MicroRNA Regulation of Cbx7 Mediates a Switch of Polycomb Orthologs during ESC Differentiation

Cell Stem Cell

Article

MicroRNA Regulation of Cbx7 Mediates a Switchof Polycomb Orthologs during ESC DifferentiationAna O’Loghlen,1,2,12 Ana M. Munoz-Cabello,1,2,12 Alexandre Gaspar-Maia,5 Hsan-Au Wu,5 Ana Banito,1,2

Natalia Kunowska,2,3 Tomas Racek,6 Helen N. Pemberton,6 Patrizia Beolchi,1,2 Fabrice Lavial,7 Osamu Masui,8

Michiel Vermeulen,9,10 Thomas Carroll,2 Johannes Graumann,9,11 Edith Heard,8 Niall Dillon,2,3 Veronique Azuara,7

Ambrosius P. Snijders,4 Gordon Peters,6 Emily Bernstein,5 and Jesus Gil1,2,*1Cell Proliferation Group2Epigenetics Section3Gene Regulation and Chromatin Group4Biomolecular Mass Spectrometry and Proteomics LaboratoryMRC Clinical Sciences Centre, Imperial College London, Hammersmith Campus, London W12 0NN, UK5Department of Oncological Sciences, Mount Sinai School of Medicine, 1425 Madison Avenue, New York, NY 10029, USA6Molecular Oncology Laboratory, CRUK London Research Institute, London WC2A 3PX, UK7Institute of Reproductive and Developmental Biology, Imperial College, London W12 0NN, UK8Institut Curie, 26 rue d’Ulm, Paris F-75248, France9Department of Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, 82152 Martinsried, Germany10Department of Molecular Cancer Research, University Medical Center Utrecht, The Netherlands11Weill Cornell Medical College in Qatar, Qatar Foundation, Education City, Doha, Qatar12These authors contributed equally to this work

*Correspondence: [email protected]

DOI 10.1016/j.stem.2011.12.004

SUMMARY

The Polycomb Group (PcG) of chromatin modifiersregulates pluripotency and differentiation. Mamma-lian genomes encode multiple homologs of the Poly-comb repressive complex 1 (PRC1) components,including five orthologs of the Drosophila Polycombprotein (Cbx2, Cbx4, Cbx6, Cbx7, and Cbx8). Wehave identified Cbx7 as the primary Polycomb ortho-log of PRC1 complexes in embryonic stem cells(ESCs). The expression of Cbx7 is downregulatedduring ESC differentiation, preceding the upregula-tion of Cbx2, Cbx4, and Cbx8, which are directlyrepressed by Cbx7. Ectopic expression of Cbx7inhibits differentiation and X chromosome inactiva-tion and enhances ESC self-renewal. Conversely,Cbx7 knockdown induces differentiation and dere-presses lineage-specific markers. In a functionalscreen, we identified the miR-125 and miR-181 fami-lies as regulators of Cbx7 that are induced duringESC differentiation. Ectopic expression of thesemiRNAs accelerates ESC differentiation via regula-tion of Cbx7. These observations establish a criticalrole for Cbx7 and its regulatory miRNAs in deter-mining pluripotency.

INTRODUCTION

The Polycomb Group (PcG) family of transcriptional repressors

plays important roles in the epigenetic regulation of pluripotency,

differentiation, X chromosome inactivation, and senescence

(Sparmann and van Lohuizen, 2006). PcG proteins operate in

multicomponent complexes, the best characterized of which

are termed Polycomb repressive complexes 1 (PRC1) and 2

(Morey and Helin, 2010). PRC2 catalyzes the trimethylation of

histone H3 on lysine 27 (H3K27me3), leading to recruitment of

PRC1, which ‘‘reads’’ H3K27me3 and ubiquitylates H2AK119

(Simon and Kingston, 2009). In Drosophila, PRC1 is composed

of stoichiometric amounts of Polycomb (Pc), Posterior sex

combs (Psc), Polyhomeotic (Ph), and Sex combs extra (Sce)

proteins; however, because mammalian genomes encode five

Pc, six Psc, three Ph, and two Sce orthologs, there are many

possible combinations of PRC1 components (summarized in

Figure 2A) (Whitcomb et al., 2007).

The reasons for such PcG diversification and the interplay

between the different orthologs remain unclear. Biochemical

analyses suggest that PRC1 complexes contain a single repre-

sentative of each subunit, and chromatin immunoprecipitation

(ChIP) studies in human fibroblasts have revealed that multiple

Pc and Psc orthologs, and therefore multiple PRC1 complexes,

can bind simultaneously at the INK4a/ARF locus (Maertens et al.,

2009). While the two Pc orthologs Cbx7 and Cbx8 are both

capable of repressing the INK4a/ARF locus in this cell model

(Dietrich et al., 2007; Gil et al., 2004), the role of each subunit

is also likely to be context dependent, reflecting the combina-

tions expressed in different cell lineages. For example, the

striking phenotype of Bmi1�/� mice, which have hematological

and neurological defects due to a failure in stem cell self-renewal

(Sparmann and van Lohuizen, 2006), implies that the other

members of the Psc family are unable to compensate for the

absence of Bmi1.

Genome-wide ChIP analyses in human and mouse embryonic

stem cells (ESCs) have shown that PRC1 and PRC2 localize to

the promoters of a subset of genes encoding transcription

factors (TFs) required for lineage specification (Boyer et al.,

Cell Stem Cell 10, 33–46, January 6, 2012 ª2012 Elsevier Inc. 33

mailto:[email protected]

http://dx.doi.org/10.1016/j.stem.2011.12.004

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Figure 1. Cbx7 Expression Is Associated with Pluripotency

(A) 3D visualization of Cbx7 binding to H3K27 versus H3K27me3 in a histone peptide PD experiment with SILAC-labeled mouse ESC nuclear extracts. The x axis

represents the mass-to-charge ratio of the peptides (m/z), the chromatographic retention time (t) is plotted on the y axis, and intensity of the peptides is in the

z axis. The identified Cbx7 peptide is �8 times more abundant in the ‘‘heavy’’ form compared to the ‘‘light’’ form, indicating specific H3K27me3 binding.

(B and C) H3K27me3 is bound preferentially by Cbx7 in ESCs and by Cbx2 and Cbx8 in differentiated ESCs and MEFs. H3K27me3 histone peptide PDs were

performedwith SILAC-labeled ESC (heavy) and differentiated ESC (B, light) orMEF (C, light) nuclear extracts. A negative value corresponds to preferential binding

in differentiated ESCs (B) or MEFs (C), and a positive value, to preferential binding on ESCs. The H/L spectral count is shown. Peptides corresponding to Cbx4 or

Cbx6 were not detected. Peptide PD followed by western blot against Cbx7 and Cbx8 was performed to confirm the MS results. Streptavidin immunobloting or

PonceuS staining revealing the biotinylated peptides that served as loading controls. I, input; U, unmethylated Histone H3 peptide; me3, H3K27me3 peptide.

Cell Stem Cell

ESC Differentiation via miRNA Regulation of Cbx7

34 Cell Stem Cell 10, 33–46, January 6, 2012 ª2012 Elsevier Inc.

Cell Stem Cell


2006; Lee et al., 2006). These promoters contain binding sites for

pluripotency-associated TFs, such as Oct4, Sox2, Nanog, and

Sall4 (Boyer et al., 2005, 2006; Lee et al., 2006), and are enriched

for both histone H3K4me3 and H3K27me3 marks (Azuara et al.,

2006; Bernstein et al., 2006a). This so-called bivalent mark

enables pluripotent cells to respond rapidly to differentiation

signals, and ESCs that lack critical PRC1 or PRC2 components

display global derepression of these target genes (Azuara et al.,

2006; Boyer et al., 2006). As a result, these ESCs are unstable

and more prone to spontaneous differentiation in culture (Boyer

et al., 2006), but the simultaneous ablation of PRC1 and PRC2

suggests that their activities are also required for full differentia-

tion (Leeb et al., 2010).

Decreased H3K27me3 and PcG binding has been observed

at lineage-specific genes during neural differentiation (Bracken

et al., 2006), keratinocyte maturation (Sen et al., 2008), and

skeletal muscle differentiation (Caretti et al., 2004), and in neural

progenitor cells (Mikkelsen et al., 2007). Several mechanisms

have been proposed to account for these changes. For example,

induction of the H3K27me3 histone demethylase Jmjd3 occurs

during differentiation in the epidermis (Sen et al., 2008) and

the expression of the PRC2 catalytic component Ezh2 is down-

regulated during skeletal muscle differentiation (Caretti et al.,

2004) in a manner that is at least partially dependent on

miR-214 expression (Juan et al., 2009). MicroRNAs (miRNAs)

are a class of small noncoding RNAs that function as post-

transcriptional regulators of gene expression (Brodersen and

Voinnet, 2009; He and Hannon, 2004). miRNAs are involved in

fine-tuning the expression of key target genes and play impor-

tant roles in the regulation of ESC pluripotency and differentia-

tion (Melton and Blelloch, 2010). However, to our knowledge,

there have been no reports describing the regulation of PRC1

components by miRNAs during ESC differentiation.

In this study, we identify Cbx7 as the specific Pc ‘‘reader’’ of

the H3K27me3 mark in ESCs. Cbx7 is expressed at high levels

in ESCs and teratocarcinoma cells while its levels decrease

during differentiation. This is in contrast to the expression

pattern of other Pc proteins, such as Cbx2, Cbx4, and Cbx8,

which are negatively regulated by Cbx7. Cbx7 has a critical

role in maintaining ESC self-renewal and inhibiting differentiation

and X-inactivation. We further identify miRNAs of the miR-125

and miR-181 families as key regulators of Cbx7 during ESC

differentiation. Taken together, our results suggest distinct,

context-dependent roles for individual PRC1 subunits and high-

light the importance of Cbx7 and its regulatory miRNA network

in ESC self-renewal and differentiation.

RESULTS

The Pc Ortholog Cbx7 Is Associated with PluripotencyA defining feature ofDrosophila Pc and its mammalian orthologs,

Cbx2, Cbx4, Cbx6, Cbx7, and Cbx8, is the ability to bind to

(D) Expression of Cbx6, Cbx7, and Cbx8 in different mouse cell types was assess

at passage 2 (p2) or 4 (p4).

(E) Cbx7 expression decreases upon neural differentiation of 46C ESCs.

(F) qRT-PCR analyses of the expression of Pc orthologs in 46C cells at days 0, 6

(G–I) The expression of Cbx7 decreases during embryoid body (EB) differentia

differentiation of P19 mouse teratocarcinoma cells.

H3K27me3 via sequences in the conserved chromodomain. To

investigate which of the Pc orthologs is responsible for PRC1

function in mouse ESCs, we took a quantitative proteomic

approach (Vermeulen et al., 2010) to identify proteins that bind

specifically to H3K27me3. One of the main binders, and the

only Pc ortholog detected, was Cbx7 (Figure 1A). This was in

sharp contrast to the H3K27me3 interactome of HeLa cells,

which identified Cbx2, Cbx4, and Cbx8, but not Cbx7 (Vermeu-

len et al., 2010). To assess whether the binding of Cbx7 to

H3K27me3 was specific for ESCs, we performed H3K27me3

pulldown (PD) assays using nuclear extracts from mouse ESCs

grown in ‘‘heavy’’ medium and differentiated ESCs or mouse

embryo fibroblasts (MEFs), grown in ‘‘light’’ medium. Mass

spectroscopy analysis revealed that while Cbx7 was the only

Pc ortholog recovered from ESCs, Cbx2 and Cbx8 were the

primary binders of this modification detected in differentiated

ESCs orMEFs (Figures 1B and 1C and Table S1 available online).

These results were confirmed by peptide PD followed by

immunoblot detection of Cbx7 or Cbx8 (Figures 1B and 1C

and Figure S1A).

To explore the biology underlying the differences in Cbx

affinity for H3K27me3, we analyzed the expression dynamics

of Cbx7 and the other Pc proteins during the differentiation of

ESCs (46C cells). These cells contain a Sox1-GFP transgene

and upon neural differentiation, expression of the Sox1 reporter

is induced concomitant with the downregulation of pluripotency

genes that include Oct4 and Nanog (data not shown). Immuno-

blotting showed that while Cbx7 is expressed in the ESCs,

very low levels were detected in the differentiated cells or in other

cell types (Figures 1D and 1E). Expression of Cbx6 did not

change significantly, whereas Cbx8 was absent in ESCs but

induced upon differentiation (Figures 1D and 1E). Analysis of

mRNA levels by qRT-PCR confirmed a sharp decline in Cbx7

expression during ESC differentiation as well as lower levels in

NSCs (Figure 1F and Figure S1B). This was in stark contrast to

the expression dynamics of other Pc paralogs, which were either

unchanged (Cbx6) or upregulated (Cbx2, Cbx4, and Cbx8)

during ESC differentiation (Figure 1F). We noted similar changes

in Cbx7 expression when 46C ESCs were induced to differen-

tiate into embryoid bodies (EBs) in an independent ESC line

(Oct4-GIP ESC), and during retinoic acid-induced differentiation

of P19 murine teratocarcinoma cells (Figures 1G–1I and Figures

S1C–S1E). Taken together, these findings suggest that among

the mammalian Pc homologs, Cbx7 is specifically associated

with pluripotency.

PRC1 Complex Composition Changes during ESCDifferentiationCbx7 is one of five Pc orthologs that can be present in the core

PRC1 complex (Figure 2A). Given the dynamic expression of

Cbx7, we investigated the composition of Cbx7-containing

PRC1 complexes in ESCs and during differentiation. To this

ed by immunoblotting. NSC, neural stem cells; MEF, mouse embryo fibroblasts

, 8, and 10 of neural differentiation.

tion of 46C ESCs, neural differentiation of Oct4-GiP ESCs, and RA-induced


A GFP IP/MS

PcPh

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Cbx2Cbx4Cbx6Cbx7Cbx8

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Figure 2. Changes in PRC1 Composition during ESC Differentiation(A) Putative PRC1members inmouse cells: five Polycomb (Pc), six Posterior sex comb (Psc), three Polyhomeiotic (Ph), and two Sex combs extra (Sce) paralogs in

mouse cells.

(B) Cbx7-EGFP (heavy) and EGFP (light) ESCs were SILAC-labeled and subjected to GFP IP followed byMS analysis. The H/L peptide count is represented to the

right.

(C) ESCs (heavy) and differentiated ESCs (light) were SILAC-labeled and subjected to IP using aRing1b antibodies followed byMS analysis. Proteins preferentially

binding to Ring1b in ESCs show a positive log2(H/L) value, and those that are preferentially associated to Ring1b in differentiated ESCs show a negative value.

H/L spectral count is presented to the right. A cutoff of two detected peptides was used to reliably quantify protein ratios.

(D) Cellular extracts were prepared from ESCs (d0) or ESCs subjected to neural differentiation (d5), and used for IP using aRing1b antibodies. Inputs and Ring1b

IPs were subjected to immunoblots (IBs) to detect Ring1b and Cbx7.

(E) The expression of PRC1 components during neural differentiation of 46C ESCs was analyzed by qRT-PCR. A different scale is used in the inset to better note

how the expression of Phc1 and Mblr decreases upon ESC differentiation.

Cell Stem Cell


end, we generated ESCs (derived from PGK12.1) expressing

a Cbx7-EGFP protein (Figure S2A) and used a SILAC strategy

to identify proteins that copurify with Cbx7-EGFP on GFP nano-

trap beads. We detected two Psc orthologs (Mel18 and Mblr),

two Ph proteins (Phc1 and Phc2), and one Sce protein (Ring1b),

consistent with formation of canonical PRC1 complexes (Fig-

ure 2B and Table S2). As an alternative strategy, we used

a monoclonal antibody against Ring1b to immunoprecipitate

endogenous PRC1 complexes from ESCs prior to and following

differentiation. Nuclear extracts from ESCs grown in heavy

medium were compared to extracts from differentiated ESCs

grown in light medium. Importantly, Cbx7 was the only Pc

ortholog associated with Ring1b in undifferentiated ESCs. The

differential association of Cbx7 with Ring1b was confirmed by

immunoblot (Figure 2D). Consistent with the GFP IPs, Mblr and

Phc1 were also found preferentially associated with Ring1b in

ESCs. In contrast, Cbx2, Cbx8, Bmi1, and others were more


prominently associated with Ring1b in differentiated cells.

Finally, other PRC1 components such as Mel18 and Phc2 bound

Ring1b to a similar extent in ESCs and differentiated cells. Both

of these proteins were also detected in association with Cbx7

in ESCs (Figure 2C and Table S2).

To further understand the differential composition of

PRC1 complexes, we analyzed the expression of PRC1

components during neural differentiation of 46C ESCs. Interest-

ingly, we observed that the expression of Phc1 and Mblr,

which were associated with Ring1b preferentially in ESCs,

declined during ESC differentiation, thus mimicking the

changes in Cbx7 expression (Figure 2E, inset). In contrast,

the expression of other PRC1 members, most notably Bmi1

and Phc2, increased during neural differentiation (Figure 2E).

These results are consistent with our observations in NSCs,

an independent ESC line, and teratocarcinoma cells (Figures

S2B–S2D).

Cell Stem Cell


Dynamic Interplay between Cbx7 and Cbx8 during ESCDifferentiationThe changes we observed above suggested a degree of antag-

onism in the expression of some PRC1 components during

differentiation, particularly among the Pc orthologs. To substan-

tiate this idea, we analyzed the levels of Cbx7 andCbx8 in a panel

of ESCs induced to differentiate into EBs (Figure 3A) or with ret-

inoic acid (Figure S3A). Again, we observed that the Pc paralog

expressed in ESCs is Cbx7, while Cbx8 is expressed upon

differentiation. To further dissect the implications of this switch,

we performed ChIP to determine the occupancy of Cbx7 and

Cbx8 at four targets genes (Gata4, Sox3, Neurog2, and Nr2f2)

in ESCs and their differentiated counterparts (Figure 3B and

Figure S3B). Whereas Cbx7 was present at the promoter of

each target gene in ESCs, it was barely detectable in differenti-

ated ESCs, neural stem cells (NSCs), or MEFs. Cbx8 showed

a reciprocal binding pattern, being absent from the promoters

studied in ESCs but present in some of the differentiated cells

in a gene-specific manner (Figure 3B and Figure S3B).

Analysis of published data sets (Marson et al., 2008) sug-

gested that Cbx7 is unique among the Pc orthologs in being

a target for the TFs of the pluripotency network, Sox2, Oct4,

and Nanog (Figure S3C). ChIP studies using ESCs showed that

indeed Sox2, Oct4, and Nanog all bind to a region upstream of

the transcription start site (TSS) of Cbx7 (Figure 3C). Taking

advantage of ZHBTc4 ESCs, which contain a tetracycline-

regulated Oct4 transgene, we could show that depletion of

Oct4 resulted in downregulation of Cbx7 expression (Figure 3D),

consistent with Cbx7 being under control of the pluripotency TF

network. We also observed a modest upregulation of Cbx8 in

these cells. This is consistent with ChIP studies demonstrating

that Cbx7 is marked by H3K4me3 in ESCs, while Cbx8 is marked

by H3K27me3 (Figure 3E). An independent data set (Mikkelsen

et al., 2007) also indicated that Cbx8 is a PcG target in ESCs

(data not shown). Our ChIP assays confirmed that the promoter

of Cbx8, and also those of Cbx2 and Cbx4, are occupied by

Cbx7 in ESCs (Figure 3F), whereas neither Cbx7 nor Cbx6

register as a PcG target in the cell types examined. Finally,

lentiviral shRNA-mediated knockdown of Cbx7 in ESCs resulted

in increased expression of Cbx2, Cbx4, and Cbx8 (Figure 3G),

suggesting that the expression of these Pc paralogs observed

upon ESC differentiation may be directly caused by loss of

Cbx7-mediated repression.

Cbx7 Contributes to the Maintenance of Pluripotencyin Mouse ESCsTo directly assess the role of Cbx7 in ESC pluripotency, we

knocked down Cbx7 using two independent siRNAs (Fig-

ure S4A). The extent of the knockdown was validated by western

blot and qRT-PCR (Figure 4A) and resulted in a clear and repro-

ducible phenotype with a higher proportion of ESC colonies dis-

playing a flattened or spread morphology indicative of a loss of

ESC characteristics (Figure 4B). This phenotype was similar to

the differentiation effects observed with siRNAs against Oct4

and Nanog (Figure 4B). In addition, the number of cells that

were positive for alkaline phosphatase (AP), a marker of undiffer-

entiated cells, was reduced upon Cbx7 knockdown (data not

shown). Similar results were observed with two additional

shRNAs delivered by lentiviral vectors (Figures S4B and S4C).

To ensure that off-target effects were not responsible for these

phenotypes, we used an shRNA (pLKO-shCbx7.1) that targets

the 30UTR of Cbx7 and repeated the experiment in ESCs ex-

pressing the Cbx7-EGFP protein (expression of this construct

lacking the 30UTR was unaffected by this shRNA) (Figure 4C).

Consistent with a Cbx7-mediated phenotype, infection with the

pLKO-shCbx7.1 lentivirus resulted in differentiation of control

ESCs but not of Cbx7-EGFP-expressing ESCs (Figure 4C). To

confirm by an alternative method that Cbx7 contributes to ESC

pluripotency, we obtained two independent clones of ESCs in

which one allele of Cbx7 has been inactivated (Wellcome Trust

Sanger Institute’s knockout mouse project, http://www.komp.

org). As expected, these Cbx7+/� ESC clones (B05 and F05)

expressed lower levels of Cbx7 as assessed by qRT-PCR and

immunoblot (Figure 4D). We observed a lower proportion of

AP-positive cells among Cbx7+/� ESCs than in control Cbx7+/+

ESCs (Figure 4D). Interestingly, delivery of a FLAG-tagged

version of Cbx7 into Cbx7+/� ESC resulted in an increased

number of colonies with a compact ESC morphology (Figures

S4D and S4E).

Finally, in order to understand how the depletion of Cbx7

affected ESCs, we analyzed the expression of several Pc target

genes associated with differentiation, and observed that their

expression increased upon Cbx7 knockdown, particularly that

of ectoderm-lineage-associated genes (Figure 4E), whereas

several pluripotency-related genes remained relatively unaf-

fected at both the mRNA (Figure 4E) and protein (Figure 4F

and Figure S4F) level. This suggests that although depletion of

Cbx7 results in a degree of spontaneous differentiation, ESCs

that have low levels of Cbx7 can still self-renew.

Cbx7 Expression Promotes ESC Self-Renewal bySuppressing Differentiation and X-InactivationThe fact that Cbx7 levels decreased during ESC differentiation

prompted us to test whether ectopic expression of Cbx7 could

sustain pluripotency. When cultured under ESC conditions,

ESCs overexpressing Cbx7 displayed lower levels of sponta-

neous differentiation, as judged by the number of colonies with

a compact morphology (Figure 5A) or stained positively for

Nanog (not shown). When cells were placed under differentiation

conditions, we noticed striking effects resulting from ectopic

Cbx7 expression. First, Cbx7 expression resulted in the dramatic

inhibition of X chromosome inactivation during ESC differentia-

tion, as measured by quantification of nuclear Xist RNA expres-

sion in several independent female ESC clones (Figure 5B and

Figure S5A). The cells also retained ESC characteristics when

subjected to neural differentiation or EB formation, as evaluated

by AP, Oct4, and Nanog staining as well as qRT-PCR analysis

(Figures 5C and 5D and Figures S5B–S5D).

To exclude the possibility that these results were clone

specific or due to excessive levels of Cbx7, we expressed

a FLAG/HA-tagged version of Cbx7 in ESCs and derived both

a pool, which expressed lower overall levels of Cbx7, and a clone

expressing higher levels of Cbx7 (Figure 5E). These ESCs, which

were subjected to EB differentiation and replating into ESC

conditions, also gave rise to significantly more AP-positive

colonies than did the corresponding controls (Figure 5F and

Figure S5E). We also analyzed the expression of several plu-

ripotency and lineage-specific genes at different times during


http://www.komp.org

http://www.komp.org

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hIP/

Inpu

t

Gata4 Neurog2

d0 d7 NSCMEFESC

d0 d7 NSC MEFESC

d0 d7 NSCMEFESC

d0 d7 NSCMEFESC

% C

bx8

ChI

P/In

put

% C

bx7

ChI

P/In

put

Amido Black

Figure 3. Dynamic Interplay between Cbx7 and Cbx8 Expression during Differentiation

(A) Male (E14, CCE) ESCs underwent EB differentiation over 12 days and the levels of Oct4, Cbx7, Cbx8, and histone H3 in the chromatin fraction were

assessed by IB.

(B) ChIP analysis showing binding of Cbx7 and Cbx8 at the Gata4 and Neurog2 loci in ESCs, differentiated ESCs (d7), NSCs, and MEFs. Similar trends

were observed with multiple primer sets for each locus (data not shown; primer sets listed in the Supplemental Information).

(C) ChIP data showing Nanog, Oct4, and Sox2 binding upstream of Cbx7, but not Cbx8, in ESCs. NOS, positive control region 200 bp upstream of the Nanog

gene bound by Nanog, Oct4, and Sox2.

(D) IB for Oct4, Cbx7, Cbx8, and H3 following doxycycline (Dox)-induced repression of Oct4 in ZHBTc4 ESCs. Amido Black staining of the membrane is shown

as a loading control.

(E) ChIP data showing H3K4me3 and H3K27me3 histone modifications on the Cbx7 and Cbx8 genes in ESCs.

(F) ChIP analysis of Cbx7 binding at the promoters of Pc paralogs in ESCs.

(G) Knockdown of Cbx7 in ESCs using shRNA results in upregulation of Cbx2, Cbx4, and Cbx8 mRNA expression.

Cell Stem Cell



siGLO

siCbx7.2 siCbx7.3

siOct4 siNanog

Scrambled

A

% o

f col

onie

s

C

vect

orsh

Cbx

7.1

Control

B

Scra

mbl

ed

siG

LOsi

Cbx

7.2

siC

bx7.

3

Cbx7-tubulin

Cbx7

-tubulin

GFP

D

0

40

80

120

Scr. siGLO siC7.2 siC7.3

mR

NA

leve

ls Cbx7

AP staining

0

30

60

90

Scr. siGLO siC7.2 siC7.3 siOct4 siNanog

vect

orsh

Cbx

7.1

Cbx7-EGFPESC:

Cbx7+/+

B05 F05

Cbx7

-tubulin

ControlCbx7

+/-

AP staining

B05

F05

Cbx7+/+

Cbx7+/-

Cbx7+/-

0

50

100

% A

P +

col

onie

s

Cbx7+/+ B05 F05Cbx7+/-

CompactFlatSpread

20

40

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100 CompactFlatSpread

% o

f col

onie

s

vector shCbx7.1 vector shCbx7.1Control Cbx7-EGFPESC:

40

80

120

Cbx7+/+ B05 F05Cbx7+/-

Cbx

7 m

RN

A le

vels

vector

shCbx7.1

shCbx7.2

Oct3/4

F

NanogIF:

Oct3/4DAPI

NanogDAPI

E

0

100

200

300

400

Oct

4

Nan

og

Sox

2

Fgf4

FoxA

2

Gat

a4

Gat

a6N

kx2.

2

Nkx

2.9

Olig

1

Rho

x5

Rho

x9

Dac

h1

Irx3

Mat

h1

Sox

3

Neu

rog1

Neu

rog2

Hox

a4B

rach

yury

Goo

seco

id

Rel

ativ

e m

RN

A le

vels Scrambled

siCbx7.2siCbx7.3

Pluripotency Endoderm Ectoderm Mesoderm

Figure 4. Cbx7 Levels Contribute to Maintenance of Pluripotency in ESCs

(A) ESCs were transfected with siRNAs targeting Cbx7, and 3–5 days after transfection, Cbx7 expression was quantified by IB (upper panel) and qRT-PCR (lower

panel).

(B) Three days after transfection of ESCs with the indicated siRNAs, pluripotency was examined by AP staining. Representative images are shown. siGLO and

a scrambled siRNA (AllStars) were used as a negative controls. Percentage of AP-positive colonies that showed a compact, flat, or spreading morphology is

represented.

(C) A lentiviral shRNA targeting Cbx7 30UTRdoes not induce differentiation of Cbx7-EGFP ESCs. Control or Cbx7-EGFP ESCswere infected with an empty vector

(vector) or a lentiviral shRNA targeting Cbx7 in its 30UTR (shCbx7.1). Immunoblot showing the expression of the EGFP-Cbx7 fusion, endogenous Cbx7, or

b-tubulin is shown (upper panel). Percentage of AP-positive colonies that showed a compact, flat, or spread morphology in the experiments are plotted.

(D) Two independentCbx7+/� ESC clones show spontaneous ESC differentiation. The expression of Cbx7 was assessed by IB (top panel) and qRT-PCR (middle).

The number of AP-positive colonies was quantified (bottom). Representative pictures are shown (right).

(E) Expression of a subset of pluripotency-associated and PcG target genes in ESCs transfected with Cbx7 siRNAs was monitored by qRT-PCR.

(F) The expression of Nanog, Oct3, and Oct4 remains unchanged in ESCs infected with lentiviral vectors targeting Cbx7, as assessed by immunofluorescence.

Cell Stem Cell



A

C

% A

P+

Col

onie

s/ce

ll pl

.

Control Cbx7-EGFP

D EB to ESC conditions

E

G

F

H

0

20

40

60

80

100

ESCControl

Cbx7-EGFP

compactflatspread

% C

olon

ies

AP staining

Control Cbx7-EGFP

Control Cbx7-EGFP

AP stainingAP staining

B

% C

ells

0 20406080

100

Control Cbx7-EGFP

Xist RNA FISH

2 Xist signals

1 Xist signal

1 Xist domain

LF2

LF2 E

GFPLF

2 Cbx

7 poo

lLF

2 Cbx

7 clon

e

Cbx7

Cbx7Cbx7-Flag/HA

0

2

4

6

% A

P+

Col

onie

s/ce

ll pl

.

Control Cbx7-EGFP

Neural differentiation

Control Cbx7-EGFP

LF2 LF2 EGFP

AP staining

% A

P+

Col

onie

s/ce

ll pl

.

EB to ESC conditions

* **

LF2 Cbx7pool LF2 Cbx7clone

Figure 5. Cbx7 Expression Blocks ESC Differentiation and X-Inactivation

(A) PGK12.1 ESCs expressing Cbx7-EGFP and control ESCs were kept in ESC media under nondifferentiation conditions. Cells were stained with AP.

Representative images and the percentage of AP-positive colonies showing a compact, flat, or spread morphology are shown.

(B) Control and Cbx7-EGFP-expressing ESCs were subjected to 5 days of retinoic acid treatment to induce differentiation. Xist RNA was detected by RNA FISH,

and more than 100 cells were counted for each clone. Represented is the percentage of cells showing two (yellow) or one (gray) punctate Xist RNA signals,

or a properly inactivated Xist RNA domain (blue).

Cell Stem Cell



Cell Stem Cell


differentiation (Figures 5G and 5H). While Cbx7 did not affect

the basal expression of pluripotency-associated genes in the

ESCs (Figure 5G), it prevented or delayed the induction of

most of the differentiation-associated genes representative of

endoderm, mesoderm, and ectoderm (Figure 5H). Collectively,

ectopic expression of Cbx7 results in increased ESC self-

renewal and prevents ESC differentiation and X chromosome

inactivation.

miRNA Families miR-125 and miR-181 Are Bona FideCbx7 RegulatorsIn identifying a key role for Cbx7 in pluripotency, it was important

to consider the mechanisms responsible for the downregulation

of Cbx7 during differentiation. Although this could obviously

reflect transcriptional control, we speculated that Cbx7 could

additionally be subjected to posttranscriptional regulation by

miRNAs.

To identify miRNAs regulating Cbx7 expression, we used

a mouse Cbx7-30UTR reporter (psiCHECK2-Cbx7-30UTR) anda miRNA expression library comprising 371 miRNAs (Voorhoeve

et al., 2006) to perform a reporter screen in single-well format

(Figure 6A). We conducted two independent screens and Fig-

ure 6B shows the results obtained in one of the replicas. We

set the Z-score threshold at < �2 to select miRNAs significantly

downregulating the Cbx7 reporter. The candidates weremiRNAs

of the miR-125 (miR-125a and miR-125b) and the miR-181

(a vector expressing miR-181a and miR-181b and another

expressing miR-181c and miR-181d) families. miRNAs from

these families have two predicted binding sites in the 30UTR of

Cbx7 (see scheme in Figure S6A) that are conserved among

vertebrates (data not shown). To verify the results from the

screening, we retested vectors expressing miR-125a, miR-

125b, miR-181a/b, and miR-181c/d and confirmed their ability

to reduce the luciferase activity when cotransfected with

psiCHECK2-Cbx7-30UTR (Figure 6C). Members of the miR-125

and miR-181 families also regulated human CBX7 in similar

reporter assays (Figure S6B).

To determine which of the putative miRNA target sites were

responsible for the regulation of Cbx7, we generated reporter

constructs bearing specific mutations (Figure S6A). While

mutation of both miR-125 target sites was required to preclude

downregulation of the Cbx7-30UTR reporter by miR-125 (Fig-

ure 6D), only the second miR-181 target site was important for

downregulating Cbx7 (Figure 6E). We also generated a Cbx7-

30UTR reporter in which the four putative miR-125 and miR-181

target sites were mutated (Cbx7-30UTR mut). This mutant

reporter was resistant to downregulation by miRNAs of both

the miR-125 and miR-181 families (Figure 6F). Similarly, deletion

of the seed sequences of miRNA-181a/b (miR-181a/b mut)

(C) Control and Cbx7-EGFP ESCs were subjected to neural differentiation for 4 da

of AP-positive colonies of the total cells plated is represented.

(D) Control and Cbx7-EGFP-expressing ESCs were cultured in nonattachmen

dissociated and plated back in ESC medium at day 20. Numbers of ESC-like co

(E) LF2 ESCs, expressing EGFP (control), and Cbx7-FLAG/HA (pool and clone) w

(F) LF2 cells described above were assayed as in (D). Percentage of AP-positive

(G and H) Cbx7 expression in ESCs prevents induction of lineage markers up

LF2 ESCs (wt) and LF2 ESCs expressing EGFP (control) and Cbx7-FLAG/HA (p

and ectoderm markers in EBs derived from above cells analyzed by qRT-PCR a

or miRNA-125b (miR-125b mut) abrogated the ability of

these constructs to downregulate the Cbx7-30UTR reporter

(Figure S6C).

Expression of miRNA-181a/b Induces Senescenceby Targeting Cbx7One of the best-defined functions of Cbx7 is its ability to delay

senescence through PRC1-mediated repression of INK4a. In

human fibroblasts, shRNA-mediated knockdown of endogenous

CBX7 impairs cell growth and induces premature senescence

(Gil et al., 2004). To investigate whether the miR-125 and

miR-181 families can act as bona fide regulators of Cbx7, we

assessed their ability to cause p16INK4a-dependent senescence

in human fibroblasts, either by transfecting the cells with

miRNA mimics (Figure 6G) or by retroviral infection (Figure 6H

and Figures S6D–S6G). Expression of miR-181a or miR-181b

caused downregulation of Cbx7 (Figure 6G) accompanied by

induction of p16INK4a expression (Figure 6G and Figure S6F).

Specificity was confirmed by using variants of miR-181a and

miR-181b with a mutated seed sequence. These variants had

no effect on Cbx7 levels or senescence induction (Figure 6I

and Figure S6D). In similar experiments, miR-125b also caused

a seed-sequence-dependent arrest correlated with upregulation

of p16INK4a (Figures S6E–S6G). Collectively, these results

suggest that the two miRNA families identified in our screen

are bona fide regulators of Cbx7 expression.

miR-125 and miR-181 Regulate ESC Differentiationvia Cbx7Given these findings in fibroblasts, we next investigated whether

members of the miR-125 and miR-181 families have a role in

ESCs. Interestingly, the expression of most members of the

miR-125 and miR-181 families is low or undetectable in 46C

ESCs, but becomes upregulated upon neural differentiation

(Figure 7A). The increased expression of these miRNAs upon

differentiation was also observed in other ESC lines, during EB

formation and upon differentiation of P19 teratocarcinoma cells

(data not shown). The inverse correlation between the expres-

sion of the miR-125 and miR-181 families and Cbx7 suggested

that these miRNAs could contribute to the regulation of Cbx7

levels during ESC differentiation.

Consistent with this hypothesis, expression of miR-125b or

miR-181a/b in ESCs promoted differentiation as assessed

by increased colonies with a flat or spread morphology (Fig-

ure 7B) and a reduced frequency of AP-positive colonies (Fig-

ure S7A). To confirm and extend these findings, we transfected

mimics for miR-125b, miR-181a, or miR-181b into ESCs. The

levels of the transfected miRNAs were similar to the levels of

these miRNAs during ESC differentiation (Figure S7B) and

ys and stained with AP. Representative images were taken and the percentage

t conditions without leukemia inhibitory factor to form EBs. EBs were then

lonies were analyzed by AP staining after 5 days.

ere probed for levels of endogenous and exogenous Cbx7 protein.

colonies formed per cells plated is represented. *p < 0.005, **p < 0.00005.

on differentiation. (G) mRNA expression of Cbx7 and pluripotency factors in

ool and clone) by qRT-PCR. (H) mRNA expression of endoderm, mesoderm,

t days 6, 12, and 20.


A BCbx7 3 UTR

luciferase reporter

miRNA library

Luciferase assay

C

00.20.40.60.8

11.21.41.6

pRS 125a 125b 181a/b 181c/d 221

3' UTR CBX73' UTR p27

mouse Cbx7 3 UTR

Rel

ativ

e lu

cife

rase

act

ivity

-4

-2

0

2

4

miRNA clones

Z- s

core

D

0

0.2

0.4

0.6

0.8

1

1.2

pRS 125a 125b 181a/b

mCbx7 3 UTR 125.1

0

0.2

0.4

0.6

0.8

1

1.2

pRS 125a 125b 181a/b

0

0.2

0.4

0.6

0.8

1

1.2

pRS 125a 125b 181a/b0

0.20.40.60.8

11.21.41.61.8

pRS 125a 125b 181a/b

0

0.2

0.4

0.6

0.8

1

1.2

pRS 125a 125b 181a/b

mCbx7 3 UTR 125.2 mCbx7 3 UTR 125.1/2

mCbx7 3 UTR 181.1 mCbx7 3 UTR 181.2

Rel

ativ

e lu

cife

rase

act

ivity

Rel

ativ

e lu

cife

rase

act

ivity

3 UTR mut

00.20.40.60.81

1.21.41.61.8

pRS 125b 181a/b

3 UTR wt E F

Rel

ativ

e lu

cife

rase

act

ivity

Rel

ativ

e lu

cife

rase

act

ivity

Rel

ativ

e lu

cife

rase

act

ivity

Rel

ativ

e lu

cife

rase

act

ivity

x xxx

x x

G

0

20

40

60

80

100

120

siGLO 181a 181b

CBX7

mR

NA

leve

ls

0

0.5

1

1.5

2

2.5

siGLO 181a 181b

INK4a

mR

NA

leve

ls

0

10

20

30

40

50

60

siGLO181a 181b 34a% B

rdU

pos

itive

cel

ls

BrdUH

vector miR-181a/b

I

mouse Cbx7 3 UTR

vector miR-181a/b miR-181a/b mut

0

10

20

30

40

vector 181a/b 181a/bmut

% B

rdU

+ c

ells

Figure 6. The miR-125 and miR-181 miRNA Families Are Bona Fide Cbx7 Regulators

(A) A mouse Cbx7-30UTR luciferase reporter was cotransfected in HEK293T cells with a miRNA library in 96-well format to screen for Cbx7 regulatory miRNAs.

(B) Results of the miRNA screen were plotted and Z-scores were calculated. miRNAs with Z-scores lower than �2 were chosen for retesting.

(C) Validation of miRNAs identified in the screen confirms the miR-125 and miR-181 families as regulators of mouse Cbx7 30UTR in a luciferase reporter assay.

miR-221 regulation of a p27-30UTR reporter is included as a control.

(D) Luciferase assays using reporters in which the two putative miR-125 target sites of the Cbx7 30UTR have been mutated individually or combined.

(E) Luciferase assays using reporters in which two putative miR-181 target sites in the Cbx7 30UTR have been mutated.

(F) A Cbx7-30UTR reporter with all putative miR-125 and miR-181 sites mutated (30UTR mut) is resistant to regulation by the miR125 and miR181 families.

Cell Stem Cell



Cell Stem Cell


downregulated Cbx7 expression (Figure 7C). Upon transfection

with mimics for miR-125b, miR-181a, or miR-181b, we ob-

served a loss of ESC characteristics (Figure 7E, see transfec-

tion in control ESCs, and Figure S7C). Furthermore, ESCs

transfected with miRNA mimics for miR-125b, miR-181a, and

miR-181b displayed increased expression of a subset of Pc

target genes involved in lineage specification (Figure 7D).

To understand whether Cbx7 is a critical target of the miR-125

and miR-181 families during ESC differentiation, we compared

the effect of transfecting miR-125b, miR-181a, or miR-181b

mimics in control ESCs or those expressing Cbx7-EGFP lacking

its 30UTR, which are therefore resistant to miRNA regulation. In

contrast to their effects in control ESCs, these miRNAs did not

enhance the differentiation of Cbx7-EGFP ESCs (Figure 7E),

even when the cells were switched to neural differentiation

conditions (Figures S7D and S7E). Similar results were obtained

upon infection of Cbx7-EGFP ESCs with retroviral vectors ex-

pressing miR-125b or miR-181a/b (Figure S7D). These results

demonstrate that miR-125b and miR-181a/b are induced

during ESC differentiation and contribute to this process by

downregulating Cbx7 expression through direct targeting of its

30UTR.

DISCUSSION

The rationale for the evolutionary expansion of PcG genes,

particularly those encoding PRC1 components, is not well

understood (Whitcomb et al., 2007). Here, we hypothesized

that the numerous potential combinations of PRC1 in mammals

might have specialized or context-dependent roles. In the few

cases where PRC1 composition has been examined, such as

at the INK4a/ARF locus in human fibroblasts, several variants

of PRC1 have been found to colocalize, yet each component

appears to contribute to the regulation of INK4a (Maertens

et al., 2009). However, the variable phenotypes associated

with genetic ablation of PRC1 components in mice imply that

they are not functionally equivalent (Sparmann and van Lohui-

zen, 2006). In this study, we investigated which of the five

mammalian orthologs of Pc is required for PRC1 function and

maintenance of pluripotency in mouse ESCs.

In contrast to somatic cells or differentiated ESCs, Cbx7 is the

Pc ortholog that reads H3K27me3 in ESCs. Although this could

be explained by the high affinity of Cbx7 for H3K27me3 (Bern-

stein et al., 2006b; Yap et al., 2010), this is likely not the case,

as we identified Cbx2 and Cbx8 as the Pc orthologs interacting

with H3K27me3 in MEFs and differentiated ESCs. In addition,

analysis of the expression of Cbx7 and the other Pc orthologs

during ESC differentiation showed very different dynamics.

While Cbx7 is expressed in ESCs and is sharply downregulated

during differentiation, the expression of the other Pc orthologs

did not change significantly (Cbx6) or in contrast to Cbx7,

increased upon differentiation (Cbx2, Cbx4, and Cbx8). Interest-

ingly, among the different Pc orthologs, the expression of Cbx7

(G) IMR90 cells were transfected with miR-181a, miR-181b, or miR-34 mimics, a

qRT-PCR. The percentage of BrdU-positive cells in the same experiment was m

(H) Expression of miR-181a/b in human IMR90 fibroblasts results in decreased c

(I) IMR90 cells were infected with miR-181a/b or a version in which the seed sequ

BrdU incorporation (left) and phase microscopy (right).

was clearly associated with pluripotency; we observed that it is

highly expressed in ESCs and teratocarcinomas. The expression

of other PRC1 members, most notably Phc1 and Mblr, seem to

also be associated with pluripotency, while others like Bmi1

and Phc2 were more highly expressed in differentiated ESCs

or other cell types. As a result, subunit composition of PRC1

complexes changes in a dynamic fashion during ESC differenti-

ation, as highlighted in our mass spectrometry (MS) experi-

ments. Given the multiple possible combinations of PRC1

subunits and the potential difficulty in detecting some members

due to experimental bias of the technique used and/or a lack of

antibodies, we believe a combination of MS, gel filtration, immu-

noblotting, and other approaches should be used to thoroughly

investigate the changes in composition and coexistence of

different PRC1 complexes during ESC differentiation in the

future.

Direct evidence for Cbx7 being a critical factor in pluripotency

came from the finding that knockdown of Cbx7 accelerated ESC

differentiation and correlated with increased expression of

lineage-specific PcG targets. Conversely, ectopic expression

of Cbx7 impaired ESC differentiation and X chromosome inacti-

vation. This blockade suggested a prominent role for Cbx7

overexpression in repressing PRC1 target genes during ESC

differentiation. A possible explanation for the predominance of

Cbx7 in stem cells is that its expression is under the control of

the pluripotency network of TFs. Consistent with this hypothesis,

depletion of Oct4 in ESCs caused downregulation of Cbx7, and

ChIP studies demonstrated direct occupancy by Oct4, Sox2,

and Nanog. Interestingly Cbx2, Cbx4, and Cbx8, which show

a reciprocal pattern of expression to that of Cbx7, are direct

targets of Cbx7. The importance of Cbx7 in pluripotency is

therefore underscored by its role in restraining other Pc ortho-

logs in ESCs. Whether other PRC1 components are subjected

to similar regulation remains to be investigated.

We suspected that posttranscriptional mechanisms might

contribute to the downregulation of Cbx7 during ESC differenti-

ation. A number of miRNAs have been implicated in regulating

the balance between self-renewal and differentiation of ESCs,

including those of the miR-302 and let-7 families (Melton and

Blelloch, 2010). Moreover, Ezh2, the enzymatic component of

the PRC2 complex, is regulated by miR-214 during differentia-

tion of ESCs and muscle cells (Juan et al., 2009). Here we iden-

tified members of the miR-125 and miR-181 families as bona

fide regulators of Cbx7. The expression of either miR-125b or

miR-181a/b in human fibroblasts resulted in upregulation of

p16INK4a and senescence in agreement with a recent report

showing that miR-125b can cause senescence in human

melanoma cells (Glud et al., 2011). Importantly, miR-125b,

miR-181a, and miR-181b are not expressed (or are expressed

at low levels) in ESCs and are sharply induced during ESC

differentiation. Previous reports have shown that miR-125 is

induced in NSCs and targets lin28 to allow processing of let-7

(Rybak et al., 2008) and that regulation of multiple miR-125b

nd the expression of CBX7 (left) and INK4a mRNA (middle) was monitored by

onitored by immunofluorescence and quantified (right).

ell growth as assessed by crystal violet staining.

ences had been mutated. Their ability to induce senescence was assessed by


D

B

0

1

2

3

4

Rel

ativ

e ex

pres

sion

siGLOmiR-125bmiR-181amiR-181b

0

30

60

90

siGLO 125b 181a 181b siGLO 125b 181a 181b

compactflatspread

% o

f col

onie

s

ControlESC: Cbx7-EGFP

vector miR-125b miR-181a/b

0

10

20

30

40

50

60

70

vector miR-125b miR-181a/b

compactflatspread

% o

f col

onie

s5

A miR-125a

Rel

ativ

e ex

pres

sion

0

1.0

2.0

3.0

ESC d6 d8 d10

4.0miR-125b

ESC d6 d8 d100

20406080

100120140

miR-181a

ESC d6 d8 d10050

100150200250300350400

miR-181b

ESC d6 d8 d100

50

100

150

200

C

Cbx

7 m

RN

A le

vels

0

20

40

60

80

100

120

siGLO siCbx7.2 siCbx7.3 125b 181a 181b

E

siGLO miR-125b miR-181a miR-181b

Control

Cbx7-EGFP

ESC

Gata4 FoxA2 Nkx2.2 Nkx2.9 Sox3 Olig1 Rhox9 Irx3 Math1 Dach1

Figure 7. The miR-181 and miR-125 Families Regulate Cbx7 Expression and Influence ESC Differentiation

(A) The expression of miR-125 and miR-181 families is upregulated during neural differentiation of 46C ESC as measured using Taqman probes.

(B) Expression of miR-125b or miR-181a/b by retroviral infection causes a loss of ESC properties. Representative images of AP-stained cells (left) and quan-

tification of colonies (right) are presented.

(C) Transfection of ESCs with miRNA mimics for miR-125b, miR-181a, or miR-181b results in Cbx7 downregulation.

(D) Expression of a subset of PcG target genes associatedwith differentiation in ESCs transfectedwithmiR-125b,miR-181a, ormiR-181bmimics weremonitored

by qRT-PCR.

(E) Control ESCs (PGK12.1) or Cbx7-EGFP ESCs (PGK12.1 Cbx7-EGFP) were transfected with siGLO or synthetic mimics for miR-125b, miR-181a, and

miR-181b. Cells weremaintained in ESCmedia and stained with AP. Representative images (left) and the percentage of AP-positive colonies showing a compact,

flat, or spreading morphology are shown (right).

Cell Stem Cell


targets is required for neural differentiation (Le et al., 2009). Simi-

larly, miR-181 is upregulated during differentiation to myoblast

and hematopoietic lineages (Chen et al., 2004; Naguibneva

et al., 2006) and plays an active role in driving both processes.

miR-181 targeting of HoxA11, which itself represses MyoD, is

a key facet of myoblast differentiation (Naguibneva et al.,

2006). Whether control of PRC1 function through targeting of


Cbx7 could also play a role in these processes is presently

unknown.

In conclusion, we have identified a prominent role for Cbx7 in

pluripotency. Cbx7 function in ESCs is critical to suppress differ-

entiation. A complex mechanism is clearly in place in order to

fine-tune the expression of Cbx7 and its orthologs both in

ESCs and upon differentiation. This involves transcriptional

Cell Stem Cell


control of Cbx7 by the pluripotency network of TFs in ESCs as

well as induction of miRNAs that target Cbx7 during differentia-

tion. Future studies will explore how expression of the miR-125

and miR-181 families is controlled in ESC differentiation, and

whether other PRC1 members are also regulated in a similar

fashion.

EXPERIMENTAL PROCEDURES

Plasmids

The miR-Vec library and control vector have been described by (Voorhoeve

et al., 2006). The generation of the miRNA mutants, reporter plasmids, and

Cbx7-derived plasmids used in this study is described in the Supplemental

Information.

Cell Culture and Differentiation Assays

P19, HEK293T, and IMR90 cells were maintained in Dulbecco’s modified

Eagle’s medium (Invitrogen) with 10% fetal bovine serum (PAA) and 1%

antibiotic-antimycotic solution (Invitrogen). Mouse ESC lines CCE, E14,

ZHBTc4, LF2, PGK12.1, 46C, and Oct4-GIP, and Cbx7+/� lines (B05 and

F05), were cultured as described in detail in the Supplemental Information.

Cbx7+/� ESCs (B05 and F05) were obtained from the Wellcome Trust Sanger

Institute.

Retroviral and Lentiviral Infection

Virus production and infection have been described elsewhere (Banito et al.,

2009).

Mass Spectroscopy

This was performed essentially as described in Vermeulen et al. (2010). Details

are mentioned in the Supplemental Information.

Peptide PD and GFP and Ring1b Immunoprecipitation

These assays were conducted using standard protocols that are described in

detail in the Supplemental Information.

Colony Formation Assay after EB Disaggregation

ESCs were differentiated to form EBs under nonadherent conditions. At

days 12 and 20, EBs were washed with PBS, trypsinized, and resuspended

in ESCmedia. Cells were plated at 5,000, 10,000 or 15,000 cells per gelatinized

plate. After 5–10 days, colonies were fixed and stained for AP. The number of

colonies were counted from scanned images using Image J software, and

plotted as percentage of AP-positive colonies per cells plated.

AP Staining

ESCs were plated (5 3 104) in 6-well plates and fixed with 4% paraformalde-

hyde for 1–2 min. Staining was performed using the Alkaline Phosphatase

Detection Kit (Millipore or Stemgent) according to manufacturers’ protocol.

Reverse Transfection and Luciferase Assay

For the luciferase screening, HEK293T cells were reverse transfected using

Polyethylenimine (PEI, Sigma) to individually transfect 371 clones from the

miR-Vec library in a 96-well plate format. A 9:1 ratio of miR-Vec to luciferase

reporter construct was used. miR-Vec-Ctrl was used as control vector. A 3:1

ratio of PEI to DNA was used, and after incubation of reagent-DNA

complexes for 30 min, cells were added. Firefly and Renilla luciferase activ-

ities were measured using the Dual-Luciferase Reporter Assay system

(Promega) 48 hr after transfection. Values were expressed as the number

of median-adjusted standard deviations (Z-score value) and a threshold

was established (Z-score < �2) to identify miRNAs that caused a significant

reduction of luciferase expression when cotransfected with psiCHECK2-

Cbx7-30UTR. miRNAs with a Z-score value lower than �1 when cotrans-

fected with empty psiCHECK were discarded.

BrdU Assay and Crystal Violet Staining

BrdU labeling was performed for 16 hr. Crystal violet staining was performed

as previously described (Banito et al., 2009).

Immunofluorescence and Immunoblotting

Immunofluorescence was performed using an InCell Analyzer 1000 (GE).

Image processing and quantification was performed using InCell Investigator

software (GE). Immunoblotting was performed following standard procedures,

and, when indicated, from chromatin fractions prepared as described

(Bernstein et al., 2006b). Donkey anti-rabbit HRP (GE Healthcare) and sheep

anti-mouse HRP (GE Healthcare) -conjugated antibodies were used and

signals were detected by ECL (GE Healthcare). Antibodies are listed in the

Supplemental Information.

qRT-PCR Analysis

qRT-PCR was performed as described previously (Banito et al., 2009). A list of

primers and Taqman probes used is presented as part of the Supplemental

Information.

RNA Interference and miRNA Transfection

IMR90 or 46C ESCs were transfected with 30 nM siRNA for IMR90 and 100 nM

for 46C ESCs in 6-well plates. A 3.5% solution of HiPerFect transfection

reagent (QIAGEN) was prepared in serum-free DMEM and then mixed with

the siRNA. The mix was incubated for 30 min at room temperature and then

added to the cells. Medium was changed on the following day and cells

were either fixed for immunofluorescence or harvested for RNA extraction

24–96 hr later. The Cy3-labeled siGLO cyclophilin B siRNA (Dharmacon) was

used to monitor transfection efficiency and as a negative control. A scrambled

siRNA (AllStars) or Silencer Select Negative Control #1 and #2 siRNA (Ambion)

were included as additional negative controls in most experiments. For a list of

siRNAs used see the Supplemental Information.

ChIP

ChIP experiments were performed as previously described (Maertens et al.,

2009). A detailed explanation of the protocol and a list of the primers used

for the ChIP is provided in the Supplemental Information.

Xist RNA FISH

RNA FISH was performed as described in (Masui et al., 2011) and under

‘‘protocols’’at the following URL: http://www.epigenesys.eu/.

SUPPLEMENTAL INFORMATION

Supplemental Information for this article includes seven figures, two tables,

and Supplemental Experimental Procedures and can be found with this article

online at doi:10.1016/j.stem.2011.12.004.

ACKNOWLEDGMENTS

We thank Mathias Mann for his support; Elizabeth Duncan and David Allis for

sharing the LF2 Cbx7 ESCs; and Yen-Sin Ang, Kajan Ratnakumar, Haruhiko

Koseki, Adrian Bracken, Meng Li, Tristan Rodriguez, Cynthia Fisher, and

Sam Wormwald for advice and reagents. This work was supported by an

NYSTEM IDEA Award C024285 to E.B. E.H. and O.M. received support from

the ANR and ERC Advanced Investigator award. Core support from the

MRC and grants from MRC Technology, CRUK, and AICR went to fund

J.G.’s research. A.M. was funded by Fundacion Ramon Areces, and A.B., by

the Portuguese FCT. J.G. is also supported by the EMBO Young Investigator

Programme.

Received: June 6, 2011

Revised: October 11, 2011

Accepted: December 2, 2011

Published: January 5, 2012

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MicroRNA Regulation of Cbx7 Mediates a Switch of Polycomb Orthologs during ESC Differentiation

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