Interdependência entre Xist RNA e Complexos de Polycomb

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Article
XistDeletional Analysis Re
veals an Interdependency
between Xist RNA and Polycomb Complexes for
Spreading along the Inactive X
Graphical Abstract
Xist locus: Cas9
XistΔB
HNRNPK KD
PRC1/2 KO
Wild type
PRC2
PRC1
Xi chromatin
Xist RNA
B
Xi chromatin
B
K
K119ub
K27me3
K27me3
K119ub
*
*
*
*
*
* *
Interdependent
spreading of
Xist and 
Polycomb
Xist
Highlights
d Repeat B is required to spread Xist RNA, silence genes, and
alter Xi 3D conformation
d Deleting Repeat B compromises recruitment of PRC1
and PRC2
d Reciprocally, ablating PRC1 or PRC2 impairs Xist spreading
d Xist, PRC1, and PRC2 spread across the Xi in an
interdependent manner
Colognori et al., 2019, Molecular Cell 74, 101–117
April 4, 2019 ª 2019 Elsevier Inc.
https://doi.org/10.1016/j.molcel.2019.01.015
Authors
David Colognori, Hongjae Sunwoo,
Andrea J. Kriz, Chen-Yu Wang,
Jeannie T. Lee
Correspondence
lee@molbio.mgh.harvard.edu
In Brief
This study demonstrates that Repeat B of
Xist RNA is essential for spreading Xist,
PRC1, and PRC2 along the inactive X.
Spreading of Xist, PRC1, and PRC2 is
interdependent. Deleting Repeat B
compromises PRC1/2 recruitment; loss
of PRC1/2 in turn impairs Xist
spreading—suggesting a positive
feedback mechanism for RNA-protein
propagation.
mailto:lee@molbio.mgh.harvard.�edu
https://doi.org/10.1016/j.molcel.2019.01.015
http://crossmark.crossref.org/dialog/?doi=10.1016/j.molcel.2019.01.015&domain=pdf
Molecular Cell
Article
Xist Deletional Analysis Reveals an Interdependency
between Xist RNA and Polycomb Complexes
for Spreading along the Inactive X
David Colognori,1,2,3,4 Hongjae Sunwoo,1,2,3,4 Andrea J. Kriz,1,2,3 Chen-Yu Wang,1,2,3 and Jeannie T. Lee1,2,3,5,*
1Howard Hughes Medical Institute
2Department of Molecular Biology, Massachusetts General Hospital, Boston, MA 02114, USA
3Department of Genetics, Harvard Medical School, Boston, MA 02115, USA
4These authors contributed equally
5Lead Contact
*Correspondence: lee@molbio.mgh.harvard.edu
https://doi.org/10.1016/j.molcel.2019.01.015
SUMMARY
During X-inactivation, Xist RNA spreads along
an entire chromosome to establish silencing. How-
ever, the mechanism and functional RNA elements
involved in spreading remain undefined. By per-
forming a comprehensive endogenous Xist deletion
screen, we identify Repeat B as crucial for spreading
Xist andmaintainingPolycomb repressivecomplexes
1 and 2 (PRC1/PRC2) along the inactive X (Xi). Unex-
pectedly, spreading of these three factors is inextri-
cably linked. Deleting Repeat B or its direct bind-
ing partner, HNRNPK, compromises recruitment of
PRC1 and PRC2. In turn, ablating PRC1 or PRC2 im-
pairs Xist spreading. Therefore, Xist and Polycomb
complexes require each other to propagate along
the Xi, suggesting a positive feedback mechanism
between RNA initiator and protein effectors. Perturb-
ing Xist/Polycomb spreading causes failure of de
novo Xi silencing, with partial compensatory downre-
gulation of the active X, and also disrupts topolog-
ical Xi reconfiguration. Thus, Repeat B is a multifunc-
tional element that integrates interdependent Xist/
Polycomb spreading, silencing, and changes in chro-
mosome architecture.
INTRODUCTION
X chromosome inactivation (XCI) has served as an epigenetic
archetype (Starmer and Magnuson, 2009; Lee, 2011; Disteche,
2016; Mira-Bontenbal and Gribnau, 2016). During XCI, the
17-kb noncoding RNA Xist spreads exclusively in cis along the
future inactive X (Xi) and induces conversion to a heterochro-
matic state (Brown et al., 1992; Clemson et al., 1996; Marahrens
et al., 1997). The functions of Xist are manifold. On one hand, Xist
acts as a modular RNA scaffold in the assembly of repressive
protein factors. Two well-known factors, Polycomb repressive
complexes 1 and 2 (PRC1/PRC2), are responsible for monoubi-
quitylating histone H2A at lysine 119 (H2AK119ub) and trime-
thylating histone H3 at lysine 27 (H3K27me3), respectively
(Schoeftner et al., 2006; Zhao et al., 2008; Chu et al., 2015;
McHugh et al., 2015; Minajigi et al., 2015; Moindrot et al.,
2015; Monfort et al., 2015). On the other hand, Xist forms a
repressive compartment by repelling transcriptional and archi-
tectural factors to establish a unique Xi chromosome conforma-
tion (Nora et al., 2012; Rao et al., 2014; Deng et al., 2015; Minajigi
et al., 2015; Giorgetti et al., 2016).
Although broad functions have been associated with Xist, spe-
cific mechanisms have not been clarified—in particular, how Xist
RNA spreads in cis. Recent work has demonstrated the impor-
tance of nuclear matrix factors in restricting Xist to the Xi territory
(Hasegawa et al., 2010; Ridings-Figueroa et al., 2017; Sunwoo
et al., 2017). On the Xi itself, Xist spreads from its site of tran-
scription to nearby contacts in 3D space, preferentially targeting
regions enriched for active genes before spreading to less active
and gene-poor regions (Engreitz et al., 2013; Simon et al., 2013).
However, the mechanism by which Xist associates with and
spreads along Xi chromatin remains unknown.
Lacking in the field is a comprehensive map of Xist functional
elements. While several essential domains have been identi-
fied—often corresponding to conserved repetitive motifs (‘‘Re-
peats A–F’’) (Wutz et al., 2002; Zhao et al., 2008; Hoki et al.,
2009; Jeon and Lee, 2011; Ridings-Figueroa et al., 2017; Sun-
woo et al., 2017; Yue et al., 2017)—these together account
for <20% of Xist’s total sequence. Prior to CRISPR/Cas9 tech-
nology (Ran et al., 2013), genetic dissection at the endogenous
locus proved challenging due to inefficiency of homologous tar-
geting, compounded by Xist’s large size and purported redun-
dant regions. Previous analyses have relied heavily on the use
of Xist transgenes and ectopic insertions, often in male cells
(Lee et al., 1999; Wutz et al., 2002; Jeon and Lee, 2011; Pinta-
cuda et al., 2017), with the caveat that these non-physiological
perturbations might not inform Xist function in the endogenous
context.
Here, we carry out a systematic deletional analysis of endog-
enous Xist and identify a specific RNA motif—Repeat B—for
RNA spreading and Polycomb targeting. In doing so, we reveal
the surprising discovery that Xist and Polycomb complexes
depend on each other to spread across the Xi.
Molecular Cell 74, 101–117, April 4, 2019 ª 2019 Elsevier Inc. 101
mailto:lee@molbio.mgh.harvard.edu
https://doi.org/10.1016/j.molcel.2019.01.015
http://crossmark.crossref.org/dialog/?doi=10.1016/j.molcel.2019.01.015&domain=pdf
Ex
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 ΔRepF ΔRepB ΔEx2-6 ΔRepE
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 ΔRepA
 Xi+/-
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D
eleted
Xi- Xi+
Xist cloud (3D STORM)
X
ist R
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 probe region
ΔR
ep
B
ΔR
ep
E
z-axis
nm
 Xi+/-
Xi-Xi+ Xi-Xi+
0
10
20
30
40
50
60
70
Xist cloud sizeμm2
p=1.1e-07
p=3.7e-12
Xi-/-
5 μm
ΔR
ep
B
ΔR
ep
E
W
T
5μm
(legend on next page)
102 Molecular Cell 74, 101–117, April 4, 2019
RESULTS
Comprehensive Deletional Analysis of Native Xist in
Female Cells
To perform a systematic CRISPR/Cas9 deletion screen, guide
RNA (gRNA) pairs were designed to remove consecutive 1-to
2-kb regions across the Xist locus in female mouse embryonic
fibroblasts (MEFs), where XCI has already been established (Fig-
ure 1A). The transformed MEFs were tetraploid (with genome
duplication after XCI) carrying two Xi’s and two Xa’s within the
same nucleus (Yildirim et al., 2011) and thus enabled isolation
of Xi+/� clones (deletion on only one Xi) and Xi�/� clones (deletion
on both Xi’s) (Figure 1B). Xi+/� cells provided an internal control
Xist cloud within the same nucleus for comparative microscopy,
while Xi�/� cells provided a homogeneous system for genomic
experiments. We screened for mutant clones by two-color
RNA fluorescence in situ hybridization (FISH)—cyan probes
external and red probes internal to each deletion—and selected
clones exhibiting cyan with no overlapping red signal (Figure 1B).
All deletions were validated by Sanger sequencing (Data S1).
We began with a visual inspection of Xist cloudmorphology by
RNA FISH. Of the 13 deletions, 7 exhibited some phenotype.
While Repeat A is known for its role in gene silencing (Wutz
et al., 2002; Zhao et al., 2008), its deletion has been reported
to cause decreased accumulation and/or loss of Xist expression
in both human and mouse cells (Chow et al., 2007; Zhao et al.,
2008; Hoki et al., 2009). A minimal Repeat A deletion allowed
us to derive clones with an intact Xist cloud and overall RNA level
(Figures 1C and 1D). However, further characterization revealed
aberrant splicing—as suggested previously (Royce-Tolland
et al., 2010)—through de-suppression of a cryptic splice donor
(Figure S1). Because this resulted in simultaneous skipping of
the majority of exon 1 in �50% of transcripts, we did not pursue
the DRepA clones further. Likewise, exon 7a or 7d deletions
caused skipping of adjacent exon 7 regions in most transcripts,
exhibiting a pattern similar to Xist’s minor splice isoform
(Figure S1).
Deleting the region containing Repeat F caused loss or signif-
icant weakening of Xist clouds (Figure 1C), consistent with other
reports (Jeon and Lee, 2011; Makhlouf et al., 2014). qRT-PCR in
DRepF Xi�/� cells confirmed a reduction in Xist RNA level (Fig-
ure 1D), which could be due to loss of expression (Makhlouf
et al., 2014), RNA stability, and/or proper ‘‘nucleation’’ (Jeon
and Lee, 2011). Deletion of the internal exons 2–6 also yielded
a weaker cloud and decreased RNA levels (Figures 1C and
1D), presumably by affecting splicing efficiency and/or transcript
stability.
Figure 1. CRISPR/Cas9 Deletion Screen Identifies Xist Functional Dom
(A) Diagram of Xist locus, repeat elements, gRNA target sites, and qPCR amplic
(B) Schematic of screening method using tandem two-color RNA FISH.
(C) Xist RNA FISH for deletions showing altered Xist cloud morphology in Xi+/� M
Right panels show 33 zoom-in of each cloud.
(D) qRT-PCR showing effect of deletions on Xist RNA levels in Xi�/� MEFs. Error
(E) 3D STORM imaging and size measurements of Xist clouds in DRepB and DRe
arrowhead indicating WT and an arrow indicating mutant Xist cloud. p values are
(F) H3K27me3 and H2AK119ub IF for deletions showing phenotype in Xi+/� MEF
See also Figures S1–S3.
Intriguingly, deleting Repeat B- or E-containing regions pro-
duced Xist clouds with aberrant morphologies. While DRepE
caused widespread dispersal of Xist throughout the nucleus
(Ridings-Figueroa et al., 2017; Sunwoo et al., 2017; Yue et al.,
2017), DRepB caused Xist clouds to appear more diffuse yet
remain localized near the Xi vicinity (Figure 1C). Morphological
aberrations were accentuated using single-molecule super-res-
olution imaging by 3D stochastic optical reconstruction micro-
scopy (3D STORM) (Figure 1E). DRepB’s diffuse Xist cloud was
not due to changes in Xist RNA level (Figure 1D), nor failure to re-
cruit the nuclear matrix protein CIZ1 as was the case for DRepE
(Figure S2A) (Ridings-Figueroa et al., 2017; Sunwoo et al., 2017).
Thus, DRepB represents a distinct mechanism of Xist RNA local-
ization. To pinpoint the specific element responsible forDRepB’s
phenotype, we generated smaller internal deletions (Figure S2B).
The�300-bp subdeletion of Repeat B itself (DRepBd) fully reca-
pitulated aberrant Xist clouds, whereas the other subdeletions
did not. These data show that the conserved repetitive
GCCCC(A/T) element itself is critical for proper Xist localization.
Next, we investigated whether any deletions might affect Xist-
dependent chromatin modifications enriched on Xi. Specifically,
we performed immunofluorescence (IF) for H2AK119ub and
H3K27me3 marks, mediated by PRC1 and PRC2, respectively
(Figures 1F and S3). The DRepA Xi showed a reduction in both
marks, which could be caused by loss of Repeat A and/or other
exon 1 regions due to the confounding splicing defect. Similarly,
DRepF and DEx2–6 showed decreased H2AK119ub and
H3K27me3 Xi enrichment, presumably due to the decrease in
Xist RNA levels. Strikingly, this was not the case for DRepB.
Although Xist still partially covered themutant Xi, there was com-
plete loss of H2AK119ub and H3K27me3. DRepB contrasted
sharply with DRepE, which caused only moderate loss of these
marks despite having an even stronger impact on Xist localiza-
tion (Figures 1C and 1E). Therefore, Xist delocalization cannot
completely account for the absence of H2AK119ub/H3K27me3
on the DRepB Xi. Henceforth, we focus on DRepB for its unique
effect on both Xist localization and Xi chromatin modifications.
Genomic Mapping Reveals Defects in Xist Spreading
and Polycomb Recruitment
DRepB’s aberrant cloud morphology suggested a defect in RNA
spreading. To visualize Xist binding sites on chromatin, we per-
formed capture hybridization analysis of RNA targets (CHART-
seq) in DRepB Xi�/� cells. Since the mutant MEFs were derived
from F1 hybrid cells in which the Xi’s are of Mus musculus (mus)
origin and Xa’s of Mus castaneus (cas) origin, SNPs allowed for
allelic analysis of sequencing results (Yildirim et al., 2011).
ains
ons
EFs. The arrowhead indicates WT, and the arrow indicates mutant Xist cloud.
bars show SD for 3 biological replicates.
pE Xi+/� MEFs. Epifluorescent images of same cells shown to the right, with an
by two-tailed t test.
s. The arrowhead indicates WT, and the arrow indicates mutant Xist cloud.
Molecular Cell 74, 101–117, April 4, 2019 103
A
WT
ΔRepB
 Xi-/-
[0-1500]
[0-1500]
[-4-1]
B
Subject to XCI
Non-expressed
Escapee
ChrX: 166 Mb Xist
C
[0-1100]
[0-1100]
[-2-1]
[0-12000]
[0-12000]
[-1-1]
WT [0-700]
[0-700]
[-5-0]
 Δ
 Δ
 Kdm5c
(Escapee)
XistTsix
ΔRepB
ΔRepB Xi-/-
comp
Xi
Xa
comp
Xi
Xa
W
T
ΔR
ep
B
 X
i-/
-
H
3K
27
m
e3
comp
Xi
Xa
comp
Xi
Xa
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T
ΔR
ep
B
 X
i-/
-
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2A
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9u
b
RefSeq
F
comp
Xi
Xa
comp
Xi
Xa
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 X
i-/
-
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3K
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m
e3
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Xi
Xa
comp
Xi
Xa
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T
ΔR
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 X
i-/
-
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2A
K
11
9u
b
RefSeq
WT
ΔRepB
X
is
t C
H
A
R
T
Δ
E
G
[0-350]
[0-110]
[0-110]
[0-350]
[0-110]
[0-110]
[0-150]
[0-60]
[0-60]
[0-150]
[0-60]
[0-60]
ChrX: 166 Mb Xist
GFP-RYBPEZH2
5μm
R
ep
B
E
xo
n 
7X
is
t R
N
A
ΔRepB Xi+/-
0
20
40
60
80
100
Xi+ Xi-
GFP-RYBPEZH2
None
Weak
Strong
%
Xi+ Xi-
n > 50
Gk
[0-800]
[0-800]
[-1-0]
[0-300]
[0-150]
[0-150]
[0-300]
[0-150]
[0-150]
[0-150]
[0-80]
[0-80]
[0-150]
[0-80]
[0-80]
Efhc2
[0-700]
[0-700]
[-2-0]
[0-350]
[0-120]
[0-120]
[0-350]
[0-120]
[0-120]
[0-120]
[0-60]
[0-60]
[0-120]
[0-60]
[0-60]
D
 Kdm6a
(Escapee)
[0-600]
[0-600]
[-1.5-1]
[0-500]
[0-200]
[0-200]
[0-500]
[0-200]
[0-200]
[0-150]
[0-80]
[0-80]
[0-150]
[0-80]
[0-80]
Klf8
RefSeq
0
50
100
Subject to XCI
 (Xa)
Subject to XCI
 (Xi)
Non-expressed
 (Xa)
Non-expressed
 (Xi)
Escapee
 (Xa)
Escapee
 (Xi)
0
500
1000
1500
X
is
t d
en
si
ty
p=0.42
H
3K
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m
e3
 d
en
si
ty
0
20
40
60
H
2A
K
11
9u
b 
de
ns
ity
RefSeq
ΔRepB Xi-/-WT ΔRepB Xi-/-WT
ΔRepB Xi-/-WT
p=0.03
p<2.2e-16
p<2.2e-16p<0.001
p<2.2e-16
p<2.2e-16
p=2e-47
p=2e-95
(legend on next page)
104 Molecular Cell 74, 101–117, April 4, 2019
Antisense oligos to Xist (avoiding our deleted area) efficiently
captured Xist RNA and associated Xi chromatin (Figure 2A).
Whereas wild-type (WT) cells displayed an expected pattern of
dense chromosome-wide Xist coverage, DRepB Xi�/� cells
showed reduced coverage across the entire Xi relative to WT.
Importantly, Xist occupancy was most strongly affected at
regions more distant from its site of synthesis—particularly at
centromeric and telomeric ends—arguing for inefficient
spreading (Figure 2A, D track). Plots of Xist density indicated
significantly diminished coverage over genes normally subject
to XCI as well as over non-expressed genes (Figure 2B). Exam-
ination of specific genes confirmed a reduction of Xist occu-
pancy across gene bodies and surrounding intergenic regions
(Figure 2C). Xist coverage was reduced to the point of being
indistinguishable from that over escapee genes, which normally
are not subject to XCI and have low-level Xist coverage
(Figures 2B and 2C, Kdm5c). Similar depletion was not seen at
the Xist locus (Figure 2C, Xist), where the nascent transcript is
naturally tethered to chromatin. Collectively, these data demon-
strate that Repeat B is necessary for proper spreading of Xist
across Xi.
We then examined DRepB’s effect on Xi chromatin modifica-
tions by performing allele-specific chromatin immunoprecipita-
tion sequencing (ChIP-seq) in DRepB Xi�/� cells. Consistent
with IF, there was near-complete loss of both H2AK119ub and
H3K27me3 on a chromosome-wide scale, specific to the Xi (Fig-
ure 2D). Depletion was uniform across the chromosome (Figures
2D–2F), in contrast to the non-uniform spreading defect of Xist
RNA as visualized by CHART-seq. This distinction supports
the idea that H2AK119ub/H3K27me3 loss is not merely a sec-
ondary effect of aberrant Xist spreading. Rather, we found it
was due to failure of Xist to recruit PRC1/2, as shown by loss
of EZH2 (the catalytic component of PRC2) and RYBP (a compo-
nent of noncanonical PRC1) from themutant Xi (Figure 2G).While
this work was in progress, another study using Xist transgenes
reported a similar result (Pintacuda et al., 2017). Thus, Repeat
B is essential for continual recruitment of Polycomb complexes
during maintenance of XCI.
Xist Spreading andPolycombRecruitment AreMediated
by HNRNPK
To identify trans factors involved in Repeat B function, we per-
formed in vitro pull-down experiments using aptamer-tagged
Repeat B RNA (Figures 3A, S4A, and S4B). Proteins signif-
icantly enriched over aptamer-only and antisense controls
Figure 2. Repeat B Is Required for Xist RNA Spreading and Polycomb
(A) Xist CHART-seq in WT and DRepB Xi�/� MEFs. Change in Xist coverage (D)
regions are masked.
(B) Boxplots showing Xist coverage over genes subject to XCI, non-expressed,
sum test.
(C) Zoom-in of CHART-seq tracks for representative regions.
(D) Allele-specific ChIP-seq for H3K27me3 and H2AK119ub in DRepB Xi�/� MEFs
locus is indicated and unmappable regions are masked.
(E) Boxplots showing H3K27me3 andH2AK119ub coverage over genes subject to
by Wilcoxon rank-sum test.
(F) Zoom-in of CHART-seq and ChIP-seq tracks for representative regions.
(G) Fluorescence microscopy showing loss of GFP-RYBP (noncanonical PRC1) o
WT, and the arrow indicates mutant Xist cloud.
were identified by liquid chromatography-tandem mass spec-
trometry (LC-MS/MS) (Figure S4C; Table S1). The top candidate
in all replicates was HNRNPK, a nuclear poly(C) RNA-binding
protein involved in RNA processing, stability, and transport
(Bomsztyk et al., 2004). Other candidates included the related
HNRNPE1–3 (PCBP1–3) proteins. HNRNPK has been impli-
cated in Xist-binding as well as interaction with PRC1 and
PRC2 (Denisenko and Bomsztyk, 1997; Chu et al., 2015; Cirillo
et al., 2016; Pintacuda et al., 2017). However, being a ubiquitous
RNA-binding protein, it was unclear how HNRNPK might be
specifically and functionally relevant to XCI. Here, we validated
HNRNPK as a bona fide Xist-interacting protein in vivo. IF using
two different antibodies demonstrated clear enrichment on Xi
(Figures 3B and S4D). Notably, enrichment of HNRNPK (but
not HNRNPE1–3) only became visible upon pre-extraction of
soluble protein prior to cell fixation (Figures S4E and S4F),
implying its tighter association with the Xi compartment.
Significantly, IF in DRepB Xi+/� cells revealed loss of HNRNPK
from the mutant Xi (Figure 3B), demonstrating Xist Repeat B is
necessary for HNRNPK’s Xi-association. We then ectopically
expressed Xist (full-length or exon 1) via transgene (Jeon and
Lee, 2011) and found this was sufficient to stabilize HNRNPK
on an autosome as well (Figure 3C). To test whether Repeat
B-HNRNPK interaction is direct, we performed electrophoretic
mobility shift assay (EMSA) using recombinant protein and a
synthetic RNA fragment (Figures 3D and 3E). Indeed, we
observed formation of multiple higher molecular weight species
with increasing HNRNPK concentrations, indicative of >1 pro-
tein:RNA stoichiometry. Taken together, these data support a
direct Repeat B-HNRNPK interaction in vitro and in vivo.
We sought whether HNRNPK is functionally relevant to Repeat
B’s roles in Xist spreading and Polycomb recruitment in post-
XCI cells. Attempts to generate stable HNRNPK knockout (KO)
clones using CRISPR/Cas9 were unsuccessful—consistent with
HNRNPK being essential in metazoans (Bomsztyk et al., 2004;
Gallardo et al., 2015). However, we were able to temporarily
ablate HNRNPK among a mixed cell population, and found defi-
ciency of HNRNPK recapitulated DRepB phenotypes (Fig-
ure S4G). To investigate the kinetics of these events, we
performed small interfering RNA (siRNA) knockdown (KD) of
HNRNPK across a 6-day time course (Figure 3F). Complete loss
of H2AK119ub from the Xi was evident by day 2, although, at
this time, Xist clouds still appeared morphologically normal. Dis-
ruptedcloudsdid not fullymanifest until day4 andwerenotdue to
decreased expression of Xist or of genes known to affect Xist
Maintenance across the Xi
is shown below as log2(DRepB/WT). Xist locus is indicated and unmappable
and escapees in WT and DRepB Xi�/� cells. p values are by Wilcoxon rank-
. Composite (comp) of all reads as well as allelic (Xi and Xa) tracks shown. Xist
XCI, non-expressed, and escapees inWT andDRepB Xi�/�MEFs. p values are
r EZH2 (PRC2) from mutant Xi in DRepB Xi+/� MEFs. The arrowhead indicates
Molecular Cell 74, 101–117, April 4, 2019 105
A
D
ay
 2
H2AK119ub H3K27me3
D
ay
 4
D
ay
 6
Xist RNA
S
cr
am
bl
e 
K
D
H
N
R
N
P
K
 K
D
5 μm
F
Xist cloud
S
cr D
2
D
4
D
6
H3K27me3
None
Dispersed
Weak
Normal/
Strong
H2AK119ub
S
cr D
2
D
4
D
6
S
cr D
2
D
4
D
6n > 100 0
20
40
60
80
100
%
: [HNRNPK] nM0 25 50 100 200400 400 4000
WT mut : Repeat B probe
10x100x : competitor
bound
unbound
HNRNPK
ΔR
ep
B
 X
i+
/-
5μm
5μm
W
T
HNRNPK
Xi+ Xi-
None
WeakStrong
n > 50
0
20
40
60
80
100
%
WT
ΔRepB
D
S
TO
R
M
X
is
t R
N
A
 F
IS
H
Scramble KD HNRNPK KDE
5μm
2μm
RepB Exon 7
Xist RNA
F
ul
l l
en
gt
h
Exon 1 Exon 7 
5μm
HNRNPK
E
xo
n 
1
X
is
t T
ra
ns
ge
ne
Xist RNA
0
20
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E
nd
og
en
ou
s
E
xo
n 
1 
T
g
F
ul
l l
en
gt
h 
T
g
♀♂
HNRNPK
B
Strept-
avidin
S1m
W
as
h
El
ut
e
LC/MS
Re
pB
Co
nt
ro
l
RepB
Scr D2 D4 D6
HNRNPK KD
GAPDH
H2AK119ub
H3K27me3
HNRNPK
C
G
H
52
76
38
31
102
150
225
kDa
His-HNRNPK
0 400
200
-200
-400
z-axis
 nm
0
5
10
15
20
25
30 p=2.7e-18
Xist cloud size
μm2
Sc
ra
m
ble
 K
D
HN
RN
PK
 K
D
E H
Figure 3. Direct Repeat B-HNRNPK Interaction Is Required for Xist Spreading and Polycomb Maintenance
(A) In vitro RNA pull-down scheme.
(B) IF showingRepeatB-dependent associationofHNRNPKwithXi inpre-extractedMEFs. Thearrowhead indicatesWT, and thearrow indicatesmutantXistcloud.
(C) IF showing HNRNPK recruitment to an ectopic full-length or exon 1 Xist transgene (arrow). Overexpressed transgenic RNA outcompetes endogenous Xist
(arrowheads) for HNRNPK-binding in female cells.
(D) Coomassie staining of SDS-PAGE demonstrating purity of recombinant His-HNRNPK.
(E) RNA EMSA showing recombinant HNRNPK directly binds a Repeat B RNA fragment in vitro. Binding is specific to WT but not mutated (mut) RNA sequence
and can be outcompeted by excess unlabeled WT RNA.
(F) Xist RNA FISH andH3K27me3/H2AK119ub IF inMEFs following scramble or HNRNPKKD for 2, 4, and 6 days. Inset shows increased contrast of boxed region.
(G) Western blot showing HNRNPK depletion does not affect global H3K27me3/H2AK119ub levels. GAPDH serves as loading control.
(H) 3D STORM imaging and size measurements of Xist clouds after scramble or HNRNPK KD. p values are by two-tailed t test.
See also Figure S4 and Table S1.
106 Molecular Cell 74, 101–117, April 4, 2019
0
400
200
-200
-400
z-axis
 nm
WT
EED KO
RING1A/B KO
RI
NG
1A
RI
NG
1B
EE
D
HN
RN
PU
HN
RN
PK
CI
Z1
Xi
st 
Ex
1-
3
Xi
st 
Ex
7-
2
0
0.2
0.4
0.6
0.8
1.0
1.2
R
el
at
iv
e 
R
N
A
 le
ve
l
A B
C D
E
E
D
 K
O
R
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G
1A
/B
 K
O
HNRNPK Xist
5μm
0
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%
None
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Strong
n > 50
RI
NG
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/B
 K
O
EE
D 
KO
0
10
20
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40
50
60
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RI
NG
1A
/B
 K
O
EE
D 
KO
Xist cloud size
μm2
W
T
p=9.9e-24
p=8.5e-42
W
T
RI
NG
1A
/B
 K
O
EE
D 
KO
RING1A/B KO
HNRNPK
[0-1000]
[-2-2]
[0-1000]
[-2-2]
 Δ
EED KO
RING1A/B KO
 Δ
0
500
1000
1500
2000
X
is
t d
en
si
ty
Subject to XCI
Non-expressed
Escapee
E
Xist cloud
0
20
40
60
80
100
%
None Dispersed
Weak Normal
n > 100
RI
NG
1A
/B
 K
O
EE
D 
KOW
T
GAPDH
H2AK119ub
H3K27me3
F G
ChrX: 166 Mb Xist
[0-20000]
[0-20000]
[-3-3]
[0-20000]
[-3-3]
[0-1000]
[0-1000]
[-1.5-2]
[0-1000]
[-1.5-2]
[0-700]
[0-700]
[-3-0]
[0-700]
[-3-0]
RefSeq
RefSeq
WT RING1A/B KO EED KO
XistTsixKlf8
 Kdm5c
(Escapee)
 Δ
EED KO
RING1A/B KO
 Δ
WT
RefSeq
 Δ
EED KO
RING1A/B KO
 Δ
WT
EED KO
2μm
p<2.2e-16
p=0.1
p=0.9
p<2.2e-16
p<2.2e-16
p<2.2e-16
[0-1000]
5μm
WT
(legend on next page)
Molecular Cell 74, 101–117, April 4, 2019 107
localization (Figure S4H). Reduction of H3K27me3 enrichment
over the Xi did not occur until day 6. No change in global
H3K27me3 or H2AK119ub was observed by western analysis at
days 2, 4, or 6 (Figure 3G), suggesting HNRNPK’s role in Poly-
comb maintenance is Xi specific. Super-resolution imaging
confirmed the diffuse Xist clouds in HNRNPK KD cells to be
highly similar to those in DRepB cells (Figure 3H, cf. to Fig-
ure 1E). Collectively, these data demonstrate the importance of
HNRNPK in mediating Xist spreading and Polycomb targeting
across the Xi.
Interdependent Spreading of Xist RNA and Polycomb
Complexes
We wondered whether the Xist spreading defect might be linked
to Polycomb loss. To test this, we generated RING1A/RING1B
(the catalytic components of PRC1) double KO or EED (a core
component of PRC2) KO MEFs and verified the total loss of
H2AK119ub or H3K27me3, respectively (Figure 4A; Data S1).
In both KO cell lines, we observed surprising delocalization of
Xist RNA reminiscent of DRepB and HNRNPK KD phenotypes,
confirmed by super-resolution imaging (Figures 4B, cf. to Figures
1E and 3H). This was true in additional RING1A/B and EED KO
clones (Figures S5A and S5B; Data S1) and was not an indirect
effect of PRC1/2 depletion on Xist RNA level or on genes known
to affect Xist localization (Figure 4C). Importantly, HNRNPK’s
association with Xist was unaffected, indicating that PRC1/2’s
effect on cloud morphology occurs downstream of Repeat
B-HNRNPK interaction (Figure 4D).
Because Polycomb is known to play a role in chromatin
compaction (Boettiger et al., 2016; Kundu et al., 2018), we asked
whether diffuse Xist clouds could be a byproduct of Xi decom-
paction, rather than a true defect in Xist spreading. Sequential
Xist RNA FISH and X chromosome painting showed Xist clouds
often exceeding the boundary of the underlying Xi territory in
RING1A/B and EED KO cells—and occurring on the DRepB
but not WT Xi in the same nucleus of DRepB Xi+/� cells (Fig-
ure S5C). This observation is consistent with decreased accu-
mulation of Xist on chromatin as seen by CHART-seq. Size
measurements of WT Xi and Xa DNA territories revealed a differ-
ence of only �20%, similar to a previous analysis in mouse cells
(Giorgetti et al., 2016). This is considerably smaller than the 2- to
4-fold difference between WT and diffuse Xist RNA clouds in
DRepB Xi+/�, HNRNPK KD, RING1A/B KO, and EED KO cells
(Figures 1E, 3H, and 4B). Moreover, we were unable to detect
any significant shift in Xi:Xa size ratio in DRepB Xi+/�, RING1A/B
KO, and EED KO cells (Figure S5C). These data suggest that the
Figure 4. Xist and Polycomb Complexes Depend on Each Other to Spr
(A) Western blot confirming the total loss of H2AK119ub and H3K27me3 in RING
(B) RNA FISH, 3D STORM imaging, and size measurements of Xist clouds in WT
(C) qRT-PCR showing no change in Xist levels or genes known to affect Xist loc
replicates.
(D) IF showing HNRNPK association with Xist is unaffected in RING1A/B and EE
(E) Xist CHART-seq in RING1A/B and EED KO MEFs. Change in Xist coverage r
unmappable regions are masked.
(F) Boxplots showing Xist coverage over genes subject to XCI, non-expressed, and
by Wilcoxon rank-sum test.
(G) Zoom-in of CHART-seq tracks for representative regions.
See also Figure S5.
108 Molecular Cell 74, 101–117, April 4, 2019
loss of Repeat B or Polycomb in post-XCI cells is not sufficient to
reverse Xi compaction at this scale.
To investigate the Xist localization defect further, we per-
formed RNA/DNA FISH on mitotic chromosomes from DRepB
Xi+/�, RING1A/B KO, and EED KO cells. In this state, all chromo-
somes are condensed and WT Xist RNA often remained associ-
ated with the Xi in cis (Figure S5D). However, DRepB Xist within
the same cell was barely detectable or completely absent
from its corresponding Xi. Similar Xist dissociation was seen in
RING1A/B and EED KO cells. These analyses indicate that a
difference in Xist’s ability to interact with chromatin, rather
than Xi decompaction, likely accounts for the cloud dispersal
phenotype.
To corroborate these findings at the molecular level, we per-
formed CHART-seq using RING1A/B and EED KO cells. In
agreement with RNA FISH analysis, Xist binding was reduced
across the Xi relative to WT in both cell lines (Figure 4E). Interest-
ingly, EEDKO showed a stronger effect than RING1A/B KO, indi-
cating that spreading of Xist RNA may depend on PRC2/
H3K27me3 more than on PRC1/H2AK119ub. Overall coverage
profiles mirrored that of DRepB Xi�/� CHART-seq (cf. to Fig-
ure 2A), exhibiting particularly diminished Xist binding at chro-
mosome extremities. Plots of Xist density again showed low
coverage over genes normally subject to XCI and non-expressed
genes (Figure 4F). Examination of specific genes confirmed a
reduction of Xist occupancy across gene bodies and surround-
ing intergenic regions, but not at the Xist locus itself or escapees
(Figure 4G). Depletion of PRC1 or PRC2 therefore phenocopies
deletion of Repeat B. Taken together, our data reveal the surpris-
ing conclusion that, while Xist RNA recruits Polycomb com-
plexes to the Xi, PRC1 and PRC2 are in turn required to properly
spread Xist.
Independent and Interdependent Recruitment of
Polycomb Complexes
An ongoing debate in the XCI field regards the order in which
PRC1 and PRC2 are recruited to chromatin (Schoeftner et al.,
2006; Zhao et al., 2008; Margueron and Reinberg, 2011; Simon
and Kingston, 2013; Cooper et al., 2016; Almeida et al., 2017;
Pintacuda et al., 2017). Given our KO cell lines, we examined
reciprocal effects of depleting PRC1 and PRC2. As shown by
IF, KO of EED expectedly caused a total loss of H3K27me3 but
also reduced H2AK119ubenrichment on the Xi (Figures 5A and
S5B). This effect is consistent with the canonical Polycomb
pathway whereby H3K27me3 facilitates PRC1 recruitment
(Cao et al., 2002; Margueron and Reinberg, 2011; Simon and
ead across Xi
1A/B and EED KO MEFs, respectively
, RING1A/B KO, and EED KO MEFs. p values are by two-tailed t test.
alization in RING1A/B and EED KO MEFs. Error bars show SD for 3 biological
D KO MEFs.
elative to WT (D) is shown below as log2(KO/WT). Xist locus is indicated and
escapees inWT,DRepB Xi�/�, RING1A/BKO, and EEDKOMEFs. p values are
A
B
DC
0
50
100
H
3K
27
m
e3
 d
en
si
ty
0
20
40
60
H
2A
K
11
9u
b 
de
ns
ity
Subject to XCI
(Xa)
Subject to XCI
(Xi)
Non-expressed
(Xa)
Non-expressed
(Xi)
Escapee
 (Xa)
Escapee
 (Xi)
WT
WT
RING1A/B KO
EED KO
p<2.2e-16
p<2.2e-16
p<0.006
p<2.2e-16
p<2.2e-16
p=0.7
Figure 5. Independent and interdependent Recruitment of PRC1 and PRC2 to Xi
(A) Xist RNA FISH and H3K27me3/H2AK119ub IF in WT, RING1A/B KO, and EED KO MEFs.
(B) Allele-specific ChIP-seq for H3K27me3 and H2AK119ub in RING1A/B and EED KO MEFs, respectively. WT tracks included for comparison. Composite
(comp) of all reads as well as allelic (Xi and Xa) tracks shown. Xist locus is indicated and unmappable regions are masked.
(legend continued on next page)
Molecular Cell 74, 101–117, April 4, 2019 109
Kingston, 2013). Similarly, RING1A/B KO caused a total loss
of H2AK119ub but also significantly reduced H3K27me3
enrichment on the Xi. This effect is consistent with the non-
canonical Polycomb recruitment pathway whereby H2AK119ub
facilitates PRC2 recruitment (Tavares et al., 2012; Kalb et al.,
2014; Cooper et al., 2016). Xist delocalization (due to Polycomb
loss) may also partly contribute toward the overall reduction
of H2AK119ub/H3K27me3 in each KO cell. If so, this would sug-
gest that the interdependency between Polycomb complexes on
Xi may in fact partially stem from their role in helping Xist to
spread.
Nevertheless, in both EED and RING1A/B KO cell lines, a
notable fraction of cells retained H2AK119ub or H3K27me3 Xi
enrichment, respectively (Figures 5A and S5B), suggesting
PRC1 and PRC2 can be recruited independently of one another
to a degree. This was unexpected given a recent model in which
PRC2 recruitment to the Xi was proposed to strictly depend on
prior H2AK119ub modification by non-canonical PRC1 (Cooper
et al., 2016; Almeida et al., 2017; Pintacuda et al., 2017). We ruled
out the possibility that residual H2AK119ub/H3K27me3 Xi enrich-
ment could be due to channel bleed-through from concurrent Xist
RNA FISH, as IF alone showed identical results (Figure S5E). To
see whether the more compact state of the Xi could cause
H2AK119ub or H3K27me3 to appear enriched above neighboring
chromatin, we performed IF using panH2A or panH3 antibodies,
respectively, inWTcells andsawnosuchenrichment (FigureS5F).
Taken together, perturbing either PRC1 or PRC2 weakened the
other’s modification of Xi chromatin, though PRC1’s effect on
H3K27me3 was stronger than PRC2’s on H2AK119ub. Yet at the
same time, the two can function independently on Xi to an extent,
sincemany cells retainedweakH2AK119ub orH3K27me3enrich-
ment when PRC2 or PRC1 was depleted, respectively.
To confirm these results with another approach, we performed
ChIP-seq for H3K27me3 in RING1A/B KO and H2AK119ub in
EED KO cells. In agreement with our IF observations, EED KO
caused significant reduction of H2AK119ub on Xi relative to
WT, and RING1A/B KO caused even stronger reduction of
H3K27me3 (Figure 5B). These effects were chromosome-wide.
Nevertheless, H2AK119ub/H3K27me3 levels remained slightly
higher on Xi over Xa in PRC2/1 KO cells, respectively, as well
as over Xi in DRepB Xi�/� cells, which showed near-complete
loss of both marks (Figures 5B–5D, cf. to Figure 2). This residual
enrichment was particularly noticeable over genes subject to XCI
(Figures 5C and 5D, Pak3) but less so over non-expressed genes
(Figures 5C and 5D, Dcx), which often already contain
H2AK119ub/H3K27me3 on Xa. Together, these data support
both independent and interdependent mechanisms of PRC1/2
recruitment during maintenance of XCI.
Failure of Xi Gene Silencing with Partial Compensatory
Downregulation of Xa
To determine the role of Repeat B in maintenance of gene
silencing, we performed RNA-seq in DRepB Xi�/�MEFs. Noma-
(C) Boxplots showing H3K27me3 and H2AK119ub coverage over genes subject t
EED KO MEFs. p values are by Wilcoxon rank-sum test.
(D) Zoom-in of ChIP-seq tracks for representative region.
See also Figure S5.
110 Molecular Cell 74, 101–117, April 4, 2019
jor changes occurred in X-linked gene expression (Figures S6A
and S6B), despite chromosome-wide depletion of H2AK119ub
and H3K27me3 (Figure 2D). These findings are consistent with
gene silencing being generally stable in post-XCI cells, indepen-
dent of Xist RNA (Brown and Willard, 1994; Yildirim et al., 2013).
To determine whether Repeat B is required for de novo silencing,
we generated the same deletion in female mouse embryonic
stem cells (ESCs) (Figure S6C; Data S1), which undergo XCI as
they differentiate. Importantly, our ESC line is a mus/cas hybrid
that selectively inactivates the mus X chromosome (Figure S6D)
(Ogawa et al., 2008), facilitating downstream allelic analysis.
When differentiated for 14 days, the DRepB ESCs recapitulated
the defective Xist cloud and H2AK119ub/H3K27me3 pheno-
types seen in MEFs (Figure 6A). Thus, Repeat B is also required
for Xist spreading and Polycomb targeting during de novo estab-
lishment of XCI.
Transcriptomic analysis at day 14 of differentiation showed an
upregulation of X-linked genes in DRepB cells compared to WT,
whereas no significant change was observed for autosomes
such as chromosome 13 (Figures 6B and 6C, ‘‘comp’’). Examina-
tion of individual alleles showed a drastic increase specific to Xi,
as evidenced by a rightward shift in cumulative distribution fre-
quency (CDF) plots and an upward deviation in scatterplots (Fig-
ures 6B and 6C, ‘‘mus’’). The fraction of reads from Xi shifted
from �5% (monoallelic) in WT to �35% (biallelic) in DRepB
ESCs (Figure 6D). This percentage remained at the expected
50% (biallelic) for reads from chromosome 13 in both WT and
DRepB cells. Genes that failed to be silenced showed no obvious
clustering along the chromosome (Figure 6E). Inspection of indi-
vidual genes confirmed the presence of reads from the Xi in
DRepB but not WT cells (Figure 6F). Interestingly, Xi upregulation
was accompanied by a slight but significant (p = 5.7e-15) and
reproducible downregulation of Xa (Figures 6B and 6C, ‘‘cas’’),
suggesting partial Xa compensation for failed Xi silencing. This
offers one possible explanation for the continued survival of
DRepB cells despite undergoing incomplete XCI. Similar results
were obtained using a second independently derived DRepB
clone (Figures S6E–S6I). Thus, Repeat B is essential during dif-
ferentiation for establishing chromosome-wide gene silencing
on the Xi—which, if compromised, may be partially rescued by
downregulating the Xa.
Deleting Repeat B Impairs Xi Topological
Reconfiguration
XCI is accompanied by architectural reconfiguration of the Xi,
characterized by weakening of topologically associating do-
mains (TADs) and formation of megadomains (Nora et al.,
2012; Rao et al., 2014; Deng et al., 2015; Minajigi et al., 2015;
Giorgetti et al., 2016). We wondered how deleting Repeat B
might broadly affect Xi chromosome structure. To address this,
we performed allele-specific in situ Hi-C in DRepB ESCs after
14 days of differentiation. Contact heatmaps at 100-kb resolu-
tion showed that, as expected, the WT Xi folded into two
o XCI, non-expressed, and escapees in WT, DRepB Xi�/�, RING1A/B KO, and
A
B
C
D F
E
(legend on next page)
Molecular Cell 74, 101–117, April 4, 2019 111
megadomains (Figure 7A, upper-left panel, ‘‘WT’’). Interestingly,megadomains were also present on DRepB Xi (Figure 7A,
upper-left panel, ‘‘DRepB’’), despite the significant defect in
XCI establishment (Figures 6 and S6E–S6I). Thus, whilemegado-
main architecture is not required for Xi gene silencing (Darrow
et al., 2016; Giorgetti et al., 2016; Froberg et al., 2018), our
data show it is neither sufficient for, nor a consequence of
gene silencing. By examining the Pearson correlation map for
interaction frequencies, the two megadomains appeared even
more pronounced on the Xi in DRepB relative to WT (Figure 7A,
lower-left panel, cf. ‘‘WT’’ and ‘‘DRepB’’). Consistent with this
observation, interaction frequency between the two megado-
mains on Xi decreased from 15% of total chromosome interac-
tions (WT) to 9% (DRepB), whereas the Xa remained unchanged
(9%). DRepB Xi�/� MEFs showed the same trend (Figure S7A)—
17% (WT) to 12% (DRepB) on Xi; Xa unchanged (12%)—sug-
gesting Repeat B might play a role in promoting long-range
chromosomal interactions spanning the megadomain border.
Next, we examined Xi TAD organization. Consistent with a
recent study from our lab (Wang et al., 2018), TADs were still pre-
sent on Xi but in a significantly attenuated state relative to Xa (Fig-
ures 7B and 7C). However, TADs were visibly less attenuated on
DRepBXi compared toWT. To quantify this, we calculated ‘‘insu-
lation scores’’ at 100-kb resolution acrossWT Xa and used these
to call TADs. Consistent with prior studies (Giorgetti et al., 2016),
�110 TADswere identified (�90 after removal of regionswith low
SNP density). We then assigned a metric to each, with a greater
‘‘TAD score’’ signifying greater TAD strength (see STARMethods
for details). Averaged across the chromosome, TADswere stron-
ger on theDRepB Xi compared toWT in day 14 ESCs (Figure 7B),
with �30 individual TADs being significantly stronger (�8 were
weaker) (Figure 7C). We did not detect any significant correlation
between strength of these TADs and corresponding gene
expression (Figure S7D), in line with our earlier observation that
genes no longer silenced in DRepB ESCs do not cluster along
the chromosome (Figures 6E and S6H). Allele-specific in situ
Hi-C in DRepB Xi�/� MEFs revealed similar, albeit subtler, im-
pacts of deleting Repeat B on maintenance of Xi chromosome
structure (Figures S7A–S7C), despite no significant change in
gene expression (Figures S6A and S6B). In particular, �20
TADswerestronger (�7wereweaker) on theDRepBXi compared
to WT (Figures S7B and S7C), similar to our previous observa-
tion in MEFs harboring a full-length Xist deletion (Minajigi et al.,
2015). Collectively, these results suggest that Repeat B is at least
partly responsible for Xist’s role in disrupting TADs during both
establishment and maintenance of XCI. Furthermore, they
demonstrate that TADs and megadomains can co-exist on the
X chromosome.
In addition to TADs, mammalian chromosomes are partitioned
into alternating A/B compartments, formed through self-associ-
Figure 6. Repeat B Is Required for Complete Xist-Mediated Gene Silen
(A) H2AK119ub/H3K27me3 IF and Xist RNA FISH in DRepB and WT ESCs.
(B and C) CDF (B) plots and (C) scatterplots depicting allele-specific expression
(D) Boxplots showing increase in the fraction of mus reads for X-linked genes in
(E) Fraction of mus reads for genes with respect to chromosomal position in DRe
(F) Zoom-in of RNA-seq tracks for representative genes.
See also Figure S6.
112 Molecular Cell 74, 101–117, April 4, 2019
ation of active gene-rich (A) and inactive gene-poor (B) regions
(Lieberman-Aiden et al., 2009; Bickmore and van Steensel,
2013; Rao et al., 2014; Bonev and Cavalli, 2016). During forma-
tion of the Xi, A/B compartments are partially merged to create
larger ‘‘S1/S2’’ transitional compartments, which are merged
further to yield the final Xi-specific macro-structure (Wang
et al., 2018). To examine potential effects of deleting Repeat
B on compartmentalization, we again examined Pearson corre-
lation maps of our Hi-C interaction matrices. As expected, the
Xa displayed a checkerboard pattern characteristic of A/B
compartmentalization (Figures 7A, lower-right panel, and S7E).
Meanwhile, the WT Xi displayed a partial checkerboard pattern
resembling S1/S2 compartmentalization (Figures 7A, lower-left
panel, ‘‘WT’’, S7E). However, our principal-component analysis
only detected megadomains, perhaps due to the late differenti-
ation time point of our dataset (S1/S2 compartmentalization
was originally reported using day 7 ESCs). This was absent on
DRepB Xi, which instead retained Xa-like features in some re-
gions (Figure 7A, arrows). Taken together, our data show that
Repeat B plays a critical role in transforming the X from an active
to inactive chromosomal structure.
DISCUSSION
Here,wehaveperformed the first comprehensive deletional anal-
ysis of Xist RNA in the native context and found that Repeat B
plays a critical role in propagating Xist RNA and Polycomb com-
plexes along the Xi. Unexpectedly, our analyses revealed a
profound inter-dependency between Xist RNA and Polycomb
complexes during the process of spreading. These findings shift
our view of the relationship between Xist and Polycomb from one
of unidirectional to bidirectional recruitment. Indeed, our study
shows that thedynamicsofPolycomb recruitment aremorecom-
plex than previously suggested. Both canonical (PRC2 action re-
cruits PRC1) and non-canonical (PRC1 action recruits PRC2)
pathways have been proposed, with prevalent models positing
that one complex depends on the other in a hierarchical fashion.
However, more recent evidence suggests a greater degree of
interdependency between PRC1 and PRC2 (Schoeftner et al.,
2006; Margueron and Reinberg, 2011; Kahn et al., 2016; Almeida
et al., 2017; Dorafshan et al., 2017). Consistently, our results for
the X chromosome show that depleting PRC2 limits but does
not abolish PRC1 recruitment, and vice versa. Thus, PRC1 and
PRC2 reinforce each other’s occupancy but can be recruited
independently of the other to a limited extent. Our model there-
fore proposes that the two pathways can work in parallel during
maintenance of XCI, though both require Xist Repeat B and
HNRNPK.
In the simplest model (Figures 7D and 7E), Xist recruits PRC1
and PRC2 to Xi chromatin. PRC2 then trimethylates H3K27 and
cing
in DRepB versus WT ESCs. p values are by Wilcoxon rank-sum test.
DRepB versus WT ESCs. p values are by two-tailed t test.
pB versus WT ESCs.
A B
C
D E
Figure 7. Repeat B Is Required for Proper Xa to Xi Topological Reconfiguration
(A) Hi-C interaction maps at 500-kb resolution for WT (above diagonal) versus DRepB (below diagonal) Xi (left) and Xa (right) in ESCs. Corresponding Pearson
correlation maps at 200-kb resolution are shown below. Arrows indicate regions of DRepB Xi exhibiting Xa-like features.
(B) Average TAD scores for Xi and Xa in WT and DRepB ESCs. Chromosome 13 is shown for comparison. p values are by Wilcoxon rank-sum test.
(C) Individual TAD scores for Xi and Xa inWT (blue) and DRepB (pink) ESCs. TADs showing significant difference are indicated above with a black dot. Zoom-in of
regions with corresponding Hi-C interaction maps is shown at 100-kb resolution.
(D) Model: Interdependent spreading of Xist RNA, PRC1, and PRC2. Xist recruits PRC1/2 to the Xi. HNRNPK is required for this process through its direct
interaction with Repeat B. PRC1/2 reinforce each other’s recruitment and spread along chromatin via canonical and noncanonical Polycomb pathways.
(legend continued on next page)
Molecular Cell 74, 101–117, April 4, 2019 113
PRC1 ubiquitylates H2AK119. The H3K27me3 and H2AK119ub
marks would then serve as binding sites for PRC1 and PRC2
through the canonical and non-canonical pathways, respectively
(Margueron and Reinberg, 2011; Tavares et al., 2012; Simon and
Kingston, 2013; Kalb et al., 2014; Cooper et al., 2016). In this
way, PRC1 and PRC2 reinforce each other’srecruitment by
Xist RNA and partially self-propagate along the chromatin in
cis. In turn, as revealed in this study, PRC1 and PRC2 recipro-
cally enable Xist to spread on the Xi, possibly by mediating con-
tact between RNA and chromatin. Alternatively, Polycomb-
mediated changes to chromatin structure (Francis et al., 2004;
Kundu et al., 2018; Oksuz et al., 2018) may allow Xist to better
interact with chromatin or spread to less accessible regions (Fig-
ure 7D). Thus, the inter-dependency between Xist and Polycomb
complexes would underlie the chromosome-wide propagation
of the silencing machinery via a positive feedback loop.
Notably,previousstudieswithPRC1/2KOsordeletionsencom-
passingRepeatBdidnotexhibit noticeableeffectson themorpho-
logical character of Xist clouds (Wutz et al., 2002; Schoeftner et al.,
2006; da Rocha et al., 2014; Almeida et al., 2017; Pintacuda et al.,
2017). A number of reasons could explain this discrepancy,
including non-native conditions employed by previous studies,
such as the use of transgenes and/or forced Xist expression. Prior
studies also did not employ super-resolution imaging. Here, all
experiments were performed in the native context and in female
cells and furthermore benefited from inclusion of an unperturbed
Xist cloud in the same nucleus for side-by-side comparison.
Another interesting result from our screen was that many of
Xist’s functional elements correspond to repetitive motifs,
which may act as multicopy binding platforms for trans factors.
As a proof of principle, we demonstrate that Repeat B can
indeed accommodate multiple molecules of HNRNPK. Previous
studies have suggested a direct interaction between HNRNPK
and PRC1 (Pintacuda et al., 2017) as well as PRC2 (Denisenko
and Bomsztyk, 1997). Because HNRNPK is a general RNA-
binding protein, it is likely to require cooperation with additional
Xi factors to carry out its role in Polycomb recruitment. Indeed,
the Repeat B pathway may be only part of the picture. Deleting
Repeat B causes substantial failure of de novo gene silencing
chromosome-wide. However, partial silencing still occurred,
likely due to intact Repeat A, a motif known to be involved in
gene silencing (Wutz et al., 2002). Thus, Repeats A and B
may work together to establish, spread, and maintain epige-
netic silencing. Unfortunately, we were unable to investigate
Repeat A’s individual contribution in this study due to the con-
founding effect on Xist splicing.
Finally, our study also demonstrates that Repeat B plays an
important role in architectural reorganization of the Xi. Deleting
Repeat B post-XCI resulted in failed attenuation of TADs but at
the same time left megadomains strongly intact. This effect
was even greater when deleting Repeat B during de novo XCI,
consistent with the accompanying failure of gene silencing.
Polycomb spreading in turn is required for proper Xist binding along chromatin. T
histones, and/or changes in local chromatin structure.
(E) Simple schematic of the Repeat B pathway, showing inter-dependency betwee
Polycomb recruitment as shown previously (Zhao et al., 2008).
See also Figure S7.
114 Molecular Cell 74, 101–117, April 4, 2019
These results indicate that TADs and megadomains are not
mutually exclusive, and furthermore that the Xi-specificmegado-
main structure does not preclude gene expression. Moreover,
deleting Repeat B led to a relative decrease in interaction fre-
quency between the two megadomains. Thus, Repeat B (and
Xist in general) may play a role in promoting long-range chromo-
somal interactions that span the two domains. Last, compart-
ment structures were also affected, with the mutant Xi lacking
patterns resembling S1/S2 compartmentalization, unlike the
WT Xi. Instead, the mutant Xi exhibited regional Xa-like features.
Thus, Repeat Bmay play a role in S1/S2 compartment formation.
Our study has thus identified a multifunctional RNA domain that
coordinates interdependent spreading of Xist and Polycomb
complexes, gene silencing, and the topological transformation
from active to Xi chromosome structure.
STAR+METHODS
Detailed methods are provided in the online version of this paper
and include the following:
d KEY RESOURCES TABLE
d CONTACT FOR REAGENT AND RESOURCE SHARING
d EXPERIMENTAL MODEL AND SUBJECT DETAILS
his
n X
B Cell lines
d METHOD DETAILS
B ES cell differentiation
B Generation of Xist deletions and HNRNPK, RING1A/B,
and EED KO cells
B Labeling of Xist oligo FISH probes
B RNA FISH
B RNA FISH/X chromosome painting
B IF/RNA FISH
B Preparation of mitotic chromosomes
B Microscopy
B 3D STORM super-resolution microscopy
B Microscopy image analysis
B Antibodies
B Western blot
B Southern blot
B In vitro RNA pulldown and mass spectrometry
B Purification of His-tagged HNRNPK
B RNA EMSA
B GFP-RYBP plasmid and HNRNPK siRNA delivery
B RT-PCR
B ChIP-seq
B CHART-seq
B ChIP-seq and CHART-seq analysis
B RNA-seq
B RNA-seq analysis
B In situ Hi-C
B In situ Hi-C analysis
may be due to Xist interaction with Polycomb complexes, modification of
ist and Polycomb complexes. Dotted arrow represents Repeat-A-mediated
d QUANTIFICATION AND STATISTICAL ANALYSIS
d DATA AND SOFTWARE AVAILABILITY
SUPPLEMENTAL INFORMATION
Supplemental Information includes seven figures, four tables, and one data file
and can be found with this article online at https://doi.org/10.1016/j.molcel.
2019.01.015.
ACKNOWLEDGMENTS
We thank all members of the Lee lab for critical comments and stimulating dis-
cussions. We acknowledge Hyun Jung Oh and Hsueh-Ping Chu for assistance
with the CHART protocol, YongWoo Lee for help with preparation of mitotic
chromosomes, Catherine Cifuentes-Rojas for characterization of EED KO
clones, and John E. Froberg and Chunyao Wei for advice on bioinformatic an-
alyses. H.S. was supported by NIH 5T32HD007396-24, A.J.K. by NSF GRFP
and Herchel Smith Fellowship, and J.T.L. by NIH RO1-GM090278 and the Ho-
ward Hughes Medical Institute.
AUTHOR CONTRIBUTIONS
D.C., H.S., and J.T.L. conceived the project, devised experiments, interpreted
data, and wrote the paper. D.C. designed and performed RNA pull-down and
HNRNPK experiments. H.S performed STORM super-resolution imaging and
quantitative image analyses. A.J.K. performed Hi-C and related bioinformatic
analyses. C.-Y.W. contributed an optimized CHART protocol. H.S. and
C.-Y.W. performed ChIP-seq, CHART-seq, and RNA-seq bioinformatic ana-
lyses. All other experiments and analyses were performed together by D.C.
and H.S.
DECLARATION OF INTERESTS
J.T.L. is a co-founder and amember of the Scientific Advisory Boards of Trans-
late Bio and Fulcrum Therapeutics.
Received: July 27, 2018
Revised: November 21, 2018
Accepted: January 10, 2019
Published: February 28, 2019
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