Regulation of the Cyclin D3 Promoter by E2F1
2003; Elsevier BV; Volume: 278; Issue: 19 Linguagem: Inglês
10.1074/jbc.m212702200
ISSN1083-351X
AutoresYihong Ma, Jing Yuan, Mei Huang, Richard Jove, W. Douglas Cress,
Tópico(s)Genomics and Chromatin Dynamics
ResumoWe have previously demonstrated that ectopic expression of E2F1 is sufficient to drive quiescent cells into S phase and that E2F1 expression can contribute to oncogenic transformation. Key target genes in this process include master regulators of the cell cycle, such as cyclin E, which regulates G1progression, and cyclin A, which is required for the initiation of DNA synthesis. In the present work, we present novel evidence that a second G1 cyclin, cyclin D3, is also potently activated by E2F1. First, an estrogen receptor-E2F1 fusion protein (ER-E2F1) potently activates the endogenous cyclin D3 mRNA upon treatment with 4-hydroxytamoxifen, which induces nuclear accumulation of the otherwise cytosolic fusion protein. Furthermore, trans-activation of cyclin D3 by ER-E2F1 occurs even in the presence of the protein synthesis inhibitor cycloheximide and thus appears direct. Second, all of the growth-stimulatory members of the E2F family (E2F1, -2, and -3A) potently activate a cyclin D3 promoter reporter, whereas growth-restraining members of the family (E2F4, -5, and -6) have little effect. Third, recombinant E2F1 binds with high affinity to the cyclin D3 promoter in vitro. Fourth, chromatin immunoprecipitation assays demonstrate that endogenous E2F1 is associated with the cyclin D3 promoter in vivo. Finally, mapping experiments localize the essential E2F regulatory element of the cyclin D3 promoter to a noncanonical E2F site in the promoter between nucleotides −143 and −135 relative to the initiating methionine codon. We conclude that in addition to cyclins E and A, E2F family members can also activate one member of the D-type cyclins, further contributing to the ability of the stimulatory E2F family members to drive cellular proliferation. We have previously demonstrated that ectopic expression of E2F1 is sufficient to drive quiescent cells into S phase and that E2F1 expression can contribute to oncogenic transformation. Key target genes in this process include master regulators of the cell cycle, such as cyclin E, which regulates G1progression, and cyclin A, which is required for the initiation of DNA synthesis. In the present work, we present novel evidence that a second G1 cyclin, cyclin D3, is also potently activated by E2F1. First, an estrogen receptor-E2F1 fusion protein (ER-E2F1) potently activates the endogenous cyclin D3 mRNA upon treatment with 4-hydroxytamoxifen, which induces nuclear accumulation of the otherwise cytosolic fusion protein. Furthermore, trans-activation of cyclin D3 by ER-E2F1 occurs even in the presence of the protein synthesis inhibitor cycloheximide and thus appears direct. Second, all of the growth-stimulatory members of the E2F family (E2F1, -2, and -3A) potently activate a cyclin D3 promoter reporter, whereas growth-restraining members of the family (E2F4, -5, and -6) have little effect. Third, recombinant E2F1 binds with high affinity to the cyclin D3 promoter in vitro. Fourth, chromatin immunoprecipitation assays demonstrate that endogenous E2F1 is associated with the cyclin D3 promoter in vivo. Finally, mapping experiments localize the essential E2F regulatory element of the cyclin D3 promoter to a noncanonical E2F site in the promoter between nucleotides −143 and −135 relative to the initiating methionine codon. We conclude that in addition to cyclins E and A, E2F family members can also activate one member of the D-type cyclins, further contributing to the ability of the stimulatory E2F family members to drive cellular proliferation. 4-hydroxytamoxifen cycloheximide electrophoretic mobility shift assay chromatin immunoprecipitation estrogen receptor wild type mutant The mammalian G1 cyclins, which include cyclins D1, D2, D3, E1 and E2, are important regulators of cellular proliferation (1Nevins J.R. Hum. Mol. Genet. 2001; 10: 699-703Crossref PubMed Scopus (745) Google Scholar, 2Payton M. Coats S. Int. J. Biochem. Cell Biol. 2002; 34: 315-320Crossref PubMed Scopus (43) Google Scholar, 3Ho A. Dowdy S.F. Curr. Opin. Genet. Dev. 2002; 12: 47-52Crossref PubMed Scopus (159) Google Scholar). Quiescent cells express the G1 cyclins at minimal levels; however, upon mitogenic stimulation, the D-type cyclins are transcriptionally activated, and their levels rise as cells traverse G1 (4Sherr C.J. Cancer Res. 2000; 60: 3689-3695PubMed Google Scholar). In the prevailing model (3Ho A. Dowdy S.F. Curr. Opin. Genet. Dev. 2002; 12: 47-52Crossref PubMed Scopus (159) Google Scholar), it is thought that the D-type cyclins direct the limited phosphorylation of Rb via their interactions with cyclin-dependent kinases (5Ezhevsky S.A. Nagahara H. Vocero-Akbani A.M. Gius D.R. Wei M.C. Dowdy S.F. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10699-10704Crossref PubMed Scopus (250) Google Scholar, 6Ezhevsky S.A. Ho A. Becker-Hapak M. Davis P.K. Dowdy S.F. Mol. Cell. Biol. 2001; 21: 4773-4784Crossref PubMed Scopus (178) Google Scholar, 7Ewen M.E. Sluss H.K. Sherr C.J. Matsushime H. Kato J. Livingston D.M. Cell. 1993; 73: 487-497Abstract Full Text PDF PubMed Scopus (918) Google Scholar). Phosphorylation of Rb by the D-type cyclin-cyclin-dependent kinases complexes permits the release of DNA-E2F-Rb-bound histone deacetylase proteins, which impart a dominant transcriptional repressing activity to promoter-bound Rb-E2F complexes (8Zhang H.S. Gavin M. Dahiya A. Postigo A.A. Ma D. Luo R.X. Harbour J.W. Dean D.C. Cell. 2000; 101: 79-89Abstract Full Text Full Text PDF PubMed Scopus (542) Google Scholar). Loss of histone deacetylase-mediated inhibition allows a modest transcriptional activation of certain Rb-silenced genes, including cyclin E (9Ohtani K. DeGregori J. Nevins J.R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 12146-12150Crossref PubMed Scopus (536) Google Scholar), but other E2F-Rb-regulated genes remain repressed (8Zhang H.S. Gavin M. Dahiya A. Postigo A.A. Ma D. Luo R.X. Harbour J.W. Dean D.C. Cell. 2000; 101: 79-89Abstract Full Text Full Text PDF PubMed Scopus (542) Google Scholar). Once stimulated, cyclin E associates with cyclin-dependent kinase 2 and adds additional phosphate modifications to Rb later in G1 (10Lundberg A.S. Weinberg R.A. Mol. Cell. Biol. 1998; 18: 753-761Crossref PubMed Scopus (859) Google Scholar). Upon its hyperphosphorylation by the cooperative efforts of the D-type cyclins and cyclin E, the Rb protein completely releases the E2F transcription factor. Liberated E2F3B and E2F4, which are the predominant E2Fs expressed (11He Y. Cress W.D. J. Biol. Chem. 2002; 277: 23493-23499Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar, 12He Y. Armanious M.K. Thomas M.J. Cress W.D. Oncogene. 2000; 19: 3422-3433Crossref PubMed Scopus (68) Google Scholar, 13Leone G. Nuckolls F. Ishida S. Adams M. Sears R. Jakoi L. Miron A. Nevins J.R. Mol. Cell. Biol. 2000; 20: 3626-3632Crossref PubMed Scopus (152) Google Scholar, 14Moberg K. Starz M.A. Lees J.A. Mol. Cell. Biol. 1996; 16: 1436-1449Crossref PubMed Scopus (304) Google Scholar) and promoter-bound (15Takahashi Y. Rayman J.B. Dynlacht B.D. Genes Dev. 2000; 14: 804-816Crossref PubMed Google Scholar, 16Wells J. Boyd K.E. Fry C.J. Bartley S.M. Farnham P.J. Mol. Cell. Biol. 2000; 20: 5797-5807Crossref PubMed Scopus (208) Google Scholar) in G0 and early G1, then activate transcription (17Johnson D.G. Ohtani K. Nevins J.R. Genes Dev. 1994; 8: 1514-1525Crossref PubMed Scopus (454) Google Scholar, 18Hsiao K.M. McMahon S.L. Farnham P.J. Genes Dev. 1994; 8: 1526-1537Crossref PubMed Scopus (222) Google Scholar, 19Sears R. Ohtani K. Nevins J.R. Mol. Cell. Biol. 1997; 17: 5227-5235Crossref PubMed Scopus (182) Google Scholar, 20Adams M.R. Sears R. Nuckolls F. Leone G. Nevins J.R. Mol. Cell. Biol. 2000; 20: 3633-3639Crossref PubMed Scopus (115) Google Scholar) of the more potent S phase-promoting E2Fs, E2F1, -2, and -3A (21DeGregori J. Leone G. Miron A. Jakoi L. Nevins J.R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7245-7250Crossref PubMed Scopus (610) Google Scholar). The combined activities of E2F1, -2, and -3A then lead to the potent activation of a large number of genes that are required for nucleotide biosynthesis, the firing of origins of replication, and the completion of replicative DNA synthesis (22Muller H. Bracken A.P. Vernell R. Moroni M.C. Christians F. Grassilli E. Prosperini E. Vigo E. Oliner J.D. Helin K. Genes Dev. 2001; 15: 267-285Crossref PubMed Scopus (633) Google Scholar, 23Ishida S. Huang E. Zuzan H. Spang R. Leone G. West M. Nevins J.R. Mol. Cell. Biol. 2001; 21: 4684-4699Crossref PubMed Scopus (500) Google Scholar, 24Ma Y. Croxton R.L. Moorer Jr., R.L. Cress W.D. Arch. Biochem. Biophys. 2002; 399: 212-224Crossref PubMed Scopus (96) Google Scholar). In this paradigm, E2F activates transcription of cyclin E, which then further activates E2F by stimulating the phosphorylation of Rb. This positive feedback loop leads to an irreversible commitment to entry into S phase at the restriction point (25Pardee A.B. Proc. Natl. Acad. Sci. U. S. A. 1974; 71: 1286-1290Crossref PubMed Scopus (1062) Google Scholar). In previous work, we showed that ectopic expression of E2F1 alone is sufficient to stimulate quiescent cells to enter cell cycle (26Johnson D.G. Schwarz J.K. Cress W.D. Nevins J.R. Nature. 1993; 365: 349-352Crossref PubMed Scopus (836) Google Scholar) and that stable expression of E2F1 can lead to oncogenic transformation (27Johnson D.G. Cress W.D. Jakoi L. Nevins J.R. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12823-12827Crossref PubMed Scopus (242) Google Scholar). Subsequently, we utilized microarray technology to screen for genes that could account for the ability of E2F to induce cellular proliferation (24Ma Y. Croxton R.L. Moorer Jr., R.L. Cress W.D. Arch. Biochem. Biophys. 2002; 399: 212-224Crossref PubMed Scopus (96) Google Scholar) and apoptosis (28Croxton R.L. Ma Y. Song L. Haura E.B. Cress W.D. Oncogene. 2002; 21: 1359-1369Crossref PubMed Scopus (125) Google Scholar, 29Croxton R.L. Ma Y. Cress W.D. Oncogene. 2002; 21: 1563-1570Crossref PubMed Scopus (22) Google Scholar). In addition to verifying several known E2F targets, our microarray screen suggested that E2F1 potently activates cyclin D3 transcription. Cyclins D1 and D2 were not activated by E2F1 expression in this screen. Here we verify that cyclin D3 is indeed a direct E2F-regulated transcript, and we localize the E2F regulatory element of the cyclin D3 promoter within a small and extremely GC-rich region in the vicinity of the major transcriptional start site. We conclude that in addition to cyclins E and A, E2F family members can also activate one member of the D-type cyclins, further contributing to the ability of E2F to drive the G1 to S phase transition. Human cyclin D3 promoter fragments were generated by PCR from genomic DNA, ligated into pGL3 basic vector, and sequenced. Initial PCR primers were designed to amplify 1023 bp (−1017/+6) of the published cyclin D3 promoter sequence (30Brooks A.R. Shiffman D. Chan C.S. Brooks E.E. Milner P.G. J. Biol. Chem. 1996; 271: 9090-9099Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar), which are numbered relative to the ATG codon at +1. The forward (−1017F) and reverse (+6R) PCR primers for the full-length promoter were 5′-GGGGACGCGTAGATCTCACAGAGTCTGTGCA-3′ and 5′-GGGGCTCGAGCTCCATACTCGGGGCAGCGAA-3′, respectively. The forward primer added a MluI site, and the reverse primer added aXhoI site to facilitate subcloning. Deletion mutants of the cyclin D3 promoter construct (−362/+6, −202/+6, −159/+6, and −115/+6) were generated using reverse primer +6R with the following forward primers: 5′-GGCCGGTACCACCTCCTAGAAAGTTCTCT-3′ (−362F), 5′-GGCCGGTACCGAGCATTCCACGGTTGCTA-3′ (−202F), 5′-GGGGGGTACCTGTCAGGGAAGCGGCGCG-3′ (−159F) and 5′-GGGGGGTACCGGATCCGCCGCGCAGTGCCAG-3′ (−115F), respectively. Each of these primers added a KpnI restriction site for subcloning. Promoter deletion mutants −202/−146, −202/−134, and −202/−112 were generated using primer −202F in combination with primers 5′-GGGGAAGCTTCCCTGACAGGCGCCCCG-3′ (−146R), 5′-CCCCAAGCTTCGCGCGCGCGCCGCTTCCCTG-3′ (−134R) and 5′-GGGGAAGCTTGGATCCCCAGCCCGCCCGCCG-3′ (−112R), respectively. Primers −146R, −134R, and −112R each added a HindIII restriction site for subcloning. Construct −159/+6(del9) was created using primer 5′-CCCCGGTACCTGTCAGGGAAGCGAGGGCGGCGGGCGGGCTGG-3′ (159del9F) and reverse primer +6R. Construct −146/−111 was created by annealing oligonucleotides 5′-CGGCGCGCGCGCGGGCGGCGGGCGGGCTGGGGATCCA-3′ and 5′-AGCTTGGATCCCCAGCCCGCCCGCCGCCCGCGCGCGCGCCGGTAC-3′ and cloning directly into the KpnI and HindIII sites of pGL3. The pBSK-ER-E2F1 plasmid, which encodes a hemagglutinin-tagged estrogen receptor E2F1 fusion protein (31Vigo E. Muller H. Prosperini E. Hateboer G. Cartwright P. Moroni M.C. Helin K. Mol. Cell. Biol. 1999; 19: 6379-6395Crossref PubMed Scopus (285) Google Scholar), was a gift from Dr. Kristian Helin (European Institute of Oncology, Milan, Italy). An EcoRI andNotI fragment from pBSK-ER-E2F1 was cloned into pcDNA3 to allow protein expression and to provide a selectable marker. H1299 cells obtained from Dr. Jiandong Chen (Moffitt Cancer Center) were cultured in Dulbecco's modified Eagle's medium supplemented with 57 fetal bovine serum. H1299 cells were transfected with pcDNA3-ER-E2F1 plasmid using the calcium phosphate method, and stable transfectants were selected with G418 (0.5 mg/ml). G418-resistant colonies were isolated after 2 weeks and expanded in the continued presence of 0.25 mg/ml G418. ER-E2F1-positive lines were identified by Western blot using anti-hemagglutinin and anti-E2F1 antibodies. H1299-pcDNA3 stable cell lines were established by the same method and served as negative controls. 4-Hydroxytamoxifen (HT1; 300 nm) was added to the medium to induce rapid ER-E2F1 nuclear accumulation of the ER-E2F1 protein. Cycloheximide (CHX) was used at a final concentration of 10 ॖg/ml to block new protein synthesis. Apoptosis and cell cycle parameters were measured by flow cytometry using an Apo-BrdU Kit (DB PharMingen) as previously described (32Ma Y. Cress W.D. Haura E.B. Mol. Cancer Ther. 2003; 2: 73-81PubMed Google Scholar). Total RNA was isolated from 5 × 106 H1299 cells using the RNAeasy mini kit (Qiagen). RNase protection assays were carried out with the Riboquant hCYC1 (cyclin family) multiprobe templates (BD PharMingen). Briefly, the multiprobe templates were synthesized by in vitro transcription with incorporation of [32P]dUTP and purified on Quick Spin RNA columns (Roche Applied Science). Labeled probe (1× 106cpm) was hybridized with 10 ॖg of total RNA through a temperature gradient of 90 to 56 °C over a 16-h period. Unprotected probe was removed by RNase digestion at 30 °C for 1 h followed by separation of protected RNA fragments on a 57 polyacrylamide-urea gel and detection using autoradiography. Transfections were performed using calcium phosphate with test DNAs totaling 20 ॖg of DNA per 100-cm dish. Transfections included 300 ng of expression plasmid (pcDNA3-based vectors), 10 ॖg of reporter firefly luciferase reporter plasmid (pGL3, Promega), 2 ॖg ofRenilla luciferase reporter plasmid (pRL-TK, Promega), and carrier DNA (sheared salmon sperm DNA) to equal 20 ॖg of total DNA in each transfection. Cells were harvested 48 h after transfection, and luciferase assays were performed using the Dual-Luciferase Reporter Assay System following the manufacturer's protocol (Promega). Experiments were done in triplicate, and the relative activities and S.E. values were determined. To control for transfection efficiency, firefly luciferase values were normalized to the values forRenilla luciferase. Western blots were performed as previously described (28Croxton R.L. Ma Y. Song L. Haura E.B. Cress W.D. Oncogene. 2002; 21: 1359-1369Crossref PubMed Scopus (125) Google Scholar) using monoclonal antibodies against cyclin D3 (14781A; BD PharMingen). Western blots were stripped and reprobed with an antibody to actin (A5441; Sigma) to ensure equivalent loading. Electrophoretic mobility shift assays (EMSAs) and antibody supershift assays were performed as previously described. Briefly, EMSA assays included 20 ॖg of total protein extract from E2F1-DP1 baculovirus-infected Sf9 or uninfected Sf9 cells. EMSA probes were generated by restriction digestion of the appropriate luciferase reporter plasmids (−202/+6,KpnI/XhoI; −202/−146,KpnI/HindIII; −202/−112,KpnI/BamHI; −159/+6,KpnI/XhoI; −115/+6,BamHI/XhoI; −159/−112,KpnI/BamHI). [α-32P]dATP was incorporated into band-purified DNA fragments using the Klenow fragment of DNA polymerase I. E2F consensus (sc-2507; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and E2F mutant consensus (sc-2508; Santa Cruz Biotechnology) competitor oligonucleotides were added at 20 ng/10-ॖl reaction (100-fold excess). An E2F1 monoclonal antibody (sc-251; Santa Cruz Biotechnology) was used to identify the putative E2F1-DP1 complex. The 15-bp competitor oligonucleotides used in Fig. 5C were desalted, annealed, and used in competition assays without further purification. The sequences of the upper strands of the competitor oligonucleotides were as follows: 1 (5′-GTACCGGCGCGCGCG-3′), 2 (5′-GGCGCGCGCGCGGGC-3′), 3 (5′-GCGCGCGGGCGGCGG-3′), 4 (5′-CGGGCGGCGGGCGGG-3′), 5 (5′-GGCGGGCGGGCTGGG-3′), 6 (5′-GCGGGCTGGGGATCC-3′), 7 (5′-CTGGGGATCCAAGCT-3′), and C15 (5′-AAGTTTCGCGCCCTT-3′). For reference, the boldface letters in each oligonucleotide represent the key E2F1 regulatory element as determined in Fig. 5 (although oligonucleotide 3 contains part of the sequence, it does not bind E2F; thus, the element is apparently too close to the 5′-end of the oligonucleotide to bind E2F. Underlined bases in the oligonucleotides listed above are not from the cyclin D3 promoter but represent cloning sites that were present in the relevant luciferase vectors. Chromatin immunoprecipitation (ChIP) assays were performed as previously described (16Wells J. Boyd K.E. Fry C.J. Bartley S.M. Farnham P.J. Mol. Cell. Biol. 2000; 20: 5797-5807Crossref PubMed Scopus (208) Google Scholar, 28Croxton R.L. Ma Y. Song L. Haura E.B. Cress W.D. Oncogene. 2002; 21: 1359-1369Crossref PubMed Scopus (125) Google Scholar). Briefly, asynchronously growing H1299 cells were treated with formaldehyde to create protein-DNA cross-links, and the cross-linked chromatin was then extracted, diluted with ChIP buffer, and sheared by sonication. After preclearing with protein A beads, blocked with 17 salmon sperm DNA and 17 bovine serum albumin, the chromatin was divided into equal samples for immunoprecipitation with either anti-E2F1 polyclonal antibody (sc-193; Santa Cruz Biotechnology), anti-RhoA polyclonal antibody (sc-179; Santa Cruz Biotechnology), or no antibody. The immunoprecipitates were pelleted by centrifugation, and heating reversed the cross-linking. After proteins and any contaminating RNA were removed by treatment with proteinase K and RNase, PCR primers (−202F and +6R) that generate a 230-bp product were used to detect the presence of specific DNA sequences. PCR primers corresponding to the human actin promoter (negative control) were previously described (15Takahashi Y. Rayman J.B. Dynlacht B.D. Genes Dev. 2000; 14: 804-816Crossref PubMed Google Scholar). Our previous microarray analysis utilized an adenovirus to express E2F1; thus, it was possible that the activation of the cyclin D3 promoter was a secondary consequence of E2F1 expression. To verify that activation of cyclin D3 by E2F1 was direct, we created a cell line that constitutively expresses a hemagglutinin-tagged estrogen receptor-E2F1 fusion protein (ER-E2F1). In this construct, E2F1 is fused to a transcriptionally inactive mutant of the murine estrogen receptor that is unable to bind estrogen yet retains high affinity for HT. In the absence of HT, the ER-E2F1 fusion protein is excluded from the nucleus and thus cannot directly affect transcription. Upon the addition of HT, however, the fusion protein rapidly enters the nucleus and induces transcription of E2F1 target genes. The advantage of this system is that, since the fusion protein is preexisting when HT is added, induction can occur in the absence of new protein synthesis. Therefore, promoters that are activated upon HT addition in the presence of the protein synthesis inhibitor CHX are most likely regulated by a direct mechanism. ER-E2F1-expressing H1299 cell lines were derived by transfecting the pcDNA3-ER-E2F1 plasmid followed by selection in G418. After G418-resistant colonies were isolated and screened by Western blot with both hemagglutinin and E2F1 antibodies (data not shown), five colonies expressing detectable levels of the ER-E2F1 protein were chosen for further experiments. For negative controls, G418-resistant cell lines were also derived with the empty pcDNA3 plasmid. To identify the ER-E2F1 cell line with the tightest HT regulation, the five ER-E2F1-expressing lines were transfected with an E2F reporter plasmid in which the adenovirus E2 promoter was fused to firefly luciferase. Following transfection, HT (or solvent control) was added to induce nuclear accumulation of ER-E2F1. Fig.1A shows that all five ER-E2F1 positive cell lines induced the reporter gene upon the addition of HT. Cell line H1299-ER-E2F1–15, which produced the most dramatic response (a 9-fold induction), was selected for subsequent experiments. As expected, H1299-pcDNA3 control cells did not activate the Ad E2 promoter in response to HT. To determine whether the ER-E2F1 fusion protein in ER-E2F1–15 was expressed at sufficient levels to alter cell growth properties, cell cycle parameters were determined before and after the addition of HT. As demonstrated in Fig. 1B, a 48-h treatment with HT resulted in a 157 increase in the fraction of cells in S phase and a corresponding decrease in cells in G0/G1. In addition to its ability to induce S phase, E2F1 is also a potent inducer of apoptosis. Not surprisingly, Fig. 1B reveals that nearly 247 of H1299-ER-E2F1–15 cells underwent apoptosis after 48 h of HT treatment, whereas less than 17 of the cells were apoptotic in the absence of HT (Fig. 1B). Thus, the ER-E2F1 fusion protein expressed in H1299-ER-E2F1–15 cells functioned as expected in the presence of HT. To verify direct activation of the cyclin D3 promoter by E2F1, the cell line ER-E2F1–15 and a control line (pcDNA3) were treated with solvent only, CHX alone, HT alone, or a combination of both CHX and HT. Following 16 h of treatment, cyclin D3 message levels were measured by an RNase protection assay. Fig.2A reveals that the cyclin D3 mRNA increased dramatically in the presence of HT alone and even more dramatically in the presence of both HT and cycloheximide. Since immediate early mRNAs such as cyclin D3 are stabilized in the presence of cycloheximide, the observed effects of cycloheximide alone or in combination with HT are expected (33Lau L.F. Nathans D. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 1182-1186Crossref PubMed Scopus (639) Google Scholar). These results suggest that the induction of the cyclin D3 mRNA is a direct effect of the ER-E2F1 fusion protein and does not involve synthesis of another protein that is activated or repressed by E2F1. Cyclin D3 protein expression was also measured by Western blot. In Fig. 2B, 48 h after drug treatment, the cyclin D3 protein was also induced in the presence of HT, reflecting the increase in mRNA. Levels of cyclin D3 mRNA and protein did not change in response to HT in the control cell line, as expected. These results demonstrate that cyclin D3 is a direct target of E2F1. In contrast to cyclin D3, cyclin D1 and D2 were repressed following HT treatment (data not shown). To determine whether the activation of cyclin D3 expression by E2F1 was mediated via the promoter, we used luciferase reporter constructs to examine the effect of E2F1 expression on the level of transcription from the cyclin D3 promoter. Previous analysis (30Brooks A.R. Shiffman D. Chan C.S. Brooks E.E. Milner P.G. J. Biol. Chem. 1996; 271: 9090-9099Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar) has mapped the cyclin D3 promoter to a region from nucleotide position −1017 to +6 relative to the ATG start codon. To identify E2F1-responsive elements, we tested luciferase reporter activity of three cyclin D3 promoter constructs (−1017/+6, −362/+6, and −202/+6). As demonstrated in Fig.3A, transient co-transfection of E2F1 resulted in strong activation of all three of the cyclin D3 promoter constructs. The smallest of these constructs (−202/+6) was fully responsive to exogenous E2F, suggesting that this small region contains the E2F1-responsive element. Previous studies using various E2F1 mutants have demonstrated that both the DNA-binding domain and the transcriptional activation/Rb-binding domains of E2F1 are required for trans-activation to occur. To determine whether both of these functional domains are required for cyclin D3 promoter activation, two previously described E2F1 mutant proteins were tested for the ability to activate the cyclin D3 promoter. The first was E2F1E132, which lacks the ability to bind DNA (34Cress W.D. Johnson D.G. Nevins J.R. Mol. Cell. Biol. 1993; 13: 6314-6325Crossref PubMed Scopus (109) Google Scholar), and the second was E2F11–284, which lacks the trans-activation domain (35Cress W.D. Nevins J.R. J. Virol. 1994; 68: 4213-4219Crossref PubMed Google Scholar, 36Cress W.D. Nevins J.R. Mol. Cell. Biol. 1996; 16: 2119-2127Crossref PubMed Scopus (59) Google Scholar). As expected, both E2F1 mutants had diminished ability to trans-activate the cyclin D3 promoter (Fig. 3B), demonstrating that activation of the cyclin D3 promoter is dependent on the ability of E2F1 to bind DNA and to activate transcription. Based upon structural and functional relatedness and on cell cycle expression pattern, the E2F family members are classified into two major subfamilies (37Trimarchi J.M. Lees J.A. Nat. Rev. Mol. Cell. Biol. 2002; 3: 11-20Crossref PubMed Scopus (968) Google Scholar): the growth-promoting E2Fs (including E2F1, -2, and -3A) and the growth-restraining E2Fs (including E2F3B, -4, -5, and -6). Growth-promoting E2Fs are primarily expressed at the G1/S boundary and are capable of driving quiescent fibroblasts into S phase upon overexpression. In contrast, the growth-restraining E2Fs are expressed constitutively during the cell cycle and induce S phase less efficiently or not at all when overexpressed. To determine which of the E2Fs most potently modulate the cyclin D3 promoter, several E2F family members were tested in luciferase reporter assays. Fig. 3C shows that the cyclin D3 promoter is most potently activated by the growth-promoting E2Fs (especially E2F1 and E2F2), whereas growth-restraining E2Fs have much smaller effects, if any. These results suggest that cyclin D3 is a target of multiple growth-promoting E2F1s and is not an E2F1-specific target. The experiments described above utilized ectopic E2F expression, which is subject to the concern that the observed regulation is not physiological. Thus, ChIP assays were performed on native H1299 cells to ascertain whether endogenous E2F1 binds to the cyclin D3 promoter in vivo. As shown in Fig.4A, PCR primers that span the region of −202 to +6 of the cyclin D3 promoter clearly detect cyclin D3 promoter DNA in ChIP samples generated using an E2F1 antibody. However, chromatin immunoprecipitations with either an irrelevant antibody (anti-RhoA) or no added antibody resulted in the absence of detectable cyclin D3 promoter DNA. PCR primers specific for the human actin promoter (negative control) did not detect actin promoter DNA in E2F1 ChIP samples, as expected from previous work demonstrating that E2F1 does not bind the actin promoter (15Takahashi Y. Rayman J.B. Dynlacht B.D. Genes Dev. 2000; 14: 804-816Crossref PubMed Google Scholar). These data demonstrate that E2F1 associates with the cyclin D3 promoter under physiological conditions. As a complementary approach to ChIP assays, an EMSA was performed to determine whether recombinant E2F1-DP1 would bind the cyclin D3 −202/+6 promoter region in vitro. Fig. 4Bdemonstrates that recombinant E2F1-DP1 present in extracts of Sf9 cells co-infected with E2F1- and DP1-expressing baculoviruses (38Tao Y. Kassatly R.F. Cress W.D. Horowitz J.M. Mol. Cell. Biol. 1997; 17: 6994-7007Crossref PubMed Scopus (117) Google Scholar) binds to the cyclin D3 −202/+6 promoter fragment, whereas no such binding activity is detectable in uninfected Sf9 cell extracts. The addition of an excess of double-stranded oligonucleotides containing a consensus E2F1 binding site (WT) abolished formation of the E2F1-DP1 complex on the cyclin D3 promoter, whereas the addition of a mutated version (MT) of the consensus oligonucleotide had no effect. The addition of an E2F1 antibody (100 ng) resulted in the retarded mobility of the putative E2F1-DP1-cyclin D3 promoter complex. Together, these data support the conclusion that E2F1 activates cyclin D3 via direct binding to the promoter in a manner dependent upon its transcriptional activation domain. To further define the E2F1-responsive region of the cyclin D3 promoter, we generated several additional promoter constructs. Constructs −202/−146, −202/−134, and −202/−112 possessed deletions from the 3′-end of the promoter. Fig.5A reveals that deletion of sequences downstream of −134 did not abolish induction by E2F1; in fact, constructs −202/−134 and −202/−112 were both activated nearly 4-fold by E2F1. However further deletion to −146 (−202/−146) abolished the E2F1 response. Constructs −159/+6 and −115/+6 had additional nucleotides deleted from the 5′-end of the promoter. Fig.5A reveals that deletion of sequences upstream of −159 (−159/+6) had little effect on the E2F1 response. In contrast, further deletion of sequences upstream of −115 (−115/+6) abolished E2F1 responsiveness. Taken together, these results demonstrate that the critical E2F1-responsive elemen
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