RBP1 Recruits Both Histone Deacetylase-Dependent and

MOLECULAR AND CELLULAR BIOLOGY,0270-7306/99/$04.0010

Oct. 1999, p. 6632–6641 Vol. 19, No. 10

Copyright © 1999, American Society for Microbiology. All Rights Reserved.

RBP1 Recruits Both Histone Deacetylase-Dependent and-Independent Repression Activities to Retinoblastoma

Family ProteinsALBERT LAI,1 JOSEPH M. LEE,1 WEN-MING YANG,2 JAMES A. DECAPRIO,3 WILLIAM G. KAELIN, JR.,3,4

EDWARD SETO,2 AND PHILIP E. BRANTON1,5*

Departments of Biochemistry1 and Oncology,5 McGill University, Montreal, Quebec, Canada H3G 1Y6;H. Lee Moffitt Cancer Center and Research Institute, Molecular Oncology Program, University of

South Florida, Tampa, Florida 336122; and Dana-Farber Cancer Institute and HarvardMedical School3 and Howard Hughes Medical Institute,4 Boston, Massachusetts 02115

Received 3 May 1999/Returned for modification 1 June 1999/Accepted 18 June 1999

Retinoblastoma (RB) tumor suppressor family proteins block cell proliferation in part by repressing certainE2F-specific promoters. Both histone deacetylase (HDAC)-dependent and -independent repression activitiesare associated with the RB “pocket.” The mechanism by which these two repression functions occupy thepocket is unknown. A known RB-binding protein, RBP1, was previously found by our group to be an activecorepressor which, if overexpressed, represses E2F-mediated transcription via its association with the pocket.We show here that RBP1 contains two repression domains, one of which binds all three known HDACs andrepresses them in an HDAC-dependent manner while the other domain functions independently of the HDACs.Thus, RB family members repress transcription by recruiting RBP1 to the pocket. RBP1, in turn, serves as abridging molecule to recruit HDACs and, in addition, provides a second HDAC-independent repressionfunction.

The retinoblastoma (RB) tumor suppressor protein pRBplays a critical role in the control of cell proliferation. Inaddition, loss of the Rb gene is known to contribute to theestablishment of a variety of cancers. pRB and the relatedproteins p130 and p107 control cell cycle progression throughinteractions with the E2F family of transcription factors (re-viewed in reference 11). Such interactions regulate transcrip-tion by mechanisms requiring the “pocket” domain of RB

family members (1, 3, 5, 18, 32, 39, 40, 46). One role of thepocket is to interact with and mask the transcriptional activa-tion domain of E2F; however, this mechanism does not explainthe repression of E2F-dependent promoters in which E2Fbinding sites act as negative elements that, if deleted, result inrelief from repression (7, 11, 20, 28). The pocket can interactsimultaneously with both E2F and certain cellular and viralproteins that bind by utilizing a conserved Leu-X-Cys-X-Glu

FIG. 1. Endogenous interactions of pRB (A) and p107 (B) with HDACs. (A) Immunoprecipitations were done in lysates from either H1299 or 293T cells. Plates(100 mm) of both cell types were lysed in low-stringency buffer. Cell extracts were incubated with 1 mg of the indicated antisera and 30 ml of a 50% slurry of proteinG-Sepharose for at least 12 h. Immunoprecipitated complexes were washed six times with the 150 mM low-stringency buffer and eluted by boiling with 23 sample buffer.Eluted proteins were subjected to SDS-PAGE with a 6% polyacrylamide gel. The presence of pRB was detected with monoclonal antibody (G3-245) against pRB(PharMingen). (B) Binding studies similar to those described for panel A were done, but coimmunoprecipitation of p107 was detected with a rabbit polyclonal antibody(C-18) against p107 (a-107) (Santa Cruz).

* Corresponding author. Mailing address: Departments of Biochem-istry and Oncology, McGill University, McIntyre Medical Building,3655 Drummond St., Montreal, Quebec, Canada H3G 1Y6. Phone:(514) 398-7268. Fax: (514) 398-7384. E-mail: [email protected].

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(LXCXE) sequence (10, 14, 25, 38, 41). Many of these cellularLXCXE-containing proteins have been shown to be transcrip-tional repressors, including RBP1 (24), HBP1 (37), RIZ (4),RBP2 (unpublished data), and histone deacetylase 1 (HDAC1)and HDAC2 (19, 42, 43). Recently, it has been proposed thatthe pockets of the pRB-E2F, p130-E2F, and p107-E2F com-plexes actively repress E2F-dependent transcription by twomechanisms, one involving the recruitment of HDAC1 (andpossibly HDAC2) (2, 16, 26, 27) and the other independent ofHDACs (26, 30). HDACs are multiprotein complexes in whichthe human homologues of the yeast RPD3 protein, HDAC1,HDAC2, and HDAC3, constitute catalytic subunits (8, 13, 35,42, 43). It is widely believed that histone deacetylation con-

denses chromatin structure, thereby shutting down transcrip-tion (reviewed in references 17, 19, 31, and 34). The recruit-ment of HDAC1 and HDAC2 to the pocket may thereforeaccount for active repression by RB family members throughdeacetylation at the promoter level, and in fact, enzymaticallyactive forms of HDACs have been detected both in vitro andin vivo in association with both the pocket and E2F (2, 26, 27).Magnaghi-Jaulin et al. (27) and Ferreira et al. (16) have shownthat a region within HDAC1 containing an IXCXE motif isimportant for interactions with RB family members because itsdeletion resulted in a reduction in binding. Thus, it was pro-posed that a direct physical interaction between the degener-ate IXCXE motif of HDAC1 and the pocket of RB familymembers occurs in a manner analogous to interactions withLXCXE-containing viral transforming proteins. One problemwith this interpretation concerns the fact that in addition to theIXCXE motif, the deletion mutants used in these studies elim-inated additional HDAC1-coding sequences. Second, the in

FIG. 2. In vivo interaction of the small pocket of pRB with HDACs. (A)cDNAs encoding the Gal4DBD fused with the pocket of pRB were overex-pressed with those encoding Flag-tagged HDAC1, HDAC2, or HDAC3 inH1299 cells. RK5C1 (anti-Gal4DBD) antibody (Santa Cruz) was used to immu-noprecipitate Gal4-tagged overexpressed proteins, and the coimmunoprecipita-tion of HDACs was detected by Western blotting with anti-Flag M2 monoclonalantibody (Sigma). WC, whole-cell extracts; IP, anti-Gal4-immunoprecipitatedproteins. Binding studies similar to those described for panel A were performedwith 293T (B) and 293 (C) cells. (D) A modified binding experiment similar tothat described for panel A was done with H1299 cells by incubating cell extractswith increasing amounts (0 to 20 mg) of E1A protein (the first exon of the genecontaining the LXCXE motif) fused to GST (GST-E1A) and synthesized in vitroin bacteria (6). The positions of migration of Flag-tagged HDAC1 (open arrow),HDAC2 (closed arrow), and HDAC3 (arrowhead) are indicated.

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vitro binding assays used by these groups utilized HDAC1translated in vitro with reticulocyte lysates. Thus, it is possiblethat other factors might function in the interaction, perhapseven serving as linkers for HDAC1. We present evidenceherein that a known RB pocket-binding protein, RBP1, linksall three HDACs to RB proteins and, in addition, provides asecond HDAC-independent repression function.

MATERIALS AND METHODS

Cell culture and transfection. Human lung carcinoma H1299 cells were grownin Dulbecco’s modified Eagle medium containing 10% fetal calf serum. 293 cells,293T cells (a variant of 293 cells that expresses simian virus 40 [SV40] large Tantigen), and Chinese hamster ovary (CHO) cells (ATCC CCL-61) were grownin a-minimal essential medium supplemented with 10% fetal calf serum. Trans-fections for binding studies were carried out with Lipofectamine reagents (NENLife Science), and transfections for chloramphenicol acetyltransferase (CAT)assays were done by the calcium phosphate precipitation method with the pGEMplasmid as the carrier DNA, as described previously (24).

Antibodies. A rabbit polyclonal antibody raised against HDAC2 was describedpreviously (23). Antibodies raised by E. Seto were prepared against peptidescorresponding to the unique carboxy termini of HDAC1 (EEKPEAKGVKEEVKLA) and HDAC3 (NEFYDGDHDNDKESDVEI), which had beencoupled to keyhole limpet hemocyanin and injected separately into New ZealandWhite rabbits. The resulting antibodies were immunoaffinity purified on peptidecolumns. LY11 and LY32 monoclonal antibodies raised by J. A. DeCaprio andW. G. Kaelin specifically against RBP1 were described previously (24). Anti-pRBantibody G3-245 was purchased from Pharmingen. Antibodies against p130(C-20), p107 (C-18), and the Gal4 DNA-binding domain (Gal4DBD) (RK5C1)were purchased from Santa Cruz. Antihemagglutinin (anti-HA) antibody HA.11was purchased from Babco, and anti-Flag antibody M2 was from Sigma.

Plasmids. A mammalian expression plasmid expressing the small pocket ofpRB as a fusion product with Gal4DBD, termed Gal4-pRB(pocket), was pro-vided by Tony Kouzarides (2). Flag-HDAC1, -HDAC2, and -HDAC3 and Gal4-HDAC1, -HDAC2, and -HDAC3 constructs have been described elsewhere (42,43). Gal4-VP16 was provided by Arnie Berk (44). Constructs expressing Gal4-RBP1, RBP1-HA, Gal4-RBP1dl-LXCXE, and RBP1dl-LXCXE-HA mutantswhich lack the LXCXE pocket-binding motif were described previously (24). TheG5TKCAT reporter construct has been described elsewhere (36, 45). TheG5MLPCAT reporter was provided by Doug Dean (26). The E2F1-luc reporterconstruct has been described elsewhere (24). Mutants of Gal4DBD-RBP1 weregenerated as follows. Gal4-dlR1 mutants were generated with two specific prim-ers close to the 39 end of the region corresponding to R1 and the end of theRBP1-coding sequence. PCR was done to generate fragments with unique re-striction sites (Bsp1107I and HindIII) which were subcloned into digested RBP1constructs, in which all the RBP1-coding sequences corresponding to betweenthe beginning of R1 and the end of the protein had been removed with the samerestriction enzymes. All carboxy-terminal-deletion mutants of Gal4-dlR1 weregenerated from fragments produced by restriction enzyme digestion of the Gal4-dlR1 plasmid DNA, and then these were subcloned into a modified pcDNA3(Invitrogen) construct containing stop codons inserted 39 of the multicloningcassette. Gal4-R2 truncation mutants were constructed by subcloning the restric-tion enzyme-digested fragments of the Gal4-RBP1-coding region that corre-spond to residues 1311 to 1404, 1314 to 1404, or 1263 to 1404 into pSG424.Mammalian expression plasmids encoding the pocket of pRB and the inactivepRB pocket mutant mRB(C706F) were described previously (22).

CAT, b-galactosidase, and luciferase assays. Transcriptional analyses involv-ing CAT, b-galactosidase, and luciferase assays were performed as describedpreviously (24, 36).

Binding assays. One microgram each of cDNAs encoding Gal4-pRB(pocket),RBP1, or RBP1 mutants was introduced with Lipofectamine (NEN Life Science)along with 1 mg each of those encoding Flag-tagged HDAC1, HDAC2, orHDAC3 into H1299 or 293T cells. In some experiments, 1 mg each of cDNAsencoding HA-tagged RBP1 or RBP1 mutants was introduced with Lipo-fectamine along with 1 mg each of those encoding Gal4DBD-fused HDAC1,HDAC2, or HDAC3 into H1299 or 293T cells. Cells were harvested 40 hposttransfection and lysed with low-stringency buffer (24). Cell extracts werediluted to 150 mM KCl in a 1-ml volume and precleared with protein G-Sepharose (Pharmacia) for 2 h. Precleared extracts were incubated with 1 mg of

Gal4DBD antibody RK5C1 (Santa Cruz) and 30 ml of a 50% slurry of proteinG-Sepharose for at least 12 h. Immunoprecipitated Gal4-tagged protein com-plexes were washed six times with lysis buffer and eluted by boiling in 23 samplebuffer. Eluted proteins were subjected to sodium dodecyl sulfate-polyacrylamidegel electrophoresis (SDS-PAGE) with a 10% polyacrylamide gel, and proteinswere then transferred to polyvinylidene difluoride membranes (Millipore) andwere probed with either anti-HA (HA.11 [Babco]) or anti-Flag (M2; Sigma)monoclonal antibody and then with horseradish peroxidase-conjugated goat anti-mouse (l light chain-specific) secondary antibody (PharMingen). Binding wasdetected by Enhanced Luminol Reagent (NEN Life Science).

RESULTSEndogenous interactions between RB family members and

HDACs. Coimmunoprecipitation experiments were conductedto determine the ability of pRB to interact with various HDACenzymes in vivo. Rabbit polyclonal antibodies that specificallyrecognize HDAC2 (23) or HDAC1 and HDAC3 (see Materi-als and Methods) were used under low-stringency conditions toimmunoprecipitate endogenous HDAC species from extractsof H1299 cells. In each case, significant amounts of corre-sponding HDAC proteins were immunoprecipitated (data notshown). The presence of pRB in these complexes was deter-mined by Western blot analysis with the G3-245 monoclonalantibody, which recognizes both hyperphosphorylated and hy-pophosphorylated forms of pRB. Figure 1A shows that thehypophosphorylated form of pRB coprecipitated with HDAC1,as reported by others (26). Interestingly, significant amounts ofhypophosphorylated pRB were also detected in associationwith HDAC2 and HDAC3. These interactions are specific, aspRB was not detected in immunoprecipitates prepared fromthe same cell extracts with antibody to CREB-binding protein,a histone acetyltransferase (data not shown). These resultssuggested that the active form of pRB is able to recruit allthree forms of the HDAC.

Previous studies showed that HDAC1 also interacts with thepRB-related family members p107 and p130 (16, 21, 33). Thus,immunoprecipitates from H1299 cells containing variousHDACs were immunoblotted with C-18 polyclonal antibody,which recognizes up to three different phosphorylated forms ofp107 in various cell lines. Figure 1B shows that p107, its un-derphosphorylated forms in particular, also coprecipitatedwith all the endogenous HDAC species, especially HDAC1.We attempted a similar experiment with p130, but as H1299cells die upon serum starvation and p130 is expressed largely atgrowth arrest, we were unable to detect sufficient quantities.Significant amounts of p130 were present in asynchronizedH1299 cells, but these p130 species were mostly hyperphos-phorylated and did not appear to associate with HDACs atsignificant levels (data not shown).

Adenovirus E1A protein and SV40 large T antigen associatewith the pRB pocket via LXCXE-binding motifs (12) and havebeen found to disrupt interactions between pRB and HDAC1 (2,26, 27). We therefore conducted a parallel series of binding stud-ies in 293T cells, which express high levels of both adenovirus type5 (Ad5) E1A proteins and SV40 large T antigen. Figure 1A showsthat no interactions were apparent between pRB and any of theHDAC enzymes, including HDAC3. Figure 1B shows that a sim-ilar effect was apparent with p107. Thus, in both cases, binding of

FIG. 3. In vivo interaction of RBP1 with HDACs. Binding studies similar to those described for Fig. 2A and B were performed with H1299 cells (A) and 293T cells(B) by using Gal4-RBP1 instead of Gal4-pRB. (C) Binding studies similar to those described for panel B were performed in 293T cells transfected with the RBP1-HAconstruct (24) and the Gal4-HDAC1, Gal4-HDAC2, and Gal4-HDAC3 constructs (42, 43). Molecular mass was determined with Rainbow Color Marker RPN-756(Amersham Life Science). (D) A binding study similar to that described for panels B and C was done with the Gal4-RBP1dl-LXCXE and RBP1dl-LXCXE-HA mutants,which lack the LXCXE pocket-binding motif (24). The positions of migration of Flag-tagged HDAC1 (open arrows), HDAC2 (closed arrow), and HDAC3 (arrowhead)are indicated. (E) Coimmunoprecipitation studies similar to those for Fig. 1 were done with both H1299 and 293T cells. In addition to the antisera described, antibodiesagainst pRB (G3-245), p130 (C-20 polyclonal) (Santa Cruz), p107 (C-18), and the HA epitope (HA.11) were used. Coimmunoprecipitation with RBP1 was detectedwith a monoclonal antibody raised specifically against RBP1 (LY32).

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all three HDAC enzymes seemed to require the pocket regiontargeted by DNA tumor virus proteins.

The small pocket interacts with different HDACs. It hasbeen proposed that HDAC1 utilizes a degenerate IXCXE mo-

tif to interact with the small pocket (residues 379 to 792) ofpRB (16, 27). To directly determine if this region is involved inthe binding of all of the HDACs, studies were carried out withextracts from H1299 cells cotransfected with plasmid DNAs

FIG. 4. Mapping of transcriptional repression domains in RBP1 and effect of TSA on RBP1 repression activity. (A) Repression by RBP1 mutants. CAT assays wereperformed with CHO cells, as described previously (24), with G5TKCAT as the reporter. (B) Illustration of the Gal4DBD-RBP1 mutants. The name of each mutantis provided, as well as the amino acid residues affected by deletions. A summary of repression results (Rep.) obtained in experiments described for panel A is shownat the left. NLS, nuclear localization signal. (C) Effect of TSA on repression. Repression assays were carried out as for panel A, except that some cells were incubatedwith 330 nM TSA for 24 h prior to harvesting and the G5MLPCAT reporter was assayed instead of the G5TKCAT reporter. (D) An experiment similar to that describedfor panel C was performed with a reporter construct consisting of the E2F-1 promoter linked to a cDNA encoding luciferase, as described previously (24), and eitherRBP1, RBP1dl-LXCXE, the pocket of pRB, or an inactive pRB pocket mutant, mRB(C706F) mutant (22). All data are the averages (6 standard errors) of at leastsix individual experiments.



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expressing Gal4-pRB(pocket) and Flag-tagged versions ofHDAC1, HDAC2, or HDAC3. Following immunoprecipita-tion with an antibody against the Gal4DBD, precipitates wereresolved by SDS-PAGE, and after transfer, the presence ofHDAC1 to HDAC3 was analyzed by Western blotting withanti-Flag antibody. Figure 2A shows that all three HDACs,which were detected at similar levels in whole-cell extracts,associated with the small pocket of pRB in vivo. Anti-Flagantibody recognized three Flag-HDAC1 species, but only theslowest-migrating form was evident in Gal4-pRB(pocket) pre-cipitates, suggesting that the faster-migrating species may bedegradation products. Flag-HDAC3 was detected as twoclosely migrating species that were both associated with theGal4-pRB(pocket). These interactions were highly specific; acDNA expressing the Gal4DBD linked to VP16, a knowntranscriptional activator, did not associate with any of theHDACs (Fig. 2A) even though Gal4-pRB(pocket) and Gal4-VP16 were expressed at comparable levels (data not shown).

We believe that these results, as well as those described abovefor antibody against CREB-binding protein, demonstrateclearly the specificity of interactions involving HDACs and ruleout the possibility that the interactions we observed were non-specific.

Figure 2B shows that in a similar experiment with 293T cells,none of the HDAC proteins were present in immunoprecipi-tates containing Gal4-pRB(pocket). Identical results were alsoobtained with 293 cells, which express only Ad5 E1A proteins(Fig. 2C). Figure 2D shows that addition prior to immunopre-cipitation of increasing amounts of in vitro-synthesized E1Aprotein fused to glutathione S-transferase (GST-E1A) (6) toextracts from H1299 cells expressing Gal4-pRB(pocket) andFlag-HDAC1 caused a decrease in the binding of HDAC1 tothe small pocket of pRB. Figure 2D also demonstrates thatpRB coimmunoprecipitated with E1A protein but not withGST. Similar results were also obtained with Flag-HDAC2 andFlag-HDAC3 (data not shown). Thus, the small pocket of pRBis important for interactions not only with HDAC1 but alsowith HDAC2 and HDAC3. Disruption of such binding by theE1A protein or T antigen also implied that the associations ofHDACs may be mediated by LXCXE-like interactions. Al-though HDAC1 and HDAC2 contain IXCXE motifs, themechanism of binding of HDAC3 was uncertain, as HDAC3lacks such sequences. Of further interest, no interaction be-tween the pocket of pRB and HDAC1 (2) or HDAC2 (32a)was observed in studies involving the yeast two-hybrid method.As all three human HDACs are highly homologous, it is pos-sible that the interactions of all three with the pocket may beindirect and may involve an additional LXCXE-containingprotein as a linker.

One possible candidate for a linker is RBP1, a known nu-clear pRB-binding phosphoprotein that interacts with thepocket via an LXCXE motif (9, 15, 22). In previous studies, itwas shown that RBP1 associates with both pRB-E2F and p130-E2F complexes following serum starvation and, if overex-pressed, induces both growth arrest and repression of E2F-dependent transcription (24). In addition, in studies involvingRBP1 fused to the Gal4DBD, RBP1 was shown to be an activerepressor containing an isolable repression domain (24),termed R1.

RBP1 interacts with HDACs in vivo. The ability of RBP1 toassociate with HDACs in vivo was tested. A binding experi-ment similar to that described above for Gal4-pRB(pocket)was performed with RBP1 fused to the Gal4DBD. Figure 3Ashows that when expressed in H1299 cells, Gal4-RBP1, but not

FIG. 5. Mapping of specific binding of HDAC3 to RBP1. Binding studiessimilar to those described for Fig. 3B were done with 293T cells by using eitherGal4-R1, Gal4-R2(1314-C), Gal4-R2(1263-C), Gal4 alone, Gal4-RBP1 (wildtype [WT]), Gal4-ARID, Gal4-dlR1, or Gal4-dl-93C(dl-R2).

FIG. 6. Model of repression of E2F-dependent promoters by RB family members and RBP1.



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Gal4-VP16, interacted with all three HDACs. Again, only theslowest-migrating Flag-HDAC1 species was evident, and Flag-HDAC3 was present as a doublet in Gal4-RBP1 precipitates.

Figure 3B shows that in 293T cells expressing E1A productsand SV40 large T antigen, both HDAC1 and HDAC3 associ-ated with Gal4-RBP1, indicating that these interactions wereunaffected by high levels of these viral pocket-binding proteins.The binding of HDAC2 was not highly evident under theseconditions. Whereas Flag-HDAC1 and Flag-HDAC3 expres-sion relied on the cytomegalovirus promoter (32a) that yieldedhigh levels of these products, the expression of Flag-HDAC2was achieved in these experiments with a hybrid SV40-humanT-cell leukemia virus type 1 promoter that produced smalleramounts of product in 293T and 293 cells (Fig. 2C). To reex-amine RBP1-HDAC binding under conditions in which allthree HDACs were expressed at high levels, constructs inwhich all three HDACs were expressed in 293T cells along withHA-tagged RBP1 as Gal4DBD-HDAC fusion products underthe control of the SV40 early promoter were prepared. Figure3C shows that all the HDACs were expressed at comparablelevels, as determined by the immunoblotting of whole-cell ex-tracts with anti-Gal4DBD antibody. Similarly, all cells ex-pressed comparable amounts of RBP1, as detected with an-ti-HA antibody. Figure 3C also shows that interactionsbetween all three HDACs and HA-RBP1 were evident follow-ing the immunoblotting of individual HDAC immunoprecipi-tates with anti-HA antibody. No such binding was observedwith Gal4 alone or with Gal4-VP16. Figure 3E shows thatinteractions between RBP1 and endogenous HDAC enzymescould also be detected in H1299 cells. With immunoprecipi-tates prepared with the same polyclonal antibodies againstindividual HDACs as employed for Fig. 1, RBP1 was found tobe in association with all three HDACs, as detected by immu-noblotting with LY32 monoclonal antibody, raised against hu-man RBP1 and found previously to interact specifically withthis polypeptide (8a). Interactions with HDAC1 clearly oc-curred at much higher levels than those with HDAC2 andHDAC3, implying that RBP1-HDAC1 complexes are the pre-dominant species in H1299 cells. This observation may suggestthat RBP1 could be a component of the HDAC1 core complex.If this is the case, much of the endogenous RBP1-HDAC1complex could be targeted to sites other than RB family mem-bers, as the levels of RBP1 detected in association with theseproteins were much lower than those observed with HDAC1.Figure 3E also shows that RBP1 associates with all members ofthe RB family, and examination of pertinent lanes in Fig. 1indicated that this association occurred preferentially with hy-pophosphorylated forms of pRB (Fig. 1A) and p107 (Fig. 1B).In addition, Fig. 3E shows that such interactions of RBP1 withpRB, p107, and p130 were disrupted in 293T cells, as expected.Taken together, these results suggest that interactions betweenRBP1 and RB family members are truly pocket dependent, asthey were disrupted by T antigen and E1A pocket-bindingproteins. On the other hand, interactions between RBP1 andHDACs are not at all sensitive to disruption by these viralpocket-binding proteins, suggesting that HDACs cannot berecruited to RBP1 via endogenous RB family members presentin 293T cells. Instead, RBP1 could mediate the associationbetween RB family members and all three HDAC enzymes.

It is unlikely that the interactions of HDACs with RBP1occur indirectly through the recruitment of RB family mem-bers by RBP1. In 293T and 293 cells, we failed to observeinteractions either between the pocket of pRB and these en-zymes (Fig. 2B and C) or between RBP1 and RB family mem-bers (Fig. 1 and 3E). In addition, we studied interactions be-tween the HDACs and an RBP1 mutant, RBP1dl-LXCXE,

that fails to interact with pRB because of the removal of theconserved LXCXE pocket-binding motif by an internal dele-tion (9, 24). Figure 3D shows that in 293T cells, both Gal4-RBP1dl-LXCXE and RBP1dl-LXCXE-HA mutants inter-acted with Flag-tagged or Gal4DBD-tagged HDACs. Similarresults were also obtained with human H1299 cells (data notshown). Thus, HDACs appear to interact with RBP1 in aregion apart from the pocket-binding motif, further supportinga role for RBP1 in bridging interactions between HDACs andpRB-E2F complexes.

RBP1 contains two independent transcriptional repressiondomains. Previous studies showed the existence of both pRB-E2F-RBP1 and p130-E2F-RBP1 complexes in growth-arrestedcells that correlated with the ability of RB family members toactively repress E2F-dependent transcription (24). These stud-ies also used Gal4-RBP1 fusion products to map a repressiondomain, R1, between residues 388 and 599 of RBP1, compris-ing an ARID sequence and a region predicted to have ana-helical structure. Figure 4A shows that Gal4-RBP1 is able torepress the expression of CAT under the control of the Gal4minimal herpesvirus thymidine kinase promoter. Similar re-pression was also seen with Gal4-R1 that contains only the R1repression domain of RBP1. Curiously, when R1 was deleted(Gal4-RBP1dl-R1), this mutant RBP1 product still repressedCAT expression at high levels (Fig. 4A), suggesting that asecond repression domain may exist towards the carboxy ter-minus of RBP1. We therefore generated a series of in-framecarboxy-terminal-deletion mutants that also lacked R1 (Fig.4B) and found that all, including Gal4-dl-R1-93C, whichlacked only 93 residues at the carboxy terminus, failed to re-press CAT expression (Fig. 4A). All mutants used in the ex-periments illustrated in Fig. 4 were shown to be expressed atsimilarly high levels (data not shown). As these data suggestedthat the second repression domain likely lies at the most car-boxy-terminal portion, three additional constructs that con-tained only various amounts of the carboxy terminus of RBP1linked to the Gal4DBD were generated. Figure 4A shows thatall three constructs, Gal4-R2(1311-C), Gal4-R2(1314-C), andGal4-R2(1263-C), repressed CAT expression. Thus, a secondRBP1 repression domain, R2, exists between residues 1314and 1404. Furthermore, repression by neither R1 nor R2 relieson the LXCXE pocket-binding motif when RBP1 is tetheredto DNA by a heterologous DNA-binding domain, like theGal4DBD.

Transcriptional repression by RBP1 is both dependent andindependent of HDAC activity. Previous studies by Luo et al.(26) suggested that the ability of the pocket to actively represstranscription relies on both HDAC-dependent and -indepen-dent mechanisms. These authors also showed that only a sub-set of promoters repressed by the pocket of pRB was sensitiveto the specific HDAC inhibitor trichostatin A (TSA). Amongthese, the only Gal4-dependent promoter/reporter constructfound to be repressed by the pocket and to be sensitive to TSAis G5MLPCAT, which contains the adenovirus major late pro-moter. Figure 4C shows that both Gal4-RBP1 and Gal4-pRB-(pocket) repressed the G5MLPCAT reporter, as did bothGal4-R1 and Gal4-R2, which contain only R1 and R2, respec-tively. Figure 4C also shows that following the treatment oftransfected cells with 330 nM TSA for 24 h prior to harvesting,repression by both Gal4-pRB(pocket) and Gal4-RBP1 waspartially relieved; however, whereas drug treatment completelyabolished repression by Gal4-R2, it had no effect on that byGal4-R1. Thus, it appears that repression by R2 depends onHDACs, whereas that by R1 does not. We also noted thatwhereas Gal4-HDAC1 repression activity is completely re-lieved by TSA (Fig. 4C), repression by Gal4-Ad5-E1B-55K, an



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adenoviral repressor known to block p53-dependent transacti-vation (36, 45), at this promoter is not affected by TSA (datanot shown), suggesting that the adenovirus major late pro-moter can be subject to both HDAC-dependent and -indepen-dent repression. These results therefore strengthen the possi-bility that RBP1 plays an important role in repression by pRB,as TSA only partially relieves repression by the pRB pocket.Interestingly, TSA had little effect on repression by Gal4-RBP1 or Gal4-R2 with either the G5TKCAT or G5SV40CATpromoter (data not shown), as determined previously withGal4-pRB(pocket) by Luo et al. (26). Studies were extendedfrom the synthetic G5MLPCAT construct to the E2F-depen-dent promoter regulating E2F-1 expression (E2F1-luc). Figure4D shows that both RBP1 and the pocket of pRB repressed theexpression of luciferase from the E2F1-luc reporter, whereasRBP1 lacking the LXCXE pocket-binding motif or a pRBpoint mutant [mRB(C706F)] did not. Members of our grouphad demonstrated previously that mutation of the E2F bindingsite in this reporter ablated repression by RBP1 (24). Additionof TSA partially relieved repression by both RBP1 and thepRB pocket. These results indicated that both the RBP1HDAC-dependent and -independent repression activities canrepress this E2F-dependent promoter via interactions with RBfamily members, and thus interactions with RBP1 could pro-vide both types of repression activities attributed to the pocketof RB family members (26).

Only one repression domain of RBP1 interacts with HDACs.We tested the ability of individual R1 and R2 transcriptionalrepression domains to interact with HDACs. Figure 5 showsresults of coimmunoprecipitation experiments using 293T cellsexpression Gal4-RBP1 constructs and Flag-HDAC3, in whichRBP1 was immunoprecipitated with anti-Gal4DBD antibodiesand HDAC3 binding was detected by immunoblotting withanti-Flag antibody. No HDAC3 binding was observed witheither all of R1 or just the ARID portion of R1, but suchbinding was clearly evident with both R2 constructs and withRBP1 lacking R1. Binding was eliminated or greatly reducedwith RBP1 lacking R2. Similar results were also obtained withFlag-HDAC1 and Flag-HDAC2 (data not shown).

DISCUSSION

The present study reports for the first time that the pocket ofpRB, and possibly other RB family members, interacts with allthree cloned human HDACs. Furthermore, the RB-bindingprotein RBP1 also binds these enzymes, but unlike pRB, thepocket-binding E1A protein and large T antigen do not affectinteractions between RBP1 and any of the HDACs. It is cur-rently believed that HDAC1 or HDAC2 interacts directly withthe pocket via degenerate IXCXE motifs, but this model doesnot account for the interactions of HDAC3, which lacks suchsequences. Our data indicated that the binding of HDAC3 toRB family members also requires the pocket, suggesting that,in this case at least, such interactions may be indirect and relyon a pocket-binding protein, such as RBP1. The previous ob-servation that RBP1 is present in pRB-E2F and p130-E2Fcomplexes following serum starvation and the effects of itsoverexpression on inducing growth arrest and the repression ofE2F-dependent transcription (24) suggest that RBP1 may befunctionally important in the repression of E2F-dependenttranscription by linking HDACs to the pockets of RB familyproteins. Figure 6 shows a model in which the repression ofE2F-dependent transcription by pRB and p130 at growth ar-rest or by p107 in G1 results from the binding of RBP1 at thepocket, thus introducing not only HDACs via interactions with

the R2 domain but also a second R1 repression domain thatfunctions by another mechanism.

At present, it is not known if HDACs bind directly to theRBP1 or if they interact indirectly via an additional R2-bindingprotein. Previous work with anti-RBP1 LY11 antibodies indi-cated the presence of several proteins that coprecipitate withRBP1, including pRB and p130 (24). The most prominentspecies was a protein of about 48 kDa. HDAC1 copurifies withRBAP48, which, along with RBAP46, plays a role in targetingHDAC1 to histones (47). It is possible that both RBP1 andeither RBAP48 or RBAP46 could coexist in a single HDACcomplex. RBAP48 binds to the so-called extended pocket, in-cluding a carboxy-terminal portion of pRB, and thus mightplay a role as a linker for HDAC1; however, it lacks theLXCXE-binding motif and thus is not targeted to the smallpocket. In addition, no interactions between the pRB pocketand HDAC1 (2) or HDAC2 (32a) have been detected by theyeast two-hybrid system, even though yeast cells contain highlevels of MSI1, a protein that is highly homologous to RBAP48and RBAP46 (47). The present results strongly suggest thatRBP1 is responsible for bridging the pocket of RB familymembers to HDAC complexes to repress a diversity of E2F-dependent promoters. RBP1 therefore appears to represent amajor component of the growth-regulatory machinery con-trolled by RB family members.

ACKNOWLEDGMENTS

We thank Tony Kouzarides for Gal4-pRB(pocket) and for helpfuldiscussions; Xiang-Jiao Yang and Brian Kennedy for critical review ofthe manuscript; Arnie Berk for Gal4-VP16, pSG424, and G5TKCAT;and Doug Dean and Don Ayer for G5MLPCAT and G5SV40CAT.We also thank Dennis Paquette for the construction of the Gal4-RBP1dl-R1 mutant.

This work was supported through grants from the National CancerInstitute of Canada and the Medical Research Council of Canada.

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RBP1 Recruits Both Histone Deacetylase-Dependent and