Coordinated regulation of transcriptional
repression by the RBP2 H3K4
demethylase and Polycomb-Repressive
Diego Pasini, Klaus H. Hansen, Jesper Christensen, Karl Agger, Paul A.C. Cloos,
and Kristian Helin1
Biotech Research and Innovation Centre (BRIC) and Centre for Epigenetics, University of Copenhagen,
2200 Copenhagen, Denmark
Polycomb group (PcG) proteins regulate important cellular processes such as embryogenesis, cell proliferation,
and stem cell self-renewal through the transcriptional repression of genes determining cell fate decisions. The
Polycomb-Repressive Complex 2 (PRC2) is highly conserved during evolution, and its intrinsic histone H3 Lys
27 (K27) trimethylation (me3) activity is essential for PcG-mediated transcriptional repression. Here, we show
a functional interplay between the PRC2 complex and the H3K4me3 demethylase Rbp2 (Jarid1a) in mouse
embryonic stem (ES) cells. By genome-wide location analysis we found that Rbp2 is associated with a large
number of PcG target genes in mouse ES cells. We show that the PRC2 complex recruits Rbp2 to its target
genes, and that this interaction is required for PRC2-mediated repressive activity during ES cell
differentiation. Taken together, these results demonstrate an elegant mechanism for repression of
developmental genes by the coordinated regulation of epigenetic marks involved in repression and activation
[Keywords: EZH2; Polycomb; histone methyl transferase; histone demethylase; epigenetics; embryonic stem
Supplemental material is available at http://www.genesdev.org.
Received January 8, 2008; revised version accepted March 20, 2008.
PcG proteins are essential regulators of development,
and their function is highly conserved during evolution
(Ringrose 2007; Schuettengruber et al. 2007; Schwartz
and Pirrotta 2007). The PcG proteins form mainly two
different Polycomb-repressive multiprotein complexes,
named PRC1 and PRC2 (Francis and Kingston 2001; Si-
mon and Tamkun 2002). PRC1 is a large-sized complex
that includes a variety of different PcG proteins, and its
composition can vary in a cell type-specific manner. The
PRC1 complex mediates the ubiquitylation of histone
H2A through the ubiquitin E3 ligase activity of the
Ring1A and Ring1B subunits and is required for tran-
scriptional silencing of target genes, possibly by mediat-
ing chromatin compaction (Shao et al. 1999; Saurin et al.
2001; de Napoles et al. 2004; Francis et al. 2004; Wang et
al. 2004). The PRC2 complex is smaller, and the three
PcG proteins EZH2, EED, and SUZ12 form the core com-
plex. The catalytic subunit EZH2 of the PRC2 complex
can modify histone H3 N-terminal tails by specifically
trimethylating (me3) Lys 27 (K27). This modification
correlates with transcriptional repression, and it has
been proposed to serve as a docking site for PRC1 re-
cruitment (Cao et al. 2002; Czermin et al. 2002;
Kuzmichev et al. 2002; Muller et al. 2002).
The three PcG proteins forming the core of the PRC2
complex are all required for EZH2 lysine methyl trans-
ferase (KMT) activity in vitro and in vivo (Cao and Zhang
2004; Pasini et al. 2004b). Moreover, consistent with a
potential essential role of H3K27me3 in regulating tran-
scriptional silencing during development, the three PcG
proteins are essential for embryonic development during
the gastrulation stage (Faust et al. 1995; O’Carroll et al.
2001; Pasini et al. 2004b; Montgomery et al. 2005).
The identification of PRC2 and PRC1 downstream
regulatory pathways by genome-wide location analysis
has demonstrated that the two complexes share a highly
significant number of target genes in different cell types
(Boyer et al. 2006; Bracken et al. 2006; Lee et al. 2006).
The analysis of PcG target genes in embryonic stem (ES)
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GENES & DEVELOPMENT 22:1345–1355 © 2008 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/08; www.genesdev.org1345
cells revealed that PcG proteins directly control the ex-
pression of a large number of genes that are essential for
determining cell fate decisions during embryonic devel-
opment (Boyer et al. 2006; Lee et al. 2006). Despite this
key role of the PcG protein in developmental control, the
mechanisms by which epigenetic silencing through
H3K27me3 occurs are poorly understood, and the path-
ways regulating PcG recruitment to target genes, the fac-
tors, and enzymatic activities involved in PcG-mediated
silencing are still unknown.
The recent isolation and characterization of enzymes
with histone lysine demethylase activity (KDM) have
shown that histone methylation is more dynamic than
previously anticipated and have introduced an additional
layer of complexity in the regulation of transcription (Shi
2007; Agger et al. 2008). Among the histone demethyl-
ases, we and others have shown that members of the
Jumonji protein family, JARID1, specifically catalyze the
demethylation of H3K4me3 and H3K4me2, regulate the
expression of HOX genes, and are required for normal
development (Christensen et al. 2007; Eissenberg et al.
2007; Iwase et al. 2007; Klose et al. 2007; N. Lee et al.
2007; Secombe et al. 2007; Tahiliani et al. 2007; Yamane
et al. 2007). However, our knowledge regarding the
mechanisms by which the JARID1 proteins contribute to
developmental control is limited, and the identification
of the downstream pathways regulated by the JARID pro-
teins together with the mechanisms by which these fac-
tors regulate transcription are important questions to ad-
The H3K4me3 mark is restricted to transcription start
sites (TSS) and most often found associated with active
promoters (Mikkelsen et al. 2007). However, recent stud-
ies have demonstrated that H3K27me3 and H3K4me3
can coexist on transcriptional silent promoters in ES
cells and have suggested that this “bivalent mark” main-
tains a promoter in a “poised” state accessible for acti-
vation during cellular commitment (Bernstein et al.
2006; Mikkelsen et al. 2007). This intriguing observation
suggests a mechanism for how transcriptional programs
are achieved during development and the existence of a
cross-talk between these two antagonistic modifications
that could require a tight regulation mediated by both
KMTs and KDMs. Consistent with this, recent studies
found that a specific H3K27me3 demethylase, UTX,
physically interacts with the MLL complex (a histone
H3K4-specific methyltransferase), and that the UTX ac-
tivity is involved in the transcriptional activation of
HOX genes in NT2 cells (Agger et al. 2007; M.G. Lee et
We investigated the functional role of the H3K4 de-
methylase RBP2 (JARID1A) and its interplay with the
PRC2 complex. We demonstrate that RBP2, in associa-
tion with the PRC2 complex, is critical for the transcrip-
tional repression of a large number of Polycomb target
genes involved in developmental control. Based on our
results, we propose a mechanism for transcriptional con-
trol as cells undergo differentiation, which involves the
coordinated regulation of H3K4 demethylation and
Identification of Rbp2 target genes in mouse ES cells
To understand how RBP2 can contribute to development
and differentiation, we identified Rbp2 target genes in
mouse ES cells by genome-wide location analysis. To
this extent, we used an Rbp2-specific antibody that in
agreement with previously published results (Benevolen-
skaya et al. 2005) specifically enriched for the BRD8 pro-
moter in human cells (Fig. 1A) and that recognized
mouse Rbp2 (Fig. 4A, below). Hence, we performed chro-
mouse ES cells and hybridized the precipitated material
to an oligonucleotide array covering ∼2.5 kb (−2 kb to
+0.5 kb with respect to the TSS) of 19,489 annotated
transcripts in the NCBI database. The analysis identified
606 promoters that were significantly bound by Rbp2 in
ES cells (Supplemental Table 1). Functional clustering of
the Rbp2-bound promoters revealed that Rbp2 is present
on the promoters of a considerable number of genes that
are involved in the regulation of development and differ-
entiation (Fig. 1B), suggesting that Rbp2 could play a di-
rect role in regulating the transcription of these genes
Previously, we and others have identified the down-
stream regulatory pathways of PcG proteins, showing
that PcGs repress the expression of a large number of
genes essential for cell fate decisions during develop-
ment (Boyer et al. 2006; Bracken et al. 2006; Lee et al.
2006). Interestingly, we observed a substantial overlap
between the genes and the pathways regulated by the
PcG proteins (Bracken et al. 2006) and Rbp2 (Fig. 1C). To
obtain statistical data for this observation, we compared
the PcG target genes identified in mouse ES cells (Boyer
et al. 2006) with our list of Rbp2 target genes. Despite the
Rbp2 and PRC2 ChIP–chip analysis were performed in
different laboratories using different platforms, this
analysis revealed that Rbp2 and the PcG proteins share a
statistically highly significant number of overlapping
target genes in ES cells (32% of targets, 195 out of 606,
Fig. 1D), suggesting a functional interplay between the
PcG proteins and Rbp2.
To validate the Rbp2 location analysis, we performed
independent ChIP experiments for 31 Rbp2 target genes
that were or were not identified previously as PcG tar-
gets with a high, medium, and low false discovery rate
(FDR) (Supplemental Table 1). Rbp2 binding was con-
firmed for most of the target genes (29/31), showing the
high accuracy of the location analysis also for target
genes with an FDR close to the cutoff (0.2) (Fig. 2A, top
To confirm that the PcG proteins share common tar-
get genes with Rbp2, we performed ChIP analysis for the
PRC2 protein, Suz12. The results from this analysis con-
firmed that Rbp2 and Suz12 are associated with the same
promoters (Fig. 2A) and, surprisingly, that Rbp2 targets
that were not identified previously as PcG target genes
(Fig. 2A, Rbp2 targets) also showed a significant binding
of Suz12. These data suggest that the binding overlap
between Rbp2 and the PcG proteins is more extensive
Pasini et al.
1346GENES & DEVELOPMENT
than suggested by the location analysis. To confirm this
possibility, we tested and found that Rbp2 is associated
with PcG targets that were not identified in the Rbp2
location analysis (Fig. 2A, PRC2 targets). These results
further support the fact that PcGs and Rbp2 share a
larger number of target genes than were identified by the
location analysis, and this is most likely due to the tech-
nical differences between the compared experiments.
To further investigate the potential functional inter-
play between Rbp2 and the PcG proteins, we speculated
that, if it exists, they should show a similar binding dis-
tribution on their target genes. Therefore, we analyzed
the binding patterns of Rbp2, Suz12, and Ezh2 at the
Irx3, Igf2, Wnt5a, and Brachyury loci by ChIP. The dis-
tribution of the real-time quantitative PCR (qPCR) am-
plicons covered a region of ∼20 kb upstream of and down-
stream from the TSS in the four loci with a higher reso-
lution (∼2 kb) within the first 10 kb upstream of the TSS,
as indicated in Figure 2B and in Supplemental Figure 1A.
This analysis revealed a perfect overlap between the
binding profiles of Suz12, Ezh2, and Rbp2 (Fig. 2B;
Supplemental Fig. 1A) and confirmed that Rbp2 and the
PRC2 complex bind to the same promoters in the prox-
imity of the TSS. In conclusion, these results suggest
that the overlap between PcG and Rbp2 target genes is
even more extensive than the data presented in Figure
Rbp2 binds directly to the PRC2 complex
The significant overlap between PcG and Rbp2 target
promoters indicates a physical interaction between the
proteins. To investigate this, we tested and showed that
ectopically expressed RBP2 associates with endogenous
components of the PRC2 complex (Fig. 3A). This asso-
ciation was confirmed also with the endogenous pro-
teins: Immunoprecipitation (IP) experiments using anti-
bodies specific to different PRC2 subunits (Ezh2 and
Suz12) and to Rbp2 showed coimmunoprecipitation of
endogenous Rbp2 with the members of the PRC2 com-
plex in both mouse ES cells and in human 293T cells
(Fig. 3A,B; Supplemental Fig. 1B,C). Moreover, the asso-
ciation between the proteins is independent of DNA,
since Rbp2 associates with the PRC2 in the presence of
ethidium bromide (Supplemental Fig. 1D).
To further support these findings, we tested if Rbp2
and the components of the PRC2 complex are present in
the same molecular weight fractions. For this, we frac-
tionated ES cells nuclear extracts on a Superose-6 gel
filtration column and analyzed the different fractions by
Western blot. Importantly Ezh2, Eed, Suz12, and Rbp2
coelute in fractions corresponding to a molecular weight
?1 MDa (Fig. 3C, top panels). This result is consistent
with previous findings showing the existence of a large-
sized complex containing PRC2 subunits and suggests
shRNA-treated 293T cells on the BRD8 promoter. ChIP with antibody against the hemagglutinin (HA) tag served as negative control.
ChIP enrichments are presented as percentage (%) of bound/input signal. (B) Clustering of Rbp2-bound promoters in mouse ES cells
into functional groups. (C) Comparison of PcG target genes and their regulatory pathways published elsewhere (Bracken et al. 2006)
with those regulated by Rbp2. (D) Statistically significant overlap between promoters bound by Rbp2 and promoters bound by PcGs
published elsewhere (Boyer et al. 2006) in ES cells. The P-value was calculated by hypergeometric probability test using Genespring
Identification of Rbp2 target genes in mouse ES cells by genome-wide location analysis. (A) ChIP assays in control and RBP2
RBP2 functionally interacts with PRC2
GENES & DEVELOPMENT1347
that this includes the H3K4 demethylase, Rbp2. To test
if Rbp2 and the components of the PRC2 complex physi-
cally interact in the high molecular weight fractions, we
incubated the different gel filtration fractions with a spe-
cific antibody to Suz12 and demonstrated that Rbp2 and
the PRC2 complex interact in fractions with complexes
larger than ∼1 MDa (Fig. 3C, bottom panels).
Finally, coexpression of recombinant RBP2, EZH2,
EED, and SUZ12 in insect cells confirmed the efficient
binding of RBP2 with the PRC2 complex (Fig. 3D). Taken
together, these results provide evidence for a strong di-
rect binding between PRC2 and RBP2 and suggest the
existence of high molecular weight biochemical struc-
tures that contain RBP2 and the PRC2 complex.
Rbp2 is required for maintaining repression of PRC2
To understand if Rbp2 plays a role in maintaining repres-
sion of PcG target genes, we tested the effects of inhib-
iting Rbp2 expression (Fig. 4A) on genes that were re-
ported previously to be activated by loss of PRC2 (Boyer
et al. 2006; Lee et al. 2006). For this, we infected ES cells
with lentiviral particles expressing specific shRNA
against Rbp2 (Fig. 4A) and isolated RNA from these cells
for qPCR expression analysis. As shown in Figure 4B,
down-regulation of Rbp2 leads to increased expression of
Irx3, Wnt5a, and Igf2. This demonstrates that Rbp2 ac-
tivity is required for the maintenance of the repression of
PRC2 target genes in ES cells. Importantly, ChIP experi-
ments performed in the same cells showed that inhibi-
tion of Rbp2 expression also correlates with decreased
binding of Rbp2 to its target promoters (Fig. 4C) and,
consistent with Rbp2 demethylase activity, to increased
levels of H3K4me3 (Fig. 4D, left panels). However, no
changes in H3K27me3 levels (Fig. 4D, right panels;
Supplemental Fig. 2B) and PRC2 binding (Supplemental
Fig. 2A) were observed upon inhibition of Rbp2 expres-
sion. Taken together, these results show that Rbp2 con-
tributes to maintenance of PcG–Rbp2 target gene repres-
sion, and that Rbp2 is not required for the binding of
PRC2 to its target genes.
first group includes unrelated control genes, the second group includes genes found in both Rbp2 and Suz12 genome-wide location
analysis, the third group provides genes found bound by Rbp2 only, and the fourth group of genes are bound by PRC2 only. Antibody
against the HA tag served as negative control. ChIP enrichments are presented as percentage (%) of bound/input signal. (B) ChIP
analysis in mouse ES cells of Rbp2, Suz12, and Ezh2 along the Wnt5a and Irx3 gene loci. ChIPs with antibody against the HA tag served
as negative control. ChIP enrichments are presented as percentage (%) of bound/input signal. The position of the qPCR amplicons
(arrows) relative to the genes (black boxes). TSS (dashed line) are also presented in the figure.
Rbp2 and PRC2 occupy overlapping target genes. (A) ChIP analysis in ES cells using antibodies against Rbp2 and Suz12. The
Pasini et al.
1348 GENES & DEVELOPMENT
The PRC2 complex recruits RBP2 to target genes
To test if the PRC2 complex is required for Rbp2 binding
to its target genes, we performed ChIP experiments in
wild-type and Suz12 knockout mouse ES cells (Fig. 5A)
as well as in mouse ES cells expressing an shRNA to
Suz12 (Supplemental Fig. 3A). As shown in Figure 5B and
Supplemental Figure 3B, Rbp2 binding to its target pro-
moters was dramatically decreased in the absence of the
PRC2 complex. Consistent with this, H3K27me levels
are reduced from the Irx3, Wnt5a, and Igf2 promoters,
while H3K4me3 are increased at the Irx3 and Wnt5a
promoters in Suz12 knockout ES cells (Supplemental
Fig. 2C). These experiments strongly suggest that the
PRC2 complex contributes to the recruitment of Rbp2 to
its target genes.
To obtain evidence that the PRC2 complex could me-
diate the recruitment of Rbp2 to target genes, we used a
heterologous reporter system in which the expression of
GAL4-EED from a tetracycline-inducible promoter re-
presses the expression of an integrated Luciferase re-
porter plasmid containing GAL4-binding sites (K.H.
Hansen, A.P. Bracken, D. Pasini, N. Dietrich, A. Monrad,
and K. Helin, in prep.). As expected, expression of GAL4-
EED, upon addition of tetracycline (Fig. 5C), led to ∼80%
genes. (A) Western blot analysis of protein lysates prepared from
mouse ES cells transduced with either control or Rbp2 shRNA
lentiviruses. (B–D) Expression levels of the Irx3, Wnt5a, and
Igf2 (B) and ChIP assays of the Irx3, Wnt5a, and Igf2 promoters
using antibodies specific for Rbp2, histone H3, H3K4me3, and
H3K27me3 (C,D) in control (Ctrl) or Rbp2 shRNA-treated mouse
ES cells. ChIPs with antibody against HA tag served as negative con-
trol. ChIP enrichments are presented as percentage (%) of bound/
input signal. ChIPs for H3K4me3 and H3K27me3 are further nor-
malized to histone H3 density. Gene expression is normalized to
Rbp2 is required for the repression of PRC2 target
using 293T cell lysates expressing HA-tagged wild-type (WT) or
enzymatically inactive RBP2 mutant showing specific coimmu-
noprecipitation of RBP2 with EZH2 and EED. (B) IPs of mouse
ES cell lysates using specific antibodies to Ezh2 and Suz12
showing specific coimmunoprecipitation of Rbp2. IP with anti-
HA-tag antibody served as negative control. (C, top panels) Size
exclusion chromatography (Superose-6, GE Healthcare) of
mouse ES cell protein lysate showing coelution of Rbp2, Ezh2,
Suz12, and Eed in high-molecular-weight fractions. (Bottom
panels) The indicated fractions were immunoprecipitated with
a Suz12-specific antibody. IP with anti-HA-tag antibody served
as negative control (Ctrl). (D) Coomassie-stained gel and West-
ern blot of protein-immunoprecipitated material from Sf9 cells
expressing recombinant RBP2, EZH2, SUZ12, and Eed with
anti-SUZ12 and anti-HA-tag (as negative control) antibodies
showing the binding of RBP2 to the PRC2 complex.
RBP2 interacts with the PRC2 complex. (A) IP assays
RBP2 functionally interacts with PRC2
GENES & DEVELOPMENT1349
repression of the Luciferase activity (Fig. 5D). ChIP
analysis performed in the same cells showed that GAL4-
EED was recruited to the Gal4 DNA-binding site present
at the promoter of the reporter system, and that this led
to the recruitment of the endogenous PRC2 components
EZH2 and SUZ12. Importantly, GAL4-EED expression
also led to the recruitment of RBP2 to the same pro-
moter, demonstrating that the PRC2 complex has the
ability to recruit RBP2 to target genes. Consistent with
this, PRC2–RBP2 recruitment to the reporter promoter
led to increased H3K27me3 levels and to reduced
H3K4me3 levels (Fig. 5E). Furthermore, RBP2 knock-
down by siRNA led to reduced GAL4-EED repression of
Luciferase activity, suggesting that RBP2 plays a role in
this repressive mechanism (Supplemental Fig. 3C).
Taken together, these results show that the PRC2 com-
plex directly recruits RBP2 activity to target genes.
Rbp2 and PRC2 activities cooperate during ES cell
The PRC2 complex is required to maintain the repres-
sive state of genes involved in development, and its dis-
placement during ES cell differentiation leads to the cor-
rect expression of important regulators of development
(Boyer et al. 2006; Bracken et al. 2006; Lee et al. 2006;
Pasini et al. 2007). Consistent with this, the expression
of Wnt5a, Irx3, and Igf2 (Fig. 6A; Supplemental Fig. 4A)
is induced during differentiation of ES cells (Supplemen-
tal Fig. 4D). Importantly, this induction correlates with
Rbp2 and PRC2 displacement from the promoters (Fig.
6B; Supplemental Fig. 4B), increased H3K4me3 levels, and
decreased H3K27me3 levels (Fig. 6C; Supplemental Fig.
4C). Moreover, the analysis of differentiated cells, such as
mouse embryonic fibroblasts (MEFs) and mouse myo-
blasts C2C12, showed that both PRC2 and Rbp2 are lost
from the same promoters (Fig. 6D; Supplemental Fig. 4E).
Previously, we have also shown that the PRC2 com-
plex is recruited to repress the expression of specific
genes during ES cell differentiation (Pasini et al. 2007).
To understand if Rbp2 is recruited together with the
PRC2 complex to promoters that are repressed during ES
cell differentiation, we performed ChIP analysis on the
Fgf4 and Otx2 promoters. As shown in Figure 6A–C and
Supplemental Figure 4A–C, Rbp2 is recruited together
with the PRC2 complex to these promoters during ES
cell differentiation, and the recruitment correlates with
increased H3K27me3 levels, reduced H3K4me3 levels,
and Fgf4 and Otx2 repression.
knockout (KO) mouse ES cells using the indicated antibodies. ?-Tubulin served as a loading control. (B) ChIP assays for Suz12, Ezh2,
and Rbp2 on the Irx3, Wnt5a, and Igf2 promoters in wild-type (WT) and knockout (KO) ES cells. ChIPs with antibody against HA tag
served as negative control. ChIP enrichments are presented as percentage (%) of bound/input signal. (C) Western blot analysis using
antibodies specific for EED, SUZ12, and EZH2 of lysates prepared from a Gal4-EED-inducible 293T cell line grown with or without
tetracycline. (D) Luciferase activity on cell lysates prepared from GAL4-EED 293T cells grown with or without tetracycline. (E) ChIP
assays of lysates prepared from GAL4-EED 293T cells grown with or without tetracycline using the indicated antibodies. ChIP with
antibody against HA tag served as negative control. ChIP enrichments are presented as percentage (%) of bound/input signal.
The PRC2 complex is required for Rbp2 recruitment to target genes. (A) Western blots from Suz12 wild-type (WT) and
Pasini et al.
1350 GENES & DEVELOPMENT
Taken together, these results demonstrate a functional
role for the PRC2 complex and Rbp2 for the coordinated
regulation of the histone methylation state of their tar-
get promoters during ES cell differentiation.
A model for repression
In this study, we show that Rbp2 (1) binds to a significant
number of PcG target genes in mouse ES cells, (2) binds
to the PRC2 complex, (3) is a critical regulator of PRC2
target genes, and (4) is recruited to its target genes by the
PRC2 complex during ES cell differentiation.
PcG proteins regulate the expression of a large number
of factors that determine cell fate decisions during de-
velopment (Boyer et al. 2006; Bracken et al. 2006; Lee et
al. 2006). The PcG proteins are required to maintain the
silencing of specific target genes in ES cells, and it is
believed that the correct transcriptional activation of a
subset of these target genes upon ES cell differentiation
is achieved, at least in part, through the dissociation of
the PRC2-repressive activity from promoters (Boyer et
al. 2006). In addition to this, we have previously shown
that the PRC2 complex can also be “actively” recruited
to target genes during differentiation to silence the ex-
pression of specific genes (Pasini et al. 2007). These re-
sults suggest that the PRC2 complex does not play only
a “passive” role during differentiation and are consistent
with the broad expression pattern of Ezh2 during devel-
opment (O’Carroll et al. 2001).
Our results show that the Rbp2 H3K4 demethylase is
important for the correct regulation of PcG target genes.
Taken together with previously published data, we have
summarized our findings in two models (Fig. 7): In the
“derepression model,” Rbp2 together with PRC2 con-
tributes to the silencing of target genes in ES cells. Dif-
ferentiation signals lead to their dissociation and recruit-
ment of transcriptional activators such as H3K4 histone
methyltransferases and H3K27 demethylases (Agger et
al. 2007; M.G. Lee et al. 2007b). In the “repression
model,” PRC2 and Rbp2 are recruited to their target pro-
moters during ES cell differentiation, while transcrip-
tional activators are dissociated. In other words, our re-
sults show that methylation of H3K27 and demethyl-
ation of H3K4 are coordinately regulated by PRC2 and
Rbp2 to maintain transcriptional repression, whereas
previous results have shown the coordinated regulation
of H3K4 methylation and H3K27 demethylation to
maintain transcriptional activation. The mechanisms
leading to the recruitment of PRC2 to target genes are
still unknown. The fact that Rbp2 is not completely dis-
placed from target genes in the absence of the PRC2
complex suggests, as is highlighted in the models (Fig. 7),
that other factors may be involved in recruiting and or
maintaining Rbp2 and PRC2 associated to target genes.
Rbp2 interaction with PRC2
In this work, we show that endogenous levels of Rbp2
and PRC2 interact and that Rbp2 and members of the
PRC2 complex coelute and interact in higher molecular
weight fractions (?1 MDa). Moreover, we show that
RBP2 interacts with high affinity with the PRC2 com-
plex when coexpressed in insect cells. Taken together,
our results suggest that a substantial fraction of Rbp2
binds to PRC2 and that a substantial fraction of PRC2
binds to Rbp2. However, these data suggest that Rbp2 is
not a core subunit of the PRC2 complex.
Implication of Rbp2 recruitment at ‘bivalent domains’
Interestingly, it has recently been proposed that genes
involved in cell fate decisions during embryonic devel-
opment are featured by the coexistence of H3K27me3
ing ES cell differentiation. (A) Real-time quantitative
expression analysis of Wnt5a and Fgf4 in ES cells before
and 48 h after ATRA-induced (1 µM) differentiation. (B)
ChIP assay in pluripotent and differentiated ES cells
presented in A for Rbp2, Suz12, and Ezh2. ChIPs with
antibody against HA tag served as negative control.
ChIP enrichments are presented as percentage (%) of
bound/input signal. (C) ChIP analysis as described in A
using antibodies for histone H3, H3K4me3, and
H3K27me3. ChIP enrichments for H3K4me3 and
H3K27me3 are normalized to histone H3 density. (D)
ChIP assays for Suz12 and Rbp2 in mouse ES, MEF, and
C2C12 cells. ChIPs with antibody against the HA tag
served as negative control. ChIP enrichments are pre-
sented as percentage (%) of bound/input signal.
Regulation of Rbp2 and PRC2 activities dur-
RBP2 functionally interacts with PRC2
GENES & DEVELOPMENT1351
and H3K4me3 marks, and that this correlates with a
poised repressed state in ES cells (Bernstein et al. 2006).
This “bivalent mark” may provide plasticity to the gene;
i.e., the gene can either be activated or maintained re-
pressed in specific cell lineages during differentiation.
The fact that both PRC2 and Rbp2 associate with these
“bivalent domains” could appear counterintuitive, but
our data, together with previous publications showing
that the H3K4me3 MLL methyltransferase complex spe-
cifically associates with the H3K27me3 UTX demethyl-
ase (Agger et al. 2007; Lan et al. 2007; M.G. Lee et al.
2007b), suggest that the cooperation between KMTs and
KDMs is a common feature in epigenetic regulation of
transcription. The fact that Rbp2 associates with pro-
moters enriched for H3K4me3 and that UTX associates
with promoters enriched for H3K27me3 (Agger et al.
2007; Lan et al. 2007; M.G. Lee et al. 2007b) strongly
suggests that these activities are not present to com-
pletely “erase” the methylation marks from promoters,
but instead are required for “fine-tuning” the regulation
of the transcriptional activity of the target genes. Con-
sistent with this, the complete loss of specific modifica-
tions during different cellular processes does not appear
to be solely dependent on an “active” process catalyzed
by demethylases, but to a high degree are also dependent
on the specific loss of KMT activities, as documented for
the H3K27me3 mark during replicative senescence of
MEFs and differentiation of myotubes (Caretti et al.
2004; Bracken et al. 2007).
Rbp2 recruitment at target genes
Interestingly, our results showed that Rbp2 binding to
target genes is dependent on the PRC2 complex. More-
over, using a heterologous reporter system, we show that
the PRC2 complex can direct recruitment of Rbp2 to
target genes. Consistent with this, Rbp2 association and
displacement from promoters during ES cell differentia-
tion always correlate with PcG binding. Rbp2 contains
an Arid/Bright domain that potentially retains the abil-
ity to directly bind DNA sequences. The DNA-binding
specificity of this domain is promiscuous, and while for
some proteins it was shown to have preference for AT-
rich motifs (Gregory et al. 1996), this specificity was not
observed for other proteins (Herrscher et al. 1995; Huang
et al. 1996; Whitson et al. 1999). In addition, recent stud-
ies have shown that the Arid domain of JARID1B/PLU1
has a preferential affinity for GC-rich motifs (Scibetta et
al. 2007), suggesting that Arid domains have DNA-bind-
ing affinities with either low or no conserved sequences.
The fact that Rbp2 down-regulation does not affect
PRC2 binding, and that Rbp2 is displaced from promot-
ers in the absence of PcG proteins, suggests that the pu-
tative DNA-binding ability of Rbp2 may not be required
for PRC2 and Rbp2 recruitment to target genes. How-
ever, it is possible that several factors contribute to the
recruitment and stabilization of the PRC2–Rbp2 com-
plex to promoters such as unidentified transcription fac-
tors, the affinity of the Arid domain for DNA, the ability
of RbAp48/46 (a subunit of the PRC2 complex) to bind
histone tails (Verreault et al. 1998), and the affinity of the
PRC2 complex for H3K27me3 (K.H. Hansen, A.P. Bracken,
D. Pasini, N. Dietrich, A. Monrad, and K. Helin, in prep.).
Rbp2 and development
H3K4 demethylase activity is essential for the develop-
ment of a number of different organisms such as Dro-
Models for Rbp2–PRC2-repressive mechanisms during ES cell differentiation.
Pasini et al.
1352 GENES & DEVELOPMENT
sophila and Caenorhabditis elegans (Gildea et al. 2000;
Christensen et al. 2007). The fact that Rbp2 knockout
mice are viable and display only minor defects in the
hematopoietic system (Klose et al. 2007) suggests that
three other members of the Jarid1 family compensate for
the lack of Rbp2. Therefore, it would be interesting to
investigate if other Jarid1 proteins play a role in the regu-
lation of PcG target genes. That this is a likely scenario
is suggested by the fact that SMCY (JARID1D), like Rbp2
(Supplemental Table 1) and the PRC2 complex (Boyer et
al. 2006), associates with the Engrailed promoter and
represses its expression through association with the
Ring6a/MBLR Polycomb-like protein (M.G. Lee et al.
PcG proteins are essential for development and regu-
late important cellular and epigenetic processes such as
proliferation, differentiation, X-chromosome inactiva-
tion, and imprinting (Delaval and Feil 2004; Pasini et al.
2004a; Heard and Disteche 2006; Schwartz and Pirrotta
2007). The identification of Rbp2 as a functional impor-
tant interaction partner of PRC2 provides important
knowledge as to how two distinct enzymatic activities
regulate transcription in ES cells. However, many impor-
tant and challenging questions remain to be addressed in
the future, such as the identification of the proteins that
mediate the recruitment of Rbp2 and PRC2, the stimuli
that can modulate their activities, and the mechanisms
by which histone post-translational modifications affect
Materials and methods
293T, MEF, and C2C12 cells were grown in DMEM (Gibco)
supplemented with 10% FBS (Gibco), Glutamax (Gibco), and
Pen/Strep (Gibco). Mouse ES cells were grown on 0.1% gelatin-
coated (Sigma) tissue culture plates (Nunc) in ES cell media as
described (Pasini et al. 2007) in the presence of 103U/mL LIF
(ESGRO). To induce ES cell differentiation, 1.5 × 106cells per
10-cm dish were plated and induced to differentiate 16 h later by
removal of LIF and stimulation with 1 µM All Trans Retinoic
Acid (ATRA) (Sigma) for the indicated times. Gal4-EED-induc-
ible 293T cells are described elsewhere (K.H. Hansen, A.P.
Bracken, D. Pasini, N. Dietrich, A. Monrad, and K. Helin, in
prep.). The expression of Gal4-EED was induced by the addition
of 1 µg/mL tetracycline (Sigma) to the cell media. For the siRNA
experiment, the GAL4-EED and parental 293T cells clones were
transfected with oligofectamine (Invitrogen) in the presence of
50 nM Cyclophilin B (Dharmacom) or RBP2 (GCACAAGGAT
GAACATTCT) siRNA oligos. Cells were transfected using the
same conditions 48 h after the first transfection and supple-
mented with 40 ng/mL tetracycline (Sigma). Cells were har-
vested 48 h later and analyzed both for protein expression and
for Luciferase activity with Passive Lysis Buffer (Promega).
shRNA constructs with the target sequences Rbp2 (5?-GAAGT
TAGCTAAAGAAGAA-3?) and Suz12 (5?-GCTGTTACCAA
GCTCCGAG-3?) were generated as described (Ivanova et al.
2006). pCMVHA-RBP2 wild type and pCMVHA-RBP2 (H483G/
E485Q) mutant have been described (Christensen et al. 2007).
Lentiviral particles were produced in 293FT cells grown in ES
media. Medium containing viral particles was filtered (0.45 µm;
Millipore) and supplemented with 10 µg/mL Polybrene (Sigma)
and 103U/mL LIF (ESGRO). ES cells were transduced with len-
tiviral particles for 16 h, washed, incubated, and selected with
200 µg/mL Hygromycin B (Invitrogen) 24 h after the transduc-
RNA preparation and qPCR
RNA was extracted from whole-cell pellets using the RNeasy
extraction kit (Qiagen) according to the manufacturer’s recom-
mendation. RNA was retrotranscribed using the TaqMan RT–
PCR kit (Applied Biosystem). qPCR was carried out with 5–10
ng of retrotranscribed RNA per reaction.
Primers for qPCR were designed using Primer Express software
with an optimal annealing temperature of 60°C. Primer se-
quences are available as Supplemental Table 2.
ChIP assays were performed as described (Pasini et al. 2007).
Genome-wide location analysis
Sample preparation for genome-wide location analysis was per-
formed as described (Bracken et al. 2006). In brief, ChIP DNA
was amplified by ligation-mediated PCR and purified with a
PCR purification kit (Qiagen). DNA was hybridized on a mouse
promoter array (MM8 RefSeq promoter) manufactured by
NimbleGen Systems, Inc. Sample labeling, hybridization, and
data analysis were carried out by NimbleGen Systems, Inc. Da-
tabase handling and statistical analysis were carried out with
GeneSpring software (Agilent).
The following antibodies were used for immunoblotting: rabbit
anti-Rbp2 (Christensen et al. 2007), mouse anti-Ezh2 (BD43)
(Pasini et al. 2004b), mouse anti-EED (Bracken et al. 2003), rab-
bit anti-SUZ12 (Upstate Biotechnologies), rabbit anti-?-Tubulin
(Santa Cruz Biotechnologies); rabbit anti-hemagglutinin (HA)
(BabCo); rabbit anti-Gal4 (Santa Cruz Biotechnologies).
The following antibodies were used for IP analysis: mouse
anti-HA (12CA5), mouse anti-EZH2 (AC22) (Bracken et al.
2006); mouse anti-SUZ12 (2AO9) (Villa et al. 2007); rabbit anti-
RBP2 (Christensen et al. 2007); rabbit anti-HA (Y11) (Santa Cruz
The following antibodies were used for ChIP analysis: rabbit
anti-Rbp2 (Christensen et al. 2007), rabbit anti-SUZ12 (Abcam),
mouse anti-EZH2 (AC22) (Bracken et al. 2006); rabbit anti-his-
tone H3 (Abcam); rabbit anti-H3K4me3 (Abcam); rabbit anti-
H3K27me3 (Upstate Biotechnologies); rabbit anti-HA (Y11)
(Santa Cruz Biotechnologies).
IP assays and size exclusion chromatography
IP assays were performed in IP(150) buffer (50 mM Tris-HCl at
pH 7.5, 150 mM NaCl, 5% glycerol, 0.2% Igepal [Sigma], Apro-
tein, Leupeptin, 100 mM PMSF, 1 mM DTT). Ethidium bromide
(20 µg/µL) was added when indicated. Size exclusion chroma-
tography was performed with ES cell nuclear extracts in a
Superose-6 10/300 gel filtration column (GE Healthcare) on an
RBP2 functionally interacts with PRC2
GENES & DEVELOPMENT1353
AKTA purifier system (GE Healthcare) in IP(300) buffer (50 mM
Tris-HCl at pH 7.5, 300 mM NaCl, 5% glycerol, 0.2% Igepal
[Sigma], Aprotein, Leupeptin, 100 mM PMSF, 1 mM DTT).
Recombinant protein expression
Generation of baculoviruses for RBP2, EZH2, SUZ12, and EED
has been described (Pasini et al. 2004b; Christensen et al. 2007).
Recombinant proteins were produced in Sf9 cells infected with
equal amounts of viral supernatants for RBP2, EZH2, SUZ12,
We thank Natalia Ivanova for providing the lentiviral vector for
expressing the shRNAs. We thank members of the Helin labo-
ratory for technical advice and support. D.P. was supported by a
post-doctoral fellowship from the Danish Medical Research
Council and P.A.C.C. was supported by a grant from the Benzon
Foundation. The work in the Helin laboratory was supported by
grants from the Danish Cancer Society, the Novo Nordisk
Foundation, the Danish Medical Research Council, the Danish
Natural Science Research Council, the Danish National Re-
search Foundation, and the International Association for Can-
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