes, 19 class B synMuv genes, two class C synMuv genes and genes encoding three
Ras-pathway modifiers. No detectable change in LIN-61 localization was noted in any of
these mutant backgrounds (data not shown), suggesting that these genes do not
regulate vulval development by modifying LIN-61 expression or subcellular localization.
126
Missense mutations in LIN-61 might disrupt protein stability: The crystal
structures for peptides containing two or three MBT repeats have been solved
(SATHYAMURTHY et al 2003; WANG et al. 2003). Both structures show that individual
repeats consist of an N-terminal arm and a C-terminal 13-barrel core region. The N-
terminal arm of one repeat interacts with the p -barrel core region of the preceding
repeat, resulting in the stabilized tertiary structure (SATHYAMURTHY et aL 2003; WANG et
al. 2003).
Given that the crystal structures demonstrate residues within the MBT repeats
interact to form a stabilized structure, we analyzed LIN-61 protein levels in strains
carrying each of the ten mutant alleles to determine if any of the mutations might result
in protein misfolding and subsequent degradation. Full-length LIN-61 was absent or
levels were greatly reduced in animals with any of the three nonsense mutations or two
splice-acceptor mutations (Figure 4B). Alternatively, a truncated protein product could
be expressed that does not contain the epitope recognized by either of the polyclonal
antibodies. lin-61(n3736), lin-61(n3807), and lin-61(n3922) animals also showed
decreases in LIN-61 protein levels as compared to the wild type, while lin-61(n3447)
and lin-61(n3624) had wild-type or nearly wild-type protein levels. Protein levels in
animals with any of the five missense mutations were analyzed by both western blots
and by immunocytochemistry (Figure 4B and data not shown). These data suggest that
the residues mutated in n3736, n3807 and n3922 might be necessary for proper protein
folding and stability. Indeed, the mutations in both n3736 and n3807 are in residues of
the P-barrel core region likely to be important for interaction with the N-terminal arm of
the preceding repeat based on the crystal structures (SATHYAMURTHY et aL 2003; WANG
et al. 2003).
The missense mutations in n3447 and n3624 mutant animals interrupt the ability
of LIN-61 to properly regulate vulval development despite having wild-type or nearly
wild-type protein levels. These residues are not required for protein stability but are
important for LIN-61 function. While the missense mutation in n3624 is located between
two MBT repeats, the missense mutation in n3447 is in the third MBT repeat and
changes a serine residue to an asparigine residue. Thus it is likely that at least the third
127
MBT repeat is important for the function of LIN-61 in regulating vulval development 
and
that, more specifically, the serine at residue 354 is important for function.
Analysis of pleiotropies associated loss of function of lin-61 or mbtr-1: The
class B synMuv genes have roles in many processes apart from the regulation of vulval
development, including the regulation of transgene expression, RNAi sensitivity, cell-
cycle control and expression of PGL-1 and LAG-2 (HSIEH et al. 1999; BOXEM and VAN
DEN HEUVEL 2002; DUFOURCQ et al. 2002; UNHAVAITHAYA et al. 2002; POULIN et al. 2005;
WANG et al. 2005). While class B synMuv genes act similarly in vulval development,
these genes do not all function similarly in the aforementioned processes. A more
complete analysis of these pleiotropies should help to elucidate which of the more than
19 identified class B synMuv genes act in similar processes and which have distinct
roles in development.
To better understand the role of lin-61 and mbtr- 1 in development and to
compare these genes to the known class B synMuv genes, we investigated whether
loss-of-function mutations in either gene or in both genes result in specific synMuv-
regulated pleiotropies (Table 3). We found in multiple experiments that lin-61(n3809) did
not show enhanced sensitivity to either hmr- 1 or cel- 1 RNAi as compared to wild type
(data not shown). In the same experiments, lin-15B(n744), rrf-3(pk1426) and
eri- 1(mg366) were RNAi hypersensitive, as has previously been reported (SIMMER et al.
2002; KENNEDY et al. 2004; WANG et al. 2005).
Using antibody staining, we did not observe any PGL-1 misexpression in
lin-61(n3809) animals. PGL-1 is expressed specifically in the germline of wild-type
animals (KAWASAKI et al. 1998) and is misexpressed in the soma of animals with loss-of-
function mutations in a number of class B synMuv genes, including lin-9, lin-13, lin-15B,
lin-35, hpl-2, and dpl-1 (UNHAVAITHAYA et al. 2002; WANG et al. 2005; Cui et al. 2006b).
Staining of lin-15B(n744) animals with the same antibody reliably showed misexpresion
of PGL-1 in the soma.
In addition to repression of PGL-1 expression, class B synMuv genes also have a
role in lag-2 repression. A lag-2.vgfp reporter that is expressed in the distal tip cells and
vulva of wild-type hermaphrodites was misexpressed in the gut and epithelia of hda- 1
128
mutant animals (DUFOURCQ et al. 2002). RNAi against many other synMuv genes also
caused lag-2.:gfp misexpression (POULIN et al. 2005). In lin-61(n3809) animals,
however, lag-2::gfp was not expressed in the gut or epithelia.
However, in lin-61(n3809) animals carrying the lag-2:.gfp reporter we observed
strong pharyngeal GFP expression that is not seen in wild-type animals. This "green
pharynx" phenotype has been previously observed for a CEM-specific GFP reporter (H.
Schwartz and H.R.H, unpublished results). The transgene misexpression was seen in
animals mutant for two additional class B synMuv genes, hpl-2 and lin-13, and one
class A synMuv gene, lin-8, but not other synMuv genes (H. Schwartz and H.R.H).
These data suggest that while lin-61 might not act to repress transgene expression in
the gut and epithelia like many other class B synMuv genes, it has a role in repression
of some transgenes in the pharynx.
By contrast to the transgene misexpression noted 
for the lag-2:.gfp transgene,
loss of function of most class B synMuv genes results in the silencing of a simple array
of the myo-3.:gfp transgene (HSIEH et al. 1999), termed the transgene array modifier
(Tam) phenotype. Neither lin-61(n3809) nor iin-61(n3922) resulted in silencing of an
extrachromasomal array carrying myo-3::gfp (ccEx6188).
lin-61 was identified in a genome-wide RNAi screen for genes that were involved
in protecting the genome from DNA instability (POTHOF et al. 2003). Using an out-of-
frame LacZ reporter that is not expressed in wild-type animals, Pothof etal. (2003)
demonstrated that RNAi directed against lin-61 and 60 other genes could cause
expression of the transgene, suggesting that loss-of-function of these genes can result
in insertion or deletion mutations that result in an in-frame LacZ. Besides lin-61, the only
other class B gene that was identified in this screen was hda- 1. We used the LacZ
reporter used in the initial screen to test if a loss-of-function allele in lin-61 could
phenocopy the effects of RNAi. LacZstaining was evident in a significant proportion of
lin-61(n3809) animals. In the same experiment, wild-type animals showed no LacZ
staining. We further tested animals with mutations in the class B synMuv genes hpl-2
and lin- 13 and showed that in these animals LacZ was expressed. Therefore these
genes might function with lin-61 in maintaining genome stability.
129
The vulval defect caused by the loss-of-function mutation in ku233, an allele of
the gene mat-3 that encodes a member of the anaphase promoting 
complex (APC), can
be suppressed by loss-of-function in the class 
B synMuv genes lin-35, lin-15B, lin-53,
dpl- 1, and efl- 1 (GARBE et al. 2004). No coding mutations were found in the ku233 allele.
The vulval defect is likely caused by two adjacent base-pair changes 400 base pairs
upstream of the mat-3 start site, which results in a 5-10 fold reduction in mat-3 RNA
levels (GARBE et al. 2004). Loss of function of lin-35 restored expression of mat-3 to
wild-type levels, suggesting that LIN-35 might repress transcription of mat-3 (GARBE et
al. 2004). lin-61(n3809) could, like loss-of-function mutations in other class B synMuv
genes, suppress the mat-3(ku233) vulval defect. 58% of mat-3(ku233) animals had
abnormal vulvas, as compared to only 3.5% of lin-61(n3809); mat-3(ku233) animals.
Thus, LIN-61 might act to repress transcription of mat-3.
Loss of function of mbtr- 1 alone did not cause any of the pleiotropies discussed
above. In addition, mbtr- 1(n4775) did not modify the abnormalities associated with loss
of function in lin-61 nor is mbtr- I1 redundantly required to cause any of the above
mentioned abnormalities. Thus, the inability to detect a role for lin-61 in a number of
synMuv-regulated pleiotropies is not the result of compensation by another MBT-repeat-
containing protein.
LIN-61 is not a core member of the DRM or NuRD-like complexes: We have
recently identified two independent complexes that are composed of class B synMuv
proteins, the DRM complex, containing eight class B synMuv proteins including LIN-35
Rb and DPL-1 DP, and a NuRD-like complex, containing at least LET-418 Mi2, HDA-1
HDAC1, and LIN-53 RbAp48 (M.M.H and H.R.H. unpublished results). We used
coimmunoprecipitation experiments to test whether LIN-61 associates with members of
either of these two complexes. We demonstrated that although LIN-61 could be
immunoprecipitated with anti-LIN-61 antibodies from wild-type but not lin-61(n3809)
embryonic extracts, none of the DRM complex members coimmunoprecipitated with
LIN-61 (Figure 5A). Reciprocally, LIN-61 failed to coimmunoprecipitate with the DRM
complex members LIN-37 and LIN-9 (Figure 5B). These data demonstrate that LIN-61 is
not a core component of the DRM complex, although it could be weakly associated with
130
the complex or associate only at certain stages of development or in specific cell types.
Additionally, HDA-1, a component of the NuRD-like complex, failed to
coimmunoprecipitate with LIN-61 (Figure 5A), suggesting that LIN-61 also is not a
member of the NuRD-like complex. Two class A synMuv proteins, LIN-8 and LIN-56,
and the class B synMuv protein HPL-2 also do not coimmunoprecipitate with LIN-61
(Figure 5A).
While our data demonstrate that LIN-61 is not a core component of the DRM
complex, LIN-61 could act to modify the activity of this complex. To partially test this
hypothesis, we used coimmunoprecipitation experiments to determine whether the DRM
complex is properly formed in a lin-61(n3809) mutant animal. All seven members of the
DRM complex coimmunoprecipitated with LIN-37 in extracts from lin-61(n3809) mutant
embryos, suggesting that LIN-61 function is not required for proper complex formation.
However, we cannot rule out that LIN-61 might be acting to control the activity of the
DRM complex through a mechanism distinct from altering complex formation.
131
DISCUSSION
Genetic screens have identified at least 31 synMuv genes, 19 of which have
been categorized as class B synMuv genes. Genetic and biochemical studies suggest
that these class B synMuv genes are not likely to regulate vulval cell-fate specification
through the same function. For example, loss-of-function mutations in the class B
synMuv genes hda-1, let-418, and lin- 13 cause a strong synMuv phenotype in
combination with loss of function in a class A synMuv gene but also cause a weak Muv
phenotype as single mutants (MELENDEZ and GREENWALD 2000; VON ZELEWSKY et al.
2000; DUFOURCQ etal. 2002). By contrast, loss of function of other class B synMuv
genes does not cause a Muv phenotype in the absence of a mutation in a class A or C
synMuv gene. Furthermore, we have shown that some of these proteins likely function
together in a NuRD-like complex that is biochemically 
distinct from the DRM complex,
which contains at least eight other class B synMuv proteins (M.M.H. and H.R.H
unpublished results).
We demonstrate that only one of the two MBT-repeat-containing proteins in C.
elegans, LIN-61, acts with other class B synMuv proteins to regulate vulval
development. We further show that the function of LIN-61 in other aspects of
development is likely to be distinct from a 
number of other class B synMuv proteins,
suggesting it can function independently from these proteins.
MBT-repeat-containing proteins are not required for C. elegans viability: C.
elegans contains only two MBT-repeat-containing proteins, MBTR-1 and LIN-61. Both of
these proteins are composed almost exclusively of MBT repeats and lack either atypical
zinc fingers or the SPM domain that are found in many MBT-repeat-containing proteins
in other organisms, including Sex Comb on Midleg and lethal(3) malignant brain tumor.
These proteins are required for the viability of Drosophila (WISMAR et al 1995;
BORNEMANN etal. 1996).
By contast, the C. elegans MBT-repeat-containing proteins are not required for
viability. In fact, mutant animals that lack both MBTR-1 and LIN-61 appear superficially
wild-type. We have shown that while LIN-61 
is required to regulate vulval development,
MBTR-1 does not have a similar function. We further demonstrated that in a number of
132
other processes there is no detectable redundancy between MBTR-1 and LIN-61. This
suggests that although these two proteins share similar domain structures they do not
function redundantly. It remains possible that mbtr- 1 could complement for the loss of
lin-61 function, but it is not expressed in the necessary tissues or at the correct time.
LIN-61 is likely involved in transcriptional repression: Studies of MBT-
repeat-containing proteins in other organisms suggest that MBT repeats function in
transcriptional repression. Notably, MBT repeats are found in the Drosophila Polycomb
group protein Sex Comb on Midleg (SCM), which is a substoichiometric component of
the Polycomb Repression Complex 1 (PRC1). PRC1 maintains transcriptional
repression of genes by binding to methylated histones (CAO et al. 2002; CZERMIN etal.
2002; MULLER et al. 2002). Genetic analysis of SCM suggests that the MBT domains are
likely to be important for protein function (BORNEMANN et al 1996; BORNEMANN et al.
1998). Human 1(3)mbt can repress transcription when artificially 
recruited to promoters,
and this transcriptional activity requires the MBT repeats, but not the zinc fingers or the
SAM domain (BoccUNI et al. 2003). In addition, Drosophila 1(3)mbt is required for
transcriptional repression of a number of endogenous genes (LEWIS et al. 2004).
Sequence and structural analysis demonstrates that 
MBT repeats are similar to Tudor,
PWWP, and chromo domains, suggesting that like these domains, MBT repeats might
also bind to modified histones (MAURER-STROH etal. 2003; SATHYAMURTHY etal. 2003;
WANG etal. 2003). More recently, the MBT repeats of human 1(3)mbt and CGI-72 have
been shown to bind histones methylated on specific residues (KIM et al. 2006). Together
these data suggest that MBT repeats in other proteins might bind to modified histones
and repress transcription. The localization of LIN-61 to condensed chromosomes, that it
functions with other transcription factors in vulval development and that it is composed
almost exclusively of MBT repeats suggest that LIN-61 functions in transcriptional
repression via the direct interaction with histones.
lin-61 functions separately from the DRM and NuRD-like complexes: In
Drosophila, the MBT-repeat-containing protein 1(3)mbt associates at substoichiometric
levels with the Myb-MuvB complex, which includes both fly pocket-proteins RBF1 and
RBF2 as well as dE2F2 and dDP (LEWIS et al. 2004). The Myb-MuvB complex
133
represses transcription of many E2F-responsive genes. However, 1(3)mbt is required to
mediate transcriptional repression of only a subset of these targets (LEWIS et al. 2004).
Thus 1(3)mbt might only function with the Myb-MuvB complex at specific promoters.
The Myb-MuvB complex is very similar to the DRM complex we identified in C.
elegans (M.M.H. and H.R.H. unpublished results). Our coimmunoprecipitation data
demonstrate that LIN-61 is not a core componenent of either the DRM or NuRD-like
complexes. Since the immunoprecipitations were from embryonic protein extracts we
cannot exclude the possibility that LIN-61 associates with these complexes at different
stages in development or in specific cell types. It remains 
possible that, like 1(3)mbt,
LIN-61 functions with the DRM complex to control specific processes, like vulval
development. However, while both 1(3)mbt and LIN-61 contain MBT repeats, LIN-61
does not contain the atypical zinc fingers or the SAM domain found in 1(3)mbt. The SAM
domain is important for protein-protein interaction and might help to mediate the
interaction of 1(3)mbt with components of the Myb-MuvB complex.
Our analysis of the pleiotropies associated with loss-of-function mutations in
lin-61 further suggests that in some biological processes lin-61 functions separately
from other class B synMuv genes, including components of the DRM complex. Most
notably, loss-of-function mutations in lin-61 cause genomic instability, result in ectopic
pharyngeal expression of a lag-2::gfp reporter and suppresses the vulval defect of
mat-3(ku233) mutant animals. Putative null alleles of lin-35 and lin- 15B share with lin-61
mutants only the ability to suppress mat-3(ku233). However, lin-35(n745) and
lin- 15B(n744) both share a number of other pleiotropies, including RNAi
hypersensitivity, PGL-1 somatic misexpression, the Tam phenotype, and ectopic
lag-2:.gfp expression in the gut and epithelia (Table 3). These data suggest that in many
biological functions, lin-61 is not functioning with lin-35 or lin-15B, although lin-35 and
lin- 15B might act together for these functions. It is interesting to note that loss of
function of all three genes both can suppress the defects in mat-3(ku233) mutant
animals and can act redundantly with the class A synMuv genes to regulate vulval
development. Perhaps, as has been proposed for LIN-35, LIN-61 is also acting to
repress transcription of mat-3.
134
We have shown that while lin-61 shares with other class B genes the ability to
regulate vulval development redundantly with the class A synMuv genes, it functions
separately from class B genes in many other biological processes. LIN-61 is also
distinguished from other class B synMuv proteins by coimmunoprecipitation
experiments, which show that LIN-61 is not a core component of the DRM complex.
While it is unclear whether LIN-61 functions alone or in a complex with other synMuv
proteins, comprehensive biochemical and genetic analyses have the potential to define
its functional partners. Future studies should help our understanding of not only how
LIN-61 functions but also how other synMuv proteins act to regulate vulval development
and more broadly, how transcriptional regulators, like Polycomb group proteins and
pocket proteins, might act separately in some biological processes and cooperate in
others.
135
ACKNOWLEDGMENTS
We thank Paul Sternberg for generously providing us with the initial lin-61 allele, sy223;
Daniel Denning and Niels Ringstad for comments about 
the manuscript; Beth Castor,
Na An and Andrew Hellman for technical assistance; members of the Horvitz laboratory
for construction of the deletion library; Alan Coulson for cosmid clones; Yuji Kohara for
cDNA clones; the C. elegans Genome Sequencing Consortium for genomic sequence;
and the C. elegans Genetics Center for some of the strains used in this work. M.M.H.
was a Howard Hughes Predoctoral Fellow. H.R.H. is the David H. Koch Professor of
Biology at M.I.T. and an Investigator of the Howard Hughes Medical Institute. This work
was supported by NIH grant GM24663.
136
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TABLE 1
lin-61 mutations cause a class B synMuv phenotype
Genotype 
Percent Muv (n)
Single mutants lin-61(sy223) 0 (260)
lin-61(n3442) 0 (327)
lin-61(n3446) 0 (217)
lin-61(n3447) 0 (334)
lin-61(n3624) 0 (242)
lin-61(n3687) 0 (252)
lin-61(n3736) 0 (234)
lin-61(n3807) 0 (278)
lin-61(n3809) 0 (269)
lin-61(n3922) 0 (82)
hda-1(e 1795) 31 (143)
hpl-2(n4274) 0 (>50)
trr-1(n3712) 18
a 
(39)
mys- I1(n368 1)b 8
a 
(36)
Class A synMuv lin-61(n3809); lin-8(n2731) 72 (414)
double mutants lin-61(n3809); lin-38(n751) 93 (175)
lin-61(n3809); lin-56(n2728) 100 (180)
lin-61(n3809), lin- 15A(n433) 14 (261)
lin-61(n3809); lin- 15A(n767) 97 (166)
Class B synMuv lin-61(n3809), lin-37(n 758) 0 (165)
double mutants lin-61(n3809); lin- 15B(n744) 0 (153)
lin-61(n3809); hda- I1(e 1795) 20 (96)
lin-61(n3809); hpl-2(n4274) 3 (172)
Class C synMuv lin-61(n3809); 
trr-1(n3712) 
17
a 
(111)
double mutants lin-61(n3809), mys- 1(n3681) 7a (45)
All animals were raised at 200C. The Muv phenotype was scored using a dissecting
microscope except in the cases noted. trr- I1 mutant homozygotes were recognized
as the non-G FP progeny of ttr- 1/mrn 1[dpy- 10 mls14] heterozygous parents.
hda-1(e 1795) homozygotes were recognixed as the non-GFP progeny of
hda- 1/n T[qls51], +/n T[qls51] heterozygous parents. lin-61(n3687) and
lin-61(n3922) were also homozygous for the linked integrated transgene n/s 133 that
includes pkd-2.gfp and a rescuing lin- 15AB construct.
a Muv, greater 
than three 
Pn.p cells 
induced.
bThese data 
are from 
Ceol and 
Horvitz (2004).
144
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Figure 1. Molecular cloning of lin-61
A) lin-61 maps between unc-14 and unc-15 on LGI. The cosmid R06C7 is shown below
as a grey bar. The rescuing Stul-Sacll fragment of R06C7 is shown below the cosmid.
Open boxes represent the exons of the predicted genes within the subclone. Arrows
indicate the direction of transcription.
B) lin-61 gene structure as determined from cDNA and genomic sequences. Shaded
boxes indicate coding sequence. Open boxes indicate the 3' untranslated region.
Predicted translation intiation and termination codons are shown along with the poly(A)
tail and the SL1 leader sequence. The positions of the mutations found in the ten lin-61
alleles are indicated above the gene structure.
C) Schematic depiction of the LIN-61 protein. Shaded boxes indicate the positions and
relative sizes of the four MBT repeats.
147
Figure 1
lin-61
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Figure 2. Identifcation of mbtr-1
A) Alignment of MBTR-1 and LIN-61. Solid boxes indicate identities between LIN-61 and
MBTR-1. Shaded boxes indicate similarities between the two proteins. Underlined
regions correspond to the four MBT repeats. Red box indicates the 15 amino acids
inserted into the second repeats of both LIN-61 and MBTR-1.
B) mbtr- 1 gene structure as determined from cDNA and genomic sequences. Shaded
boxes indicate coding sequence. Open boxes indicate 5' and 3'untranslated regions.
Predicted tranlation initiation and termination codons are shown along with the poly(A)
tail. The position of the predicted gene Y48GIA.2 that is located within the first intron of
mbtr-1 is shown. The arrow depicts the direction of transcription. The genomic region
deleted in n4775 is indicated by brackets.
C) Schematic depiction of the MBTR-1 protein. Shaded boxes indicate the positions and
relative sizes of the four MBT repeats.
149
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A) Affinity-purified antibodies raised against recombinant LIN-61 were used to blot
extracts from both wild-type and lin-61(n3809) mutant animals. The asterisks denotes
non-specific immunoreactivity. HM4077 antibodies were raised in a guinea pig. HM4078
antibodies were raised in a rabbit.
B, D, F, H) Whole-mount staining with anti-LIN-61 antisera.
B) LIN-61 is expressed in discrete foci in the nuclei of the developing embryo.
C) 4,6-Diamidino-2-phenylindole (DAPI) staining of the embryo shown in (B).
D) LIN-61 staining is absent from lin-61(n3809) embryos.
E) DAPI staining of the embryo shown in (D).
F) LIN-61 is broadly expressed in the adult hermphrodite germline and is localized to
condensed chromosomes.
G) DAPI staining of the germline shown in (F)
H) LIN-61 staining is absent from the germline of lin-61(n3809) adult hermaphrodites.
I) DAPI staining of the germline shown in (H).
WT, wild type. Scale bars, 10 pm.
151
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A) Alignment of the MBT repeats from Caenorhabditis elegans proteins LIN-61 and
MBTR-1 and Homo sapiens proteins L(3)MBT2 and L(3)MBT1. The top portion
corresponds to the N-terminal arm and the bottom portion corresponds to the b-core
region. Shaded residues indicate identities between more than eight MBT repeats.
Circled residues indicate postions of missense mutations in lin-61. The corresponding
allele is indicated above the residue. The missense mutation in n3624 is located
between the first and second repeats. Boxed region indicates the 15 amino acids
inserted in the second repeats of MBTR-1 and LIN-61.
B) Equivalent amounts of protein from each of the genotypes indicated above were
loaded per lane. Proteins were separated by SDS-PAGE and immunoblotted with the
antibodies indicated to the left. Anti-tubulin antibodies were used to asses protein
loading and transfer. The asterisk denotes non-specific immunoreactivity.
153
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A) Extract from either wild-type or lin-61(n3809) mutant embryos (as indicated above
the lanes) were precipitated with antibodies against LIN-61. Immunoprecipitations were
analyzed by immunoblots with antibodies specific to the antigen indicated 
to the left. IN,
2% of the input. IP, 100% of the immunoprecipitate.
B) Extract from wild-type embryos was precipitated with antibodies indicated above the
lanes and immunoblotted with antibodies specific to the antigens indicated 
to the left. IN,
2% of the input. IP, 100% of the immunoprecipitate.
C) Extract from lin-61(n3809) mutant embryos were precipitated with antibodies against
LIN-37 and immunoblotted with antibodies specific to the antigen indicated to the left.
IP, 100% of the immunoprecipitate.
155
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TM
CHAPTER 4
lin-8, which antagonizes C. elegans 
Ras-mediated vulval induction,
encodes a novel nuclear protein that interacts with the LIN-35 Rb
protein
Ewa M. Davison, Melissa M. Harrison, Albertha J.M. Walhout,' 
Marc Vidal,
2
and H. Robert Horvitz
Published as Davison et al. (2005) Genetics 171: 1017-1031.
My contribution to this chapter includes LIN-8/ LIN-35 in vitro binding studies shown in
Figure 4.
1
Present address: Program in Gene Function and Expression and Program in Molecular
Medicine, University of Massachusetts Medical School, Worcester, Massachusetts
01605
2
Dana Farber Cancer Institute 
and Department of Genetics, 
Harvard Medical School,
Boston, Masachusetts 02115
157
ABSTRACT
Ras-mediated vulval development in C. elegans is inhibited by the functionally
redundant sets of class A, B, and C synthetic Multivulva (synMuv) genes. Three of the
class B synMuv genes encode an Rb/DP/E2F complex that, by analogy with its
mammalian and Drosophila counterparts, has been proposed to silence genes required
for vulval specification through chromatin modification and remodeling. Two class A
synMuv genes, lin- 15A and iin-56, encode novel nuclear proteins that appear to function
as a complex. We show that a third class A synMuv gene, lin-8, is the defining member
of a novel C. elegans gene family. The LIN-8 protein is nuclear and can interact
physically with the product of the class B synMuv gene lin-35, the C. elegans homolog
of mammalian Rb. LIN-8 likely acts with the synMuv A proteins LIN-15A and LIN-56 in
the nucleus, possibly in a protein complex with the synMuv B protein LIN-35 Rb. Other
LIN-8 family members may function in similar complexes in different cells or at different
stages. The nuclear localization of LIN-15A, LIN-56, and LIN-8, as well as our
observation of a direct physical interaction between class A and class B synMuv
proteins support the hypothesis that the class A synMuv genes control vulval induction
through the transcriptional regulation of gene expression.
158
INTRODUCTION
The Retinoblastoma (Rb) gene was the first tumor 
suppressor gene to be cloned,
and the Rb pathway has been found to be a frequent target of inactivation in many
human cancers (NEVINS 2001). The nematode Caenorhabditis elegans possesses a
single homolog of Rb, lin-35, which functions in the inhibition of both cellular proliferation
and differentiation (Lu and HORVITZ 1998; BOXEM and VAN DEN HEUVEL 2001). The class
A synthetic Multivulva (synMuv) genes function redundantly with the lin-35 Rb gene to
inhibit Ras-mediated vulval induction. The analysis of the class A synMuv genes may
further our understanding of activities that interact with the Rb pathway in the regulation
of cell-fate determination and in the prevention of oncogenic transformation.
The vulva of the C. elegans hermaphrodite is the conduit through which embryos
are expelled and is also the point of entry for sperm after mating with a male. The vulva
is formed by the descendants of three of six equipotent cells, P(3-8).p. These six cells
are all able to express any one of three fates: the 10 vulval fate, the 20 vulval fate, and
the 30 non-vulval fate. Vulval development is induced by activation of a receptor tyrosine
kinase (RTK)/Ras pathway (KORNFELD 1997; STERNBERG and HAN 1998). During wild-
type larval development, signaling from the anchor cell of the somatic gonad activates
an RTK/Ras pathway, causing P6.p to adopt the 10 vulval fate and directly or indirectly
causing P5.p and P7.p to adopt the 20 vulval fate. P(5-7).p then divide to produce 22
cells that migrate and fuse to form the toroidal vulva. P3.p, P4.p, and P8.p normally
express the 30 non-vulval fate, dividing once and fusing with the hypodermis. Loss of
RTK/Ras pathway signaling results in the expression of the non-vulval 30 fate by P(5-
7).p and thus in a Vulvaless (Vul) phenotype. Vul animals lack a functional vulva and
are consumed by their internally hatched progeny. Ectopic activation of RTK/Ras
pathway signaling results in the expression of 10 or 20 vulval fates by P3.p, P4.p, and
P8.p, causing a Multivulva (Muv) phenotype. The extra vulval tissue produced in Muv
animals forms ectopic ventral protrusions.
The class A, B, and C synMuv genes act to antagonize RTK/Ras function in
potential vulval cells (FERGUSON and HORVITZ 1989; CEOL and HORVITZ 2004). As a
result of a functional redundancy among these three classes of synMuv genes, only
1-59
hermaphrodites mutant in two sets of genes exhibit the synMuv phenotype. Genetic
screens and targeted studies have identified at least four class A synMuv genes - lin-8,
lin- 15A, lin-38, and lin-56- at least 17 class B synMuv 
genes - lin-9, lin- 13, lin- 15B,
lin-35, lin-36, lin-37, lin-52, lin-53, lin-54, lin-61, dpl- 
1, efl- 1, hda- 1, hpl-2, let-418, mep- 1,
and tam- 1 - and four class C synMuv genes - trr- 1, mys- 1, epc- 1, and ssl- 1 (HORVITZ
and SULSTON 1980; FERGUSON and HORVITZ 1985, 1989; Lu and HORVITZ 1998; HSIEH et
al. 1999; SOLARI and AHRINGER 2000; VON ZELEWSKY et al. 2000; CEOL and HORVITZ
2001; COUTEAU et al. 2002; UNHAVAITHAYA et al. 2002; THOMAS et al. 2003; CEOL and
HORVITZ 2004; X. Lu, M. M. HARRISON, P. W. STERNBERG 
and H. R. HORVITZ,
unpublished results). A subset of the class B synMuv genes encode proteins that
mediate histone modification, chromatin remodeling, and transcriptional repression. In
particular, ef- 1, dpl- , lin-35, lin-53, hda- 1, let-418, and hpl-2 encode C. elegans
homologs of E2F, DP, Rb, the Rb-associated protein RbAp48, a class I histone
deacetylase (HDAC), the Mi-2 chromatin remodeling ATPase, and heterochromatin
protein 1 (HP1), respectively (Lu and HORVITZ 1998; SOLARI and AHRINGER 2000; VON
ZELEWSKY et al. 2000; CEOL and HORVITZ 2001; COUTEAU et al. 2002). The mammalian
homologs of LIN-53 RbAp48, HDA-1 HDAC, and LET-418 Mi-2 are components of the
histone deacetylase and chromatin remodeling NuRD complex, while HP1 has been
shown to function as a histone H3 methyl-lysine-9 binding protein (KNOEPFLER and
EISENMAN 1999; RICHARDS and ELGIN 2002). Because of their molecular identities, the
synMuv B genes are thought to antagonize RTK/Ras function in the vulva by silencing
transcription of vulval specification genes through chromatin modification and
remodeling. LIN-35 Rb is likely to play a pivotal role in this process, as evidence
suggests that mammalian pRb mediates the association of the sequence-specific
heterodimeric transcription factor DP/E2F with the NuRD complex components HDAC1
and RbAp48 as well as with a histone H3 K9 methyltransferase (NICOLAS et al. 2000;
NIELSEN et al. 2001; ZHANG and DEAN 2001). The resultant likely recruitment of Mi-2 and
HP1 may induce a facultative heterochromatic 
state around the targeted genes,
preventing transcription. The class C synMuv genes encode a putative C. elegans
Tip60/NuA4-like histone acetyltransferase complex; it has not yet been determined if
160
this putative complex acts in transcriptional activation or repression (CEOL and HORVITZ
2004).
The class A synMuv genes may inhibit vulval development through the regulation
of transcription. Of the four known class A synMuv genes, two - lin-56 and lin- 15A -
have been cloned and encode novel nuclear proteins that likely associate in a functional
complex in vivo (CLARK et al. 1994; HUANG et al. 1994; E. M. DAVISON, A. M. SAFFER, L.
S. HUANG, J. DEMODENA, P. W. STERNBERG and H. R. HORVITZ, manuscript in
preparation). Furthermore, LIN-56 and LIN-15A share a novel C2CH motif related to the
THAP domain, shown in the human protein THAP1 to possess zinc-dependent
sequence-specific DNA-binding activity in vitro (CLOUAIRE et al. 2005; E. M. DAVISON, A.
M. SAFFER, L. S. HUANG, J. DEMODENA, P. W. STERNBERG 
and H. R. HORVITZ,
manuscript in preparation). Here we report our characterization of a third class A
synMuv gene, lin-8.
161
MATERIALS AND METHODS
Strains and general techniques: C. elegans strains were cultivated on NGM
agar seeded with E coli strain OP50 as described by BRENNER (1974) and were grown
at 200 unless otherwise indicated. Bristol strain N2 was used as the wild-type strain. The
mutant alleles used in this study are listed below, and a description of each can be
found in RIDDLE et al. (1997) unless noted otherwise:
LG I: lin-35(n745).
LG II: lin-8(nl 11, n2376, n2378, n2403, n2731, n2738, n2739, n2741) (THOMAS et al.
2003), dpy- 10(e 128).
LG III: lin-52(n771) (FERGUSON and HORVITZ 1989; THOMAS et al. 2003).
LG X: lin- 15B(n744, n2245).
pPK5363 is a Tcl-transposon insertion polymorphism on LG II found in the NL7000 but
not the N2 strain (KORSWAGEN et al. 1996). nls128 contains a gfp transgene integrated
into LG II (H. T. SCHWARTZ and H. R. HORVITZ, unpublished results). In addition, the
following deficiencies were used: ccDf , ccDf2, and ccDfl I (CHEN et al. 1992).
Deletion and Polymorphism Mapping: To test complementation of lin-8 with
the deficiencies ccDfl, ccDf2, and ccDfl 1, lin-8(n 111) dpy- 10O(e 128); lin- 15B(n744)
hermaphrodites were mated with ccDfl+ males. The Muv phenotype of the non-Dpy
male progeny, half of which should possess lin-8 in trans to the relevant deficiency, was
scored. 0/88 and 0/84 male offspring of 
the ccDfl and ccDf2 crosses, respectively,
exhibited ventral protrusions, whereas 20/63 male offspring of the ccDf 11 cross
exhibited ventral protrusions.
The left endpoint of ccDfl was defined relative to the physical map by
determining if cosmid sequences from the interval between sup-9 and lin-31 could be
amplified by PCR from ccDfl homozygous inviable embryos. A drop of chitinase
solution (20 mg/ml chitinase, 
50 mM NaCI, 70 mM KCI, 
2.5 mM MgCI
2
, 2.5 mM CaCl
2
)
was placed over single inviable embryos, which were promptly transferred to 10 pL of
lysis buffer (50 mM KCI, 10 mM Tris, pH 8.2, 2.5 mM MgCI
2
, 0.45% NP-40, 0.45%
162
Tween-20, 0.01% gelatin, 60 pg/ml proteinase K) and subsequently frozen at -800.
Embryos were thawed and lysed by incubation at 600 for 1 hr. Proteinase K was
inactivated by incubation at 950 for 15 min. The lysate from each inviable embryo was
used for three PCR reactions: the test amplification, amplification of a sequence from
cosmid F34D6, which served as a positive control for successful lysis, and amplification
of lin-31, which served as a negative control to confirm identification of each egg as a
ccDf homozygote. Sequences from cosmids B0454, ZC239, F39E9, and W10G11 but
not from cosmids M151 or F19B10, were successfully amplified from ccDfl
homozygous inviable embryos, placing the left endpoint of ccDf between W10G11 and
M151. The primers used were as follows: F34D6.7, 5'-CACCTGAAGATTCAAGTTTAG-
3'; F34D6.11, 5'-GTGTGAGCTCAGCAGCTTC-3'; B0454 Fwd, 5'-GGTTCTCGTTAGCT-
GAGTGG-3'; B0454 Rev, 5'-GTACGGAGCCAAGATCATACG-3'; ZC239 Fwd, 5'-GCAG-
AGACGTTGGATCCTAGC; ZC239 Rev, 5'-CTTCAGGAGTCGGTGAACTCG-3'; F39E9
Fwd, 5'-CAGTCTCAGGCTAGACTTGG-3'; F39E9 Rev, 5'-GCTGAGCAGATCTCGAAT-
GG-3'; W10G11 Fwd1, 5'-GCTTCCACATTCAGTGAAGG-3'; W10G11 Rev1, 5'-CAAGC-
CAGAAGAGCAAGTCG-3'; W10G11 N1, 5'-CGAGATGTAAGCTCAGTATGG-3'; M151
Fwd, 5'-CATCGGTCTCCCATAGTTACC-3'; M151 Rev, 5'-GCTCTGGCTGCTCGAGTT-
CC-3'; F19B10 Fwd, 5'-CTGAAGCATTGGCTCAGAGG-3'; F19B10 Rev, 5'-CGTCATT-
GATGGACCATGTGC-3'; iin-31 Fwdl, 5'-GCTATTCAGGACTCTGACG-3'; 
lin-31 Revl,
5'-CCTTCCCAGGACGATCG-3'.
The pPK5363 polymorphism is an insertion of the Tcl transposon into cosmid
C17F4 present in the NL7000 but not in the N2 strain (KORSWAGEN et al. 1996). To map
lin-8 against pPK5363, lin-8(n I 11) dpy- 10(e 128) /NL7000; lin- 15B(n744) males were
crossed with lin-8(n 111) dpy- 10(e 128); lin- 15B(n744) hermaphrodites, and resulting
Muv non-Dpy and Dpy non-Muv hermaphrodite progeny were picked and used to
establish homozygous recombinant lines. PCR employing Tcl-specific (5'-GCTGATCG-
ACTCGATGCCACGTCG-3') and C1 7F4-specific (5'-CCATCAACGAGTACGATACG-3')
primers was used to determine if polymorphism pPK5363 was present. Of the Muv non-
Dpy chromosomes, 4/13 carried pPK5363, and of the Dpy non-Muv chromosomes, 2/14
163
carried pPK5363. These results placed lin-8 to the left of pPK5363, which is itself to the
left of dpy-10.
Transgenic Animals: Germline transformation by microinjection was performed
as described by MELLO et al. (1991). The coinjection marker pRF4 was injected at a
concentration of 80 ng/[l. Transgenic animals were identified using the Roller
phenotype generated by expression of the rol-6(su1006) dominant allele from pRF4.
Experimental constructs were injected at a concentration of 20 ng/[l or 50 ng/[l.
Antibody Preparation, Immunoblotting, and Immunocytochemistry: Anti-
LIN-8 antibodies were generated using purified MBP-LIN-8(aa 1-386) fusion protein.
The crude antisera were subsequently affinity purified against GST-LIN-8(aa 1-386)
fusion protein as described by KOELLE and HORVITZ (1996) and then pre-adsorbed
against an acetone precipitate of proteins prepared from lin-8(n2731) mixed-stage
worms, essentially as described by HARLOW and LANE (1988). Affinity-purified and pre-
adsorbed anti-LIN-8 antibodies HM2247 were used at a dilution of 1:1000 for western
blots. Samples for western analysis were prepared by Dounce homogenization of
mixed-stage worms in hypotonic lysis buffer 
(10 mM KCI, 1.5 mM MgCl2, 1 mM EDTA,
1 mM EGTA, 1 mM DTT, 250 mM sucrose) containing 1X Protease Inhibitor Cocktail
(800 pg/ml benzamidine HCI, 500 pg/ml phenanthroline, 500 pg/ml aprotinin, 500 pg/ml
leupeptin, 500 pg/ml pepstatin A, 50 mM phenylmethylsulfonyl fluoride, BD Biosciences,
Franklin Lakes, NJ) as well as phosphatase 
inhibitors (0.2 mM sodium orthovanadate,
50 mM sodium fluoride, 1 uM microcystin-LR).
Affinity-purified and pre-adsorbed anti-LIN-8 antibodies HM2247 were used at a
dilution of 1:100 for immunocytochemistry. Anti-a-tubulin mouse monoclonal antibody
DM1A (Sigma, St. Louis, MO) and MH27 (FRANCIS and WATERSTON 1991), which
recognizes the apical borders of C. elegans epithelial cells, were used as positive
controls for immunocytochemistry at 1:100 and 1:1000 dilutions, respectively. Embryos
were fixed in 0.8% paraformaldehyde for 20 min as described by GUENTHER and
GARRIGA (1996). Larvae and adults were 
fixed in 2% paraformaldehyde for 15 
min,
essentially as described by FINNEY and RUVKUN (1990). Images were obtained using a
164
Zeiss LSM510 laser confocal microscope and software, and processed using Adobe
Photoshop software.
Two-Hybrid and In Vitro Binding Experiments: The yeast two-hybrid screen of
a C. elegans cDNA library was performed as described by WALHOUT and VIDAL (2001).
Full-length lin-35 Rb was used as bait. 1.4 x 106 colonies of the C. elegans AD-
wrmcDNA library (WALHOUT et al. 2000b) were screened. Interaction of LIN-35 Rb and
LIN-8 could not be tested in the reverse orientation, as LIN-8 was found to self-activate
when fused to the Gal4 DNA-binding domain.
The full-length and partial MBP-LIN-8 constructs were made by subcloning
appropriate portions of the lin-8cDNA into vector pMAL-c2 (NEB, Beverly, MA). The
GST-LIN-8 (aa 175-285) construct was made by subcloning the appropriate portion of
the lin-8 cDNA into vector pGEX-2T (Amersham Biosciences, Piscataway, NJ). MBP
and GST fusion constructs were expressed in E coli BL21 (DE3) cells (STUDIER et al.
1990) and purified using amylose resin (NEB) or Glutathione Sepharose 4B (Amersham
Biosciences), respectively, as recommended by the manufacturers. The constructs
encoding LIN-35 Rb (aa 1-555) and LIN-35 Rb (aa 270-961) have been described
previously (Lu and HORVITZ 1998) and were used as templates for in vitro synthesis of
35
S-labeled protein (TNT Couple Reticulocyte Lysate 
System, Promega, Madison, WI).
In vitro binding experiments were otherwise performed as described by REDDIEN and
HORVITZ (2000), and formation of protein complexes was analyzed by SDS-PAGE and
autoradiography.
165
RESULTS
LIN-8 defines a family of novel C. elegans proteins: The class A synMuv
gene lin-8 was originally identified through the chance recovery of a Iin-8(If); Iin-9(If)
double mutant in a screen by S. BRENNER (personal communication) for animals
abnormal in morphology or behavior; the Muv phenotype of this strain was later shown
to be synthetic, as it required the presence of two unlinked mutations, lin-8(n 111) and
lin-9(n 112) (HORVITZ and SULSTON 1980). An additional eight alleles of lin-8 have since
been identified in two independent screens for synMuv A genes (THOMAS et al 2003).
lin-8 was previously mapped to the 7 map unit interval between sup-9 and lin-31 on
chromosome II (FERGUSON and HORVITZ 1985). We used 
the deficiencies ccDfl, ccDf2,
and ccDfl 1, each of which deletes lin-31 (CHEN et al. 1992), to more precisely locate
lin-8 on the physical map. We performed complementation tests and found that of these
three deficiencies, only ccDfl I deletes the lin-8 locus. As the sup-9 locus resides in
cosmid F34D6 (PEREZ DE LA CRUZ et al. 2003) and the left endpoints of ccDfl, ccDf2,
and ccDf 11 had been defined approximately by experiments using the polymerase
chain reaction (PCR) (data concerning the left endpoints of ccDf2 and ccDfl I were
generously provided to us by C. A. SPIKE and R. K. HERMAN), we placed lin-8 between
the cosmids F34D6 and M151 (Figure 1A). Further mapping using the polymorphism
pPK5363 placed Iin-8 between cosmids F34D6 and C17F4. We injected cosmids from
this interval into lin-8(nl 11); lin- 15B(n744) animals and obtained rescue of their synMuv
phenotype with cosmid C03E12 as well as with a 7.5 kb subclone of C03E12 (Figure
1 B). This minimal rescuing fragment contains a single intact predicted gene, B0454.1
(C. ELEGANS SEQUENCING CONSORTIUM 1998). We determined the sequences of the nine
alleles of lin-8 and found all to contain mutations within the B0454.1 open reading frame
(ORF) (Table 1). Furthermore, RNA-mediated interference (RNAi) of B0454.1 in
lin- 15B(n744) animals resulted in a synMuv phenotype, and expression of the B0454.1
ORF under the control of the two C. elegans heat-shock promoters (STRINGHAM et al.
1992) shortly after L1 lethargus efficiently rescued the synMuv phenotype of
lin-8(n273 1); lin- 15B(n744) animals (data not shown). We conclude that lin-8 and
B0454.1 are equivalent.
166
lin-8 encodes a novel, acidic protein of 386 amino acids (Figure 1C). Sequencing
of six lin-8 cDNA clones (courtesy of Yuji Kohara) verified the exon/intron junctions
predicted by GENEFINDER (EDGLEY etal. 1997). Extensive database searches (using
PSI-BLAST (ALTSCHUL et al. 1997), PROSITE (FALOUET et al. 2002), Pfam (BATEMAN et
al. 2002) and SMART (SCHULTZ et al. 2000)) with the LIN-8 protein sequence have
revealed no significant canonical motifs and no apparent sequence homologs in other
species. LIN-8 is, however, a member of a family of 17 C. elegans proteins (Figure 2).
This family was independently detected by the Pfam protein families database, and is
there referred to as DUF278 (BATEMAN et 
al 2002). While the scores are very weak,
LIN-8 identifies the most distant family member in BLAST searches, and vice versa. The
biological roles of the remaining 16 family members are not known. Several of the family
members, including LIN-8, possess an N-terminal proline-rich region (Figure 2)
containing at least one PxxP motif, the core sequence to which SH3 domains can bind
(KAY et al. 2000).
Characterization of lin-8 alleles: To identify null alleles of lin-8 as well as
residues important for LIN-8 function, we characterized all nine independently isolated
lin-8 alleles (Tables 1 and 2). Three lin-8 alleles - n2731, n2738, n2739- contain
nonsense mutations (Table 1). The 
remaining six lin-8 alleles - n 111, n2376, 
n2378,
n2403, n2724, n2741 - contain missense mutations (Table 1). Two of the missense
alleles, n2403 and n2724 (THOMAS etal. 
2003), contain the identical nucleotide 
change;
only n2403 was subsequently used for quantitative studies. Four of the five amino-acid
residues altered in the missense alleles are conserved in several LIN-8 family members
(Figure 2).
The nine lin-8 alleles are not easily distinguishable in combination with the strong
canonical synMuv B allele lin- 15B(n744) (data not shown). The synMuv phenotype is
inherently temperature-sensitive - both its penetrance and expressivity are usually
greater in mutants raised at 200 than at 150 (FERGUSON and HORVITZ 1989). We
therefore quantitated the penetrance of the synMuv phenotype associated with each
lin-8 allele in combination with two weak synMuv B alleles, lin- 15B(n2245) (THOMAS et
al. 2003) and lin-52(n771) (FERGUSON and HORVITZ 1989), at both 150 and 200 (Table 2).
167
The missense alleles can be placed in three categories on the basis of phenotypic
strength: weak (n2741), intermediate (n 111), and strong (n2376, n2378, n2403). The
three strong missense alleles all mutate charged amino acids in a cluster of residues
conserved in many members of the LIN-8 family. Of the nonsense mutations, n2731 and
n2738 appeared to be substantially stronger than n2739 when tested in combination
with lin-52(n771). Both n2731 and n2738 are predicted to truncate more than two-thirds
of the wild-type LIN-8 protein, whereas n2739 is predicted to leave more than two-thirds
of the wild-type LIN-8 protein intact.
On the basis of their molecular lesions, we considered n2731 and n2738 to be
candidate null alleles of the lin-8 locus. Neither nonsense allele, however, inactivated
the synMuv A pathway to the same extent as loss of the class A synMuv gene lin-56 in
the lin- 15B(n2245) mutant background (Table 2). We also observed that the strong
missense mutations n2376, n2378, and n2403 appeared more penetrant for the synMuv
phenotype than either n2731 or n2738 in combination with lin- 15B(n2245) (Table 2). To
more stringently determine if n2731 and n2738 were null alleles of the lin-8 locus, we
compared the penetrances of the synMuv phenotype of hermaphrodites homozygous
for each allele with hermaphrodites heterozygous for each allele and for ccDfl 1, a
deficiency that deletes the lin-8 locus (see Figure 1A). The penetrances of the synMuv
phenotype for both lin-8(n273 1); lin- 15B(n2245) and lin-8(n2738); lin- 15B(n2245)
homozygotes were weaker than those of lin-8(n273 1)/ccDf 11; lin- 15B(n2245) and
lin-8(n2738)/ccDf11; lin- 15B(n2245) heterozygotes, respectively (Table 2). One possible
interpretation of these observations is that neither n2731 nor n2738 completely
eliminates lin-8 function. However, the ccDfl I deficiency also eliminates several other
LIN-8 family members - B0454.9, C41 H7.3, 
C41 H7.4, C41 H7.5, C41 H7.6, F14D2.2,
F54D10.3, K08A2.1, and K08A2.4. We suspect that n2731 and/or n2738 are 
null alleles
of lin-8 and that the penetrances of their synMuv phenotypes are enhanced by a
decrease in the dosage of one or more of the lin-8 family genes deleted by ccDfl 1. This
latter hypothesis is supported by the observation that no LIN-8 protein is detected in
n2738 protein extracts (see below).
168
Unlike mutants carrying the nonsense mutations n2731 and n2738, mutants
carrying the missense mutation n2376 had an equally penetrant phenotype when
homozygous as when heterozygous over the ccDfl I deficiency (Table 2), suggesting
that n2376 may be a null allele of lin-8. However, n2376 results in the production of
stable LIN-8 protein, at least in extracts from mixed-stage animals (see below).
Furthermore, lin-8(n2376) retains wild-type lin-8 function in another assay (H. T.
SCHWARTZ and H. R. HORVITZ, unpublished results). Thus, it is likely that the n2376
allele is not a null allele of lin-8 but is instead specifically defective for lin-8 synMuv A
function.
LIN-8 is a nuclear protein expressed in many cells: To determine the
expression pattern and localization of the LIN-8 protein, we generated a rabbit
polyclonal antibody against a fusion of full-length LIN-8 with maltose-binding protein
(MBP-LIN-8). The affinity-purified and pre-adsorbed antibody recognized an apparent
doublet of approximately 50 kD in wild-type but not in lin-8(n2731) or lin-8(n2738)
protein extracts analyzed by western blots (Figure 3A); the predicted size of the LIN-8
protein is 44 kD. As the two LIN-8 proteins are approximately equal in their levels and
the six lin-8 cDNA clones (courtesy of Yuji Kohara) that we analyzed are identical in
sequence, we suspect that the LIN-8 protein may be post-translationally modified. The
n2376 missense mutation does not destabilize the full-length LIN-8 protein (Figure 3A).
Since lin-8functions with lin-15A, lin-38, and lin-56 to inhibit vulval development
(FERGUSON and HORVITZ 1989; THOMAS et al. 2003), we analyzed the impact of loss-of-
function mutations in these class A synMuv genes on the LIN-8 protein. By western blot
analysis, neither the levels nor the electrophoretic mobility of LIN-8 appears to be
altered in Iin-56(/f), lin- 15A(If), or Iin-38(If) mutants (Figure 3A). This result contrasts with
that for the class A synMuv proteins LIN-15A and LIN-56, which are dependent on each
other, but not on lin-8 or lin-38, for wild-type levels (E. M. DAVISON, A. M. SAFFER, L. S.
HUANG, J. DEMODENA, P. W. STERNBERG and H. R. HORVITZ, manuscript in preparation).
We used the same anti-LIN-8 antibody for whole-mount staining of worms. LIN-8
appeared to be predominantly localized to nuclei (Figure 3B-D). We observed LIN-8
expression in many cells in embryos, larvae, and adults, and LIN-8 staining was
169
particularly prominent in the germline as well as in neuronal nuclei of the head (Figure
3B-D). Although diffuse within the syncytium of the distal gonad arms, LIN-8 was
specifically associated with germ cell nuclei during the pachytene stage and was also
localized to oocyte nuclei (Figure 3C). No anti-LIN-8 staining was observed in any of
these somatic or germ cell nuclei in lin-8(n2731) animals of any stage (Figure 3B,C and
data not shown). Background staining in the larval midbody of lin-8(n2731) animals was
too high to examine LIN-8 expression in vulval cells.
LIN-8 interacts with LIN-35 Rb in vitro: We performed a yeast two-hybrid
analysis of a C. elegans cDNA library using full-length lin-35 Rb as bait and identified
LIN-8 as a potential LIN-35 Rb interactor (data not shown). The Gal4-based screen
made use of three reporter genes - GAL 1:.HIS3, GAL 1.:lacZ, and SPAL O1::URA3
(FIELDS and SONG 1989; WALHOUT and VIDAL 2001). Of 1.4 x 106 transformants, we
identified 11 clones that grew on selective medium in the presence of 3-aminotriazole
(3-AT). Further analysis revealed that six of these 11 clones also expressed 3-
galactosidase (P-gal) and were able to grow in the absence of uracil. The six clones that
tested positive for expression of all three reporter genes were all found to contain the
B0454.1 open reading frame encoding LIN-8. Neither LIN-35 nor LIN-8 interacted in the
yeast two-hybrid system with any of 29 other vulval proteins tested (WALHOUT et al.
2000a and data not shown). Western blot analyses (data not shown) indicate that
LIN-35 is expressed at wild-type levels in lin-8(n2731) worms and that LIN-8 is
expressed at wild-type levels in lin-35(n745) worms, which lack LIN-35 protein (Lu and
HORVITZ 1998).
To test the hypothesis that LIN-8 and LIN-35 Rb can interact and, if so, to identify
the region of LIN-35 Rb required for the interaction, we sought to determine if LIN-8
could associate in vitro with two different fragments of LIN-35 Rb. The A/B pocket
domain of mammalian pRb, p107, and p130 mediates association with many interacting
proteins (MORRIS and DYSON 2001), and in C elegans is contained within a portion of
LIN-35 Rb sufficient for interaction in in vitro pull-down experiments with LIN-53
RbAp48, HDA-1 HDAC, DPL-1 DP, and EFL-1 E2F (Lu and HORVITZ 1998; CEOL and
HORVITZ 2001). An [
35
S]methionine-labeled N-terminal 
LIN-35 Rb fragment (aa 1-555),
170
which lacks an intact pocket domain, failed to show any significant association with a
full-length MBP-LIN-8 fusion protein in in vitro pull-down assays (Figure 4A). By
contrast, an [35S]methionine-labeled C-terminal LIN-35 Rb fragment (aa 270-961), which
contains the pocket domain, interacted with MBP-LIN-8 (Figure 4A). LIN-8 and LIN-35
Rb are thus capable of interacting in both yeast two-hybrid and in vitro pull-down
assays. Furthermore, these observations suggest that, as with its other C. elegans
interactors (Lu and HORVITZ 1998; CEOL and HORVITZ 2001), LIN-35 Rb associates with
LIN-8 through its C-terminus, possibly via the pocket domain.
We next identified the domain of LIN-8 required for interaction with the C-terminal
LIN-35 Rb fragment using an in vitro pull-down assay. Progressive deletions of N- and
C-terminal LIN-8 residues revealed that amino acids 170-359 of LIN-8 were necessary
for interaction with LIN-35 Rb (data not shown; Figure 4C). Furthermore, amino acids
175-285 of LIN-8 were sufficient for interaction with the C-terminal LIN-35 Rb fragment
(Figure 4B,C). Several viral proteins interact with the pocket domain of pRb via an
LXCXE motif (HARBOUR and DEAN 2000); this sequence is not contained within amino
acids 175-285 of LIN-8. Amino acids 175-285 of LIN-8 share a small region of similarity
with other LIN-8 family members (Figure 2). None of the lin-8 missense mutations
resides within the interaction domain, and thus no existing mutation compromises the
predicted association between LIN-35 Rb and LIN-8 by directly affecting the interaction
domain.
171
DISCUSSION
The class A synMuv genes function redundantly with the C. elegans homolog of
the mammalian tumor suppressor pRb to inhibit Ras-mediated vulval development. We
have shown that the class A synMuv gene lin-8 encodes a novel nuclear protein that not
only functions redundantly but also physically interacts with C. elegans Rb. Given these
observations, we propose that the class A synMuv genes act in transcriptional
regulation. Further characterization of the mechanism by which the proteins of the class
A synMuv pathway act may well reveal molecular processes that interact with the
mammalian Rb tumor suppressor pathway both in the regulation of cell fate and in the
prevention of tumorigenesis.
Class A synMuv genes may regulate transcription: The two previously cloned
synMuv A genes - lin- 15A and lin-56 - encode novel nuclear proteins that share a novel
C2CH motif also found in the synMuv B proteins LIN-15B and LIN-36, as well as in
HIM-17, a protein required for meiotic recombination and histone H3 lysine-9
methylation in the germline (REDDY 
and VILLENEUVE, 2004; E. M. DAVISON, A. 
M. SAFFER,
L. S. HUANG, J. DEMODENA, P. W. STERNBERG and H. R. HORVITZ, manuscript in
preparation). This C2CH motif is likely related to the THAP domain (CLOUAIRE et al.
2005; ROUSSIGNE et al. 2003), and has been proposed to mediate interaction with
chromatin or chromatin-associated proteins (REDDY and VILLENEUVE, 2004; E. M.
DAVISON, A. M. SAFFER, L. S. HUANG, J. DEMODENA, P. W. STERNBERG and H. R.
HORVITZ, manuscript in preparation). The THAP domain of the human protein THAP1
has been shown to possess zinc-dependent sequence-specific DNA-binding activity in
vitro (CLOUAIRE et al. 2005). It has therefore been proposed that the synMuv A proteins
inhibit vulval development through the regulation of transcription (E. M. DAVISON, A. M.
SAFFER, L. S. HUANG, J. DEMODENA, P. W. STERNBERG and H. R. HORVITZ, manuscript 
in
preparation).
The nuclear localization of LIN-8 and the physical association between LIN-8 
and
LIN-35 Rb is consistent with the hypothesis that LIN-8 is present at the sites of
transcriptional repressor complexes. How might the proteins encoded by these three
synMuv A genes modulate transcriptional activity? Three general mechanisms seem
172
possible. First, like some synMuv B proteins (Lu and HORVITZ 1998; SOLARI and
AHRINGER 2000; VON ZELEWSKY et al. 2000; CEOL and HORVITZ 2001; COUTEAU et al.
2002), the synMuv A proteins may impact chromatin structure. Second, the synMuv A
proteins may have more direct functions in regulating the initiation, elongation, or
termination of transcription. Third, the synMuv A proteins may mediate the localization
of target genes to nuclear subdomains where their transcription could be coordinately
and efficiently regulated. The localization of genes to the vicinity of centromeric
heterochromatin, for example, may contribute to transcriptional repression in Drosophila
and mammals (GASSER 2001).
Physical interaction between synMuv A and B proteins: Indirect physical
interaction between the class A and class B proteins was proposed by WALHOUT et a/.
(2000a), who found that the synMuv A protein LIN-15A shared interactors with the
synMuv B proteins LIN-36 and LIN-37 in the yeast two-hybrid system. As RNAi analysis
has thus far not revealed a role for any of these shared interactors in the class A or
class B synMuv pathways or in antagonism of these pathways (C. J. CEOL and H. R.
HORVITZ, unpublished results), the significance of this observation remains unclear.
The direct interaction described in this manuscript between LIN-8 and LIN-35 Rb
in vitro suggests that LIN-8 and LIN-35 Rb may associate in vivo. The biological role of
such an interaction is unclear. If the putative interaction between LIN-8 and LIN-35 Rb
were to facilitate lin-8 and/or lin-35 Rb function in vulval development, then one might
expect to observe both synMuv A and B activity associated with one or both of these
genes. However, a reduction of lin-8 function does not result in a synMuv phenotype in
combination with a loss of the function of the synMuv A genes lin-15A, lin-38, or lin-56
(FERGUSON and HORVITZ 1989; THOMAS et al. 2003), suggesting that lin-8 does not
possess class B synMuv activity. Similarly, a loss of lin-35 Rb function does not result in
a synMuv phenotype in combination with a loss of the function of the synMuv B genes
lin-36, lin-37, or lin-15B (FERGUSON and HORVITZ 1989), suggesting that lin-35 Rb does
not possess class A synMuv activity.
Proteins that physically interact often work together directly in the same biological
process. By contrast, synthetic genetic interactions between null alleles of two genes
173
usually indicate that the genes affect a biological process through separate
mechanisms. Although lin-8 and lin-35 Rb function in the parallel synMuv A and synMuv
B pathways, respectively, the proteins they encode physically interact in vitro. If the
interaction between LIN-8 and LIN-35 Rb is biologically important, then three models
could explain why neither lin-8 nor lin-35 Rb appears to possess both synMuv A and
synMuv B activity. First, the functional consequence of the LIN-8/LIN-35 Rb interaction
may be redundant with another process in vulval development. For example, LIN-8 may
be independently localized to the promoters of vulval specification genes by both LIN-35
Rb and by another protein. Second, LIN-8 and LIN-35 Rb may function together in the
vulva, but in some process not required for vulval development. Third, lin-8 and lin-35
Rb may act together but not in the vulva. The widespread expression of lin-8 and lin-35
Rb (Lu and HORVITZ 1998) indicates that they could function together in other tissues.
Mutation of lin-35 Rb has indeed been shown to result not only in the synMuv B
phenotype but also in defects in cell cycle progression (BOXEM and VAN DEN HEUVEL
2001; FAY et al. 2002; GARBE et al. 2004), in defects in pharyngeal morphogenesis (FAY
et al. 2003), and in severely reduced expression of a muscle cell-specific gfp reporter
gene from repetitive transgene arrays (the Tam phenotype) (HSIEH etal. 1999).
Although current evidence suggests that lin-8 does not function with lin-35 Rb in the
regulation of either cell cycle progression or transgene expression (HSIEH etal. 1999;
BOXEM and VAN DEN HEUVEL 2002; GARBE etal. 2004; E. C. ANDERSEN and H. R.
HORVITZ, unpublished observations), the possibility remains that lin-8 and lin-35 Rb act
together in the developing pharynx or in processes not yet analyzed.
Partial redundancy in the LIN-8 family: The nonsense alleles n2731 and
n2738 appeared to be null alleles on the basis of their molecular lesions and lack of LIN-
8 protein, yet by comparison to loss of the class A synMuv gene lin-56 and by
comparison to a deficiency that removes the lin-8 locus did not appear to have lost all
synMuv A pathway activity. By contrast, three of the lin-8 missense alleles impaired
synMuv A activity almost to the extent seen upon loss of lin-56. One of these three lin-8
missense alleles, n2376, acted like a null by deficiency analysis but did not destabilize
full-length LIN-8 protein (at least in extracts from mixed-stage animals). One hypothesis
174
to account for these observations is that LIN-8 normally functions as part of a protein
complex and that other family members can partially replace LIN-8 activity within the
complex in its absence. Specifically, in mutants that lack LIN-8 protein, closely related
family members may partially substitute for LIN-8. ccDfl 1, the deletion used for
deficiency analysis, removes both lin-8 and several lin-8family members. The predicted
partial replacement of LIN-8 by other LIN-8 family members may therefore be reduced
in lin-8(null)/ccDfl 1 heterozygotes as compared to lin-8(null) homozygotes. The strong
missense alleles may encode stable LIN-8 proteins that inactivate other family members
either by direct interaction or by competition with a partner. A similar phenomenon has
been observed in S. cerevisiae for the MAP kinases Fus3 and Kssl, which in wild-type
yeast regulate the mating pheromone response and filamentation pathways,
respectively (MADHANI et al. 1997). In the complete absence of Fus3, Kssl provides
substitute MAPK activity for the mating pheromone response pathway and thus only a
slight reduction in mating efficiency is observed. By contrast, when Fus3 is rendered
catalytically inactive by a missense mutation, Kssl cannot substitute in the mating
pheromone response pathway and a much stronger mating defect results. A
comparable model has been proposed for the histone deacetylase family 
in Drosophila,
as missense but not null mutations of the histone deacetylase HDAC1 dominantly
suppress silencing caused by position effect variegation (MOTTUS et al. 2000).
The biological roles of the remaining 16 lin-8 family members are not known.
Their similarity to lin-8 suggests that these genes are also likely to have roles in
transcriptional regulation, perhaps with other components or in other cells. The high
degree of similarity shared by the lin-8 family members also suggests that redundancy
may have prevented their identification in genetic screens: more than one family
member may have to be inactivated for a mutant phenotype to be apparent.
Interactions within the class A synMuv pathway: LIN-56 and LIN-15A are
dependent on each other for wild-type protein levels and likely form a functional
complex in vivo (E. M. DAVISON, A. M. SAFFER, L. S. HUANG, J. DEMODENA, P. W.
STERNBERG and H. R. HORVITZ, manuscript in preparation). Mutation of lin-8 or lin-38
does not perturb the expression or localization of either LIN-56 or LIN-15A, indicating
175
that neither lin-8 nor lin-38 is normally required for formation or stability of the putative
LIN-56/LIN-15A complex. In this manuscript, we demonstrate that lin-56, lin- 15A, and
lin-38 do not appear to be required for expression of LIN-8. These results form a basis
upon which our understanding both of the roles of individual components of the class A
synMuv pathway and of the interactions among these components can be further
expanded.
Implications for human cancer: Mammalian tumorigenesis requires
deregulation of cell proliferation, cell differentiation, and apoptosis and is thus an
inherently synthetic process requiring multiple mutations in the proto-oncogene and
tumor suppressor pathways controlling these biological activities (HANAHAN and
WEINBERG 2000). The Rb tumor suppressor pathway likely plays a critical role in
preventing oncogenic transformation, as its inactivation is observed in many human
cancers (NEVINS 2001). That the class A synMuv genes function redundantly with the C.
elegans Rb pathway suggests that mammalian counterparts of the synMuv A genes
may well possess tumor suppressor activity. We hope that characterization of the
mechanism by which the class A synMuv genes function will lead to greater
understanding of processes that act with the mammalian Rb pathway both in cell-fate
determination and in protection from oncogenic transformation and that the redundancy
of the class A, B, and C synMuv genes in regulating C. elegans vulval cell fates will
serve as a model for the etiology of other 
synthetic processes, such as tumorigenesis,
whose manifestation requires multiple mutations.
176
ACKNOWLEDGMENTS
We thank Craig Ceol and Hillel Schwartz for critical reading of the manuscript, and
members of the Vidal lab for their support. We are grateful to Caroline Spike and Bob
Herman for data concerning the left endpoints of the ccDf , ccDf2 and ccDf 11
deficiencies. We thank Beth Castor for help with DNA sequence determination, Na An
for strain management, Yuji Kohara for EST cDNA clones, and Alan Coulson and the C.
elegans Sequencing Consortium for cosmid clones and sequences. The deficiency
strains were provided by Theresa Stiernagle 
of the Caenorhabditis Genetics Center,
which is supported by the National Institutes of Health National Center for Research
Resources. This work was supported by National Institutes of Health grant GM24663.
E.M.D. was supported by a Howard Hughes Medical Institute predoctoral fellowship.
H.R.H. is an Investigator of the Howard Hughes Medical Institute.
177
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TABLE 1
Sequences of lin-8 mutations
Allele Wild-type codon Mutant codon Substitution
nill CTG CCG L20P
n2741 GTG ATG V68M
n2376 GAG AAG El 48K
n2378 CGC TGC R 154C
n2403 GAG AAG E164K
n2724 GAG AAG E 164K
n2738 TGG TAG W79amber
n2731 CAA TAA Q 113ochre
n2739 AGA TGA R304opal
Amino acid substitutions are indicated
number, and mutant residue.
as wild-type residue, residue
186
TABLE 2
lin-8 allele strengths
Penetrance of Muv phenotype (%)
lin- 15B(n2245) lin-52(n771)
Genotypea"b 150 (n) 200 (n) 150 (n) 200 (n)
Iin-8(n 111) 29 (94) 100 (109) 49 (98) 99 (143)
lin-8(n274 1) 1 (98) 100 (101) 15 (96) 99 (146)
lin-8(n2376) 98 (99) 100 (103) 80 (96) 100 (155)
lin-8(n2378) 99 (98) 100 (101) 88 (97) 100 (142)
lin-8(n2403J 92 (101) 100 (104) 98 (100) 100 (107)
lin-8(n2738) 68 (96) 100 (103) 96 (97) 100 (139)
lin-8(n2731) 77 (74) 100 (104) 99 (97) 100 (146)
lin-8(n2739) 76 (100) 100 (102) 63 (95) 97 (119)
lin-8(n2376) dpy- 10(e 128)/ccDf 11 96 (135) nd nd nd
lin-8(n2738) dpy- 10(e 128)/ccDf 11 95 (127) nd nd nd
lin-8(n273 1) dpy- 10(e 128)/ccDf 11 94 (183) nd nd nd
lin-56(n2728f 100 (127) 100 (127) nd nd
lin-56(n3355) 100 (124) 100 (100) nd nd
187
a Animals homozygous for either a lin-8(lf) or lin-56(lf) allele were 
raised at either 150
or 200 for at least three generations before scoring.
bTo generate lin-8(If)/ccDf 
11; fin- 15B(n2245) 
animals, dpy- 1O(e 
128); lin- 15B(n2245)
hermaphrodites were mated with ccDfl 1/nls128 males. The resulting non-Dpy non-
GFP ccDfl 1/dpy- 10(e 128); lin- 15B(n2245) male offspring were then crossed with
lin-8(lf) dpy- 10O(e 128), lin- 15B(n2245) hermaphrodites, and the Muv phenotype of any
non-Dpy cross progeny of this mating was scored. All crosses were carried out at 150.
c Since n2403 and n2724 contain the same amino acid change, only n2403 was
analyzed.
d The n2728 allele 
contains a deletion 
of the entire lin-56 
locus. The n3355 
allele
contains an early nonsense mutation within the lin-56 coding sequence. (E. M.
DAVISON, A. M. SAFFER, L. S. HUANG, J. DEMODENA, P. W. STERNBERG and H. R.
HORVITZ, manuscript in preparation)
% Muv, percent animals with at least one pseudovulva on their ventral sides
n, number of animals scored
nd, not determined
188
Figure 1: Cloning of Iin-8.
(A) Physical map of the genomic region containing the lin-8 locus. Deficiency and
polymorphism mapping placed lin-8 between cosmids F34D6 and C17F4. lin-8
rescuing cosmid C03E12 is shown in bold. Solid lines indicate regions known to
be deleted by the deficiencies; broken lines indicate regions that may be deleted
by the deficiencies.
(B) Transformation rescue of lin-8. Shown are the predicted open reading frames
within rescuing cosmid C03E12, top, as well as the subclones derived from this
cosmid, bottom.
(C) Sequence of the LIN-8 protein. The region sufficient for interaction with
LIN-35 Rb is underlined.
189
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MSKIKTHSTGSKRTVPFYKLPPPVPLPPLPPPDPTRYFSTEKYIALSKDE 
50
KFKFDDYDVNDETLKKVVLNEIGKCPDIWS SRSQAAIMEHYPIVATETYR 
100
RTGLLLS IKSLKQIYKCGKDNLRNRLRVAIVSKRLTPAQVEAYMWRWEFY 
150
GFIRYYRDYTQRWEADLLKDLDVVLGLEARRASKNMEKVDSGELMEPMEP 
200
MDSTMDEMCVEEEPYEETGSNWSDPAPEPSQSKSQSPEAKYPQAYLLPEA 
250
DEVYNPDDFYQEEHESASNAMYRIAFSQQYGGGGSPAVQKPVTFSAQPAP 
300
APVREAPSPVVENVSSSSFTPKPPAMINNFGEEMNQITYQAIRIAREQPE 
350
RLKLLRKALFDVVLAFDQKEYADVGDLYRDLAQKNS 
386
Figure 2. LIN-8 defines a family of C. elegans proteins.
Alignment of LIN-8 with the other 16 members of the LIN-8 family; all of these
proteins are from C. elegans. Solid boxes indicate identity with LIN-8 in at least
three additional family members. Arrowheads indicate positions of missense
mutations found in the indicated lin-8 alleles. The proline-rich N-terminal motif
and the region of LIN-8 sufficient for interaction with LIN-35 Rb are indicated with
solid lines.
192
Figure 2
C06C3.8 MTQEMQQSMELSHEIVIANGHLFGIQGFTCNITLSLEQIGRTYNGV
C06C3.8 SMALPFVLLIISAIKKNSLRKDYVATGDVSLAGAVLTVDYINNKIVGAIN
C06C3.8 AGLKGVVIPAENGKDVKPFLKTQIKVIEVTSIVDAWSTMIIPEMPNGDSG
ASNCGSLDGVRSVTRKGKTMKTMK
n711P proline
V F L *V LP
V LL KQ*LSI AE
I V .. T TIS % PI
 " T T=T l '   :
KFSE- -
TGVY - -
REDYLVI
L QE
MNP EE
L QE
LN QE
L S PQ E
VSATRV
RMQ E
C06C3.8
K08A2.1
LIN-8
B0454.9
K08A2.4
C06C3.8
C41 H7.5
C08A9.6
C41 H7.4
C41 H7.6
C08A9.8
C41 H7.3
C08A9.7
F14D2.2
F54D10.3
K09E3.5
C08A9.9
K09E3.6
K08A2.1
LIN-8
B0454.9
K08A2.4
C06C3.8
C41 H7.5
C08A9.6
C41 H7.4
C41 H7.6
C08A9.8
C41 H7.3
C08A9.7
F14D2.2
F54D10.3
K09E3.5
C08A9.9
K09E3.6
K08A2.1
LIN-8
B0454.9
K08A2.4
C06C3.8
C41 H7.5
C08A9.6
C41 H7.4
C41 H7.6
C08A9.8
C41 H7.3
C08A9.7
Fl 4D2.2
F54D10.3
K09E3.5
C08A9.9
K09E3.6
K08A2.1
n2741M
F G DDYDVN vET
EIG Q KAEVR VL
RKVYEIKPDDRIL
L LGVQK IGSK 
V 
VL
RDYYRNATK TS
LNSVYEEATK*SA
NT ONHKTATRAPK
AKS L K TMTM LC
NRE VVNASK IIA
MK DYYRDATK AS
EHIEGTKFPM NL
T S G R Y S K K R VN
RNHVIDKKKKVI
NNYLEDATINSY
Q D DAL S SSOK -
R L KQ
-- HMN
SIVPLP
--- HMH
-GVVA
SIVPLP
-LLDAP
SGLPHRNQTDQKNEMTSMGECIGEVI
AEDTPIFGFFSRQVKTEQLVPEIKEE
P N YF5T IAESKDE
L N NLPL S FLMMKTP
AGLRITVQE MAMLKSG
PE KM HTE LQOSESP
- HQITIKE KER-- -V
-SHCITMAD LLE ---
- HQITL E KER-- ER
-IHHV MRM E MGM ---
-SHCITMAD LL ---
-EHRIHLSG RNI---H
-SFPITMDE LEK - -
-EHRITMDE KKR ---
L E
PB
TI
T
AIT
ALT
OT T
AAT
TTTSATAQQRTPS
-QVNP P RAIT
MT'NTEMSITMQ NIE-  - - G
MA TTRQSITQK CE ---
M APRRLS TF G KGTI VKE LE --- P
R LT I -- R M S V E LK T K VKL K AFNRC --- K
I
I
S
DR
DR
DH
DR
RS
NR
LY
DY
PK
NY
KY
NH
ME
---------------- SS
-.. 
- - - .
. -N- 
.
N C
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----------------- 
B AG
---------------- 
NT
NT
---------------- QN
---------------- 
DR
---------------- GM QN
---------------- KAM AP
IKTKLLKLKSYSNKYKMFSRKK
---------------- RAL IR
---------------- 
EM P
---------------- 
QN
----------------
E   L NS
---------------- 
KS
---------------- 
CF QH
---------------- QNL EY
---------------- KRRYMW
RSQAAIMEHYPI-- AT T R
KNTYNANKLYPE-- VQV 
R
EKNTNRQAEHYA-T AAV A
YKANGIYSEVAV-N --- FK
GNRFQLENWREL-G - DV Q
KAQFSAKNWQNL-G - V E
GNRFQPEKWRAL-G -DV EQ
AAKPSEDKWQKL-GA-- VFS
IYRKFLNIHCFK-FHKF -SL
VHKSPKADWEVL-G- - VFE
GGPMVAKKWQAF-GA-- HER
GNRFQTRKWRAL-GE-- VEQ
NQKIDRERWETV-GI-- LFQ
KSSGPPDEWRQI-VM-- VFK
NVRGSLDQWILV-RA-- V E
NATVVEEKWALL-GI-- VL E
KGIIDRGKKKNWTIFGA LQQ
196
27
50
42
42
246
35
42
35
36
42
37
43
37
10
18
18
27
72
75
67
66
271
60
67
60
61
92
62
68
62
35
43
43
52
96
100
92
92
293
84
91
84
85
140
86
92
86
59
67
67
76
123
VKAQ H T E Q P L T V I
Figure 2
LIN-8
B0454.9
K08A2.4
C06C3.8
C41 H7.5
C08A9.6
C41 H7.4
C41 H7.6
C08A9.8
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Figure 2
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Figue 3. LIN-8 protein is expressed broadly and localized in nuclei.
(A) Western analysis of protein extracts from 
wild-type, lin-8(lf), lin-56(n2728),
lin-38(n751), lin- 15A(n767), and lin- 15AB(e 1763) mixed-stage worms probed
with affinity-purified and pre-adsorbed anti-LIN-8 antibody. The position of the
apparent LIN-8 protein doublet is indicated by the arrow. The molecular weights
of marker proteins are indicated at left in kD.
(B-D) Whole-mount staining of wild-type animals with affinity-purified and pre-
adsorbed anti-LIN-8 antibodies (green), as well as DAPI (blue) to visualize DNA.
Staining with anti-tubulin antibody (red) is shown as a fixation control in embryos.
Staining with the MH27 antibody (red), which recognizes the apical borders of C.
elegans epithelial cells, is shown as a fixation control in adults. Scale bars, 5 pm.
(B) LIN-8 staining is observed in multiple nuclei in the wild-type but not
lin-8(n2731) embryo.
(C) LIN-8 staining is present in the wild-type but not lin-8(n2731) gonad in
pachytene nuclei and in oocytes.
(D) LIN-8 staining is observed in multiple nuclei in the wild-type adult head.
196
Figure 3
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Figure 4. LIN-8 interacts with LIN-35 Rb in vitro.
(A) LIN-35 Rb (aa 270-961) but neither LIN-35 Rb (aa 1-155) nor luciferase
interacts with full-length MBP-LIN-8 fusion protein. None of the constructs
interacts with MBP alone. 20% of the 
35
S-labeled proteins used in the binding
reactions are shown. Coomassie blue staining indicates that approximately equal
amounts of full-length MBP-LIN-8 and MBP were used in the binding reactions.
The molecular weights of marker proteins are indicated at the left in kD.
(B) LIN-35 Rb (aa 270-961) but not luciferase interacts with GST-LIN-8 (aa 175-
285) and GST-EFL-1. Neither construct binds to GST 
alone. 20% of the 
35
S-
labeled protein used in the binding reactions are shown. Coomassie blue staining
indicates that approximately equal amounts of MBP, GST, and fusion proteins
were used in the binding reactions. The molecular weights of marker proteins are
indicated at left in kD.
(C) Summary of the LIN-8 fragments used for in vitro pull-down experiments and
of their ability to interact with LIN-35 Rb (aa 270-961). +, wild-type interaction; +/-,
interaction detected but weaker than wild-type; -, no interaction.
199
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CHAPTER 5
Identification and classification of genes that act antagonistically 
to
let-60 Ras signaling in Caenorhabditis elegans 
vulval development
Craig J. Ceol', Frank Stegmeier
2
, Melissa M. Harrison and H. Robert Horvitz
Published in Genetics,
My contributions to this chapter include helping to isolate new synMuv mutants and
cloning sli- 1 by mapping and transformation rescue.
1
Present address: Howard Hughes Medical Institute, Department of
Hematology/Oncology, Children's Hospital, Boston, Massachusetts 02115
2
Present address: Howard Hughes Medical Institute, Department 
of Genetics, Harvard
Medical School, Massachusetts 02115
202
ABSTRACT
The synthetic multivulva (synMuv) genes negatively regulate Ras-mediated
vulval induction in the nematode Caenorhabditis elegans. The synMuv genes define
three classes, A, B and C, such that double mutants carrying mutations in genes of any
two classes are multivulva. The class B synMuv genes include lin-35, a homolog of the
retinoblastoma (Rb) tumor suppressor gene, as well as homologs of genes that function
with Rb in transcriptional regulation. We screened for additional synMuv mutations
using a strategy different from that of previous synMuv genetic screens. Some of the
mutations we recovered affect new synMuv genes. We present criteria for assigning
synMuv mutations into different genetic classes. We also describe the molecular
characterization of the class B synMuv gene lin-65.
203
INTRODUCTION
A fundamental issue in developmental biology is how cells that are initially
equivalent in developmental potential ultimately adopt different fates. Genetic studies
have indicated that cells within a developmental equivalence group often adopt different
fates in response to the combined action of multiple and sometimes competing signals
(reviewed by FREEMAN and GURDON 2002). For example, the initial step of R8
photoreceptor specification in ommatidial development in Drosophila melanogaster uses
both positive and negative signals to properly select presumptive R8 photoreceptors
from a field of developmentally equivalent cells in the eye imaginal disc (reviewed by
FRANKFORT and MARDON 2002). An overlay of such signals can make a response in
binary cell-fate decisions more precise or can increase the number of fates available to
a particular cell.
Vulval development in the nematode Caenorhabditis elegans involves a set of
ectodermal Pn.p cells that initially have similar developmental potentials but ultimately
adopt different fates (KIMBLE 1981; STERNBERG and HORVITZ 1986). The specification of
Pn.p cells that eventually make vulval tissue occurs in two steps, each of which involves
the selection of a subset of Pn.p cells from a larger Pn.p field (SULSTON and HORVITZ
1977). First, in the L1 larval stage shortly after the 12 Pn.p cells are generated, the
P1.p and P2.p anterior and P(9-11).p posterior cells fuse with the syncytial hypodermis.
Each of the six remaining unfused midbody cells P(3-8).p has the capacity to adopt a
vulval cell fate (STERNBERG and HORVITZ 
1986). Second, three of these six cells,
P(5-7).p, adopt vulval fates and undergo three rounds of division to generate seven
(P5.p and P7.p) or eight (P6.p) descendants. P3.p, P4.p and P8.p adopt non-vulval
fates, typically dividing only once to generate two descendants that eventually fuse with
the syncytial hypodermis. The decision to adopt vulval cell fates occurs during the L2
and early L3 larval stages and is followed by cell divisions and differentiation in the L3
and L4 larval stages, respectively (KIMBLE 1981; STERNBERG and HORVITZ 1986;
FERGUSON etal. 1987).
Many genes that control the specification of Pn.p fates have been identified.
Some of these genes act in a spatially-restricted fashion to select Pn.p cells for vulval
204
development. The homeobox gene lin-39 is expressed in the midbody and regulates
the sequential steps of fusion and vulval cell-fate specification of the Pn.p cells in this
region (CLARK et al. 1993; WANG et al. 1993; MALOOF and KENYON 1998). Strong loss-
of-function lin-39 mutations result in ectopic P(3-8).p cell fusion during the L1 stage. In
partial loss-of-function lin-39 mutants, unfused P(5-7).p cells are sometimes observed
and often show vulval-to-non-vulval cell-fate transformations (CLARK etal. 1993). lin-39
activity therefore promotes unfused cell fates in the L1 stage and vulval cell fates in the
L2 and early L3 stages. Genes in the let-60 Ras signaling pathway also regulate the
specification of Pn.p fates (BEITEL et al. 1990; HAN and STERNBERG 1990). In addition to
let-60 Ras, this pathway includes the receptor tyrosine kinase let-23, the SH2/SH3
adaptor sem-5 and the MAP kinase mpk- 1, all of which are broadly conserved in Ras
signaling systems (reviewed by MOGHAL and STERNBERG 2003). The role of let-60 Ras
signaling in the specification of vulval cell fates is well characterized. In wild-type
animals, the let-60 Ras pathway is specifically activated in P(5-7).p in response to an
EGF-like signal, encoded by lin-3, that is produced by the neighboring gonadal anchor
cell (HILL and STERNBERG 1992). Mutations that reduce let-60 Ras pathway activity
prevent P(5-7).p vulval cell-fate specification, resulting in a vulvaless (Vul) phenotype.
Mutations that abnormally activate this pathway cause P(3-8).p all to adopt vulval cell
fates, resulting in a multivulva (Muv) phenotype (BEITEL et al. 1990; HAN and STERNBERG
1990; EISENMANN and KIM 1997). Increases in let-60 Ras pathway activity may promote
vulval cell fates in part by upregulating lin-39 expression (MALOOF and KENYON 1998).
The activities of lin-39 and genes in the let-60 Ras pathway are antagonized by
the synthetic multivulva (synMuv) genes. The synMuv genes define three redundant
classes, A, B and C (FERGUSON and HORVITZ 1989; THOMAS et al. 2003; CEOL and
HORVITZ 2004). Animals carrying mutations affecting any two classes of synMuv genes
are Muv, but animals with a mutation in one synMuv gene or in multiple synMuv genes
of a single class undergo wild-type vulval development. All three classes of genes
promote the expression of non-vulval cell fates by P(3-8).p. At present it is unknown
whether the synMuv mutations cause an increase of let-60 Ras pathway activity in these
cells or cause these cells to be more sensitive to normal levels of let-60 Ras pathway
205
activity. Roles for synMuv genes in regulating Pn.p fusion have also been described.
Some class B genes, but no class A genes, antagonize lin-39-mediated cell fusion of at
least one Pn.p cell, P3.p (CHEN and HAN 2001).
Many synMuv genes have been molecularly characterized. The class B synMuv
protein LIN-35 is similar to the mammalian tumor suppressor pRb (Lu and HORVITZ
1998). Other class B synMuv proteins include DPL-1 and EFL-1, which are similar to
mammalian DP and E2F proteins and, by analogy to their mammalian counterparts,
likely function to target LIN-35 Rb to DNA (CEOL and HORVITZ 2001). The class B
synMuv protein HDA-1 is similar to class I histone deacetylases (Lu and HORVITZ 1998)
and may be targeted to specific genes by a DPL-1/EFL-1/LIN-35-containing protein
complex.
As lin-35 Rb can act in the surrounding hypodermis to regulate P(3-8).p fates, the
genes targeted by a DPL-1/EFL-1/IIN-35-containing complex may function non-cell
autonomously to regulate the specification of vulval cell fates (MYERS and GREENWALD
2005). Other class B synMuv proteins also are components of this complex (M.M.H. and
H.R.H., unpublished observations), and complexes purified from Drosophila extracts
containing DP, E2F and Rb homologs contain 
homologs of the synMuv proteins LIN-9,
LIN-37, LIN-52, LIN-53 RbAp48, LIN-54 and LIN-61 (KORENJAK, et al. 2004; LEWI et al.
2004). These Drosophila complexes can repress transcription of DP/E2F target genes
and can inhibit genome-wide DNA replication in ovarian somatic follicle cells.
The class C synMuv genes encode components of a putative histone
acetyltransferase complex similar to the human Tip60 and yeast NuA4 histone
acetyltransferase complexes (CEOL and HORVITZ 2004). The molecular identities of
class B and class C synMuv genes suggest that chromatin remodeling and modification
are important in specifying P(3-8).p fates. The class A synMuv gene lin-15A encodes a
novel protein (CLARK et al. 1994; HUANG et al. 1994). Little is known about the
mechanism of action of the class A synMuv genes.
Previous synMuv genetic screens required that mutant isolates be fertile for the
recovery of synMuv mutations. We used a screening approach that allowed the
recovery of synMuv mutations that cause recessive sterility. We describe the
206
characterization of new synMuv mutations and criteria used to distinguish new and
previously described classes of synMuv genes.
207
MATERIALS AND METHODS
Strains and general techniques: Strains were cultured as described by
BRENNER (1974) and grown at 200C unless otherwise indicated. The wild-type parent of
all Caenorhabditis elegans strains described in this study was the Bristol strain N2,
except that some multi-factor mapping experiments used the polymorphic wild-type
strains RW7000 (WILLIAMS et al. 1992) and CB4856 (WICKS et al. 2001). We also used
strains containing the following mutations:
LGI: bli-3(e767), lin-17(n677), unc- 11(e47), 
unc-73(e936), lin-44(n 1792), unc-38(x20),
dpy-5(e61), lin-35(n745), unc-13(e1091), lin-53(n833) 
(FERGUSON and HORVITZ 1989),
unc-54(e 1092) (DIBB et al. 1985).
LGII: lin-31(n301), dpy-10(e 128), tra-2(q276), rol-6(e 187), dpl-1(n2994) (CEOL and
HORVITZ 2001; THOMAS et al. 2003), let-23(sy10, 
sy97), unc-4(e120), unc-53(n569),
mex- 1(it9), rol- 1(e91), lin-38(n751).
LGIII: dpy-17(e164), Ion-1(e185), 
lin-13(n770) (FERGUSON and HORVITZ 
1989),
lin-37(n758), lin-36(n766), unc-36(e251), lin-9(n112), unc-32(e189), lin-52(n771)
(FERGUSON and HORVITZ 1989), dpy-18(e364).
LGIV: lin-l(e1275), unc-5(e53), unc-24(e138), mec-3(e1338), lin-3(n378), sem-3(n1900)
(M.J. STERN and H.R.H., unpublished 
results), dpy-20(e 1282), unc-22(e66),
dpy-26(n 198), ark-1(sy247) (HOPPER 
etal. 2000), unc-31(e169), unc-30(e 
191),
lin-54(n223 1) (THOMAS et al. 2003), dpy-4(e 1166).
LGV: tam- 1(cc567) (HSIEH et al. 1999), 
unc-46(e 177), let-418(s1617), dpy- 
11(e224),
rol-4(sc8), unc-76(e911), efl- 1(n3318) (CEOL and HORVITZ 2001), dpy-21(e428).
LGX: egl-17(e1313), sli-1(sy143), aex-3(ad418), 
unc-1(e1598 n1201) (PARK and H.R.H.,
unpublished results), dpy-3(e27), gap-1(ga133) 
(HAJNAL et al. 1997), unc-2(e55),
Ion-2(e678), unc- 10(e 102), dpy-6(e 
14), unc-9(e 101), unc-3(e 151), lin- 15B(n744),
lin- 15A(n 767), lin- 15AB(n765).
Unless otherwise noted, the mutations used are described by RIDDLE (1997). In
addition, we used strains containing the following chromosomal aberrations: mnDf57 II
(SIGURDSON et al. 1984), mnDf90 II(SIGURDSON et al. 1984), mnDf29 //II (SIGURDSON et al.
1984), mnDf87 II (SIGURDSON et al. 1984), mln1[dpy-10(e 128) mls14] // (EDGLEY and
208
RIDDLE 2001), mnCi[dpy- 10(e 128) unc-52(e444)] II (HERMAN 1978), nDf40 III
(HENGARTNER et al. 1992), qCl[dpy-19(e1259) glp- 
1(q339)] III (AUSTIN and KIMBLE,
1989), sDf63 IV(CLARK and BAILLIE 1992), sDf62 IV(CLARK and BAILLIE 1992), sDf l0 IV
(ROGALSKI et al. 1982), hT2[qls48] (1,111) (L. MATHIES and J. KIMBLE, personal
communication), eTi(III/V) (ROSENBLUTH and BAILLIE 1981), nT1(IV;V) (FERGUSON and
HORVITZ 1985), nTI(n754) (IV;V), nTl[qls51] (IV;V) (L. MATHIES and J. KIMBLE, personal
communication). n754 causes a dominant Unc phenotype, allowing nTi(n754)-
containing larvae and adults to be scored (E.L. FERGUSON and H.R.H., unpublished
results). mls14, an integrated transgene linked to the chromosomal inversion mini
(EDGLEY and RIDDLE 2001), and qls48 and qls51, integrated transgenes linked to the
reciprocal translocations hT2(I;II) and nTI(IV;V), respectively (L. MATHIES and J.
KIMBLE, personal communication), consist of GFP-expressing transgenes that allow
mls14, qls48 or qls51-containing animals to be scored beginning at the 4-cell stage of
embryogenesis.
Isolation of new alleles: We mutagenized lin- 15A(n767) hermaphrodites with
ethyl methanesulfonate (EMS) as described by BRENNER (1974). We allowed these
animals to recover on food for between 15 minutes and one hour and then transferred
individual P
0 
larvae in L4 lethargus to 50 mm Petri plates. After three to five days, 20 F,
L4 larvae per P
0 
were individually transferred to 50 mm plates, and F
2 
animals on these
plates were subsequently screened for a Muv phenotype. We screened the progeny of
3380 F, animals using this procedure.
Linkage group assignment: We mapped newly isolated synMuv mutations to
linkage groups using standard methods (BRENNER 1974), except for some mutations
that we mapped using the polymorphisms present in the wild-type strain RW7000
(WILLIAMS etal. 1992).
Complementation tests: We performed complementation tests as described by
FERGUSON and HORVITZ (1989). Hemizygous lin-15B(n371 1) lin- 15A(n767) males could
not mate. To perform complementation tests with this mutation, we mated tra-2(q276);
lin- 15B(n371 1) lin- 15A(n767)/++ XX males with marked lin- 15AB hermaphrodites and
scored cross progeny.
209
Construction of deficiency heterozygotes: To construct trr- 1(n3712)
heterozygotes with the mnDf57, mnDf90O and mnDf29 deletions, Df/mln 1, lin- 15A(n767)
males were generated. These males were mated with rol-6 trr-1(n3712)/mlnl;
lin-15A(n767) hermaphrodites, and non-Rol, non-Gfp cross-progeny were scored.
mnDf87heterozygous males do not mate, so in this case we generated trr-
1(n3712)/mnDf87; lin- 15A(n767) animals by mating trr- 1(n3712)/mn 1/n lin- 15A(n767)
males with unc-4 mnDf87/mln 1; lin- 15A(n767) hermaphrodites. mep- 1/Df animals were
constructed by mating Df/nTi; +/nTI males with dpy-20 mep- 1; fin- 15A(n767)
hermaphrodites and scoring non-Dpy cross-progeny.
Construction of single mutant and unlinked double mutant strains: The
synMuv mutations listed below were balanced in trans by the specified double mutant
combinations or chromosomal aberrations in constructing strains with a single synMuv
mutation or strains carrying two unlinked synMuv mutations:
lin-65(n344 1): bli-3(e767) lin- 17(n677), h T2[qls48] (I;//l)
lin(n3628): unc- 11(e47) dpy-5(e61), hT2[qls48](I;,1/1)
lin-35(n745): dpy-5(e61) unc- 13(e 1091), hT2[qls48] (111)
trr- 1(n3712): mln I[dpy- 10(e 128) mIls14]
lin-38(n751): mnC 1[dpy- 10 O(e 128) unc-52(e444)]
mep-1(n3703): dpy-20(e1282) unc-30(e 191), nTi n754 (IV;V), nTi[qls51] (IV;V)
ark- 1(n3701): dpy-20(e 1282) unc-30(e191), nT1 n754 (IV;V), nTl[qls51] (IV;V)
mys- 1(n368 1): unc-46(e 177) dpy- 1 1(e224), nT n754 (IV;V), nTl[qls51] (IV;V)
let-23(sy97) was balanced with mln i[dpy- 10O(e 128) mls14].
A sli- I single mutant was constucted by generating + sli- 1 + lin- 15A / egl- 17 +
unc-1 + hermaphrodites and identifying non-mutant progeny that segregated only Egl
Unc non-Muv and non-Egl non-Unc non-Muv animals. Non-Egl non-Unc non-Muv
animals were isolated, and sli-1 homozygotes were identified as those that did not
segregate Egl Unc non-Muv progeny. Double mutant strains containing an X-linked
mutation in either sli-1, gap- 1, lin-15A or lin- 15B and an autosomal mutation were
constructed essentially as described by FERGUSON and HORVITZ (1989).
210
To ensure that mutations were not lost by recombination, several independent
lines were isolated for each strain. Some double mutant strains that exhibited a strong
synMuv phenotype were constructed based on their Muv phenotype without the use of
balancers.
Construction of linked double mutant strains: To construct an n3628 lin-35
double mutant, hermaphrodites of genotype n3628 dpy-5 + + /+ + lin-35 unc- 13; lin- 15A
were generated. Muv non-Dpy non-Unc progeny that segregated only Muv non-Dpy
non-Unc, Muv Dpy non-Unc and Muv Unc non-Dpy animals were selected. Muv Unc
non-Dpy animals of the genotype n3628 lin-35 unc- 13; lin- 15A were isolated, and the
lin- 15A mutation was crossed out using unc- 11 dpy-5 as a balancer.
To construct a sli-1 lin-15B double mutant, + sli-1 + fin-15A /egl-17 + unc-1 lin-
15AB hermaphrodites were generated. Muv non-Egl non-Unc progeny that segregated
only Muv non-Egl non-Unc and Muv Egl Unc animals were selected. Muv non-Egl non-
Unc animals of the genotype sli-1 lin- 15AB were isolated. From these animals, + sli- I +
lin- 15AB /egl- 17+ unc- 1 lin- 15B animals were generated. non-Muv non-Egl non-Unc
progeny that segregated only non-Muv non-Egl non-Unc and Egl Unc non-Muv animals
were identified, and non-Muv non-Egl non-Unc animals of the genotype sli- 1 lin- 15B
were isolated. A gap- I lin- 15B double mutant was similarly constructed using dpy-3
unc-2 as a balancer.
A sli- 1 gap-i double mutant was constructed by generating sli- I + dpy-3 + /+
unc-1 + gap-i hermaphrodites and individually isolating non-Dpy non-Unc progeny.
Progeny that segregated only non-Dpy non-Unc and Dpy non-Unc animals were
identified, and non-Dpy non-Unc animals of the genotype sli- I gap-1 were subsequently
isolated.
Because trr- 1(n3712) and let-23(sy97) cause recessive sterility and highly
penetrant larval lethality, respectively, we could not isolate trr- I or let-23 homozygotes
in our construction of a trr- 1 let-23 double mutant. For this reason, we built this double
mutant by first generating + rol-6 + trr- 1 /let-23 + unc-4 +; lin- 15A males and mating
them with mlnl[dpy-10(e 128) rn/s4]; lin-15A hermaphrodites. non-Dpy cross progeny
were individually isolated. non-Dpy progeny with broods consisting of dead larvae, Vul
211
Unc Gro non-Muv non-Rol non-Gfp and Gfp non-Vul non-Unc non-Gro non-Rol animals
were identified. The presence of trr- 1 in these broods, as judged by the trr- 1-associated
growth-rate abnormality (Gro), was later confirmed by complementation testing. lin-15A
was crossed out to generate a let-23 unc-4 trr- 1 /mln [dpy- 1 O(e 128) mls 14] strain.
Assay for P(3-8).p vulval cell fates: Cell fates were scored in L4
hermaphrodites using Nomarski microscopy by counting the number of descendants
that had been produced by individual P(3-8).p cells. Scores of 1, 0.5 and 0, were
assigned to cells that fully, partially, or not at all adopted vulval cell fates, respectively.
P(3-8).p cells that partially adopt a vulval cell fate have one daughter that divides to
produce two to four descendants and another daughter that remains undivided (AROIAN
and STERNBERG 1991)
RNA-mediated interference: Templates for in vitro transcription reactions were
made by PCR amplification of cDNAs and their flanking T3 and T7 promoter sequences.
In vitro transcribed RNA was denatured for 10 minutes and subsequently annealed prior
to injection.
lin-65 rescue: Using Gateway in vitro recombination technology (Invitrogen), we
cloned the open reading frame encoding the 728 amino acid LIN-65 protein from a
pENTR201/in-65entry clone into the pMB1 and pMB7 destination vectors. pMB1 and
pMB7 (kindly provided by M. Boxem and S. van den Heuvel) are designed to express
inserted sequences under the control of the C. elegans heat-shock protein promoters
Phsp
1
6-2 and Phspl6-41, respectively. We performed transformation 
rescue (MELLO et al.
1991) using the green fluorescent protein-expressing plasmid pTG96 (kindly provided
by Min Han) as a coinjection marker. Transgenic animals were heat-shocked as L1 and
L2 larvae for one hour at 330C and scored as adults. Control transgenic animals were
not heat-shocked.
Allele sequence: We used PCR-amplified regions of genomic DNA as templates
in determining gene sequences. For each gene investigated, we determined the
sequences of all exons and splice junctions. Whenever observed, the sequence of a
mutation was confirmed using an independently-derived PCR product. All sequences
were determined using an automated ABI 373 DNA sequencer (Applied Biosystems).
212
RESULTS
Isolation of new synMuv mutants: A severe reduction of class B synMuv gene
function is often associated with sterility: 1) In a genetic screen for alleles that did not
complement the synMuv phenotype of lin-9(n 112), FERGUSON and HORVITZ (1989)
recovered two lin-9 alleles, n942 and n943, that caused recessive sterility. 2) Gene-
dosage studies indicate that, in comparison to the wild type, lin-52(n771)/Df and
dpl- l1(n2994)/Df heterozygotes have markedly reduced brood sizes (CEOL and HORVITZ
2001; THOMAS et al. 2003). 3) Deletion mutations of some synMuv genes recovered
using a PCR-based screening approach show recessive sterility, e.g., mutations of
lin-53 (Lu 1999), efl-1 and dpl-I (CEOL and HORVITZ 2001).
Previous genetic screens for synMuv 
mutants (FERGUSON and HORVITZ 1989;
THOMAS et al. 2003) were performed before a connection between loss of synMuv gene
function and sterility was well established. These screens required that isolates be
fertile and viable for the recovery of mutant alleles and failed to recover mutations of the
class B synMuv genes efl- 1 and let-418, both of which can mutate to cause a sterile
phenotype (voN ZELEWSKY et al. 2000; CEOL and HORVITZ 2001). These results
suggested that additional synMuv genes might be identified in a screen that allowed the
recovery of homozygous sterile mutations.
To screen for new synMuv 
mutants, we examined the 
progeny of individual F,
animals after EMS mutagenesis of their lin- 15A(n767) parents. We screened the
progeny of 3380 F, animals (6760 haploid genomes) for mutations that either alone or in
combination with lin-15A(n767) caused a recessive Muv phenotype. Mutations that
caused recessive sterility in addition to a Muv phenotype were recovered from their
heterozygous wild-type siblings present on the same Petri plate. Using this strategy we
identified 95 Muv mutations, 24 of which we maintained as heterozygotes because of a
recessive sterility that cosegregated with the Muv phenotype. Three mutations caused
a Muv phenotype in the absence of lin-15A(n767) and were found to affect the
previously studied genes lin- and lin-31, both of which function downstream of let-60
Ras in vulval induction (FERGUSON et 
al. 1987). These mutations, lin-1(n3443),
lin-1(n3522) and lin-31(n3440), were not characterized further. Thirty mutations when in
213
combination with iin-15A(n767) caused a weakly penetrant (< 30%) Muv phenotype.
We were unable to convincingly map these mutations to linkage groups. The remaining
62 mutations were assigned to 20 complementation groups (see below). Five of these
mutations affect the synMuv gene lin-61 and will be described elsewhere (M.M.H., X. Lu
and H.R.H., manuscript in preparation).
Phenotypes of new mutants: We characterized the penetrance of the Muv
phenotype of each strain at 150C and 200C (Table 1). At 25
0
C the penetrance of each
strain was between 98 and 100% (n>25), except 
for gap- 1(n3535); lin-15A(n767) (91%,
n=l 11) and lin(n3542) lin-15A(n767) (90%, n=42). Since a heat-sensitive Muv
phenotype is characteristic of most synMuv strains, including those with null mutations
in synMuv genes, it is likely that many individual synMuv mutations are not temperature-
sensitive but rather that the synMuv genes regulate a temperature-sensitive process
(FERGUSON and HORVITZ 1989).
As described in Table 1, many of these synMuv strains also exhibited a sterile
phenotype. In these strains, the sterile phenotype cosegregated with the Muv
phenotype during backcrosses and two- and three-factor mapping experiments. For efl-
1, let-418 and certain lin-9 and lin-53 mutations, we found that our new mutations did
not complement the sterile phenotypes caused by previously isolated allelic synMuv
mutations (data not shown). Mutations defining new synMuv loci likewise failed to
complement each other for the sterile phenotype: mep- 1(n3702) did not complement
mep- 1(n3703), and none of the other five trr- 1 mutations complemented trr- 1(n3712) for
the sterile phenotype. These observations indicate that the sterile and Muv phenotypes
of these strains were caused by the same mutation.
New synMuv genes: Using two-factor crosses and X chromosome transmission
tests (see MATERIALS AND METHODS), we mapped the new mutations to linkage groups.
We then determined if each mutation failed to complement mutations in known synMuv
genes on the same linkage group. In these tests we identified 41 alleles of known
synMuv genes: one dpl-1, one efl-1, seven let-418, three lin-9, four lin-13, ten lin-15B,
two lin-35, three lin-36, one lin-52, four lin-53 and five lin-61 mutations. We isolated one
mutation in gap- I and three in sli- 1, two genes that were originally identified in screens
214
for mutations that suppress the Vul phenotype caused by a reduction in let-60 Ras
pathway signaling (JONGEWARD et al. 1995; HAJNAL et al. 1997). We also identified two
mutations in ark- 1, a gene first identified in a screen for mutations that cause ectopic
vulval cell fates in a sli- I mutant background (HOPPER et al. 2000). gap- 1, sli- I and
ark- 1 single mutants were previously found to have no (sli- 1, gap- 1) or subtle (ark- 1)
defects in vulval development. Our results indicate that sli-1, gap- I and ark- act
redundantly with lin- 15A to negatively regulate let-60 Ras signaling.
Mutations that were not assigned to known synMuv complementation groups
were tested against unassigned mutations on the same linkage group for
complementation. These tests defined five new synMuv 
loci: lin-65, lin(n3628), mep-1,
mys- 1 and trr- 1. (lin(n3542) may define another new synMuv locus, but since we have
not separated lin(n3542) from /in- 15A(n767), we do not know whether lin(n3542) is a
synMuv mutation or whether it causes a Muv phenotype on its own.) We used multi-
factor crosses (Table 2) and deficiency heterozygotes (Table 3) to map these new
synMuv genes on their respective linkage groups. While our studies were in progress,
mep- I and Iin-65 were independently identified and reported to have a loss-of-function
synMuv phenotype (UNHAVAITHAYA et al. 2002; POULIN et al. 2005). Our detailed
characterization of the class C synMuv genes mys- I and trr- 1 is presented elsewhere
(CEOL and HORVITZ 2004). We separated lin-65, lin(n3628) and mep-1 mutations from
the parental lin- 15A(n767) mutation and found that these mutations alone do not cause
extra vulval cells to be produced (Table 4). Thus, these mutations synergize with
lin-15A(n767) and are synMuv mutations.
Interactions with other synMuv mutations: 
Since mutations affecting lin-65,
lin(n3628), mep-1, gap-1, sli- I and ark- interact synthetically with a class A synMuv
mutation, lin-15A(n767), these genes may either be class B or class C synMuv genes or
they may define a new synMuv gene class that shares some but not all properties with
class B or class C genes. To distinguish between these possiblities, we built double
mutant strains and measured synthetic interactions with lin-65, lin(n3628), mep-1, gap-
1, sli- 1 and ark- I mutations. We used the strongest available mutation for each of these
genes in these strain constructions. ga133 rather than gap- l(n3535) was used as the
215
gap- I mutation, because ga 133 is a deletion and is considered a null mutation (HAJNAL
et al. 1997). For the sake of brevity, gap- 1(ga133) will be referred to as a "new" synMuv
mutation hereafter. We quantified synthetic interactions by directly examining the fates
of individual P(3-8).p cells (see MATERIALS AND METHODS). In wild-type animals three
cells invariably adopt vulval fates, whereas in Muv mutants more than three cells adopt
vulval fates.
We first measured synthetic interactions with the class A mutation lin-38(n751)
and the class B mutations lin-15B(n744) and lin-35(n745) (Table 4). The new synMuv
mutations interacted synthetically not only 
with lin-15A(n767) but also with lin-38(n751),
suggesting a general redundancy with the class A synMuv genes. With lin-15B(n744)
and lin-35(n745) the new mutations showed weak to no synthetic interaction.
We also investigated whether the new mutations interacted synthetically with the
class C mutation trr-1(n3712) (Table 5). In trr-1(n3712) single mutants, P8.p adopts a
vulval cell fate at a low but detectable penetrance (CEOL and HORVITZ 2004). We
monitored synthetic interactions with trr- 1(n3712) for P(3-8).p but report synthetic effects
only for P8.p, as this cell is particularly sensitive to cell-fate transformation. lin-
65(n344 1), mep- 1(n3703), gap- 1(ga 133) and sli- 1(n3538) but not lin(n3628) and ark-
1(n3701) showed a strong synthetic interaction with trr- 1(n3712). In further tests, ark-
1(n3701) but not lin(n3628) interacted synthetically with the class C mutation mys-
1(n3681): ark- 1(n3701); mys- 1(n3681) double mutants had a strong synthetic P8.p
vulval fate defect (80%, n=41) as compared to ark- 1(n3701) (0%, n=33) and mys-
1(n3681) (8.3%, n=36) single mutants, whereas the P8.p vulval-fate defect of
lin(n3628); mys-1(n3681) (6.7%, n=30) double mutants was low, like that of lin(n3628)
(0%, n=37) and mys-1(n3681) single mutants. Why ark-1(n3701) interacted with one
class C mutation but not another is unclear. It is possible that the synthetic interaction
with ark-1(n3701) is sensitive to maternally-provided levels of class C synMuv activity
and mys-1(n3681), which can be maintained in homozygous strains, provided less
maternal activity than did trr- 1(n3712), which because of its recessive sterility requires
that homozygotes be generated from heterozygous parents.
216
Most of the new mutations interacted synthetically with class A and class C but
not with class B mutations, which indicates that these new mutations are neither class A
nor class C mutations. The synthetic interaction of lin(n3628) with class A but not class
B or class C mutations is unusual and will be discussed below.
Suppression of let-23 mutations: Are lin-65, lin(n3628), mep- 1, gap-1, sli-1
and ark- 1 class B synMuv genes? Neither in combination with class A mutations
(FERGUSON et al. 1987; Lu and HORVITZ 1998; THOMAS and HORVITZ 1999; CEOL and
HORVITZ 2001) nor on their own (Table 6) do class B mutations suppress the Vul
phenotype caused by strong loss-of-function let-23 receptor tyrosine kinase mutations.
However, previous studies showed that gap- I or sli- 1 mutations alone can suppress the
let-23 Vul phenotype (JONGEWARD et al. 1995; HAJNAL et al. 1997). Together these
findings distinguish gap- and sli-1 from class B synMuv genes and indicate that let-23
suppression may be used as a criterion in classifying synMuv mutations. We found that
mutations affecting lin-65, lin(n3628), mep- 1 and ark- I did not suppress the let-23 Vul
phenotype (Table 6), suggesting that these genes are not in the same class as gap- 1
and sli- 1.
Interactions with ark-1, gap-1 and sli-1 mutations: gap- I and sli- 1 mutations
interact synthetically to produce extra vulval cells (Table 7). Furthermore, an ark- 1
mutation interacts synthetically with these gap-1 and sli-1 mutations, suggesting that all
three genes act in parallel in regulating vulval cell fates. Similar synergism of an ark-1
mutation with gap- 1 and sli- 1 mutations was observed previously (HOPPER et al. 2000).
By contrast, we observed that the class B synMuv mutations lin-15B(n744) and lin-
35(n745) did not interact synthetically with gap-1 or sli- I mutations (Table 4). This lack
of synergism is likely not the result of using weak alleles, as the lin-15B(n744), lin-
35(n745), gap- (ga 133) and sli- 1(n3538) mutations used in these studies are strong
loss-of-function, and possibly null, mutations of their corresponding genes. These
results distinguish ark- from the class B genes lin- 15B and lin-35 Rb and suggest that
these class B genes do not act with 
ark- 1 in antagonizing Ras pathway activity. 
lin-65,
lin(n3628) and mep- 1 mutations also did not interact synthetically with gap-1(ga 133) or
217
sli- l(n3538) (Table 7), revealing a further similarity between lin-65, lin(n3628) and mep-
1 mutations and lin- 15B and lin-35 Rb class B synMuv mutations.
Molecular identification of lin-65: We mapped the synMuv gene lin-65to a
small interval between the C. elegans strain CB4856 polymorphisms Y71G12B. 17and
Y71G12B. 18 (Figure 1A). This interval contains four complete predicted genes, one of
which is a microRNA gene, and portions of three other genes, two of which overlap (C.
ELEGANSSEQUENCING CONSORTIUM 1998). We performed RNA-mediated interference
(RNAi) to determine if inactivation of any of the three complete, protein-encoding genes
would result in a synMuv phenotype. RNAi of Y71G12B.9 caused a Muv phenotype in a
lin-15A(n767) but not in a wild-type or lin-15B(n744) background (data not shown).
POULIN et al. independently found that RNAi of Y71G12B.9 caused a synMuv phenotype
(2005). We obtained six cDNAs (kindly provided by Yuji Kohara and coworkers) and
compared the sequences of these clones with genomic sequence to determine a gene
structure for Y71G12B.9 (Figure 1 B). One clone had an SL1 and two had an SL2
splice-leader sequence. The presence of an SL2 splice leader suggests that
Y71G12B.9 is a downstream gene in an operon (ZORIO et al. 1994). The predicted
initiator methionine codon of the SL2-spliced Y71G12B.9 cDNAs lies just downstream of
the trans-splice site. The open reading frame beginning with this initiator methionine
encodes a 728 amino acid protein (Figure 2). The SL1 trans-splice site is downstream
from that of SL2, and the single SL1-spliced cDNA lacks the initiator methionine
corresponding to the 728 amino acid predicted protein. The open reading frames
defined by the first three potential initiator methionine codons of the SL1-spliced cDNA
are all short ( :16 codons). If the fourth potential initiator methionine codon were used, a
691 amino acid protein lacking the first 37 amino acids of the 728 amino acid protein
described above would be synthesized. Expression under the control of the C. elegans
heat-shock promoters of a cDNA predicted to encode the 728 amino acid protein
rescued the Muv phenotype of lin-65 mutants: two transgenic lines of lin-65(n3441); lin-
15A(n767) mutants containing Phs:/in-65 transgenes were 0% (n=73) and 2.0% (n-49)
non-Muv without heat shock but were 
71% (n-68) and 67% (n=30) non-Muv,
respectively, following heat-shock treatment.
218
We determined the sequence of Y71G12B.9 in lin-65(n3441), lin-65(n3541) and
lin-65(n3543) mutants. lin-65(n3441) and lin-65(n3541) contain identical nonsense
mutations predicted to truncate the Y71G12B.9 protein after 533 of the 728 amino acids.
It is unlikely that lin-65(n344 1) and lin-65(n354 1) were caused by the same mutational
event, since they were isolated from independently mutagenized and screened P
0
animals. lin-65(n3543) contains a missense mutation that changes a polar serine
residue to a non-polar leucine (S720L). The map position, RNAi phenocopy, cDNA
rescue data as well as the mutant allele sequences indicate that Y71G12B.9 is lin-65.
The 728 amino acid LIN-65 protein is rich in acidic amino acids (Figure 2). Over
7% and 10% of the total number of 
amino acids are aspartates and glutamates,
respectively, and these acidic amino acids are found both in clusters and dispersed
throughout LIN-65. BLAST searches (ALTSCHUL et al. 1990) with LIN-65 identified
proteins from mammalian and other species that are similarly acid-rich. Because the
similarity between LIN-65 and these proteins is primarily limited to acidic residues and
not to specific protein domains (data not shown), it is difficult to predict whether these
proteins are functional orthologs of LIN-65. As described above, mutations in lin-65 and
lin-35 Rb show similar genetic interactions, suggesting that lin-65 is a class B synMuv
gene that acts in the lin-35 Rb pathway. Protein complexes purified from Drosophila
extracts and analogous to a class B synMuv complex (M.M.H. and H.R.H., unpublished
observations) have not been reported to contain LIN-65-like proteins (KORENJAK et al.
2004; LEWIS etal. 2004). It is possible that LIN-65 and LIN-65 orthologs act upstream of
class B synMuv and analogous complexes to promote complex activity or act
downstream as effectors of these complexes.
Sequences of synMuv mutations: We determined DNA sequences of 41
mutant synMuv genes identified in our screen; four of these mutant genes had two
distinct mutations (Table 8). The 41 
include all of the dpl- 1, efl- 1, let-418, 
lin-9, lin-13,
lin-36, lin-52, lin-53, lin-65, mep- 1, mys- 1, sli- 1 and trr- 1 alleles and one of two lin-35
alleles identified in our screen. Forty of 45 mutations are GC-to-AT transitions, which
are characteristic of EMS mutagenesis (ANDERSON 1995). Many of these mutations are
predicted to truncate the corresponding synMuv proteins. The truncations predicted by
219
efl- (n3639), let-418(n3719), lin-52(n3718) and trr-1(n3704) are particularly severe, and
the synMuv and sterile abnormalities caused by these mutations likely represent the null
phenotypes of these genes. In addition, we found missense mutations that disrupt
predicted functional domains of synMuv proteins. For example, n3536, n3626, n3629
and one of the two mutations of n3636 affect the ATPase/helicase domain of LET-418.
LET-418 is a member of the Mi-2 family of ATP-dependent chromatin remodeling
enzymes (SOLARI and AHRINGER 2000; VON ZELEWSKY et al. 2000), and the LET-418
missense mutations suggest that LET-418 function is dependent on ATP hydrolysis. At
least one mutation affecting the LIN-13 protein, n3642, is predicted to disrupt a
canonical zinc-finger motif. This missense mutation, along with those isolated
previously (THOMAS et al. 2003), indicate that at least some of the 24 LIN-13 zinc fingers
are important for LIN-13 synMuv activity. Missense mutations affecting other synMuv
proteins are not as easily linked to the disruption of predicted functional domains.
These mutations may provide useful starting points for identifying functional motifs
within synMuv proteins that are not predicted by sequence comparisons.
220
DISCUSSION
Frequency of mutant isolation: The rate at which we isolated synMuv
mutations was much higher than that observed in previous screens. Considering
screens that were conducted in class A synMuv mutant backgrounds, we recovered one
synMuv mutation per 109 haploid genomes screened as compared with one per 750
(FERGUSON and HORVITz 1989), one per 400 (THOMAS et al 2003) and one per 667
(THOMAS et al 2003) in previous screens. We believe the reasons for this difference are
three-fold. First, our screen design allowed the isolation of synMuv mutations that also
caused sterility. Numerous sterile synMuv mutants had been observed in previous
screens, but in general the mutations responsible were not recovered. Second, our
parental strain carried a strong class A mutation, lin-15A(n767). The penetrance of the
Muv phenotype of a synMuv strain is dependent on the combined strengths of the
individual synMuv mutations (C.J.C. and H.R.H., unpublished 
observations). Therefore,
even weak mutations could be identified in a strong synMuv background such as
lin- 15A(n767). Such weak mutations may not have been recovered in the three
previous screens described above, all of which were performed in partial loss-of-
function synMuv backgrounds. Third, by screening Petri plates with many F
2 
progeny
derived from a single F, animal, we observed many genotypically identical animals for
each haploid genome screened. Such screening can efficiently recover partially
penetrant synMuv mutations.
Of the 62 mutations described in this study, 24 caused recessive sterility. The 38
mutations that did not cause sterility were recovered at one mutation per 178 haploid
genomes screened, a frequency higher than that of previous screens. The difference in
the rate of recovery of non-sterile mutants is likely a consequence of the second and
third differences in screening described above.
Given that the average gene mutates to loss of function at a rate of about 5x10-4
under the conditions of EMS mutagenesis we used (BRENNER 1974; MENEELY and
HERMAN 1979; GREENWALD and HORVITZ, 1980), our observed rate of 10-2 suggests that
about 20 genes can mutate by loss of function to cause a synMuv phenotype in
combination with a class A synMuv mutation. Including the genes we identified in this
221
study, a total of 25 such genes have been described to date. Three or fewer alleles of
15 of these genes have been recovered in synMuv screens, indicating that screens for
such genes are not saturated.
Different synMuv gene classes likely act in parallel to antagonize let-60 Ras
pathway activity: Class A synMuv mutations synergize with class B mutations but not
with other class A mutations, whereas Class B synMuv mutations synergize with class A
synMuv mutations but not with other class B synMuv mutations. Such genetic behavior
led to the hypothesis that the A and B classes of synMuv genes encode components of
two functionally redundant pathways that negatively regulate vulval development
(FERGUSON and HORVITZ 1989). Consistent with this hypothesis, a subset of class B
synMuv gene products have been shown to physically interact and their homologs are
known function together in other organisms (Lu and HORVITZ 1998; CEOL and HORVITZ
2001; UNHAVAITHAYA et al 2002; KORENJAK et al. 2004; LEWIS et al. 2004).
Because we conducted our screen using a class A synMuv background, we
anticipated recovering mutations that affected class B synMuv genes. Indeed, 47 of the
62 mutations we isolated affected previously known and newly described class B
synMuv genes. However, we discovered that some new mutations define new classes
of synMuv genes. synMuv mutations previously were categorized by testing for
synergism with class A and class B mutations. From such tests we discovered that
some of our new mutations synthetically interacted with both class A and class B
mutations; such mutations defined the class C genes trr- 1 and mys- 1 (THIS STUDY and
CEOL and HORVITZ 2004). Other new mutations interacted like class B mutations in
these standard tests but were distinguished from class B mutations by additional tests.
For example, like class B mutations sli-1(n3538) synthetically interacted with class A but
not class B mutations yet, unlike class B mutations synthetically interacted with ark-1
and gap- I and suppresses the let-23 Vul phenotype. These results led us to adopt two
criteria when classifying synMuv mutations: (1) if two mutations synthetically interact to
cause a Muv phenotype, then they are in different classes, (2) if two mutations do not
synthetically interact but interact differently with other classes of synMuv mutations or
with let-23, then they are in different classes. Since we have found that interaction tests
222
with only class A and class B mutations 
are insufficient to classify some synMuv genes,
we suggest that previously described synMuv genes should be tested more extensively
to establish their classifications.
Using more extensive genetic interaction tests and additional criteria to interpret
these interactions, we define six classes of genes, synMuv A, synMuv B, synMuv C,
gap-1, sli-1 and ark-1, that seem to act in parallel to each other to negatively regulate
Ras-mediated vulval development (Table 9). Some of these classes, such as gap- 1, sli-
1 and ark- 1, likely interface directly with Ras pathway components (see below). The
point at which the synMuv A, synMuv B and synMuv C classes interface with Ras
signaling is unknown.
Different synMuv gene classes control distinct biochemical activities: A
synthetic genetic interaction implies functional redundancy between two sets of genes.
There are many possible mechanisms by which two sets of genes might appear
redundant. These possibilities include: (1) two sets of genes encode similar sets of
proteins with corresponding proteins of each set controlling the same biochemical
activity, and hence each set controls the same biological process; (2) two sets of genes
encode distinct sets of proteins with each set controlling distinct biochemical activities
but the same biological process; and (3) two sets of genes encode distinct sets of
proteins with each set regulating distinct but redundant biological processes.
The first of these mechanisms likely does not apply to the different classes of
synMuv genes, as no cloned gene in one synMuv class is similar to any gene of another
class. Furthermore, many of the cloned synMuv genes, including the class A gene lin-
15A, the class B gene lin-35 Rb, the class 
C genes trr- 1 and epc- 1, and ark- I and 
sli- 1,
encode the sole C. elegans member of their respective gene families.
The redundancy exhibited among sli- 1, gap- 1 and ark- I likely exemplifies the
second mechanism. sli- 1, ark- I and gap-1 are thought to directly down-regulate Ras
pathway activity, and, as might be predicted based on their synthetic interactions, each
is proposed to act upon a different Ras pathway component. sli- I encodes a homolog
of the c-Cbl proto-oncoprotein, which is thought to down-regulate receptor tyrosine
kinase levels though ubiquitin-mediated degradation (YOON et al 1995; LEVKOWITZ et al.
223
1999). ark- I encodes a protein that interacts with the SEM-5 SH2/SH3 adaptor protein
and is predicted to be a cytoplasmic tyrosine kinase (HOPPER et al. 2000). Since sem-5
acts downstream of the let-23 receptor tyrosine kinase, ark- 1 is proposed to inhibit let-
60 Ras signaling downstream of let-23. gap- 1 is a member of the GTPase-activating
protein (GAP) family (HAJNAL et al. 1997). GAPs enhance the catalytic function of Ras
family GTPases such as let-60 Ras, thereby facilitating the switch from active GTP-
bound to inactive GDP-bound Ras. The genetic suppression of let-23(sy97) by and the
molecular identities of sli- 1 and gap- 1 support their action downstream of the let-23
RTK. Although ark-1 mutations do not suppress let-23(sy97), HOPPER et al. (2000)
found that an ark-1 mutation suppressed the Vul phenotypes caused by weaker let-23
mutations and by sem-5 mutations. Based on these suppression data and on the
molecular data described above, these authors argued that ark- I acts downstream of
let-23, although its negative regulation of the let-60 pathway may not be as great as that
of sli- 1 or gap- 1. The redundancy displayed by sli- 1, gap- 1 and ark- 1 suggests that a
mutation affecting one of these genes only mildly affects Ras pathway activity whereas
mutations affecting two genes elevate pathway activity to a level that inappropriately
transforms vulval cell fates. That these genes converge on the same signaling pathway
implies that they regulate the same biological process.
The class A, B and C synMuv genes may or may not act similarly. It is possible
that these classes act on components of the let-60 Ras pathway. Since at least some
class A and B synMuv genes are thought to act in the hypodermis, an effect on let-60
Ras signaling is likely indirect (HERMAN and HEDGECOCK 1990; HEDGECOCK and HERMAN
1995; MYERS and GREENWALD 2005). Alternatively, as in the case of the third
mechanism, these classes may regulate entirely distinct biological processes. For
example, the class B genes, some of which encode components of a putative histone
deacetylase complex, may repress transcription of genes that indirectly promote P(3-
8).p cell division. By contrast, the class C genes, which encode components of a
putative histone acetyltransferase complex, 
may activate the transcription of genes,
different from those targeted by class B genes, that promote differentiation of P(3-8).p
descendants into hypodermal and not vulval cells. A better understanding of synMuv
224
target genes should help to resolve whether different synMuv classes regulate the same
or distinct biological activities.
225
ACKNOWLEDGMENTS
We thank Beth Castor, Na An and Andrew Hellman for expert technical
assistance and Erik Andersen and Adam Saffer for critical reading of this manuscript.
We thank Yuji Kohara for providing cDNA clones, Mike Boxem and Sander van den
Heuvel for providing pMB1 and pMB7, Min Han for providing pTG96 and Neil Hopper
and Paul Sternberg for providing the ark- 1(sy247) strain. Some of the strains used in
this work were provided by Theresa Stiernagle of the Caenorhabditis Genetics Center,
which is supported by the National Institutes of Health National Center for Research
Resources. This work was supported by National Institutes of Health grant GM24663 to
H.R.H., a Koch Graduate Fellowship to C.J.C. and a Howard Hughes Medical Institute
Predoctoral Fellowship to M.M.H. H.R.H. is the David H. Koch Professor of Biology at
M.I.T. and an Investigator of the Howard Hughes Medical Institute.
226
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234
TABLE 1
Phenotypes of synMuv mutant strains
Percent Muv (n)
Genotype 150 C 200 C Additional abnormalities
ark- 1(n3524); lin- 15A(n 767)
ark- 1(n3701); lin-15A(n767)
dpl- 1(n3643); lin- 15A(n767)a
efl- 1(n3639), lin-15A(n767)a
gap- 1(n3535) lin- 15A(n 767)
let-418(n3536); lin-15A(n767)
let-418(n3626); lin- 15A(n767)
let-4 18(n3629); lin- 15A(n767)
let-4 18(n3634); lin-15A(n767)
let-4 18(n3635); lin- 15A(n767)
let-4 18(n3636); lin-15A(n767)
let-4 18(n3 719); lin-15A(n767)
lin-9(n363 1); lin-15A(n767)
lin-9(n3675); lin-15A(n767)
0(251)
12 (190)
99 (154)
93 (74)
1(143)
0 (201)
2 (62)
0 (52)
0 (87)
0 (76)
0 (77)
0(101)
100 (42)
43 (166)
80(171)
95(160)
100 (252)
100 (78)
50 (236)
55 (183)
97 (76)
86 (58)
92 (48)
71 (70)
92 (78)
100 (60)
100 (72)
100 (105)
Ste
hs Ste
Ste
Ste
Ste
Ste
Ste
Ste
Ste
235
lin-9(n3767), lin- 15A (n767)
lin-13(n3642); lin- 15A(n 767)
lin- 13(n3673); lin-15A(n 767)
lin- 13(n3674); lin- 15A (n 767)
lin- 13(n3726); lin- 15A(n 767)
lin-15B(n3436) lin-15A(n767)
lin- 15B(n3676) lin- 15A (n 767)
lin- 15B(n3677) lin- 15A(n 767)
lin-15B(n3711) lin-15A(n767)
lin-15B(n3 760) lin-15A (n 767)
lin- 15B(n3 762) lin- 15A(n 767)
lin-15B(n3 764) lin-15A(n767)
lin- 15B(n3 766) lin- 15A (n 76 7)
lin- 15B(n3 768) lin-15A(n 767)
lin-15B(n3772) lin-15A(n767)
lin-35(n3438); lin- 15A(n 767)
lin-35(n3763), lin- 15A(n 767)
lin-36(n3671); lin-15A(n 767)
100 (67) 100 (56)
3 (60)
61 (145)
78(131)
31 (225)
100 (193)
18(167)
99 (111)
100 (186)
32(171)
63 (113)
96 (232)
55 (132)
80 (159)
100 (220)
100 (153)
100 (108)
65(191)
100 (63)
97(129)
100 (191)
99 (149)
100 (212)
72(130)
100 (122)
100 (156)
100 (150)
97 (116)
100 (199)
100 (173)
100 (302)
100 (191)
100 (126)
100(160)
100 (151)
Ste
Ste
hs Ste
hs Ste
partial Ste at 200C, Rup
partial Ste at 200C, Rup
236
lin-36(n3672); lin- 15A(n 767)
lin-36(n3 765); lin- 15A(n 767)
lin-52(n3718); lin- 15A(n767)b
lin-53(n3448); lin-15A(n 767)
lin-53(n3521); lin-15A(n 767)
lin-53(n3622); lin-15A(n 767)
lin-53(n3623); lin- 15A(n 767)
lin-65(n344 1); lin-15A(n 767)
lin-65(n3541); lin- 15A(n 767)
lin-65(n3543), lin- 15A(n 767)
lin(n3628); lin-15A(n 767)
lin(n3542) lin-15A(n767)
mep- 1(n3680), lin- 15A(n 767)
mep- 1(n3 702); lin- 15A(n 767)
mep- 1(n3703); lin- 15A(n 767)
mys- 1(n368 1); lin- 15A(n767)C
sli- 1(n3538) lin- 15A(n 767)
sli- 1(n3544) lin- 15A(n 767)
98 (198) 100(178)
0 (184)
100 (41)
67 (130)
100 (34)
85 (61)
24 (55)
80 (165)
79 (242)
85 (177)
3 (103)
0(127)
5(122)
30 (61)
25 (72)
0(214)
4 (138)
5(153)
37 (202)
100 (82)
100 (211)
100(125)
100 (66)
100 (51)
99 (195)
98(137)
100 (121)
84 (188)
35(218)
97(105)
100 (141)
100 (107)
72 (192)
90 (173)
80 (265)
Ste
partial Ste at 20'C
partial Ste at 200C
Ste
Ste
hs Ste
Ste
Ste
cs embryonic lethality
237
5 (80) 88(148)
trr-1(n3630); lin-15A(n767)c 3(131) 85 (212) Ste, Gro
trr-1(n3637); lin-15A(n767)c 1 (92) 80 (200) Ste, Gro
trr-1(n3704); lin- 15A(n767)c 3 (96) 79 (244) Ste, Gro
trr-1(n3708); lin-15A(n767)c 2 (151) 84 (228) Ste, Gro
trr- I (n3709); lin- 15A(n767)c 1 (97) 77 (154) Ste, Gro
trr-1(n3712); lin-15A(n767)c 6 (121) 77 (192) Ste, Gro
The penetrance of the Muv phenotype was determined after synMuv mutant
strains grew at the indicated temperature for two or more generations. For most
strains for which a fully penetrant sterile phenotype was associated with the Muv
phenotype, we scored the penetrance of the Muv phenotype by examining sterile
progeny of heterozygous mutant parents. For trr-1 mutant strains, we scored the
penetrance of the Muv phenotype by examining non-Gfp progeny of trr- 1 /
mlnl[dpy-10O(e128)mls14]; lin-15A(n767) heterozygous parents. All strains were
backcrossed to lin- 15A(n767) twice prior to phenotypic characterization. In
addition to the phenotypes described above, many of the strains exhibited heat-
sensitive inviability as a consequence of rupture and/or general sickness. Ste,
sterile; Gro, growth rate abnormal; Rup, rupture at the vulva; cs, cold-sensitive;
hs, heat-sensitive. The characterization of some of these strains was previously
238
sli- 1(n3683) lin-15A(n767) cs embryonic lethality
described by: a(CEOL and HORVITZ 2001); b(THOMAS et al. 2003); c(CEOL and
HORVITZ 2004).
239
TABLE 2
Three- and four-factor crosses
Phenotype Genotype 
of selected
of selected recombinants (with respect to
Gene Genotype of heterozygote recombinants unselected markers)
+ lin-65 + /bli-3 + lin-17; lin-15A(n767)
bli-3 + lin-65/+ spe-15 +; lin-15A(n767)
+ lin-65 lin-17/spe-15+ +; lin-15A(n767)
bli-3+ + + lin-65+ /+ Y73E7.2 Y71G12B.2 Y71G12B. 17
+ Y71G12B. 18; lin-15A(n767)
Lin-17
Muv
Lin-17
Muv
Muv
Muv
Muv
+ lin-65+ ++ lin-17/Y71G12B. 17+ Y71G12B. 18
Y71G12B.27 MO 1D7.2 +; lin-15A(n767) Lin-17
Lin-17
Lin-17
Lin-17
9/19 lin-65 /+
10/18 spe-15 /+
11/11 spe-15 /+
4/30 Y73E72 /+
2/30 Y71G12B.2/+
1/30 Y71G12B. 17/+
0/30 Y71G12B. 18 /+
17/23 MOID7.2/+
18/23 Y71G12B.27/+
21/23 Y71G12B. 18 /+
23/23 Y71G12B. 17/+
lin(n3542) + + lin(n3542) lin-15A(n767) /unc- 10 dpy-6 + lin-15A(n767)
+ lin(n3542) + lin- 15A(n767) /dpy-6 + unc-9 lin-15A(n767)
lin(n3628) lin(n3628) + + /+ dpy-5 unc- 13; lin-15A(n767)
Unc
Unc
Dpy
Unc
8/8 lin(n3542) /+
4/40 lin(n3542) /+
0/6 lin(n3628) /+
6/6 lin(n3628) /+
240
lin-65
+ lin(n3628) + /unc-11 + dpy-5; lin-15A(n767)
unc-11 + + lin(n3628) /+ unc-73 lin-44 +; lin-15A(n767)
+ + lin(n3628) dpy-5 /unc-73 lin-44 + +; lin-15A(n767)
lin(n3628) + dpy-5 /+ unc-38 +; lin-15A(n767)
unc- 11 lin(n3628) + /+ + unc-38; lin-15A(n767)
+ mep-1 + /unc-5+ dpy-20; lin-15A(n767)
mep-1 + + /+ dpy-20 unc-30; lin-15A(n767)
+ + mep-1 + /unc-24 mec-3 + dpy-20; lin-15A(n767)
Dpy
Muv
Muv
Muv
Muv
Unc
Dpy
Dpy
Unc
5/11 lin(n3628) /+
3/9 unc-73 lin-44/+ +
0/21 unc-73 lin-44 /+ +
3/7 unc-38 /+
0/9 unc-38 /+
56/57 mep-1 /+
2/61 mep-1 /+
0/51 mep-1/+
58/58 mep-1 /+
Unc Mec 10/12 mep-1 /+
Unc 17/17 mep-1 /+
Mec Dpy 0/8 mep-1 /+
Dpy 2/8 mep-1 /+
+ mep-1 dpy-20 + /Iin-3 + + unc-22; lin-15A(n767) Dpy 5/5 lin-3 /+
Vul 3/10 mep-I /+
+ + mep-1+ /mec-3 sem-3 + dpy-20; lin-15A(n767) Mec
Dpy
mep-1
241
17/17 mep-1/+
6/13 mep-1 /+
Unc 1/11 lin(n3628)/+
mys- I1 + mys-1 + /unc-46 + dpy- 1; lin-15A
Dpy 7/11 mys-1 /+
+ rol-6 + trr-1 /dpy-lO + unc-4 +; lin-15A(n767)
+ trr-1 + /dpy-lO+ rol-i; lin-15A(n767)
+ + trr-1 /dpy-1O unc-53 +; lin-15A(n767)
+ trr-1 + /unc-53+ rol-1; lin-15A(n767)
Rol 3/14 unc-4 /+
Dpy 3/3 trr-1 /+
Unc 0/8 trr-1 /+
Rol 9/20 trr- 1/+
Unc
Unc
0/17 trr-1 /+
7/10 trr- 1/+
+ trr-1 + rol-1 /unc-4 + mex-1 +; lin-15A(n767)
Rol 7/10 trr- I/+
Rol 12/14 mex-1/+
Three- and four-factor crosses were performed using standard methods (BRENNER
1974). We mapped lin-65using the Y73E7.2, 
Y71G12B.2, Y71G12B. 17,
Y71G12B. 18, Y71G12B.27, MO1D7.2 DNA sequence polymorphisms present in the
CB4856 strain.
242
trr-1
Unc 3/7 mys-1/+
TABLE 3
Deficiency heterozygote mapping
Gene Genotype of heterozygote Phenotype of heterozygote
mep-1 mep-1/ sDf63 unc-31; lin-15A(n767) /+ Pvl Ste
mep-1 / sDf62 unc-31; lin-15A(n767) /+ Pvl Ste
mep-1/ sDflO; lin-15A(n767) /+ WT
trr- I1 rol-6 trr- 1 /mnDf57; lin- 15A(n767) WT
rol-6 trr- 1 /unc-4 mnDf90; lin-15A(n767) WT
rol-6 trr- 1 /mnDf29; lin-15A(n767) WT
trr-1 / unc-4 mnDf87; lin-15A(n767) Muv
Deficiency heterozygotes were constructed as described in MATERIALS AND METHODS.
WT, wild type; Pvl, protruding vulva; Ste, sterile.
243
v
V
C)
?D
v
C
V
cD
v
Oc
co
'3)
:3
p
02
02
(D
0
:E
3
02
>
a.
o m
Xh
5- .r
02
0
a)
ca
Oi
02
Cn
C
02
0
02
N
CD.
(0.
CD
O
N
lo(0
0
Co
(5
CD
0
CD
0
-5
0
(a
0
-h
CD
-o
cD
0
N
0
0
-D
CD
CD
CD
cD
O
0
0
CD
CD
N
0
Co
0
CD
-I
CD
e0
CD3
O
O
N
Co
CO
-I
0.
o
O
cD
0
(a
CD
0
9b
m
I-
U)
z
o
m
-H
I
0
0
c
0
3
cD
o
2
(D
Co
CD
3
CO
0
o
CD
(o
CD
CD
Co
N
2
0
Co
z
C3
* z
C3
CD
()
CD
0
3
C-
o-
O
0
m
O
r
I
O
N
rN
0C
CO
TABLE 5
Interactions of new mutations with the class C synMuv mutation trr-1(n3712)
Percentage of animals with P8.p vulval fate
trr- 1 (+) trr- 1(n3712)
0 (many) 13 (89)
a
New mutation
Class A
lin-65(n344 1)
lin(n3628)
mep- 1(n3703)
ark-1(n3701)
gap- 1(ga 133)
sli- 1(n3538)
lin-15A(n767)
lin-38(n751)
lin- 15B(n 744)
lin-35(n 745)
Class B
0 (35) 45 (31)
0(37)
2.5 (40)
0 (33)
3.1 (32)
0 (25)
4.2 (24)
Letb
13(24)
37 (38)
32 (37)
0(24)
0 (27)
0 (20)
a
0 (48)
a
28 (54)
36(14)
50 (38)a
64 (41)
a
247
Double mutant strains were constructed as described in MATERIALS AND METHODS.
mep- 1(n3703) homozygotes were recognized as the non-Unc progeny of mep-
1(n3703)/nTI n754 heterozygous parents or the sterile progeny of mep- 1(n3703)
/ dpy-20(e 1282) unc-30(e 191) heterozygous parents. trr- 1(n3712) homozygotes
were recognized as the non-Gfp progeny of trr-1(n3712) /mlnl[dpy-10(e 128)
mls14] heterozygous parents.
a These data 
are from Table 
1 of CEOL 
and HORVITZ 
(2004).
b We interpret this synthetic lethality as indicating redundancy between mep- 1
and trr- 1.
248
TABLE 6
Suppression of the let-23 vulvaless phenotype
Ave. no. vulval fates (?SE, n)
let-23(+) let-23(sy97)
3.0 (many) 0 (?0, 36)
New mutation
Class A
lin-65(n344 1)
lin(n3628)
mep-1(n3703)
ark- 1(n3701)
gap- 1(ga 133)
sli- 1(n3538)
lin- 15A(n767)
lin-38(n 751)
3.0 (+0, 35)
a
0 (+0, 30)
3.0 (+0, 37)a
3.01 (?0.01, 40)
a
3.0 (?0, 33)a
3.02 (?0.02, 32)"
3.0 (?0, 25)a
3.0 (?0, 24)b
3.0 (+0, 27) b
Let d
Let
0.10 (?0.05, 34)
3.0 (+0, 26)
3.0 (+0, 31)
0 (?0, 21)
ND
lin- 15B(n744)
lin-35(n 745)
3.0 (?0, 20)"
3.0 (?0, 48)a
0.23 (?0.08, 26)
0.20 (?0.06, 38)
249
Class B
mys-1(n3681) 3.06 (?0.03, 36)c
trr-1(n3712) 3.10 (?0.03, 89)b 0.28 (?0.07, 46)
mep- 1(n3703) homozygotes were recognized as the non-Unc progeny of mep-
1(n3703)/nT1 n754 heterozygous parents or the sterile progeny of mep- 1(n3703)
/dpy-20(e 1282) unc-30(e 191) heterozygous parents. trr- 1(n3712) homozygotes
were recognized as the non-Gfp progeny of trr- 1(n3712) /mln [dpy- 10(e 128)
mls 14] heterozygous parents. let-23(sy97) was marked with unc-4(e 120), and
let-23(sy97) homozygotes were recognized as the Unc non-Gfp progeny of let-
23(sy97) unc-4(e 120) /mln l[dpy- 10(e128) 
mls14] heterozygous parents. ND,
not determined because of linkage of these mutations.
a These 
data 
are from 
TABLE 
1.
b These data 
are from Table 
1 of CEOL 
and HORVITZ 
(2004).
0 These data are from Table 3 of CEOL and HORVITZ (2004).
d Because of 
the lethality of 
these animals, 
we measured 
the abilities 
of
lin(n3628) and mep- 1(n3703) to suppress the Vul phenotype caused by sylO, a
let-23 allele that is weaker than sy97. lin(n3628) and mep- 1(n3703) were unable
to suppress the Vul phenotype of let-23(sy10): lin(n3628); let-23(sy10) double
mutants averaged 0.11 vulval fates (n=27), let-23(sy10); mep- 1(n3703) double
mutants averaged 0.06 vulval fates (n=24) and let-23(sy10) single mutants
averaged 0.14 vulval fates (n=21).
250
Class C 1.47 (?0.15, 31)
TABLE 7
Interactions of new mutations with gap-1 and sli-1 mutations
Double mutant with ap- 1 (ga 133)
% >3 vulval Ave. no. vulval
fates (n) fates (+SE)
Double mutant with sli- 1(n3538)
% >3 vulval Ave. no. vulval
fates (n) fates (?SE)
New mutation lin-65(n344 1)
lin(n3628)
mep- 1(n3703)
ark- 1(n3701)
0 (31)
6.1 (33)
8.3 (36)
83 (40)
3.0 (?0)
3.05 (?0.03)
3.07 (?0.04)
4.18 (?0.14)
gap- 1(ga 133)
0 (34)
0 (36)
5.6 (36)
48 (29)
46 (35)
3.0 (+0)
3.0 (?0)
3.03 (?0.02)
3.48 (+0.11)
3.51 (?0.11)
Double mutant strains were constructed as described in MATERIALS AND METHODS.
mep- 1(n3703) homozygotes were recognized as the sterile progeny of 
mep-
1(n3703)/dpy-20(e1282) unc-30(e191) heterozygous parents. trr- 1(n3712)
homozygotes were recognized as the non-Gfp progeny of trr- 1(n3712) /
mln 1[dpy- 10O(e 128) mls 14] heterozygous parents.
251
TABLE 8
Sequences of new mutations of class B and C synMuv proteins a
A. Features of synMuv proteins
ClassProtein
DPL-1
EFL-1
252
--
LET-41 8
LIN-9
B
B
B
B
B
B
B
C
sli- 1
LIN-13
LIN-35
LIN-36
LIN-52
LIN-53
LIN-65
MEP-1
MYS-1
SLI-1
TRR-1
No. amino acids
598
342
1829
LIN-9L: 644
LIN-9S: 642
2248
961
962
161
417
728
853
458
582
Protein similarities and domainsb
Similar to DP family transcription factors; contains
DNA- and E2F-binding domains
Similar to E2F family transcription factors; contains
DNA-, DP- and Rb-binding domains
Similar to Mi-2 family ATP-dependent chromatin
remodeling enzymes; contains chromodomains, PHD
finger motifs and a helicase domaino
Similar to Drosophila Mipl30 DNA replication and Aly
cell cycle regulators and mammalian proteins of
unknown function
24 predicted Zn-finger motifs
Similar to Retinoblastoma (pRb) family transcriptional
regulators; contains "pocket" interaction domain
THAP domain, C/H-rich and Q-rich regions
Similar to Drosophila and mammalian proteins of
unknown function
Similar to Drosophila p55, mammalian RbAp48
subunits of chromatin remodeling and histone
deacetylase complexes; contains WD repeats
Acid-rich
Six Zn finger motifs
Similar to MYST family histone acetyltransferases;
contains chromodomain and acetyltransferase domain
Similar to Cbl family ubiquitination-promoting 
proteins;
Contains SH2 domain and RING finger motif
Similar to mammalian TRRAP transcriptional regulator
B. Allele sequences
Substitution, splice Domain affected
Wild-type Mutant site change or by missense
Mutation sequence sequence aberration mutation
dpl- 1(n3643)e
efl- 1(n3639)'
let-418(n3536)
let-418(n3626)
let-418(n3629)
let-418(n3634)
let-418(n3635)
let-418(n3636)
let-418(n3719)
lin-9(n363 1)
TAT
GGC
CAA
CCT
GGT
TCC
TGG
CAG
ACT
TGG
TGG
CAA
TAA
CGC
TAA
CTT
AGT
TTC
TAG
TAG
TCT
TGA
TAG
TAA
Y341ochre
G533R unknown
Q175ochre
P675L
G1006S
S925F
helicase/ATPase
helicase/ATPase
helicase/ATPase
W1128amber
Q1594amber
T807S helicase/ATPase
W1329opal
W295amber
LIN-9L: Q594ochre
LIN-9S: Q592ochre
LIN-9L: D305N
LIN-9S: D303N
lin-9(n3675) GAT AAT unknown
unknown
253
LIN-9L: Q509amber
LIN-9S: Q507amber
lin-13(n3642)
lin-13(n3673)
lin-13(n3674)
lin- 13(n3 726)
lin-35(n3763)
g
CAT
CAG
CGA
GGA
GCA
TAT
TAG
TGA
GAA
GTA
H832Y
Q1988amber
R 1250opal
G229E
A555V
TTG AAA AAG TTG AAA AAA G
K594frameshift and
truncation after 611 a.a.
lin-36(n3671)
lin-36(n3672)
lin-36(n3765)
h
lin-52(n3718)'
lin-53(n3448)
lin-53(n3521)
lin-53(n3622)
lin-53(n3623)
CAT
GAA
CAG
GCT
CAG
AGT
GAA
AAG/gtatgtgt
TGG
H284P
E424K
CCT
AAA
TAG
GTT
TAG
ATT
AAA
C/H-rich region
unknown
Q467amber
A242V C/H-rich region
Q31 amber
S3841
E174K
AAG/atatgtgt
TAG
Exon 1 donor
W337amber
254
lin-9(n3767) CAG TAG
Zn finger
unknown
Pocket
WD repeat
WD repeat
TGA W534amber
lin-65(n354 1)
lin-65(n3543)
mep-1(n3680)
mep- (n3702)
mep-1(n3703)
mys- 1(n368 1y
sli- 1(n3538)
sli- 1(n3544)
sli- 1(n3683)
trr- 1(n3630)k
trr- 1(n3637)k
trr- 1(n3704)k
trr- 1(n3 708)k
trr- 1(n3709)k
trr- 1(n3712)k
TGG
TCG
AGT
CAG
CTT/gtaagttt
GGA
TCA
ttttccag/AAA
ttttttag/GAT
TGG
CAG
CAA
CGA
CGA
TGG
TGA
TTG
AAT
TAG
CTT/ataagttt
AGA
TTA
ttttccaa/AAA
ttttttaa/GAT
TAG
TAG
TAA
TGA
TGA
TAG
W534amber
S720L
S309N
Q706amber
Exon 3 donor
G341 R
S305L
acetyltransferase
SH2
Exon 6 acceptor
Exon 4 acceptor
W2064amber
Q3444amber
Q694ochre
R 12480pal
R2550opal
W2505amber
255
unknown
unknown
lin-65(n3441) TGG
In the "Wild-type sequence" and "Mutant sequence" columns, exon and intron
sequences are denoted by uppercase and lowercase script, respectively.
Nucleotides altered by the mutation are underlined.
a The synMuv proteins described in this table are limited to those for which we
obtained mutant allele sequence; this table is not a comprehensive listing of
synMuv proteins.
b Molecular descriptions 
of the proteins 
listed were 
obtained from 
the following
sources: DPL-1, EFL-1 (CEOL and HORVITZ 2001; PAGE et al. 2001); LET-418
(SOLARI and AHRINGER 2000; VON ZELEWSKY et al. 2000); LIN-9 (BEITEL et al.
2000); LIN-13 (MELENDEZ and GREENWALD 2000); LIN-35, LIN-53 (Lu and
HORVITZ 1998); LIN-36 (THOMAS and HORVITZ 1999; REDDY and VILLENEUVE
2004); LIN-52 (THOMAS etal. 2003); MEP-1 (BELFIORE etal. 2002); SLI-1 (YOON
etal. 1995); MYS-1, TRR-1 (CEOL and HORVITZ 2004).
c The predicted LET-418 protein contains a sequence that is annotated as a
helicase domain (see www.wormbase.org). This domain was originally identified
in helicases but has since been found in non-helicase proteins. Many of these
proteins share a common ATPase activity, and this domain contains residues
that are important for ATP binding and hydrolysis.
d Because of alternative 
splicing, trr- 
1 encodes proteins 
that may range 
in length
between 4054 and 4064 amino acids (CEOL and HORVITZ 2004).
e These data 
are from Figure 
1 of CEOL and 
HORVITZ (2001).
'These data are from Figure 4 of CEOL and HORVITZ (2001).
256
9 The adenosine inserted by the lin-35(n3763) frameshift mutation is not
underlined, because it is unclear which adenosine in the adenosine repeat was
inserted.
h In addition to the 
missense mutation 
described, we 
found an additional 
mutation
associated with lin-36(n3765). This mutation, AG/gtaagaagaaaagc to
AG/gtaagaagaaaagt, is present in the third intron of lin-36 and creates a possible
splice-donor sequence. If this splice-donor were used, an in-frame ochre (TAA)
stop codon would be encountered, truncating the LIN-36 protein after 261 amino
acids.
'These data are from Figure 3 of THOMAS et al (2003).
' These data are from Figure 2 of CEOL and HORVITZ (2004).
kThese data are from Figure 1 of CEOL and HORVITZ (2004).
257
R)
(0
00
0
:3
- -s
CC Z3
C CC 
C C
0 0 0 0 0
=3 :3
< < < <
:3
0 0
0 0
< < < < < <
3 :3
0 0
33 :3
0 0
S4z 3
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Figure 1. Molecular cloning of lin-65
A) The genetic map location of lin-65 on linkage group I (top) and the physical
map interval between the C. elegans strain CB4856 polymorphisms Y71G12B. 17
and Y71G12B. 18 and including lin-65 (bottom).
B) iin-65 gene structure as derived from cDNA and genomic sequences. Shaded
boxes indicate coding sequence and an open box indicates the 3' untranslated
region (lin-65 transcripts also contain a 5' untranslated region that is too small to
be viewed in this representation). Predicted translation initiation and termination
codons and the poly(A) tail are shown. Arrows above indicate the positions of
the lin-65(n3441), iin-65(n3541) and lin-65(n3543) mutations. The fourth exon of
the cDNA yk1279h11 is smaller than that of the other five lin-65 cDNAs (the end
of the yk1 279h11 fourth exon is indicated by a dashed line). The use of an
alternative splice donor may have created this shorter fourth exon. However, if
the end of the yk1279h11 fourth exon were the site of alternative splicing, a CA
and not the typical GT splice donor would have been used. In addition, the end
of the yk1279h11-specific fourth exon and the beginning of the fifth exon encode
multiple glutamine residues and are highly similar in DNA sequence (see Figure
2). The intervening sequence between two regions of highly similar sequence
can be lost because of recombination in bacteria (ROBINETT et al. 1996). For
these reasons we speculate that the apparent alternative splice site at the end of
the fourth exon in yk1279h11 may be artifactual and have resulted from an error
during the generation or maintenance of this cDNA clone. In support of this
possibility: 1) we failed to amplify a shorter-than-wild-type yk1279h11 product in a
high stringency RT-PCR using oligonucleotide primers that flank the putative
alternative splice junction, and 2) we failed to amplify any RT-PCR products
when an oligonucleotide spanning the putative yk1279h1 1 alternative splice
junction was used in a PCR.
261
Figure 1
l- in-65 -I
Nf
lin-65
,J)~
n3441, n3541
W534AMBER
ATG
I /ATG
n3543
S720L
I TGA
SI , A,.
1 a~i.iL]*'`
\V V 'V/ 7 3'
A) LGI
U
1 m.u.
B)
50 kb
CIQV
3
r
c
c
0.5 kb
s
SL5' SL1
SL2 SL1
Figure 2. lin-65cDNA sequence
lin-65 cDNA sequence indicating differences among individual lin-65 cDNAs.
SL1 and SL2 splice-leader sequences are italicized. The SL1 leader, as
observed with one cDNA, is spliced two nucleotides downstream of the site at
which the SL2 leader is spliced, as observed with two independently-derived
cDNAs. Intron positions are indicated by carats. The translation termination
codon is underlined. Sites of alternative polyadenylation are indicated with
closed arrowheads. The predicted LIN-65 protein is shown beneath. The SL2-
spliced cDNAs are predicted to encode a 728 amino acid protein. The SL1-
spliced cDNA cannot encode the predicted initiator methionine of the 728 amino
acid protein; it may use the underlined methionine codon to initiate synthesis of a
691 amino acid protein. The alternatively spliced cDNA yk1279h1 1 is predicted
to encode a protein lacking amino acids 421 through 481 of the 728 amino acid
protein, although as described in the legend to Figure 1, the alternative splicing of
yk1279h11 is likely to be artifactual. The site at which the putative fourth exon in
ykl 279h1 1 ends is indicated with an open arrowhead. This end is juxtaposed to
the beginning of the fifth exon to give a CAGCAACAA/CAACAAAAT junction
sequence.
263
Figure 2
SL1 GTTTAA TACCCAAGTTTGG TGTCAGAAGTAATCGACGAAAGTATCTTAAATACAGAAGCTTCAGATGATCCAATACCTCCATTAAATGATGATCAGATTGCTGAGCTT 
113
SL2 GGTTTTAA.CCCATACTCAAGAA S E V I D E S I L N T E A S D D P I P P L N D D Q I A E L 30
SA
TTGGGTGAAGATGGAGAAATTATGGAGATAACTGAGCAGAAAGACGAATCAGATGATGTGGTGATGCTGGACGACGATGATGACGACACTCCGGAACCGATTCTCGTGATTGATATGGAT 233
L G E D G E I M E I T E Q K D E S D D V V M L D D D D D D T P E P I L V I D M D 70
GAGGATGAGGATGTTACTACAGATGGTCCTGAATCTCAGGAAGAGCTGGCTGCAGATGCTCCTCCGGCTCCAGGAGCTCCAGAAGCTTCAGCTCCAGCTCAAGAAGCCTCAGAAGCTTCAGCT 353
E D E D V T T D G P E S Q E E L A A D A P A P G A P E A S A P A Q E A S E A S A 110
CCGGATCAAGAAGCTCCAGAAGTGTAGGATGTTCCGGATTCTTCGGGAGCTCCAGATGCTTCAGCTCAGGCTTCAGAGGCTTCTGATGCTTCAGCTCCAGAAGTTCCAGGATCTACAGAA 473
P D Q E A P E V Q D V P D S S G A P D A S A Q A S E A S D A S A P E V P G S T E 150
GCTCAGGATGCTCAGGATGTTCCGGATTCTTTGGGAGCTTCAGATGCTTCAGCTCAAGAAATTICCCCCAGAGAAGCTCCAAGCTCCAGAAATCGCCGCTGAAATCGACGAAGAA 593
A Q D A Q D V P D S L G A S D A S A Q E I P E A P E A P E A P E I A A E I D E E 190
GTGCTGCTCGCCGAGCAAAATGGAGTTTTGGACGAAGGATTTGATGAGACTGACGATATTATCATAGAAGAAGAAGAA GCTGTAGAAGAAGCTGAAGCCGTGGAGCCACCAATTAACACTGAA 713
V L L A E Q N G V L D E G F D E T D D I I I E E E A V E E A E A V E P P I N T E 230
AATCAGGAAAACGCGCTGGAAATGCTCGAAGAGCGCCTCAAGAAGAATGAAGAAAAGGAAATTGTGGAGAAAAGTGATGTGAAGCCAGAGGATGAAGATATTATACATATGGAGACGGAT 833
N Q E N A L E M L E E R L K K N E E K E I V E K S D V K P E D E D I I H M E T D 270
A
TCAGTTGAAACGTCAAGCCGCAAACGTACTGGCGGAGCCACAAGTCCGCGGAGCCCGGCTCAAAAACGACCAAAACGACGTGTTCAAACGTTATTAAAGATGCGTCAGAATGCAATTGAA 953
S V E T S S R K R T G G A T S P R S P A Q K R P K R R V Q T L L K M R Q N A I E 310
CTATTGACACGACTTTATGGCTCATGGGATGCACAATTGAGCCTCTCAAATCTTGAGACAATTCGATTGTTGGGTGTCAATAATAATAGGAAGCTTATCGAAATTTTTGAGGAGAATGAG 1073
L L T R L Y G S W D A Q L S L S N L E T I R L L G V N N N R K L I E I F E E N E 350
A
CAAGTGCTCAAACAAAAGTGTCCGCACTGACAGAAGAGCTGAAAAGGAGAAGCTGGCTCACGCGGGAAC CCGTTCAGCATTGAAAGAATTGACTAATGAAATAACTGGAATGCGTGTA 1193
Q V L K Q K V S A L T E E L K K E K L A H A G T R S A L K E L T N E I T G M R V 390
CAAATGAATAAACTACGTTCAATGGTCACTCAGCCTACGACTTCGAAAATTATTGATAGTTTTGTTCAACGTCATCAGGCTTTCGAGCAGCAACAACAATTCCAACACCAACACCACCAA 1313
Q M N K L R S M V T Q P T T S K I I D S F V Q R H Q A F E Q Q Q Q F Q H Q H H Q 430
CACCGACCAATAATGTTGGCTCCACGTCATCATCCGCCGCCCCCCCGCATTTTACACCGAATCAACGGGCGGCGGCTCCGTATCATCCGAATATGGTTCAACCGAATCGTCTTGCTGCT 1433
H R P I M L A P R H H P P P P P H F T P N Q R A A A P Y H P N M V Q P N R L A A 470
A
ATGCCACATAGAAGACCGATTATTGGAATGCAGCAACAAAATTCGGCTCCACCACAATTCAACGGTCACCAAGCTCTCGTCCCATCACCTCAATCATCATCTGCATTTTCTCGTCCACCA 1553
M P H R R P I I G M Q Q Q N S A P P Q F N G H Q A L V P S P Q S S S A F S R P P 510
(A n3441,n3541 W534AMBER
CCAACTCAACTTGCAACACAGAGAAGAGCTCCACCATTGGCAAGTACCGGCCTTCCGGCAACAGTCAGATGGGAAGCAATTCCACCGCCAAAATCCGAATCC GTCGGGCACAATC GAGCCA 1673
P T Q L A T Q R R A P P L A S T G L P A T V R W E A I P P P K N P N V G H N E P 550
A
CCGCT AACAATGGAGGCCGTGCACAGCCACTAATCGATAATACACGTGTACACGACAATACAATTATGCTGTGTGTACCACTTGTCTCCACTGCAAATACAATATCATCGGGCGATTCG 1793
P L N N G G R A Q P L I D N T R V H D N T I M L C V P L V S T A N T I S S G D S 590
ACACGTCTACCAAAAGTACCACGAATCTACGAGAATCTCACGGCAAATCCCGATTTGAGTGTGACGATTCATTCGAGTGCACAGGATTTCCGAGAGAATTATCAAATTGGTGGAAAGATT 1913
T R L P K V P R I Y E N L T A N P D L S V T I H S S A QDFRENYQIGGKI 630
A
AACTATGAATATCTCGGAGGATTTGATCAATATAATATCCAAGTGTTCGTCCAAGTGTCATCTCTTAAATTCACTGGAATGAACGGTTACCCGGATCCAGAAGATCGTATATCAATTGAC 2033
N Y E Y L G G F D Q Y N I Q V F V Q V S S L K F T G M N G Y P D P E D R I S I D 670
TGGGGATGCTCGAAATTGTGGCCTTGTAAGCCGAAATCTCATCACAAATTCCGTGTACGCTTCCATCAAGCACAACTGCTGCCGAAGAACGATCGAATTACGATTGTGGCTGTGGCGAAG 2153
W G C S K L W P C K P K S H H K F R V R F H Q A Q L L P K N D R I T I V A V A K 710
n3543 S720L 
T I A
GATAAAACTAGCGGAATTATTCACATTTCGCAGCCCACCTTCATCACTCTCGAAATCGATCTCTTCACGTCAAATGCACT TCTG GATTTTTTTGTTAAAAAATTTGAAATTCTC 2273
DKTSGIIHISQPTF I T L E 728
GTGTTTTTTCTTCTGAAAAATTGCTTG TTTGATTTTCTGTAA TTTTTTTTTTGTTGATTTTCTAATTTT AATTCAAAAAATCTTTTTCATCTCTTTCTCTCTCTCTCTGAA 2393
TCTCAATTT CCTGAATTTCCCCGTTTTTTTCTGATAATTTTCAATATTTCTCTGAATTTTTCTATTCCCCCCGTGTAATGCCAAAATATGTGGTAATTTCTCCCCATTTTCGCT 2513
TTATTACTATTTATTCTATTCAATTGGTGCCTCTCTCAATGTGTTGTATGAAAAACACTGTTTTATGGAGGTTTTGG 2590
1 "
CHAPTER 6
Future Directions
265
synMuv proteins form multiple complexes that likely regulate transcription levels
At least 31 synMuv genes have been identified and based on their genetic
interactions they have been placed into three classes. Among the class B synMuv
proteins are homologs of a histone deacetylase and the RB, DP, E2F complex that
could recruit the histone deacetylase to DNA. Based on these similarities to mammalian
proteins, a model has been proposed suggesting that the class B synMuv proteins act
through the recruitment of a histone deacetylase complex to the promoters of vulval
genes and regulate cell-fate specification through 
transcriptional repression. However,
both work discussed in this thesis and data from other laboratories have demonstrated
that the proteins encoded by the class B synMuv genes are not in a single complex. The
DRM complex consists of at least eight class 
B synMuv proteins: LIN-9, LIN-35, LIN-37,
LIN-52, LIN-53, LIN-54, DPL-1, and EFL-1. While the DRM complex includes the only C.
elegans pocket protein, it is separable from the histone deacetylase-containing NuRD-
like complex, which likely consists of at least HDA-1, LET-418, LIN-53, and MEP-1
(UNHAVAITHAYA et al. 2002). LIN-61 is not found as a core member of either of these
complexes, and LIN-36 and HPL-2 are unlikely to be members of the DRM complex.
Studies of the interactions among the synMuv proteins must be extended to other class
B synMuv proteins like LIN-15B, LIN-65, MET-2, and TAM-1 for which suitable
antibodies do not exist currently.
The identification of these distinct complexes suggests a modified model for how
the class B synMuv proteins might act together to maintain transcriptional repression.
The NuRD-like complex could be recruited to the promoters of genes that cause vulval
induction and deacetylate the histones in that promoter region. Subsequently, the DRM
complex could bind to these histones and maintain them in their deacetylated state
(Figure 1 A). Alternatively, a methyltransferase such as the class B synMuv protein
MET-2 could methylate the now deacetylated histone allowing HPL-2 to bind to the
methyl mark and recruit LIN-13 to the promoter (Figure 1B). Detailed studies of
identified target genes should be able to distinguish between these models. It is
possible that each model is correct and that the class B synMuv genes regulate multiple
266
target genes by different mechanisms. Of course, other models also are possible.
Perhaps each complex is acting individually at different target genes.
According to the first model, binding of the DRM complex to promoters would
require the histone deacetylase activity of the NuRD-like complex. Thus, in mutants
lacking NuRD complex activity DRM would no longer be able to bind to those regions of
DNA. If instead MET-2 acts to methylate 
these histones prior to HPL-2 binding,
methylation at specific promoters would be dependent on NuRD complex components.
Each of these predctions should be testable using chromatin immunoprecipitation
(ChIP) in various mutant backgrounds once genes regulated by the synMuv proteins are
identified. ChIP experiments will also allow us to monitor the histone modifications in
regions of the genome to which various synMuv proteins have been recruited. For
example, it might be expected that at promoters that are repressed by the NuRD-like
complex the histones will be hypoacetylated, and that when components of the complex
are removed by mutation the histones will become hyperacetylated. Antibodies against
many specific histone modifications have been characterized and are commercially
available.
Genetic experiments analyzing redundancy among the class B synMuv proteins
should also help in refining the proposed models. Loss of function of many class B
synMuv genes if combined with a weaker class A synMuv mutant or if double mutants
are raised at lower temperatures do not result in a fully penetrant synMuv phenotype. In
such backgrounds we can look for enhancement of the weak synMuv phenotype by
mutations in other class B synMuv genes. Such enhancement would suggest that the
two class B synMuv genes provide redundant functions in regulating vulval
development.
synMuv proteins could regulate vulval development
through transcriptional repression of lin-3
While most class A and class B synMuv proteins are likely functioning to repress
transcription, for many of these proteins how they cause this repression is still unknown.
While the DRM complex contains at least eight synMuv proteins, none of these complex
267
components has known chromatin-remodeling activities. Thus it will be important to
determine how this protein complex is regulating gene expression. In addition, it is has
been unclear whether the redundancy among the different synMuv classes results from
functional redundancy in regulating transcription of the same target genes or whether
the classes act on different processes. Studies of how the synMuv proteins are
cooperating to regulate expression of specific target genes should help us understand
how these proteins are functioning.
Recently, transcription of lin-3, which encodes an EGF-like signal known to be
important in vulval induction, has been shown to be regulated redundantly by the class
A and class B synMuv genes (Cui etal. 2006a). Loss of function in either the class A
synMuv genes lin-8 or lin- 15A or the class B synMuv genes lin-35, lin-36 or lin- 15B as
single mutants did not result in upregulation of lin-3 mRNA levels. However, in animals
mutant for both a class A and class B synMuv gene lin-3 levels were significantly
upregulated. Thus, at least for regulation of lin-3, the class A and B synMuv genes are
acting redundantly by both repressing transcription of a single locus. It has yet 
to tested
if regulation of lin-3 levels is directly mediated by binding of synMuv proteins to the lin-3
promoter. Experiments to determine if this is the case are an obvious next step.
Studies of how the class A and B synMuv proteins are cooperating to repress
transcription of specific target genes, like lin-3, should give us insight into how these
proteins are functioning. ChIP experiments first can be used to determine if the synMuv
proteins are directly regulating a given promoter. If this is the case, these experiments
should allow us to identify approximately where in the promoter region of specific target
genes the synMuv proteins are binding. Given that both class A and B synMuv proteins
can repress transcription from the lin-3 promoter, ChIP experiments could be useful in
elucidating if the binding sites for the class A and B synMuv proteins in the promoter are
distantly spaced from each other. In addition, it might be possible to show whether all or
most of the proteins from a single synMuv class bind to the same region of the
promoter.
Binding sites could be verified genetically. If the binding sites for different synMuv
proteins are distantly spaced in the promoter, it might be possible to identify 
deletion
268
alleles that specifically remove one, but not the other binding site. Animals with these
mutations should therefore require loss of function in only one class of synMuv genes to
result in a Muv phenotype. For example, a missense allele upstream of lin-3 causes a
strong Muv phenotype when combined 
with loss of function in class B synMuv 
genes,
but not class A synMuv genes, suggesting that it might target binding of class A synMuv
proteins (A. Saffer and H.R.H. unpublished results.) Binding sites could be identified
more specifically using transgenic animals. The regions of the genome that have been
shown to bind specific synMuv proteins might be able to repress transcription when
inserted upstream of a transgene. By systematically removing or mutating portions of
this region, it should be possible to narrow down the specific protein-binding site.
Class B synMuv genes do not all function similarly
to regulate vulval development
Biochemical data demonstrate that the class B synMuv proteins are not forming a
single complex, suggesting that these proteins might not all be functioning together in
vulval development. Indeed, genetic analyses of these genes similarly indicate that not
all class B synMuv genes are acting identically in the regulation of vulval development.
hda-1, let-418, and lin-13 have all been categorized as class B synMuv genes
(MELENDEZ and GREENWALD 2000; voN ZELEWSKY et al 2000; DUFOURCO et al 2002).
However, unlike loss of function in other class B synMuv genes, loss of function in these
three genes cause Muv phenotypes as single mutants (MELENDEZ and GREENWALD
2000; voN ZELEWSKY et al. 2000; DUFOURCQ et al. 2002). In addition, mutations in other
genes that have been categorized as class B synMuv genes, like hpl-2, have synthetic
defects when combined with mutations in other class B synMuv genes, suggesting
functional redundancy among these genes (COUTEAU et al 2002; COUSTHAM et a.
2006). Synthetic interactions between members of the same synMuv class have not
been extensively tested and might reveal additional redundancies.
Studies of lin-3 levels in animals mutant for let-418, hda- 1, and lin- 13, which
result in a weak Muv phenotype as single mutants (MELENDEZ and GREENWALD 2000;
VON ZELEWSKY et al. 2000; DUFOURCQ et al. 2002), should help in elucidating whether
269
there are additional genes important for vulval induction regulated by the protein
products of these genes. The fact that as single mutants loss of function of these genes
causes a Muv phenotype suggests that if lin-3 is the only target they should cause some
derepression of lin-3 even in the absence of a class A synMuv mutation. If no
derepression of lin-3 is evident, it is likely that these proteins repress additional target
genes that contribute to the vulval phenotype.
synMuv proteins regulate many processes in addition to vulval development
Identification of target genes will further our understanding of how the large
number of transcriptional regulators that have been identified genetically for their roles
in vuval development can act together in some biological processes but not in others.
Loss of function of synMuv genes results in a large number of abnormalities in addition
to vulval defects, and these defects are not shared by all identified synMuv genes. As
discussed in Chapter 3, animals mutant for lin-35 and lin- 15B cause RNAi
hypersensitivity, LAG-2 misexpression, PGL-1 somatic expression, and modification of
expression from simple transgenes, but these pleiotropies are not caused by loss of
function of lin-61. Thus it is probable that there are genes specifically repressed by
LIN-35 and LIN-15B but not LIN-61. Detailed studies of the promoter regions of verified
target genes should help us understand why at some 
promoters, like pro-vulval genes,
LIN-61 might be functioning with LIN-35 and LIN-15B while at others these proteins do
not function together. Further genetic characterization of the pleiotropies associated with
loss of function of additional class B genes along with analysis of the genes they
regulate should help in understanding how an array of different transcription factors can
coordinately control expression of a large number of genes.
Antibodies exist against a significant number of class B synMuv genes.
Antibodies against LIN-9, LIN-37, LIN-52, LIN-53, LIN-54, and LIN-61 are all able to
specifically immunoprecipitate the protein they were raised against from C. elegans
protein extract. These antibodies could be used for ChIP experiments. By hybridizing
the genomic fragments that coprecipitate with these proteins to DNA arrays tiled with
intragenic sequences, ChIP experiments can identify genomic regions that are bound by
270
synMuv proteins and, by extension, can find additional target genes. Unfortunately, at
the current time intragenic arrays are not available so standard microarrays could be a
useful alternative in identifying additional gene targets of the synMuv proteins. In these
experiments, mRNA isolated from both single and double synMuv mutant strains would
be hybridized to arrays and, by comparison to the wild type, those mRNAs that are more
or less abundant in the mutants can be identified. Standard gene arrays, as opposed to
intragenic arrays, provide information on whether mRNA levels are upregulated or down
regulated. It will be important in analyzing these data to recognize the possibility that the
synMuv proteins might not be acting solely as transcriptional repressors but could
potentially also have roles in transcriptional activation. By comparison between results
obtained with single synMuv mutants and with double synMuv mutants it should be
possible to identify genes that are specifically regulated by a single class of synMuv
proteins or genes that are regulated by the redundant functions of multiple classes.
In C. elegans, the ease of transgenics, RNAi, and screening for targeted
deletions, can be used to determine the biological functions of target genes. For
example, loss of function of a target gene might result in the suppression specific
pleiotropies associated with loss of function in synMuv genes. This would suggest that
the increased expression of this gene in a synMuv mutant resulted in the abnormality.
Such detailed studies of these target genes could possibly identify additional processes
that are regulated by the synMuv genes.
Different target genes are likely to be regulated by
different sets of synMuv proteins
Another potential target for repression by the synMuv genes is mat-3, which
encodes a component of the anaphase-promoting complex. An allele of mat-3, ku233,
that results in defects in vulval formation contains two adjacent base-pair changes
approximately 400 base pairs upstream of the transcriptional start site. These mutations
are likely to interfere with transcription of the mat-3 locus as mat-3 mRNA levels are 5-
10 fold reduced in ku233 mutant animals. Loss of function of either lin-35 or dpl- 1, but
not lin-36 restores mat-3 mRNA levels in ku233 mutant animals. In addition, mutations
271
in the class B synMuv genes lin- 15B, lin-35, lin-53, lin-61, dpl- 1, and efl- 1 can suppress
the vulval defects caused by mat-3(ku233), but neither RNAi directed against lin-36 nor
a loss-of-function mutation in lin-36, can suppress these defects (GARBE et al 2004).
These data suggest that only a subset of the class B synMuv proteins regulate
expression of mat-3. This transcriptional regulation might be directly caused by binding
of synMuv proteins to the mat-3 promoter as an E2F-binding site exists 130 base pairs
upstream of the transcriptional start site (GARBE et al. 2004). It is not known currently
whether the class A synMuv proteins act to regulate mat-3 expression levels. However
since loss of function in lin- 15A and lin-8 fail to suppress the vulval defects of
mat-3(ku233) animals, it is unlikely that the class A proteins are regulating mat-3
expression levels. Thus regulation of mat-3 appears to be distinct from the regulation of
lin-3 in two ways. First, mat-3 levels are not redundantly controlled by both class A and
class B synMuv proteins, and second, lin-3, but not mat-3, is likely regulated by LIN-36.
Thus individual synMuv target genes are likely to be controlled by distinct combinations
of synMuv proteins.
As discussed in the Introduction to this thesis, pocket proteins interact with a
diverse array of chromatin-remodeling factors to regulate gene expression. Perhaps in a
similar manner different synMuv proteins are cooperating to regulate expression of
individual target genes. The NuRD-like complex and the DRM complex could be
cooperating to regulate transcription of one set of target genes while the NuRD-like
complex could act with MET-2 and HPL-2 at other target genes. Detailed studies of
which synMuv proteins are recruited the promoters of specific target genes and which
synMuv mutants cause deregulation of these target genes should provide insight into
how the different sets of transcription factors are cooperating to control the levels of a
diverse array of genes.
Multiple proteins likely act to recruit synMuv complexes to DNA
Given that LIN-36 is unlikely to regulate mat-3 levels and that each synMuv gene
seems to control a different set of pleiotropies, it is unlikely that all synMuv proteins are
272
regulating the same set of target genes. Perhaps some of this control is mediated by the
recruitment of different protein complexes to individual promoters.
How are these various complexes recruited to specific regions of the genome?
The DRM complex contains at least three proteins that are involved in sequence-
specific DNA binding, EFL-1, DPL-1, and LIN-54, and it is likely that the complex is
being recruited to promoters by the activities of these proteins. No DNA-binding protein
is a component of the NuRD-like complex. However, MEP-1 binds to the transcription
factor LIN-1 when LIN-1 is sumoylated, suggesting that MEP-1 mediates interactions
between the NuRD-like complex and transcription factors (LEIGHT et al 2005). LIN-36
contains a THAP domain, and these domains are capable of binding to DNA in a
sequence-specific manner (CLOUAIRE et al. 2005). Thus LIN-36 might also act to recruit
other class B synMuv proteins that are not members of the DRM complex to specific
regions of the genome. The class A synMuv proteins LIN-15A and LIN-56 also contain
THAP domains.
ChIP experiments should allow us to test, 
using various single mutant strains,
which of the synMuv proteins are required to recruit other synMuv proteins to promoter
regions. For example as it is expected that LIN-54 and the heterodimer of EFL-1 and
DPL-1 bind DNA and recruit the DRM complex to promoter regions, it is likely that in
animals that lack some or all of these proteins other members of the DRM complex will
not be recruited to promoters. Furthermore, we can determine if these two sequence-
specific DNA-binding activities are cooperating to recruit the DRM complex to promoters
containing binding sites for both proteins or whether each activity is required individually
to recruit the complex. Finally, we can test whether 
other proteins, like LIN-36 or LIN-1,
are required to recruit class B synMuv proteins to promoters.
How do the coordinated functions of different transcription factors cooperate to
regulate development?
More detailed understanding of the functions of the synMuv genes and the genes
they target for transcriptional repression will help us to better understand how
transcription factors coordinate their functions to control different biological processes.
273
In addition, a large number of proteins that are predicted to regulate chromatin have
been found to suppress the synMuv phenotype (Cui et al. 2006b). Loss of function of
some of these genes also suppresses abnormalities beyond vulval development that
are associated with loss of function of synMuv genes. RT-PCR or northern blots should
help to identify whether these suppressors are acting on the same target genes as the
synMuv proteins or whether the suppression is a secondary effect from regulation of
other gene targets. For example, do mutations in the suppressors reduce lin-3 mRNA
levels to wild type in an animal mutant for both a class A and class B synMuv gene.
Furthermore, it will be possible to test whether the suppressors are directly
counteracting chromatin modifications by the synMuv proteins. A histone
acetyltransferase complex was identified among the suppressors (Cui etal. 2006b), and
this protein complex could directly counteract the histone deacetylase function of the
NuRD-like complex.
Conclusions
The DRM complex is a conserved pocket protein-containing complex that likely
acts to repress target genes, possibly including lin-3 and mat-3. As all components of
this complex have homologs in mammals, a similar complex might exist in humans and
provide yet another mechanism by which pocket proteins can repress their
transcriptional targets. As discussed in Chapter 1, pocket proteins can interact with
many different chromatin-remodeling enzymes to repress transcription. Detailed studies
of individual promoters should help in elucidating how the DRM complex represses
transcription and whether chromatin-remodeling enzymes are required for the
repression at specific times or in certain tissues. Future studies of the function of pocket
proteins in regulating genes necessary for development rather than cell cycle might
demonstrate that a DRM-like complex is specifically required for this regulation.
Many additional synMuv genes have been identified that are not components of
the DRM complex, but are also likely to act 
as transcriptional repressors. In the future,
studies will need to focus on both identifying the transcriptional targets of the synMuv
proteins and on how these proteins function to cause transcriptional repression. Such
274
analyses should provide insight into the mechanism by which transcriptional regulators
are cooperating to regulate the transcription of a large number of target genes.
Acknowlegments
I thank Michael Hurwitz and Adam Saffer for helpful comments about this chapter.
27-5
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Figure 1. Schematic indicating two possible models for how complexes among the
class B synMuv proteins might regulate transcription during vulval development. Genes
that promote the expression of vulval cell fates (red box) are transcriptionally active
when the histones in the region of their promoters are acetylated. The NuRD-like
deacetylates these histones, causing transcriptional repression. (A) The DRM complex
maintains transcriptional repression by binding to histones that have been deacetylated
by the activity of the NuRD-like complex. (B) The putative methyltransferase MET-2 can
methylate deacetylated histones. The methylated histones are subsequently bound by
HPL-2, which can recruit LIN-13.
278
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