# A B-myb promoter corepressor site facilitates in vivo occupation of the adjacent E2F site by p107-E2F and p130-E2F complexes

S. Catchpole, F. Tavner, L. Le Cam, C. Sardet, R. J. Watson

► To cite this version:

S. Catchpole, F. Tavner, L. Le Cam, C. Sardet, R. J. Watson. A B-myb promoter corepressor site facilitates in vivo occupation of the adjacent E2F site by p107-E2F and p130-E2F complexes. Journal of Biological Chemistry, 2002, 277 (41), pp.39015-39024. 10.1074/jbc.M202960200. hal-02197519

HAL Id: hal-02197519

https://hal.science/hal-02197519

Submitted on 27 May 2021

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>
# A *B*-myb Promoter Corepressor Site Facilitates *in Vivo* Occupation of the Adjacent E2F Site by p107·E2F and p130·E2F Complexes*\*

Received for publication, March 27, 2002, and in revised form, July 9, 2002
Published, JBC Papers in Press, July 29, 2002, DOI 10.1074/jbc.M202960200

Steven Catchpole‡, Fiona Tavner‡, Laurent Le Cam§, Claude Sardet§, and Roger J. Watson‡¶

From the ‡Ludwig Institute for Cancer Research and the Section of Virology and Cell Biology, Imperial College of Science, Technology and Medicine, Faculty of Medicine, Norfolk Place, London W2 1PG, United Kingdom and the §Institut de Génétique Moléculaire, UMR 5535, CNRS, 1919 route de Mende, 34293 Montpellier Cedex 5, France

Transcription from the *B-myb* (*MybL2* gene) promoter is strictly cell cycle-regulated by repression mediated through an E2F site during G₀/early G₁. We report here the characterization of a corepressor site (downstream repression site (DRS)) required for this activity that is closely linked to the E2F site. Systematic mutagenesis of the DRS enabled a consensus to be derived, and it is notable that this sequence is compatible with cell cycle gene homology region sequences associated with cell cycle-dependent elements in the cyclin A, *cdc2*, and *CDC25C* promoters. The *B-myb* promoter is inappropriately active during G₀ in mouse embryo fibroblasts lacking the p107 and p130 pocket proteins, and we show that the ability of transfected p107 and p130 to re-impose repression on the promoter is dependent on the DRS. In contrast, transfected Rb was unable to repress the *B-myb* promoter. Consistent with the notion that Rb-E2F complexes are unable to bind the *B-myb* promoter E2F site *in vivo*, footprinting showed that this site is unoccupied in cells lacking p107 and p130. Chromatin immunoprecipitation assays showed a requirement for the DRS in recruiting p107 and p130 complexes to the *B-myb* promoter, indicating that *in vivo* the DRS governs the occupancy of the adjacent E2F site by transcriptional repressors.

It has become well established that E2F transcription factor activity, through its interactions with the retinoblastoma family of pocket proteins, plays a major role in coordinating gene transcription during the mammalian cell cycle. E2F activity is a heterogeneous entity made up of heterodimers from two distantly related protein families, E2F and DP (1, 2). Each component of the E2F protein family, E2F-1 through E2F-6, appears to be functional in association with either member of the related DP protein family, DP-1 and DP-2 (3). E2F-1, E2F-2, and E2F-3 heterodimers form complexes preferentially with the retinoblastoma protein Rb, whereas the related pocket proteins p107 and p130 associate preferentially with E2F-4 and E2F-5. This demarcation is not absolute, however; and it has been reported that Rb-E2F-4 complexes are the predominant species in the nuclei of actively dividing cells during G₁ (4). E2F-6 lacks the C-terminal domain that, in the other species, has been implicated in transactivation function and interaction with the pocket proteins (5–8).

The diversity present within the E2F and pocket protein families suggests that different E2F complexes may play distinct roles in cell cycle regulation of gene transcription. In support of this notion, it was found that “knockouts” of specific pocket protein genes by homologous recombination have quite different effects on mouse embryonic development. Whereas Rb⁻/⁻ mice die *in utero* with defects in the liver, central nervous system, and ocular lens and a profound reduction in definitive erythropoiesis (9), p107⁻/⁻ and p130⁻/⁻ mice develop normally (10). Inactivation of both p130 and p107 results, however, in neonatal lethality, with defects in bone and cartilage arising from hyperproliferation of chondrocytes (10). Differences in pocket protein function are also manifest at the level of cell cycle-regulated transcription in mouse embryo fibroblasts (MEFs)<fnref n="1" /> derived from mutant animals (11). Whereas elimination of either p107 or p130 had no discernible effect on cell cycle-regulated transcription, knockout of both p107 and p130 genes resulted in deregulated expression of a set of genes (*B-myb*, *cdc2*, E2F-1, thymidylate synthase, *RRM2*, and cyclin A2) distinct from those deregulated in Rb⁻/⁻ MEFs (cyclin E and p107). These phenotypic differences between Rb⁻/⁻ and p107⁻/⁻/p130⁻/⁻ MEFs are unlikely to be due to gene dosage effects, as studies with established knockout fibroblasts showed that deregulation of the *B-myb* promoter in p107⁻/⁻/p130⁻/⁻ cells could not be recapitulated in either p107⁻/⁻/Rb⁻/⁻ or p130⁻/⁻/Rb⁻/⁻ cells (12). It is therefore evident that p107 and p130 have a functionally redundant role in E2F-mediated gene regulation, which is distinct from that of Rb.

It is currently unclear how different pocket protein-E2F complexes discriminate between the genes they regulate. One possibility is that they demonstrate some sequence specificity for the E2F binding site in the promoters of these genes. Although most *in vitro* binding studies have failed to show this, repetitive selection of redundant binding sites by the CASTing procedure (13) suggests that different E2F complexes do have inherent preferences for particular E2F sites. An additional possibility is that occupation of the E2F site *in vivo* is influenced by factors binding to adjacent sites or that the ability of E2F complexes to regulate transcription, whether by repression or activation, is dependent upon interactions with these accessory factors. The contribution of a putative accessory binding site to E2F-dependent transcription is exemplified by *B-myb* (the *MybL2* gene), in which it was found that cell cycle regulation is influenced by a distinct promoter site located immediately down-

<footnote>\* This work was supported in part by Biotechnology and Biological Sciences Research Council Project Grant 28/C08517. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.</footnote>

<footnote>¶ To whom correspondence should be addressed. Tel.: 44-20-7563-7711; Fax: 44-20-7724-8586; E-mail: r.watson@ic.ac.uk.</footnote>

<footnote n="1">¹ The abbreviations used are: MEFs, mouse embryo fibroblasts; DRS, downstream repression site; CDE, cell cycle-dependent element; CHR, cell cycle gene homology region; mE2F, mutant E2F site; mDRS, mutant downstream repression site; ChIP, chromatin immunoprecipitation; RbNP, non-phosphorylatable Rb; EMSAs, electrophoretic mobility shift assays.</footnote>
stream of the E2F site (17, 32). Mutations in either the E2F site or the adjacent site (downstream repression site (DRS)) abolish transcriptional repression in G₀/G₁. Significantly, *in vivo* footprinting studies revealed that the B-myb promoter E2F site is occupied in quiescent cells, but becomes unoccupied in mid G₁ preceding the induction of B-myb transcription (14). As E2F abundance actually increases at the G₁/S boundary, it is possible that the role of the DRS is to stabilize interactions of repressor complexes at the adjacent E2F site specifically in G₀/early G₁.

Transcriptional repression is a common mechanism to extinguish expression of periodically regulated genes during cell cycle arrest and quiescence (15–28); however, there is evidence that the regulation of certain of these genes is not directly dependent on E2F. Most notably, transcriptional repression of *CDC25C* in G₀/early G₁ involves bipartite elements termed CDE/CHR (23, 29). The *CDC25C* CDE/CHR elements are partially homologous to the B-myb E2F/DRS sites and, moreover, have an identical spatial relationship (30); however, E2F binds weakly if at all to CDE/CHR. Compared with E2F/DRS, the CDE/CHR elements confer a subtly different cell cycle kinetics on promoters: both sets of elements permit derepression of transcription in the latter part of G₁; however, induction is somewhat prolonged through S/G₂ when regulated by CDE/CHR (30). To gain further understanding of the relationship between the downstream DRS and DCE elements, we have extensively characterized the B-myb DRE by mutagenesis. We have also studied the role played by B-myb E2F/DRS sites in transcriptional regulation *in vivo* and conclude that the DRS facilitates interaction of repressive p107-E2F and p130-E2F complexes with the adjacent E2F site.

## EXPERIMENTAL PROCEDURES

**Cell Culture and Flow Cytometry**—MEFs from control p107<sup>+/-</sup> and gene knockout p107<sup>-/-</sup>/p130<sup>-/-</sup> animals were kindly provided by Dr. Nick Dyson (Massachusetts General Hospital Cancer Center, Charlestown, MA) and were expanded and used at passage 4. MEFs and Swiss 3T3 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. Established 3T3 cell lines were derived from MEFs by splitting cultures every 3 days according to standard procedures. These established 3T3 and NIH 3T3 cells (obtained from Dr. René Bernards) were maintained in Dulbecco's modified Eagle's medium supplemented with 10% newborn calf serum. Flow cytometry was performed on ethanol-fixed cells stained with propidium iodide as described previously (22).

**B-myb Promoter/Reporter Plasmids and Preparation of Stably Transfected Cell Lines**—The mouse B-myb promoter/reporter cassette was made using PCR with Pfu DNA polymerase (Stratagene) to introduce *AatII* and *BclI* sites 5' and 3', respectively, of the B-myb promoter E2F/DRS sites in the pGL2(-536) luciferase reporter (15). Double-stranded DNA oligonucleotides containing 5'-*AatII* and 3'-*BclI* ends were then inserted using T4 DNA ligase to replace the wild-type sequence with variant transcriptional control sites. The sequences of these oligonucleotides (sense strand only) were as follows: mE2F, CCT-TGTATGGAGATAGGAAAGT; mDRS, CCTTGGCGGGAGATAGGCC-TGT; CDE/CHR, CGCTGGCGAAGGTTTGAATGT; E2F/CHR, CCTT-GGCGGGAGATTTGAATGT; CDE/DRS, CGCTGGCGAAGGTAGGA-AAGT; E2F/DRS+2, CCTTGGCGGGAGATATAGGAAAGT; and E2F/DRS+4, CCTTGGCGGGAGATATATAGGAAAGT. Additional mutations in the DRS were introduced as indicated in Fig. 1B.

To insert wild-type, mE2F, and mDRS B-myb promoter sequences into the pAG/EluW luciferase reporter, which contains flanking scaffold/matrix attachment regions from the human interferon-β gene to attenuate positional effects in chromosomally integrated plasmids (31), fragments with terminal *Hind*III sites were generated by PCR with Pfu DNA polymerase using pGL2(-536), pGL2(-536)mut1, and pGL2(-536)mut9 as templates (32). The primers used (5'-AGCTAAGCTTC-CAGTCTTTGCTATGTGTGTG and 3'-ACGTAAGCTTCGAGCCGCTC-CGGGCCCCAGG) amplified promoter sequences from -533 to -88 with respect to the start of the protein coding sequence, and these *Hind*III fragments replaced the SV40 promoter in pAG/EluW. Plasmids were transfected into NIH 3T3 cells using calcium phosphate coprecipitation, and stable transfectants were selected with 0.5 µg/ml G418

using the conditions described to limit copy number (31). Clones carrying approximately five copies of each transgene were selected for chromatin immunoprecipitation (ChIP) analysis.

**Pocket Protein Expression Plasmids**—An expression plasmid encoding untagged p107, pCMVp107, was kindly provided by Dr. Liang Zhu. pcDNA3 (Invitrogen) expression plasmids encoding Myc epitope-tagged wild-type Rb and non-phosphoryatable Rb (RbNP; in which 14 of 15 cyclin-dependent kinase sites were mutated to alanine) were kindly provided by Dr. Sybille Mittnacht (33). Expression vectors encoding Myc epitope-tagged p107 and p130 proteins were constructed by fusing p107 and p130 coding sequences lacking the initiating methionine codon, generated by a combination of PCR and restriction endonuclease digestion, downstream of the Myc epitope coding sequence in pcDNA3(9E10). A combination of PCR and restriction endonuclease digestion was also used to generate coding sequences specifying fragments of p130. These were cloned downstream of the Myc epitope coding sequence in pcDNA3(9E10): mutant N417 (amino acids 417–1139) contains the A and B pockets, the spacer region, and the entire C-terminal domain; mutant C793 (amino acids 2–793) is C-terminally truncated within the spacer; mutant C1065 (amino acids 2–1065) is C-terminally truncated immediately after the B pocket; and mutant dl622–818 contains a deletion of most of the spacer. PCR-generated sequences were checked by DNA sequencing.

**Cell Transfection and Reporter Assays**—MEFs and 3T3 derivatives were transfected according to the manufacturer's instruction in 60-mm Petri dishes (10⁵ cells) using 20 µl of LipofectAMINE (Invitrogen) and 4 µg of DNA (3 µg of luciferase reporter + 1 µg of β-galactosidase plasmid). After 5 h, the LipofectAMINE was removed, and cells were allowed to recover in Dulbecco's modified Eagle's medium containing 10% fetal calf serum overnight before replacing the medium with Dulbecco's modified Eagle's medium containing 0.5% fetal calf serum. Cell lysates were made 60 h later, and luciferase and β-galactosidase assays were performed as described (15). Where cell extracts were prepared from transfected 3T3 derivatives, cells were transfected in 100-mm dishes using 60 µl of LipofectAMINE and a total of 12 µg of DNA.

**Northern and Western Blots**—RNA was prepared from p107<sup>+/-</sup> and p107<sup>-/-</sup>/p130<sup>-/-</sup> 3T3 cells using RNAzol B (Biogenesis) as described by the manufacturer and resolved on 1% agarose gels containing 2.2 M formaldehyde. Northern blots were probed with a ³²P-labeled murine B-myb cDNA probe. Western blotting was performed under standard conditions using anti-c-Myc antibody 9E10 (Santa Cruz Biotechnology sc-40) to detect the tagged pocket proteins.

**Electrophoretic Mobility Shift Assays**—Nuclear and cytoplasmic protein fractions were prepared from MEFs and transfected 3T3 cell lines after lysing in a hypotonic buffer containing 0.4% Nonidet P-40 essentially as described (34). Equivalent amounts of nuclear and cytoplasmic fractions (~10 µg) were used in electrophoretic mobility shift assays (EMSAs) using an oligonucleotide probe encompassing the mouse B-myb promoter E2F/DRS sites as described previously (15). Where appropriate, antibodies to p107 (monoclonal antibody SD15; a gift of Dr. Nick Dyson), E2F-4 (polyclonal; a gift of Dr. Eric W.-F. Lam), p130 (polyclonal; a gift of Dr. Antonio Giordano), Rb (monoclonal antibody 21C9; a gift of Dr. Sybille Mittnacht), or c-Myc (monoclonal antibody 9E10) were added to supershift specific complexes. Whole cell extracts were prepared from NIH 3T3 cells stably transfected with the wild-type or mDRS B-myb promoter as described previously (15). These were used in EMSAs as described above; however, the antibodies used in super-shifts were the same as those used in ChIP assays (rabbit anti-p107 polyclonal antibody, Santa Cruz Biotechnology sc-318; rabbit anti-p130 polyclonal antibody, Santa Cruz Biotechnology sc-317; and anti-Rb monoclonal antibody 21C9).

**In Vivo Footprinting and Chromatin Immunoprecipitation**—Footprints were obtained by linker-mediated PCR on DNA alkylated *in vivo* by addition of 0.2% dimethyl sulfate to cell cultures as described previously (22). The B-myb primers used were as published (14).

ChIP assays were performed using essentially the published method (35). Sonicated chromatin from 10⁷ cells treated *in vivo* with 1% formaldehyde for 10 min at ambient temperature was immunoprecipitated with 2 µg of each antibody (control (normal rabbit serum), rabbit anti-p107 polyclonal, and rabbit anti-p130 polyclonal antibodies and anti-Rb monoclonal antibody 21C9) and collected by mixing for 1 h at 4 °C with 45 µl of protein A-Sepharose 4B beads (50% suspension in TE buffer (20 mM Tris-HCl (pH 8.1) and 1 mM EDTA) preadsorbed with sonicated salmon sperm DNA (10 µg/ml). Rabbit anti-mouse IgG (2 µg; Sigma M7023) was added to monoclonal immunoprecipitations for 1 h before addition of protein A beads. The beads were collected by brief microcentrifugation, and the supernatant of the control antibody sample was retained for use as an input control. The beads were washed
successively for 10 min at 4 °C with TSE buffer (1% Triton X-100, 0.1% SDS, 2 mM EDTA, and 20 mM Tris-HCl (pH 8.1)); TSE buffer containing 150 mM NaCl; TSE buffer containing 500 mM NaCl; buffer containing 250 mM LiCl, 1% Nonidet P-40, 1% deoxycholate, 1 mM EDTA, and 10 mM Tris-HCl (pH 8.1); and TE buffer. Immunoprecipitated DNA was eluted by washing the beads three times with 150 µl of 1% SDS and 100 mM NaHCO₃, and then 10 µg of salmon sperm DNA was added to the pooled eluates. The cross-links were reversed by heating at 65 °C for 4 h, and then the DNA was phenol-extracted twice and ethanol-precipitated. DNA was recovered by centrifugation and dissolved in 350 µl of buffer containing 100 mM NaCl, 10 mM Tris-HCl (pH 8.1), and 1 mM EDTA. After reprecipitation with ethanol, the final pellet was dissolved in 30 µl of TE buffer. PCR amplification was carried out for 25 or 26 cycles on 2-µl amounts using Taq polymerase. Separate reactions were carried out to detect the transgene and the endogenous B-myb gene; transgene reactions used a 5'-B-myb primer (CCTTCCGTATGCTCCGCC) and a 3'-primer to the luciferase gene (TCATAGCCTTATGCAGTTGCTCTCC), whereas endogenous reactions used 5'- and 3'-B-myb primers (ATTGAATCCCTAAGGTAGGTGTATCTG and TGGTCGCACGTTCCCCAG, respectively). PCR products were resolved on 1.5% agarose gels, Southern-blotted, and detected by hybridization with a 5'-³²P-labeled oligonucleotide probe (TTCTGTGCGCTCCCTAGGCAGCTGCAGTTCCTGGGAACG). Hybridization was quantitated by phosphorimaging.

RESULTS

*Identification of Functionally Critical DRS Nucleotides*—To characterize the precise sequence requirement for transcriptional repression of the B-myb promoter, each nucleotide within the previously delimited DRS motif (GGAAA, nucleotides −198 to −194 relative to the start of the coding sequence) was mutated individually to each of the three alternatives. To facilitate this, AatII and BclI restriction sites were first introduced into the B-myb promoter at sequences immediately flanking the E2F/DRS elements by substituting 2 and 3 nucleotides, respectively (Fig. 1A). Previous studies have shown that the regions mutated do not influence cell cycle-regulated transcription (32). Double-stranded oligonucleotides containing DRS mutations could then be quickly transferred into the modified reporter plasmid, the so-called B-myb promoter cassette, by direct ligation (see “Experimental Procedures”). The functions of the wild-type and mutant DRS elements were then assayed in transient transfection assays of NIH 3T3 cells. Following transfection, the cells were arrested in G₀ by serum deprivation, and cell extracts were made from these cells and from duplicates that were induced to enter S phase by serum stimulation for 16 h. The results of a typical experiment (Fig. 1B) show that the central 3 nucleotides in the DRS (GGAAA, nucleotides −197 to −195) are particularly critical for E2F-mediated transcriptional repression. Thus, mutation of either A residue to any other nucleotide resulted in a significant increase in promoter activity in both G₀ and S phase cells. Mutation of the central G residue to A had no effect upon promoter activity, whereas either C or T at this position resulted in a very significant increase in promoter activity. It is pertinent that the equivalent DRS sequence in the human B-myb promoter is GAAAA, consistent with the evidence that either G or A at nucleotide −197 is permissive for repression function. In contrast, mutation of the first G residue of the DRS (nucleotide −198) had only a marginal effect on transcriptional derepression; mutation to A led to a slight loss of repression, whereas mutation to C actually resulted in a slight increase in repression. Mutation of the ultimate A residue (nucleotide −194) to C or T had little or no effect upon DRS function, whereas substitution by G led to a substantial loss of transcriptional repression (Fig. 1B). Comparison of a DRS consensus sequence derived from this analysis with CHR sequences located downstream of the cyclin A2, cdc2, and CDC25C CDE sites (Fig. 1C) revealed that all three CHRs are actually compatible with the DRS sequence requirements.

<figure><img src="image_2.png" /></figure>

<figure><img src="image_3.png" /></figure>

FIG. 1. Characterization of nucleotides required for DRS function. A, nucleotides flanking the E2F/DRS sites in the B-myb promoter/reporter pGL2(−536) were mutated as indicated in lowercase to generate AatII and BclI restriction sites. B, double-stranded oligonucleotides containing the DRS mutations indicated in lowercase were introduced into the pGL2(−536) cassette by replacement of the AatII/BclI fragment. The wild-type and mutant promoter/reporters were transiently transfected into NIH 3T3 cells, and luciferase activities were measured in cells arrested in G₀ and in parallel cultures that were restimulated by re-addition of serum (S phase). Flow cytometry indicated that 62% of the cells were measurably in S phase at the time the serum-induced samples were collected. C, shown is a comparison of the mouse B-myb E2F/DRS sequences (underlined) with the bipartite CDE/CHR sequences (also underlined) in the human cyclin A (CCNA), CDC2, and CDC25C genes. With respect to the major transcription start sites, the sequences presented are B-myb (−49/−31), CCNA (−38/−20), CDC2 (−25/−7), and CDC25C (−19/−1). Nucleotide changes permissible for DRS function are indicated above the DRS sequence.

It is notable that promoters in which the critical GAA core of the DRS is mutated were substantially induced in S phase (Fig. 1B), suggesting that the E2F site can mediate transcriptional activation in S phase when DRS function is compromised. To test this possibility, the activity of a promoter in which both sites have been mutated was compared with those of reporters containing single E2F or DRS mutations, and this assay confirmed that activation of the mDRS promoter was indeed abolished when the adjacent E2F site was also mutated (Fig. 2A). As the mDRS promoter was significantly more active in S phase than the wild-type promoter, it appears that transactivating E2F complexes are able to bind to the B-myb E2F site only in the absence of the DRS. It may therefore be suggested that the DRS specifically prevents activating E2F species from binding to the adjacent E2F site while conversely facilitating binding of repressive E2F complexes.

To determine whether the *CDC25C* CHR could indeed substitute for a functional B-myb DRS and, conversely, whether the B-myb DRS could substitute for the *CDC25C* CHR, the E2F/DRS sites in the B-myb promoter cassette were replaced
Function of an E2F Corepressor Site in the B-myb Promoter

<figure><img src="image_4.png" /></figure>

FIG. 2. Characterization of DRS function. A, the B-myb promoter/luciferase reporter cassette containing the wild-type (wt) sequence or variants in which the wild-type sequence was replaced by E2F/DRS elements containing mutations in the E2F site (mE2F, CTTGtatG-GAGATAGGAAAG), the DRS (mDRS, CTTGGCGGGAGATAGGcctG), or both (mE2F/mDRS, CTTGtatGGAGATAGGcctG) were transiently transfected into NIH 3T3 cells, and luciferase activities were measured in cells arrested in G₀ and in parallel cultures that were restimulated by re-addition of serum for 16 h (S phase). B, the B-myb promoter/luciferase reporter cassette containing the wild-type sequence (E2F/DRS, CTTGGCGGGAGATAGGAAAG) or variants in which the wild-type sequence was replaced by the CDC25C CDE/CHR sequence (CDE/CHR, GCTGGCGGAAGGTTTTGAATG) or combinations of these elements (E2F/CHR, CTTGGCGGGAGATTTGAATG; and CDE/DRS, GCTG-GCGGAAGGTAGGAAAG) were transiently transfected into NIH 3T3 cells, and luciferase activities were compared as described for A. C, the B-myb promoter/luciferase reporter cassette containing the wild-type sequence or variants in which the wild-type sequence was replaced by E2F/DRS elements containing intervening iterations of either +2 or +4 nucleotides (CTTGGCGGGAGAtaTAGGAAAG and CTTGGCGG-GAGAtataTAGGAAAG, respectively) were transiently transfected into NIH 3T3 cells, and luciferase activities were compared as described for A.

with wild-type *CDC25C* CDE/CHR or hybrid E2F/CHR and CDE/DRS sites (Fig. 2B). Transfection into NIH 3T3 cells showed that the *CDC25C* CDE/CHR conferred appropriate cell cycle regulation on the *B-myb* promoter in that the promoter was repressed in G₀ cells and derepressed in S phase (Fig. 2B). Significantly, the hybrid E2F/CHR element conferred more rigorous regulation on the *B-myb* promoter than either E2F/ DRS or CDE/CHR, whereas in contrast, substitution by the hybrid CDE/DRS element led to complete deregulation (Fig. 2B). These findings indicate that the DRS and CHR are not absolutely equivalent. Whereas both elements are functional in the context of the E2F site, the CHR also has a specific function in the context of the CDE, which cannot be substituted by the DRS.

To determine whether the relative positions of the B-myb
E2F/DRS sites are important for their functional interaction,
small iterations of 2 and 4 nucleotides were introduced between
these sites in the reporter cassette, and transient transfections

<figure><img src="image_5.png" /></figure>

FIG. 3. Transcriptional repression of the B-myb promoter by p107-E2F complexes requires the DRS. A, the wild-type (wt) B-myb promoter/reporter pGL2-(−536) or derivatives containing mutations in either the E2F (mE2F) or DRS (mDRS) site were transfected into primary MEFs derived from control p107<sup>+/−</sup> or knockout p107<sup>−/−</sup>/p130<sup>−/−</sup> animals. Luciferase was assayed in transfected cells deprived of serum for 60 h. B, knockout p107<sup>−/−</sup>/p130<sup>−/−</sup> MEFs were transfected with the B-myb promoter/reporters described for A together with either the empty pCMV expression vector (control (Con)) or vector encoding p107 or Myc epitope-tagged p130. Luciferase was assayed in transfected cells deprived of serum for 60 h. Data are presented as -fold repression, calculated by dividing the average luciferase value obtained with the control empty vector by the average value obtained for that reporter with the p107 or p130 expression vector. The control for each reporter was assigned a value of unity.

assays were performed as before. Displacing the DRS from the
E2F site by 2 nucleotides resulted in significant transcriptional
derepression in G₀ (Fig. 2C), and this effect was even more
pronounced when the sites were displaced by 4 nucleotides.
These results indicate that the spatial relationship between the
E2F and DRS sites is essential for their cooperation.

The DRS Is Required for p107-mediated Repression of B-myb
Transcription—Repression of B-myb transcription is substan-
tially deregulated in quiescent MEFs derived from embryos
lacking both the p107 and p130 genes, although it is unaffected
in MEFs lacking either of these pocket proteins alone or in
MEFs lacking Rb (11). These data suggest that p107-E2F and
p130-E2F complexes have a redundant function in repressing
the B-myb promoter during G₀, which cannot be compensated
by Rb-E2F. Consistent with this notion, reintroduction of p107
and, to a lesser extent, p130 into p107⁻/⁻/p130⁻/⁻ MEFs by
transient transfection results in repression of a cotransfected
B-myb promoter (11), and this activity depends on an intact
E2F site. The p107⁻/⁻/p130⁻/⁻ cells therefore provide a con-
venient system in which to investigate further the interactions
between pocket protein-E2F complexes and the bipartite E2F/
DRS site. To determine first the contribution of the DRS in this
system, we performed additional transient transfection exper-
iments in p107⁻/⁻/p130⁻/⁻ and control p107⁺/⁻ MEFs. These
studies confirmed that the wild-type B-myb promoter is signif-
icantly deregulated in quiescent p107⁻/⁻/p130⁻/⁻ MEFs com-
pared with control cells (Fig. 3A). It is notable, however, that
deregulation of the wild-type promoter was incomplete in the
knockout cells compared with a mE2F site promoter, suggest-
ing that quiescent p107⁻/⁻/p130⁻/⁻ MEFs actually contain di-
minished rather than no E2F repressor activity. It is notable
also that the DRS mutation had similar effects in both quies-
cent p107⁻/⁻/p130⁻/⁻ and control MEFs, being somewhat de-
<figure><img src="image_6.png" /><figcaption>FIG. 4. The DRS is required for occupation of the B-myb E2F site with p130 and p107 complexes. A, stable NIH 3T3 transfectants were established with luciferase reporter genes driven by either the wild-type (wt) B-myb promoter or mutant B-myb promoters with nonfunctional E2F (mE2F) or DRS (mDRS) sites. The reporter genes were in each case flanked by scaffold/matrix attachment regions from the human interferon-β gene to attenuate positional effects, and clones were selected with approximately five transgene copies. Luciferase activities were assayed in cells arrested in G₀ by serum deprivation for 60 h and after restimulation with serum for 16 h (S phase). B, EMSA was performed with whole cell extracts prepared from NIH 3T3 cell clones stably transfected with the wild-type and mDRS B-myb promoter/luciferase reporters. Pocket protein-E2F complexes were supershifted with antibodies (Ab) against p107, p130, or Rb as indicated. Control reactions (Con) contained preimmune rabbit serum. C, NIH 3T3 cell clones stably transfected with the wild-type and mDRS B-myb promoter/luciferase reporters were arrested in G₀ by serum deprivation, and then protein-DNA complexes were formaldehyde-cross-linked in vivo. Chromatin fragments from these cells were subjected to immunoprecipitation (IP) with control serum or with antibodies to p107, p130, or Rb as indicated. After cross-link reversal, the co-immunoprecipitated DNA was amplified by PCR and detected by Southern blotting. Separate primers were used to detect the transgene and the endogenous gene. D, shown is the quantitation by phosphorimaging of the results shown in C for the wild-type promoter-transfected cell transgene (wt/Tr) and endogenous gene (wt/En) and the mDRS transgene (mDRS/Tr) and endogenous gene (mDRS/En).</figcaption></figure>

repressed compared with the wild-type promoter in control MEFs, but not further deregulated in p107⁻/⁻/p130⁻/⁻ MEFs (Fig. 3A). These findings suggest that residual E2F repressor activity in p107⁻/⁻/p130⁻/⁻ MEFs is impervious to the influence of the DRS.

As reported previously (11), we found that reintroducing p107 and p130 by transfection into quiescent p107⁻/⁻/p130⁻/⁻ MEFs resulted in repression of the wild-type B-myb promoter (Fig. 3B). Consistent with the previous study (11), we found that p107 repressed the wild-type B-myb promoter more strongly than p130, although this may simply reflect the relative expression levels of these proteins in these cells. Significantly, the ability of p107 and p130 to repress B-myb promoter activity was dependent upon both an intact E2F site and DRS (Fig. 3B). Stimulation by p107 of both the mE2F site and mDRS promoters observed in this experiment presumably reflects an effect on constitutive activators binding to sites upstream of the cell cycle control elements. Overall, it is clear that the DRS is essential for the imposition of transcriptional repression mediated by p107-E2F complexes through the adjacent E2F site.

The DRS Is Required for in Vivo Interactions with p130-E2F and p107-E2F Complexes—Although it is clear from this study that the DRS is required for B-myb transcriptional repression by p130 and p107, previous *in vitro* binding assays did not indicate any influence of the DRS on binding of E2F complexes containing these pocket proteins to the B-myb promoter site (32). To explain this conundrum, we set out to determine whether the DRS influences occupation of the adjacent E2F site *in vivo*. To facilitate this analysis, the wild-type and mutant B-myb promoters were cloned into a luciferase reporter gene that incorporates scaffold/matrix attachment sites to

attenuate positional effects on the chromosomally integrated promoter (31). Stably transfected NIH 3T3 cells expressing these transgenes were then established using low DNA inputs to reduce plasmid copy number, and clones containing approximately five copies were selected for analysis. It is evident that the wild-type and mutant promoters were regulated in these clones in a manner similar to that in transient transfections (Fig. 4A). Thus, the wild-type B-myb promoter was activated 8.5-fold as serum-arrested cells entered S phase, whereas in contrast, the mE2F site promoter was completely derepressed in G₀. Significantly, the mDRS promoter was substantially deregulated in G₀, but was induced ~1.5-fold in S phase (Fig. 4A). EMSAs using extracts prepared from wild-type and mDRS promoter-transfected NIH 3T3 cell lines showed a predominance of p130-E2F complexes during G₀ (Fig. 4B). Lesser amounts of p107 complexes were also present, but no Rb complexes were detectable (Fig. 4B).

Previous studies of these cell lines using ChIP assays have shown that the wild-type promoter transgene is bound by E2F-4 complexes in quiescent cells and that this association is abrogated by mutation of the E2F site (37). We adopted a similar approach to study whether the DRS influences occupation of the adjacent E2F site. Protein-DNA complexes were formaldehyde-cross-linked in cells stably expressing the wild-type and mDRS promoter/luciferase reporters, and sites bound by pocket protein-E2F complexes were immunoprecipitated with control, anti-p107, anti-p130, or anti-Rb antibodies. PCR primer pairs were designed to detect selectively either the B-myb transgene (the 3'-primer is homologous to luciferase sequences) or the endogenous B-myb gene. We found that the transfected wild-type B-myb promoter was bound predomi-
<figure><img src="image_7.png" /><figcaption>**FIG. 5.** Knockout p107<sup>-/-</sup>/p130<sup>-/-</sup> embryos contain abundant nuclear Rb-E2F complexes. Cytoplasmic (CYT) and nuclear (NUC) extracts were prepared from knockout p107<sup>-/-</sup>/p130<sup>-/-</sup> and control p107<sup>+/-</sup> MEFs that were arrested in G<sub>0</sub> by serum deprivation for 60 h. EMSA was performed with these extracts using a <sup>32</sup>P-labeled oligonucleotide probe containing the B-myb E2F/DRS sites. Control (Con; preimmune rabbit serum), anti-p130, anti-Rb, anti-p107, or anti-E2F-4 antibodies were added to EMSA reactions as indicated. Arrowheads indicate the positions of various E2F complexes and antibody (Ab) supershifts. A, p107<sup>-/-</sup>/p130<sup>-/-</sup> MEF cytoplasmic extracts; B, p107<sup>+/-</sup>/p130<sup>-/-</sup> MEF nuclear extracts; C, p107<sup>+/-</sup> MEF cytoplasmic extracts; D, p107<sup>+/-</sup> MEF nuclear extracts.</figcaption></figure>

nantly by p130 complexes and, to a lesser extent, by p107 complexes, but little Rb was cross-linked to this site (Fig. 4, C and D). The endogenous promoter in these cells showed a very similar pattern of occupancy. The relative levels of p130 and p107 binding to the B-myb promoter detected in the ChIP assays therefore reflected their levels associated with E2F complexes as detected by EMSAs (Fig. 4B). Significantly, p130 and p107 complexes were only weakly associated with the mDRS promoter, although interactions of these proteins with the endogenous promoter were still detected in these cells (Fig. 4, C and D). These experiments therefore demonstrate that the DRS has an important role in promoting binding of p130-E2F and p107-E2F complexes to the adjacent E2F site.

*In Vitro Analysis of E2F Complexes in p107<sup>-/-</sup>/p130<sup>-/-</sup> MEFs*—In contrast to NIH 3T3 cells, quiescent p107<sup>-/-</sup>/p130<sup>-/-</sup> MEFs contain abundant Rb-E2F complexes, which are readily detectable by EMSAs using an adenovirus E2A promoter probe (11). It is therefore unclear why these species are unable to fully repress the B-myb promoter in p107<sup>-/-</sup>/p130<sup>-/-</sup> cells. To begin to address this issue, it was important to determine whether Rb-E2F complexes in quiescent p107<sup>-/-</sup>/p130<sup>-/-</sup> MEFs are located in the nucleus, as at least certain E2F complexes are found sequestered in the cytoplasm (4). Moreover, it was necessary to confirm that these Rb-E2F complexes are able to recognize the distinctive B-myb E2F site. To these ends, nuclear and cytoplasmic extracts were prepared from quiescent p107<sup>-/-</sup>/p130<sup>-/-</sup> MEFs, and EMSAs were performed using a B-myb E2F site oligonucleotide. The addition of specific antibodies to the EMSAs revealed that the nuclei of p107<sup>-/-</sup>/p130<sup>-/-</sup> MEFs contained Rb-E2F complexes, which were able to bind the B-myb E2F site *in vitro* (Fig. 5B); however, these higher order complexes were absent from the cytoplasmic fractions (Fig. 5A). It is notable that the cytoplasmic species in these extracts was composed exclusively of free E2F-4; however, the anti-E2F-4 antibody failed to recognize the majority of the nuclear Rb complex (Fig. 5B). Although it is possible that the E2F-4 epitope is masked by association with Rb, it is more likely that Rb is complexed predominantly with other E2F species. Control EMSAs using extracts from quiescent p107<sup>+/-</sup> MEFs showed that both cytoplasmic and nuclear fractions contained p130-E2F species, whereas Rb-E2F complexes were again found exclusively in the nuclear fraction (Fig. 5, C and D).

*Transfected Rb Forms E2F Complexes, but Inefficiently Represses the B-myb Promoter*—Although the *in vitro* binding assays described above suggest that Rb complexes in p107<sup>-/-</sup>/p130<sup>-/-</sup> MEFs are at least as abundant as the p130-E2F complexes, which repressed B-myb transcription in control p107<sup>+/-</sup> cells (Fig. 5B), it remains possible that their levels are insufficient. To address this question, we wished to compare the activities of Rb and p107 when these proteins were reintroduced into p107<sup>-/-</sup>/p130<sup>-/-</sup> cells by transfection. As primary MEFs are poorly transfectable, we first established immortalized p107<sup>-/-</sup>/p130<sup>-/-</sup> 3T3 lines to facilitate the analysis. Northern blotting demonstrated that B-myb was abnormally expressed in the p107<sup>-/-</sup>/p130<sup>-/-</sup> 3T3 cells during G<sub>0</sub> compared with control p107<sup>+/-</sup> 3T3 cells (Fig. 6A). Some further increases in B-myb mRNA levels were seen when p107<sup>-/-</sup>/p130<sup>-/-</sup> 3T3 cells were induced to enter S phase, similar to findings with primary p107<sup>-/-</sup>/p130<sup>-/-</sup> fibroblasts (11), presumably reflecting the increased metabolism of these cells when serum-stimulated. Nonetheless, it is clear that even after establishment of an immortalized line, the dependence upon p107 and p130 for repression of the B-myb promoter in G<sub>0</sub> is maintained (Fig. 6A).

Significantly, transfection of p107<sup>-/-</sup>/p130<sup>-/-</sup> 3T3 cells with a p107 expression plasmid resulted in repression of the B-myb promoter (Fig. 6B), whereas in contrast, wild-type Rb and a mutant Rb (RbNP, in which the major cyclin-dependent kinase phosphorylation sites have been eliminated) demonstrated little activity in this assay. Notably, the ability of p107 to repress the B-myb promoter in these cells was largely dependent upon an intact DRS (Fig. 6B).

Cell extracts made from parallel transfections were assayed by EMSAs. Antibody supershifts showed that similar levels of E2F complexes were formed with transfected p107 and Rb (Rb and RbNP were Myc epitope-tagged to distinguish these complexes from endogenous Rb complexes) (Fig. 6C). These experiments therefore indicate that p107 is intrinsically more active than Rb in repressing the B-myb promoter. Moreover, the fact that RbNP is also a weak repressor of B-myb transcription indicates that this differential activity cannot be explained by differences in the sensitivity of p107 and Rb to inactivation by cyclin-dependent kinases.

*Repression of B-myb Transcription by p130 Requires Specific Protein Domains*—To enable a more systematic comparison between the transcriptional repressive properties of the individual Rb family members on the B-myb promoter, further experiments were performed using Myc epitope-tagged derivatives of all three pocket proteins. By probing Western blots using the common epitope tag, it was evident that Rb was the most highly expressed of these proteins in transfected p107<sup>-/-</sup>/p130<sup>-/-</sup> 3T3 cells (Fig. 7B), whereas p107 was relatively poorly expressed (a faint p107 band was detectable just below the background cellular band in Fig. 7B). Additional
<figure><img src="image_8.png" /></figure>

FIG. 6. The B-myb promoter can be transcriptionally repressed by transfected p107 (but not Rb) in immortalized p107⁻/⁻/p130⁻/⁻ 3T3 cells. A, shown is a Northern blot probed for B-myb expression in 3T3 cells derived from control p107⁺/⁺ and knockout p107⁻/⁻/p130⁻/⁻ MEFs. Cells were deprived of serum for 60 h to arrest in G₀. Flow cytometry showed that both cell lines were quiescent at this time. The cells were then stimulated by re-addition of serum for 18 h (S phase), by which stage >60% were judged to be in S phase by flow cytometry. B, knockout p107⁻/⁻/p130⁻/⁻ 3T3 cells were transfected in triplicate with the wild-type (wt) B-myb promoter/luciferase reporter pGL2-(−536) or with variants carrying mutations in the DRS (mDRS) or mutations in both the E2F/DRS sites (mE2F/mDRS) together with a control pCMV plasmid or an expression vector encoding p107 or Myc epitope-tagged Rb and RbNP. Cells were arrested in G₀ by serum deprivation for 60 h, and luciferase activities were assayed in cell extracts. The results are presented as -fold repression of transcription by these pocket proteins, where the average luciferase activity obtained with the control empty expression plasmid transfection for each re-porter was given a value of unity. C, EMSA was performed with ex-tracts from knockout p107⁻/⁻/p130⁻/⁻ 3T3 cells transfected with the control pCMV vector (CON) or with an expression vector encoding p107, Rb, or RbNP. To detect E2F complexes containing the ectopically ex-pressed pocket proteins, antibody (Ab) supershifts were done with anti-p107 antibody SD15 or antibody 9E10, which recognizes the Myc epitope tag present on the transfected Rb and RbNP proteins.

experiments (data not shown) showed that the levels of expres-
sion that could be obtained with Myc epitope-tagged p107 were
severalfold lower than those obtained with untagged p107 used
in previous experiments (Figs. 3 and 6). Despite this, tagged
p107 was more active than Rb in repressing the B-myb pro-
moter in quiescent p107⁻/⁻/p130⁻/⁻ 3T3 cells, and notably,
p130 had the greatest activity in this assay (Fig. 7A). These
findings therefore emphasize that E2F complexes containing
Rb are weakly active in transcriptional repression of B-myb in
comparison with p107 and p130 complexes.

To define the domains of p130 required for transcriptional

repression of B-myb, a number of deletion mutants were em-
ployed. These experiments showed that deletion of virtually the
entire spacer region (as in mutant dl622–818) had no effect
upon the ability of p130 to repress B-myb transcription,
whereas in contrast, removal of the B pocket and C-terminal
sequences (as in mutant C793) completely abolished activity.
The lack of mutant C793 activity can clearly be attributed to its
inability to bind E2F. The activity of a less extensively deleted
C-terminal mutant (mutant C1065) was impaired to a lesser
extent (Fig. 7C), and this defect may be attributed to loss of a
potential C-terminal nuclear localization signal or its inability
to bind histone deacetylase-1 (36). Most notably, deletion of
sequences N-terminal to the pocket domain (as in mutant
N417) significantly reduced transcriptional repression (Fig.
7C), although the truncated protein was expressed at much
higher levels than wild-type p130 (Fig. 7D). The N-terminal
domain is not required for interaction with E2F, and the re-
duced activity of mutant N417 indicates that this region con-
tributes a novel functional requirement for transcriptional re-
pression. It may be significant that p130 and p107 display
significant homology in this N-terminal sequence, but little
homology to Rb.

The B-myb Promoter E2F Site Is Unbound in p107⁻/⁻/p130⁻/⁻ Cells—A number of explanations could account for the inability of Rb-E2F complexes to repress B-myb transcription in p107⁻/⁻/p130⁻/⁻ MEFs: (i) the conformation of the promoter *in vivo* may preclude strong interactions with Rb-E2F complexes while permitting binding of p107-E2F and p130-E2F complexes; (ii) binding of Rb-E2F complexes may be competed by higher levels of free E2F/DP heterodimers resulting from deregulation of E2F gene transcription (11); and (iii) Rb-E2F complexes may bind the site, but be unable to repress transcription. To help distinguish between these possibilities, we used the *in vivo* footprinting technique to study whether complexes are bound to the E2F site in quiescent p107⁻/⁻/p130⁻/⁻ MEFs as would be predicted from Explanations ii and iii. Previous studies in NIH 3T3 cells demonstrated that the B-myb promoter E2F site is occupied in G₀, but becomes unoccupied in G₁ concurrently with derepression of transcription (14). Using Swiss 3T3 cells initially, we confirmed that the E2F site was partially protected when intact cells were treated with dimethyl sulfate (Fig. 8A), but this degree of protection was lost when cells were stimulated by serum to reenter the cell cycle (16- and 24-h time points). Incomplete protection is a characteristic of this procedure and does not necessarily imply that the site is incompletely occupied. No protection of the DRS was observed, consistent with previous results (14), suggesting that interactions at this site are not made in the major groove. We next compared occupation of the E2F site in serum-starved MEFs derived from p107⁻/⁻/p130⁻/⁻ and control (wild-type) embryos; as a further control, *in vivo* footprints were also obtained with quiescent Rb⁻/⁻ MEFs. It was apparent that the E2F site was partially protected in both wild-type and Rb⁻/⁻ MEFs (Fig. 8C); however, this site was completely unprotected in the p107⁻/⁻/p130⁻/⁻ MEFs. These findings are therefore inconsistent with occupation of the E2F site by free E2F species or inactive Rb-E2F complexes and strongly imply that only p107-E2F and p130-E2F complexes are able to make stable interactions *in vivo*.

DISCUSSION

We have demonstrated here that the sequence requirements
for B-myb promoter DRS corepressor function overlap with
that of the CHR site, which is part of a bipartite element
required for cell cycle regulation of the CDC25C, cyclin A, and
cdc2 promoters (38). Notably, the CDE/CHR element was ca-
pable of regulating cell cycle transcription in place of the E2F/
DRS sites in the B-myb promoter; however, the DRS was un-
<figure><img src="image_9.png" /><figcaption>FIG. 7. The p130 N terminus is required for transcriptional repression of the B-myb promoter. A, knockout p107⁻/⁻/p130⁻/⁻ 3T3 cells were transfected in triplicate with the wild-type B-myb promoter/luciferase reporter pGL2(−536) together with 1- and 2-µg amounts as indicated of a control pcDNA3 vector (CON) or pcDNA3 encoding Myc epitope-tagged Rb, p107, or p130. Cells were arrested in G₀ by serum deprivation for 60 h, and luciferase activities were assayed in cell extracts. The results are presented as -fold repression by these proteins on the reporter, where the average luciferase activity obtained with the control transfection was given a value of unity. B, Western blotting was carried out using antibody 9E10 as the primary antibody, and the results show the relative levels of expression of Rb, p107, and p130 in transfected p107⁻/⁻/p130⁻/⁻ 3T3 cells at inputs of 1 and 2 µg of expression vector. Note that p107 appears as a faint band immediately below the major background band obtained in all lanes. The background band is indicated with an *asterisk*. C, transcriptional repression of the B-myb promoter by p130 requires the E2F binding domain and an N-terminal domain. Knockout p107⁻/⁻/p130⁻/⁻ 3T3 cells were transfected in triplicate with the wild-type B-myb promoter/luciferase reporter pGL2(−536) together with 0.2-, 0.5-, and 1 µg-amounts as indicated of a control pcDNA3 vector or pcDNA3 encoding Myc-epitope-tagged p130 and p130 mutants N417 (amino acids 417–1139), C793 (amino acids 2–793), C1065 (amino acids 2–1065), and dl622–818 (deletion of amino acids 622–818 within the spacer). Cells were arrested in G₀ by serum deprivation for 60 h, and luciferase activities were assayed in cell extracts. The results are presented as -fold repression by these proteins on the reporter, where the average luciferase activity obtained with the control transfection was given a value of unity. D, Western blotting was performed using antibody 9E10 as the primary antibody, and the results show the relative levels of expression of wild-type and mutant p130 proteins in transfected p107⁻/⁻/p130⁻/⁻ 3T3 cells at 1-µg input of expression vector. *Lane 1*, wild-type p130; *lane 2*, mutant N417; *lane 3*, mutant C793; *lane 4*, mutant C1065; *lane 5*, mutant dl622–818. The major background band is indicated with an *asterisk*. *E*, shown is a schematic representation of Myc epitope-tagged p130 and p130 deletion mutants. Myc epitope-tagged p130 contains amino acids 2–1139 of wild-type p130, and the amino acid numbers defining the boundaries of the A and B pockets and the intervening spacer are indicated. Sequences retained in the p130 deletion mutants are indicated below.</figcaption></figure>

able to substitute for the CHR in this context (Fig. 2B). This suggests that, although the E2F/DRS and CDE/CHR elements are similar in many respects, there is some homology between the respective elements, and their relative spacing is identical, there are certain functional differences between them. In the case of the cyclin A and *cdc2* CDE/CHR elements, where the CDE can clearly double as an E2F site, these bipartite elements may act analogously to the *B-myb* E2F/DRS elements in binding p107-E2F and p130-E2F complexes in G₀ and early G₁. Potentially, the cyclin A and *cdc2* elements may then have a dual role, acting analogously to the *CDC25C* CDE/CHR elements to prolong active transcription into G₂.

E2F complexed to each of the three pocket proteins can bind the *B-myb* E2F site in *in vitro* binding assays, and this is not influenced by the presence of the DRS (32). These assays clearly do not reflect the specificity of the *B-myb* promoter for binding p107-E2F and p130-E2F complexes as evidenced in this study by *in vivo* footprinting (Fig. 8) and repression assays (Figs. 6 and 7). Notably, previous ChIP assays have also led to

the conclusion that the *B-myb* promoter is bound specifically by p107-E2F and p130-E2F complexes *in vivo* and that whereas Rb binds to other E2F-regulated promoters, these complexes are absent from *B-myb* (39). Indeed, this study suggested that E2F-4 complexes exclusively regulate *B-myb* in cells synchronized by serum starvation. In further distinction to other E2F-regulated promoters, the *B-myb* promoter is not occupied by E2F complexes during S phase (39, 40). Altogether, published evidence and our current findings suggest that *B-myb* displays a distinctive mode of regulation that utilizes a closely linked DRS corepressor site to selectively bind transcriptionally repressive p130-E2F and p107-E2F complexes. A role for the DRS in excluding binding of transcriptionally active “free” E2F complexes is also suggested by our finding that mutation of the DRS led to activation of the promoter through the adjacent site in S phase (Fig. 2A).

Clearly, a full understanding of the way in which DRS and CHR elements co-regulate transcription during the cell cycle requires identification of the factors that bind these sites. We
<figure><img src="image_10.png" /></figure>

FIG. 8. The B-myb E2F site is unoccupied in p107⁻/⁻/p130⁻/⁻ MEFs. A, Swiss 3T3 cells were arrested in G₀ by serum deprivation for 60 h and then restimulated to reenter the cell cycle by addition of serum. Cells taken at 0, 16, and 24 h after restimulation as indicated were methylated *in vivo* with dimethyl sulfate. Footprints obtained by linker-mediated PCR on DNA extracted from the dimethyl sulfate-treated cells were compared with those of Swiss 3T3 DNA methylated *in vitro* (IV). The sequence of the (+)-strand shown spans the B-myb promoter E2F/DRS sites in a 5' to 3' direction, top to bottom. B, shown are the results from flow cytometry of propidium iodide-stained Swiss 3T3 cells used in A at 0, 16, and 24 h after serum stimulation. C, MEFs obtained from wild-type (WT), Rb⁻/⁻, and p107⁻/⁻/p130⁻/⁻ embryos were arrested in G₀ by serum deprivation for 72 h and then methylated *in vivo* with dimethyl sulfate. Footprints obtained by linker-mediated PCR on DNA extracted from the dimethyl sulfate-treated cells were compared with those of wild-type MEF DNA methylated *in vitro* (IV). The sequence of the (+)-strand shown spans the B-myb promoter E2F/DRS sites and a putative SP-1 site (which was occupied *in vivo* in all the cells tested) located 5' to the regulatory elements.

have been unable to detect proteins binding to the DRS in *in vitro* binding assays, although in the presence of spermine, a novel lower mobility p130-E2F complex was detected in G₀ extracts from various rodent fibroblasts that was absent on probes in which the DRS was mutated.<fnref n="2" /> A protein (CHF) that can bind the *B-myb* DRS has been purified (41); however, evidence that this binding correlates with the sequence requirements for transcriptional corepression is lacking. Evidence from *in vivo* footprinting that protein contacts are made with the *CDC25C* CHR in the minor groove suggests that minor groove contacts are also made with the *B-myb* DRS. The CHR and DRS may therefore act in some respects analogously to high mobility group I(Y) binding sites for assembly of an NF-κB complex on the human interferon-β gene promoter (42). In this respect, it is of interest that the DRS sequence (GGAAA) is present within the PRDII site, which binds high mobility

group I(Y) (42); however, the sequence specificity for high mobility group I(Y) binding within the interferon-β site is inconsistent with sequence requirements within the *B-myb* DRS.

Rb-E2F complexes were abundant in p107⁻/⁻/p130⁻/⁻ cells (Fig. 5), yet were unable to repress efficiently the *B-myb* promoter. Moreover, transfected Rb was unable to substitute efficiently for the loss of p107 and p130 function in these cells (Figs. 6 and 7). The latter deficiency cannot be ascribed to hyperphosphorylation of Rb in the p107⁻/⁻/p130⁻/⁻ cells, as RbNP was similarly inactive in repressing *B-myb* transcription (Fig. 6). It is also apparent that Rb-E2F complexes could be formed in the transfected p107⁻/⁻/p130⁻/⁻ cells (Fig. 6). It can be concluded that Rb-E2F complexes have relatively low affinity for the *B-myb* promoter E2F site *in vivo* in comparison with p107-E2F and p130-E2F complexes. This conclusion is consistent with a previous ChIP analysis of E2F target gene specificity in NIH 3T3 cells (39), in which Rb could be detected on the promoters of a number of E2F-regulated genes (dihydrofolate reductase, thymidine kinase, *cdc2*, and cyclin E), albeit at different stages of the cell cycle, whereas in contrast, only p130 and p107 occupied the *B-myb* promoter. It is also notable that *B-myb* was found to have a singular mode of regulation by E2F in that it was the only promoter not to bind E2F complexes in S phase (39). As DRS mutations enabled *B-myb* transcription to be activated through the adjacent E2F site in S phase (Fig. 2A), it may be suggested that the DRS promotes binding of transcriptionally repressive p107-E2F and p130-E2F complexes while preventing binding of transactivating E2F species.

Although corepressor CHR sites have been described in the promoters of several genes that are maximally activated during S/G₂, such as *CDC25C*, cyclin A, polo-like kinase, cyclin B2, and *survivin* (38, 43–45), sites directly analogous to the *B-myb* DRS have not been described in other G₁/S-regulated genes. This again points to the singular nature of *B-myb* regulation and illustrates how the precise positioning and context of an E2F site can influence cell cycle transcriptional control. It is notable that *B-myb* transcription was found to be the most strongly deregulated of ~20 genes tested in p107⁻/⁻/p130⁻/⁻ MEFs (11), possibly reflecting the absolute dependence of p107-E2F and p130-E2F complexes, under the influence of the DRS corepressor, for transcriptional repression of *B-myb*.

In view of the significant role played by transcriptional repression in regulating *B-myb* mRNA levels, it is surprising that induction of *B-myb* expression is severely impaired in MEFs derived from E2F-3⁻/⁻ mouse embryos (46), implying that E2F-3 function is required to transcriptionally induce *B-myb* at the G₁/S transition. This conclusion is not consistent with our finding that mutation of the *B-myb* promoter E2F site resulted in constitutive highly active transcription in stably transfected cells that contained a low copy number of the transgene (Fig. 4A), indicating that in this context, at least the E2F site is not required for activation by E2F-3. Moreover, ChIP data indicate that this promoter is not bound by E2F-3 or other E2F species at the G₁/S transition (39, 40), although binding of E2F-1 and E2F-3 was detected transiently in late G₁ (40). The failure of *B-myb* transcription to be fully induced following serum induction of quiescent E2F-3⁻/⁻ MEFs may therefore reflect the persistence of p130-E2F and/or p107-E2F repressor complexes under these conditions or, alternatively, a role for E2F-3 transactivation through distal promoter sites that are not retained in the *B-myb* promoter/reporters used. Analysis of *B-myb* promoter occupancy in E2F-3⁻/⁻ MEFs using the ChIP assay should help to resolve this issue.

The *B-myb* promoter has been used in many studies as a model for E2F regulation. Our current findings indicate that occupancy of this E2F site *in vivo* by transcriptionally repre-

<footnote n="2">² R. J. Watson, unpublished data.</footnote>
sive complexes is governed by the adjacent DRS corepressor
site. To date, we have been unable to identify proteins binding
to the DRS, and we can only speculate on the mechanism
whereby it acts to promote binding of complexes to the E2F site.
Recent studies of the E2F-1 promoter have identified a nucleo-
some proximal to the E2F binding site (37), which may provide
a target for histone acetylases and deacetylases to modulate
transcription initiation. Possibly, the DRS is involved in nu-
cleosome positioning, thereby influencing the ability of repres-
sive E2F complexes to interact with the adjacent E2F site. The
recognition that this corepressor site plays a prominent role in
occupancy of the B-myb promoter by transcriptionally repres-
sive p107-E2F and p130-E2F complexes will add significantly to
future studies using this system as a model for E2F regulation.

*Acknowledgments*—We thank Dr. Sybille Mittnacht for kindly providing pcDNA3(9E10) and plasmids encoding epitope-tagged Rb proteins and Dr. Nick Dyson for providing knockout and control MEFs.

REFERENCES

1. Girling, R., Partridge, J. F., Bandara, L. R., Burden, N., Totty, N. F., Hsuan, J. J., and La Thangue, N. B. (1993) *Nature* **362**, 83–87

2. Helin, K., Wu, C.-L., Fattaey, A. R., Lees, J. A., Dynlacht, B. D., Ngwu, C., and Harlow, E. (1993) *Genes Dev.* **7**, 1850–1861

3. Wu, C.-L., Zukerberg, L. R., Ngwu, C., Harlow, E., and Lees, J. A. (1995) *Mol. Cell. Biol.* **15**, 2536–2546

4. Verona, R., Moberg, K., Estes, S., Starz, M., Vernon, J. P., and Lees, J. A. (1997) *Mol. Cell. Biol.* **17**, 7268–7282

5. Morkel, M., Wenkel, J., Bannister, A. J., Kouzarides, T., and Hagemeier, C. (1997) *Nature* **390**, 567–568

6. Cartwright, P., Muller, H., Wagener, C., Holm, K., and Helin, K. (1998) *Oncogene* **17**, 611–623

7. Gaubatz, S., Wood, J. G., and Livingston, D. M. (1998) *Proc. Natl. Acad. Sci. U. S. A.* **95**, 9190–9195

8. Trimarchi, J. M., Fairchild, B., Verona, R., Moberg, K., Andon, N., and Lees, J. A. (1998) *Proc. Natl. Acad. Sci. U. S. A.* **95**, 2850–2855

9. Jacks, T., Fazeli, A., Scmidtt, E. M., Bronson, R. T., Goodell, M. A., and Weinberg, R. A. (1992) *Nature* **359**, 295–300

10. Cobrinik, D., Lee, M.-H., Hannon, G., Mulligan, G., Bronson, R. T., Dyson, N., Harlow, E., Beach, D., Weinberg, R. A., and Jacks, T. (1996) *Genes Dev.* **10**, 1633–1644

11. Hurford, R. K., Cobrinik, D., Lee, M.-H., and Dyson, N. (1997) *Genes Dev.* **11**, 1447–1463

12. Classon, M., Salama, S., Gorka, C., Mulloy, R., Braun, P., and Harlow, E. (2000) *Proc. Natl. Acad. Sci. U. S. A.* **97**, 10820–10825

13. Tao, Y., Kassatly, R. F., Cress, W. D., and Horowitz, J. M. (1997) *Mol. Cell. Biol.* **17**, 6994–7007

14. Zwicker, J., Liu, N., Engeland, K., Lucibello, F. C., and Müller, R. (1996) *Science* **271**, 1595–1597

15. Lam, E. W.-F., and Watson, R. J. (1993) *EMBO J.* **12**, 2705–2713

16. Sugarman, J. L., Schonthal, A. H., and Glass, C. K. (1995) *Mol. Cell. Biol.* **15**, 3282–3290

17. Liu, N., Lucibello, F., Engeland, K., and Müller, R. (1998) *Oncogene* **16**, 2957–2963

18. Zhu, L., Zhu, L., Xie, E., and Chang, L.-S. (1995) *Mol. Cell. Biol.* **15**, 3552–3562

19. Neumann, E., Flemington, E. K., Sellers, W. R., and Kaelin, W. G. (1994) *Mol. Cell. Biol.* **14**, 6607–6615

20. Hsiao, K.-M., McMahon, S. L., and Farnham, P. J. (1994) *Genes Dev.* **8**, 1526–1537

21. Henglein, B., Chenivesse, X., Wang, J., Eick, D., and Brechot, C. (1994) *Proc. Natl. Acad. Sci. U. S. A.* **91**, 5490–5494

22. Le Cam, L., Polanowska, J., Fabrizio, E., Olivier, M., Philips, A., Ng Eaton, E., Classon, M., Geng, Y., and Sardet, C. (1999) *EMBO J.* **18**, 1878–1890

23. Lucibello, F. C., Truss, M., Zwicker, J., Ehlert, F., Beato, M., and Müller, R. (1995) *EMBO J.* **14**, 132–142

24. Zwicker, J., and Muller, R. (1997) *Trends Genet.* **13**, 3–6

25. Furukawa, Y., Iwase, S., Kikuchi, J., Nakamura, M., Yamada, H., and Matsuda, M. (1999) *Oncogene* **18**, 2003–2014

26. Chen, X., and Prywes, R. (1999) *Mol. Cell. Biol.* **19**, 4695–4702

27. Iavarone, A., and Massague, J. (1999) *Mol. Cell. Biol.* **19**, 916–922

28. Zhang, H. S., Postigo, A. A., and Dean, D. C. (1999) *Cell* **97**, 53–61

29. Liu, N., Lucibello, F. C., Körner, K., Wolfraim, L. A., Zwicker, J., and Müller, R. (1997) *Nucleic Acids Res.* **25**, 4915–4920

30. Lucibello, F. C., Liu, N., Zwicker, J., Gross, C., and Müller, R. (1997) *Nucleic Acids Res.* **25**, 4921–4925

31. Klehr, D., Maass, K., and Bode, J. (1991) *Biochemistry* **30**, 1264–1270

32. Bennett, J. D., Farlie, P. G., and Watson, R. J. (1996) *Oncogene* **13**, 1073–1082

33. Chew, Y. P., Ellis, M., Wilkie, S., and Mittnacht, S. (1998) *Oncogene* **17**, 2177–2186

34. Moberg, K., Starz, M. A., and Lees, J. A. (1996) *Mol. Cell. Biol.* **16**, 1436–1449

35. Weinmann, A. S., Bartley, S. M., Zhang, T., Zhang, M. Q., and Farnham, P. J. (2001) *Mol. Cell. Biol.* **21**, 6820–6832

36. Stiegler, P., De Luca, A., Bagella, L., and Giordano, A. (1998) *Cancer Res.* **58**, 5049–5052

37. Rayman, J. B., Takahashi, Y., Dannenberg, J. H., Catchpole, S., Watson, R., te Riele, H., and Dynlacht, B. D. (2002) *Genes Dev.* **16**, 933–947

38. Zwicker, J., Lucibello, F. C., Wolfraim, L. A., Gross, C., Truss, M., Engeland, K., and Müller, R. (1995) *EMBO J.* **14**, 4514–4522

39. Wells, J., Boyd, K. E., Fry, C. J., Bartley, S. M., and Farnham, P. J. (2000) *Mol. Cell. Biol.* **20**, 5797–5807

40. Takahashi, Y., Rayman, J. B., and Dynlacht, B. D. (2000) *Genes Dev.* **14**, 804–816

41. Liu, N., Lucibello, F. C., Zwicker, J., Engeland, K., and Muller, R. (1996) *Nucleic Acids Res.* **24**, 2905–2910

42. Thanos, D., and Maniatis, T. (1992) *Cell* **71**, 777–789

43. Uchiumi, T., Longo, D. L., and Ferris, D. K. (1997) *J. Biol. Chem.* **272**, 9166–9174

44. Otaki, M., Hatano, M., Kobayashi, K., Ogasawara, T., Kuriyama, T., and Tokuhisa, T. (2000) *Biochim. Biophys. Acta* **1493**, 188–194

45. Lange-zu Dohna, C., Brandeis, M., Berr, F., Mossner, J., and Engeland, K. (2000) *FEBS Lett.* **484**, 77–81

46. Humbert, P. O., Verona, R., Trimarchi, J. M., Rogers, C., Dandapani, S., and Lees, J. A. (2000) *Genes Dev.* **14**, 690–703