JOURNAL OF CELLULAR PHYSIOLOGY 196:541–556 (2003)

Tumor Suppressor pRB Functions as a Co-Repressor of theCCAAT Displacement Protein (CDP/Cut) to Regulate Cell

Cycle Controlled Histone H4 Transcription

SUNITA GUPTA,1 MAI X. LUONG,1 SYLVIA A. BLEUMING,1 ANGELA MIELE,1 MICHAEL LUONG,1

DANIEL YOUNG,1 ERIK S. KNUDSEN,2 ANDRE J. Van WIJNEN,1 JANET L. STEIN,1 AND GARY S. STEIN1*1Department of Cell Biology and Cancer Center,

University of Massachusetts Medical School, Worcester2Department of Cell Biology, University of Cincinnati College of Medicine,

Cincinnati, Ohio

The CCAAT displacement protein (CDP-cut/CUTL1/cux) performs a key prolifera-tion-related function as theDNA binding subunit of the cell cycle controlledHiNF-D complex. HiNF-D interacts with all five classes (H1, H2A, H2B, H3, and H4) ofthe cell-cycle dependent histone genes, which are transcriptionally and coordi-nately activated at the G1/S phase transition independent of E2F. The tumorsuppressor pRB/p105 is an intrinsic component of the HiNF-D complex. However,the molecular interactions that enable CDP and pRB to form a complex and thusconvey cell growth regulatory information onto histone gene promoters must befurther defined. Using transient transfections, we show that CDP represses the H4gene promoter and that pRB functions with CDP as a co-repressor. Direct physicalinteraction between CDP and pRB was observed in glutathione-S-transferase(GST) pull-down assays. Furthermore, interactions between these proteins wereestablished by yeast and mammalian two-hybrid experiments and co-immunopre-cipitation assays. Confocal microscopy shows that subsets of each protein are co-localized in situ. Using a series of pRB mutants, we find that the CDP/pRBinteraction, similar to the E2F/pRB interaction, utilizes the A/B large pocket (LP) ofpRB. Thus, several converging lines of evidence indicate that complexes betweenCDPandpRB repress cell cycle regulatedhistone gene promoters. J. Cell. Physiol.196: 541–556, 2003. � 2003 Wiley-Liss, Inc.

Cell proliferation is regulated by a complex and inter-dependent series of biochemical events involving cellcycle-stage specific modifications in gene expression.The S-phase specific expression of histone genes repre-sents one of the earliest characterized examples of cellcycle dependent gene regulation and provides a para-digm for understanding gene regulatory signalingmechanisms operative at the G1/S transition (Prescott,1966; Stein et al., 1996). Histone gene expression inmammalian cells is both temporally and functionallycoupled with DNA replication (Stein et al., 1984; Osley,1991; Stein et al., 1996; Dominski and Marzluff, 1999).Cell cycle dependent modulations of histone genetranscription provide the initial rate-limiting step inthe induction of histone gene expression at the G1/Sphase transition.

Cell cycle control of histone H4 gene transcriptiondoes not depend on E2F and requires a critical multi-partite promoter element, Site II, that interacts withthree distinct histone nuclear factors (HiNFs) (vanWijnen et al., 1989, 1992; Ramsey-Ewing et al., 1994;Aziz et al., 1998b; van der Meijden et al., 1998; Vaughanet al., 1998; Xie et al., 2001). Several other promoterelements, transcription factors, and/or co-factors alsocontribute to the regulation of histone H4 gene tran-

scription in the context of a dynamic and transcription-ally active chromatin organization (Stein et al., 1996;Last et al., 1998, 1999a; Staal et al., 2000; Mitra et al.,2001; Hovhannisyan et al., 2003). Overlapping recogni-tion sequences within Site II for HiNF-M (IRF-2), HiNF-P, and HiNF-D (CDP-cut) together modulate H4 genetranscription levels by at least an order of magnitude.This composite organization of Site II supports respon-siveness to multiple signaling pathways that modulatethe activities of H4 gene transcription factors during thecell cycle. HiNF-M is identical to the oncoprotein IRF-2(Vaughan et al., 1995), HiNF-P is a 65 kDa Zn finger

� 2003 WILEY-LISS, INC.

Sunita Gupta and Mai X. Luong contributed equally to this study.

Contract grant sponsor: NIH; Contract grant number: GM32010.

*Correspondence to: Gary S. Stein, Department of Cell Biology,Rm S3-310, University of Massachusetts Medical School, 55 LakeAvenue North, Worcester, MA 01655.E-mail: gary.stein@umassmed.edu

Received 7 March 2003; Accepted 28 March 2003

DOI: 10.1002/jcp.10335

protein that links the growth factor dependent NPAT/Cyclin E/CDK2 pathway to cell cycle control of H4gene transcription (Mitra et al., 2002), and HiNF-D is acomplex of the homeodomain protein CDP and the cellcycle regulators pRB, CDK1/CDC2, and cyclin A (vanWijnen et al., 1989; van Wijnen et al., 1994, 1996, 1997;Shakoori et al., 1995). Gene knock-out, ectopic forcedexpression, and anti-sense interference strategies haveestablished roles for HiNF-M (IRF-2), HiNF-P, andHiNF-D (i.e., CDP/cut/Cux1) in cell cycle regulation ofhistone H4 gene expression and/or control of cell growthand differentiation (Quaggin et al., 1997; Vaughan et al.,1998; Ellis et al., 2001; Luong et al., 2002; Mitra et al.,2002; Xie et al., 2002). Taken together, these find-ings indicate that HiNFs are components of an E2F-independent mechanism that mediates control of cellproliferation.

The functional interactions of CDP (and/or HiNF-D)with the promoters of histone (Barberis et al., 1987;van den Ent et al., 1994, 1996; Wu and Lee, 2002), c-Myc(Dufort and Nepveu, 1994), p21 (Coqueret et al., 1998a),c-Mos (Higgy et al., 1997), TGFb type II receptor(Jackson et al., 1999), and thymidine kinase (TK) (Kimet al., 1997) genes indicate that this factor is a majorcomponent of a phylogenetically conserved gene regul-atory mechanism that controls cell growth (Nepveu,2001). CDP contains four independent DNA bindingdomains (Aufiero et al., 1994; Harada et al., 1995; Maillyet al., 1996; Moon et al., 2000) and as a result may exhibitmultiple conformational modes to support promoterrecognition. In addition, the DNA binding and/or tran-scriptional activities of CDP may be regulated by inter-actions with different members of the pRB family (i.e.,p105 and p107) (van Wijnen et al., 1996, 1997; van Gurpet al., 1999) and cyclin/CDK proteins (van Wijnen et al.,1994; Shakoori et al., 1995). CDP activity can also becontrolled by phosphorylation (Coqueret et al., 1996;Coqueret et al., 1998b; Santaguida et al., 2001), dephos-phorylation (van Wijnen et al., 1991; Santaguida et al.,2001), and proteolytic processing (Moon et al., 2001,2002). Furthermore, the ability of CDP to bind nucleo-somal DNA (Last et al., 1999b) and to recruit histonemodifying proteins (e.g., the histone H1 kinase cyclinA/CDK1 and the histone acetyl transferases P/CAF andCBP) (van Wijnen et al., 1994; Li et al., 2000) suggeststhat the protein may contribute to modifications in thechromatin architecture of its target genes.

CDP interacts with all five classes (i.e.,H1,H2A,H2B,H3, and H4) of histone genes and consequently maycoordinately regulate their transcriptional activation atthe G1/S phase transition or their repression in mid- tolate- S phase (Barberis et al., 1987; van den Ent et al.,1994; van Wijnen et al., 1996; Wu and Lee, 2002). It hasbeen well-documented that the interaction of the CDPcontaining HiNF-D complex with histone gene regula-tory elements is proliferation-specific and cell cycleregulated with respect to S phase (Holthuis et al., 1990;van Wijnen et al., 1992, 1997; Wright et al., 1992;Shakoori et al., 1995; Last et al., 1998). Our laboratoryhas shown that this interaction is important for thetiming of maximal histone H4 gene transcription duringthe cell cycle (Aziz et al., 1998a). When cell growth isstimulated by growth factors, up-regulation of HiNF-DDNA binding activity occurs subsequent to hyperphos-

phorylation of pRb and an increase in cyclin A and CDK1(van Wijnen et al., 1997), while CDP protein levelsremain constant during the cell cycle (van Wijnen et al.,1997; Last et al., 1998). To understand the molecularbasis of the interactions between CDP and pRB ascomponents of the multi-subunit HiNF-D complex thatcontrols histone gene expression, we performed yeastand mammalian two-hybrid experiments, glutathione-S-transferase (GST) pull down assays, co-immunopre-cipitations, and transient transcriptional assays. Ourstudies indicate that CDP forms protein/protein com-plexes with pRB in the absence of DNA and thatpRB functions with CDP as a co-repressor of H4 genetranscription. Our data support the concept that com-plexes between CDP and pRB are important cell cycleregulators of transcription during S phase.

MATERIALS AND METHODSCell culture

All cell lines used in our studies were propagatedaccording to culture conditions suggested by the Amer-ican Type Culture Collection (Manassas, VA; http://www.atcc.org). In brief, actively proliferating cultures ofNIH/3T3, HeLa, COS-7, and PANC-1 cells were main-tained at sub-confluency in Dulbecco’s modified Eagle’smedium, supplemented with 10% fetal calf serum,100 U/ml penicillin, 100 mg/ml streptomycin, and 0.2 mML-glutamine, at 378C in humidified air containing 5%CO2.

HeLa cells are HPV-18 transformed human cervicalcarcinoma cells that express wild type pRB (Scheffneret al., 1991), but expression of the HPV-18 derived E7protein alters the cell growth regulatory functions of thepRB protein (Chellappan et al., 1992). COS-7 cells areSV40 transformed monkey kidney fibroblasts thatexpress wild-type SV40 T antigen, which is known tointeract with endogenous pRB and alter its function(Hamel et al., 1990). NIH/3T3 cells are derived frommouse embryo cultures, have normal cell growth chara-cteristics, and express endogenous pRB (Banks et al.,1990). Apart from differences in pRB status, HeLa, COS-7, and NIH/3T3 cells all contain detectable levels of CDPbinding activity as reflected by detection of the CDP-containing HiNF-D complex [(van Wijnen et al., 1996;van der Meijden et al., 1998); and unpublished observa-tions]. PANC-1 cells are pancreatic duct-derived humanepithelial carcinoma cells (Lieber et al., 1975) that do nothave detectable levels of CDP by western blot analysis(Li et al., 1999) but express a phosphorylatable form ofpRB (Hirai et al., 1996).

Construction of expressionand reporter gene vectors

All oligonucleotides used in this study were syn-thesized for cloning purposes using a Beckman 1000Msynthesizer and all inserts were subjected to automatedsequencing (Applied Biosytems ABI Model 377, FosterCity, CA) to verify correct orientation and absence ofchemical synthesis related mutations. Plasmids forexpression of recombinant proteins in mammalian cellswere prepared as follows. To create a full-length Myc-epitope tagged CDP protein, we isolated the full lengthcoding sequence of the CDP cDNA from MT2-CDP(kindly provided by Ellis Neufeld, Children’s Hospital,

542 GUPTA ET AL.

Boston) (Neufeld et al., 1992) as a NotI/XhoI fragmentwhich was inserted into the NotI/XhoI sites of pcDNA3.1containing a myc epitope tag. At a ClaI site near the 50

end of the CDP insert, we inserted a 14 basepairoligonucleotide (50 CGA GCA AGC TTG CT; eliminatesthe ClaI site upon insertion) to place the CDP codingsequence in frame with the Myc tag. To clone the Myc-tagged CDP (CR2-Cterm) protein (a 110 kDa deletionmutant that encompasses Cut repeat 2 (CR2) to theC-terminus of CDP), we isolated CDP coding sequencesas an EcoRI/XhoI fragment from the GST/CDP (CR2-Cterm) construct (kindly provided by Ellis Neufeld,Children’s Hospital, Boston) (Lievens et al., 1995). Thefragment was sub-cloned into the EcoRI/XhoI sites ofpcDNA3.1 Myc.

Expression vectors for yeast and mammalian two-hybrid assays were generated as follows. The vectorexpressing a LexA/pRB fusion protein was constructedby cloning the full length coding sequence of a CMVdriven pRB construct (kindly provided by Jean Wang,University of California, San Diego) into the BamHI siteof the pEG202 yeast vector which contains the codingsequence for the LexA DNA binding domain. A vectorexpressing a C-terminal portion of CDP (CR2-Cterm)fused to the B42 activation domain was based on theCDP coding sequences in the GST/CDP (CR2-Cterm)construct. The CDP (CR2-Cterm) fragment was insertedin-frame with the B42 coding sequences using the EcoRIand XhoI sites of pJG4-5 (kindly provided by RogerBrent, Massachusetts General Hospital, Boston). Thevector pRB/pCMXGal4/N (kindly provided by PaulRobbins, University of Pittsburgh, PA) (Adnane et al.,1995), which expresses a GAL4/pRB fusion protein geneproduct, was used for mammalian two-hybrid assays.The full-length CDP cDNA was cloned into the NotI siteof pCMXVP16 to generate a vector expressing a VP16/CDP fusion protein.

Reporter gene transcription experiments

NIH/3T3, HeLa, and COS-7 cells were seeded in 6-wellculture plates at a density of 1.5� 105 cells/well andwere transiently transfected by the Superfect transfec-tion method (Qiagen, Valencia, CA) 22–24 h afterplating at �50% confluency. Transient transfectionswith PANC-1 cells were performed with FuGENE 6(Roche, Indianapolis, IN) according to the manufac-turer’s instructions. For standard transient transfectionassays to measure H4 gene promoter activity, we co-transfected 1 mg of H4 promoter/luciferase (Luc) repor-ter gene construct or a promoterless luciferase reporterconstruct (pGL2) and different amounts of expressionvectors as indicated in the figure legends (e.g., Myc-tagged CDP expression vector; empty vector, vectorbackbone of CDP and pRB expression vectors; pRB largepocket; phosphorylation site mutant spanning the LP ofpRB). The amount of DNA in each well was maintainedat a constant level by supplementing the transfectionmixture with the empty expression vector. Co-transfec-tion experiments with CDP and pRB proteins wereperformed with 1 mg of each vector with the exception ofvectors for pRB LP (500 ng). Cell lysates were preparedfor luciferase assay or for western blot analysis 22–24 hafter transfection. Each transfection experiment was

performed in triplicate and repeated at least threetimes.

Reporter gene activity was measured by luciferaseassays. Cells were washed twice with 1� PBS bufferand lysed with 1�Lysis buffer (Promega, Madison, WI).Luciferase assays were carried out according to themanufacturer’s specifications using a dual luciferasereporter assay system (Promega) and a Monolight 2010Luminometer (Analytical Luminescence Laboratory,San Diego, CA).

Yeast two-hybrid assay

The yeast strain EGY48 (generously provided byErica Golemis, Fox Chase Cancer Center, Philadelphia,PA) contains two reporter genes, Leucine2 (LEU2) andthe Lac Z gene. Both reporter genes are under thecontrol of LexA operator sequences. Transcriptionalactivation is observed when interaction between LexA/pRB and B42/CDP occurs, resulting in the recruitmentof the B42 activation domain to the promoter. Expres-sion of the B42/CDP fusion protein is under a galactose-responsive promoter. Yeast two-hybrid assays wereperformed as described (Ausubel et al., 1997) usingthe lithium acetate method for transformation. Cellswere selected for the presence of the LexA/pRB constructby growth on plates containing medium lacking histi-dine (�H) and for the B42/CDP construct on mediumlacking tryptophan (�T). Expression of the fusion pro-teins was tested by western blot analysis. LexA/pRB andB42/CDP yeast expression vectors were transformedtogether into the yeast strain EGY48 and cells wereplated on appropriate media to select for cells expressingboth constructs. Yeast colonies were scraped and in-cubated in galactose-containing medium to induce ex-pression of B42/CDP fusion protein. Cells were re-platedon selective media lacking leucine to detect transactiva-tion of the LEU2 reporter. We performed an X-Gal filterlift assay (BD Biosciences, Santa Cruz, CA) by growingyeast on a filter membrane (Millipore, Bedford, MA) inX-gal medium to detect the reporter activity of LacZ.

Mammalian two-hybrid assays

Mammalian two-hybrid assays were performed bytransient transfection of a HeLa stable cell line (HeLaGAL-5-Luc) (Zeng et al., 1998) carrying an integratedluciferase gene under control of five tandem Gal4binding sites and a minimal TATA-box. HeLa GAL-5-Luc cells were seeded in 6-well culture plates at adensity of 1.2�105 cells/well and transfected at about60% confluency using the Superfect transfection method(Qiagen) with 0.5mg of each mammalian 2-hybrid vector.The amount of DNA was kept constant at 1 mg bysupplementing with empty GAL4 and VP16 expressionvectors when VP16/CDP and GAL4/pRB were expressedseparately. Luciferase activities were determined 24 hpost-transfection. Each transfection was performed intriplicate.

Native polyacrylamide gel electrophoresis

To determine the molecular weight of HiNF-D, weperformed electrophoretic mobility shift assays (EMSA)and native gel electrophoresis. EMSA was carried out asdescribed previously (van Wijnen et al., 1992). The

CDP/pRB INTERACTIONS 543

histone H4/Site II probe is an EcoRI-HindIII insert fromplasmid pFP202. Protein/DNA binding reactions withH4/SiteII were performed by combining 32P-labeledprobe DNA (10 fmole), non-specific competitor DNA(1 mg poly G/C and 100 ng poly I/C), and nuclear extract(1.5 mg) prepared from proliferating HeLa cells. In thesame polyacrylamide gel (4.4%) used in EMSA, urease(4 mg) and a-macroglobulin (1 mg) were subjected toelectrophoresis. The gel was cut and the portion cont-aining the EMSA reaction was dried and exposed to filmto detect the HiNF-D complex. Protein markers werevisualized with Coomassie dye (Ausubel et al., 1997).

GST pull down assays

GST pull down assays were performed using a GSTprotein fused to the CDP (CR2-Cterm) protein. The GSTand GST–CDP fusion proteins were expressed in thebacterial strain BL21 and conjugated to glutathione–Sepharose beads (Ausubel et al., 1997). The resinscontaining the bound GST and GST–CDP (CR2-Cterm)fusion proteins were each incubated with endogenous,over-expressed, or in vitro translated 35S-labeledproteins.

Nuclear extract from proliferating HeLa cells waspassed through a phospho-cellulose column and elutedwith a KCl 200–400 mM buffer as described previously(van Wijnen et al., 1992; Vaughan et al., 1995). ThisKCl fraction (3 mg/ml) was diluted one-fold with buffer X(18.2 mM dibasic sodium phosphate, 3.4 mM monobasicsodium phosphate, 2% Nonidet P-40, 1% sodium deoxy-cholate, and 0.2% SDS).

HeLa cells were transfected with CMV vectorsexpressing wild type pRB, LP pRB, and PSM7 mutantLP pRB. Superfect (Qiagen) transfection methods wereused to transfect HeLa cells. Whole cell extraction wasachieved with RIPA buffer (1� PBS, 1% Nonidet P-40,0.5% sodium deoxycholate, and 0.1% SDS) supplemen-ted with Complete Protease Inhibitor Cocktail (Roche).

In vitro translated proteins were prepared as follows:the expression constructs pcDNA pRB A/B and CMVcyclin A were subjected to coupled in vitro transcriptionand translation with [35S] methionine in a rabbitreticulocyte lysate according to the manufacturer’sinstructions (Promega). The lysate was diluted one-foldwith buffer X.

Protein preparations were pre-cleared with gluta-thione–Sepharose beads (Amersham Pharmacia Bio-tech; Uppsala, Sweden) prior to incubation with 2 mg ofGST or GST–CDP (CR2-Cterm) glutathione–Sephar-ose beads at 48C for 16 h. Beads were then centrifugedand washed three times with Wash buffer (10 mMTris-HCl/pH 8, 50 mM NaCl, 2 mM EDTA/pH 8, and0.2% NP-40). Bound proteins were resuspended in 2�Laemmli gel loading buffer, separated by SDS–PAGE,and transferred to polyvinylidene difluoride membranes(Immobilon-P; Millipore Corp.). Proteins were detectedusing several different primary antibodies (mousemonoclonal anti-pRB, anti-Cdk1, and anti-cyclin A IgG,as well as rabbit polyclonal anti-cdk2 and anti-Sp1 IgG)(Santa Cruz Biotechnology, Santa Cruz, CA). Primaryantibodies (Santa Cruz Biotechnology) were used at a1:1,000 dilution. [35S]-labeled samples were detected byautoradiography.

Co-immunoprecipitation assays

Cell extracts from COS-7 cells were prepared 30 hafter transfection, using RIPA buffer (Ausubel et al.,1997) containing 1� protease inhibitor cocktail (Roche).Two 100 mm plates of cells were used for each co-immu-noprecipitation assay, which was carried out basicallyas recommended (Santa Cruz Biotechnology) (Ausubelet al., 1997). We used 20 ml packed beads per 100 mmplate of cells. Cell lysates were pre-cleared for 30 min at48C and antibody incubation was carried out overnightat 48C. Protein A/G Agarose beads were washed fourtimes with low stringency buffer (20 mM Tris-HCl/pH7.5, 50 mM NaCl, 0.5% NP40, and 1� protease inhibitorcocktail), and resuspended in an equal volume of loadingbuffer (62.5 mM Tris-HCl/pH 6.8, 10% glycerol, 2% SDS,2% b-mercaptoethanol, and bromophenol blue). Thesamples were boiled for 5 min and subjected to 6%SDS–PAGE. Proteins were transferred to Immobilon-Pmembranes. The membranes were saturated withphosphate-buffered saline containing 0.05% Tween 20(1� PBS–T buffer) and 5% fat free dry milk (Ausubelet al., 1997) for 1 h at room temperature and incubatedovernight with primary antibodies at 1:1,000 dilution(anti-Myc [Zymed Laboratories, Inc., San Francisco,CA], pRB, CDK2, CDK1, cyclin A and actin polyclonalrabbit antibodies were purchased from Santa CruzBiotechnology; the pRB antibody was used at a 1:200dilution) in 1% fat free dry milk in 1� PBS–T buffer.After washing with 1� PBS–T buffer containing 1%milk, filters were washed three times with wash bufferplus 0.5% Tween 20 and incubated with 1:5,000 anti-mouse or anti-rabbit antibodies conjugated to horse-radish peroxidase (Santa Cruz Biotechnology). Blotswere then washed four times with the same buffer beforevisualization of immunoreactive protein bands byenhanced chemiluminescence detection (ECL kit; Amer-sham Pharmacia Biotech, Inc., Piscataway, NJ).

Immunofluorescence microscopy

Briefly, cells were rinsed twice with ice-cold PBS andfixed in 3.7% formaldehyde in PBS for 10 min on ice.Cells were then permeabilized in 0.1% Triton X-100 inPBS and rinsed twice with PBSA (0.5% bovine serumalbumin in PBS) followed by antibody staining. Cellswere incubated with mouse monoclonal antibody forc-myc (1:1,000), IgG-purified guinea pig antibodyagainst full-length CDP (1:100), or two mouse mono-clonal antibodies against pRB (1:100; Santa Cruz or BDBiosciences) for 1 h at 378C. Coverslips were rinsed fourtimeswithPBSAbefore incubationwithcyanred donkeyanti-guinea pig antibody (1:200) or Alexa 428 goat anti-mouse antibody (1:400; Molecular Probes, Eugene, OR)for 1 h at 378C. Cells were rinsed four times with PBSAand then stained with 40, 6-diamidino-2-phenylindole(DAPI in 0.1% Triton X-100 in PBSA (PBSAT)) for 1 min.Coverslips were washed once with PBSAT and twicewith PBS. Immunofluorescent signals were detectedusing an epifluorescence microscope attached to a CCD-camera, and the digital images were analyzed with theMetamorph software programs. Endogenous CDP andpRB proteins were examined using the Leica TrueConfocal Scanning Spectrophotometer.

Image analysis was performed using the MATLABimage processing toolbox (Mathworks, Inc., Natick,

544 GUPTA ET AL.

MA). Cross-correlation, which measures the degree ofsignal overlap between two images, was calculatedessentially as described (van Steensel et al., 1996). Here,the correlation is expressed as the average percentcolocalization of several image pairs. To determine thatthe observed correlation is not due to random signaloverlap, one image from each image pair was rotated tendegrees and the cross-correlation was calculated. If theobserved colocalization were random, rotation of theimage would not change the degree of signal overlap.Differences between rotated and unrotated cross-corre-lation values were assessed by two-tailed pairedStudent’s t-test.

RESULTSN-terminus of CDP is required for fullrepression of histone H4 transcription

Cell-cycle regulation of histone H4 transcription ismediated by the cell cycle element (Site II). The HiNF-Dcomplex interacts with Site II in S phase and has CDP asits DNA binding subunit (Fig. 1). CDP has been shown torepress H4 promoter activity in COS-7 cells (van Wijnenet al., 1996), and the C-terminal region of CDP thatencompasses its DNA binding domains is involved intranscriptional inhibition of CDP-responsive promoters(Mailly et al., 1996; Moon et al., 2000). To test whetherCDP mediates repression of histone H4 transcriptionin cells with normal growth characteristics and endo-genous pRB function, we performed transient transfec-tion assays in NIH/3T3 cells using constructs for ahistone H4 promoter-driven luciferase reporter andMyc-tagged full length CDP. We also tested HeLa cells

in which HiNF-D was initially characterized (vanWijnen et al., 1989). The results show that full-lengthCDP represses H4 promoter activity in NIH/3T3(Fig. 2A) and HeLa cells (data not shown).

CDP has been shown to undergo proteolytic cleavagein S phase (Moon et al., 2001). The cleavage product,CDP (CR2-Cterm) protein, lacks the N-terminus of CDPand spans Cut Repeat 2 (CR2) to the C-terminus (Cterm)(Fig.1C).ToassesswhetherCDP(CR2-Cterm)represseshistone H4 gene transcription, we performed reportergene assays with constructs expressing Myc-tagged full-length CDP or CDP (CR2-Cterm) in NIH/3T3 (Fig. 2B)and COS-7 cells (data not shown). Both CDP proteinsare expressed in a dose-dependent manner and arelocalized to the nucleus (Fig. 2, Parts C and D). The datashow that CDP (CR2-Cterm) mediates repression but toa lesser extent than the full-length protein (Fig. 2B).Thus, theN-terminal sequencescontribute to therepres-sive potential of CDP on histone H4 gene transcription.The N-terminus contains a coiled-coil protein–proteininteraction motif as well as one of the DNA-binding CutRepeats. Hence, absence of the N-terminus may affectCDP protein-protein and/or protein–DNA interactions.

CDP interacts with pRB, Cyclin A,and CDK1 in vitro

The HiNF-D complex represents an electrophoreti-cally stable protein/DNA complex that is immuno-reactive with antibodies against CDP, as well as thecell cycle regulatory factors pRB, cyclin A, and CDK1/CDC2 (van Wijnen et al., 1994). HiNF-D can bechromatographically fractionated over multiple ion

Fig. 1. Organization of the H4 gene proximal promoter. Part A:Promoter organization of the human histone H4 gene designated H4/n(FO108). Part B: Schematic diagram of the H4 promoter–luciferasereporter constructs used in this study. The construct spans the wildtype H4 promoter encompassing Sites I and II; Site II contains a

conserved H4-specific cell cycle element (CCE). Part C: Structureof the two CDP proteins tested in our studies. The full-length proteinspans a coiled-coil (CC) domain, three Cut Repeats (CR), and ahomeodomain (HD).

CDP/pRB INTERACTIONS 545

Fig. 2.

546 GUPTA ET AL.

exchange resins indicating that this factor may form adistinct biochemical entity in the absence of DNA (vanWijnen et al., 1992). However, it has not been possibleyet to purify the HiNF-D complex to homogeneity, andthe size of the complex is not known. To determine themolecular weight of HiNF-D, we performed EMSAs withHeLa nuclear extract and a 32P-labeled probe spanninghistone H4 Site II. Protein markers were electrophor-esed in the same gel and detected by Coomassie dye(Fig. 3A). Specific HiNF-D activity was verified by oligo-nucleotide competition assays. Based on the migrationof HiNF-D and the marker proteins, the observedmolecular weight of HiNF-D is greater than 340 kDa(Fig. 3A), consistent with the estimated sum (400 kDa)of the molecular weights of the HiNF-D components[i.e., CDP (190 kDa), pRB (110 kDa), cyclin A (60 kDa),and CDK1 (34 kDa)]. Thus, the previously identifiedcomponents of HiNF-D can account for the observedmolecular weight.

To test whether CDP can form stable protein/proteincomplexes with the non-DNA binding partner proteinsin the HiNF-D complex, we performed pull-down assaysusing GST protein fused to CDP (CR2-Cterm), theS phase-specific cleavage product of CDP. We note thatto date expression of full-length CDP-cut, as a recombi-nant GST fusion protein has not been possible. Weanalyzed in vitro interactions of endogenous pRB, cyclinA, and CDK1/CDC2 present in HeLa nuclear extractwith recombinant GST–CDP (CR2-Cterm). Interactingproteins were eluted and subjected to western blotanalysis using a panel of antibodies against the knownHiNF-D subunits. We find that pRB, cyclinA, and CDK1/CDC2 are all capable of binding to GST–CDP (CR2-Cterm), but these proteins do not interact with GSTalone (Fig. 3B). For comparison, SP1 does not bind toGST–CDP (CR2-Cterm), thus demonstrating that theinteractions detected in our experiments are selective.Furthermore, p107 and CDK2 show limited binding toGST–CDP (CR2-Cterm) as compared to pRB and CDK1,respectively. Hence, the GST pull-down assays establishthat the S phase-specific cleavage product of CDP, CDP(CR2-Cterm), supports interactions with pRB, cyclin A,and CDK1/CDC2.

CDP (CR2-Cterm) interaction with thepRB LP is phosphorylation-site independent

The A/B large pocket (LP, amino acids 379–928) is theminimal growth-suppressing domain of pRB, and nearlyall of the germline tumor-derived mutations of pRBoccur within this region. In addition, most of the knownpRB interactions with other proteins require an intactpocket domain (Morris and Dyson, 2001). The A/Bpocket of pRB binds free E2F and proteins containing

the LXCXE motif such as D cyclins. To test whether CDPinteracts with the LP of pRB, we performed pull-downassays with GST–CDP (CR2-Cterm) and HeLa lysatescontaining exogenously expressed full-length pRB orproteins spanning the LP. Our results indicate that theCDP C-terminus interacts with full-length pRB and LPto a similar extent (Fig. 3C). Thus, the pRB LP issufficient to mediate interaction between CDP and pRB.

When hyperphosphorylated by cyclin/Cdk complexesduring G1 phase, pRB can no longer sequester E2F orexert its growth suppressing activity through E2F.PSM7 LP (PSM7-LP) has mutations in seven of ninephosphorylation sites. To determine whether phosphor-ylation of the LP is required for interaction with CDP,we performed GST–CDP pull-down assays with over-expressed PSM7-LP. We find that CDP (CR2-Cterm)interacts with PSM7-LP (Fig. 3C), suggesting thatthe CDP-pRB interaction is not phosphorylation-sitedependent.

Direct interaction between CDP and pRB

Our data show that CDP (CR2-Cterm) can interactwith pRB LP in the absence of cell signaling-dependentpost-translational modifications. However, it is unclearwhether the observed GST–CDP interaction with pRBLP is direct or whether it is mediated by bridging pro-teins present in the HeLa nuclear extract. To determinewhether GST–CDP (CR2-Cterm) interacts directly withpRB or cyclin A, we performed pull-down assays withGST–CDP (CR2-Cterm) and in vitro translated 35S-A/Bpocket or 35S-cyclin A. Our data show that GST–CDP(CR2-Cterm) specifically binds the pRB minimal A/Bpocket but not cyclin A (Fig. 3D). Thus the CDP (CR2-Cterm)/cyclin A interaction observed with HeLa nuclearproteins (Fig. 3B) may be mediated through an acces-sory factor (e.g., pRB). More importantly, CDP (CR2-Cterm) and the pRB A/B pocket are sufficient for directinteraction between CDP and pRB

In vivo interactions of CDP with pRB

To assess whether pRB interacts with CDP in vivo, weperformed yeast two-hybrid assays with chimeric pro-teins containing the LexA DNA binding domain fusedto pRB and the B42 activation domain fused to CDP.Transcription of B42/CDP is galactose-dependent.Expression and integrity of the LexA/pRB and B42/CDP fusion proteins were confirmed by western blotanalysis (data not shown). Transformed yeast colonieswere evaluated for two diagnostic markers under thecontrol of LexA binding sites; the LEU2 selectablemarker supports growth on leucine-deficient mediaand the b-galactosidase reporter enzyme converts thechromogenic substrate X-gal. We first established that

Fig. 2. The N-terminus of CDP is required for full repression of H4promoter activity. Part A: CDP-dependent repression of H4 promoteractivity. The functional activity of CDP on the wild type H4 promoterwas tested in transient transfection studies with NIH/3T3 cells. Cellswere co-transfected with a luciferase construct with (H4/Luc; 1 mg)or without (ev/Luc; 1 mg) the wild type H4 promoter and a CMV drivenexpression vector with (CMV/CDP; 1 mg) or without (CMV/ev; 1 mg) thefull length CDP cDNA. Luciferase assays were performed 24 h aftertransfection. Part B: NIH/3T3 cells were cotransfected with the H4/Luc reporter construct (1 mg) and increasing amounts (0.1–1 mg) of theCMV driven vectors expressing myc-tagged full length CDP or the

CDP (CR2-Cterm) deletion mutant. Luciferase assays were performed24 h after transfection. Part C: The same samples used in theluciferase assays (see Part B) were analyzed by western blotting of a6% SDS polyacrylamide gel. CDP was detected with the myc antibodyand CDK2 was detected with a specific antibody as an internal controlfor protein loading. Part C: Nuclear localization of Myc-tagged wildtype CDP and CDP (CR2-Cterm) mutant CDP proteins was assessedby immunofluorescence microscopy in HeLa cells using a monoclonalantibody against c-myc. Chromatin is visualized by DAPI staining.The bar represents 10 mm. The graph in Parts A and B showrepresentative data (n¼3) from three independent experiments.

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Fig. 3. The CDP C-terminus interacts with H4 Site II-associatedproteins in vitro. Part A: HiNF-D is greater than 340 kDa. Left: EMSAwas performed with 32P-labeled ds-oligos spanning the Site II/cellcycle element of histone H4 promoter, and nuclear extract (1.5 mg)prepared from proliferating HeLa cells. Right: In the same poly-acrylamide gel (4.4%) used in EMSA, urease (4 mg) and a-macro-globulin (1 mg) were subjected to native polyacrylamide electrophoresis(PAGE) and protein markers were detected with Coomassie dye. PartB: CDP (CR2-Cterm) specifically and selectively binds pRB, cyclin A,CDK1. HeLa nuclear proteins (690 mg) were used in pull-down assayswith 2 mg of either glutathione-S-transferase (GST) or the GST–CDP(CR2-Cterm) fusion protein. Bound proteins were separated by SDS–PAGE and subjected to western blot analysis with antibodies to pRB,

p107, cyclin A, CDK1, CDK2, and SP1. Part C: CDP (CR2-Cterm)interaction with pRB LP is phosphorylation-site independent. HeLacell lysates (1 mg) containing over-expressed pRB proteins were usedin pull-down assays with GST or GST–CDP (CR2-Cterm) proteins.Wild type pRB (WT RB), pRB large pocket (LP, amino acids 379–928)and a phosphorylation-site deficient LP protein (PSM7-LP). Part D:CDP (CR2-Cterm) interacts directly with the minimal pRB A/Bpocket. GST pull-down assays were performed by mixing GST or GST-fusion proteins with in vitro translated 35S-labeled cyclin A or pRB(A/B) pocket (amino acids 379–772). Protein from the input (Inp),bound and unbound (UB) fractions were separated by SDS–PAGE anddetected by autoradiography. Input represents 5% (Part B) and 3%(Parts C and D) of the bound samples.

cells expressing either LexA/pRB or B42/CDP do notgrow on leucine-deficient media nor show any b-galactosidase activity (data not shown). Yeast coloniesexpressing both the LexA/pRB and B42/CDP fusionproteins exhibit growth on selective media lackingleucine and form diagnostic blue colonies (Fig. 4A) inthe presence of galactose. However, transactivation ofthe LEU2 and b-galactosidase reporters was notobserved in cells containing both LexA/pRB and B42/CDP constructs in the absence of galactose (glucose-containing media), when expression of B42/CDP is notinduced. Hence, the yeast two-hybrid assays demon-strate that CDP and pRB participate in functionalprotein/protein interactions in vivo.

To assess the functional interaction between CDP andpRB in a more physiological setting, we performed amammalian two-hybrid assay. Vectors expressingGAL4/pRB and VP16/CDP fusion proteins and the corre-sponding vectors which express only GAL4 or VP16,were transiently transfected into HeLa GAL-5-Luc cells.As expected, cotransfection of both vectors expressingGAL4 or VP16 without the fusion moieties (i.e., pRB orCDP) did not activate the stably integrated luciferasereporter driven by tandem GAL4 binding sites. No

transactivation of the reporter was observed in cellsexpressing VP16/CDP and GAL4. Cells co-transfectedwith VP16 and GAL4/pRB exhibited increased reportergene expression, indicating that GAL4/pRB has intrin-sic activation potential under our experimental condi-tions. More importantly, when GAL4/pRB and VP16/CDP were co-expressed, we observed a dramatic activa-tion of reporter gene expression (Fig. 4B). A similarpattern of transcriptional activation was observed inNIH/3T3 cells and Saos-2 cells (data not shown). Theseresults suggest that CDP and pRB are capable offunctional interactions for transcriptional regulationin mammalian cells.

Formation of stable CDP/pRBcomplexes in transfected cells

We performed co-immunoprecipitation assays to fur-ther assess formation of CDP/pRB complexes withinthe intact cell. Interactions of both endogenous and co-expressed pRB with CDP were assessed in COS-7 cellstransiently expressing Myc-tagged CDP protein. Im-munoprecipitates were analyzed by western blot usingantibodies against the Myc tag or the pRB protein. Theresults show that pRB co-immunoprecipitates with full

Fig. 4. CDP CR2/C term interacts with pRB in vivo. Part A: Yeasttwo-hybrid assays were performed with LexA/pRB and B42/CDPconstructs. The two constructs were transformed into the EGY48yeast strain, which contains the dual reporters LEU2 and Lac Z undercontrol of the LexA binding site. The interaction of CDP with pRB inyeast cells is reflected by growth in the absence of leucine on galactosebut not on glucose containing plates. Detection of X-gal conversion wascarried out using filter lift assays. Part B: Mammalian two-hybridassays also reveal that CDP and pRB functionally interact. HeLa

Gal-5-Luc cells containing an integrated GAL4 responsive promoterwere transiently transfected with 0.5 mg of each different expressionconstructs encoding the GAL4 DNA binding domain (GAL4), GAL4fused to pRB (GAL4/pRB), the VP16 trans-activation domain (VP16),or VP16 fused to CDP (VP16/CDP). The total amount of DNAtransfected was kept constant at 1 mg with either GAL4 or VP16. Luci-ferase assays were performed 24 h later. Data are shown for arepresentative experiment performed in triplicate.

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length CDP (Fig. 5, Parts A and B) and to a lesser extentwith CDP (CR2-Cterm) (Fig. 5, Parts C and D). Specificimmunoprecipitation was not observed with normalmouse IgG that was used as a negative control. Inaddition, the pRB related protein p107, which formselectrophoretically stable complexes with CDP on thegp91phox (van Wijnen et al., 1994) and osteocalcin (vanGurp et al., 1999) promoters, is also capable of forming acomplex with full length CDP (Fig. 5E). Thus, optimalCDP–pRB interaction requires the CDP N-terminus,which includes a coiled-coil domain, a putative protein–protein interaction motif. Taken together, these dataestablish that CDP and pRB, as well as CDP and p107,form specific protein/protein complexes in COS-7 cells.

Subsets of pRB and CDP co-localize in situ

Because pRB and CDP exist in a complex in vivo andinteract functionally to transactivate heterologouspromoters, their association in situ within the nucleus

was assessed. Expression of endogenous CDP and pRBproteins was detected by in situ immunofluorescenceand analyzed by confocal microscopy. Colocalization oftwo proteins was assessed with the cross-correlationfunction, which establishes the degree of signal overlapbetween two images (see Materials and Methods).Endogenous pRB and CDP are approximately 35%colocalized (Fig. 6). To determine whether the observedcorrelation is due to random signal overlap, one imagefrom each image pair was rotated ten degrees and thecross-correlation within the remaining area of overlapwas measured. If the observed colocalization wererandom, rotation of the image would not change thedegree of signal overlap. Differences between rotatedand unrotated cross-correlation values were assessed bypaired Student’s t-test. Our analysis shows that theobserved colocalization of CDP and pRB in both HeLa(Fig. 6) and T98G glioblastoma cells (data not shown) isnot due to random signal overlap (P<0.00001). Thus,

Fig. 5. Interactions of CDP with the pRB family proteins pRB andp107 by co-immunoprecipitations. COS-7 cells were cotransfectedwith Myc-tagged full length CDP or CR2-Cterm CDP to detectinteraction with endogenous (Parts A and B) or co-expressed (Parts Cand D) pRB by co-immunoprecipitation assays. Cells were harvested32 h after transfection and co-immunoprecipitation was performedusing myc antibody (Parts A and C) or the pRB antibody (Parts B andD). Samples were analyzed by western blot with the respectiveantibodies. Input represents 3% of bound sample (Parts A and B), or10% of the bound sample (Part C). Interaction of CDP with the pRBfamily protein p107 by Co-IP (Part E). COS-7 cells were cotransfectedwith expression constructs for Myc-tagged full length CDP and p107.Cells were harvested 32 h after transfection and co-immunoprecipi-tation was performed using anti-myc and anti-pRB antibodies.Samples were analyzed by western blot with respective antibodies.Input represents 10% of the bound sample.

550 GUPTA ET AL.

endogenous CDP and pRB have significant nonrandomco-localization in situ within the nucleus.

Co-repression of H4 promoteractivity by pRB and CDP

To understand the mechanistic role of pRB in tran-scriptional regulation of histone genes, we co-expressedpRB together with CDP and measured H4 promoteractivity in HeLa, COS-7, and NIH/3T3 cells, as well asPANC-1 cells which do not express detectable levels ofCDP by western blot analysis (Li et al., 1999). The datashow that expression of pRB or CDP alone reduces H4promoter activity by four-fold to seven-fold (Fig. 7).When pRB is co-expressed with CDP, we observe thatreporter gene expression is inhibited 15–25 fold. Themagnitude of co-repression by pRB is comparable inthe four different cell lines tested (Fig. 7). Thus pRBand CDP interact functionally to repress histone H4transcription.

The LP of pRB functionally interactswith CDP to repress histone H4 transcription

The CDP C-terminus interacts with the wild type (LP)and phosphorylation mutant LP (PSM7-LP) of pRBin vitro (Fig. 3C). To test whether the LP of pRB is suffi-cient for co-repression of histone gene transcription, wecotransfected NIH/3T3 cells with the H4/luciferasereporter construct plus CDP and/or pRB constructs(Fig. 8). Because prolonged overexpression of pRBproteins in certain cell lines may result in a G1 cell cycleblock (Angus et al., 2002), we performed flow cytometric

analysis in parallel with these experiments. Our trans-fection efficiency in NIH/3T3 was consistently above50%. Thus, perturbation of cell cycle distribution wouldbe detected by flow cytometric analysis. However, nosignificant effect on the cell cycle distribution resultedfrom expression of pRB proteins for 22 h (data notshown). Immunofluorescence microscopy was used todetermine that the overexpressed pRB mutant proteinswere localized to the nucleus (data not shown). We findthat H4 promoter activity is strongly repressed by forcedexpression of CDP (�17 fold), whereas overexpressionof the LP or mutant LP (PSM-7-LP) alone causes amoderate repression of H4 gene transcription (�6 and�4 fold, respectively) similar to wild type pRB (�4 fold)(Fig. 8). Co-expression of CDP with pRB or wild typepRB LP results in increased repression of the H4-drivenluciferase reporter (�26 and �35 fold, respectively).

Taken together, our results suggest that pRB LP issufficient for co-repression with CDP. To determinewhether CDK phosphorylation sites in the LP arerequired for this interaction, we co-expressed CDP withthe phosphorylation site-deficient mutant PSM7-LP.We find that PSM7-LP and CDP function together asstrong repressors of H4 gene transcription (�67 fold)(Fig. 8). Thus,the CDP functional interaction with pRBLP is independent of LP phosphorylation sites. Thisobservation is consistent with a model in which pRB isavailable to interact with CDP only upon release fromE2F (Fig. 9). We postulate that upregulation of H4gene transcription requires coactivation by NPAT andHiNF-P and that the CDP/pRB interaction mediates

Fig. 6. Subsets of CDP/Cut and pRB co-localize within the nucleus.HeLa cells were analyzed by confocal microscopy. Cells grown ongelatin-coated coverslips were fixed, permeabilized, and incubatedwith antibodies against full-length CDP, and pRB, and fluorescentlytagged secondary antibodies. Bar represents 10 mm. (a pRB IF8)mouse monoclonal antibody raised against full-length pRB (Santa

Cruz Biotechnology); (a pRB C36) mouse monoclonal antibody raisedagainst amino acids 300–380 of pRB; (no 18 ab) no primary antibodieswere added to these coverslips; (DIC) differential interference con-trast. Merged inset is percent colocalization calculated as described inMaterials and Methods. The number of cells analyzed with a pRB IF8and a pRB C36 was 7 and 4, respectively.

CDP/pRB INTERACTIONS 551

Fig. 7. CDP and pRB co-repress H4 promoter activity. HeLa (Part A), NIH/3T3 (Part B), COS-7 (Part C),and PANC-1 (Part D) cells were co-transfected with the H4/Luc promoter reporter construct and a fixedamount (1 mg) of expression plasmids for CDP and pRB. Cells were harvested 28 h after transfection andlysates were used in luciferase assays. The data plotted in each part are from a representative triplicateexperiment that was repeated three times.

Fig. 8. The pRB LP is sufficient for CDP dependent co-repression ofH4 transcription. NIH/3T3 cells were transfected with a promoterlessluciferase reporter construct (ev/Luc) or an H4 promoter-driven luci-ferase construct (H4-Luc) and cotransfected with expression vectors forCDP and/or pRB proteins. Cells were harvested 22 h after transfection

andtotalcell lysatewasassayedforluciferaseactivity.Luciferasevalues(Part A) and fold repression (Part B) are shown. Error bars representstandard error of the mean where n¼12. (E.V.) empty vector backbonefor CDP and pRB expression constructs; (WT pRB) wild type pRB; (LP)pRB large pocket; (PSM7-LP) phosphorylation mutant pRB LP.

552 GUPTA ET AL.

downregulation of the H4 gene transcription in late Sphase when the demand for histone synthesis decreases.

DISCUSSION

In this study, we have presented multiple lines ofevidence indicating that CDP and pRB functionally andphysically interact as a protein/protein complex. Thisfinding provides a molecular mechanism for the inte-gration of the activities of CDP and pRB in the HiNF-Dprotein/DNA complex that interacts with Site II ofhistone H4 genes. Converging data obtained by thecombined application of GST pull-down assays, immu-noprecipitations, yeast and mammalian two-hybridanalyses, as well as immunofluorescence microscopyall indicate that CDP and pRB form a complex bothin vitro and in live cells. Functional assays show thatpRB acts as a co-repressor of CDP to modulate H4 genetranscription. Previous data from our laboratory haveindicated that CDP forms promoter-selective protein/DNA complexes in vitro with pRB on the cell cyclecontrolled histone H4, H3,and H1 promoters or with thepRB-related protein p107 on the cell type specificosteocalcin and gp91phox promoters (van Wijnen et al.,1996; van Gurp et al., 1999). Based on these results, wepropose that complexes between CDP and pRB are

biologically significant and perform important cell cyclerelated gene-regulatory functions.

The pRB tumor suppressor modulates cell cycleprogression through transcriptional regulation of genesrequired for the G1 to S transition (Morris and Dyson,2001). The protein can directly bind to and inactivatecertain promoter bound transcription factors, the mostnotable of which is E2F. Our data indicate that CDPprovides an E2F-independent mechanism for a cellgrowth regulatory function of pRB in HeLa, COS-7,PANC-1 and NIH/3T3 cells. Although it is well docu-mented that E2F is deregulated as a result of viral orcellular modifications in pRB, previous findings fromour laboratory show that the level of the HiNF-Dcomplex (i.e., the protein/DNA complex that is observedwith probes spanning the Site II cell cycle element) iselevated in HeLa and COS-7 cells, as well as in othertransformed(or tumor-derived) cell lines (Holthuisetal.,1990; van Wijnen et al., 1992). Thus, it appears thatformation of CDP/pRB complexes are not adverselyaffected by viral modifications of pRB. Consistent withthis concept, we find that pRB repression of histone H4promoter activity occurs in tumor-derived and/or virallytransformed cell types. It has previously been shownthat paired-like homeodomain proteins (e.g., Pax-3,

Fig. 9. Hypothetical model of CDP/pRB interaction in different cellcycle stages. We propose that HiNF-P and CDP-cut (as part of theHiNF-D complex) interactions with the Site II cell cycle regulatorysequences of the H4 gene integrate temporally distinct cell cycleregulatory signals. The growth factor dependent activation of cyclinE/CDK2 kinase complexes releases E2F from pRB at the restriction

(R) point. Concomitant activation of NPAT by cyclin E/CDK2 supportsthe subsequent HiNF-P dependent induction of the histone H4 gene atthe G1/S phase transition (Mitra et al., 2002). The pRB protein inter-acts with CDP/cut, cyclin A,and CDK1 to form the HiNF-D complexwhen cells progress through later stages of S phase.

CDP/pRB INTERACTIONS 553

MHox, Chx10) can interact with pRB through theirconserved homeodomains (Wiggan et al., 1998; Cveklet al., 1999; Eberhard and Busslinger, 1999). Our find-ings that the interaction of the CDP/cut protein withpRB requires a segment encompassing its cut-relatedhomeodomain are consistent with these observations.

The temporal correlation in S phase between maximalHiNF-D interaction with H4 Site II (Shakoori et al.,1995; Last et al., 1998; Stein et al., 1998) and thepresence of the CR2-Cterm cleavage product of CDP(Santaguida et al., 2001) suggests that CR2-Cterm maybe a component of HiNF-D. Our data show that the CDP(CR2-Cterm) moiety is sufficient for interaction withHiNF-D subunits cyclin A, CDK1, and pRB, and fordirect interaction with the pRB minimal A/B pocket.However, reporter transcriptional assays and co-immu-noprecipitation experiments indicate that the N-termi-nus of CDP, which contains a coiled-coil proteininteraction motif and the Cut Repeat 1 DNA-bindingdomain, is required for full repression of histone H4transcription and optimal levels of complex formationwith pRB in vivo. Thus, it is likely that full-length CDP,rather than the CR2-Cterm isoform, is present in theHiNF-D complex.

The CDP-dependent transcriptional repressor func-tion of pRB on histone genes operates independently ofE2F, because H4 gene promoters do not contain E2Fbinding sites (van Wijnen et al., 1996). Our results showthat the structural determinants of pRB that supportthe CDP/pRB interaction are similar to those requiredfor E2F. Specifically, both CDP and E2F interact withthe pRB LP, and neither protein depends strictly oncontacts with CDK phosphorylation sites in the pRB LP(Knudsen and Wang, 1997). Because of similarities inthe binding characteristics of E2F and CDP with pRB,these proteins may compete for binding to pRB inbiological conditions where these proteins are expressedsimultaneously. However, during cell growth stimula-tion (e.g., IL-3 dependent cell cycle entry of FDC-P1hematopoietic progenitor cells) when cells progress intoS phase, the levels of CDP/cut remain constant, thelevels of pRB are slightly elevated and the levels of theHiNF-D protein/DNA complex are dramatically ele-vated following hyperphosphorylation of pRB in late G1

(Shakoori et al., 1995; van Wijnen et al., 1997). Thisfinding suggests that the disruption of E2F/pRB com-plexes by pRB phosphorylation may increase theavailability of pRB for interaction with CDP/cut.

The CDP/pRB interaction may be of broader signifi-cance in regulating transcription during the cell cycle.Apart from regulating human histone H4 gene tran-scription, CDP represses the hamster TK promoter(Wiggan et al., 1998) and complexes containing pRB-related proteins interact with the TK promoter (Kimet al., 1996). Recent data by Lee and colleagues haveprovided evidence suggesting that the combined actionsof CDP and pRB at the histone H3.2 and TK promotersmay contribute to cell cycle control of transcription (Kimet al., 1996, 1997; Wu and Lee, 1998, 2002). However, incontrast to our findings that show a direct protein–protein interaction between CDP-cut and pRB, Lee andcolleagues presented data indicating that AP-2 may actas a bridging protein between pRB and CDP-cut byinteracting with pRB and with the cut-repeats in the

C-terminal half of CDP (Wu and Lee, 2002). Hence,there may be different mechanisms by which pRB andCDP are recruited to cell cycle controlled promoters. TheH4, H3.2, and TK genes are transiently activated at theG1/S phase transition, while the levels of the DNAbinding complex between CDP and pRB (i.e., HiNF-Dcomplex) are maximal in mid- to late- S phase (vanWijnen et al., 1997; Last et al., 1998). Therefore, wepropose that CDP/pRB co-repression may attenuatetranscription of the H4, H3.2, and TK genes during thelater stages of S phase when DNA synthesis rates aredecreasing and hence the demand for histones andnucleotides is diminishing.

ACKNOWLEDGMENTS

We thank all the members of our research group forstimulating discussions and valuable suggestions,Beata Paluch and Rosa Mastrototaro for technicalassistance, and Judy Rask for assistance with thepreparation of this manuscript. We also thank JeffreyNickerson and the 3-Dimensional Microscopy Labora-tory for assistance with confocal microscopy experi-ments. We are grateful to Ellis Neufeld (Children’sHospital, Boston, MA) for providing reagents and enthu-siastic support throughout the course of these studies.We also thank Drs. Jean Wang (University of California,San Diego), Roger Brent (Massachusetts GeneralHospital, Boston), Paul Robbins (University of Pitts-burgh, Pittsburgh), Keiko Ozato (National Instituteof Child Health and Human Development, NIH,Bethesda), and Erica Golemis (Fox Chase CancerCenter, Philadelphia) for kindly providing miscella-neous constructs or yeast strains that were useful forour studies.

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