Position-Dependent Transcriptional Regulation of the Murine ...

MOLECULAR AND CELLULAR BIOLOGY, Apr. 1997, p. 1966–1976 0270-7306/97/$04.0010 Copyright q 1997, American Society for Microbiology

Vol. 17, No. 4

Position-Dependent Transcriptional Regulation of the Murine Dihydrofolate Reductase Promoter by the E2F Transactivation Domain CHRISTOPHER J. FRY, JILL E. SLANSKY,†

AND

PEGGY J. FARNHAM*

McArdle Laboratory for Cancer Research, University of Wisconsin Medical School, Madison, Wisconsin 53706 Received 19 November 1996/Returned for modification 12 December 1996/Accepted 18 December 1996

Activity of the dihydrofolate reductase (dhfr) promoter increases at the G1-S-phase boundary of the cell cycle. Mutations that abolish protein binding to an E2F element in the dhfr promoter also abolish the G1-S-phase increase in dhfr transcription, indicating that transcriptional regulation is mediated by the E2F family of proteins. To investigate the mechanism by which E2F regulates dhfr transcription, we moved the E2F element upstream and downstream of its natural position in the promoter. We found that the E2F element confers growth regulation to the dhfr promoter only when it is proximal to the transcription start site. Using a heterologous E2F element, we showed that position-dependent regulation is a property that is promoter specific, not E2F element specific. We demonstrated that E2F-mediated growth regulation of dhfr transcription requires activation of the dhfr promoter in S phase and that the C-terminal activation domains of E2F1, E2F4, and E2F5, when fused to the Gal4 DNA binding domain, are sufficient to specify position-dependent activation. To further investigate the role of activation in dhfr regulation, we tested other transactivation domains for their ability to activate the dhfr promoter. We found that the N-terminal transactivation domain of VP16 cannot activate the dhfr promoter. We propose that, unlike other E2F-regulated promoters, robust transcription from the dhfr promoter requires an E2F transactivation domain close to the transcription start site. to a region which overlaps the transactivation domain of E2F (for reviews, see references 32 and 43). In addition, Rb has been shown to repress transactivation mediated by other transcriptional activator proteins when recruited to a promoter by the Gal4 DNA binding domain (56, 57). E2F-pocket protein complexes are most abundant in G0 and early G1 phase. In late G1 and early S phase, the pocket protein is phosphorylated by cyclin D- and cyclin E-dependent kinases and subsequently released from E2F-DP complexes. Thus, it is proposed that E2F mediates differential levels of transcription in G0 versus S phase by recruiting an Rb family member which represses transcription in G0 phase. However, previous studies suggest that E2F-mediated repression cannot account for the growth regulation of the dhfr promoter. We have shown that mutation of the dhfr E2F element results in only a two- to threefold increase in promoter activity in quiescent cells (50). Also, unlike the B-myb promoter, in vivo footprinting analysis of the hamster dhfr promoter demonstrated that protein binding to one strand of the E2F element correlates with the increase in promoter activity in late G1 and early S phase; the other strand is constitutively occupied (58). We have further investigated the possibility that dhfr may be regulated differently than other E2F elementcontaining promoters, and we now present evidence that growth regulation of the murine dhfr promoter is achieved by position-dependent E2F-mediated activation of transcription in S phase.

The E2F family of heterodimeric transcription factors plays an important role in regulating gene expression at the G1-Sphase transition of the mammalian cell cycle. Seven members of this family have been identified: five different E2F proteins (E2F1 to E2F5) and two different DP proteins (DP1 and DP2). E2F and DP proteins bind to DNA as a heterodimer to form a functional E2F complex that contributes to the regulation of many promoters (for recent reviews, see references 10 and 51). Cellular promoters regulated by E2F include genes required for DNA synthesis (dihydrofolate reductase [dhfr], thymidine kinase, and DNA polymerase a) and transcriptional regulators of cell growth (N-myc, c-myc, B-myb, cdc2, E2F1, retinoblastoma [Rb], p107, and cyclins E and A) (5, 20, 44, 48, 51, 60). E2F is believed to confer growth regulation to the E2F1, B-myb, and cdc2 promoters by mediating transcriptional repression in the G0 phase of the cell cycle (11, 23, 25, 33, 34, 42, 61). For example, mutation of the E2F element in the murine B-myb promoter results in constitutively high levels of transcription in mouse fibroblasts and human keratinocytes (33, 34). Also, in vivo footprinting analysis of the B-myb promoter shows occupation of the E2F element only in G0-phase cells when the B-myb promoter is inactive (61). This transcriptional repressor activity of E2F is attributed to its interaction with the Rb family of pocket proteins. E2F1, E2F2, and E2F3 bind to the Rb protein, E2F4 binds to Rb and the related p107 and p130 proteins, and E2F5 binds to the p107 and p130 proteins (for a recent review, see reference 8). Transcriptional activation by E2F can be masked by pocket proteins, since they bind

MATERIALS AND METHODS Plasmids. Standard cloning techniques were used for all plasmid constructions (38). All dhfr promoter reporter plasmids contain promoter fragments cloned upstream of the luciferase cDNA in the vector pAAlucA (21). The pMaeWT and pMaeDE2F reporter constructs contain dhfr sequences from positions 2270 to 120 and have been described previously as pWTluc and pNWluc, respectively (41). pMaeE2F@2375 was created by inserting a double-stranded oligonucleotide containing the wild-type dhfr promoter sequence from 220 to 19 plus a PvuII site for screening and SacII sites at both ends into SacII-digested pMaeDE2F. The sequence of the top strand of the oligonucleotide reads 59-CA

* Corresponding author. Mailing address: McArdle Laboratory for Cancer Research, 1400 University Ave., University of Wisconsin Medical School, Madison, WI 53706-1599. Phone: (608) 262-2071. Fax: (608) 262-2824. E-mail: [email protected]. † Present address: Johns Hopkins University Oncology Center, Johns Hopkins University School of Medicine, Baltimore, MD 212052196. 1966

VOL. 17, 1997 GCTGCTGCGATTTCGCGCCAAACTTGACGCCGC-39, with the E2F element indicated in boldface type. The E2F element was placed in the same orientation as in the wild-type dhfr promoter. The same cloning scheme was used to create pMae2xE2F@2375, which contains two copies of the oligonucleotide in the same orientation, and pMaeF2E@2375, which contains one copy of the oligonucleotide in the orientation opposite that in pMaeE2F@2375. The same cloning scheme was also used to create pMaeB-myb@2375 and pMaebymB@2375 by inserting a double-stranded oligonucleotide (top strand reads 59-G GCACTTGGCGGGAGATACCGC-39), which contains the E2F element from the B-myb promoter (indicated in boldface type) and SacII sites at both ends. pMaeB-myb@2375 contains the E2F element in the same orientation as in the B-myb promoter, while pMaebym-B@2375 contains the E2F element in the opposite orientation. To create p410WT, the pST410 plasmid which contains dhfr sequences from positions 2365 to 161 in pUC9 (15) was digested with EcoRI and HindIII, and the 410-bp fragment was placed into EcoRI- and HindIII-cut pBSM13 (Stratagene). Then a double-stranded PstI linker oligonucleotide (top strand reads 59-AATTCCTGCAGC-39), which contains a PstI site for cloning and EcoRI and XmaI sites at the 59 and 39 ends, respectively, was placed into the EcoRI- and XmaI-digested pBSM13 plasmid containing the dhfr fragment. Finally, the pBSM13 plasmid containing the dhfr fragment and PstI linker was digested with PstI, and the fragment containing the dhfr sequence was placed into PstI-digested pAAlucA. The resultant p410WT reporter construct contains dhfr promoter sequences from 2356 to 161. To create p410E2F@114, the PstI linker used to clone p410WT was placed into EcoRI- and XmaI-digested pSTU114mp19 plasmid (40). The pSTU1 14mp19 plasmid containing the PstI linker was then digested with PstI, and the fragment containing the dhfr sequence was placed into PstI-digested pAAlucA. The p410E2F@114 reporter construct is identical to p410WT, except that it contains a 14-bp linker oligonucleotide (top strand reads 59-CTAGTCTAGAC TAG-39) inserted into the StuI site. p410E2F@142 and p410E2F@166 were created by inserting two copies of the 21-bp oligonucleotide 59-CTAGTCTAG ACTAGGTCGACT-39 (top strand) and two copies of the 33-bp oligonucleotide 59-TCTAGTCTAGACTAGCTAGTCTAGACTAGCAGG-39 (top strand) into StuI-digested p410WT. To create p410bym-B, p410WT was digested with StuI and HindIII to remove the 217 to 161 region of the dhfr promoter. This region was then replaced by a double-stranded oligonucleotide (top strand reads 59-CCTGCGATCTCCCGCC AAGCTTGACGGCAATCCTAGCA-39), which contains StuI and HindIII sites at the 59 and 39 ends and the dhfr promoter sequence from positions 217 to 120, but the dhfr E2F element is replaced precisely with the E2F element from the B-myb promoter (indicated in boldface type). This construct contains the B-myb E2F element in the orientation opposite that in the B-myb promoter. DHFRGal4 was created by replacing the 217 to 161 region of p410WT with a double-stranded oligonucleotide (top strand reads 59-CCTCGGAAGACTCTC CTCCGCAcACTTGACGGCAATCCTAGCA-39) which contains dhfr sequence from positions 217 to 120, with the E2F element replaced precisely with one Gal4 element (indicated in boldface type). DHFRGal4 contains a different transcription start nucleotide than p410WT (shown in lowercase letters) which has previously been shown not to affect the position of the start site or the level of transcription (39). pseudoWT was created by replacing the 217 to 161 region of p410WT with a double-stranded oligonucleotide (top strand reads 59-CCTC GGAAGACTTTTCGCGCCAAACTTGACGGCAATCCTAGCA-39) containing dhfr sequence from 217 to 120, including the dhfr E2F element (indicated in boldface type). Both DHFRGal4 and pseudoWT contain an additional 5 bp between the proximal Sp1 element and the transcriptional start site. DHFRTATAGal4 was created by replacing the XmaI/StuI fragment of DHFRGal4, which contains nucleotides 2356 to 217 of the dhfr promoter, with the XmaI/StuI fragment of dhfr1TATA, which contains the same nucleotides but with changes made to create a consensus TATA box centered at 230 (6). The pG5TIluc synthetic reporter construct contains five Gal4 elements upstream of the adenovirus major late promoter TATA box and murine terminal deoxynucleotidyltransferase initiator element (Inr) cloned upstream of the luciferase cDNA in the vector pGL2Basic (Promega). DHFRGal4@2375 was created by inserting a double-stranded oligonucleotide (top strand reads 59-GGAGGCCTCGGAAGACTCTCCTCCGGTACCGC39) into SacII-digested pMaeDE2F. This oligonucleotide contains a Gal4 element (indicated in boldface type), a StuI restriction site for screening, and SacII sites at both ends. Gal4-E2F1(368-437), Gal4-E2F4(276-412), and Gal4-E2F5(222346) all contain human E2F sequence and have been described previously (17, 22, 26). The Rb-binding-deficient Gal4-E2F1 contains human E2F1 sequence and has been described previously as Gal4-E2F1(368-437)(d423-427) (17). The Gal4 fusion to the N terminus of E2F1 has been described previously as Gal4E2F1(1-163) (17). Gal4-VP16 (49) and Gal4(1-147) (17) have been described previously. Cell culture and transfection. NIH 3T3 cell cultures were maintained as described previously (52). Calcium phosphate transfections were performed as described previously (41) with the following alterations. One day prior to transfection, 1.0 3 105 (growing cell experiments) to 1.3 3 105 (serum starvation and stimulation experiments) cells were seeded into 60-mm-diameter dishes. For serum starvation and stimulation experiments, each dish of cells was transfected with 5 mg of reporter DNA and 10 mg of sonicated salmon sperm DNA. For

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growing cell experiments, each dish of cells was transfected with 5 mg of reporter DNA, 5 mg of Gal4 fusion expression DNA, and 5 mg of sonicated salmon sperm DNA. Each dish received a total of 15 mg of DNA as a precipitate in 450 ml of transfection buffer (41) and 50 ml of 1.0 M CaCl2. At 6 h after transfection, cells were rinsed with rinse medium (Dulbecco’s modified Eagle’s medium [DMEM] [GIBCO]–100 U of penicillin per ml–100 mg of streptomycin [GIBCO] per ml), shocked for 4 min with 1 ml of 15% (vol/vol) glycerol in transfection buffer, rinsed twice with rinse medium, and incubated in the appropriate medium. For serum starvation and stimulation experiments, the cells were induced to growth arrest by immediate incubation in starvation medium (DMEM plus 0.5% [vol/ vol] bovine calf serum [Hyclone]) for 48 to 60 h. The cells were then stimulated to reenter the proliferative cell cycle by replacing the starvation medium with stimulation medium (DMEM plus 10% [vol/vol] bovine calf serum). Cells were harvested at 0 h (starved cells) and 12 h (S-phase cells with peak levels of dhfr transcription) after serum stimulation, and total cell lysates were assayed for luciferase activity. For growing cell experiments, the cells were incubated in growth medium (DMEM plus 5% [vol/vol] bovine calf serum) for 40 to 48 h before harvesting, and total cell lysates were assayed for luciferase activity. Each transfection was repeated at least two times with duplicate samples and multiple DNA preparations. For luciferase assays, cells were rinsed once with phosphate-buffered saline, scraped from the plates in phosphate-buffered saline containing 1 mM EDTA, and pelleted for 4 min at 48C in a microcentrifuge at 14,000 rpm. Cell pellets were resuspended in 100 ml of luciferase lysis buffer (Promega), lysed on ice for 7 min, and spun at 14,000 rpm in a model 5415C Eppendorf Microfuge at 48C for 6 min. Luciferase assays were performed with 35 to 50 ml of total cell lysate and 100 ml of luciferin substrate buffer (Promega) in a Monolight 2010 Luminometer (Analytical Luminescence Laboratory). In vitro transcription. Templates for in vitro transcriptions were prepared as follows: p410WT, p410E2F@166, DHFRGal4, pMaeWT, and pMaeDE2F were digested with SacII and Bsu36I. pMaeE2F@2375 (same as pMaeDE2F but contains an E2F element placed at position 2375) was digested with PvuII and Bsu36I. The promoter fragments were isolated by polyacrylamide gel electrophoresis followed by electroelution. All of the templates contain the promoter sequence indicated and additional vector sequence including some multiple cloning region and 642 bp of luciferase cDNA. HeLa cell nuclear extracts used for in vitro transcriptions were prepared from frozen cells as described previously (4, 40). In vitro transcription reactions were performed as described previously (16) with the following modifications. Each reaction mixture (final volume, 25 ml), containing 16 nM template DNA, was incubated for 15 min at 248C with 3.9 mg of HeLa nuclear extract per ml in 6 mM MgCl2–24 mM Tris hydrochloride (pH 7.4)–12% (vol/vol) glycerol–60 mM KCl– 0.12 mM EDTA–0.3 mM dithiothreitol–0.12 mM phenylmethylsulfonyl fluoride. Nuceloside triphosphates, including 10 mCi of [a-32P]GTP (800 Ci/mmol) were then added to final concentrations of 600 mM (for GTP, CTP, and UTP) and 200 mM (for ATP). After an additional incubation for 30 min at 248C, the reactions were stopped, and the products were phenol-chloroform extracted and ethanol precipitated. The precipitates were resuspended in formamide plus tracking dyes and loaded onto an 8 M urea–4% polyacrylamide gel. Products were sized by comparison with molecular weight markers produced by BstNI digestion of plasmid pRI25 (18).

RESULTS The E2F element confers growth regulation to the dhfr promoter by position-dependent activation of transcription. Although E2F-mediated growth regulation has been proposed to result from G0-specific repression of transcription, our initial studies of the mouse dhfr promoter did not support this hypothesis. Mutation of the E2F element in the murine dhfr promoter resulted in only a two- to threefold increase in transcriptional activity in G0-phase cells (50), suggesting that repression may not mediate dhfr regulation. However, since the E2F element overlaps the transcription start site, the change in promoter activity could have been influenced (positively or negatively) by changes in the initiator region. To further investigate the mechanisms by which E2F regulates the dhfr promoter, we have now examined the influence of position on the ability of the E2F element to confer regulation to the core dhfr promoter. To determine if the dhfr E2F element could confer growth regulation from an upstream position, we placed one or two copies of the E2F element 375 bp upstream of the transcription start site of a core dhfr promoter containing a mutated E2F element in the wild-type position (Fig. 1A). NIH 3T3 cells were transfected with the dhfr-luciferase reporter constructs and

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FIG. 1. The position of the E2F element is critical for growth regulation of the dhfr promoter. Schematics of the wild-type (WT) and modified dhfr-luciferase reporter constructs containing E2F elements upstream (A) or downstream (C) of the wild-type position are shown. All of the constructs contain the E2F element in the same orientation as in the wild-type dhfr promoter, with the exception of pMaeF2E@2375, which contains the E2F element in the opposite orientation. Growth regulation of the dhfr-luciferase reporter constructs containing the E2F element moved upstream (B) and downstream (D) of the wild-type position was compared to that of the wild-type reporter constructs in transiently transfected NIH 3T3 cells in serum starvation and stimulation experiments. Each reporter construct (5 mg) was transiently transfected into 1.3 3 105 NIH 3T3 cells which were induced to enter quiescence by incubation in serum starvation medium for 48 h. The cells were then incubated in serum stimulation medium for 12 h, causing the cells to enter the S phase of the cell cycle. Luciferase values are reported as a ratio of the activity of each reporter construct 12 h after serum stimulation relative to the activity of the same reporter construct in quiescent cells. Bars represent standard errors of the means.

induced to enter quiescence by using serum starvation medium. The starvation medium was then replaced with serum stimulation medium, causing the cells to reenter the proliferative cycle as a synchronous population. Cells were harvested and assayed for luciferase activity at 0 h (quiescent cells) and 12 h (S-phase cells) after serum stimulation, and the increase in activity of each reporter construct after serum stimulation is shown as fold S-phase induction (Fig. 1B). The activity of pMaeWT, which contains a functional E2F element, increased approximately 14-fold after serum stimulation, while the activity of pMaeDE2F, which contains a mutated E2F element, increased only 4-fold (Fig. 1B). This residual activation of pMaeDE2F is conferred by the four Sp1 elements positioned upstream of the E2F element (47). The activity of the reporter constructs with one or two copies of the E2F element posi-

tioned at nucleotide 2375 increased only three- to fourfold (Fig. 1B). Thus, these constructs do not show the E2F-mediated increase in transcription levels after serum stimulation. This finding demonstrates that the E2F element cannot confer growth regulation from a distance upstream of the core dhfr promoter. To determine if the E2F element could confer growth regulation from a position downstream of the core dhfr promoter, we moved the E2F element 14, 42, and 66 bp downstream of the wild-type position (Fig. 1C). NIH 3T3 cells were transfected with the dhfr-luciferase reporter constructs, serum starved, and stimulated as described above for the upstream reporter constructs. The activity of the wild-type dhfr promoter increased approximately 12-fold after serum stimulation (Fig. 1D). As the E2F element was moved further downstream from

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FIG. 2. The position of the E2F element does not influence the position of the transcriptional start site. In vitro transcription analysis of templates corresponding to the pMaeWT, pMaeDE2F, and pMaeE2F@2375 reporter constructs (A) and the p410WT and p410E2F@166 reporter constructs (B) was performed with HeLa cell nuclear extracts. The numbers to the left of each gel indicate the positions of size markers, while the numbers to the right indicate the sizes (in nucleotides) of the transcripts produced by the indicated templates. Schematics of the templates and sizes of the transcripts produced are shown below each gel.

the wild-type position, the S-phase induction of dhfr promoter activity gradually decreased (Fig. 1D). For example, the activity of the dhfr promoter construct having an E2F element at 166 increased only twofold after serum stimulation (Fig. 1D). Taken together, our results indicate that the E2F element cannot confer growth regulation to the dhfr promoter from a distance, either upstream or downstream of the wild-type position. Although our results suggest that loss of growth regulation of the dhfr promoter is due to the movement of the E2F element, it was possible that loss of regulation was actually due to the movement of the site of transcription initiation. Based on the transcriptional activity of a dhfr promoter with the E2F element deleted, we previously proposed that protein binding to the E2F element positioned the start site of transcription (40). However, the E2F element overlaps a consensus initiator element (a sequence that spans the transcription start site of many promoters and may play a critical role in the positioning of transcription) located at nucleotides 22 to 16 in the dhfr promoter (28, 40, 46). Subsequent experiments using several different point mutations showed that mutation of the E2F element only alters the transcription start site when the mutation also alters the 21 to 12 region of the promoter (28, 46), suggesting that E2F is not critical for positioning of the transcription start site. Other studies have implicated Sp1 in the positioning of transcription initiation, demonstrating that an Sp1 element can substitute for a TATA box as the primary positioning element and direct transcription initiation approx-

imately 50 bp downstream (2, 29). Therefore, it is likely that the Sp1 elements, in combination with the 21 to 12 region in the murine dhfr promoter direct efficient transcription at the initiation site (14, 19, 40, 53). Although we expected the Sp1 elements to specify the position of the start site in our dhfr promoter-reporter constructs, we felt it necessary to demonstrate that the position of the transcription start site did not change when the E2F element was moved. Therefore, we analyzed three non-growth-regulated dhfr promoter constructs by using an in vitro transcription assay with HeLa cell nuclear extract. pMaeDE2F and pMaeE2F@2375, both of which contain the wild-type 21 to 12 region of the dhfr promoter (CAA), produced RNA transcripts the same length as the transcript produced by pMaeWT (Fig. 2A). p410E2F@166 produced an RNA transcript approximately 66 nucleotides longer than the RNA transcript produced by p410WT (Fig. 2B), indicating that even though the E2F element was moved 66 bp downstream, the approximate position of the transcription start site relative to the upstream Sp1 elements was maintained. These results indicate that the approximate position of the transcription start site relative to the core dhfr promoter region does not change when the E2F element is moved upstream or downstream of its wild-type position. Also, the transcriptional activity of pMaeE2F@2375 and p410E2F@166 was similar to the activity of their wild-type counterparts, indicating that movement of the E2F element did not cause a decrease in levels of basal transcription in vitro.

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FIG. 3. Loss of regulation of the dhfr promoter is not due to derepression. Promoter activities of the p410WT and p410E2F@166 reporter constructs in serum-starved (G0-phase) and serum-stimulated (S-phase) cells are shown for the starvation and stimulation experiments described in the legend to Fig. 1. Luciferase values are reported as relative luciferase units (Rlu), which directly correspond to the transcriptional activities of the reporter constructs. Bars represent standard errors of the means.

Our experiments indicate that although the E2F element is not involved in the positioning of the start site, the position of the E2F element in the dhfr promoter is critical for growth regulation. Others have shown that an E2F element can confer Rb-mediated transcriptional repression to the simian virus 40 (SV40) enhancer from a distance of 2 kb upstream, suggesting that repression is independent of the position of the E2F element (56). Therefore, our results suggest a different role for E2F in the regulation of the dhfr promoter. Support for the hypothesis that dhfr is not regulated by G0-specific repression comes from a comparison of actual promoter activities of growth-regulated versus non-growth-regulated dhfr promoter constructs. We find that loss of growth regulation resulting from the displacement of the E2F element is not due to increased promoter activity in G0-phase cells (which would be expected if loss of repression occurred) but rather is due to decreased promoter activity in S-phase cells (Fig. 3). Taken together, our results indicate that the E2F element confers growth regulation to the dhfr promoter by mediating positiondependent activation of transcription in S phase. The mechanism by which E2F mediates growth regulation is core promoter specific, not E2F element specific. Although our studies suggest that the E2F element is necessary for high levels of transcription from the dhfr promoter, E2F elements in other promoters, including B-myb, are thought to regulate activity through the cell cycle by conferring G0-specific transcriptional repression (33, 34, 61). To determine if these distinct mechanisms of E2F-mediated growth regulation are specified by the E2F element or by the core promoter, we replaced the E2F element in the dhfr promoter with the E2F element from the murine B-myb promoter (Fig. 4A). Given that an E2F element can confer Rb-mediated repression from a distance of 2 kb upstream of a core promoter (56), the B-myb E2F element should be able to repress the dhfr promoter when positioned at nucleotide 2375. Therefore, if G0-phase repression is an intrinsic property of the E2F element, the B-myb “repressor” E2F element should confer growth regulation to the dhfr promoter from both the wild-type and upstream positions. In contrast, if position-dependent S-phase-specific activation of

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transcription is specified by the core dhfr promoter, regulation of the dhfr promoter by the B-myb E2F element should be identical to regulation by the dhfr E2F element. For a positive control, we first determined if the B-myb E2F element could confer growth regulation to the dhfr promoter if it is proximal to the transcription start site (Fig. 4B and C). Both the dhfr and B-myb E2F elements consist of two overlapping and inverted E2F consensus binding sites. Thus, the orientation of the B-myb element should not affect E2F activity. However, the sequences flanking either side of the B-myb E2F element are different. Therefore, we placed the B-myb E2F element in the orientation that creates a natural transcription start element (CAA from positions 21 to 12). NIH 3T3 cells were transfected with dhfr promoter constructs having the dhfr E2F element, B-myb E2F element, or no E2F element and then serum starved and stimulated as described above. The activity of p410WT, which contains the dhfr E2F element, increased 14-fold after serum stimulation, while the activity of p410DE2F, which contains a mutated E2F element, increased only 4-fold (Fig. 4C). The activity of p410bym-B, which contains the B-myb E2F element, increased approximately 10-fold after serum stimulation (Fig. 4C). Deviation of the B-myb E2F binding sites from the consensus E2F binding site may account for the lower increase in promoter activity after serum stimulation. Thus, the B-myb E2F element can confer growth regulation to the dhfr promoter if placed proximal to the transcription start site. To determine if the B-myb E2F element can confer growth regulation from a position upstream of the core dhfr promoter, we placed the B-myb E2F element in both orientations 375 bp upstream of the transcriptional start site of a core dhfr promoter containing a mutated E2F element in the wild-type position (Fig. 4D). NIH 3T3 cells were transfected with the dhfr-luciferase reporter constructs, serum starved, and stimulated as described above. The activity of pMaeWT increased 21-fold after serum stimulation, while the activity of pMaeDE2F, which contains a mutated E2F element, increased only 5-fold (Fig. 4E). The activities of the dhfr promoters having the B-myb E2F element positioned at nucleotide 2375, increased only 4- to 5-fold after serum stimulation (Fig. 4E). These results demonstrate that the B-myb E2F element cannot confer growth regulation from a distance upstream of the core dhfr promoter. Thus, our experiments indicate that the position-dependent S-phase-specific activation of the dhfr promoter by E2F is specified by the core dhfr promoter and not by the E2F element. The C-terminal transactivation domain of E2F can mediate position-dependent activation of the dhfr promoter. Several properties have been ascribed to the various members of the E2F family of transcription factors. For example, E2F1, E2F2, and E2F3 contain regions near the N terminus that bind to Sp1 and cyclin A (27, 30, 31, 36, 59). E2F1 has been shown to bend DNA (9); the other family members may share this property, since DNA bending is mediated by a region common to all five E2F proteins. Furthermore, all five E2F family members contain activation domains at the carboxyl terminus of the protein (17, 22, 26, 35). To determine which domain of the E2F proteins was responsible for the position-dependent activation of dhfr transcription in S phase, we created a Gal4-responsive dhfr promoter-reporter construct by replacing the E2F element with a Gal4 element (Fig. 5A). This replacement deleted dhfr promoter sequences from positions 120 to 161 and inserted an additional 5 bp between the transcription start site and upstream Sp1 elements. To control for any effects on the position of the start site or transcriptional activity of the promoter, we also created the pseudoWT reporter construct (Fig. 5A), which contains the 5-bp insertion and ends at 120 but still

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FIG. 4. The dhfr and B-myb E2F elements confer similar regulation to the dhfr promoter. (A) Sequence comparison of the dhfr and B-myb E2F elements. The overlapping E2F binding sites in each element are indicated in boldface type. The E2F binding sites in the dhfr E2F element are both consensus binding sites (TTTSSCGC, with S being C or G) while the two binding sites in the B-myb E2F element deviate from the consensus binding sites (deviations indicated by lowercase type). Schematics of the dhfr-luciferase reporter constructs containing the B-myb E2F element at the wild-type (B) and upstream (D) positions are shown. The B-myb E2F element in pMaeB-myb@2375 is in the same orientation as in the B-myb promoter, while the E2F element in p410bym-B and pMaebym-B@2375 is in the orientation opposite that in the B-myb promoter. Growth regulation of the dhfr-luciferase reporter constructs containing the dhfr or B-myb E2F elements at the wild-type (C) and upstream (E) position was examined in transiently transfected NIH 3T3 cells in serum starvation and stimulation experiments, as described in the legend to Fig. 1. Luciferase values are reported as a ratio of the activity of each reporter construct 12 h after serum stimulation relative to the activity of the same reporter construct in quiescent cells. Bars represent standard errors of the means.

contains an E2F element. Before analysis of these promoter constructs in cells, the start sites and activity of the p410WT, pseudoWT, and DHFRGal4 constructs were analyzed by in vitro transcription reactions with HeLa cell nuclear extract. As expected due to the loss of dhfr sequences and vector sequences (see Materials and Methods), DHFRGal4 and pseudoWT produced RNA transcripts approximately 61 bp shorter than the RNA transcript produced by p410WT (Fig. 5B). Also, the transcriptional activities of DHFRGal4 and pseudoWT were similar to the activity of p410WT, indicating

that the basal levels of transcription for DHFRGal4 and pseudoWT are comparable to that of p410WT. Our experiments using Gal4-E2F fusion constructs were performed as cotransfection assays using asynchronously growing NIH 3T3 cells. For a control, we examined the ability of all Gal4 fusions to activate pG5TI, a promoter construct containing five Gal4 elements cloned upstream of a TATA box and an initiator element (Fig. 6A). We found that the N terminus of E2F1, when fused to the Gal4 DNA binding domain, could not activate transcription from DHFRGal4 or pG5TI (data not

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FIG. 5. Cloning and characterization of a Gal4-responsive dhfr-luciferase reporter construct. (A) The DHFRGal4 reporter was constructed by replacing the E2F element in p410WT with one Gal4 binding element. DHFRGal4 contains an additional 5 bp between the transcriptional start site and upstream Sp1 elements because of the different sizes of the E2F and Gal4 elements; therefore, pseudoWT was constructed by inserting the same 5 bp of DNA directly upstream of the E2F element in p410WT. Templates with the sequence changes and expected transcript sizes are shown. (B) In vitro transcription analysis of the p410WT, pseudoWT, and DHFRGal4 templates was performed with HeLa cell nuclear extracts. The numbers to the left of the gel indicate the positions of size markers, while the numbers to the right indicate the sizes (in nucleotides) of the transcripts produced by the indicated templates.

shown). However, Gal4 fusions to the carboxyl-terminal domains of E2F1 (amino acids 368 to 437), E2F4 (amino acids 276 to 412), and E2F5 (amino acids 222 to 346) were all potent transactivators, resulting in 5,000-, 2,400-, and 1,000-fold activation of pG5TI transcription, respectively (Fig. 6A). These fusion proteins could also activate the DHFRGal4 reporter construct (Fig. 6B), with Gal4-E2F1, Gal4-E2F4, and Gal4E2F5, giving 150-, 120-, and 20-fold activation of transcription, respectively. Therefore, we concluded that the C-terminal activation domains of E2F1, E2F4, and E2F5 can all activate the dhfr promoter from the wild-type E2F element position. To determine if the E2F activation domains can activate transcription from a position upstream of the core dhfr promoter, we placed one Gal4 element 375 bp upstream of the transcription start site of a core dhfr promoter containing a mutated E2F element in the wild-type position (Fig. 6C). NIH 3T3 cells were cotransfected with this DHFRGal4@2375 reporter construct and the Gal4-fusion expression plasmids. Figure 6C shows that cotransfection of DHFRGal4@2375 with Gal4-E2F1, Gal4-E2F4, and Gal4-E2F5 resulted in only a twoto threefold activation of transcription; therefore, the C-termi-

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nal activation domains of E2F1, E2F4, and E2F5 cannot activate transcription from a distance upstream of the core dhfr promoter. The E2F transactivation domain contains a special region that allows it to activate the dhfr promoter. Our results suggest that growth regulation of the dhfr promoter involves positiondependent activation of transcription by the E2F transactivation domain. We next wished to determine if other transactivation domains displayed a similar position dependence with the dhfr promoter. We first examined the ability of several Gal4 fusions to activate the pG5TI promoter in asynchronously growing NIH 3T3 cells. Cotransfection of pG5TI or DHFR Gal4 with Gal4-MyoD, Gal4-Ets2, Gal4-YY1 (amino acids 1 to 200 or 1 to 414), Gal4-p53, Gal4-Sp1 (amino acids 83 to 621), Gal4-ATF1, Gal4-ATF2, and Gal4-EBNA1 expression plasmids all resulted in very minimal activation of transcription (data not shown), indicating that these Gal4 fusion proteins are very weak activators of both the pG5TI and dhfr promoters. However, we found that the E2F1 C-terminal and VP16 N-terminal transactivation domains are equally potent activators of pG5TI, both resulting in approximately 4,000-fold activation of transcription (Fig. 7A). Therefore, we tested the ability of Gal4-VP16 to activate the dhfr promoter from a position at the transcription start site and upstream of the core promoter. Surprisingly, we found that Gal4-VP16 could not activate the dhfr promoter from either position (Fig. 7B). Previously, it has been demonstrated that Gal4-VP16 is a much more efficient activator of TATA-containing promoters than TATA-less promoters both in vitro and in vivo (7, 12). The dhfr promoter does not contain a consensus TATA element (46); therefore, it is possible that the inability of Gal4-VP16 to activate the dhfr promoter is due to the lack of a TATA element. To determine if the addition of a TATA element to the dhfr promoter could alter the ability of Gal4-VP16 to activate transcription, we employed a dhfr promoter-reporter construct containing a TATA element centered at 230 in the DHFRGal4 promoter. For a control, we also examined the ability of Gal4-E2F1 to activate this promoter. We found that Gal4-E2F1 gave an 85-fold activation of transcription, but Gal4-VP16 gave only a 4-fold activation of transcription (Fig. 7C). These results demonstrate that the inability of the Nterminal transactivation domain of VP16 to activate the dhfr promoter is not due to the lack of a TATA element. Taken together, our results suggest that S-phase activation of dhfr transcription requires the recruitment of a specific transactivation domain (i.e., that from an E2F family member) to a position proximal to the transcription start site of the dhfr promoter. DISCUSSION Many promoters that contain E2F elements display differential activity in G0 versus S phase. A model for E2F-mediated regulation of transcription has been previously proposed in which G0-phase repression of transcription is conferred by E2F-pocket protein (Rb, p107, or p130) complexes. According to this model, the increase in transcription observed in S phase is due to release of the transcriptional repressor pocket protein from E2F proteins, which occurs upon phosphorylation of the pocket proteins by cyclin-cdk complexes. This model is consistent with mutational analyses of promoters such as B-myb that show constitutively high activity in G0 and S phase upon mutation of the E2F element (33, 34). However, this model does not sufficiently explain the regulation of the dhfr promoter. We now propose a model in which growth regulation of dhfr transcription involves the S-phase-specific activation of the dhfr

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FIG. 6. The transactivation domains of E2F1, E2F4, and E2F5 show position-dependent activation of the dhfr promoter. The ability of the Gal4-E2F fusion proteins to activate the pG5TI (A), DHFRGal4 (B), and DHFRGal4@2375 (C) reporter constructs was examined in transiently transfected asynchronously growing NIH 3T3 cells. Each reporter plasmid and Gal4 fusion expression plasmid (5 mg of each) were transfected into 105 cells, which were then incubated in growth medium for 48 h. Schematics of the pG5TI, DHFRGal4, and DHFRGal4@2375 reporter constructs are shown above each graph. Luciferase values are reported as a ratio of the activity of each reporter construct in the presence of the specified Gal4 fusion expression plasmid relative to the activity of the same reporter construct in the presence of the Gal4(1-147) expression plasmid. Bars represent standard errors of the means.

promoter by an E2F transactivation domain that must be positioned proximal to the transcription start site. This model is based on the following observations. (i) Mutation or displacement of the E2F element causes a decrease in S-phase levels of transcription, not an increase in G0-phase levels of transcription. These results are in contrast to studies done on the B-myb promoter which have shown that E2F confers G0-phase repression of transcription (33, 34). (ii) Movement of the E2F element to an upstream (2375) or downstream (166) position abolishes growth regulation of the dhfr promoter, indicating that E2F can regulate the dhfr promoter only from a position proximal to the transcription start site. These results are in contrast to other studies showing that an E2F element can confer repression to the SV40 promoter from a distal position (56). We show that two dhfr E2F ele-

ments cannot confer growth regulation to the dhfr promoter from a distal upstream position (2375), even though the same two dhfr E2F elements can confer growth regulation when positioned at a distance upstream of the SV40 promoter (52). We also show that the B-myb E2F element, which has been suggested to confer G0-phase repression to the B-myb promoter, is able to confer growth regulation to the dhfr promoter only when located proximal to the transcription start site. Thus, the position dependence of E2F-mediated growth regulation is specified by the core dhfr promoter and not by the characteristics of a particular E2F element. (iii) The transactivation domains of E2F proteins display position-dependent activity at the dhfr promoter. Other studies have suggested that E2F proteins can be stabilized on promoter DNA by interaction with Sp1 and NFY (27, 36, 55). Although we have changed the distance between the E2F and Sp1 elements in our experiments, we do not believe that DNA binding is specifying position dependence in the dhfr promoter. This is because the DNA binding requirement of E2F for interaction with other proteins can be relieved when binding is mediated via the Gal4 DNA binding domain (55). We note that the Gal4 DNA binding domain has also been shown to bend DNA (45). However, we determined that the Gal4 DNA binding domain alone does not activate the dhfr promoter (data not shown). Thus, the position-dependent activation is mediated by a property inherent to the Gal4-E2F fusion proteins that is not present in the Gal4 DNA binding domain. We do not rule out the involvement of Sp1 or DNA bending in the regulation of the endogenous dhfr promoter. However, the use of a heterologous DNA binding domain has allowed us to

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FIG. 7. The N-terminal activation domain of VP16 cannot activate the dhfr promoter. We compared the activation of the pG5TI (A), DHFRGal4 and DHFRGal4@2375 (B), and DHFRTATAGal4 (C) reporter constructs by the Gal4-VP16(411-454) and Gal4-E2F1(368-437) expression plasmids, as described in the legend to Fig. 6. Schematics of the pG5TI, DHFRGal4, DHFRGal4@2375, and DHFRTATAGal4 reporter constructs are shown above each graph. Luciferase values are reported as a ratio of the activity of each reporter construct in the presence of the specified Gal4 fusion expression plasmid relative to the activity of the same reporter construct in the presence of the Gal4(1-147) expression plasmid. Bars represent standard errors of the means.

examine the post-DNA-binding requirements for E2F-mediated activation of dhfr transcription, and our results suggest that the transactivation domain alone confers the dependence on position. Our model requires that an E2F transactivation domain be brought near the dhfr transcription start site for high-level activity from the dhfr promoter (Fig. 8). Since E2F proteins are bound to at least one strand of the dhfr promoter throughout the cell cycle, but dhfr transcription levels are high only in S

phase, interactions between the core dhfr promoter and E2F must be productive only in S phase. We suggest that the dhfr promoter has the potential to be responsive to the E2F transactivation domain throughout the cell cycle but that low promoter activity in G0 phase is due to the masking of the critical E2F activation domain by the pocket proteins. This masking is distinguished from the repression of a strong promoter (such as B-myb) because the dhfr promoter is not active in the absence of an E2F protein. Thus, unlike other promoters, the increase in activity of the dhfr promoter in S phase requires the presence of an active E2F complex; it is not sufficient to simply release a transcriptional repressor pocket protein. We have shown that the dhfr promoter has a special requirement for the transactivation domain of E2F proteins that is not observed in most E2F-regulated promoters. This critical requirement of the dhfr promoter for transactivation by E2F proteins is emphasized by the fact that the potent N-terminal transactivation domain of VP16 cannot substitute for the E2F activation domain. Several properties have been ascribed to the E2F transactivation domain, including binding to the TATA-binding protein, TFIIH, the CREB-binding protein, and the Rb family of pocket proteins (3, 13, 26, 54). However, the N-terminal transactivation domain of VP16 has been shown to bind TFIID and TFIIB (24, 37), suggesting that the binding of TFIID and/or TFIIB is not sufficient for the activation of the dhfr promoter. We have determined that an E2F1 transactivation domain shown to be deficient in Rb binding (17) can still efficiently activate the dhfr promoter (data not shown), suggesting that the binding of Rb is not critical for


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FIG. 8. A model for E2F-mediated regulation of the dhfr promoter. Schematics of the dhfr reporter constructs containing a mutated E2F element (DE2F), and the E2F element in the wild-type (WT), downstream (E2F@166), and upstream (E2F@2375) positions are shown. Black vertical ovals represent Sp1 proteins. Curved arrows represent E2F-mediated activation of transcription. We have provided evidence that high levels of dhfr promoter activity occur only when an E2F transactivation domain is bound near the transcription start site. Differential activity is conferred to E2F by the pocket proteins which bind to and mask the function of the E2F transactivation domain in G0 phase, resulting in low levels of dhfr transcription. Mutation or displacement of the E2F element results in constitutively low levels of dhfr promoter activity. Thus, the increase in S-phase activity requires the direct activation of the dhfr promoter by the E2F transactivation domain, and not just the release of a repressor pocket protein.

activation of dhfr transcription (although it may play a role in masking the E2F transactivation domain in G0 phase). It remains possible that the binding of TFIIH and/or CREB-binding protein by E2F is critical for the activation of dhfr transcription. For example, it has previously been shown that transcription from the dhfr promoter in vitro requires the Cterminal domain (CTD) of the largest subunit of RNA polymerase II and the CTD-kinase activity of TFIIH, suggesting that dhfr transcription may be regulated at the level of promoter clearance (1, 6). Thus, it is possible that E2F activates the dhfr promoter by recruiting or stimulating the TFIIH CTDkinase activity and increasing levels of promoter clearance. In summary, we have provided evidence that the dhfr promoter is highly active only when an E2F transactivation domain is positioned at the transcription start site and unmasked from the pocket proteins. We suggest that the E2F transactivation domain binds a specific factor that is critical for the activation of the dhfr promoter. Further experiments will allow us to identify the factor required for dhfr activation and characterize the mechanism by which E2F activates transcription. ACKNOWLEDGMENTS We are grateful to those who kindly provided the Gal4 fusion expression constructs: Paul Robbins for Gal4-MyoD, Gal4-p53, Gal4Sp1, Gal4-ATF1, and Gal4-ATF2; Christopher Denny for Gal4-Ets2; Michael Atchison for Gal4-YY1; and Bill Sugden for Gal4-EBNA1. We thank Paul van Ginkel for generous communication of unpublished data and Stephanie M. Bartley for excellent technical assistance. We thank the rest of the Farnham lab for all of their helpful suggestions and critical reading of this manuscript. This work was supported by grants from the National Institutes of Health (CA45240, CA07175, and CA09135). REFERENCES 1. Akoulitchev, S., T. P. Makela, R. A. Weinberg, and D. Reinberg. 1995. Requirement for TFIIH kinase activity in transcription by RNA polymerase II. Nature 377:557–560.

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