The PRC1 Polycomb group complex
interacts with PLZF/RARA to mediate
Hanane Boukarabila,1,2,3,10Andrew J. Saurin,4,5,10,12Eric Batsche ´,6Noushine Mossadegh,1,2,3
Maarten van Lohuizen,7Arie P. Otte,8Jacques Pradel,4,5,9Christian Muchardt,6Michael Sieweke,1,2,3,9
and Estelle Duprez1,2,3,9,11
1Centre d’Immunologie de Marseille-Luminy (CIML), Universite ´ de la Me ´diterrane ´e, Campus de Luminy, 13288 Marseille Cedex
09, France;2Institut National de la Sante ´ et de la Recherche Me ´dicale (INSERM) U631, 13288 Marseille, France;3Centre National
de la Recherche Scientifique (CNRS), UMR 6102, 13288 Marseille, France;4Institut de Biologie du De ´veloppement de Marseille
Luminy, Universite ´ de la Me ´diterrane ´e, Campus de Luminy, 13288 Marseille Cedex 09, France;5Centre National de la Recherche
Scientifique (CNRS), UMR 6216, 13288 Marseille, France;6Unit of Epigenetic Regulation Avenir INSERM, URA2578 CNRS
Institut Pasteur, 75015 Paris, France;7Division of Molecular Genetics, Netherlands Cancer Institute, 1066 CX Amsterdam, The
Netherlands;8Swammerdam Institute for Life Sciences, University of Amsterdam, 1098 SM Amsterdam, The Netherlands
Ectopic repression of retinoic acid (RA) receptor target genes by PML/RARA and PLZF/RARA fusion proteins
through aberrant recruitment of nuclear corepressor complexes drives cellular transformation and acute
promyelocytic leukemia (APL) development. In the case of PML/RARA, this repression can be reversed through
treatment with all-trans RA (ATRA), leading to leukemic remission. However, PLZF/RARA ectopic repression is
insensitive to ATRA, resulting in persistence of the leukemic diseased state after treatment, a phenomenon that is
still poorly understood. Here we show that, like PML/RARA, PLZF/RARA expression leads to recruitment of the
Polycomb-repressive complex 2 (PRC2) Polycomb group (PcG) complex to RA response elements. However, unlike
PML/RARA, PLZF/RARA directly interacts with the PcG protein Bmi-1 and forms a stable component of the
PRC1 PcG complex, resulting in PLZF/RARA-dependent ectopic recruitment of PRC1 to RA response elements.
Upon treatment with ATRA, ectopic recruitment of PRC2 by either PML/RARA or PLZF/RARA is lost, whereas
PRC1 recruited by PLZF/RARA remains, resulting in persistent RA-insensitive gene repression. We further show
that Bmi-1 is essential for the PLZF/RARA cellular transformation property and implicates a central role for PRC1
in PLZF/RARA-mediated myeloid leukemic development.
[Keywords: Leukemia; PLZF/RARA; Polycomb group; gene repression; retinoic acid]
Supplemental material is available at http://www.genesdev.org.
Received October 28, 2008; revised version accepted April 3, 2009.
Regulation of gene expression is achieved through the
control of transcriptional activators or repressors and
through the epigenetic regulation of chromatin structure.
Dysfunction of either of these two regulatory compo-
nents disturbs the cellular homeostasis causing numer-
ous diseases. The Polycomb group (PcG) family of pro-
teins are chromatin factors whose role is to maintain
the repressed transcriptional state of their target genes.
The stable and heritable epigenetic gene repression
afforded by the PcG regulates not only body patterning,
but epigenetic cellular memory, stem cell renewal, and
cancer development (for review, see Sparmann and van
Lohuizen 2006). At the molecular lever, PcG proteins
function as Polycomb-repressive complexes (PRCs), of
which the best studied are termed PRC1 and PRC2. PRC2
is involved in the initiation of gene repression, through
trimethylation of Lys 27 of histone H3 (H3K27me3). This
epigenetic mark is recognized by the chromodomain
of Polycomb (Pc) in the PRC1 complex, an event that is
believed to aid in the recruitment of PRC1 for the
maintenance of gene repression. How the PcG is initially
recruited to silence specific target genes is unclear,
although certain sequence-specific DNA-binding pro-
teins are capable of recruiting PcG complexes to specific
target loci leading to PcG-dependent repression. Thus,
the concerted actions of sequence-specific DNA-binding
factors and epigenetic chromatin modification are likely
to play a fundamental role for stable and heritable gene
9These authors are considered cosenior authors.
10These authors contributed equally to this work.
11E-MAIL email@example.com; FAX 33(0)49126919430.
12E-MAIL firstname.lastname@example.org; FAX 33(0)491820682.
Article is online at http://www.genesdev.org/cgi/doi/10.1101/gad.512009.
GENES & DEVELOPMENT 23:1195–1206 ? 2009 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/09; www.genesdev.org1195
repression by the PcG (Grimaud et al. 2006), with non-
coding RNAs contributing an additional layer of control
(Lempradl and Ringrose 2008).
Deregulation of this essential PcG control system
contributes to aberrations in development and can lead
to different types of cancer. Gene amplification or over-
expression of PRC1 or PRC2 components results in
a broad spectrum of cancers believed to arise from
aberrant silencing of PcG target genes (for review, see
Sparmann and van Lohuizen 2006; Rajasekhar and
Begemann 2007). However, cancer development can also
result from the ectopic recruitment of the PcG to genes
not normally under its control. Recently, the ectopic
recruitment of PRC2 by the oncogenic transcription
factor PML/RARA to retinoic acid (RA)-responsive genes
was found to play a fundamental role in the development
of acute promyelocytic leukemia (APL) (Villa et al.
2007). Ectopic repression of genes by a PcG complex
leading to cancer development is an interesting onco-
genic mechanism, particularly in the case of APL. APL
originates from illegitimate recombination of the RA
receptor a (RARA) gene with one of five different partner
genes, creating an oncogenic X/RARA fusion protein
(Licht 2006). APL patients presenting the chimeric fusion
protein PML/RARA are remarkably sensitive to pharma-
cological doses of all-trans RA (ATRA) and undergo
complete remission, while the PLZF/RARA-associated
APL is more severe, presenting a poor prognosis due to
a nonresponse to ATRA treatment (Licht et al. 1995).
In the case of PML/RARA, therapeutic doses of ATRA
leads to the release of corepressor complexes and to the
loss of ectopic PRC2 recruitment to RA target genes,
reinducing their expression and redifferentiating the
leukemic blast (Villa et al. 2007). In the case of PLZF/
RARA, however, it is still not understood how the fusion
protein induces ATRA-insensitive stable and heritable
To better understand the molecular mechanisms un-
derlying PcG involvement in ATRA-insensitive APL, we
studied the role of PcG complexes in ATRA-insensitive
PLZF/RARA transformation. It has been reported pre-
viouslythat theDNA-binding protein PLZF is involvedin
the stable repression of Hox genes during mouse de-
velopment and recruits the PcG protein Bmi-1 and the
associated PRC1 complex to the HoxD locus (Barna et al.
2002). These observations prompted us to investigate
whether there was a role for PRC1 in PLZF/RARA-
mediated repression. We show that, unlike PML/RARA,
PLZF/RARA is capable of interacting with Bmi-1 and can
form an integral component of PRC1. These interactions
lead to the PLZF/RARA-dependent in vitro and in vivo
recruitment of PRC1 to RA response elements (RAREs) in
an ATRA-insensitive manner, leading to PcG-dependent
transformation of the cell. The interaction between
PLZF/RARA and PRC1 provides new insight into how
the fusion protein may induce leukemogenesis, and how
the ectopic recruitment of PRC1 can play a role in
determining cellular transformation. Identifying the fac-
tors capable of targeting PcG complexes and the molec-
ular differences between ATRA-sensitive and ATRA-
insensitive gene repression by RARA fusion proteins is
essential to understand disease progression.
PLZF/RARA interacts with Bmi-1 through its
The domains mediating all of the known interactions
between PLZF and its partners are located in the
N-terminal half of the protein, which is retained in the
PLZF/RARA chimera. As Bmi-1 interacts with PLZF
(Barna et al. 2002), we tested whether PLZF/RARA
retained the ability to associate with Bmi-1. GST ‘‘pull-
down’’ assays showed that PLZF/RARA interacts with
Bmi-1, whereas PML/RARA, the other major APL onco-
genic fusion protein, does not (Fig. 1A). Through domain
deletion experiments, we found that the interaction is
mediated by the PLZF BTB domain, in conjunction with
the two first PLZF zinc finger domains (Fig. 1A). While
the exact function of these two zinc fingers is unknown,
they are dispensable for DNA binding but essential for
repression (Dong et al. 1996).
BTB-POZ domains are linked to the assembly of
macromolecular transcriptional repression complexes
that are often involved in hematological cancers (Kelly
and Daniel 2006). Point mutations (D35N-R49Q and
Y88A) within the PLZF BTB domain diminish its di-
merization properties and abolish the transformation
potential of PLZF/RARA (Puccetti et al. 2005; Kwok
et al. 2006). However, these mutations do not perturb
its interaction between HDAC and SMRT–NCoR core-
pressors, suggesting that transformation by PLZF/RARA
is not due solely to its interactions with these corepressor
complexes. We found that disrupting PLZF/RARA di-
merization through the Y88A or the D35N-R49Q muta-
tions abrogates Bmi-1 interaction (Fig. 1A,B). The
interaction between PLZF/RARA and Bmi-1 was further
investigated in vivo through coimmunoprecipations (co-
extracts. Western blot analysis on proteins coprecipitat-
ing with Flag-tagged Bmi-1 demonstrates that PLZF/
RARA associates with Bmi-1 in vivo and that, like in
vitro, this interaction requires the dimerized form of
PLZF/RARA (Fig. 1B). This interaction is PLZF/RARA-
specific, since no interaction was observed with RARA or
PML/RARA (Fig. 1C), and is also observed with endoge-
nous Bmi-1 when PLZF/RARA is expressed conditionally
using the U937-B412 cell line (Fig. 1D; Ruthardt et al.
1997). These results demonstrate that PLZF/RARA inter-
acts directly with Bmi-1 and correlates loss of PLZF/
RARA transforming potential, through the D35N-R49Q
mutation, to loss of Bmi-1 interaction.
PLZF/RARA incorporates in PRC1 and increases its
inhibitory activity on chromatin containing RAREs
Bmi-1 is a core component of the mammalian PRC1
complex (Lavigne et al. 2004). Thus, to address whether
PLZF/RARA is capable of recruiting the PRC1 complex
Boukarabila et al.
1196GENES & DEVELOPMENT
to chromatin, we reconstituted the mammalian PRC1
core complex (mPCC) (Fig. 2A) from baculovirus-infected
Sf9 cells (Lavigne et al. 2004). Coinfecting the cells with
a recombinant virus encoding either PLZF or PLZF/
RARA, we found that both PLZF and PLZF/RARA
associate efficiently with the PcG proteins in the mPCC
mPCC–PLZF/RARA PcG complexes (Fig. 2). These data
demonstrate thatboth PLZF and PLZF/RARA are integral
components of a mammalian PRC1 complex.
The recruitment of PRC1 and PCC to chromatin
renders the chromatin refractory to chromatin remodel-
ing, creating a transcriptionally nonpermissive environ-
ment (Francis et al. 2001). Direct association of sequence-
specific DNA-binding proteins with PCC enhances this
effect when therecognition sequenceof theDNA-binding
protein is present in the chromatin DNA sequence,
demonstrating sequence-specific targeting of PCC com-
plexes in vitro (Mulholland et al. 2003). To determine
whether the RARA moiety of PLZF/RARAwas capable of
increasing the chromatin-repressing activity of mPCC
through recruitment of the complex to RARE-containing
chromatin, we performed restriction enzyme accessibil-
ity (REA) assays on in vitro reconstituted 5S chromatin
arrays (Fig. 3A). Using reconstituted 5S-Gal4 control
chromatin, containing repetitive Gal4-binding sites but
no RARE (Fig. 3A), we found that mPCC, mPCC–PLZF,
and mPCC–PLZF/RARA all maximally inhibited chro-
matin remodeling at ;1 nM active protein concentration,
with 50% maximal inhibition observed at 0.2 nM (Fig.
Bmi-1 through its BTB-POZ domain. (A)
GST and GST-Bmi-1 pull-down of
labeled wild-type and mutant isoforms of
Structures of PLZF, PLZF/RARA, and
PML/RARA isoforms used in GST pull-
down and co-IP experiments are shown. (B)
Nuclei of 293T cells cotransfected with
Flag-Bmi-1 and the PLZF or PLZF/RARA
constructs were immunoprecipitated with
anti-Flag beads and were immunoblotted
with either an anti-PLZF or anti-Bmi-1
antibody. (IP) 5% of the input; (FT) the
flow-through. (C) Nuclei of 293T cells
cotransfected with Fl-Bmi-1 and RARA,
PLZF/RARA, or PML/RARA were immu-
noprecipitated with anti-Flag beads and
immunoblotted with either an anti-RARA
or anti-Flag antibody. (IP) 2.5% of the in-
put. (D) Nuclei of U937-MT (control) of
U937-B412 (PLZF/RARA) cells pretreated
with zinc were immunoprecipitated with
anti-RARA (top panels) or anti-Bmi-1 anti-
bodies (bottom panels) and immunoblotted
for PLZF/RARA (a-RARA) or Bmi-1 (a-
Bmi-1). Mock IP represents immunoprecip-
itation without antibody; RARA IP and
Bmi-1 IP represent 2.5% of input.
Recruitment of PRC1 by PLZF/RARA
GENES & DEVELOPMENT1197
3B), which is in accordance with published results for
mPCC (Lavigne et al. 2004). On a 5S-RARE-DR5 array,
these values remained largely unchanged for mPCC and
mPCC–PLZF (;0.75 nM) (Fig. 3B). In contrast, we ob-
served a marked increase in inhibitory activity when
using mPCC–PLZF/RARA complex. In this case, maxi-
mal inhibition of chromatin remodeling by mPCC–PLZF/
RARA was observed at ;0.2 nM active complex, with
50% inhibition achieved using twofold less complex
than for mPCC and mPCC–PLZF (see Fig. 3B). No protein
or complex had any effect on HhaI activity on naked
DNA (Supplemental Fig. S1A), and the individual DNA-
binding proteins had no effect on SWI/SNF remodeling
on either template (Supplemental Fig. S1B,C). It is note-
worthy, however, that while the inclusion of PLZF/
RARA into the complex increases the inhibitory activ-
ity of PCC by at least twofold, simply adding purified
PLZF/RARA to mPCC appears to have little additional
effect (see Supplemental Fig. S1B,C), suggesting that
PLZF/RARA is required to be incorporated into the
PRC1 complex to achieve enhanced inhibitory activity
on SWI/SNF chromatin remodeling in the presence of
the RARE-DR5 sequence, which is similar in effect to
the Drosophila PCC–Zeste complex (Mulholland et al.
Thus, PLZF/RARA can directly associate with PRC1,
and this association is sufficient to recruit the complex to
RAREs, which renders the chromatin refractory to chro-
matin remodeling events.
PLZF/RARA recruits PRC1 to RAREs in vivo
The RARA moiety of PLZF/RARA and PML/RARA
retains its ability to bind RAREs, leading to recruitment
of PML/RARA and PLZF/RARA to RARA target genes in
vivo. Chromatin immunoprecipitation (ChIP) of PLZF/
RARA conditionally expressed in U937-B412 cells
showed that PLZF/RARA is present at the upstream
RARE of the RARb2 promoter (P2), a model target of
RARA, but not in the P1 region, which is not RA-
responsive (Fig. 4B). Recently, it was shown that PML/
RARA interacts with and recruits the PRC2 complex to
the P2 promoter (Villa et al. 2007). To compare the
recruitment of PcG complexes to P2 by PLZF/RARA
and PML/RARA fusion proteins, we examined the pres-
ence of PRC1 and PRC2 complexes at the P2 promoter in
cells conditionally expressing PLZF/RARA (U937-B412)
or PML/RARA (U937-PR9) from a zinc-inducible pro-
moter (Ruthardt et al. 1997). Expression of either PML/
RARA or PLZF/RARA leads to PRC2 enrichment, iden-
tified by EZH2 and its trimethylated Lys 27 of histone H3
(H3K27me3) modification (Fig. 4D). However, we found
that Bmi-1 and Ring1, major components of PRC1, were
specifically enriched on the P2 promoter only upon
expression of PLZF/RARA and not PML/RARA (Fig.
4C). These data demonstrate that expression of either
PML/RARA or PLZF/RARA leads to PRC2 enrichment at
an endogenous target gene, whereas PLZF/RARA expres-
sion distinguishes itself by the additional recruitment of
PRC1 recruitment is ATRA-insensitive
The recruitment of PRC2 to P2 by PML/RARA can
be reversed upon treating the cells with ATRA (Villa
et al. 2007). To assess the effect of ATRA treatment on
tuted PRC1 complex. (A) Colloidal Coomassie blue-stained gel
of purified mPCC (lane 1), mPCC–PLZF/RARA (lane 2), mPCC–
PLZF (lane 3), and mock (lane 4) complexes. Asterisks highlight
the bands corresponding to PLZF/RARA (lane 2) and PLZF (lane
3) verified by Western analyses. The mock complex corresponds
to anti-Flag purification of extracts from Sf9 cells infected with
mPh1, PLZF/RARA, M33, and Ring1a viruses. Densitometry
analyses of the Coomassie-stained complexes show that the
various mPCC complexes are ;55%–65% pure, when compared
with the mock purified complex. (B) Western blots of the
complexes shown in A. PLZF/RARA and PLZF were detected
by an anti-His tag antibody, and Bmi-1 was revealed by an anti-
Flag tag antibody. (C) Western analyses of the sedimentation
profiles of Bmi-1, PLZF/RARA, and mPh1 following fraction of
the mPCC–PLZF/RARA complex by glycerol gradient sedimen-
tation, showing that PLZF/RARA remains associated with the
PLZF and PLZF/RARA associate with the reconsti-
Boukarabila et al.
1198 GENES & DEVELOPMENT
PLZF/RARA-mediated recruitment of PcG complexes,
we performed ChIP analyses on the RARb2 promoter in
U937-B412 cells treated with ATRA or untreated in the
presence or absence of PLZF/RARA (Fig. 5B). Treatment
of PLZF/RARA-expressing cells leads to degradation of
the fusion protein (Fig. 5A; Koken et al. 1999; Rego et al.
containing RARE elements. (A) REA assays on in
vitro reconstituted 5S chromatin arrays. Phosphor-
Imager scans of HhaI digestion products following
chromatin incubation in the presence or absence of
SWI/SNF and PCC complexes with the 5S-Gal4
template (top panel) or the 5S-RARE-DR5 template
(bottom panel). Percentages of template undigested
by HhaI are given. The concentrations of PCC
complexes (in nanomolar) used in the reactions are
the measured concentration of active DNA-binding
molecules. Schematic representation of 5S array
templates used in the REA assays are shown above
the data. ‘‘5S-Gal4’’ contains five Gal4-binding sites
downstream from the unique HhaI restriction site,
whereas ‘‘5S-RARE-DR5’’ contains three RARE DR5
elements. (B) Data from REA assays shown in A are
graphically represented as percentage of inhibition
(Francis et al. 2001) as a function of ln (active protein
concentration) (top graph) and active protein con-
centration (bottom graph), showing increased effi-
ciency of the PCC–PLZF/RARA complex to inhibit
chromatin remodeling when RARE DR5 elements
are present in the chromatin. (Bottom graph) The
linear section of the data is shown, where the
quantities of active complex required to achieve
50% inhibition of chromatin remodeling are high-
lighted (dotted line), demonstrating that PCC–PLZF/
RARA is approximately twofold more efficient at
inhibition of chromatin remodeling than PCC and
PCC–PLZF when RARE DR5 elements are present.
PLZF/RARA recruits PRC1 to chromatin
Recruitment of PRC1 by PLZF/RARA
GENES & DEVELOPMENT1199
2000), although PLZF/RARA can still be detected at the
P2 promoter (Fig. 5B), indicating that some chromatin-
bound PLZF/RARA is protected from degradation. We
observed that ATRA treatment strongly reverses the
levels of EZH2 and H3K27me3 at the P2 promoter
without affecting the PLZF/RARA-mediated Bmi-1 re-
cruitment (Fig. 5B). PRC1 and PRC2 recruitment was not
affected by PLZF/RARA expression at either the PcG-
targeted HOXD4 promoter (Fig. 5C), the CYP26A1 pro-
moter thathasbeen described as a RARAand a PcGtarget
in F9 embryonal carcinoma cells (Fig. 5D; Gillespie and
Gudas 2007), theRARb2 P1 promoter (data not shown), or
the unrelated GAPDH promoter (Fig. 5E). No changes in
expression patterns of either HOXD4 or CYP26A1 were
observed under any conditions tested (Fig. 5F). These
data correlate the ATRA-insensitive recruitment of
PRC1 to the RARb2 promoter with inhibition of the
ATRA-stimulated expression of RARb2 (Fig. 5F). Con-
sistent with repression of RARb2, no increase in histone
H3 acetylation at the RARb2 promoter was observed
following ATRA treatment in the presence of PLZF/
RARA (Supplemental Fig. S2). These data implicate
PRC1 in the PLZF/RARA-mediated repression of ATRA-
stimulated gene expression.
Bmi-1 is required for PLZF/RARA-mediated
In APL, the expression pattern of PLZF/RARA mimics
that of PLZF, since PLZF/RARA expression is under the
control of the PLZF promoter. Thus, to determine
whether PRC1 can play a role in the PLZF/RARA-
mediated diseased state, we first determined in which
hematopoietic compartments PRC1 components and
PLZF are expressed. Expression profiling of PRC1 PcG
components and PLZF in hematopoietic stem cells
showed that all of these components are expressed
in the same bone marrow (BM) compartment (Supple-
mental Fig. S3), in accordance with previously published
data for PcG protein expression patterns (Iwama et al.
Next, we assessed whether Bmi-1 is required for trans-
formation of hematopoietic progenitors by PLZF/RARA.
In methylcellulose culture, normal progenitors exhaust
their proliferative capacity after the second passage,
while transformed progenitors form colonies for more
than three passages (Lavau et al. 1997). We used this assay
of transformation to compare the replating capacity of
wild-type and Bmi-1?/?BM progenitor cells (Bel et al.
1998) when transduced by PLZF/RARA or PML/RARA.
Even though Bmi-1?/?hematopoietic progenitor com-
partments are reduced (Supplemental Fig. S4A) due to
defects in proliferation and self-renewal capacity (Lessard
and Sauvageau 2003; Park et al. 2003), they can still be
transformed (Supplemental Fig. S4). Empty vector con-
trol-infected wild-type cells lose colony-forming capacity
after the second plating, while PLZF/RARA- or PML/
RARA-infected wild-type cells continue to form colonies
(Fig. 6A). In Bmi-1?/?progenitor cells, the two fusion
proteins display differences in colony formation: While
PML/RARA transduction yields a significant number of
third-round colonies, PLZF/RARA transduction cannot
impose extended colony formation capacity on Bmi-1?/?
cells (Fig. 6A).
The tumor suppressor locus Ink4a/Arf can be repressed
by Bmi-1, and its derepression in the absence of Bmi-1
results in defects in proliferation (Jacobs et al. 1999).
To test whether the loss of PLZF/RARA transforma-
tion potential in Bmi-1?/?progenitors was the result of
expression of Ink4a/Arf, we infected BM progeni-
with PLZF/RARA. We found that PLZF/RARA-infected
PRC2 to RARb2 promoter. (A) Representa-
tion of the RARb2 promoter. P1 and P2
indicate the regions amplified by PCR. (B)
PLZF/RARA on RARb2 promoter was ana-
lyzed by quantitative ChIP (qChIP) with
an anti-PLZF antibody on chromatin frag-
ments prepared from U937-B412 cells pre-
treated with zinc or untreated. (**) P < 0.01
(C,D) qChIP analyses of the PRC1 subunits
Bmi-1 and Ring1a (C), or EZH2, H3K27me3,
and histone H3 (D) at RARb2 promoter in
the absence (MT) or in the presence of either
PLZF/RARA or PML/RARA. Error bars rep-
resent standard deviations obtained from
at least two independent experiments. (**)
P < 0.01; (***) P < 0.001.
PLZF/RARA targets PRC1 and
Boukarabila et al.
1200GENES & DEVELOPMENT
Bmi-1?/?/Ink4a?/?/Arf?/?progenitors gave only a small
number of colonies that were diffuse and small in size,
and that could not sustain successive replatings (Supple-
mental Fig. S5). These data suggest that the requirement
of Bmi-1 for PLZF/RARA transformation of BM cells is
independent of Ink4a/Arf expression. Additionally, these
effects are not due to loss of PLZF/RARA expression in
Bmi-1?/?cells, as quantitative RT–PCR (qRT–PCR) and
Western blot analysis show that PLZF/RARA expression
is maintained after the third passage in Bmi-1?/?cells
Morphological analyses of BM cells from third-
round colonies from both PLZF/RARA- and PML/
RARA-transduced Bmi-1?/?progenitors showed that
prepared from PLZF/RARA-expressing cells (+Zinc) at various times of ATRA treatment. Proteins were blotted and probed with
antibodies to PLZF, EZH2, Bmi-1, and b-tubulin. (B–E) Analyses of the effects no stimulation (0) or 18 h of ATRA stimulation (18) on the
enrichment of PLZF/RARA, Bmi-1, EZH2, and H3K27me3 in untreated B412 cells or B412 cells expressing PLZF/RARA (PLZF/RARA
panels) at the RARb2 promoter (B), the HOXD4 promoter (C), the CYP26A1 promoter (D), or the GAPDH promoter (E). Data are
presented as percentage of bound/input and error bars indicate the standard deviation obtained from two or three independent
experiments. White bars represent mock immunoprecipitation; black bars represent specific immunoprecipitation with the indicated
antibodies. (F) The expression levels of RARb2, HOXD4, and CYP26A1 were analyzed by quantitative real time PCR (qRT–PCR). RNA
from U937-B412 cells (white bars) or zinc-pretreated U937-B412 cells (black bars; +zinc), either unstimulated or stimulated with 1 mM
ATRA for 18 h (+ATRA) was extracted and 1–2 mg of RNA was reverse-transcribed. Gene expression of each gene is shown relative to
Effect of ATRA treatment on PLZF/RARA-mediated PRC1 and PRC2 recruitment. (A) Western analyses of nuclear extracts
Recruitment of PRC1 by PLZF/RARA
GENES & DEVELOPMENT 1201
whereas colonies from PML/RARA-transduced Bmi-1?/?
cells were as large and compact as wild-type-transduced
cells, the rarely seen PLZF/RARA-infected Bmi-1?/?
colonies remained diffuse and smaller in size (Fig. 6C).
Giemsa and FACS analyses showed that cells from PLZF/
RARA-transduced Bmi-1?/?-derived colonies were fully
mature, reflected by their morphology (Fig. 6D) and their
pronounced reduction in expression of the c-kit progen-
itor marker, together with increased expression of the
myeloid differentiation marker Gr-1 (Fig. 6E). In contrast,
cells from PML/RARA-transduced Bmi-1?/?-derived col-
onies, while morphologically more mature than the wild-
type-transduced counterparts, display more blast-like
features compared with PLZF/RARA (Fig. 6D) and retain
expression of c-kit progenitor marker (Fig. 6E).
Taken together, these data show that Bmi-1 is neces-
sary for transformation of myeloid progenitors by PLZF/
RARA, but not by the related PML/RARA oncogenic
fusion, supporting our model that PRC1 plays a key role
in PLZF/RARA-mediated transformation.
PLZF/RARA associates with PRC1
X/RARA APL fusion proteins represent a model of altered
transcription factors that function as dominant-acting
oncoproteins. Their ability to form oligomers and to
recruit corepressors is critical for inducing oncogenic
transcriptional repression (So and Cleary 2004), which is
the result of ectopic epigenetic modification of RA target
genes (Di Croce et al. 2002). Forced oligomerization of
RARA, which in APL is due to the fusion partner protein,
is necessary and sufficient to induce cellular transforma-
tion (Kwok et al. 2006; Sternsdorf et al. 2006). Besides
inducing oligomerization, the fusion partner has a decisive
impact on the diseased state and sensitivity to the thera-
peutic effects of ATRA, due to ectopic recruitment of its
interacting proteins to RA-responsive genes. We show
that, through the PLZF moiety, PLZF/RARA can interact
with the PcG protein Bmi-1. Bmi-1 is a member of the
PRC1 complex that can inhibit chromatin remodeling
RARA transformation. (A) Bar charts indi-
cate the number of colonies formed in
methylcellulose cultures of lin BM trans-
duced with the empty vector (MSCV),
MSCV-PML/RARA (PML/RARA). Data rep-
resent the number of colonies from wild-
type and Bmi-1?/?BM during three succes-
sive replatings. Error bars are standard de-
viation of the mean from five (PLZF/RARA)
or two (PML/RARA) independent replating
experiments. (B) Transgene expression was
assessed by qRT-PCR (panel i) and immu-
noblotting (panel ii) from wild-type (WT) or
Bmi-1?/?BM cells infected with either
PLZF/RARA or PML/RARA viruses har-
vested after the third passage. Fusion pro-
teins were detected in panel ii using
antibodies against PLZF (PLZF/RARA) or
RARA (PML/RARA). (C,D) Typical mor-
phology (C) and MGG staining (D) of the
second-round (II) or third-round (III) colonies
generated from wild-type or Bmi-1?/?cells
transduced with empty vector, PLZF/RARA,
or PML/RARA. Bars: C, 200 mm; D, 10 mm.
(E) Surface marker expression analyzed by
FACS of the second-round colonies gener-
ated from wild-type or Bmi-1?/?cells trans-
duced with PLZF/RARA or PML/RARA.
Bmi-1 is required for PLZF/
Boukarabila et al.
1202 GENES & DEVELOPMENT
(Francis et al. 2001; Mulholland et al. 2003) and tran-
scription (King et al. 2002) through the compaction of
chromatin structure (Francis et al. 2004). The role of
sequence-specific binding factors in targeting PRC1 to
chromatin passes by one of two mechanisms: binding of
the transcription factor to DNA, thereby recruiting the
complex, as is the case for the GAGA factor; or incorpo-
ration of the transcription factor in the protein complex,
thereby recruiting the complex to DNA, as is the case for
Zeste (Mulholland et al. 2003) and Pho (Mohd-Sarip et al.
2005). We show that PLZF/RARA is incorporated into the
mPCC (mPCC–PLZF/RARA) (Fig. 2). The DNA-binding
domains of RARA are preserved in PLZF/RARA, which
serves to recruit mPCC–PLZF/RARA to RARE-containing
chromatin in vitro (Fig. 3) and in vivo (Fig. 4), providing
essential mechanistic insight into how recruitment is
achieved. The REA assays (Fig. 3) demonstrate that the
effect of incorporation of PLZF/RARA into PRC1 leads to
the RARA-mediated recruitment of the complex to
RAREs, and that the recruitment is not occurring solely
via some histone mark.
Divergence in PRC recruitment between PLZF/RARA
Recently, PRC2 activity was shown to play a key role in
PML/RARA-mediated oncogenesis, in which ectopic re-
cruitment and methylation activities of PRC2 and DNA
methyltransferases by PML/RARA lead to oncogenic
repression of RARA-controlled genes (Villa et al. 2007).
The histone H3K27me3 modification laid down by PRC2
is believed to aid in the recruitment of PRC1 (Fischle
et al. 2003; Min et al. 2003). This idea fits well with
the model of hierarchical recruitment of PcG complexes,
in which the establishment of robust epigenetic silencing
of Hox genes requires a transient interaction between
PRC2 and PRC1 components that is then maintained by
PRC1 complexes (Poux et al. 2001). However, while
H3K27me3 serves a role in aiding in PRC1 binding, it is
not the sole deciding factor for PRC1 recruitment, since
a large number of genes displaying H3K27me3 do not
have bound PRC1 (for review, see Ringrose and Paro
2007), and in Drosophila cells, PRC1 binds to discrete
sites, whereas H3K27me3 covers large genomic domains
(Beisel et al. 2007). Indeed, while expression of PML/
RARA leads to ectopic PRC2 recruitment at RA response
genes (Villa et al. 2007), the H3K27me3 mark alone is
insufficient to recruit PRC1 (Fig. 4). Conversely, we found
that expression of PLZF/RARA leads to ectopic recruit-
ment of both PRC1 and PRC2. While reconstitution
experiments and immunoprecipitations demonstrate
that recruitment of PRC1 by PLZF/RARA is through
interactions between PLZF/RARA and Bmi-1, recruit-
ment of PRC2 upon expression of PLZF/RARA is likely
to be due to the known interactions between RARA and
PRC2 (see Epping et al. 2005; Villa et al. 2007). Thus, we
observe a fundamental difference in recruitment of PcG
complexes between PML/RARA and PLZF/RARA, due to
the additional interactions between PLZF/RARA and
PRC1 is insensitive to RA treatment and is essential
for PLZF/RARA transformation
Treatment of PML/RARA-expressing cells with ATRA
leads to degradation of the fusion protein (Rego et al.
2000), loss of PRC2 recruitment, and derepression of RA
response genes (Villa et al. 2007). Treatment of PLZF/
RARA cells with ATRA also leads to degradation of the
fusion protein, but with very little change in the RA
response gene repressed state (He et al. 1998; Lin et al.
1998). Our data show that while the soluble nuclear pool
of PLZF/RARA is degraded upon ATRA treatment (Fig.
5A), some of the chromatin-bound fraction remains
protected from degradation (Fig. 5B)—protection that we
attribute to either the incorporation of PLZF/RARA into
the PRC1 complex (Fig. 2), or the highly compact nature
of the chromatin once targeted by PRC1 (Francis et al.
2004). Like PML/RARA, we found that treatment of
PLZF/RARA cells with ATRA leads to a stark reversal
in recruited PRC2 and its H3K27me3 mark, whereas
PRC1 recruited by PLZF/RARA was unaffected. This
suggests that PRC1 plays a role in the continued main-
tenance of ectopic silencing of PLZF/RARA, helping us to
understand why PLZF/RARA-targeted genes respond
poorly to ATRA.
If the recruitment of PRC1 by PLZF/RARA to its target
genes plays a direct role in ectopic gene repression, then
circumventing its recruitment would have profound
effects on PLZF/RARA oncogenic potential. Bmi-1 is
a major component of the PRC1 complex (Levine et al.
2002), and likely mediates the liaison between PRC1 and
PLZF/RARA (see Fig. 1). When this interaction can no
longer occur in myeloid progenitors lacking Bmi-1, PLZF/
RARA loses its ability to transform. PML/RARA, how-
ever, is still able to transform Bmi-1?/?progenitors,
which is in agreement with our model that PRC1 is
required for PLZF/RARA-mediated but not PML/RARA-
PRCs: a deciding factor in the efficiency
of a therapeutic RA response?
While PcG complexes play a central role in epigenetic
gene repression, the repressive state imposed can either
be stable, as in Hox repression, or more dynamic, as with
certain lineage-determining transcription factors in stem
cells. We found that both PRC2 and PRC1 are ectopically
recruited to RA response genes through PLZF/RARA
expression, whereas only PRC2 is recruited when PML/
RARA is expressed. This difference is due to a unique
interaction between PLZF/RARA and Bmi-1 that recruits
the PRC1 complex to RA response genes and is essential
for the transformation ability of PLZF/RARA. Thus,
while PRCs play a major role in both PLZF/RARA-
mediated and PML/RARA-mediated transformation, we
propose a model explaining how the different PcG com-
plexes involved have a decisive impact on RA response:
PML/RARA interacts with and ectopically recruits PRC2
to RA response genes, whereas PLZF/RARA interacts
with and ectopically recruits both PRC1 and PRC2.
Through the effect of the PcG on chromatin, these
Recruitment of PRC1 by PLZF/RARA
GENES & DEVELOPMENT1203
recruitments participate toward repression of the RA
response gene. Upon treatment with pharmacological
doses of RA, PRC2 recruitment by either PML/RARA or
PLZF/RARA is lost. In the case of PML/RARA, this leads
to reactivation of the response gene. However, PRC1
recruited by PLZF/RARA is unaffected by RA treatment
and continues to provide neomorphic Bmi-1-dependent
repression of PLZF/RARA target genes. Due to the re-
quirement of Bmi-1 in mediating the interaction between
designed to disrupt the PLZF/RARA–PRC1 interaction
in vivo will prove critical in understanding the molecular
mechanisms driving this persistent leukemic state and
toward developing a therapeutic response.
Materials and methods
Antibodies and constructs
Antibodies usedwereagainstBmi(H-99),M2(Sigma),His tag(BD
Biosciences), M33 (Abcam), PLZF (H-300), RARA (C-20) (Santa
Cruz Biotechnologie), b-Tubulin1 (Sigma), and Ring1 and Ph1
(Satijn et al. 1997) for Western blot; and Bmi-1 (1.T.21, Abcam),
PLZF (H-300, Santa Cruz Biotechnologies), EZH2 (AC22), H3ac
(06599) (Upstate Biotechnologies), H3K27me3 (ab8898), H3
(ab1791) (Abcam), Flag (M2, Sigma), and Ring1 (Satijn et al.
1997) for immunoprecipitation or ChIP. cDNAs used in this
study are described in the Supplemental Material.
Protein interaction assays
GST pull-downs were performed as described previously (Kwok
et al. 2006). For co-IP experiments, 293Tcells were cotransfected
with Flag-Bmi-1 and the following constructs: PLZF, PLZF/
RARA, RARA, or PML/RARA. Nuclear protein was extracted
and complexes were immunoprecipitated with M2 anti-Flag
beads (Sigma Aldrich), washed three times with PBS, and eluted
with Flag peptide (0.5 mg/mL). For endogenous co-IP in U937
cells, PLZF/RARA was induced with zinc for 24 h, nuclear
proteins were extracted, and endogenous Bmi-1 was immuno-
precipitated with anti-Bmi-1 and PLZF/RARA was immunopre-
cipitated with anti-RARA overnight at 4°C. Immunoprecipitates
were washed with PBS and eluted with SDS sample buffer.
Purification of mPCC complexes
Sf9 cells were coinfected simultaneously with recombinant
baculovirus encoding Flag-Bmi1, M33, mPh1, Ring1A (mPCC;
Lavigne et al. 2004) and either His-PLZF (mPCC–PLZF) or His-
PLZF/RARA (mPCC–PLZF/RARA). Volumes of each high-titer
virus were determined empirically as the minimum required to
infect all cells determined by cell cycle arrest. Forty-eight hours
post-infection, cells were harvested, nuclear extracts were per-
formed, and complexes were purified according to Lavigne et al.
(2004) using 15 column volumes of 1.2 M KCl buffer for the high-
stringency wash, and including 10 mM ZnCl2 in all buffers
(PCCB). Complexes were quantitated by Bradford analysis using
BSA as the standard, and active DNA-binding protein concen-
trations were determined by filter-binding analyses using line-
arized DNA (Francis et al. 2001). A mock purification was
performed by M2 anti-Flag purification of cells infected with
M33, mPh1, Ring1A, and His-PLZF/RARA viruses.
mPCC–PLZF/RARA was further fractionated using glycerol
gradient sedimentation. One-hundred microliters of mPCC–
PLZF/RARA complex were applied to a 10%–40% glycerol
gradient in PCCB200 and centrifuged for 21 h at 30,000 rpm in
a Beckman SW50 rotor. Two-hundred-microliter fractions were
collected and resolved by 8% SDS-PAGE, blotted to nitrocellu-
lose membranes, and probed with either anti-mPh1, anti-His
(BD Biosciences; PLZF/RARA), or M2 anti-Flag (Sigma; Bmi?)
5S array assemblies and assays
5S-RARE-DR5 DNA was constructed by replacing the PstI–XbaI
fragment containing the Gal4 DNA-binding sites from the 5S-
G5E4 vector with a 91-base-pair (bp) fragment containing three
RA receptor-binding sites (DR5T-GGTTCACCGAAAGTTCA).
Nucleosomearrayswere assembled and analyzed by gradient salt
dialysis and REA assays were performed as described (Francis
et al. 2001). Radioactive 5S nucleosome arrays (1 nM) were
incubated with varying concentrations of mPCC complexes in
12 mM HEPES, 0.1 mM EDTA, 10 mM Tris, 100 ng/mL BSA, 0.2
mM DTT, 4 mM MgCl2, and 10 mM ZnCl2for 20 min at 30°C
prior to addition of purified SWI/SNF (Sif et al. 1998) at concen-
trations determined to be saturating for chromatin remodeling
(typically 250 ng), and 4 U HhaI (New England Biolabs). Reac-
tions were allowed to proceed for 60 min at 30°C and were
stopped by the addition of 2 mL of DSB (50 mM Tris, 100 mM
of proteinase K for 15 min at 50°C, and DNA was resolved on
0.8% TAE agarose gels. Gels were vacuum dried and exposed to
a PhosphorImager (Fuji, SLA-5000) for quantitation (Image
Human myelomonoblastic cell lines U937-MT, U937-PR9, or
U937-B412 were maintained at exponential growth in RPMI
supplemented with 10% fetal calf serum. U937-PR9 and U937-
B412 contain, respectively, PML/RARA cDNA and PLZF/RARA
cDNA under the control of the zinc-inducible human metal-
lothionein promoter (Ruthardt et al. 1997).For proteininduction,
cells were prestimulated with 0.1 mM ZnSO4for at least 24 h.
For ATRA stimulation, cells were treated with 1 mM ATRA for
the indicated times.
ChIP was performed as described before in Batsche et al. (2006)
with some modifications. Cells were treated with 1% formalde-
hyde (10 min, room temperature), and the reaction was stopped
by the addition of glycine at the final concentration of 0.125 M.
After two washes in PBS, cells were resuspended in 10 mM Tris-
HCl (pH 8), 10 mM EDTA, 0.5 mM EGTA, 10 mM, and 0.25%
Triton X-100; the soluble fraction was eliminated by centrifuga-
tion; and chromatin was extracted with 250 mM NaCl, 50 mM
Tris-HCl (pH 8.0), 1 mM EDTA, and 0.5 mM EGTA for 20 min on
ice. Chromatin was resuspended in 10 mM Tris-HCl (pH 8.0),
1 mM EDTA, 0.5 mM EGTA, and 0.5% SDS, and sonicated for
15 min using Diagenode Bioruptor (full power, 20 sec on 30 sec
off). DNA fragment size (<1 kb) was verified by agarose gel
electrophoresis. For ChIP using Bmi-1, Ring1a, PLZF, or Ezh2
antibodies, chromatin extracted from 1 3 107cells per condition
was used. For ChIP using H3 or H3K27me3, chromatin from 2 3
106cells per condition was used. Chromatin was diluted five
times in 150 mM NaCl, 10 mM Tris-HCl (pH 8.0), 1% Triton
X-100, 0.1% sodium deoxycholate (NaDOC), 1 mM EDTA, and
0.5 mM EGTA. Chromatin, precleared 1 h with protein G-coupled
magnetic beads, was incubated overnight at 4°C using the
Boukarabila et al.
1204 GENES & DEVELOPMENT
protein G-coupled magnetics beads (Dynabeads, Invitrogen).
Immunocomplexes were washed with 13 buffer 1 (10 mM
Tris-HCl at pH 8.0, 1% Triton X-100, 0.1% NaDOC, 150 mM
NaCl), 13 buffer 2 (10 mM Tris-HCl at pH 8, 1% NP-40, 0.1%
NaDOC, 150 mM KCl), 13 buffer 3 (10 mM Tris-HCl at pH 8.0,
0.5%Triton X-100, 0.1% NaDOC, 300 mM NaCl), 13 buffer 4 (20
mM Tris-HCl at pH 8.0, 0.5% NP-40, 0.5% NaDOC, 250 mM
LiCl, 1 mM EDTA), and 13 buffer 5 (20 mM Tris-HCl at pH 8.0,
0.1% NP-40, 150 mM NaCl, 20 mM Tris-HCl at pH 8.0, 1 mM
EDTA). Elution, reverse cross-linking, and purification were
performed using Chelex (Bio-Rad). Beads were eluted in 100 mL
of water containing of 10% Chelex by boiling for 10 min, then
were incubated with proteinase K for 30 min at 55°C and reboiled
for 10 min.
Amplifications (40 cycles) were performed using quantitative
real-time PCR using Brilliant SYBR Green Master Mix (Strata-
gene) according to the manufacturer’s instructions. IgG control
‘‘cycle over the threshold’’ Ct values were subtracted to Input or
immunoprecipitation Ct values and converted into bound value
by 2(?[IP Ct or input Ct ? IgG IP Ct]). Data are expressed as percentage
Primers used are given in the Supplemental Material.
Statistical analyses were performed with the Student’s t test
using GraphPad and two-tailed P values are given as follows:
(*) P < 0.1; (**) P < 0.01; and (***)P < 0.001.
Retroviral infection and replating assay
BM cells were harvested from 3-wk-old littermate wild-type or
Bmi-1?/?mice. Hematopoietic progenitors and stem cells were
negatively selected for lineage expression by magnetic activated
cell sorting (Miltenyi) and prestimulated for 48 h before in-
fection. Viral supernatants were used to infect hematopoietic
progenitorsand stem cells by spinoculation at 2000 rpm for 2 h at
32°C. Infections were repeated twice and transduced cells were
plated in methylcellulose supplemented with mSCF (50 ng/mL;
R&D), mIL6(20 ng/mL),mIL3, and GM-CSF(10 ng/mL;Abcys)in
the presence of selective drugs. Colonies were counted after 7–
9 d and replated three times at 10,000 cells when possible. One
independent experiment represents the mean obtained from
parallel platings of infected cells taken from a pool of BM cells
obtained from up to two animals. After the second replating,
cellular morphology of the colonies were analyzed by Wright-
Giemsa staining of cytospin. Immunophenotypic analyses were
performed by FACS using fluorochrome-conjugated antibodies to
c-Kit (clone eB149), Mac-1 (clone M1; eBioscience), and Gr-1
(clone RB6-8C5; Becton Dickinson).
We thank A. Pietersen and C. Pritchard for isolating the Ink4a?/?/
Arf?/?/Bmi1?/?-deficient BM, A. Iwama for the Bmi-1 expression
vector, M. Ruthard for the gift of U937 and derivative cells and
PLZF/RARA mutants, M. Djabali for providing the Bmi?/+mice
and helpful discussions, R. Kingston and M. Lavigne for pro-
viding the mPCC baculovirus, and H. de The ´ for MSCV-PML/
RARA DNA. This work was supported by CNRS, INSERM, and
grants from Association pour la Recherche sur le Cancer (ARC)
to E.D., A.S., J.P., and M.S., from AICR to M.S., and from
CEFIPRA to A.S. and J.P. H.B. was supported by a fellowship
from the CNRS and the socie ´te ´ Franc xaise d’he ´matologie (SFH).
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