Binding of pRB to the PHD Protein RBP2 Promotes Cellular Differentiation

Molecular Cell, Vol. 18, 623–635, June 10, 2005, Copyright ©2005 by Elsevier Inc. DOI 10.1016/j.molcel.2005.05.012

Binding of pRB to the PHD Protein RBP2Promotes Cellular Differentiation

Elizaveta V. Benevolenskaya,1,5 Heather L. Murray,2

Philip Branton,3 Richard A. Young,2

and William G. Kaelin, Jr.1,4,*1Dana-Farber Cancer Institute andBrigham and Women’s HospitalHarvard Medical SchoolBoston, Massachusetts 021152Whitehead Institute for Biomedical Research9 Cambridge CenterCambridge, Massachusetts 021423Department of BiochemistryMcGill UniversityMontreal, Quebec H3G 1Y6Canada4Howard Hughes Medical Institute4000 Jones Bridge RoadChevy Chase, Maryland 20815

Summary

pRB can enforce a G1 block by repressing E2F-responsive promoters. It also coactivates certain non-E2F transcription factors and promotes differentia-tion. Some pRB variants activate transcription andpromote differentiation despite impaired E2F bindingand transcriptional repression capabilities. We iden-tified RBP2 in a screen for proteins that bind to suchpRB variants. RBP2 resembles other chromatin-asso-ciated transcriptional regulators and RBP2 bindingtracked with pRB’s ability to activate transcriptionand promote differentiation. RBP2 and pRB colocalizeand pRB/RBP2 complexes were detected in chroma-tin isolated from differentiating cells. RBP2 siRNAphenocopied restoration of pRB function in coactiva-tion and differentiation assays, suggesting that pRBprevents RBP2 from repressing genes required fordifferentiation. In addition, two bromodomain-con-taining proteins were identified as RBP2 targets thatare transcriptionally activated by pRB in an RBP2-dependent manner. Our results suggest that promo-tion of differentiation by pRB involves neutralizationof free RBP2 and transcriptional activation of RBP2targets linked to euchromatin maintenance.

Introduction

The RB-1 tumor-suppressor gene product, pRB, regu-lates many processes that are deregulated in cancerincluding cell-cycle progression, apoptosis, and cellu-lar differentiation. Most human cancers harbor muta-tions that directly or indirectly compromise pRB func-tion (Sellers and Kaelin, 1997). Examples of the latterinclude mutations that cause pRB hyperphosphoryla-tion, such as p16INK4A homozygous deletion or Cdk4

*Correspondence: [email protected]
5 Present address: Department of Biochemistry and Molecular Ge-netics, University of Illinois at Chicago, Chicago, Illinois 60607.
amplification. Hyperphosphorylation, like most tumor-associated RB-1 mutations, impairs pRB’s ability tobind to specific cellular proteins. pRB phosphorylationis normally linked to the presence of specific mito-genic signals.

Over 100 pRB binding proteins have been reported,although E2F family members have attracted the mostinterest (Morris and Dyson, 2001). E2Fs, when boundto a DP family member, bind to specific DNA sequencesand transcriptionally regulate genes linked to cell-cycleprogression and apoptosis (reviewed in Frolov and Dy-son [2004]). E2F1, E2F2, and E2F3a are transcriptionalactivators, whereas E2F3b and E2F4–7 act primarily astranscriptional repressors. pRB binds directly to, andtranscriptionally silences, the activating E2Fs. More-over pRB, when recruited to DNA via an E2F, is a potenttranscriptional repressor due at least partly to its abilityto bind histone deacetylases and histone methylases(Ferreira et al., 2001; Harbour and Dean, 2000). Un-timely activation of E2F target genes accelerates Sphase entry and in some settings causes apoptosis(Sellers and Kaelin, 1997). Hence, derepression of E2Ftarget genes likely accounts for abnormal proliferationexhibited by cells with compromised pRB, especiallywhen apoptosis is suppressed by mutations at otherloci.

Restoration of pRB function in certain human tumorcells leads to a G1/S block. This activity is tightly linkedto pRB’s ability to repress E2F-responsive promoters.Conversely, activating E2F variants that escape re-cognition by pRB can bypass a pRB-induced cell-cycleblock (Qin et al., 1995; Shan et al., 1996). These obser-vations, as well as genetic experiments in model organ-isms, indicate that E2F is a critical downstream targetof pRB.

Several lines of evidence implicate pRB in cellulardifferentiation. Differentiation is typically coupled tocell-cycle exit and hence is usually accompanied bydephosphorylation of pRB. In some models, however,one also observes an increase in total pRB coincidentwith differentiation (Bergh et al., 1997; Ji et al., 2002).Inactivation of pRB impairs differentiation in vitro andin vivo (Classon and Harlow, 2002; de Bruin et al., 2003;Lipinski and Jacks, 1999; Maione et al., 1994; Slack etal., 1995; Thomas et al., 2001). Conversely, overproduc-tion of pRB promotes differentiation in certain models.Studies such as these have implicated pRB in neuronal,myogenic, adipocyte, osteogenic, hematopoietic, andlens cell differentiation, especially with respect to theacquisition of late differentiation markers.

Most pRB binding proteins interact with a region ofpRB called the “large pocket” (LP), encompassing pRBresidues 379–928. This region is sufficient to promotedifferentiation and is also sufficient to bind to E2F andrepress transcription (Sellers and Kaelin, 1997). How-ever, control of differentiation by pRB is unlikely to sim-ply reflect its ability to repress E2F-responsive promot-ers. Using linker scanning mutagenesis, we previouslyidentified pRB variants that were defective with respectto E2F binding and transcriptional repression but re-

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tained the ability to promote differentiation and senes- �acent cell morphology in vitro (Sellers et al., 1998). The

products of partially penetrant RB-1 alleles, which are ppassociated with a low risk of retinoblastoma, behaved

similarly. Conversely, a chimera containing the E2F1 RlDNA binding domain fused to the pRB transrepression

domain could induce a G1/S block but did not promote tidifferentiation and senescent changes (Sellers et al.,

1998). Recent studies indicate that pRB can become epacetylated (Chan et al., 2001) and that this modification

is required for pRB to promote differentiation but not Tnto form pRB/E2F transcriptional repressor complexes

(Nguyen et al., 2004). Collectively, these observations ussuggest that differentiation control by pRB involves

E2F-independent pathways.bpRB activates transcription in concert with a variety

of transcription factors, including MyoD, C/EBPs, tiCBFA1, and GRα, and this activity tracks with its ability

to promote differentiation (Lipinski and Jacks, 1999; wcClasson and Harlow, 2002; Thomas et al., 2001). How

pRB enhances transcriptional activation by such tran- Ldscription factors is, however, unclear. The pRB binding

protein EID1 is a potent inhibitor of the histone acety- f1lases p300 and CBP and blocks differentiation (Mac-

Lellan et al., 2000; Miyake et al., 2000). Dysregulation fAof EID1 might therefore contribute to abnormal differen-

tiation in pRB-defective cells. However, some pRB vari- ptants that cannot bind to EID1, such as pRB�663, can

still promote differentiation (S. Miyake and W.G.K., un- aepublished data), suggesting the existence of additional

pRB targets linked to differentiation. 2RdResultss

To identify such targets, we screened for cellular pro-teins that can bind to pRB LP �663, which can promote C

Idifferentiation, but not to pRB LP �ex22, which cannot,using a dual bait yeast two-hybrid system. The �663 w

dmutation decreases binding to E2F as well as to canon-ical LXCXE pRB binding motifs, such as those found i

Sin EID1 (for example, see Figures 1A and 1B). It alsoneutralizes pRB’s transcriptional repression domain p

s(Sellers et al., 1998). A total of 6 proteins (HPV E7,RBP2, PLZF, AK000050, E2F2, and HIRIP4), all of which w

care known or suspected to be nuclear, were identifiedin this way after screening three different cDNA librar- l

fies. The E7 cDNAs were likely a contaminant of one ofthe libraries. Notably, E7 contains an LXCXE motif as w

cwell as a second, LXCXE-independent pRB binding do-main (Huang et al., 1993). The latter might account for p

pthe residual binding to �663 (Figure 1A). We also iso-lated E2F2 in this screen despite the fact that �663 o

hdoes not bind to recombinant E2F1 (Figure 1A) anddoes not bind to E2F in gel-shift assays conducted with (

aRB−/− mammalian cell extracts (Sellers et al., 1998).However, binding of �663 to recombinant E2F2 was not

ttested before and E2F2 is a minor component of E2Fin most mammalian cells. �

wNext, these assays were repeated for five of theseproteins using full-length pRB as bait. HIRIP4 is a n

scochaperone for Heat Shock Cognate 70 (Hsc70) pro-tein and was not studied further. Among the pRB vari- o

nants tested were four proteins (�663, �651, 661W, and

ex4) that can promote differentiation despite impairedbility to form pRB/E2F transcriptional repressor com-lexes (Sellers et al., 1998). 661W and �ex4 are theroducts of two naturally occurring, partially penetrantB-1 alleles, and �651, like �663, contains a flexible

inker substitution. The pRB binding protein SV40 T an-igen was tested in parallel as a positive control. Bind-ng of all five proteins to �663, �651, 661W, and �ex4xceeded their ability to bind to �ex22, which is theroduct of a null allele (Figure 1D and data not shown).he partially penetrant pRB mutants, like their engi-eered counterparts, were temperature sensitive (Fig-res 1C and 1D), in keeping with an earlier study (Otter-on et al., 1999).We focused on RBP2 for several reasons. First, its

inding to pRB in the yeast two-hybrid assays was par-icularly robust compared to the other interactors wesolated (Figure 1 and data not shown). Second, RBP2as among the first pRB binding proteins identified,ontains two potential pRB binding sites (a canonicalXCXE motif and a second noncanonical non-T/E1Aomain), and binds to pRB with high affinity in vitro (De-

eo-Jones et al., 1991; Fattaey et al., 1993; Kim et al.,994). Third, RBP2 contains multiple motifs frequentlyound in transcriptional regulators, including Jumonji,RID, and PHD domains (as determined by ProScanrogram [srs@embl-ebi]; Figure 1E), and RBP2 or-hologs in Drosophila, S. pombe, and Ustilago maydisre believed to regulate chromatin structure (Fukumotot al., 2002; Gildea et al., 2000; Quadbeck-Seeger et al.,000). Few papers have been published on the pRB-BP2 interaction, however, because earlier attempts toetect native pRB-RBP2 complexes in cells were un-uccessful.

olocalization of pRB and RBP2mmunostaining of WI-38 human diploid fibroblastsith two rabbit polyclonal antibodies directed againstifferent regions of RBP2 showed that RBP2 is primar-

ly a nucleolar protein (Figure 1F and data not shown).imilar results were obtained with two different fixationrotocols and in multiple cell types (data not shown;ee also Figure S1 in the Supplemental Data availableith this article online). Immunostaining of these sameells with anti-pRB antibodies showed that the subcel-

ular distribution of pRB was sensitive to serum growthactors (Figure 1F). Under serum rich conditions, pRBas largely excluded from nucleoli. Under low serumonditions, which favor the accumulation of hypophos-horylated or “active” pRB and promote cell-cycle exit,RB concentrated in nucleoli in either a “pannucleolar”r “nucleolar rim” pattern. Nucleolar accumulation ofypophosphorylated pRB has been reported by others

Angus et al., 2003; Cavanaugh et al., 1995; Hannan etl., 2000; Kennedy et al., 2000; Rogalsky et al., 1993).Next, SAOS-2 RB−/− human osteosarcoma cells were

ransfected to produce pRB wild-type (WT), �663, orex22. Both WT and �663 displayed nucleolar staining,hich again exhibited either a pannucleolar pattern orucleolar rim pattern, while �ex22 showed diffusetaining throughout the cell (Figure S2). The percentagef wild-type pRB-producing cells showing the pan-ucleolar pattern relative to the rim pattern increased

Binding of pRB to RBP2 Promotes Differentiation625

Figure 1. Interaction of the Nucleolar Protein RBP2 with pRB in Yeast Two-Hybrid Assays

(A and D) Chromogenic GusA assays of yeast coproducing the cI DNA binding domain fused to the indicated large pocket (LP) pRB (A) andfull-length pRB (D) variants (listed on top), and indicated prey cDNAs fused to B42 transactivation domain (listed on side). Control preysindicated in italics. In (D), clones were replica plated and assayed after growth at 23°C, 30°C, or 37°C. (B and C) Immunoblot assays showingbait proteins in (A) and (D). (E) Schematic representation of RBP2. Two pRB binding domains are shown in red. (F) Confocal immunofluores-cence images of WI 38 fibroblasts grown in the presence of 0.1% or 10% fetal bovine serum (FBS). All cells grown at 0.1% had exited thecell cycle as shown by failure to incorporate BrdU (data not shown). ANA positive was used as a positive control for nucleolar staining.

when cells were stained with an antibody that preferen-tially recognizes unphosphorylated pRB (Figure S1).RBP2 staining in SAOS-2 cells was again nucleolar.Therefore, �663, which retains the ability to activatetranscription and promote differentiation, also retainsthe ability to colocalize with RBP2.

Detection of Endogenous pRB/RBP2 ComplexesIn parallel, we performed biochemical assays of simi-larly transfected SAOS-2 cells. Immunoblot analysis ofwhole cell extracts confirmed that WT, �663, and �ex22were produced at comparable levels (Figure 2B). Incontrast, immunoblot analysis of isolated nuclei showedthat nuclear retention of �663 was diminished relativeto WT and undetectable for �ex22 (Figures 2A and 2C).We also noted that release of pRB and RBP2 from iso-lated nuclei was salt sensitive. In particular, essentiallyno RBP2 was released from nuclei at salt concentra-tions less that 250 mM NaCl (Figure 2C). This mightexplain the inability to detect endogenous pRB/RBP2complexes under standard cell lysis conditions. Con-siderable amounts of pRB and RBP2 remained in nucleieven after extraction at 500 mM NaCl. WT and �663were detected in anti-RBP2 immunoprecipitates, but

not control immunoprecipitates, of the 250 mM NaClnuclear extracts (Figure 2D). The interaction of pRB andRBP2 was not mediated by DNA because coimmuno-precipitation of pRB with RBP2 was insensitive toethidium bromide and DNaseI (data not shown). Thedecrease in the amount of �663 bound to RBP2 relativeto WT mirrored the decrease in nuclear �663 comparedwith WT, suggesting that WT and �663 bind to RBP2with comparable affinity or that RBP2 contributes to thenuclear tethering of pRB. In support of the former, bind-ing of WT and �663 to RBP2 was comparable when theamount of pRB was normalized by pooling the cyto-solic, nucleosolic, and salt-extractable nuclear frac-tions prior to RBP2 immunoprecipitation (Figure 2D). Incontrast, �22 did not bind to RBP2 in these assays.To ask whether endogenous pRB binds to endogenousRBP2, U937 RB+/+ human leukemia cells were inducedto differentiate with TPA. Under these conditions, RBP2levels do not change and hypophosphorylated pRB ac-cumulates after 12–27 hr (Figure S3). pRB was againdetected in anti-RBP2 immunoprecipitates of the 250mM NaCl nuclear extracts (Figure 2E). This signal wasspecific because it was not detected in anti-RBP2 im-munoprecipitates of SAOS-2 RB−/− nuclear extracts or

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Figure 2. Salt Extraction of RBP2 and pRBfrom Nuclei

(A) Schema.(B–D) SAOS-2 (RB−/−) cells were transientlytransfected with plasmids encoding WT,�663, or �ex22 pRB. Aliquots of the cellswere lysed directly (B) or processed as in (A).(B and C) Immunoblot assays with the indi-cated antibodies. Triangle in (C) represents0.15, 0.25, and 0.50 M NaCl fractions. “R”indicates remaining fraction resistant to highsalt extraction. (D) 0.25 M NaCl nuclear ex-tracts (indicted by asterisks in [C]) or pooledcytosolic, nucleosolic, and salt-extractableproteins were immunoprecipitated with anti-RBP2 or control antibodies and immunoblot-ted with anti-pRB antibody. RBP2 is not seenin input due to dilution (data not shown).(E) 0.15, 0.25, and 0.50 M NaCl fractions, asindicated by the triangle, of differentiatedU937 RB+/+ leukemic cells were immuno-precipitated with anti-RBP2 antibody andimmunoblotted with anti-pRB antibody. Cy-tosolic (C) and nucleosolic (N) fractions wereloaded on the same gel for comparison tothe salt-extractable material.(F) U937 cells grown in the presence or ab-sence of the differentiation agent TPA andSAOS-2 RB−/− were analyzed as in (D).

undifferentiated U937 nuclear extracts and was not de- ictected in immunoprecipitates obtained with control IgG

(Figure 2F). pspRB can associate with chromatin and RBP2 is likely

to do so also based on its predicted subdomains and pRthe characteristics of RBP2 orthologs in lower organ-

isms. To study the behavior of pRB and RBP2 further,U937 cells were again induced to differentiate (Figure K

P3). At various time points thereafter isolated nuclei weretreated with limiting amounts of micrococcal nuclease T

Rto release “S1” chromatin, which is in an open or acces-sible configuration (Rose and Garrard, 1984). After low t

nspeed centrifugation the nuclei were then lysed inEGTA/EDTA to release “S2” chromatin, which is en- n

Rriched for repressed chromatin. After an additional cen-trifugation step the remaining pellet (P) was solubilized (

pusing SDS. P is enriched for actively transcribed genes(Reyes et al., 1997). Interestingly, changes in chromatin w

mcompartmentalization were observed for both pRB andRBP2 in a seemingly choreographed manner. In undif- o

eferentiated cells, RBP2 and pRB were present in bothS2 and P (Figure 3C). Twelve to twenty-seven hours af- a

fter the addition of TPA, there was a noticeable increasein the amount of pRB in S1. The pRB paralogs p107 p

uand p130, as well as the nucleolar proteins fibrillarinand C23, also entered S1 during this time period (Figure s

pS4). Twenty-seven hours post TPA, corresponding tothe peak appearance of pRB in S1, RBP2 left S2 and c

nentered S1. By comparison, another nuclear pRB bind-ing protein, RBP1, remained in the P compartment. At e

rthis time point pRB could be detected in anti-RBP2 im-munoprecipitates prepared from S1 but not S2 (Figure d

m3D). Similarly, WT and �663, but not �22, were detected

n anti-RBP2 immunoprecipitates of S1 (and not S2)hromatin prepared from SAOS-2 cells transfected toroduce these proteins (Figure S5). These observationsuggest that successful execution of a differentiationrogram is linked to physical association of pRB andBP2.

nockdown of RBP2 in RB−/− Cellsromotes Differentiationo probe RBP2 function, we identified four differentBP2 siRNAs that efficiently downregulate RBP2 pro-

ein levels (Figure 4A). Interestingly, all four siRNAs, butot various control siRNAs, induced a “senescent” phe-otype when introduced into SAOS-2 RB−/− and U-2OSB+/+ human osteosarcoma cells, which lack p16INK4A

Figures 4B and S6). It was shown before that wild-typeRB and �663, but not �22, induce a similar phenotypehen reintroduced into SAOS-2 cells and that theseorphological changes correlate with the reexpression

f markers indicative of osteogenic differentiation (Hindst al., 1992; Huang et al., 1988; Qin et al., 1992; Sellers etl., 1998; Templeton et al., 1991). SAOS-2 cells trans-ected with RBP2 siRNA, like SAOS-2 cells ectopicallyroducing wild-type pRB, stopped proliferating (Fig-res S7A–S7C). Cessation of cell growth with RBP2iRNA correlated with increased accumulation of p130,21, and p27, and diminished levels of pRB, p107, andyclin E (Figure S7D). The senescent cell phenotype isot simply a consequence of cell-cycle blockade, how-ver, because it is not observed in SAOS-2 cells ar-ested with a chimera containing the E2F1 DNA bindingomain fused to the pRB transrepression domain, a do-inant-negative DP1 protein, or p21 (Sellers et al.,


Figure 3. RBP2 and pRB Interact and Redistribute in Chromatin Fractions during Differentiation

(A) Schema.(B) Ethidium bromide-stained agarose gel after electrophoresis of chromatin isolated as in (A). Nuclease digestion was omitted where indi-cated in (B) and (C). Note that under limiting conditions micrococcal nuclease digests chromatin in hypersensitive regions, which have anopen configuration. S1 is composed of mononucleosomal-sized DNA and S2 is di-, tri- and higher oligonucleosomes.(C) Immunoblot analysis of S1, S2, and P chromatin fractions from U937 cells induced to differentiate with TPA for the indicated periods oftime. As cells differentiate, there is a shift of RBP2 from S2 to S1 fraction coincident with peak appearance of pRB in S1.(D) S1 and S2 fractions from the indicated time points in hours (C) were immunoprecipitated with anti-RBP2 or control antibodies andimmunoblotted with anti-pRB antibody.

1998; Wu et al., 1996). Likewise, these three agents donot phenocopy pRB’s ability to activate transcriptionand promote differentiation.

pRB can cooperate with transcription factors such asGRα and CBFA1 when reintroduced into RB−/− cells.Similarly, GRα function was enhanced in SAOS-2 cellstransfected with RBP2 siRNA, but not control siRNA(Figure 4C). In these assays, the effect of pRB andRBP2 siRNA were additive. Among several possibilities,this might reflect suboptimal suppression of RBP2 bypRB and RBP2 siRNA under these experimental condi-tions (see also Discussion). As expected, a null pRBmutant (567L) was seemingly inert in these assays.Comparable levels of GRα and comparable levels ofpRB were produced in these assays (Figure 4D). In con-trast to the effects observed here, RBP2 siRNA did notaffect transcriptional repression by pRB and did not no-ticeably affect the Renilla luciferase control reporterplasmid included in all of the transfection mixtures(data not shown). These studies suggest that RBP2plays a specific role in transcriptional activation, butnot repression, by pRB.

It was recently reported that WT, but not 661W, aug-

ments transcriptional activation by CBFA1 (Thomas etal., 2001). However, in titration experiments we notedthat both 661W and �663 cooperated with CBFA1 inreporter gene assays, in contrast to null pRB mutantssuch as �651 and 567L (Figure 4E). In these experi-ments and those that follow, CBFA1 levels were not al-tered in the presence of RBP2 siRNA or pRB (Figure 4Hand data not shown). RBP2 siRNA, but not controlsiRNA, also enhanced CBFA1 transcriptional activationin RB−/− cells and had effects that were additive tothose observed with wild-type pRB or �663 (Figures 4Fand 4G). Augmentation of GRα and CBFA1 function byRBP2 siRNA was due specifically to downregulation ofRBP2 because it was observed with multiple indepen-dent RBP2 siRNAs and was reversed when cells weretransfected with an RBP2 plasmid containing three mu-tations in the cognate siRNA recognition sequence(Figures 4I and 4J and data not shown).

The RBP2 siRNA results suggested that endogenousRBP2 represses CBFA1-dependent transcription. In-terestingly, CBFA1-dependent transcription was in-creased when RB−/− cells were transfected with RBP2variants that cannot bind to pRB (Figure 5A) due to mis-

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Figure 4. Knockdown of RBP2 with siRNA Potentiates GRα and CBFA1 Activity

(A) Immunoblot analysis of SAOS-2 cells transfected with the indicated siRNAs. (B) Phase-contrast microscopy of SAOS-2 cells 5 days aftertransfection with siRNA against luciferase (GL3), RBP2 (siRNAs 1 and 4), or scrambled RBP2 siRNA (4sc) showing induction of “flat” or“senescent” phenotype. (C) Normalized firefly luciferase values for SAOS-2 cells transfected with a GRα expression plasmid (50 ng), a GRE-driven firefly luciferase reporter plasmid (100 ng), and a renilla luciferase expression plasmid (5 ng). Where indicated, transfection mix alsocontained 20 ng of a pRB expression plasmid (wild-type or 567L) and/or 2 nM RBP2 siRNA (“2” or scrambled “2sc”). 24 hr after transfection,cells were split 1 to 2 and grown in the presence of dexamethasone. 48 hr later the cells were lysed. Normalized firefly luciferase values areexpressed as percentage of the values obtained in the absence of pRB or siRNA (“none”). Error bars indicate mean ± SEM. (D) Immunoblotanalysis of SAOS-2 cells transfected as in (C). ([E], [F,] [G], and [I]) Normalized firefly luciferase values for SAOS-2 cells transfected with aCBFA1 expression plasmid (50 ng), an OSE-driven firefly luciferase reporter plasmid (100 ng), and a renilla luciferase expression plasmid(5 ng). Where indicated, transfection mix also contained an expression plasmid for pRB (wild-type or mutant; 50 ng except where indicatedby triangle representing 10 and 200 ng plasmid DNA) and/or RBP2 siRNA (“1,” “2,” or scrambled versions thereof; 2 nM except whereindicated by triangle representing 0.2, 1, and 4 nM). In panel (I), an RBP2 expression plasmid containing an RBP2 cDNA with silent mutationsin the RBP2 oligo 1 site was included as indicated by the triangle (8 and 80 ng). Normalized firefly luciferase values are expressed aspercentage of the values obtained in the absence of pRB, RBP2, or siRNA (“none”) ([E], [F], and [G]) or with scrambled siRNA (I). Error barsindicate mean ± SEM.(H) Immunoblot analysis of SAOS-2 cells transfected as in (E) and (F).(J) Immunoblot analysis of SAOS-2 cells transfected as in (I).

sense mutation of the RBP2 LXCXE motif (LXCXA) or mvdeletion of the RBP2 C terminus, which contains a non-

canonical pRB binding motif (�C) (Figure 5B). This sug- apgests that these RBP2 variants are dominant-negative

utants. For example, they might bind to DNA and pre-ent the binding of endogenous RBP2 and an associ-ted corepressor (“R” in Figure 5E). pRB has been re-orted to bind to CBFA1 and to physically associate


Figure 5. pRB Displaces RBP2 from the OCPromoter

(A) Decreased binding of pRB to RBP2 mu-tants. Anti-RBP2 (top) and anti-Flag (bottom)immunoblot assays of SAOS-2 cells trans-fected to produce Flag-pRB (where indi-cated by “+”) and the indicated RBP2 vari-ants. C, cytosolic fraction; N, nucleosolicfraction; NaCl fr, 0.25 M NaCl fraction (lanes6–10) or pooled NaCl-extractable material(lanes 11–30); IP, anti-RBP2, anti-Flag, orcontrol IgG immunoprecipitates.(B) Cells were transfected with expressionplasmids that encode the indicated RBP2variants (that contain the silent oligo 1 sitemutations) and RBP2 siRNA (1 or 1sc).Normalized firefly luciferase values areexpressed as percentage of the valuesobtained in the absence RBP2 or siRNA(“none”). Error bars indicate mean ± SEM.(C and D) Autoradiograms of radiolabeledPCR products using primers for the indi-cated promoter regions and chromatin im-munoprecipitated with RBP2 antibodies,anti-Flag, or control IgG from SAOS-2 cellstransfected to produce CBFA1 and, whereindicated, Flag-tagged wild-type or mutantpRB.(E) Model for transcriptional repression byRBP2 and antagonism by pRB. For simplic-ity, RBP2 is shown binding directly to DNA.

with endogenous CBFA1-responsive genes such asOsteocalcin (OC). In chromatin immunoprecipitation(CHIP) experiments we detected endogenous RBP2bound to the OC promoter in SAOS-2 cells (Figure 5C)transfected to produce CBFA1. pRB, and not RBP2,was detected bound to this promoter when such cellswere transfected to produce WT (Figure 5C). �663, butnot �22, also displaced RBP2 from the OC promoter(Figure 5D). The decreased RBP2 signal observed inthe presence of WT or �663 is unlikely to reflect epitopemasking by pRB because these assays were performedwith a polyclonal antibody that immunoprecipitatespRB-RBP2 complexes (Figure 5A). These findings, to-gether with earlier work, suggest that pRB can bind tocertain promoters in conjunction with DNA bindingtranscription factors such as CBFA1 (“TF” in Figure 5E)while preventing the formation of a productive RBP2repressor complex (Figure 5E).

Restoration of pRB function in RB−/− cells promotesadipocyte and myogenic differentiation in vitro and pRBhas been clearly tied to myogenic differentiation in vivo(Classon and Harlow, 2002; de Bruin et al., 2003). RBP2siRNAs, but not control siRNA, likewise induced RB+/+

and RB−/− 3T3 cells to undergo adipocyte differentia-tion in vitro when grown in appropriate media (FigureS8). RBP2 siRNA also promoted adipocyte differentia-tion in p107−/−p130−/− 3T3 cells. Therefore, induction ofdifferentiation by RBP2 siRNA is not solely due to indi-rect effects of RBP2 levels on p107 and p130, whichare known to antagonize pRB’s ability to promote dif-ferentiation (Classon et al., 2000).

As expected, WT and �663, but not �ex22, induced

the formation of multinucleated, myosin heavy chain(MHC)-positive, myotubes when reintroduced into RB−/−

MEFs in the presence of a MyoD expression plasmid(Figure 6). Notably RBP2 siRNA, but not control siRNA,could effectively substitute for pRB in these assays. Incontrast, p21 and dominant-negative cdk2 blockedproliferation but did not promote myogenic differentia-tion (Figure 6C and data not shown). Collectively, thesestudies suggest that differentiation control by pRB islinked, at least partly, to inhibition of RBP2.

RBP2 Binds Directly to Genes Linked to HomeoticGene ExpressionWe used genome-scale location analysis to identifyother promoters bound by RBP2. First, we enrichedRBP2-DNA complexes using ChIP. Next, the boundchromatin fragments were hybridized to a DNA microar-ray containing the w19,000 proximal promoter regions(i.e., the region spanning 700 base pairs upstream and200 base pairs downstream of the transcription startsite) of w10,000 human genes. The results of this ex-periment revealed that a number of promoters werebound by RBP2 in U937 cells (E.V.B. and W.G.K., un-published data). However, when the genes were or-dered according to their confidence values (p values),BRD2 had the highest confidence value. A gene for asecond bromodomain-containing protein belonging tothe same subfamily, BRD8, was also among the 50 geneswith the highest confidence values (i.e., p value <0.0005). Specific binding of RBP2 to the BRD2 andBRD8 promoter regions was confirmed in conventionalCHIP assays of differentiating U937 cells (Figures 7A

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Figure 6. Knockdown of RBP2 Cooperates with MyoD to Promote RDifferentiation b(A) Representative photomicrographs of RB−/− mouse embryo fi- fbroblasts (MEFs) transfected with a plasmid encoding MyoD and, wwhere indicated, a plasmid encoding pRB (WT, �663, or �22) or

wRBP2 siRNA (“2” or scrambled “2sc”). “Mock” indicates MyoDHplasmid alone. After 3 days of growth in differentiation media, thegcells were fixed and stained with anti-MHC (myosin heavy chain)

antibody (red), which is a marker of myogenic differentiation, and acounterstained with the nuclear stain DAPI (blue).(B) Approximately 200 cells from each transfection (2 slides per

Dtransfection) were scored for the presence of multiple nuclei. Errorbars indicate mean ± SEM.

W(C) Representative photomicrographs of RB−/− mouse embryo fi-broblasts (MEFs) transfected with a plasmid encoding p21 or domi- cnant-negative cdk2 and processed as in (A). c

cE
wand 7B). We also detected pRB and p130 bound to
these promoters. Peak pRB binding was reproducibly ladetected w27 hr after TPA addition (Figure 7B), consis-

tent with the timing of pRB entry into S1 and associa- tption with RBP2 (see Figures 3C and 3D). Interestingly,

peak 130 binding occurred somewhat later (Figure 7B). ttRBP2 bound to the BRD2 and BRD8 promoters in RB−/−

cells indicating that recruitment of RBP2 to these re- iagions does not require pRB (Figure 7B).

To ask whether BRD2 and BRD8 are differentially ex- papressed during differentiation, we performed RT-PCR

assays on U937 cells at various time points after addingtTPA (Figure 7C). Consistent with previous observations

on myelopoiesis, c-Myc mRNA decreased during differ- (Lentiation (Duan and Horwitz, 2003), and p21 mRNA in-

creased during differentiation (Liu et al., 1996). Two EpRBP2 mRNA isoforms exist based on examination of

EST sequences. Using isoform specific primers we de- 1etected a transient induction of the shorter RBP2 mRNA,

encoding a protein with all three PHD fingers, during cdU937 cell differentiation. Notably, both BRD2 and BRD8

expression increased during differentiation, with BRD2 F

xpression peaking at 24–30 hr after the TPA additionFigure 7C). This, and the chromatin immunoprecipita-ion and biochemical data, suggested that pRB mightooperate with, rather than antagonize, RBP2 in thisontext to promote transcription. Consistent with this

dea, introduction of WT into RB−/− cells increasedRD2 and BRD8 mRNA levels (Figure 7D) whereasBP2 siRNA decreased BRD2 protein levels (Figure 7E).Both the BRD2 and BRD8 genomic regions present

n the microarray displayed promoter activity whensed to drive the expression of firefly luciferase in tran-ient transfection experiments in RB−/− SAOS-2 cellsFigure 8 and data not shown). This activity was en-anced by exogenous RBP2 and was attenuated byBP2 siRNA (Figure 8A and data not shown). pRB vari-nts capable of promoting differentiation enhancedRD2 and BRD8 promoter activity (Figure 8B). In con-

rast, p21 did not activate the BRD2 promoter (FigureC), despite inducing a G1/S block (data not shown),rguing that the effect of pRB in these assays is notimply due to a cell-cycle block. Notably, activation ofhe BRD2 promoter was blocked by simultaneous ad-inistration of RBP2 siRNA (Figure 8B and data not

hown), suggesting that maximal transcriptional activa-ion of this promoter requires the formation of a pRB/BP2 complex. Consistent with this idea, wild-type E7,ut not a pRB binding defective mutant, prevented pRB

rom activating the BRD2 promoter (Figure 8D). Like-ise, �663 cooperated with wild-type RBP2 but notith pRB binding defective RBP2 mutants (Figure 8E).ence pRB prevents RBP2 from repressing certainenes (Figure 5E) while augmenting RBP2’s ability toctivate others, such as BRD2 and BRD8 (Figure 8F).

iscussion

e screened for proteins that can bind to �663, whichan promote differentiation, but not to �ex22, whichannot. Only a small number of proteins satisfied theseriteria, including the known pRB interactors E7 and2F2. In addition, three of the remaining four proteinse identified are, like pRB, nuclear proteins (the subcel-

ular localization of the fourth protein is unknown) andre known, or can be inferred, to play roles in transcrip-ional regulation. Although it is possible that all of theseroteins interact with pRB under physiological condi-ions, we focused on RBP2 because it bound robustlyo pRB in the yeast two-hybrid assays and in vitro bind-ng assays. Notably, binding of pRB variants (naturalnd engineered) to RBP2 in yeast tracked with theirreviously characterized ability to promote differenti-tion.RBP2 and the unrelated nuclear protein RBP1 were

he first two cellular pRB binding proteins identifiedDefeo-Jones et al., 1991). Both contain canonicalXCXE pRB binding motifs, similar to those found in7, E1A, and SV40 Large T antigen, and bind avidly toRB in vitro (Defeo-Jones et al., 1991; Fattaey et al.,993; Kim et al., 1994). Interest in RBP2 waned, how-ver, due to inability to detect endogenous pRB/RBP2omplexes in cells. We provide several lines of evi-ence that RBP2 is a bona fide pRB-interacting protein.irst, we found that RBP2 and pRB colocalize in


Figure 7. Binding of RBP2 and pRB to BRD2and BRD8 Promoters Coincides with In-creased BRD2 and BRD8 Expression

(A) Schematics of BRD2 and BRD8 promoterregions. Thick lines show PCR fragmentsgenerated in ChIP experiments (B) relative toBRD2 and BRD8 transcription start sites (ar-rows). Thin lines (tick marks = 0.25 kb) showgenomic DNA fragments subcloned up-stream of firefly luciferase cDNA to makeBRD2 and BRD8 reporter plasmids (used inFigure 8).(B) Autoradiograms of radiolabeled PCRproducts using primers for the indicated pro-moter regions, including control Hoxa5 pro-moter, and chromatin immunoprecipitatedwith the indicated antibodies from U937cells treated with TPA for the indicated timeperiods. Also shown are PCR products usinginput DNA (no ChIP). Results shown are re-presentative of four independent experi-ments and did not use the enriched DNA thatwas subjected to LM-PCR and promotermicroarray analysis.

(C and D) Ethidium stained gel showing RT-PCR products obtained with primers for the indicated genes from U937 cells induced to differenti-ate with TPA for the indicated amount of time (C) or SAOS-2 cells (D). SAOS-2 cells were transfected to produce pRB where indicated.(E) Immunoblot analysis of SAOS-2 cells transfected with the indicated RBP2 siRNAs.

duction of wild-type pRB into RB tumor cells includ-chromatin extraction methods. Third, both RBP2 and

Figure 8. pRB Cooperates with RBP2 to Acti-vate BRD2 Promoter.

(A–E) Normalized firefly luciferase values ofSAOS-2 cells transfected with the BRD2 re-porter plasmid (50 ng) together with a renillaluciferase expression plasmid ([A], left panel,[B], [C], and [D]) or a LacZ expression plas-mid ([A], right panel, and [E]). Where indi-cated, transfection mix also contained anexpression plasmid for pRB (wild-type ormutant; 50 ng), RBP2 (wild-type or mutant;500 ng), E7 (wild-type or mutant; 5 or 50 ngas indicated by the triangle), p21 (wild-typeor mutant; 20 ng), and/or RBP2 siRNA (“2” orscrambled “2sc;” 1 nM). Error bars indicatemean ± SEM.(F) Model for cooperation between pRB andRBP2. For simplicity, RBP2 is shown bindingdirectly to DNA.

nucleoli under conditions that favor the accumulationof hypophosphorylated pRB, which is the active formof pRB. RBP2 appears to be constitutively nucleolar,although it is possible that a functionally important sub-population of RBP2 resides outside nucleoli. Second,we detected pRB in anti-RBP2 immunoprecipitatesprepared from growth-arrested cells using two different

pRB enter the same chromatin compartment, repre-senting transcriptionally active chromatin, during celldifferentation in a seemingly choreographed manner.Fourth, as described below, inhibition of RBP2 withsiRNA recapitulates certain phenotypes ascribed topRB and affects pRB function in intact cells.

Several phenotypes have been reported after reintro-−/−

Molecular Cell632

ing cell-cycle arrest, senescent morphologic changes, wcand differentiation. The ability of pRB to induce an

acute G1/S block appears to be due, at least partly, to ptits ability to form pRB/E2F complexes, which repress

transcription of E2F-responsive cell-cycle regulatory imgenes and can be recapitulated using certain domi-

nant-negative versions of E2F (Sellers et al., 1998; Wu ttet al., 1996). In contrast some pRB variants, such as

�663, are defective with respect to E2F binding and sAunable to repress transcription when recruited to DNA,

and yet can induce senescent changes and promote dtdifferentiation (Sellers et al., 1998). Remarkably, these

phenotypes were induced by RBP2 siRNA, suggesting fwthat inhibition of RBP2 by pRB contributes to its ability

to induce these phenotypes. RB−/− cells transfected mlwith RBP2 siRNA, like cells transfected to produce pRB

variants such as �663 (Sellers et al., 1998), do ulti- ewmately stop proliferating, despite the lack of pRB/E2F

transcriptional repressor complexes, probably due to ocinduction of the cdk inhibitors p21 and p27. Induction

of p21 and p27 by pRB has been described previously Bt(Alexander and Hinds, 2001; Mitra et al., 1999). None-

theless, senescent morphology and induction of differ- ipentiation are not simply consequences of cell-cycle ar-

rest because they are not seen in cells arrested with vvp21 or the dominant-inhibitory E2Fs described above.

The ability of pRB to induce differentiation correlates atwith its ability to activate (rather than repress) tran-

scription in concert with transcription factors such as BbMyoD, CBFA1, and GRα. Inhibition of RBP2 with siRNA

also enhanced the activity of these transcription factors wcin RB−/− cells. Hence, the ability of RBP2 siRNA or pRB

to enhance transcriptional activation function by cell sefate determining transcription factors correlates with

their ability to induce differentiation. Genetic evidence pcin fission yeast and corn fungus, as well as study of

Gal4-RBP2 fusion proteins in human cells (R. Côté andpP.B., unpublished data; E.V.B and W.G.K., unpublished

data), indicate that RBP2 can repress transcription. In eBthe simplest model the ability of pRB to activate tran-

scription and induce differentiation would reflect its Spability to prevent RBP2 from repressing transcription.

Our data so far suggest that pRB can displace RBP2 itfrom specific promoters. In addition, we found that pRB

binding defective RBP2 mutants activate promoters cathat are repressed by wild-type RBP2. This suggests

that pRB also competes with one or more corepressors tafor binding to the RBP2 C terminus. Conceivably, p107

and p130 are such corepressors as they antagonize atpRB’s ability to promote differentiation and their loss

leads to enhanced differentiation (Classon et al., 2000). auControl of differentiation by pRB and RBP2, however,

is not likely to reflect a simple linear pathway in which mspRB neutralizes RBP2. First, it is probable that control

of differentiation by pRB in vivo also involves RBP2-tindependent activities (such as control of E2F). Second,

we discovered another class of RBP2 targets, typified dtby BRD2 and BRD8, where pRB and RBP2 cooperate

to activate transcription rather than antagonizing one eRanother.

Could these other RBP2 targets be another link be- tctween pRB and differentiation? In Drosophila embryos,

cell fate determination is linked to the regulation of ho- scmeotic gene expression by trithorax gene products,

hich serve as transcriptional activators, and Poly-omb gene products, which serve as transcriptional re-ressors, through changes in local and global chroma-in structure. RBP2 contains a number of domains,ncluding PHD domains, ARID domain, and Jumonji do-

ains, found in proteins that regulate transcriptionhrough changes in chromatin structure, including tri-horax and Polycomb members, and our biochemicaltudies indicate that RBP2 associates with chromatin.putative RBP2 ortholog in Drosophila, little imaginal

iscs (lid), was identified in a genetic screen for tri-horax genes (Gildea et al., 2000) and the BRD2 ortholog,emale sterile homeotic (fsh), interacts synergisticallyith trithorax loci. In particular, fsh mutants display ho-eotic transformations that are similar to those seen in

id mutants (Gans et al., 1980). Thus, there is geneticvidence that RBP2 and BRD2 are in the same path-ay. We found that BRD2 and BRD8 are direct targetsf, and are activated by, RBP2 and that pRB mutantsapable of promoting differentiation activate BRD2 andRD8 in an RBP2-dependent manner. Collectively,

hese findings place RBP2, a trithorax gene, and BRD2n a linear pathway, and suggest that pRB/RBP2 com-lexes promote differentiation, at least in part, by acti-ating the expression of homeotic genes through acti-ation of genes such as BRD2. The appearance of pRBnd RBP2 in open chromatin during cellular differentia-ion, coincident with increased expression of BRD2 andRD8, is consistent with this view. Although pRB haseen best studied as a transcriptional repressor, earlierork suggested that pRB can associate with SWI/SNFomplex proteins, which can positively regulate tran-cription (Harbour and Dean, 2000), and there are manyxamples of transcriptional regulators that can bothositively and negatively regulate transcription in aontext-dependent manner.The continued expression of genes responsible for a

articular cell fate in postmitotic cells requires that theyscape heterochromatinization. For example, the yeastRD2 ortholog Bdf1 competes with the deacetylaseir2 for binding to acetylated histone H4 and therebylays a role in euchromatin maintenance and antisilenc-

ng (Ladurner et al., 2003). Mouse BRD2, in contrast tohe majority of transcription factors, remains bound tohromatin during mitosis and may also play a role inntisilencing (Kanno et al., 2004). BRD2 and BRD8 con-ain bromodomains, which bind to acetylated histonesnd protect them histone deacetylases. Histone de-cetylation is linked to chromatin condensation andranscriptional silencing. Hence, proteins such as BRD2nd BRD8 might help insure that genes linked to partic-lar cell differentiation programs remain active in post-itotic cells by maintaining them in a euchromatic

tate.But if pRB/RBP2 complexes promote differentiation

hrough activation of homeotic gene expression, whyoes downregulation of RBP2 in cells promote differen-

iation and activate transcription in assays that mirrornhanced differentiation? We hypothesize that freeBP2 serves to inhibit differentiation by repressing

ranscription and participating in a “differentiationheckpoint.” Negation of this checkpoint is apparentlyufficient for continuously cycling cells to exit the cellycle and at least partially differentiate. It is possible,


for example, that transcriptional activation of homeoticgenes by pRB/RBP2 complexes is not required in thiscontext due to the absence of other activities that di-rectly or indirectly promote heterochromatin formation.In our model the binding of pRB to RBP2 in responseto differentiation signals would convert RBP2 from aninhibitor of differentiation to a stimulator of differentia-tion, by converting it from a transcriptional repressorto a transcriptional activator. This is supported by ourdiscovery of genes that are repressed by RBP2 andgenes that are activated by RBP2, as well as by ourfinding that RBP2 shifts from inactive S2 to active S1chromatin fractions during differentiation. Conceivably,there are direct RBP2 target genes linked to differentia-tion for which free RBP2 and pRB/RBP2 complexeshave antagonistic effects (for example, repression vs.activation), although this remains to be tested. Thereare clear parallels between these ideas and the currentview of the interaction between pRB and E2F. Free E2Fserves as a transcriptional activator and promotes cellproliferation. Binding to pRB converts E2F from an acti-vator to a repressor and thereby promotes cell-cycleexit. Differentiation is usually coupled to cell-cycle exit.This linkage might reflect the coordinated actions ofpRB on RBP2 and E2F.

Experimental Procedures

Dual Bait Yeast Two-Hybrid ScreenDual bait yeast two-hybrid screening was performed as described(Serebriiskii et al., 1999; Serebriiskii et al., 2002). Detailed descrip-tions of the two-hybrid plasmids and protocols used in thesestudies are available upon a request. The cDNA libraries used were:(1) human 18- to 24-week-old fetal liver cDNA library in pB42AD(B42 activation domain, Clontech), (2) human 22-week-old fetalbrain cDNA library in pJG4-5 (B42 activation domain, OriGeneTech.), and (3) human 37-week-old fetal brain cDNA library inpPC86 (GAL4 activation domain, Gibco BRL). Positive clones wereisolated and authenticated using pRB baits with swapped heterolo-gous DNA binding domains.

Miscellaneous PlasmidsThe pCMV-RB plasmids (Qin et al., 1992), pSG5L-HA-RB plasmids(Sellers et al., 1998), pRS-hGRα (Giguere et al., 1986), pRC/CMV-p21 (Adams et al., 1996), pcDNA3-HA-CBFA1 and 6OSE2-luc(Thomas et al., 2001) were described previously. phRL SV40 Renillaluciferase plasmid was purchased from Promega. pCMV-16E7 andpCMV-�DLYC16E7 were gifts from Karl Munger and the Cdk2-dnplasmid was a gift of Sander van den Heuvel. pMTV-GRE-Luc wasa gift from William Chin. pcDNA3-HA-RBP2 was mutated with aQuickChange site-directed mutagenesis kit according to the manu-facturer’s instructions (Stratagene) and the following primers(sense strand; mutations in bold): siRNA number 1 binding site mu-tant (core siRNA binding site underlined); 5#-CGAGAAGCCTTTGGATTTGAACAGGCAGTTCGAGAGTATACAC-3# and LXCXA; TGAACCCAATCTTTTTTGTGATGCAGAGATTCCCATCAAATCC-3#. TheRBP2 C-terminal truncation mutant was made by digestion ofpcDNA3-HA-RBP2 with Xba I and Bgl II (partial digest) followed byfill-in and recircularization. The proximal promoter regions for BRD2and BRD8 were PCR amplified using U937 genomic DNA and prim-ers 5#-GGGGTACCAGAGGCTTTGAGCCATGGAG-3# and 5#-GTGGATCCAGCTTTCCGAACGTTCCTG-3# (for BRD2) or 5#-GGGGTACCTGGCCTAGCAGGAAACCTAC-3# and 5#-GTGAGATCTACCTGCTCAGATACTCACTGCC-3# (for BRD8), which introduced 5# Kpn I and 3#BamH I (or 3# Bgl II for BRD8) restriction sites. The PCR productswere restricted and inserted into pGL3-Basic (Promega).

siRNAssiRNA transfections were performed in 6-well plates with 2.5 µl ofLipofectamine 2000 (Invitrogen) according to the manufacturer’s in-

structions. siRNA duplexes (Dharmacon Research, Inc.) were(sense sequences):

GL3 5#-CUUACGCUGAGUACUUCGATT-3# (Elbashir et al., 2001);RBP2 No. 1 5#-GCUGUACGAGAGUAUACACTT-3#; 1sc 5#-CGAUGUAGCGUGAACAUCATT-3#; RBP2 No. 2 5#-GCCAAGAACAUUCCAGCCUTT-3#; 2sc GCCUUCACAAGACUACCAGTT-3#; RBP2 No.3 5#-CUUCUGUACUGCUGACUGGTT-3#; RBP2 No. 4 5#-CUUGAGGCAAUGACCAGAGTT-3#; 4sc 5#GCGACUAGGUCAUAGAGACTT-3#. RBP2 siRNAs 2 and 3 target sequences that are identicalin human and mouse.

Cell Culture and DifferentiationEarly passage wild-type and RB−/− mouse embryo fibroblasts(MEFs) were a gift of James DeCaprio. Wild-type, Rb−/− and p107−/−

p130−/− 3T3 cells were a gift of Marie Classon and Ed Harlow.SAOS-2 human osteosarcoma cells, WI-38 human diploid fibro-blasts, and MEFs were grown in DMEM (CellGro) supplementedwith 10% fetal bovine serum (FBS). 3T3 cells were grown in DMEMwith 10% calf serum. U-2OS human osteosarcoma cells weregrown in DMEM with 10% fetalclone (Hyclone). U937 human leuke-mia cells were grown in RPMI 1640 (CellGro) with 10% fetalclone.Differentiation was induced using established protocols (Classonet al., 2000; Miyake et al., 2000; Sellers et al., 1998).

AntibodiesAnti-RBP2 antibodies 2410 and 2411 are affinity-purified rabbitpolyclonal antibodies directed against glutatione S-transferase(GST)-RBP2 (1416-1447) (corresponding to the amino acid posi-tions from 1416 to 1447 of the RBP2 sequence Acc. No. NP_005047[Fattaey et al., 1993]) and the affinity-purified rabbit polyclonal anti-body 1416 was generated against peptide 1416–1434 (designed byEric W. McIntush [Bethyl]). Rabbit IgG (Sigma) was used as respec-tive control. The anti-RBP2 polyclonal antisera 2471 were raisedin rabbits against glutatione S-transferase (GST)-RBP2(1655–1698)and used without further purification. Preimmune serum from thesame rabbit was used as a control. Mouse monoclonal anti-pRBantibodies were from BD Biosciences (G3-245) and Santa Cruz (sc-102). Anti-nucleolar (ANA positive) antibodies, mouse monoclonalanti-skeletal myosin (clone MY-32), and mouse monoclonal (M2)and rabbit polyclonal anti-Flag antibodies were from Sigma. Mousemonoclonal antibody anti-RBP1 (Y11) was a gift of Jim DeCaprio,anti-HA was from Covance (HA11). Anti-ORC2 antibody was a giftfrom Anindya Dutta. Goat polyclonal antibody to BRD2 (ab3718)was from Abcam. Rabbit polyclonal anti-Lamin A (Cell Signaling),anti-RB (sc-50), anti-p107 (sc-318), anti-p130 (sc-317), and anti-GRα (sc-8992) were from Santa Cruz.

Chromatin ExtractionHypotonic cell lysis was performed as described in (Hancock,1974) and salt extraction was as in (Kreitz et al., 2001). Approxi-mately 5 × 106 cells were washed briefly with 5 ml of ice-cold BufferA (10 mM Hepes/K+ [pH 7.5], 20 mM KCl, 0.25 mM EDTA) andswollen/disrupted in 1 ml of Buffer A. Nuclei were pelleted by lowspeed centrifugation and lysed in 500 �l of 10 mM Hepes/K+ (pH7.5), 70 mM NaCl, 20 mM KCl, 5 mM MgCl2, 2 mM CaCl2, 0.3%–0.5% NP-40. Chromatin bodies were isolated by centrifugationthrough a sucrose cushion and sequentially extracted in 100 �l of20 mM Hepes/K+ (pH 7.5), 0.5 mM MgCl2, 0.3 M sucrose containing0.15 M, 0.25 M, or 0.50 M NaCl. The salt-resistant pellet was resus-pended in 100 �l of 50 mM Tris-HCl (pH 8.0), 10 mM EDTA, 1%SDS (“R” fraction), which was then diluted 1:10 in PBS LSB (PBS,25 mM MgCl2, 10 mM EDTA, 10% glycerol, 1 mM DTT) prior toanalysis.

The separation of S1, S2, and P chromatin fractions was per-formed as described (Kreitz et al., 2001; Rose and Garrard, 1984).Nuclei isolated as above were incubated for 5 min at 16°C in thepresence of micrococcal nuclease (0.03–0.1 units) (Sigma) to obtainthe S1 and S2 fractions, which were diluted 1:4 in PBS LSB priorto analysis. After collection of the S2 fraction, the pellet was solubi-lized by vortexing in 100 �l of 50 mM Tris-HCl (pH 8.0), 10 mMEDTA, 1% SDS. The supernatant was saved as the P fraction andwas diluted 1:10 in PBS LSB.

Molecular Cell634

Luciferase Assays RRFor transcriptional assays, cells were transfected in 24-well plates.

24 hr later, the cells were released by trypsinization, aliquoted to APtwo fresh wells, and placed in the indicated media. 72 hr after

transfection, cells were lysed and luciferase activity was measuredusing Dual-Luciferase Reporter Assay System (Promega). Lucifer- Rase activity of the BRD2 and BRD8 reporters was normalized toeither pRL-CMV Renilla luciferase control reporter or pCMV-β-Gal Awith similar results. β-galactosidase assays were performed using KAssay System from Promega. m

i

Chromatin Immunoprecipitation AChromatin immunoprecipitation (ChIP) and data analysis were per- rformed as described previously (Odom et al., 2004). Briefly, w2.5 × 3107 cells were fixed with 1% final concentration formaldehyde for A10–20 min at room temperature, harvested and rinsed with PBS. sThe cell pellet was sonicated, and DNA fragments were immuno- nprecipitated with 5 �g of antibody consisting of rabbit IgG, poly- Mclonal antibodies against RBP2 (2411 or 1416), Flag, p130, or a

Bpool of anti-RB monoclonals (G3-245, sc-50 and sc-102) preboundBto Protein G Dynabeads (Dynal Biotech). After reversal of cross-elinking, the enriched DNA was amplified using ligation-mediatedmPCR (LM-PCR), and fluorescently labeled. 1:105-1:106 of the inputmDNA sample was also subjected to LM-PCR and labeled with aCdifferent fluorophore. The ChIP-enriched and unenriched pools ofGlabeled DNA were combined and hybridized to a DNA microarrays(Odom et al., 2004). A whole-chip error model was used to calculate1confidence values (p values) for each spot on the microarray, which

indicated the significance of enrichment for each DNA species on Cthe array. Alternatively, DNA recovered by CHIP was PCR amplified Lwith gene-specific primers for BRD2 5#-TGAGGCAGGAGGTCAG tCAC-3# and 5#-CTCGCCTCTCCATACGAGTTC-3#; for BRD8 5#-CTT CCAGCAGCCAACTCCTG-3# and 5#-CGAAGTCTCCAACCCTGAGG- p3#; for OC (Thomas et al., 2001), or for HOXA5 5#-GGAAATGACTGGGA

CCATGTACTTG-3# and 5#-TCCACCCAACTCCCCTATTAG-3#, labeledpwith α-33P dCTP, separated by 7.5% PAGE and detected by autora-Ndiography.dW

RT-PCR iTotal RNA was isolated by NucleoSpin RNA purification kit (BD Bio- Rsciences). RT-PCR was performed as a two-step procedure using

DRETROscript kit (Ambion). cDNA was primed with Oligo(dT), fol-Glowed by semiquantitative PCR with primers based on GenBankcgenomic sequences: RBP2 5#-GCTGCTGCAGCCAAAGTTG-3# andp5#-AGCATCTGCTAACTGGTCTC-3#; BRD2 5#-TGTCAGCGGACAGDCTCAATTCTAC-3# and 5#-ACTGCAGAGCCAGCTCTCCTAGAG-3#;pBRD8 5#-CAAACATCCGAGTCTGGGATCAG-3# and 5#-AGGCAATT5GTGCTACTCCAACTCTC-3#; and other primers as referenced in

(Duan and Horwitz, 2003). Amplification of constitutively expressed ES15 mRNA (Ambion) was used as a control. a

R4

Supplemental Data Fc

Supplemental Data include eight figures and can be found with this Darticle online at http://www.molecule.org/cgi/content/full/18/6/623/ RDC1/. F

BcAcknowledgments

FWe thank Ilya Serebriiskii and Erica Golemis for the Dual Bait Sys- dtem; Stine Kraeft, Gaelle Even and Brian Chen for help with confo- 1cal microscopy; Jim DeCaprio, William Sellers, Marie Classon, Nick

FDyson, Fred Kaye, Karl Munger, Julian Sage, Phil Hinds, and Ga-

abriel Gutierrez for valuable reagents; Isabella Mueller and Bruce

sSpiegelman for the adipocyte differentiation protocol; and Myles

aBrown and David Livingston for critical reading of the manuscript.

GSpecial gratitudes to Qin Yan for help with generation of RBP2 anti-abodies. This work was supported by Concept Award DAMD 17-01-u1-0512 from the Department of Defense Breast Cancer ResearchGProgram of the CDMRP (E.V.B.) and an NIH RO1 (W.G.K.). W.G.K is

a Howard Hughes Medical Institute investigator. G

eceived: September 24, 2004evised: February 2, 2005ccepted: May 16, 2005ublished: June 9, 2005

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Binding of pRB to the PHD Protein RBP2 Promotes Cellular Differentiation