Resource
eveals Gradual Chromatin
during
Highlights
d Soft X-ray tomography reveals chromatin networks in
olfactory neurons
d Chromatin compaction increases during olfactory
neurogenesis
g
Authors
Mark A. Le Gros, E. Josephine Clowney,
Angeliki Magklara, ..., Manolis Kellis,
Stavros Lomvardas, Carolyn A. Larabell
Correspondence
carolyn.larabell@ucsf.edu
In Brief
As part of the IHEC consortium, Le GrosLe Gros et al., 2016, Cell Reports 17, 2125–2136
November 15, 2016 ª 2016 The Author(s).
http://dx.doi.org/10.1016/j.celrep.2016.10.060d HP1b regulates reorganization of chromatin in mature
neuronsd Condensed chromatin moves to nuclear core durin
differentiationCompaction and Reorganization
Neurogenesis In Vivo
Graphical AbstractSoft X-Ray Tomography Ret al. characterize nuclear organization in
mouse olfactory neurons throughout
differentiation. Quantitative 3D x-ray
reconstructions reveal distinct chromatin
compartments that form an
interconnected network that spans the
nucleus and persists during nuclear
reorganization. Explore the Cell Press
IHEC webportal at www.cell.com/
consortium/IHEC.
Cell Reports
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aINTRODUCTION
Cellular differentiation is a stepwise process characterized by
et al., 2013; Solovei et al., 2013). These spatial translocations can
be extremely specific, highly coordinated, and critical for proper
development. For example, the recruitment of specific genes toloss of transcriptional pluripotency and commitment to irrevers-
ible expression programs. It is postulated that the loss of nu-
the nuclear lamina of differentiating neuroblasts in Drosophila is
essential for the silencing of specific transcription factors and theThe realization that nuclear distribution of DNA, RNA,
and proteins differs between cell types and develop-
mental stages suggests that nuclear organization
serves regulatory functions. Understanding the logic
of nuclear architecture and how it contributes to dif-
ferentiation and cell fate commitment remains chal-
lenging. Here, we use soft X-ray tomography (SXT)
to image chromatin organization, distribution, and
biophysical properties during neurogenesis in vivo.
Our analyses reveal that chromatin with similar bio-
physical properties forms an elaborate connected
network throughout the entire nucleus. Although
this interconnectivity is present in every develop-
mental stage, differentiation proceeds with concom-
itant increase in chromatin compaction and re-distri-
bution of condensed chromatin toward the nuclear
core. HP1b, but not nucleosome spacing or phasing,
regulates chromatin rearrangements because it gov-
erns both the compaction of chromatin and its inter-
actions with the nuclear envelope. Our experiments
introduce SXT as a powerful imaging technology for
nuclear architecture.
by epigenetic repression and chromatin-mediated silencing of
genes that preserve multipotency (Waddington, 1957). Indeed,
epigenetic changes during embryonic stem cell differentiation
are consistent with cellular commitment relying on the stable
repression of genes that promote pluri- and multi-potency, as
well as genes compatible with alternate differentiation pro-
grams, through progressive chromatin restriction (Hawkins
et al., 2010; Li et al., 2012b; Zhang et al., 2012; Zhu et al.,
2013). Genes governing cellular differentiation, on the other
hand, are not stably silenced in progenitor cells; rather, they
are retained in a repressed but ‘‘poised’’ or bivalent epigenetic
state until external cues allow their expression (Bernstein et al.,
2006). Consequently, fully differentiated or lineage committed
cells have a more restrictive epigenetic landscape than embry-
onic stem cells.
In addition to differentiation-dependent epigenetic restric-
tions, recent findings reveal that cellular differentiation also in-
volves general and gene-specific rearrangements in nuclear
organization (Bickmore and van Steensel, 2013; Cavalli and Mis-
teli, 2013; Dekker and Mirny, 2016; Van Bortle and Corces,
2013). The nuclear lamina appears to play an organizing role
for these rearrangements, because cell-type-dependent varia-
tions in its protein composition determines the recruitment of
specific genomic domains toward the nuclear envelope with
various transcriptional consequences and effects on differentia-
tion (Clowney et al., 2012; Gonzalez-Sandoval et al., 2015; KohwiResource
Soft X-Ray Tomography Rev
Gradual Chromatin Compac
and Reorganization during N
Mark A. Le Gros,1,2,3,4 E. Josephine Clowney,5 Angeliki Mag
Bradley Colquitt,9 Markko Myllys,10 Manolis Kellis,7,8 Stavro
1Department of Anatomy, University of California San Francisco, San
2Physical Biosciences Division, Lawrence Berkeley National Laborato
3National Center for X-Ray Tomography, University of California San
4Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA
5Program in Biomedical Sciences, University of California San Franci
6Division of Biomedical Research, Institute of Molecular Biology and
Ioannina, Greece
7Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA
8Computer Science and Artificial Intelligence Laboratory, MIT, Camb
9Program in Neurosciences, University of California San Francisco, S
10Department of Physics, University of Jyva¨skyla¨, Jyva¨skyla¨ 40014, F
11Present address: Columbia University Department of Biochemistry
and Behavior Institute, New York, NY 10027, USA
12Lead Contact
*Correspondence: carolyn.larabell@ucsf.edu
http://dx.doi.org/10.1016/j.celrep.2016.10.060
SUMMARYCell Repor
This is an open access article under the CC BY-Neals
ion
eurogenesis In Vivo
lara,6 Angela Yen,7,8 Eirene Markenscoff-Papadimitriou,9
Lomvardas,1,5,9,11 and Carolyn A. Larabell1,2,3,12,*
rancisco, CA 94158, USA
y, Berkeley, CA 94720, USA
rancisco, San Francisco, CA 94158, USA
o, San Francisco, CA 94158, USA
iotechnology, Foundation for Research and Technology-Hellas,
ge, MA 02139, USA
n Francisco, CA 94158, USA
land
nd Molecular Biophysics, Zuckerman Mind, Brain,
clear plasticity that accompanies differentiation is mediatedts 17, 2125–2136, November 15, 2016 ª 2016 The Author(s). 2125
C-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
developmental transition to subsequent differentiation stages
(Kohwi et al., 2013).
These observations suggest that cellular differentiation and
fate commitment proceed, via substantial overhaul of the
biochemical properties and 3D distribution of chromatin in vivo,
to generate nuclei specialized for the transcriptional needs of
each cell type (Alexander and Lomvardas, 2014). However, the
lack of available technologies that can resolve both the biophys-
ical state and the conformation of the genome with high spatial
precision has prevented a comprehensive and quantitative
experimental demonstration of this concept. These technical
limitations can be circumvented by soft X-ray tomography
(SXT), which allows imaging of intact eukaryotic cells in a near-
native state at a resolution of 
50 nm (Le Gros et al., 2012;
McDermott et al., 2009, 2012a, 2012b). Because SXT was
used successfully to image olfactory sensory neurons (Clowney
et al., 2012), we sought to use the continuously regenerating
main olfactory epithelium (MOE) as an in vivo model for the study
of chromatin alterations during neurogenesis and differentiation.
SXT on multipotent stem cells, neuronal progenitors, and termi-
nally differentiated post-mitotic neurons from the MOE revealed
gradual chromatin compaction and increase of heterochromati-
nization during differentiation. Surprisingly, the increase of chro-
matin condensation is not dependent upon significant changes
in nucleosome phasing or spacing, but relies on the function of
heterochromatin binding protein HP1b, because loss of function
experiments revealed substantial loss of chromatin condensa-
tion in HP1b knockout (KO) post-mitotic neurons.
Our SXT data reveal distinct compartmentalization of chro-
matin into two regions that have different degrees of ‘‘crowding’’
or ‘‘compaction.’’ Global 3D views of the nucleus reveal that
chromatin regions with similar compaction profiles are con-
nected and form a continuous network throughout the entire nu-
cleus. Finally, our analysis demonstrates that HP1b contributes
not only to the increased condensation of heterochromatin but
also to the tethering of condensed chromatin to the nuclear en-
velope corroborating the hypothesis that nuclear architecture is
organized by the interplay of histonemodifications, their readers,
and nuclear envelope proteins that interact with them (Clowney
et al., 2012; Kind et al., 2013; Pinheiro et al., 2012; Solovei
et al., 2013; Towbin et al., 2012; Worman et al., 1988; Ye et al.,
1997; Ye and Worman, 1996).
RESULTS
Imaging Intact Cells in 3D
We used SXT to image three cell types of the olfactory epithe-
lium, horizontal basal cells (HBCs), globose basal cells
(GBCs), and mature olfactory sensory neurons (mOSNs) that
correspond to multipotent stem cells, neuronal progenitors,
and terminally differentiated neurons, respectively. SXT images
are collected using photons within the ‘‘water window’’ (284–
543 eV), where biomolecules absorb X-rays an order of magni-tude more than the surrounding water. Because absorption
adheres to the Beer-Lambert Law, it is linear with biochemical
composition and concentration, generating a unique X-ray linear
absorption coefficient (LAC) measurement for each voxel (3D
pixel analog), as shown in Figure 1 (Larabell and Le Gros,
2126 Cell Reports 17, 2125–2136, November 15, 20162004; McDermott et al., 2009, 2012b). Heterochromatin, due
to an increased biomolecular concentration, appears darker
than euchromatin in computer-generated SXT orthoslices (vir-
tual sections) through the nucleus (Figure 1B) and is shown to
map to the high-LAC voxels of the nucleus using the software
program, Amira (Figure 1A, blue peak). To demonstrate the
reproducible, quantitative capabilities of SXT LAC measure-
ments, we measured the LAC of increasing concentrations of
BSA and hemoglobin in vitro (Hanssen et al., 2012) and in vivo;
the LAC of alcohol oxidase crystals in yeast is 0.626 mm1 (Fig-
ure 1C), which is almost identical to the predicted LAC values
based on theoretical calculations (0.625 mm1) (Henke et al.,
1993). We used the distinct SXT LAC values to automatically
segment and color-code select cellular structures for 3D visual-
ization of spatial information during differentiation, revealing
changes in nuclear organization and chromatin topology (Fig-
ure 1E; Movies S1, S2, and S3).
Measuring Chromatin Compaction during
Differentiation
The fact that chromatin has a rather stable macromolecular
composition of only nucleic acids and proteins, but has various
degrees of compaction (i.e., various concentrations of these
macromolecules per voxel), makes SXT an ideal method to
quantify changes in chromatin compaction during olfactory
epithelium differentiation. In all cells imaged, the mean hetero-
chromatin LAC is 0.32 ± 0.02 mm1 and the mean euchromatin
LAC is 0.22 ± 0.02 mm1, indicating that heterochromatin is

30% more compacted than euchromatin. Heterochromatin
LAC remains fairly constant during differentiation, whereas
euchromatin LAC increases from 0.18 ± 0.01 mm1 in stem cells
to 0.21 ± 0.02 mm1 in neuronal progenitors and 0.23 ±
0.01 mm1 in mature neurons (Table S1). At the same time,
the relative ratio of heterochromatin to euchromatin increases,
suggesting an increasingly restrictive epigenetic landscape.
Heterochromatin proportions increase from 0.33 ± 0.01 in
stem cells to 0.36 ± 0.02 in progenitors and 0.41 ± 0.01 in
mature neurons (Figures 2A–2D; Table S1). This gradual in-
crease is consistent with biochemical experiments in fluores-
cence-activated cell sorting (FACS) cells from the olfactory
epithelium, showing that 40 MB of the olfactory receptor
subgenome is not heterochromatic in stem cell populations of
the olfactory epithelium, but instead heterochromatinization of
receptor clusters initiates at the progenitor cell stage and is fully
established in mature olfactory neurons (Magklara et al., 2011).
The LAC values at all stages of differentiation are significantly
lower than the LAC values of more tightly packed sperm
chromatin (0.71 mm1; Figure 1D) or the previously described
olfactory receptor aggregates in mature neurons (0.55 mm1)
(Clowney et al., 2012). Interestingly, only the aggregates
approach the theoretical LAC values of the 30 nm fiber
(0.44 mm1), in agreement with recent cryo-electron spectro-
scopic imaging describing 10 but not 30 nm fibers in nuclearsections (Fussner et al., 2012; Nishino et al., 2012).
Previous reports suggest that transition from euchromatin to
heterochromatin in vivo coincides with increased nucleosome
phasing (Danzer and Wallrath, 2004). To obtain structural insight
on the potential mechanisms resulting in differences of LAC
values between euchromatin and heterochromatin, we per-
formed genome-wide nucleosome mapping in the three cell
types. Stem cells, neuronal progenitors, and mature olfactory
sensory neurons were isolated by FACS and treated with micro-
coccal nuclease (MNase). DNA isolated from mono-nucleo-
somes was subjected to Illumina sequencing (see the Supple-
mental Information). To correlate LAC values with chromatin
structure, we examined the phasing of nucleosomes over
highly transcribed (top 25%of geneswith fragments per kilobase
of transcript per million [FPKM] > 0) and completely silent
(FPKM = 0) genes, which we used to represent euchromatic
and heterochromatic regions. In stem and progenitor cells, silent
and active genes have almost indistinguishable nucleosome
phasing, and only in mature neurons do we detect phasing or
spacing differences between heterochromatin and euchromatin
Moreover, despite an
values in mature cell
phasing or spacing do
entiated cells, sugges
has minimal influenc
compaction in interph
and compaction of ol
transition from stem
et al., 2011), nucleoso
same in the three cell
HP1b Regulates He
Mature Olfactory Ne
Because changes in
correlate with differen
Cell Report(B) One orthoslice, or virtual section, from the X-ray
tomographic reconstruction showing the nucleus
(outlined with red) and surrounding cytoplasm.
Visualization software (Amira) reveals the high-
contrast (dark) heterochromatinmaps to the higher
LAC voxels of the nucleus histogram (blue) and
lower contrast (light) euchromatin maps to the
low-LAC regions (green).
(C) Orthoslice fromSXT of the yeast,Pichia stipites,
showing an alcohol oxidase crystal (arrow). The
measured LAC value of the crystal is 0.626 mm1Figure 1. Nuclear Organization and Chro-
matin Topology in Olfactory Epithelial Cells
(A) Histogram plotting the volume (number of
voxels) in an olfactory sensory neuron at each
linear absorption coefficient (LAC) value. There is a
bimodal distribution of nuclear voxels, with regions
containing fewer biomolecules per voxel (lower
LAC values) color-coded shades of green and the
more crowded regions color-coded shades of blue
(higher LAC values); the cytoplasmic voxels are
plotted in shades of gray.and the calculated LAC value (based on the atomic
composition of the crystal) is 0.625 mm1, con-
firming LACmeasurements in vivo are quantitative.
(D) Sperm chromatin has a LAC value = 0.71 mm1,
indicating it is 
50 times more densely packed
than the most dense portions of heterochromatin.
(E) Reorganization of cells obtained from mouse ol-
factory epithelium—multipotent stem cell, neuronal
progenitor cell, and mature olfactory sensory
neuron—during differentiation, segmented, and
color-coded to show heterochromatin (blue),
euchromatin (green), and mitochondria (copper).
Three orthogonal views through the nucleus (3D
cutaway) reveal the shift of pericentromeric hetero-
chromatin to the nucleus center. Scale bar, 1 mm.
(Figures 2E–2H and S1A). Notably, these
differences do not stem only from an in-
crease in heterochromatin phasing but
also from a decrease in euchromatin
phasing, likely related to the significant in-
crease of gene body hydroxymethylation
in highly transcribed genes of mature
olfactory neurons (Colquitt et al., 2013).
overall increase of X-ray absorption (LAC)
s, genome-wide analysis of nucleosome
es not reveal any differences fromundiffer-
ting that primary nucleosome architecture
e on the tertiary chromatin folding and
ase. Indeed, although DNase protection
factory receptor loci increases during the
cell to the neuronal lineage (Magklara
me phasing on the receptors remains the
populations (Figure S1B).
terochromatin Compaction in
urons
nucleosome phasing and spacing do not
ces in chromatin compaction, we sought
s 17, 2125–2136, November 15, 2016 2127
other factors that might influence the three-dimensional packing
of chromatin fibers. Recent studies revealed that HP1 proteins
have homo-polymerization properties essential for heterochro-
matin spreading (Canzio et al., 2011, 2013), which may also
assist the proper association and folding of heterochromatin in
trans. Because HP1b expression pattern in the olfactory epithe-
lium follows the differentiation-dependent increase in chromatin
compaction that we describe here, i.e., its expression starts in
progenitors and peaks in mature neurons (Clowney et al.,
2012), we sought to analyze the role of HP1b in chromatin
compaction. We analyzed mature neurons from HP1b KO
(Skarnes et al., 2011) and wild-type littermates. The analysis
was performed at E17.5 because the HP1b KO die perinatally,
however, mature neurons at this age have the same LAC proper-
ties and nuclear organization as those from adult mice. Loss of
HP1b has profound effects on chromatin compaction in olfactory
tween the fraction of
ure 3D; Table S1).
Imaging Chromatin
To determine whether
chromatin compaction
architecture, we mapp
euchromatin during di
nuclei examined, a 20
envelope contains pre
described for many ot
and S7). However, the
dramatically different
80%–97%of the chrom
is heterochromatin, wh
is mostly euchromatin
2128 Cell Reports 17, 2125–2136, November 15, 2016apart in each set of genes. Expressed genes in each
cell type had FPKM >1 and log2 fold change in that
cell type. Silent genes have FPKM = 0. Cell types
shown are stem cells (E), neuronal progenitors (F),
andmatureneurons (G), respectively. (H)Normalized
phasograms for all three cell types, genome-wide.
sensory neurons, resulting in a substantial
increase of euchromatin and proportional
decrease of heterochromatin to 0.23 ±
0.02 based on relative distribution of
absorption (LAC) values (Figures 3A–3C;
Table S1). These changes are due
to chromatin reorganization rather than
a decrease in HP1b protein content
because the total absorption values for
HP1b KO olfactory neurons are not signif-
icantly different from that of the wild-type
neurons. In addition, the volume of HP1b
KO olfactory neurons is double the vol-
ume of wild-type neurons, mimicking theFigure 2. Chromatin Compaction during
Neurogenesis
(A–D) Histograms plotting the number of voxels in
the nucleus with a measured linear absorption
coefficient (LAC) value show the increase in
percent volume of heterochromatin from 33% in a
stem cell (A) to 36% in a neuronal progenitor (B)
and 41% in a mature olfactory sensory neuron (C),
and overlay of all three cell types (D) plus the
concomitant decreases in euchromatin and total
nuclear volume during differentiation (D).
(E–H) Phasograms showing normalized counts of
the number of nucleosome pairs at the distanceeffects of Lamin B receptor (LBR) overex-
pression in mature neurons, in which
chromatin compaction is also markedly
reduced (Clowney et al., 2012). These
observations suggest that the proportion
of euchromatin is a determining factor of
the overall nuclear volume, a hypothesis
supported by the linear relationship be-
euchromatin and nuclear volumes (Fig-
Distribution in 3D
the developmentally regulated increase of
also reflects changes in the 3D chromatin
ed the relative distribution of hetero- and
fferentiation. This analysis shows that in all
0-nm thick region just beneath the nuclear
dominantly heterochromatin, as has been
her cell types (Figure 4; Movies S4, S5, S6,
relative distribution of heterochromatin is
between the three cell types. Between
atin in the nuclear periphery of stem cells
ereas in the inner regions of the nucleus it
(Figure 4A; Movie S4). In contrast, mature
neurons have an inverted chromatin ratio, with 70%–80% het-
erochromatin in the nuclear center due to the large mass of
pericentromeric heterochromatin that moves inward during dif-
ferentiation (Figure 4C; Movie S6), which is consistent with
LBR downregulation during olfactory neurogenesis (Clowneyet al., 2012). Neuronal progenitors have an intermediate chro-
matin distribution between the other two cell types (Figure 4B;
Movie S5). Importantly, in the HP1b KO olfactory neurons, the
remaining heterochomatin is completely detached from the nu-
clear envelope (Figure 4D; Movie S7), supporting the hypothesis
that HP1 proteins mediate attachment of heterochromatin to the
nuclear lamina (Worman et al., 1988; Ye and Worman, 1996).
SXT Reveals an Interconnected Network of
Heterochromatin
Visually tracking heterochromatin and euchromatin through
an animation of consecutive orthoslices reveals a striking, highly
interconnected network of chromatin with similar absorption
values. Statistical analyses of voxel connectivity indicate that
98.4% ± 1.7% of the heterochromatin and 99.9% ± 0.1% of the
euchromatin in all nuclei examined is connected. The intercon-
nectivity ismoreeasily seenafter using ‘‘skeletonization’’ software
that ‘‘shrinks’’ hetero- and euchromatin regions to backbone
structures, revealing the underlying topological organization of
chromatin (Figure 5; Movies S8, S9, S10, and S11). Remarkably,
these connections are not dependent upon a specific spatial dis-
tribution of heterochromatin, and even the large central mass of
pericentromeric heterochromatin ofmatureneurons remains con-
nected to the peripheral heterochromatin (Figure 5). These data
suggest that chromosomes are organized in a way that brings
some portion of the heterochromatin of adjacent chromosomes
in very close proximity; similarly, euchromatin regions of adjacent
chromosomes also must be contiguous—a structural organi-
zation that would facilitate inter-chromosomal interactions.
Although this continui
study, it is unlikely to r
same loci in every ce
populations have not r
ciations (Dixonet al., 2
and single cell analyse
3D chromatin organiz
gano et al., 2013). Imp
fibers is not disrupted
the proportion of hete
maining heterochrom
envelope.
Developmental Cha
A well-characterized
chromosomal associa
an organizational hub
ribosomal genes from
detection of nucleoli,
shape, and organizat
(Figure 6). For exam
described (Thiry et al.
(Figure 6A; Movie S12
posed to the pericen
apart. In these nucle
called the granular co
(Boisvert et al., 2007
0.01 mm1 (Table S1).
Cell Reportn in interphase nuclei (Tjong et al., 2016).
ty is observed in all cells analyzed for this
eflect stereotypic association between the
ll, because Hi-C experiments in multi-cell
evealed extensive interchromosomal asso-
012;Sanyal et al., 2012;Sextonet al., 2012),
s are consistent with extensive variability inFigure 3. HP1b Regulates Heterochromatin
Compaction in Mature Sensory Neurons
(A) Histogram plotting the number of voxels with a
measured linear absorption coefficient (LAC) value
in a control mature olfactory sensory neuron.
(B) Histogram of LAC values in an HP1beta KO
mature neuron shows the percent volume of het-
erochromatin decreased to 23%.
(C) Three-dimensional cutaway through the X-ray
tomogram showing three orthogonal orthoslices
(virtual sections) through the nucleus reveals
loss of the central mass of pericentromeric het-
erochromatin.
(D) Plotting the volume of heterochromatin and
euchromatin with respect to total nuclear volume of
stem, progenitor, and mature cells shows that
euchromatin is proportional to nuclear volume.
Indeed, recent analyses in olfactory neu-
rons revealed that euchromatic sequences
embedded in heterochromatic olfactory
receptor gene clusters form frequent inter-
chromosomal interactions (Markenscoff-
Papadimitriou et al., 2014) that are
completely dependent upon the aggregation of the receptor
clusters. Thus, these data suggest that the organization of hetero-
chromatin in a continuous lattice-like structure serves as an
architectural platform that organizes parts of euchromatin, as
we previously postulated for the organizational role of pericentro-
meric heterochromatiation between cells (Kind et al., 2015; Na-
ortantly, the continuity of heterochromatin
by deletion of HP1b, despite the fact that
rochromatin is vastly reduced and the re-
atin has lost contacts with the nuclear
nges in Nucleolar Architecture
example of stereotypic and robust inter-
tions is the nucleolus, which constitutes
of a large number of active and repressed
multiple chromosomes. SXT allows the
which have tremendous variability in size,
ion among the three differentiation stages
ple, the tripartite organization previously
, 2011) is only apparent in mature neurons
), which typically have two nucleoli juxta-
tromeric heterochromatin located 
180
oli, the outermost and innermost zones,
mponent and fibrillar centers, respectively
), have the highest absorption of 0.27 ±
The central zone of the tripartite nucleolus
s 17, 2125–2136, November 15, 2016 2129
forms a cup-shaped structure, the ‘‘dense fibrous core,’’ which,
based on SXT segmentation, has two sub-zones of differing ab-
sorption values. The inner sub-zone in mature neurons has the
lowest LAC in the nucleolus at 0.19 ± 0.01 mm1 (Table S1), sug-
gesting that this is the region where DNA is least compacted and
transcription is likely to occur. The greatest variability in nucle-
olus organization is seen in neuronal progenitors and stem cells
(Figures 6D and 6E), with volumes ranging from 0.3 mm3 to
2.2 mm3 and many oval rather than round nucleoli. This likely re-
flects that these are populations of cells at different stages of the
cell cycle and differentiation, which is supported by the observa-
tion that the organization of different nucleoli in the same nucleus
is remarkably similar (Figure 6E). Nucleoli in progenitor and stem
cells also have varying numbers of highly absorbing discrete
Figure 4. Spatial Distribution of Chromatin
Distribution of chromatin in a multipotent stem cell (A), neuronal progenitor (B), m
orthoslices (virtual sections) from the tomographic reconstructions and segmente
views, color-coded to represent heterochromatin (blue) and euchromatin (green).
(green) at increasing distances from the nuclear envelope quantify the reorganizat
Hp1b KO (D). Scale bar, 2 mm.
2130 Cell Reports 17, 2125–2136, November 15, 2016aggregates with absorption values between 0.29–0.33 mm1
that probably are recently transcribed RNAs moving toward
the nucleoplasm. Because nucleolar genes reside in multiple
chromosomes, it is possible that the variability in the number
of nucleoli and their architecture in less differentiated cells re-
flects the transitional state of heterochromatin and euchromatin
in these cells, which only settles in its final configuration in post-
mitotic mature neurons. Moreover, because nucleoli on their
own play organizational roles for the surrounding chromatin
and contribute to the formation of nucleolar-associated
domains, it is possible that these architectural fluctuations
are causal and not consequential for the overall chromatin orga-
nization (Matheson and Kaufman, 2016; Ne´meth and La¨ngst,
2011).
ature olfactory sensory neuron (C), and Hp1b KO mature neuron (D) seen in
d views of the nuclei, both surface-rendered and 3D cutaway volume-rendered
Plots showing the percent volume of heterochromatin (blue) and euchromatin
ion of chromatin during differentiation (A–C) and loss of heterochromatin in the
Because SXT images whole cells rather than thin sections, it
affords accurate 3D segmentation of various nucleolar struc-
tures and reveals continuity between the granular component
and fibrillar center of mature neurons (Figure 6A; Movie S12).
In addition, SXT reveals that nucleoli are not fully immersed in
heterochromatin as previously thought, with between 12% to
72%contact between nucleolar surface and euchromatin (Movie
S12). Retaining some connectivity with the less dense euchro-
matin likely provides a route for rRNA diffusion and efficient nu-
clear export.
A 3D Heatmap of the mOSN Nucleus
The demonstration that regions with intense transcriptional ac-
tivity correspond to voxels with the lowest absorption values
prompted us to search for additional low absorbing regions in
the mature neuron nucleus. For this reason, we generated heat-
maps where the absorption plots are transformed to 3D repre-
sentations of LAC values (Figure 7A), with an emphasis on the
least absorbing nuclear regions. This analysis reveals a large
number of nuclear ‘‘speckles’’ (30–41) with LAC%0.19 mm1 in
the euchromatic, but not heterochromatic, region (Figure 7B)
that might correspond to nuclear factories of robust transcription
(Eskiw et al., 2010). To test that, we performedDNA fluorescence
in situ hybridization (FISH) with a probe constructed from a cDNA
library prepared from mature neurons (Figures 7C and 7D). The
signal from such a complex probe will be dominated by the
most abundant cDNAs, therefore it represents a good method
to detect the location of intense transcription in these nuclei.
As seen in Figures 7C and 7D, the number (22–39) and distribu-
in vivo. Because X-ra
no need for chemica
imaging of intact cel
plethora of artifacts i
alization of only thin s
imaging of each cell is
like cells can be ave
measures that may no
high-resolution imagi
sorption depends upo
each voxel, SXT is ‘‘po
cellular structures bu
about their composit
with a predominantly n
grees of compaction a
an ideal biological spe
As a proof-of-princ
distinct differentiation
Our analysis shows th
tion occur with a con
tion in vivo, as we als
esis (Ugarte et al., 20
nucleus-wide phenom
this trend and follow
tion; recent findings i
specific expression is
entiation-dependent
Hobert, 2012). Surp
properties did not c
Cell ReportSXT: A Powerful Method for Nuclear
Imaging
SXT provides a powerful method to study
chromatin and nuclear architecture
ys can penetrate the whole cell, there is
l fixation and sectioning. Thus, it allows
ls under native conditions, preventing a
ntroduced either by fixatives or by visu-
ections. Moreover, because tomographicFigure 5. Heterochromatin Continuity in
Nucleus
Chromatin masses of the nuclei shown in Figure 4
were computationally reduced to skeletonized
structures in the stem cell, neuronal progenitor,
mature neuron and HP1b KO mature neuron to
reveal the interconnected networks. The surface
colormap of the skeletonized heterochromatin
ranges from red through yellow to white; red re-
flects a thinner portion of heterochromatin, yellow
thicker, and white the thickest region of hetero-
chromatin (the pericentromeric heterochromatin is
the white mass in the center of the mature neuron).
The colormap of the skeletonized euchromatin
ranges from blue (thinnest) to red (thickest); see
colormaps. The starburst-like pattern seen in the
HP1b KO euchromatin reveals the abnormally
large mass of euchromatin in this nucleus. Scale
bar, 2 mm.
tion of these transcription factories are
comparable to the ones revealed by SXT.
DISCUSSIONcompleted in minutes, data from multiple
raged, providing statistically significant
t be practical or even feasible with other
ng methods. Finally, because X-ray ab-
n the concentration of organic material in
lychromatic’’ and not only detectsmultiple
t also provides quantitative assessments
ion or structure. As a result, chromatin
ucleoprotein composition, but various de-
nd an elaborate 3D architecture, provides
cimen for this technology.
iple experiment, we imaged cells from
stages of mouse olfactory epithelium.
at cellular differentiation and specializa-
comitant increase of chromatin compac-
o observed occurring during hematopoi-
15). Without a doubt, although this is a
enon, some genomic loci may escape
the inverse chromatin compaction direc-
n C. elegans demonstrated that neuron-
achieved by lineage-specific and differ-
chromatin decompaction (Cochella and
risingly, changes in X-ray absorption
orrelate with changes in the primary
s 17, 2125–2136, November 15, 2016 2131
Figure 6. Nucleolus Organization in Olfactory Epithelial Cells
(A and B) Immuno-FISH was used to image rDNA (red) and nucleolin (green) in the nucleolus of a mature neuron and SXT to image the native-state structure of
nucleoli. Orthoslices and surface views were obtained by semi-automatic segmentation using linear absorption coefficient (LAC) values. A thin shell of the
heterochromatin in contact with the nucleolus is shown in pale blue.
(C) Second nucleolus from same neuron.
(D and E) Nucleoli from a neuronal progenitor (D) and stem cell (E); these nucleoli have numerous aggregates (dark blue) with absorption (LAC) values
0.32 mm1.
Scale bars, 500 nm.
(F) Histogram of LAC values in nucleoli shown in (A) and (B).
(G) Histogram of LAC values in nucleoli shown in (D).
(H) Histogram of LAC values in nucleoli shown in (E).
2132 Cell Reports 17, 2125–2136, November 15, 2016
chromatin architecture, because nucleosomal phasing and
spacing appear essentially identical between the three cell
types examined; this is consistent with recent findings
describing similar nucleosomal structure between interphase
and meiotic chromosomes in yeast (Zhang et al., 2011). In
contrast, our data support a role of HP1b in the generation
of highly absorbing chromatin structures. This result is in
agreement with recent data suggesting that HP1 family mem-
bers have homo-polymerization properties that may assist in-
ter-strand associations of heterochromatic fibers resulting
in the formation of higher-order heterochromatic structures
(Canzio et al., 2011, 2013).
Differentiation-Dependent Associations with the
Nuclear Lamina
In addition to changes in the biophysical properties of chro-
matin, our analyses also describe the gradual relocation of
heterochromatin from the nuclear periphery, where it is predom-
inantly located in stem cells, to the nuclear core in mature
neurons. This relocation, which was previously thought to be a
specific retinal adaptation for nocturnal animals (Solovei et al.,
2009), appears to be a general theme observed in many differ-
entiating tissues including the mouse olfactory epithelium
(Clowney et al., 2012; Solovei et al., 2013). What other ap-
proaches fail to detect is that a small portion of heterochromatin
remains in contact with the nuclear lamina, and this contact re-
lies on the function of HP1b. Thus, the exact network of interac-
tions between the nuclear lamina and the genome depends on
the protein components of the nuclear envelope, the epigenetic
state of the genome, and the availability of specific proteins that
extensive interactions
might have significant
nuclear processes. A
co-regulated genomic
tween loci with shared
effective way of modu
separating compacte
(Bantignies et al., 201
padimitriou et al., 20
maintenance of a ce
the convergence of c
mosomal association
2012; Markenscoff-Pa
chastic nature may be
tolerate, or even seek
immune systems (Dek
vardas, 2015; Proudh
Nevertheless, non-ste
loci (Osborne et al.,
scription factories (Li
co-regulation and the
population level.
Our analyses demo
for the study of chr
cellular differentiation
combining SXT with g
imaging as we showed
valuable insight towa
principles and molec
compartments.
Cell Reportuous nature of like-chromatin, which is
evident even in the nucleolus, supports
the existence of non-stereotypic but
between different chromosomes, which
regulatory roles in transcription and other
lthough sequence-specific association of
loci is appealing, random interactions be-
chromatin properties might be an equally
lating transcription levels. In that regard,
d from non-compacted chromatin fibers
1; Clowney et al., 2012; Markenscoff-Pa-
14) could be a more urgent task for the
ll-type-specific expression program thanFigure 7. Low LAC Nuclear ‘‘Speckle’’ Re-
gions of the Nucleus
(A) Heatmap of linear X-ray absorption coefficient
(LAC) values of a mature cell nucleus color-coded
from green (euchromatin) to blue (heterochromat-
in); lowest LAC values were color-coded red.
(B) SXT 3D cutaway view of the volume-rendered
mature neuron nucleus color-coded to reflect the
chromatin distribution shown in the heatmap in (A);
nucleolus (color-coded orange) is circled in yellow.
Scale bar, 1 mm.
(C) cDNA-labeled transcription sites in olfactory
epithelial cells. Scale bar, 5 mm.
(D) High-magnification view of cDNA sites in a
single nucleus. Scale bar, 1 mm.
interpret these epigenetic signals and
mediate interactions with the nuclear
lamina.
Global Chromatin Connectivity
The 3D segmentation of high-resolution
images provided by SXT reveals an un-
appreciated interconnectivity between
distinct forms of chromatin. The contin-o-regulated loci. In this regard, interchro-
of co-regulated loci (Clowney et al.,
padimitriou et al., 2014) due to its sto-
suitable only for biological systems that
, stochasticity like the olfactory and the
ker and Mirny, 2016; Monahan and Lom-
on et al., 2015; Raviram et al., 2016).
reotypic convergence of multiple gene
2007) and regulatory sequences in tran-
et al., 2012a) may also contribute to their
ir transcription at similar rates at a cellular
nstrate that SXT is a powerful technology
omatin and nuclear architecture during
in vivo. Moreover, as shown here,
enetic manipulations, or with fluorescence
previously (Smith et al., 2014), generates
rd the understanding of the organizing
ular composition of nuclear chromatin
s 17, 2125–2136, November 15, 2016 2133
EXPERIMENTAL PROCEDURES
Mice
Mice were housed under standard conditions in accordance with IACUC reg-
ulations; HP1b KO mice were obtained from Eucomm (details in the Supple-
mental Experimental Procedures).
Nucleosome Mapping and Analysis
Mapping Reads
Paired-end reads were trimmed so that all datasets had reads of the same
length: for reads longer than 51 base pairs, the first 51 base pairs of the
read were retained. Paired-end reads were mapped to themm9 genome using
the Burrows-Wheeler Alignment (Li and Durbin, 2009) algorithm in backtrack
paired-end mode. Potential PCR duplicates were removed using samtools
(Li et al., 2009), which removed reads with identical external coordinates.
Reads that had a quality score of <30 were filtered out. Additionally, we
required that reads be properly paired, with correct orientation and of reason-
able insert size, based on an estimated inset size distribution.
Phasograms
Phasograms were calculated using a similar method to Valouev et al. (2011).
Midpoints of the remaining high-quality read pairs were used to approximate
the nucleosome positions. For each genomic position, we tallied the number
of nucleosomemidpoints that were found for each cell type (stem cell, neuronal
progenitor, andmature neuron), filtering on any conditions as appropriate. Spe-
cifically, for silentgeneswe lookedonly atmidpointswithingeneswithFPKM>1-
and 2-fold expression in the specified cell type compared to the other cell types.
We set the ‘‘pile’’ argument equal to 3, as previously suggested (Valouev et al.,
2011), filtering out any midpoint positions that had <3 reads with midpoints at
thatposition. Finally, bycalculating thedistancebetweeneverypair of remaining
midpoint locations, we obtained raw phasograms.
To compare phasograms across cell types or conditions, we normalized each
phasogram. For the phasograms showing nucleosome midpoints in known
olfactory receptor (OR) genes, we calculated the best linear regression fit to
the data for between 101 and 750 base pairs and then divided the raw counts
by the corresponding point in the linear model. This was done to compensate
for the relatively lowpresenceofMNase-Seq reads inORgenes,causingastrong
decreasing linear correlation between the number of nucleosomemidpoints and
their distance apart. The range of 101 and 750 base pairs was chosen because
deltas of 0–100 base pairs resulted in very noisy counts, whereas deltas of
more than 750 base pairs resulted in counts that quickly dropped to 0. For all
other phasograms, there was a high coverage of nucleosome midpoints in the
given regions, so the linear correctionwas not needed. Therefore, the raw counts
weredividedby itsbasemidpoint count,whichwas themeannumberofmidpoint
pairs at a distance between 1,000 and 2,000 base pairs apart. All comparison
phasograms (across condition or cell type) show normalized phasograms.
Periodicities
Nucleosome periodicities were calculated by quantifying the periodicity of the
phasograms, as done previously (Valouev et al., 2011). We calculated a linear
regression of the peaks in the phasograms using an estimated prior periodicity
of 200 base pairs. We assume a peak at a distance of 0 and then identify the
locations of the remaining peaks as the maximum in each 200 base pair win-
dow, starting at a position of 100 base pairs. This is repeated until a distance of
1,300 and then a linear model is fitted to all of the identified peaks. The slope
of the best-fit line is the estimated periodicity, while the R2 value (coefficient of
determination) is used to estimate the fit of the model.
Soft X-Ray Tomography
Neurons were rapidly frozen in liquid nitrogen cooled propane and imaged
at 517 eV using a soft X-ray microscope as described previously (Le Gros
et al., 2005; McDermott et al., 2009). LAC values were determined as
described previously (Weiss et al., 2001). See also the Supplemental Experi-
mental Procedures.Soft X-Ray Tomography Data Analyses
Nuclear Segmentation
Nuclear volumes were manually segmented from full tomographic reconstruc-
tions for each cell and filtered (3Dmedian filter using MATLAB medfilt, window
2134 Cell Reports 17, 2125–2136, November 15, 2016size 4). LAC histograms were calculated generating a bimodal LAC histogram
for each nucleus. To accurately locate the peaks of the bimodal LAC distribu-
tion, a cubic interpolation routine (MATLAB code) was used to addmore points
close to the histogram maxima. The local maxima of euchromatin and hetero-
chromatin were obtained from the interpolated histogram. The LAC threshold
value corresponding to the transition between euchromatin and heterochro-
matin was found by taking the average of the euchromatin and heterochromat-
in local maxima. This definition of the euchromatin-heterochromatin transition
LAC was the threshold value used to automatically segment the nuclear
volume of each nucleus into the euchromatic and heterochromatic regions.
Voxels with a LAC above the threshold value were assigned to heterochromat-
in; those below the threshold were assigned to euchromatin. These assign-
ments agree with those obtained by manual segmentation and are consistent
with traditional classifications from transmission electron microscopy (TEM)
micrographs of stained plastic sections.
Connectivity
Connectivity was estimated by calculating the fraction of the largest con-
nected region from the total volume for either heterochromatin or euchromatin.
For the heterochromatin fraction, the largest connected region from the total
volume was 0.984 ± 0.017 and for euchromatin it was 0.9989 ± 0.0011.
Skeletonization
Using the program Amira (Visualization Sciences Group), a binarized volume
was created from the segmented dataset by assigning unity to each voxel of
heterochromatin and zero to all other voxels in the reconstruction. A distance
transform (Blum, 1973) was applied to the binarized volume; this transform as-
signs a gray level value to each non-zero voxel that is proportional to the
closest distance of that voxel to the boundary of heterochromatin. A medial
axis transform (MAT) (Du and Hong, 2004) is then applied to the distance
map; this transform selects voxels that are the locus of local maxima of the dis-
tance transform. The MAT of the distance map is similar to a skeletonization of
the heterochromatic binarized volume, where the volume has been uniformly
eroded to leave a skeleton-like structure with a minimum width of one voxel.
However, theMAT of the distancemap replaces the voxel values of the hetero-
chromatin skeletonwith the distance of a skeleton voxel to the edge of the orig-
inal binarized heterochromatic volume. The MAT is then visualized by assign-
ing a tubular surface to each branch of the MAT skeleton where the diameter
and color of the tube represents the minimum distance of the MAT skeleton to
the boundary of the heterochromatic and euchromatic regions. This visualiza-
tion conveys both the connectivity and thickness of all heterochromatin in the
nucleus. Euchromatin was skeletonized using the same approach.
Sub-nuclear and Cytoplasmic Structure Analysis and Visualization
The outer boundary of each nucleolus was obtained by manual segmentation.
Segmentation of nucleolar substructure was performed using semi-automatic
segmentation based on the LAC range determined for each component within
each cell type (see Table S1). The transcription regions in the euchromatic re-
gion of the nucleus were automatically segmented using a LAC value derived
from the transcriptionally active region of the nucleolus, specifically LAC range
%0.19 mm1. After segmentation, the assigned regions were visualized using
standard solid or transparent surface rendering (Rosenblum et al., 1994) and
3D volume rendering (Peng et al., 2010) using the software package Amira.
Immuno-DNA FISH
DNA FISH experiments and confocal images were generated as described
previously (Clowney et al., 2012) with modifications described in the Supple-
mental Experimental Procedures.
SUPPLEMENTAL INFORMATION
Supplemental Information includes Supplemental Experimental Procedures,
one figure, one table, and twelve movies can be found with this article online
at http://dx.doi.org/10.1016/j.celrep.2016.10.060.AUTHOR CONTRIBUTIONS
Conceptualization, S.L. and C.A.L.; Cell Preparation, DNA FISH, IF, E.J.C. and
E.M.-P.; ChiPS, Mnase-seq, A.M.; Nucleosome Mapping, A.Y., B.C., and
M.K.; SXT Data Collection and Analysis, M.A.L.G., M.M., and C.A.L.; Writing &
Editing, S.L., M.A.L.G., and C.A.L.; Funding Acquisition, S.L. and C.A.L.
ACKNOWLEDGMENTS
Research reported in this publication was supported by grants from NIH
(R01DA030320 and U01DA040582 to S.L. and C.A.L.). The National Center
for X-ray Tomography is supported by NIH (P41GM103445) and DOE’s Office
of Biological and Environmental Research (DE-AC02-5CH11231).
Received: January 22, 2016
Revised: August 28, 2016
Accepted: October 12, 2016
Published: November 15, 2016
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