Article
Sirtuin 1 Promotes Deacetylation of Oct4
and Maintenance of Naive Pluripotency
Graphical Abstract
Highlights
d SirT1 is required for the maintenance of naive pluripotency
d SirT1 deacetylates Oct4 in the naive state
d Oct4 is hyper-acetylated during the transition to primed
pluripotency
d Deacetylation of Oct4 inhibits Otx2 induction
Authors
Eric O. Williams, Amy K. Taylor,
Eric L. Bell, Rachelle Lim, Daniel M. Kim,
Leonard Guarente
Correspondence
leng@mit.edu
In Brief
transition from naive to primed
pluripotency. Williams et al. show that
Sirt1 deacetylates Oct4 in the naive state,
preventing Oct4-dependent induction of
Otx2, thus inhibiting the induction ofWilliams et al., 2016, Cell Reports 17, 809–820
October 11, 2016 ª 2016 The Authors.
http://dx.doi.org/10.1016/j.celrep.2016.09.046primed pluripotency.
Accession Numbers
GSE81494reorganization of Oct4 during theOtx2 induction is required for the
Cell Reports
a
l
,
io
INTRODUCTION
more than naive cells (Brons et al., 2007; Rossant, 2008; Tesar
et al., 2007). In addition, human induced pluripotent stem cellswith the ability to contribute to all embryonic lineages (Nichols
and Smith, 2009; Rossant, 2008). Embryonic stem cell lines
derived from pre-implantation mouse blastocysts exhibit char-
specification. Lastly, naive human embryonic stem cells are
capable of contributing to cross-species chimeras (Gafni et al.,
2013), a tool that will likely be useful for the creation of human-acteristics of naive pluripotency (Evans and Kaufman, 1981;
Nichols and Smith, 2011). These include a rounded morphology,
ized animal models. Several attempts have been made to stably
reprogram human embryonic stem cells to the naive state usingDuring embryogenesis, an exquisite program of ordered events
enables a single zygote to develop into a multicellular organism
with diverse cell types. A critical step in this process is the ability
of pluripotent epiblast cells to coordinate the balance between
self-renewal and lineage specification (Kunath et al., 2007;
Niwa, 2007; Silva and Smith, 2008). This task is thought to be
at least partially accomplished by the transition of these cells
through at least two distinct pluripotent states (Brons et al.,
2007; Tesar et al., 2007).
Pre-implantation epiblast cells exhibit naive or ground state
pluripotency. This state has been defined as fully unrestricted
(iPSCs) exhibit features of the primed state of pluripotency (Ta-
kahashi et al., 2007; Yu et al., 2007), while mouse iPSCs naturally
revert to the naive state (Takahashi and Yamanaka, 2006). While
the reasons for these mouse-human differences remain unclear,
there are a number of practical advantages that make the naive
state amore desirable research tool. Pluripotent naive stem cells
are characterized by a more open chromatin structure (Murtha
et al., 2015), allowing for more efficient genetic manipulation
(Buecker et al., 2010; Zwaka and Thomson, 2003). In addition
primed cells are prone to greater heterogeneity in gene expres-
sion (Bernemann et al., 2011; Gafni et al., 2013; Osafune et al.,
2008), making it difficult to obtain unbiased lineage-specificlate epiblast cell state. implantation blastocysts resemble primed murine stem cellsArticle
Sirtuin 1 Promotes Deacetyl
and Maintenance of Naive P
Eric O. Williams,1 Amy K. Taylor,1 Eric L. Bell,1 Rachelle Lim
1Glenn Laboratory for the Science of Aging and the Department of B
MA 02139, USA
2Lead Contact
*Correspondence: leng@mit.edu
http://dx.doi.org/10.1016/j.celrep.2016.09.046
SUMMARY
The enhancer landscape is dramatically restructured
as naive preimplantation epiblasts transition to
the post-implantation state of primed pluripotency.
A key factor in this process is Otx2, which is upregu-
lated during the early stages of this transition and ul-
timately recruits Oct4 to a different set of enhancers.
In this study, we discover that the acetylation status
of Oct4 regulates the induction of the primed plurip-
otency gene network. Maintenance of the naive state
requires the NAD-dependent deacetylase, SirT1,
which deacetylates Oct4. The activity of SirT1 is
reduced during the naive-to-primed transition; Oct4
becomes hyper-acetylated and binds to an Otx2
enhancer to induce Otx2 expression. Induction of
Otx2 causes the reorganization of acetylated Oct4
and results in the induction of the primed pluri-
potency gene network. Regulation of Oct4 by SirT1
may link stem cell development to environmental
conditions, and it may provide strategies to manipu-Ce
This is an open access article undtion of Oct4
uripotency
1 Daniel M. Kim,1 and Leonard Guarente1,2,*
logy, Massachusetts Institute of Technology, Cambridge,
the maintenance of self-renewal through Jak/Stat3 and Bmp4
(Matsuda et al., 1999; Niwa et al., 2009; Ying et al., 2003), sin-
gle-cell clonogenicity, and efficient contribution to chimeras
(Han et al., 2010).
During embryo implantation, cells of the epiblast layer transi-
tion from the naive state to the primed pluripotency state (Arnold
and Robertson, 2009; Brons et al., 2007; Kojima et al., 2014; Te-
sar et al., 2007). While still capable of contributing to all three
germ lineages, primed epiblast stem cells are thought to be
more developmentally restricted than naive embryonic stem
cells (ESCs), and they undergo X chromosome inactivation
(Bao et al., 2009; Guo et al., 2009; Heard, 2004; Silva et al.,
2008). Indeed, chimeric contribution of pluripotent primed cells
is significantly less efficient than that of pluripotent naive cells
(Brons et al., 2007; Han et al., 2010; Tesar et al., 2007). Primed
cells can be cultured by obtaining epiblast cells of the early
post-implantation mouse embryo (Nichols and Smith, 2011).
The primed state also can be induced in vitro by the addition
of Fgf and Activin A to naive cells (Brons et al., 2007; Tesar
et al., 2007; Thomson et al., 1998). In the primed state, cultured
stem cells are morphologically flat and exhibit low single-cell
clonogenicity.
Interestingly, human embryonic stem cells derived from pre-ll Reports 17, 809–820, October 11, 2016 ª 2016 The Authors. 809
er the CC BY license (http://creativecommons.org/licenses/by/4.0/).
both genetic (Takashima et al., 2014; Wang et al., 2011) and
chemical methodologies (Chan et al., 2013; Duggal et al.,
2015; Gafni et al., 2013; Hanna et al., 2010; Theunissen et al.,
2014; Valamehr et al., 2014; Ware et al., 2014). These studies
have beenmet with varying levels of success, but the robustness
of these protocols with regard to the long-term culture of the
naive state is still being evaluated (Dodsworth et al., 2015).
Understanding the molecular mechanisms driving the naive-
to-primed transition is crucial for comprehending the regulation
of mammalian development and the optimization of experi-
mental protocols for stem cell therapy. One important obser-
vation is the dramatic difference in the enhancer chromatin
landscape between the naive and primed states (Buecker
et al., 2014; Factor et al., 2014; Sohni et al., 2015; Tesar et al.,
2007; Yang et al., 2014; Yeom et al., 1996). Indeed, recent epige-
nomic analysis concluded that enhancer usage is the most
distinguishing factor between these two states. Interestingly,
enhancer usage changes are observed in both differentially
expressed genes and in genes with similar expression levels
between these two states (Factor et al., 2014). An example is
Oct4, a transcription factor whose expression levels promote
self-renewal and inhibit differentiation of stem cells (Nichols
et al., 1998; Radzisheuskaya et al., 2013).Oct4 enhancer activity
shifts from a distal enhancer dependent on Oct4 itself in naive
cells to a proximal, Oct4-independent enhancer in primed cells,
without affecting the total expression level of the gene (Buecker
et al., 2014; Yang et al., 2014).
Oct4 reorganization is driven by the upregulation of the
transcription factor Otx2 early in the transition from naive to
primed states. Oct4 is required for the early upregulation of
Otx2, and Otx2 is required for the recruitment of Oct4 to primed
enhancers (Acampora et al., 2013; Buecker et al., 2014; Yang
et al., 2014). Importantly, the induction of Otx2 expression by
Oct4 is sufficient to induce the transition from the naive to the
primed state (Buecker et al., 2014; Yang et al., 2014).
Here we find that Oct4 is post-translationally modified by
acetylation in mouse embryonic stem cells and that Oct4
acetylation status is a key component of the naive-to-primed
transition. During this transition, activity of the NAD+-dependent
deacetylase SirT1 decreases, and, concomitantly, the acetyla-
tion of Oct4 increases. Indeed, knocking out SirT1 in naive
ESCs increases Oct4 acetylation and results in the partial
upregulation of the primed pluripotency network. Importantly,
the hyper-acetylated Oct4 in SirT1-knockout ESCs displays
increased occupancy at anOtx2 enhancer element and, thus, in-
duces Otx2 expression. Inactivation of SirT1 in wild-type ESCs
therefore provides a mechanism for the induction of Otx2 early
in the naive-to-primed transition. Lastly, knocking down Otx2
expression in SirT1-knockout cells reverses the induction of
the primed network, thus linking the SirT1-Oct4-Otx2 axis to
the naive-to-primed transition of epiblasts.
RESULTSSirT1 Activity Decreases during the Transition from
Naive to Primed Pluripotency
Previous reports indicate that SirT1 is expressed at high levels
in pluripotent stem cells and these levels decline considerably
810 Cell Reports 17, 809–820, October 11, 2016during lineage specification (Calvanese et al., 2010; McBurney
et al., 2003a, 2003b; Sakamoto et al., 2004). We decided to use
mouse embryoid bodies to model the differentiation that nor-
mally occurs in early post-implantation embryos (Desbaillets
et al., 2000). Consistent with previous findings, we observed
a decrease in SirT1 protein levels as pluripotent mouse em-
bryonic stem cells transitioned from self-renewal (high Oct4
expression) toward lineage specification (low Oct4 expression)
(Figure 1A).
Interestingly, the levels of SirT1 protein closely mimicked that
of Oct4, the master pluripotency transcription factor (Figure 1A).
To determine the extent of SirT1/Oct4 co-regulation, we per-
formed immunocytochemistry in mouse embryonic stem cells
grown in serum/LIF conditions. Mouse embryonic stem cells
grown in serum/LIF exist in multiple stages of pluripotency and
exhibit heterogeneous transcription factor expression (Torres-
Padilla and Chambers, 2014; Ying et al., 2008). We identified
colonies within our cultures with heterogeneous expression of
Oct4. We observed that cells expressing the highest levels of
Oct4 also had the highest levels of SirT1 expression (Figure 1B,
yellow arrows). Conversely, cells with lower expression of Oct4
also had low levels of SirT1 (Figure 1B, white arrows), indicating
a degree of co-regulation betweenSirT1 andOct4. Thismay be a
conserved mechanism between humans and mice, as loss of
Oct4 expression in human embryonic stem cells also has been
reported to reduce SirT1 expression (Zhang et al., 2014). We
also found that SirT1 is localized within the nucleus, as evi-
denced by its colocalization with DAPI and the activating histone
modification (H3K4me3) (Heintzman et al., 2007) (Figure 1D). No
SirT1 signal was detected in SirT1-null embryonic stem cells,
demonstrating the specificity of the antibody (Figure 1B).
To assess SirT1 expression during the naive-to-primed transi-
tion, we employed the previously described epiblast-like cell
(EpiLC) differentiation protocol (Buecker et al., 2014; Hayashi
et al., 2011; Yang et al., 2014). Mouse embryonic stem cells
were grown under serum-free 2i LIF conditions (MEK/ERK inhib-
itor, GSK3b inhibitor, and recombinant mouse LIF) to promote
the naive state. Primed pluripotency was induced by stimulating
with Fgf2 and Activin A. Within 48 hr, naive embryonic stem cells
transitioned to primed EpiLCs. Consistent with previous studies,
we found little to no change in Oct4 expression but dramatically
reduced expression of Nanog in the primed state (Silva et al.,
2009) (Figure 1C). Genome-wide transcriptional analysis of naive
ESCs and EpiLCs revealed significant overlap with previously
characterized primed pluripotency datasets (Kim et al., 2013;
Buecker et al., 2014) (Figures S1A–S1D). While SirT1 levels re-
mained unchanged between the two states, SirT1 activity was
significantly reduced in primed EpiLCs. p53, a canonical target
of SirT1 deacetylation (Vaziri et al., 2001), was found to be
hyper-acetylated at Lysine 379 in EpiLCs (Figure 1C), indicating
a reduction in SirT1 activity during the transition to primed
pluripotency. Quantification of p53 acetylation levels revealed
a 1.67- to 1.95-fold increase in EpiLCs (Figure S1E). To confirmSirT1 dependence of p53 acetylation in ESCs, we tested p53
acetylation in SirT1-null and wild-type ESCs grown in naive
conditions (2i + LIF). p53 was found to be 1.63- to 2.11-fold
more acetylated in SirT1-null ESCs (Figure S1F). To rule out
the possibility that decreased HDAC1 levels in EpiLCs and
SirT1-null ESCs could be causing increased p53 acetylation at
lysine 379 (Ito et al., 2002), we performed western blots on
HDAC1. We found no difference in HDAC1 protein levels among
wild-type ESCs, SirT1-null ESCs, and EpiLCs (Figure S1G).
SirT1 Interacts with and Deacetylates Oct4 in the
Naive State
To gain insights into the function of SirT1 in naive pluripotency,
we assessed SirT1 occupancy at Oct4 enhancer domains. The
regulation of Oct4 expression within the naive and primed states
is well characterized. Activation of the Oct4 distal enhancer is
associated with the promotion of the naive state, while Oct4
proximal enhancer activity is associated with primed pluripo-
tency (Tesar et al., 2007; Theunissen et al., 2014; Yeom et al.,
1996) (Figure 2A). Chromatin immunoprecipitation (ChIP)-qPCR
analysis of these enhancer regions revealed an enrichment of
SirT1 protein within the distal enhancer of Oct4 in naive ESCs
(Figure 2B). Interestingly, the enrichment of SirT1 was strongest
at region 3, which previously was characterized to have strong
Oct4/Sox2 binding (Chew et al., 2005). Indeed, ChIP qPCR of
Figure 1. SirT1 Expression and Activity in Mouse Embryonic Stem Cel
(A) SirT1 expression during embryoid body differentiation. Embryoid bodies we
denoted with an arrow. SirT1 and Oct4 expression declines coordinately during
(B) Immunocytochemistry of SirT1 and Oct4 in mouse embryonic stem cells gro
protein levels of Oct4 and SirT1. White arrows highlight areas with relatively low p
type cells at 53magnification (scale bar, 200 mm); row 2, wild-type cells at 633ma
bar, 15 mm).
(C) SirT1 expression and activity during the transition from naive pluripotency to p
primed EpiLC conditions. SirT1 andOct4 protein levels are similar between naive a
acetylation at Lys379 is increased in EpiLCs.
(D) Immunocytochemistry of SirT1 and H3K4me3 is shown. DAPI, blue; SirT1, reOct4 and Sox2 revealed similar occupancy patterns as SirT1
(Figure 2C).
To determine if SirT1 physically interacts with the Oct4 com-
plex, we performed co-immunoprecipitation (coIP) experiments.
Lysates prepared from mouse embryonic stem cells grown in
naive conditions were immunopurified with a SirT1 antibody.
Immunoblotting with an Oct4 antibody revealed Oct4 coIP (Fig-
ure 2D). SirT1-Oct4 coIP was not observed in SirT1-null cells,
verifying the specificity of the interaction (Figure 2D). Conversely,
immunopurification of Oct4 yielded the coIP of SirT1. This signal
also was abolished in SirT1-null cells (Figure 2D). To determine if
Oct4 is a target of SirT1-mediated deacetylation, we used a
pan-acetyl lysine antibody to assess Oct4 acetylation in
Oct4-immunopurified lysates. This analysis revealed that Oct4
is hyper-acetylated in SirT1-null cells compared to wild-type
cells (Figure 2D). Quantification of Oct4 acetylation levels
revealed a 1.45-fold increase in the SirT1-null ESCs (Figure S2A).
Furthermore, Oct4 hyper-acetylation was observed in wild-type
ESCs treated with the SirT1 inhibitor Ex-527 (Figures S2C and
S2D) (Napper et al., 2005; Solomon et al., 2006). Consistent
ls
re differentiated for 0, 2, 4, 6, 8, 10, and 12 days. Full-length SirT1 protein is
differentiation.
wn in serum/LIF conditions. Yellow arrows highlight areas with relatively high
rotein levels of Oct4 and SirT1. DAPI, blue; Oct4, red; SirT1, green; row 1, wild-
gnification (scale bar, 15 mm); row 3, SirT1-null cells at 633magnification (scale
rimed EpiLCs. Wild-type mouse embryonic stem cells were grown in naive and
nd primed EpiLC cells. Nanog protein levels are reduced in the EpiLC state. p53
d; H3K4me3, green. Scale bar, 5 mm.
Cell Reports 17, 809–820, October 11, 2016 811
with the observed reduction in SirT1 activity in primed EpiLCs
(Figure 1C), we found that Oct4 acetylation levels increased
during the transition to primed EpiLCs (Figure 2E). Quantification
of Oct4 acetylation levels revealed a 1.39- to 1.64-fold increase
in acetylation in EpiLCs (Figure S2B). To rule out the possibility
that HATs or other deacetylases present in the primed state re-
sulted in the increased Oct4 acetylation, we assessed whether
SirT1 could deacetylate Oct4 in transfected HEK293T cells.
Myc-tagged Oct4, SirT1, and catalytically inactive SirT1H355A
(Rodgers et al., 2005) were co-transfected. Co-expression of
Figure 2. SirT1 Deacetylates Oct4 and Binds to the Oct4 Distal Enhanc
(A) Genomic structure of themouseOct4 gene. The distal enhancer is highlighted
qPCR amplicons used in qPCR analysis are identified.
(B) SirT1 ChIP qPCR of the proximal and distal enhancers ofOct4. Wild-type ESC
represent SEM.
(C) Oct4 and Sox2 ChIP qPCR of the proximal and distal enhancers of Oct4. All s
ChIP is represented in black. Error bars represent SEM.
(D) CoIP of SirT1 and Oct4 and analysis of acetylated Oct4. Top two rows: pull-do
(western blot with SirT1 antibody in the first row and Oct4 antibody in the second r
SirT1-null ESCs are shown (western blot with SirT1 antibody in the third row and O
antibodies in wild-type and SirT1-null ESCs are shown (western blot with pan-ac
(E) Acetylation of Oct4 in naive ESCs and primed EpiLCs. Pull-downs with Oct4 (
type primed EpiLCs are shown (western blot with pan-acetyl lysine antibody on
(F) Deacetylation of Oct4 in HEK293T cells. HEK cells were co-transfected withMy
b-actin are on the left andmyc-immunopurified samples are on the right. Myc-imm
Oct4, as shown.
812 Cell Reports 17, 809–820, October 11, 2016SirT1, but not catalytically inactive SirT1H355A, reduced Oct4
acetylation in transfected HEK293T cells (Figure 2F). Collec-
tively, these results suggest that Oct4 is a direct target of
SirT1-mediated deacetylation in naive cells and that Oct4 acety-
lation increases in EpiLCs due to decreased activity of SirT1.
Loss of SirT1 in the Naive State Induces Partial
Activation of the Primed Pluripotency Network
Morphologically, SirT1-knockout ESCs were found to adopt a
slightly flatter morphology when grown in 2i LIF, but they were
er
in orange and the proximal enhancer is highlighted in blue. The locations of nine
s are represented in blue and SirT1-null ESCs are represented in red. Error bars
amples are from wild-type ESCs. Oct4 ChIP is represented in brown and Sox2
wns with SirT1 (or IgG) antibodies in wild-type and SirT1-null ESCs are shown
ow). Middle two rows: pull-downs with Oct4 (or IgG) antibodies in wild-type and
ct4 antibody in the fourth row). Bottom two rows: pull-downs with Oct4 (or IgG)
etyl lysine antibody in the fifth row and Oct4 antibody in the sixth row).
or IgG) antibodies in wild-type naive ESCs grown in naive conditions and wild-
the top row and Oct4 antibody on the bottom row).
c-taggedOct4, SirT1, and SirT1355A.Western blot of inputs for SirT1, Oct4, and
unopurified sampleswere blotted with antibodies against pan-acetyl lysine and
indistinguishable from wild-type cells when grown in serum with
mouse embryonic fibroblasts (MEFs) (data not shown). The cell-
autonomous growth rate of SirT1-knockout ESCs was found to
be slightly slower than wild-type cells (Figure S3A), but no
observable differences were identified in apoptosis or cell-cycle
progression (Figures S3B–S3D).
Clonogenicity is a distinguishing factor between the naive and
primed states. Naive ESCs are able to form colonies from single
ESCs, while single primed cells either differentiate or undergo
apoptosis (Brons et al., 2007; Tesar et al., 2007). We found that
SirT1-knockout ESCs exhibited similar clonogenicity as wild-
type when grown in naive conditions. However, SirT1-knockout
ESCs lost clonogenicity faster than wild-type ESCs during the
transition from the naive state to the primed state (Figure S3E).
In addition, loss of SirT1 inhibited the reprogramming of primed
cells to the naive state as measured by clonogenicity (Fig-
ure S3F). To determine if the SirT1-Oct4 interaction might play
a role in the regulation of pluripotency networks, we performed
RNA sequencing (RNA-seq) transcriptome analysis in SirT1-
null embryonic stem cells. Wild-type and SirT1-null ESCs were
grown in naive conditions. In addition, wild-type cells were differ-
entiated into EpiLCs in order to define gene networks induced
during the naive-to-primed transition. We identified 2,616 genes
as significantly differentially expressed between the naive andFigure 3. Loss of SirT1 in Naive ESCs Induces
the Primed Pluripotency Gene Network
(A) Overlap of genes changed in SirT1-null cells and
primed datasets. Green circles contain all genes
with significant differential expression between
SirT1-null cells grown in naive conditions and wild-
type cells grown in naive conditions. Purple circles
contain all genes with significant differential
expression in primed versus naive datasets. Top
row: purple circle contains differential expression
generated by comparing our wild-type differentiated
EpiLCs and wild-type naive ESCs. Middle row:
primed dataset is defined by Buecker et al., 2014.
Data were generated by RNA-seq and compare
differentiated EpiLC and naive ESCs. Bottom row:
primed dataset is defined by Kim et al., 2013. Data
were generated from microarrays comparing mouse
epiblast stem cells and mouse embryonic stem
cells.
(B) Correlation of expression changes in SirT1-null
ESCs and primed EpiLCs. All genes exhibiting
significant differential expression in differentiatedprimed EpiLC states. These primed genes exhibited extensive
overlap with previously published datasets (Buecker et al.,
2014; Kim et al., 2013) (Figures S1A–S1D). Comparison of
wild-type naive and SirT1-null embryonic stem cells revealed
1,535 differentially expressed genes. Of these genes, 931
overlapped with the differential expression observed during the
transition fromwild-type naive ESCs towild-type primed EpiLCs.
Similar overlap also was observed between the expression
changes induced by the loss of SirT1 and previously published
datasets comparing the naive and primed states (Buecker
et al., 2014; Kim et al., 2013) (Figure 3A). The positive correlation
between the gene network induced during the transition from
naive to primed EpiLCs and the expression changes caused by
the loss of SirT1 had a Pearson product-moment correlation
coefficient of R = 0.669 (Figure 3B).
Consistent with a shift toward primed pluripotency in SirT1-
null ESCs, our RNA-seq analysis revealed changes in the
expression of important markers of primed pluripotency. These
included the downregulation of Nanog and Klf2 (Silva et al.,
2009; Yeo et al., 2014), upregulation of Fgf5 andOtx2 (Acampora
et al., 2013; Buecker et al., 2014; Nichols and Smith, 2009; Yang
et al., 2014), and the maintenance of Oct4 expression levels be-
tween the two states. The expression changes of primed
markers were confirmed by qRT-PCR (Figure S4A). In addition,
wild-type EpiLC/wild-type naive ESCs are plotted
against the gene expression changes comparing
SirT1-null/wild-type naive cells. Pearson product-
moment correlation coefficient R = 0.669 (n = 2,616).
(C) Western blot analysis of canonical primed
pluripotency markers in SirT1-knockout ESCs.
Tamoxifen-inducible SirT1-knockout cell lines are
indicated in lanes 3–6. The addition of 1 mM
tamoxifen induces loss of catalytic exon 4 of SirT1
for these lines, and it can be observed by the
downward shift in SirT1 immunoreactivity (black
arrow). Lane 8 contains SirT1-null ESCs. Quantifi-
cation of the average band intensity for each marker
is indicated (average SirT1-knockout intensity/
average wild-type intensity). All quantification mea-
surements are normalized to b-actin.
Cell Reports 17, 809–820, October 11, 2016 813
Figure 4. Loss of SirT1 Alters Expression of Transcripts Regulated by Oct4 in an Otx2-Dependent Manner
(A) ChIP qPCR of the proximal and distal enhancers ofOct4. Wild-type ESCs (blue), SirT1-knockout ESCs (red), and wild-type ESCs treated with 1 mMEx-527 for
48 hr (purple) were analyzed after IP with Oct4 antibody (left, Oct4 enhancer regions, as described in Figure 2A; right, Otx2 enhancer regions). Student’s t tests
with a p value less than 0.05 are indicated. Error bars represent SEM.
(legend continued on next page)
814 Cell Reports 17, 809–820, October 11, 2016
western blots were performed in both SirT1-null and homozy-
gous SirT1DEx4 ESCs. To acutely remove exon 4 of SirT1,
a domain required for SirT1 deacetylase activity (Cheng et al.,
2003), we employed a tamoxifen-inducible cre. Using fluores-
cent reporters, we estimated that 90%–95% of cells excised
exon 4 of SirT1 (data not shown). In all samples with SirT1
Otx2 induction and Oct4 binding increases at an Otx2 enhancer
in the primed state (Buecker et al., 2014; Yang et al., 2014). We
thus tested whether loss of SirT1 resulted in increased binding of
Oct4 at this enhancer (enhancer 1). Indeed, increased Oct4
binding was observed in SirT1-knockout ESCs and wild-type
ESCs treated with the SirT1 inhibitor Ex-527 (Figure 4A, right),
lu
a
ar
ki
ndeleted, there were reductions in Nanog and Klf2 proteins and
increases in Fgf5 and Otx2 proteins (Figure 3C). These data indi-
cate that the loss of SirT1 induces gene expression changes
consistent with the induction of the primed pluripotency gene
network.
Loss of SirT1 Causes Oct4 Reorganization at Naive
and Primed Enhancers
The shift from naive to primed pluripotency is characterized by a
dramatic reorganization of Oct4 occupancy at enhancer regions
(Buecker et al., 2014; Factor et al., 2014; Yang et al., 2014). To
test if loss of SirT1 and subsequent increased Oct4 acetylation
affects Oct4 occupancy at naive enhancers, we performed
ChIP qPCR in wild-type and SirT1-knockout embryonic stem
cells grown in naive conditions (2i-LIF). We found that Oct4 oc-
cupancy at the Oct4 naive enhancer (region 3 of the distal
enhancer) was significantly reduced in SirT1 knockout ESCs
and wild-type ESCs treated with the SirT1 inhibitor Ex-527 (Fig-
ure 4A, left). To determine if decreasedOct4 binding affectsOct4
distal enhancer activity, we performed Oct4 enhancer luciferase
reporter assays in wild-type and SirT1-knockout ESCs (Tesar
et al., 2007; Yeom et al., 1996). Consistent with previous find-
ings, the distal enhancer of Oct4 was more active in the naive
state than in the primed state. Moreover, the distal enhancer of
Oct4 was less active in SirT1-knockout ESCs grown in naive
conditions (Figure 4B; Figure S4B), indicating that the loss of
Oct4 binding results in decreased Oct4 distal enhancer activity.
Under conditions of primed pluripotency, the loss of Oct4 distal
enhancer activity is compensated for by increased activity of the
proximal enhancer, thus maintaining equivalent expression
levels of Oct4 (Tesar et al., 2007). Likewise, the proximal
enhancer activity of Oct4 was increased in SirT1-knockout cells
(Figure 4B), thus helping to maintain the levels of Oct4 expres-
sion (Figures 3C and 4C). In support of these data, loss of
SirT1 also facilitated changes in activities of the distal and
proximal enhancers during the naive-to-primed transition, and
it inhibited the reverse changes during the primed-to-naive
transition (Figure S4B).
An early event in Oct4 reorganization is the induction of Otx2, a
transcription factor whose overexpression is sufficient to induce
the primed pluripotency network. Oct4 expression is required for
(B) Luciferase activity of the Oct4 distal and proximal enhancers. Wild-type (b
transfection with luciferase reporter constructs driven by the distal (left) or proxim
time points for this experiment. Student’s t tests with a p value less than 0.05
(C) Relative expression of primed pluripotency markers. Average fragment per
grown in naive conditions, wild-type differentiated EpiLCs, SirT1-null ESCs grow
conditions are shown. Error bars represent SEM.(D) Expression of genes activated or repressed by Oct4. Genes activated or repr
(Buecker et al., 2014) and changes in RNA expression in the EpiLC state. Left: gre
cells (compared to naive wild-type ESCs); purple circles indicate the fold repressio
ESCs). Right: green circles indicate the fold activation in RNA expression within
purple circles indicate the fold repression in RNA expression within SirT1-knockconsistent with the increased expression of Otx2 described
above (Figures 3C and 4C). SirT1 itself was not found to be
enriched at this Otx2 enhancer (Figure S4C).
Otx2 directly binds to Oct4 in the primed state and is required
for Oct4 reorganization (Yang et al., 2014). If the upregulation of
Otx2 in SirT1-knockout cells is sufficient to induce Oct4 re-
organization, we expected those genes that are activated or
repressed by Oct4 to be upregulated or downregulated in the
context of SirT1 loss. To define genes whose transcripts are acti-
vated or repressed by Oct4, we used previously reported Oct4
ChIP sequencing (ChIP-seq) data in the naive and EpiLC states
(Buecker et al., 2014) and our RNA-seq expression data in the
wild-type naive and primed EpiLC states. Genes activated by
Oct4 were defined by increased Oct4 occupancy and increased
expression in the primed EpiLC state or decreased Oct4 occu-
pancy and decreased EpiLC expression in the primed EpiLC
state. Genes repressed by Oct4 were defined by increased oc-
cupancy and decreased expression in the primed EpiLC state
or decreased occupancy and increased expression in the
primed EpiLC state. We found that genes that were activated
or repressed by Oct4 exhibited directionally concordant expres-
sion in SirT1-knockout cells (Figure 4D, left). These results indi-
cate that loss of SirT1 deacetylase activity may drive naive cells
toward the primed state by promoting Oct4 binding at the Otx2
promoter.
Knockdown of Otx2 Restores the Naive Transcription
Network in SirT1-Knockout ESCs
To test whether the induction of Otx2 is required for the transition
to primed pluripotency networks in theSirT1-knockout ESCs, we
employed a short hairpin RNA (shRNA)-mediated silencing
approach to downregulate Otx2. Wild-type and SirT1-knockout
ESCs were infected with lentivirus expressing Otx2-targeting
shRNA or scrambled controls. Three Otx2 constructs consis-
tently reduced Otx2 expression levels in SirT1-knockout cells
(Figure S5A). RNA-seq analysis on one of these shRNA lines
(Otx2-3) also confirmed that Otx2 expression was restored to
wild-type levels in SirT1-null ESCs (Figure 4C).
Downregulation of Otx2 to wild-type levels in SirT1-knockout
ESCs was sufficient to rescue the expression levels of canonical
markers of primed pluripotency. Genes such as Nanog and Klf2,
e) and SirT1-knockout (red) ESCs grown in naive or primed conditions after
l (right)Oct4 enhancer are shown. Figure S4B contains additional differentiation
e indicated. Error bars represent SEM.
lobase of exon per million fragments mapped (FPKM) values in wild-type cells
in naive conditions, and SirT1-null and Otx2-knockdown ESCs grown in naiveessed by Oct4 were defined by changes in Oct4 occupancy in the EpiLC state
en circles indicate the fold activation in RNA expression within SirT1-knockout
n in RNA expressionwithinSirT1-knockout ESCs (compared to naive wild-type
SirT1-knockout, Otx2-knockdown ESCs (compared to naive wild-type ESCs);
out, Otx2-knockdown ESCs (compared to naive wild-type ESCs).
Cell Reports 17, 809–820, October 11, 2016 815
which are downregulated in the primed state and SirT1-
knockout ESCs, were restored to wild-type levels (Figure 4C).
Conversely, Fgf5, which is induced in the primed state and
SirT1-knockout ESCs, also was restored to wild-type levels.
These expression changes were confirmed by western blot anal-
ysis (Figure S5B). Numerous other genes showed a similar
pattern of expression (Figure S4A). In addition, the distal and
proximal enhancer activity of the Oct4 promoter returned to
wild-type naive levels in SirT1-knockout Otx2-knockdown
ESCs (Figure S5C).
Our previous analysis revealed that loss of SirT1 induced tran-
scriptional networks associated with the primed state (Figures
3A and 3B). We used this transcriptome data to study the role
of Otx2 by first grouping transcripts that were downregulated
or upregulated in wild-type primed EpiLCs compared to wild-
type naive ESCs (Figures 5A and 5B). Next we compared the
relative expression of SirT1-knockout naive ESCs, which clearly
displayed a shift in the direction of primed wild-type EpiLCs (Fig-
ures 5A and 5B). We then tested the hypothesis that upregulation
of Otx2 was responsible for this shift by knocking down Otx2 in
cells (Figures 5A an
Otx2 per se helps d
SirT1-knockout ESC
Otx2 is thought to
ing and genomic reor
is required for Oct4
analyzed the expres
naive-to-EpiLC trans
duction in SirT1-knoc
pression of these Oc
supporting the role o
a causative factor un
DISCUSSION
In this report, we des
Oct4 in embryonic s
First, SirT1 and Oct4
onic stem cells and a
entiation. Second, S
816 Cell Reports 17, 809–820, October 11, 2016primed EpiLC transition. Genes exhibiting up- or
downregulation during the naive and primed EpiLC
states were defined in (A). Upregulated genes were
defined as exhibiting significant increased expres-
sion in the naive state. Downregulated genes were
defined as exhibiting significant decreased expres-Figure 5. Otx2 Knockdown in SirT1-Null ES
Cells Rescues Induction of Primed EpiLC
Gene Network
(A) Heatmap comparing relative expression of genes
up- or downregulated in the EpiLC state. Genes
were chosen based on their significant differential
expression between the naive ESC and primed
EpiLC state. The relative expression of these genes
was compared among wild-type ESCs grown in
naive conditions, wild-type EpiLCs, SirT1-null ESCs
grown in naive conditions, and SirT1-null and Otx2-
knockdown ESCs grown in naive conditions. Blue
signal indicates low relative expression. Red signal
indicates high relative expression.
(B) Boxplots of expression changes in SirT1
knockout and SirT1 knockout Otx2 knockdown for
genes up- or downregulated during the naive-to-sion in the naive state.
(C) The role of SirT1, Oct4, and Otx2 during the
naive-to-primed transition. In the naive state, SirT1
deacetylates Oct4 at naive enhancers (left panels).
Naive enhancers are defined as enhancers that are
active in the naive state. During the naive-to-primed
transition, SirT1 becomes inactive, resulting in the
acetylation of Oct4. Acetylated Oct4 binds an Otx2
enhancer and drives Otx2 expression (middle
panels). In the primed state, Otx2 binds acetylated
Oct4 and directs it to primed enhancers. Primed
enhancers are defined as enhancers that are active
in the primed state.
SirT1-knockout naive ESCs and analyzing
the expression of these transcripts. There
was a clear normalization toward the wild-
type naive transcriptome profile in these
d 5B), indicating that the upregulation of
rive the shift toward the primed state in
s.
promote the primed state through the bind-
ganization of Oct4. To test if Otx2 induction
reorganization in SirT1-knockout ESCs, we
sion of genes regulated by Oct4 during the
ition. We found that suppression of Otx2 in-
kout ESCs prevented the activation or sup-
t4-regulated genes (Figure 4D, right), further
f Otx2 induction in SirT1-knockout ESCs as
derlying the shift to the primed state.
cribe a close association between SirT1 and
tem cells, both physically and functionally.
are highly expressed in pluripotent embry-
re coordinately turned off during cell differ-
irT1 and Oct4 co-immunoprecipitate, and
cells lacking SirT1 have hyper-acetylated Oct4, suggesting that
Oct4 is a substrate for deacetylation by SirT1 in stem cells.
Indeed, expression of SirT1, but not a catalytically inactive
mutant, results in Oct4 deacetylation in HEK293 cells. Third,
SirT1 and Oct4 co-occupy the same distal enhancer region in
the Oct4 promoter, along with Sox2. To probe the functional sig-
nificance of the SirT1-Oct4 interaction, we took a cue from the
fact that SirT1 activity declines during the transition from naive
to primed stem cells, and we queried whether SirT1 helps to
maintain cells in the naive state. Importantly, knocking out
SirT1 in naive cells phenocopies the transition to primed cells,
as indicated by the increase in expression of the primed cell-
induced proteins, Otx2 and Fgf5, and a decrease in the primed
cell-repressed proteins, Nanog, and Klf2. At the genome-wide
level, the transcriptome of naive cells is globally shifted toward
that of primed cells by knocking out SirT1, further indicating
that this Sirtuin helps maintain the naive state. At the molecular
level, enhancer sites bound by Oct4 also shift in cells lacking
SirT1 to mimic what happens in primed cells. At the Oct4 pro-
moter, Oct4 binding shifts from the distal to the proximal
enhancer, as does the relative activities of these two regions.
In addition, Oct4 binding to theOtx2 enhancer is greatly elevated
by knocking out SirT1, corresponding to a large increase in Otx2
expression.
What is the mechanism by which SirT1 maintains cells in the
naive state? A key step in driving cells toward the primed state
is the induction of Otx2, which interacts with Oct4 to alter the en-
hancers bound by this pluripotency factor and to generate the
primed transcriptome (Buecker et al., 2014; Yang et al., 2014).
We tested whether induction of Otx2 in SirT1-lacking cells is a
key determinant of priming by using shRNA to knock down
Otx2 back to the low levels normally observed in wild-type naive
cells. This intervention normalized the transcriptome of SirT1-
lacking cells back toward that of naive cells, strongly indicating
that the induction of Otx2 is required for inducing the primed
phenotype in SirT1-lacking cells. Since the acetylation status
of Oct4 also is increased in cells lacking SirT1 or in normal
primed cells, we suggest a model in which acetylation of Oct4
may facilitate its binding to an Otx2 enhancer to increase its
expression, followed by the redistribution of Oct4-Otx2 to
enhancer sites to generate the primed transcriptome (Figure 5C).
In addition, acetylated Oct4 may be a better binding partner for
induced Otx2 during priming. Consistent with this model, naive
reprogramming efficiency is reduced in SirT1-null cells. We
conclude that the inactivation of SirT1 is a driver of Otx2 induc-
tion and possibly the binding of Otx2 and Oct4 in the transition
from the naive to the primed state (Figure 5C).
Our findings are consistent with previous studies, which indi-
cate a role for SirT1 in induced pluripotency (Mu et al., 2015).
Loss of SirT1 decreased reprogramming efficiency of mouse
embryonic fibroblasts, an effect that was shown to involve
the SirT1-dependent deacetylation of Sox2. While acetylation
of both Sox2 (Mu et al., 2015) and Oct4 increases in ESCslacking SirT1, only Oct4 occupancy of the Otx2 enhancer
was significantly elevated, consistent with the model that acet-
ylation of Oct4 may be driving the naive-to-primed transition.
However, an important role for Sox2 in this process is certainly
possible.What is the logic in having SirT1 as an important factor in
maintaining the naive state of embryonic stem cells? It is clear
that naive cells are more robust than primed cells, both in their
growth rate and in their ability to contribute to the differentiated
cell population in injected blastocycts (Brons et al., 2007; Dods-
worth et al., 2015; Silva et al., 2008; Tesar et al., 2007). Indeed,
we found that knocking outSirT1 also slowed the growth of naive
cells and, by some assays, altered their morphology to resemble
primed cells. One key may rest in the fact that SirT1 generally
promotes cell survival, for example, by deacetylating and down-
regulating p53 and, thus, inhibiting programmed cell death. SirT1
is also critical in DNA repair, aiding both single-strand and dou-
ble-strand repair by multiple mechanisms. We suggest that, as
precursors for all the cells of the developed animal, naive cells
must be robust in their potential for duplication in a pluripotent
state and capacity for differentiation by combining extremely
high fidelity of genome maintenance and rapid cell division.
These features may be favored by high SirT1 activity in naive
cells. Our findings in this report indicate that, surprisingly, these
benefits are coupled to the cell fate decision itself between naive
and primed cells by the mechanism in Figure 5C. This coupling
may be rationalized because it would allow culling of cells with
low SirT1 activity from the naive pool and prevent failures in qual-
ity control early in development from being incorporated into the
composition of the fully developed animal.
To wit, SirT1 may be suitable as a factor that maintains fidelity
as well as the naive state of embryonic stem cells because it re-
sponds to environment cues, including food availability and
stress. It is possible that unfavorable conditions during early
stages of development can repress SirT1 activity and impose a
developmental checkpoint. For example, toxic environmental
agents could triggerDNAdamage, activation ofPARPs, depletion
ofNAD,and inactivationofSirT1, thereby imposinga roadblock to
continued embryonic development. While speculative, such a
mechanism would explain why SirT1 may be an ideal coupler of
the naive state to favorable conditions in embryonic stem cells.
The decline in SirT1 activity in the transition of naive to primed
wild-type stem cells appears to occur post-translationally.
Possible mechanisms include the induction of a SirT1 inhibitor
and a reduction in NAD levels, although the known protein inhib-
itor of SirT1, DBC1, is not induced during this transition. In this
case of NAD, we recently have observed that the addition of
an NAD precursor can facilitate the conversion of primed cells
into naive cells and inhibit the reverse process. In summary,
we have shown that SirT1 is an important factor in maintaining
the naive state of embryonic stem cells and inhibiting their
conversion into primed cells. This control of cell state may in-
crease the potential of naive cells to contribute to the body
composition with high fidelity and couple their maintenance to
favorable environmental conditions.
EXPERIMENTAL PROCEDURES
Cell Culture, Differentiation Assays, and Otx2 KnockdownNaive mouse ESCs were cultured in N2B27 media containing 2i inhibitors
(PD0325901 and CHIR99021) and LIF. ESCs grown in MEF-serum conditions
where grown on irradiated DR4 MEFs in media containing 15% fetal bovine
serum (FBS) and LIF. Embryoid bodies were formed in 10-cm Costar Ultra
Low Attachment (Corning) plates.
Cell Reports 17, 809–820, October 11, 2016 817
Differentiation of naive mouse ESCs to primed EpiLCs was performed as
previously described (Buecker et al., 2014; Hayashi et al., 2011). The mouse
ESCs were passaged three to five times in N2B27 2i + LIF and transferred
to fibronectin-coated plates. Differentiating cells were stimulated with
12 ng/mL Fgf2 (Invitrogen), 20 ng/mL Activin A (Sigma), and 1% KnockOut
Serum Replacement (Invitrogen) for 48 hr. For Otx2 knockdown, ESCs were
infected for 24 hr with lentivirus expressing shRNAs targeting Otx2 or
scrambled control, and then they were switched to selection media containing
1 mg/mL puromycin for 3–4 days.
Immunocytochemistry
ESCs were grown on coverslips and fixed with fresh 4% paraformaldehyde
(Sigma) for 15 min. For antigen retrieval, cells were incubated in PBS with
0.25% Triton X-100 for 10min. Primary antibody was applied to cells overnight
at 4
C. See Table S1 for antibody information.
Western Blot Assays
Whole-cell lysates were collected from ESCs, EpiLCs, and transfected
HEK293T cells in radio-immunoprecipitation assay (RIPA) buffer. HEK293T
cells were transfected by calcium phosphate using the following plasmids:
pCAG-Myc-Oct4-IP (Addgene 13460), pAD-Track FLAG-SIRT1 (Addgene
8438), and pAD-Track FLAG-SIRT1 H355A (Addgene 8439). CoIPs were
performed using Pierce’s direct coIP kit. See Table S1 for antibody
information.
RNA-Seq
RNA was extracted with Trizol and purified using Direct-zol RNA miniprep kit
(Zymo Research). RNA-seq libraries were sequenced using the Illumina
Hi-seq 2000 platform. FASTQ files were aligned to the NCBI37/mm9 reference
genome using the Tophat aligner. The Cuffdiff tool from the Cufflinks package
was used to detect differential expression between datasets. Significant
differential expression was defined as genes with Cuffdiff q values <0.05
and >2-fold differential expression.
ChIP qPCR
ChIPs were performed using the SimpleChIP Enzymatic Chromatin IP kit
(Cell Signaling Technology). All samples were normalized to input for qPCR
analysis. See Table S1 for oligo and antibody information.
Oct4 Enhancer Luciferase Reporter Assay
Mouse Oct4 proximal and distal enhancers were cloned into pGL3 as
previously characterized (Tesar et al., 2007). Constructs were co-transfected
with pRL-TK (Renilla) using Xfect mouse ESC transfection reagent (Clontech
Laboratories) and incubated for 48 hr. Luciferase activity was assessed using
the Dual-Glo Luciferase Assay System (Promega). Oct4 enhancer activity
(Firefly) was normalized to Renilla.
Statistical Methods
Unpaired two-tailed Student’s t tests were used to assess statistical signifi-
cance for experiments involving qPCR, flow cytometry, luciferase assays,
cell growth analysis, and clonogenicity assays. Differential expression within
RNA-seq datasets was defined as genes with Cuffdiff q values <0.05 and
>2-fold differential expression. Statistical significance of correlated gene
expression profiles was assessed by Pearson correlation.
Mice
All animal experiments were performed in accordance with protocols
approved the Institutional Animal Care and Use Committee of Massachusetts
Institute of Technology.
For more detailed descriptions, see the Supplemental Experimental
Procedures.ACCESSION NUMBERS
The accession number for the RNA-seq data reported in this paper is GEO:
GSE81494.
818 Cell Reports 17, 809–820, October 11, 2016SUPPLEMENTAL INFORMATION
Supplemental Information includes Supplemental Experimental Procedures,
five figures, and one table and can be found with this article online at http://
dx.doi.org/10.1016/j.celrep.2016.09.046.
AUTHOR CONTRIBUTIONS
Conceptualization, E.O.W. and L.G.; Methodology, E.O.W., E.L.B., and L.G.;
Formal Analysis, E.O.W.; Investigation, E.O.W., A.K.T., R.L., and D.M.K.;
Writing – Original Draft, E.O.W. and L.G.; Writing – Review & Editing, E.O.W.
and L.G.; Funding Acquisition, E.O.W. and L.G.; Supervision, E.O.W. and L.G.
ACKNOWLEDGMENTS
We thank the Jaenisch lab for luciferase constructs and Michael Williams for
bioinformatics and statistical advice. This work was supported by grants
from the NIH and The Glenn Foundation for Medical Research (L.G.) and the
American Cancer Society (E.O.W.). L.G. is a founder of Elysium Health and
consults for GSK, Segterra, and Chronos (Oxford).
Received: February 23, 2016
Revised: June 14, 2016
Accepted: September 15, 2016
Published: October 11, 2016
REFERENCES
Acampora, D., Di Giovannantonio, L.G., and Simeone, A. (2013). Otx2 is an
intrinsic determinant of the embryonic stem cell state and is required for
transition to a stable epiblast stem cell condition. Development 140, 43–55.
Arnold, S.J., and Robertson, E.J. (2009). Making a commitment: cell lineage
allocation and axis patterning in the early mouse embryo. Nat. Rev. Mol. Cell
Biol. 10, 91–103.
Bao, S., Tang, F., Li, X., Hayashi, K., Gillich, A., Lao, K., and Surani, M.A.
(2009). Epigenetic reversion of post-implantation epiblast to pluripotent
embryonic stem cells. Nature 461, 1292–1295.
Bernemann, C., Greber, B., Ko, K., Sterneckert, J., Han, D.W., Arau´zo-Bravo,
M.J., and Scho¨ler, H.R. (2011). Distinct developmental ground states of
epiblast stem cell lines determine different pluripotency features. Stem Cells
29, 1496–1503.
Brons, I.G.M., Smithers, L.E., Trotter, M.W.B., Rugg-Gunn, P., Sun, B., Chuva
de Sousa Lopes, S.M., Howlett, S.K., Clarkson, A., Ahrlund-Richter, L., Peder-
sen, R.A., and Vallier, L. (2007). Derivation of pluripotent epiblast stem cells
from mammalian embryos. Nature 448, 191–195.
Buecker, C., Chen, H.-H., Polo, J.M., Daheron, L., Bu, L., Barakat, T.S.,
Okwieka, P., Porter, A., Gribnau, J., Hochedlinger, K., and Geijsen, N.
(2010). A murine ESC-like state facilitates transgenesis and homologous
recombination in human pluripotent stem cells. Cell Stem Cell 6, 535–546.
Buecker, C., Srinivasan, R., Wu, Z., Calo, E., Acampora, D., Faial, T., Simeone,
A., Tan, M., Swigut, T., and Wysocka, J. (2014). Reorganization of enhancer
patterns in transition from naive to primed pluripotency. Cell Stem Cell 14,
838–853.
Calvanese, V., Lara, E., Sua´rez-Alvarez, B., Abu Dawud, R., Va´zquez-Chan-
tada, M., Martı´nez-Chantar, M.L., Embade, N., Lo´pez-Nieva, P., Horrillo, A.,
Hmadcha, A., et al. (2010). Sirtuin 1 regulation of developmental genes during
differentiation of stem cells. Proc. Natl. Acad. Sci. USA 107, 13736–13741.
Chan, Y.-S., Go¨ke, J., Ng, J.-H., Lu, X., Gonzales, K.A.U., Tan, C.-P., Tng,
W.-Q., Hong, Z.-Z., Lim, Y.-S., and Ng, H.-H. (2013). Induction of a human
pluripotent state with distinct regulatory circuitry that resembles preimplanta-tion epiblast. Cell Stem Cell 13, 663–675.
Cheng, H.-L., Mostoslavsky, R., Saito, S., Manis, J.P., Gu, Y., Patel, P., Bron-
son, R., Appella, E., Alt, F.W., and Chua, K.F. (2003). Developmental defects
and p53 hyperacetylation in Sir2 homolog (SIRT1)-deficient mice. Proc. Natl.
Acad. Sci. USA 100, 10794–10799.
Chew, J.L., Loh, Y.H., Zhang, W., Chen, X., Tam, W.L., Yeap, L.S., Li, P., Ang,
Y.S., Lim, B., Robson, P., and Ng, H.H. (2005). Reciprocal transcriptional regu-
lation of Pou5f1 and Sox2 via the Oct4/Sox2 complex in embryonic stem cells.
Mol. Cell. Biol. 25, 6031–6046.
Desbaillets, I., Ziegler, U., Groscurth, P., and Gassmann, M. (2000). Embryoid
bodies: an in vitro model of mouse embryogenesis. Exp. Physiol. 85, 645–651.
Dodsworth, B.T., Flynn, R., and Cowley, S.A. (2015). The current state of naı¨ve
human pluripotency. Stem Cells 33, 3181–3186.
Duggal, G., Warrier, S., Ghimire, S., Broekaert, D., Van der Jeught, M., Lier-
man, S., Deroo, T., Peelman, L., Van Soom, A., Cornelissen, R., et al. (2015).
Alternative routes to induce naı¨ve pluripotency in human embryonic stem cells.
Stem Cells 33, 2686–2698.
Evans, M.J., and Kaufman,M.H. (1981). Establishment in culture of pluripoten-
tial cells from mouse embryos. Nature 292, 154–156.
Factor, D.C., Corradin, O., Zentner, G.E., Saiakhova, A., Song, L., Chenoweth,
J.G., McKay, R.D., Crawford, G.E., Scacheri, P.C., and Tesar, P.J. (2014).
Epigenomic comparison reveals activation of ‘‘seed’’ enhancers during transi-
tion from naive to primed pluripotency. Cell Stem Cell 14, 854–863.
Gafni, O., Weinberger, L., Mansour, A.A., Manor, Y.S., Chomsky, E., Ben-Yo-
sef, D., Kalma, Y., Viukov, S., Maza, I., Zviran, A., et al. (2013). Derivation of
novel human ground state naive pluripotent stem cells. Nature 504, 282–286.
Guo, G., Yang, J., Nichols, J., Hall, J.S., Eyres, I., Mansfield, W., and Smith, A.
(2009). Klf4 reverts developmentally programmed restriction of ground state
pluripotency. Development 136, 1063–1069.
Han, D.W., Tapia, N., Joo, J.Y., Greber, B., Arau´zo-Bravo, M.J., Bernemann,
C., Ko, K., Wu, G., Stehling, M., Do, J.T., and Scho¨ler, H.R. (2010). Epiblast
stem cell subpopulations represent mouse embryos of distinct pregastrulation
stages. Cell 143, 617–627.
Hanna, J., Cheng, A.W., Saha, K., Kim, J., Lengner, C.J., Soldner, F., Cassady,
J.P., Muffat, J., Carey, B.W., and Jaenisch, R. (2010). Human embryonic stem
cells with biological and epigenetic characteristics similar to those of mouse
ESCs. Proc. Natl. Acad. Sci. USA 107, 9222–9227.
Hayashi, K., Ohta, H., Kurimoto, K., Aramaki, S., and Saitou, M. (2011). Recon-
stitution of the mouse germ cell specification pathway in culture by pluripotent
stem cells. Cell 146, 519–532.
Heard, E. (2004). Recent advances in X-chromosome inactivation. Curr. Opin.
Cell Biol. 16, 247–255.
Heintzman, N.D., Stuart, R.K., Hon, G., Fu, Y., Ching, C.W., Hawkins, R.D.,
Barrera, L.O., Van Calcar, S., Qu, C., Ching, K.A., et al. (2007). Distinct and
predictive chromatin signatures of transcriptional promoters and enhancers
in the human genome. Nat. Genet. 39, 311–318.
Ito, A., Kawaguchi, Y., Lai, C.-H., Kovacs, J.J., Higashimoto, Y., Appella, E.,
and Yao, T.-P. (2002). MDM2-HDAC1-mediated deacetylation of p53 is
required for its degradation. EMBO J. 21, 6236–6245.
Kim, H., Wu, J., Ye, S., Tai, C.-I., Zhou, X., Yan, H., Li, P., Pera, M., and Ying,
Q.-L. (2013). Modulation of b-catenin function maintains mouse epiblast stem
cell and human embryonic stem cell self-renewal. Nat. Commun. 4, 2403.
Kojima, Y., Kaufman-Francis, K., Studdert, J.B., Steiner, K.A., Power, M.D.,
Loebel, D.A., Jones, V., Hor, A., de Alencastro, G., Logan, G.J., et al. (2014).
The transcriptional and functional properties of mouse epiblast stem cells
resemble the anterior primitive streak. Cell Stem Cell 14, 107–120.
Kunath, T., Saba-El-Leil, M.K., Almousailleakh, M., Wray, J., Meloche, S., and
Smith, A. (2007). FGF stimulation of the Erk1/2 signalling cascade triggers tran-
sition of pluripotent embryonic stem cells from self-renewal to lineage commit-
ment. Development 134, 2895–2902.
Matsuda, T., Nakamura, T., Nakao, K., Arai, T., Katsuki, M., Heike, T., and
Yokota, T. (1999). STAT3 activation is sufficient to maintain an undifferentiated
state of mouse embryonic stem cells. EMBO J. 18, 4261–4269.McBurney, M.W., Yang, X., Jardine, K., Bieman,M., Th’ng, J., and Lemieux, M.
(2003a). The absence of SIR2alpha protein has no effect on global gene
silencing in mouse embryonic stem cells. Mol. Cancer Res. 1, 402–409.
McBurney, M.W., Yang, X., Jardine, K., Hixon, M., Boekelheide, K., Webb,
J.R., Lansdorp, P.M., and Lemieux, M. (2003b). The mammalian SIR2alphaprotein has a role in embryogenesis and gametogenesis. Mol. Cell. Biol. 23,
38–54.
Mu, W.-L., Wang, Y.-J., Xu, P., Hao, D.-L., Liu, X.-Z., Wang, T.-T., Chen, F.,
Chen, H.-Z., Lv, X., and Liu, D.-P. (2015). Sox2 deacetylation by Sirt1 is
involved in mouse somatic reprogramming. Stem Cells 33, 2135–2147.
Murtha, M., Strino, F., Tokcaer-Keskin, Z., Sumru Bayin, N., Shalabi, D., Xi, X.,
Kluger, Y., and Dailey, L. (2015). Comparative FAIRE-seq analysis reveals
distinguishing features of the chromatin structure of ground state- and
primed-pluripotent cells. Stem Cells 33, 378–391.
Napper, A.D., Hixon, J., McDonagh, T., Keavey, K., Pons, J.-F., Barker, J.,
Yau, W.T., Amouzegh, P., Flegg, A., Hamelin, E., et al. (2005). Discovery of in-
doles as potent and selective inhibitors of the deacetylase SIRT1. J. Med.
Chem. 48, 8045–8054.
Nichols, J., and Smith, A. (2009). Naive and primed pluripotent states. Cell
Stem Cell 4, 487–492.
Nichols, J., and Smith, A. (2011). The origin and identity of embryonic stem
cells. Development 138, 3–8.
Nichols, J., Zevnik, B., Anastassiadis, K., Niwa, H., Klewe-Nebenius, D.,
Chambers, I., Scho¨ler, H., and Smith, A. (1998). Formation of pluripotent
stem cells in the mammalian embryo depends on the POU transcription factor
Oct4. Cell 95, 379–391.
Niwa, H. (2007). How is pluripotency determined and maintained? Develop-
ment 134, 635–646.
Niwa, H., Ogawa, K., Shimosato, D., and Adachi, K. (2009). A parallel circuit of
LIF signalling pathways maintains pluripotency of mouse ES cells. Nature 460,
118–122.
Osafune, K., Caron, L., Borowiak, M., Martinez, R.J., Fitz-Gerald, C.S., Sato,
Y., Cowan, C.A., Chien, K.R., and Melton, D.A. (2008). Marked differences in
differentiation propensity among human embryonic stem cell lines. Nat.
Biotechnol. 26, 313–315.
Radzisheuskaya, A., Chia Gle, B., dos Santos, R.L., Theunissen, T.W., Castro,
L.F., Nichols, J., and Silva, J.C. (2013). A defined Oct4 level governs cell state
transitions of pluripotency entry and differentiation into all embryonic lineages.
Nat. Cell Biol. 15, 579–590.
Rodgers, J.T., Lerin, C., Haas, W., Gygi, S.P., Spiegelman, B.M., and
Puigserver, P. (2005). Nutrient control of glucose homeostasis through a
complex of PGC-1alpha and SIRT1. Nature 434, 113–118.
Rossant, J. (2008). Stem cells and early lineage development. Cell 132,
527–531.
Sakamoto, J., Miura, T., Shimamoto, K., and Horio, Y. (2004). Predominant
expression of Sir2a, an NAD-dependent histone deacetylase, in the embryonic
mouse heart and brain. FEBS Lett. 556, 281–286.
Silva, J., and Smith, A. (2008). Capturing pluripotency. Cell 132, 532–536.
Silva, J., Barrandon, O., Nichols, J., Kawaguchi, J., Theunissen, T.W., and
Smith, A. (2008). Promotion of reprogramming to ground state pluripotency
by signal inhibition. PLoS Biol. 6, e253.
Silva, J., Nichols, J., Theunissen, T.W., Guo, G., van Oosten, A.L., Barrandon,
O., Wray, J., Yamanaka, S., Chambers, I., and Smith, A. (2009). Nanog is the
gateway to the pluripotent ground state. Cell 138, 722–737.
Sohni, A., Bartoccetti, M., Khoueiry, R., Spans, L., Vande Velde, J., De Troyer,
L., Pulakanti, K., Claessens, F., Rao, S., and Koh, K.P. (2015). Dynamic
switching of active promoter and enhancer domains regulates Tet1 and Tet2
expression during cell state transitions between pluripotency and differentia-
tion. Mol. Cell. Biol. 35, 1026–1042.
Solomon, J.M., Pasupuleti, R., Xu, L., McDonagh, T., Curtis, R., DiStefano,
P.S., and Huber, L.J. (2006). Inhibition of SIRT1 catalytic activity increasesp53 acetylation but does not alter cell survival following DNA damage. Mol.
Cell. Biol. 26, 28–38.
Takahashi, K., and Yamanaka, S. (2006). Induction of pluripotent stem cells
from mouse embryonic and adult fibroblast cultures by defined factors. Cell
126, 663–676.
Cell Reports 17, 809–820, October 11, 2016 819
Takahashi, K., Tanabe, K., Ohnuki, M., Narita, M., Ichisaka, T., Tomoda, K.,
and Yamanaka, S. (2007). Induction of pluripotent stem cells from adult human
fibroblasts by defined factors. Cell 131, 861–872.
Takashima, Y., Guo, G., Loos, R., Nichols, J., Ficz, G., Krueger, F., Oxley, D.,
Santos, F., Clarke, J., Mansfield,W., et al. (2014). Resetting transcription factor
control circuitry toward ground-state pluripotency in human. Cell 158, 1254–
1269.
Tesar, P.J., Chenoweth, J.G., Brook, F.A., Davies, T.J., Evans, E.P., Mack,
D.L., Gardner, R.L., and McKay, R.D. (2007). New cell lines from mouse
epiblast share defining features with human embryonic stem cells. Nature
448, 196–199.
Theunissen, T.W., Powell, B.E., Wang, H., Mitalipova, M., Faddah, D.A.,
Reddy, J., Fan, Z.P., Maetzel, D., Ganz, K., Shi, L., et al. (2014). Systematic
identification of culture conditions for induction and maintenance of naive
human pluripotency. Cell Stem Cell 15, 471–487.
Thomson, J.A., Itskovitz-Eldor, J., Shapiro, S.S., Waknitz, M.A., Swiergiel, J.J.,
Marshall, V.S., and Jones, J.M. (1998). Embryonic stem cell lines derived from
human blastocysts. Science 282, 1145–1147.
Torres-Padilla, M.-E., and Chambers, I. (2014). Transcription factor heteroge-
neity in pluripotent stem cells: a stochastic advantage. Development 141,
2173–2181.
Valamehr, B., Robinson, M., Abujarour, R., Rezner, B., Vranceanu, F., Le, T.,
Medcalf, A., Lee, T.T., Fitch, M., Robbins, D., and Flynn, P. (2014). Platform
for induction and maintenance of transgene-free hiPSCs resembling ground
state pluripotent stem cells. Stem Cell Reports 2, 366–381.
Vaziri, H., Dessain, S.K., Ng Eaton, E., Imai, S.-I., Frye, R.A., Pandita, T.K.,
Ware, C.B., Nelson, A.M., Mecham, B., Hesson, J., Zhou, W., Jonlin, E.C.,
Jimenez-Caliani, A.J., Deng, X., Cavanaugh, C., Cook, S., et al. (2014). Deriva-
tion of naive human embryonic stem cells. Proc. Natl. Acad. Sci. USA 111,
4484–4489.
Yang, S.-H., Kalkan, T., Morissroe, C., Marks, H., Stunnenberg, H., Smith, A.,
and Sharrocks, A.D. (2014). Otx2 and Oct4 drive early enhancer activation
during embryonic stem cell transition from naive pluripotency. Cell Rep. 7,
1968–1981.
Yeo, J.C., Jiang, J., Tan, Z.Y., Yim, G.R., Ng, J.H., Go¨ke, J., Kraus, P., Liang,
H., Gonzales, K.A.U., Chong, H.C., et al. (2014). Klf2 is an essential factor that
sustains ground state pluripotency. Cell Stem Cell 14, 864–872.
Yeom, Y.I., Fuhrmann, G., Ovitt, C.E., Brehm, A., Ohbo, K., Gross, M., H€ubner,
K., and Scho¨ler, H.R. (1996). Germline regulatory element of Oct-4 specific for
the totipotent cycle of embryonal cells. Development 122, 881–894.
Ying, Q.-L., Nichols, J., Chambers, I., and Smith, A. (2003). BMP induction of Id
proteins suppresses differentiation and sustains embryonic stem cell self-
renewal in collaboration with STAT3. Cell 115, 281–292.
Ying, Q.-L., Wray, J., Nichols, J., Batlle-Morera, L., Doble, B., Woodgett, J.,
Cohen, P., and Smith, A. (2008). The ground state of embryonic stem cell
self-renewal. Nature 453, 519–523.
Yu, J., Vodyanik, M.A., Smuga-Otto, K., Antosiewicz-Bourget, J., Frane, J.L.,
Tian, S., Nie, J., Jonsdottir, G.A., Ruotti, V., Stewart, R., et al. (2007). Induced
pluripotent stem cell lines derived from human somatic cells. Science 318,
1917–1920.Guarente, L., and Weinberg, R.A. (2001). hSIR2(SIRT1) functions as an
NAD-dependent p53 deacetylase. Cell 107, 149–159.
Wang, W., Yang, J., Liu, H., Lu, D., Chen, X., Zenonos, Z., Campos, L.S., Rad,
R., Guo, G., Zhang, S., et al. (2011). Rapid and efficient reprogramming of so-
matic cells to induced pluripotent stem cells by retinoic acid receptor gamma
and liver receptor homolog 1. Proc. Natl. Acad. Sci. USA 108, 18283–18288.820 Cell Reports 17, 809–820, October 11, 2016Zhang, Z.N., Chung, S.K., Xu, Z., and Xu, Y. (2014). Oct4 maintains the
pluripotency of human embryonic stem cells by inactivating p53 through
Sirt1-mediated deacetylation. Stem Cells 32, 157–165.
Zwaka, T.P., and Thomson, J.A. (2003). Homologous recombination in human
embryonic stem cells. Nat. Biotechnol. 21, 319–321.