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E2F-3B Is a Physiological Target of Cyclin A

2002; Elsevier BV; Volume: 277; Issue: 26 Linguagem: Inglês

10.1074/jbc.m202629200

ISSN

1083-351X

Autores

Yiwen He, W. Douglas Cress,

Tópico(s)

Genomics and Chromatin Dynamics

Resumo

The E2F family of transcription factors controls the expression of numerous genes that are required for the G1/S transition. Among the mechanisms that modulate the activity of the E2F proteins, cyclin A has been found to be important for the down-regulation of E2F-1, -2, and -3A activity after cells have progressed through G1/S. Specifically, phosphorylation of these E2F proteins by cyclin A/Cdk2 ultimately results in their necessary degradation as cells progress through S phase. E2F-3B was recently identified as an alternatively spliced form of E2F-3A that was predicted to lack a functional cyclin A binding domain. In this paper, we present considerable evidence that contradicts this prediction. First, we demonstrate binding of cyclin A to E2F-3B as bacterially expressed proteins in vitro. Second, we demonstrate binding of cyclin A to E2F-3B in mammalian cellsin vivo. Third, we show that co-expression of cyclin A with E2F-3B significantly reduces E2F-3B-mediated transcriptional activity. Finally, in synchronized cells, we observe down-regulation of E2F-3B protein expression coincident with the up-regulation of cyclin A. We conclude that E2F-3B is a physiological target of cyclin A. The E2F family of transcription factors controls the expression of numerous genes that are required for the G1/S transition. Among the mechanisms that modulate the activity of the E2F proteins, cyclin A has been found to be important for the down-regulation of E2F-1, -2, and -3A activity after cells have progressed through G1/S. Specifically, phosphorylation of these E2F proteins by cyclin A/Cdk2 ultimately results in their necessary degradation as cells progress through S phase. E2F-3B was recently identified as an alternatively spliced form of E2F-3A that was predicted to lack a functional cyclin A binding domain. In this paper, we present considerable evidence that contradicts this prediction. First, we demonstrate binding of cyclin A to E2F-3B as bacterially expressed proteins in vitro. Second, we demonstrate binding of cyclin A to E2F-3B in mammalian cellsin vivo. Third, we show that co-expression of cyclin A with E2F-3B significantly reduces E2F-3B-mediated transcriptional activity. Finally, in synchronized cells, we observe down-regulation of E2F-3B protein expression coincident with the up-regulation of cyclin A. We conclude that E2F-3B is a physiological target of cyclin A. glutathione S-transferase immunoprecipitation electrophoretic mobility shift assay The E2Fs represent a family of transcription factors whose activity plays a critical role in cell growth control (1Nevins J.R. Hum. Mol. Genet. 2001; 10: 699-703Crossref PubMed Scopus (735) Google Scholar, 2Harbour J.W. Dean D.C. Genes Dev. 2000; 14: 2393-2409Crossref PubMed Scopus (955) Google Scholar, 3Dyson N. Genes Dev. 1998; 12: 2245-2262Crossref PubMed Scopus (1962) Google Scholar). Specifically, the E2F family controls the expression of genes required for DNA synthesis at the G1/S phase boundary (4Muller 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 (627) Google Scholar, 5Ishida S. Huang E. Zuzan H. Spang R. Leone G. West M. Nevins J.R. Mol. Cell. Biol. 2001; 21: 4684-4699Crossref PubMed Scopus (494) Google Scholar, 6Kalma Y. Marash L. Lamed Y. Ginsberg D. Oncogene. 2001; 20: 1379-1387Crossref PubMed Scopus (62) Google Scholar, 7Ma Y. Croxton R. Moorer R.L., Jr. Cress W.D. Arch. Biochem. Biophys. 2002; 399: 212-224Crossref PubMed Scopus (95) Google Scholar). Presently, the E2F family can be divided into two functional groups. The first group includes E2F-1, -2, and -3A. These factors represent the growth stimulatory segment of the family, since they are potent transcriptional activators and are required for the entrance of cells into S phase (8Humbert P.O. Verona R. Trimarchi J.M. Rogers C. Dandapani S. Lees J.A. Genes Dev. 2000; 14: 690-703PubMed Google Scholar, 9Wu L. Timmers C. Maiti B. Saavedra H.I. Sang L. Chong G.T. Nuckolls F. Giangrande P. Wright F.A. Field S.J. Greenberg M.E. Orkin S. Nevins J.R. Robinson M.L. Leone G. Nature. 2001; 414: 457-462Crossref PubMed Scopus (489) Google Scholar, 10Leone G. DeGregori J. Yan Z. Jakoi L. Ishida S. Williams R.S. Nevins J.R. Genes Dev. 1998; 12: 2120-2130Crossref PubMed Scopus (308) Google Scholar, 11Leone G. Sears R. Huang E. Rempel R. Nuckolls F. Park C.H. Giangrande P., Wu, L. Saavedra H.I. Field S.J. Thompson M.A. Yang H. Fujiwara Y. Greenberg M.E. Orkin S. Smith C. Nevins J.R. Mol Cell. 2001; 8: 105-113Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar). Members of this group are expressed at low levels in G0 and early G1, and their expression is highly induced in late G1 (10Leone G. DeGregori J. Yan Z. Jakoi L. Ishida S. Williams R.S. Nevins J.R. Genes Dev. 1998; 12: 2120-2130Crossref PubMed Scopus (308) Google Scholar, 12Hsiao K.M. McMahon S.L. Farnham P.J. Genes Dev. 1994; 8: 1526-1537Crossref PubMed Scopus (221) Google Scholar, 13Johnson D.G. Ohtani K. Nevins J.R. Genes Dev. 1994; 8: 1514-1525Crossref PubMed Scopus (452) Google Scholar, 14Neuman E. Flemington E.K. Sellers W.R. Kaelin W.G., Jr. Mol. Cell. Biol. 1994; 14: 6607-6615Crossref PubMed Scopus (234) Google Scholar, 15Sears R. Ohtani K. Nevins J.R. Mol. Cell. Biol. 1997; 17: 5227-5235Crossref PubMed Scopus (177) Google Scholar, 16Flores A.M. Kassatly R.F. Cress W.D. Oncogene. 1998; 16: 1289-1298Crossref PubMed Scopus (16) Google Scholar). Structural characteristics of this group include an extended N-terminal region of unknown function, a nuclear localization sequence (NLS), and overlapping the NLS, a cyclin A binding domain (17Magae J., Wu, C.L. Illenye S. Harlow E. Heintz N.H. J. Cell Sci. 1996; 109: 1717-1726Crossref PubMed Google Scholar, 18Allen K.E. de la Luna S. Kerkhoven R.M. Bernards R. La Thangue N.B. J. Cell Sci. 1997; 110: 2819-2831Crossref PubMed Google Scholar, 19Muller H. Moroni M.C. Vigo E. Petersen B.O. Bartek J. Helin K. Mol. Cell. Biol. 1997; 17: 5508-5520Crossref PubMed Scopus (172) Google Scholar, 20Verona R. Moberg K. Estes S. Starz M. Vernon J.P. Lees J.A. Mol. Cell. Biol. 1997; 17: 7268-7282Crossref PubMed Scopus (178) Google Scholar, 21Krek W. Ewen M.E. Shirodkar S. Arany Z. Kaelin W.G., Jr. Livingston D.M. Cell. 1994; 78: 161-172Abstract Full Text PDF PubMed Scopus (412) Google Scholar, 22Xu M. Sheppard K.A. Peng C.Y. Yee A.S. Piwnica-Worms H. Mol. Cell. Biol. 1994; 14: 8420-8431Crossref PubMed Scopus (257) Google Scholar, 23Kitagawa M. Higashi H. Suzuki-Takahashi I. Segawa K. Hanks S.K. Taya Y. Nishimura S. Okuyama A. Oncogene. 1995; 10: 229-236PubMed Google Scholar). The second group includes E2F-4, -5, and -6, which lack these three functional domains and induce S phase inefficiently. This group of E2Fs appears necessary for growth arrest and differentiation rather than S phase entry (24Persengiev S.P. Kondova I.I. Kilpatrick D.L. Mol. Cell. Biol. 1999; 19: 6048-6056Crossref PubMed Scopus (57) Google Scholar, 25Cartwright P. Muller H. Wagener C. Holm K. Helin K. Oncogene. 1998; 17: 611-623Crossref PubMed Scopus (163) Google Scholar, 26Gaubatz S. Wood J.G. Livingston D.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9190-9195Crossref PubMed Scopus (149) Google Scholar).In normal cells, transcriptional activation by the E2F family appears to be largely restricted to late G1 and the G1/S boundary. In G0 and early G1, E2F activity is negatively regulated by one or more members of the pRb protein family (27Chellappan S.P. Hiebert S. Mudryj M. Horowitz J.M. Nevins J.R. Cell. 1991; 65: 1053-1061Abstract Full Text PDF PubMed Scopus (1093) Google Scholar, 28Qin X.Q. Livingston D.M. Ewen M. Sellers W.R. Arany Z. Kaelin W.G., Jr. Mol. Cell. Biol. 1995; 15: 742-755Crossref PubMed Google Scholar). Once cells reach late G1, the Rb family members become phosphorylated and release the E2Fs. There is then a surge of E2F activity (primarily E2F-1, E2F-2, and E2F-3A) that drives the expression of genes that are required for DNA synthesis. Once in S phase, E2F activity is no longer needed, and the cyclin A protein directs the phosphorylation of the three growth-promoting members of the E2F family by the bound Cdk2, leading to their degradation (21Krek W. Ewen M.E. Shirodkar S. Arany Z. Kaelin W.G., Jr. Livingston D.M. Cell. 1994; 78: 161-172Abstract Full Text PDF PubMed Scopus (412) Google Scholar, 22Xu M. Sheppard K.A. Peng C.Y. Yee A.S. Piwnica-Worms H. Mol. Cell. Biol. 1994; 14: 8420-8431Crossref PubMed Scopus (257) Google Scholar, 23Kitagawa M. Higashi H. Suzuki-Takahashi I. Segawa K. Hanks S.K. Taya Y. Nishimura S. Okuyama A. Oncogene. 1995; 10: 229-236PubMed Google Scholar,29Dynlacht B.D. Flores O. Lees J.A. Harlow E. Genes Dev. 1994; 8: 1772-1786Crossref PubMed Scopus (331) Google Scholar). This down-regulation of E2F activity by cyclin A is required for orderly S-phase progression (30Logan T.J. Evans D.L. Mercer W.E. Bjornsti M.A. Hall D.J. Cancer Res. 1995; 55: 2883-2891PubMed Google Scholar, 31Krek W., Xu, G. Livingston D.M. Cell. 1995; 83: 1149-1158Abstract Full Text PDF PubMed Scopus (315) Google Scholar, 32Jordan-Sciutto K.L. Hall D.J. Biochem. Cell Biol. 1998; 76: 37-44Crossref PubMed Scopus (7) Google Scholar), and in its absence, apoptosis occurs (33Chen Y.N. Sharma S.K. Ramsey T.M. Jiang L. Martin M.S. Baker K. Adams P.D. Bair K.W. Kaelin W.G., Jr. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4325-4329Crossref PubMed Scopus (306) Google Scholar, 34Lees J.A. Weinberg R.A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4221-4223Crossref PubMed Scopus (39) Google Scholar).Although the E2F family can be divided into two functional groups, the newest member of the E2F family, E2F-3B, is not easy to classify. E2F-3B lacks the N-terminal domain present in E2F-3, which is also referred to as E2F-3A (35He Y. Armanious M.K. Thomas M.J. Cress W.D. Oncogene. 2000; 19: 3422-3433Crossref PubMed Scopus (66) Google Scholar, 36Leone 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 (149) Google Scholar). Thus, it resembles the non-growth-promoting group of E2Fs in structure. The expression pattern of E2F-3B is also consistent with its having a role in growth restraint, since E2F-3B is expressed at its highest levels in G0, where it associates with Rb, and its levels drop as cells enter S phase. This pattern of expression is the opposite of that of E2F-1, -2, and -3A (35He Y. Armanious M.K. Thomas M.J. Cress W.D. Oncogene. 2000; 19: 3422-3433Crossref PubMed Scopus (66) Google Scholar, 36Leone 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 (149) Google Scholar). However, E2F-3B differs from the growth-restraining E2Fs because it clearly encodes a nuclear localization sequence, as do the growth-promoting E2Fs (35He Y. Armanious M.K. Thomas M.J. Cress W.D. Oncogene. 2000; 19: 3422-3433Crossref PubMed Scopus (66) Google Scholar).Before the work described herein, it has not been clear whether E2F-3B contains a functional cyclin A binding domain. Fig. 1 Ahighlights the nuclear localization sequence and putative cyclin A binding domain of E2F-3A and E2F-3B. E2F-3B transcription uses an alternative promoter and an alternative initiation exon (exon 1b) compared with E2F-3A (exon 1a) and shares the same exons from exon 2 onward (36Leone 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 (149) Google Scholar). Nevins and co-workers (36Leone 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 (149) Google Scholar) predict that E2F-3B will not interact with cyclin A, based upon experiments that mapped the E2F-1 cyclin A binding domain to a region that includes the 21 amino acids highlighted in Fig. 1 A (21Krek W. Ewen M.E. Shirodkar S. Arany Z. Kaelin W.G., Jr. Livingston D.M. Cell. 1994; 78: 161-172Abstract Full Text PDF PubMed Scopus (412) Google Scholar, 37Dynlacht B.D. Moberg K. Lees J.A. Harlow E. Zhu L. Mol. Cell. Biol. 1997; 17: 3867-3875Crossref PubMed Scopus (95) Google Scholar). If true, this model would suggest that the cyclin A binding domain is encoded in part by exon 1a and in part by exon 2, which seems unlikely. Furthermore, Kaelin and co-workers (38Adams P.D. Sellers W.R. Sharma S.K., Wu, A.D. Nalin C.M. Kaelin W.G., Jr. Mol. Cell. Biol. 1996; 16: 6623-6633Crossref PubMed Scopus (313) Google Scholar) show that a significantly shorter sequence in E2F-1 (PAKRRLEL) is sufficient to bind to cyclin A. Because the shorter region is present in both E2F-3A and -3B, we have hypothesized that E2F-3B can still bind cyclin A.In the present work, we test this hypothesis and present several lines of in vivo and in vitro evidence that clearly show that E2F-3B is a physiological target of the cyclin A protein. Because a number of cyclin-dependent kinase inhibitors are in various stages of clinical trials for the treatment of cancer (39Senderowicz A.M. Oncogene. 2000; 19: 6600-6606Crossref PubMed Scopus (115) Google Scholar,40Sedlacek H.H. Crit. Rev. Oncol. Hematol. 2001; 38: 139-170Crossref PubMed Scopus (217) Google Scholar), we anticipate that E2F-3B may contribute to the activity of these drugs. Thus, the findings of this report may have important clinical ramifications.DISCUSSIONThe promoter of E2F-3A is regulated by Myc and E2F, and is activated in late G1 when cells start to progress into S-phase. E2F-3B, however, is transcribed from an alternative promoter, which is equally active throughout the cell cycle (49Adams M.R. Sears R. Nuckolls F. Leone G. Nevins J.R. Mol. Cell. Biol. 2000; 20: 3633-3639Crossref PubMed Scopus (111) Google Scholar). Thus, the observed regulation of E2F-3B is apparently at the post-transcriptional level. E2F-3B protein level is the highest in G0, and it rapidly decreases as cells enter S phase. E2F-3A, however, is expressed only at the G1/S transition, and its level goes down after cells progress into S phase. We demonstrate here that cyclin A likely accounts for the observed down-regulation of E2F-3B during S phase. We note that the down-regulation of E2F-3A in S phase appears to lag behind the down-regulation of E2F-3B. We explain this observation in terms of competing synthesis and degradation steps. E2F-3B is synthesized constitutively throughout the cell cycle. Thus, its down-regulation is apparent immediately following up-regulation of cyclin A. In contrast, E2F-3A synthesis occurs in a surge only at the G1/S boundary. Thus, early in S phase, transcriptional up-regulation of E2F-3A temporarily overcomes post-transcriptional down-regulation by cyclin A. Later in S phase, when the cyclin A protein level is high and E2F-3A expression is no longer activated, two levels of down-regulation take over and a rapid decrease in E2F-3A activity is observed.E2F-3A and -3B have particularly important roles in the regulation of the G1/S transition. For example, E2F-3 null mice are the only E2F knockout animals in which defects in cellular proliferation are observed (8Humbert P.O. Verona R. Trimarchi J.M. Rogers C. Dandapani S. Lees J.A. Genes Dev. 2000; 14: 690-703PubMed Google Scholar, 50Cloud J.E. Rogers C. Reza T.L. Ziebold U. Stone J.R. Picard M.H. Caron A.M. Bronson R.T. Lees J.A. Mol. Cell. Biol. 2002; 22: 2663-2672Crossref PubMed Scopus (73) Google Scholar). Likewise, microinjection of E2F-3A antibodies demonstrates that E2F-3A is the only member of the E2F family essential for S phase induction under physiological expression levels (10Leone G. DeGregori J. Yan Z. Jakoi L. Ishida S. Williams R.S. Nevins J.R. Genes Dev. 1998; 12: 2120-2130Crossref PubMed Scopus (308) Google Scholar). Because E2F-3A and -3B share an identical DNA binding domain, it is likely that they bind and regulate the same subset of E2F-regulated promoters that are not efficiently recognized by other members of the E2F family. Recent studies contribute to a model for E2F activity in which E2F·Rb complexes in G0 serve to actively repress transcription of the promoters to which they are bound (2Harbour J.W. Dean D.C. Genes Dev. 2000; 14: 2393-2409Crossref PubMed Scopus (955) Google Scholar, 3Dyson N. Genes Dev. 1998; 12: 2245-2262Crossref PubMed Scopus (1962) Google Scholar). In late G1, the E2F·Rb complexes dissociate (due to Cdk phosphorylation), and E2F-regulated promoters are then bound by activating members of the E2F family. Within this general model it is clear that different members of the E2F family have unique roles (3Dyson N. Genes Dev. 1998; 12: 2245-2262Crossref PubMed Scopus (1962) Google Scholar,51Lavia P. Jansen-Durr P. Bioessays. 1999; 21: 221-230Crossref PubMed Scopus (143) Google Scholar). Promoter binding analysis has shown that distinct complexes of E2F and pRb family members mediate activation or repression throughout the cell cycle (52Takahashi Y. Rayman J.B. Dynlacht B.D. Genes Dev. 2000; 14: 804-816PubMed Google Scholar).Based upon its G0 expression pattern and association with pRb, E2F-3B likely fits into this model primarily as a transcriptional repressor but could also contribute in the activation of genes, including E2F-1, -2, and -3A, in late G1. In contrast, E2F-3A likely serves exclusively as a potent transcriptional activator of genes essential for S phase entry during its brief appearance late in G1. Because E2F-3B has a DNA binding domain that is identical to E2F-3A, its major function may be to make sure that specific E2F-3A-responsive genes will not be active in G0by bringing pRb to the promoter. If E2F-3B is a repressor in G0, inhibition of E2F-3B at G1/S transition will be as important as activation of E2F-3A for G1/S transition. Because both forms are transcriptional activators when not bound by pRb (see Fig. 4), they must be destabilized and removed during S phase lest they induce apoptosis. Finally, once S phase is completed and cyclin A levels drop, E2F-3B levels rebound due to its constitutive synthesis (note the 27- and 30-h time points of Fig. 5). This rebounding of E2F-3B activity is likely necessary to serve as a tether for pRb as cells enter the next G1. The E2Fs represent a family of transcription factors whose activity plays a critical role in cell growth control (1Nevins J.R. Hum. Mol. Genet. 2001; 10: 699-703Crossref PubMed Scopus (735) Google Scholar, 2Harbour J.W. Dean D.C. Genes Dev. 2000; 14: 2393-2409Crossref PubMed Scopus (955) Google Scholar, 3Dyson N. Genes Dev. 1998; 12: 2245-2262Crossref PubMed Scopus (1962) Google Scholar). Specifically, the E2F family controls the expression of genes required for DNA synthesis at the G1/S phase boundary (4Muller 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 (627) Google Scholar, 5Ishida S. Huang E. Zuzan H. Spang R. Leone G. West M. Nevins J.R. Mol. Cell. Biol. 2001; 21: 4684-4699Crossref PubMed Scopus (494) Google Scholar, 6Kalma Y. Marash L. Lamed Y. Ginsberg D. Oncogene. 2001; 20: 1379-1387Crossref PubMed Scopus (62) Google Scholar, 7Ma Y. Croxton R. Moorer R.L., Jr. Cress W.D. Arch. Biochem. Biophys. 2002; 399: 212-224Crossref PubMed Scopus (95) Google Scholar). Presently, the E2F family can be divided into two functional groups. The first group includes E2F-1, -2, and -3A. These factors represent the growth stimulatory segment of the family, since they are potent transcriptional activators and are required for the entrance of cells into S phase (8Humbert P.O. Verona R. Trimarchi J.M. Rogers C. Dandapani S. Lees J.A. Genes Dev. 2000; 14: 690-703PubMed Google Scholar, 9Wu L. Timmers C. Maiti B. Saavedra H.I. Sang L. Chong G.T. Nuckolls F. Giangrande P. Wright F.A. Field S.J. Greenberg M.E. Orkin S. Nevins J.R. Robinson M.L. Leone G. Nature. 2001; 414: 457-462Crossref PubMed Scopus (489) Google Scholar, 10Leone G. DeGregori J. Yan Z. Jakoi L. Ishida S. Williams R.S. Nevins J.R. Genes Dev. 1998; 12: 2120-2130Crossref PubMed Scopus (308) Google Scholar, 11Leone G. Sears R. Huang E. Rempel R. Nuckolls F. Park C.H. Giangrande P., Wu, L. Saavedra H.I. Field S.J. Thompson M.A. Yang H. Fujiwara Y. Greenberg M.E. Orkin S. Smith C. Nevins J.R. Mol Cell. 2001; 8: 105-113Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar). Members of this group are expressed at low levels in G0 and early G1, and their expression is highly induced in late G1 (10Leone G. DeGregori J. Yan Z. Jakoi L. Ishida S. Williams R.S. Nevins J.R. Genes Dev. 1998; 12: 2120-2130Crossref PubMed Scopus (308) Google Scholar, 12Hsiao K.M. McMahon S.L. Farnham P.J. Genes Dev. 1994; 8: 1526-1537Crossref PubMed Scopus (221) Google Scholar, 13Johnson D.G. Ohtani K. Nevins J.R. Genes Dev. 1994; 8: 1514-1525Crossref PubMed Scopus (452) Google Scholar, 14Neuman E. Flemington E.K. Sellers W.R. Kaelin W.G., Jr. Mol. Cell. Biol. 1994; 14: 6607-6615Crossref PubMed Scopus (234) Google Scholar, 15Sears R. Ohtani K. Nevins J.R. Mol. Cell. Biol. 1997; 17: 5227-5235Crossref PubMed Scopus (177) Google Scholar, 16Flores A.M. Kassatly R.F. Cress W.D. Oncogene. 1998; 16: 1289-1298Crossref PubMed Scopus (16) Google Scholar). Structural characteristics of this group include an extended N-terminal region of unknown function, a nuclear localization sequence (NLS), and overlapping the NLS, a cyclin A binding domain (17Magae J., Wu, C.L. Illenye S. Harlow E. Heintz N.H. J. Cell Sci. 1996; 109: 1717-1726Crossref PubMed Google Scholar, 18Allen K.E. de la Luna S. Kerkhoven R.M. Bernards R. La Thangue N.B. J. Cell Sci. 1997; 110: 2819-2831Crossref PubMed Google Scholar, 19Muller H. Moroni M.C. Vigo E. Petersen B.O. Bartek J. Helin K. Mol. Cell. Biol. 1997; 17: 5508-5520Crossref PubMed Scopus (172) Google Scholar, 20Verona R. Moberg K. Estes S. Starz M. Vernon J.P. Lees J.A. Mol. Cell. Biol. 1997; 17: 7268-7282Crossref PubMed Scopus (178) Google Scholar, 21Krek W. Ewen M.E. Shirodkar S. Arany Z. Kaelin W.G., Jr. Livingston D.M. Cell. 1994; 78: 161-172Abstract Full Text PDF PubMed Scopus (412) Google Scholar, 22Xu M. Sheppard K.A. Peng C.Y. Yee A.S. Piwnica-Worms H. Mol. Cell. Biol. 1994; 14: 8420-8431Crossref PubMed Scopus (257) Google Scholar, 23Kitagawa M. Higashi H. Suzuki-Takahashi I. Segawa K. Hanks S.K. Taya Y. Nishimura S. Okuyama A. Oncogene. 1995; 10: 229-236PubMed Google Scholar). The second group includes E2F-4, -5, and -6, which lack these three functional domains and induce S phase inefficiently. This group of E2Fs appears necessary for growth arrest and differentiation rather than S phase entry (24Persengiev S.P. Kondova I.I. Kilpatrick D.L. Mol. Cell. Biol. 1999; 19: 6048-6056Crossref PubMed Scopus (57) Google Scholar, 25Cartwright P. Muller H. Wagener C. Holm K. Helin K. Oncogene. 1998; 17: 611-623Crossref PubMed Scopus (163) Google Scholar, 26Gaubatz S. Wood J.G. Livingston D.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9190-9195Crossref PubMed Scopus (149) Google Scholar). In normal cells, transcriptional activation by the E2F family appears to be largely restricted to late G1 and the G1/S boundary. In G0 and early G1, E2F activity is negatively regulated by one or more members of the pRb protein family (27Chellappan S.P. Hiebert S. Mudryj M. Horowitz J.M. Nevins J.R. Cell. 1991; 65: 1053-1061Abstract Full Text PDF PubMed Scopus (1093) Google Scholar, 28Qin X.Q. Livingston D.M. Ewen M. Sellers W.R. Arany Z. Kaelin W.G., Jr. Mol. Cell. Biol. 1995; 15: 742-755Crossref PubMed Google Scholar). Once cells reach late G1, the Rb family members become phosphorylated and release the E2Fs. There is then a surge of E2F activity (primarily E2F-1, E2F-2, and E2F-3A) that drives the expression of genes that are required for DNA synthesis. Once in S phase, E2F activity is no longer needed, and the cyclin A protein directs the phosphorylation of the three growth-promoting members of the E2F family by the bound Cdk2, leading to their degradation (21Krek W. Ewen M.E. Shirodkar S. Arany Z. Kaelin W.G., Jr. Livingston D.M. Cell. 1994; 78: 161-172Abstract Full Text PDF PubMed Scopus (412) Google Scholar, 22Xu M. Sheppard K.A. Peng C.Y. Yee A.S. Piwnica-Worms H. Mol. Cell. Biol. 1994; 14: 8420-8431Crossref PubMed Scopus (257) Google Scholar, 23Kitagawa M. Higashi H. Suzuki-Takahashi I. Segawa K. Hanks S.K. Taya Y. Nishimura S. Okuyama A. Oncogene. 1995; 10: 229-236PubMed Google Scholar,29Dynlacht B.D. Flores O. Lees J.A. Harlow E. Genes Dev. 1994; 8: 1772-1786Crossref PubMed Scopus (331) Google Scholar). This down-regulation of E2F activity by cyclin A is required for orderly S-phase progression (30Logan T.J. Evans D.L. Mercer W.E. Bjornsti M.A. Hall D.J. Cancer Res. 1995; 55: 2883-2891PubMed Google Scholar, 31Krek W., Xu, G. Livingston D.M. Cell. 1995; 83: 1149-1158Abstract Full Text PDF PubMed Scopus (315) Google Scholar, 32Jordan-Sciutto K.L. Hall D.J. Biochem. Cell Biol. 1998; 76: 37-44Crossref PubMed Scopus (7) Google Scholar), and in its absence, apoptosis occurs (33Chen Y.N. Sharma S.K. Ramsey T.M. Jiang L. Martin M.S. Baker K. Adams P.D. Bair K.W. Kaelin W.G., Jr. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4325-4329Crossref PubMed Scopus (306) Google Scholar, 34Lees J.A. Weinberg R.A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4221-4223Crossref PubMed Scopus (39) Google Scholar). Although the E2F family can be divided into two functional groups, the newest member of the E2F family, E2F-3B, is not easy to classify. E2F-3B lacks the N-terminal domain present in E2F-3, which is also referred to as E2F-3A (35He Y. Armanious M.K. Thomas M.J. Cress W.D. Oncogene. 2000; 19: 3422-3433Crossref PubMed Scopus (66) Google Scholar, 36Leone 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 (149) Google Scholar). Thus, it resembles the non-growth-promoting group of E2Fs in structure. The expression pattern of E2F-3B is also consistent with its having a role in growth restraint, since E2F-3B is expressed at its highest levels in G0, where it associates with Rb, and its levels drop as cells enter S phase. This pattern of expression is the opposite of that of E2F-1, -2, and -3A (35He Y. Armanious M.K. Thomas M.J. Cress W.D. Oncogene. 2000; 19: 3422-3433Crossref PubMed Scopus (66) Google Scholar, 36Leone 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 (149) Google Scholar). However, E2F-3B differs from the growth-restraining E2Fs because it clearly encodes a nuclear localization sequence, as do the growth-promoting E2Fs (35He Y. Armanious M.K. Thomas M.J. Cress W.D. Oncogene. 2000; 19: 3422-3433Crossref PubMed Scopus (66) Google Scholar). Before the work described herein, it has not been clear whether E2F-3B contains a functional cyclin A binding domain. Fig. 1 Ahighlights the nuclear localization sequence and putative cyclin A binding domain of E2F-3A and E2F-3B. E2F-3B transcription uses an alternative promoter and an alternative initiation exon (exon 1b) compared with E2F-3A (exon 1a) and shares the same exons from exon 2 onward (36Leone 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 (149) Google Scholar). Nevins and co-workers (36Leone 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 (149) Google Scholar) predict that E2F-3B will not interact with cyclin A, based upon experiments that mapped the E2F-1 cyclin A binding domain to a region that includes the 21 amino acids highlighted in Fig. 1 A (21Krek W. Ewen M.E. Shirodkar S. Arany Z. Kaelin W.G., Jr. Livingston D.M. Cell. 1994; 78: 161-172Abstract Full Text PDF PubMed Scopus (412) Google Scholar, 37Dynlacht B.D. Moberg K. Lees J.A. Harlow E. Zhu L. Mol. Cell. Biol. 1997; 17: 3867-3875Crossref PubMed Scopus (95) Google Scholar). If true, this model would suggest that the cyclin A binding domain is encoded in part by exon 1a and in part by exon 2, which seems unlikely. Furthermore, Kaelin and co-workers (38Adams P.D. Sellers W.R. Sharma S.K., Wu, A.D. Nalin C.M. Kaelin W.G., Jr. Mol. Cell. Biol. 1996; 16: 6623-6633Crossref PubMed Scopus (313) Google Scholar) show that a significantly shorter sequence in E2F-1 (PAKRRLEL) is sufficient to bind to cyclin A. Because the shorter region is present in both E2F-3A and -3B, we have hypothesized that E2F-3B can still bind cyclin A. In the present work, we test this hypothesis and present several lines of in vivo and in vitro evidence that clearly show that E2F-3B is a physiological target of the cyclin A protein. Because a number of cyclin-dependent kinase inhibitors are in various stages of clinical trials for the treatment of cancer (39Senderowicz A.M. Oncogene. 2000; 19: 6600-6606Crossref PubMed Scopus (115) Google Scholar,40Sedlacek H.H. Crit. Rev. Oncol. Hematol. 2001; 38: 139-170Crossref PubMed Scopus (217) Google Scholar), we anticipate that E2F-3B may contribute to the activity of these drugs. Thus, the findings of this report may have important clinical ramifications. DISCUSSIONThe promoter of E2F-3A is regulated by Myc and E2F, and is activated in late G1 when cells start to progress into S-phase. E2F-3B, however, is transcribed from an alternative promoter, which is equally active throughout the cell cycle (49Adams M.R. Sears R. Nuckolls F. Leone G. Nevins J.R. Mol. Cell. Biol. 2000; 20: 3633-3639Crossref PubMed Scopus (111) Google Scholar). Thus, the observed regulation of E2F-3B is apparently at the post-transcriptional level. E2F-3B protein level is the highest in G0, and it rapidly decreases as cells enter S phase. E2F-3A, however, is expressed only at the G1/S transition, and its level goes down after cells progress into S phase. We demonstrate here that cyclin A likely accounts for the observed down-regulation of E2F-3B during S phase. We note that the down-regulation of E2F-3A in S phase appears to lag behind the down-regulation of E2F-3B. We explain this observation in terms of competing synthesis and degradation steps. E2F-3B is synthesized constitutively throughout the cell cycle. Thus, its down-regulation is apparent immediately following up-regulation of cyclin A. In contrast, E2F-3A synthesis occurs in a surge only at the G1/S boundary. Thus, early in S phase, transcriptional up-regulation of E2F-3A temporarily overcomes post-transcriptional down-regulation by cyclin A. Later in S phase, when the cyclin A protein level is high and E2F-3A expression is no longer activated, two levels of down-regulation take over and a rapid decrease in E2F-3A activity is observed.E2F-3A and -3B have particularly important roles in the regulation of the G1/S transition. For example, E2F-3 null mice are the only E2F knockout animals in which defects in cellular proliferation are observed (8Humbert P.O. Verona R. Trimarchi J.M. Rogers C. Dandapani S. Lees J.A. Genes Dev. 2000; 14: 690-703PubMed Google Scholar, 50Cloud J.E. Rogers C. Reza T.L. Ziebold U. Stone J.R. Picard M.H. Caron A.M. Bronson R.T. Lees J.A. Mol. Cell. Biol. 2002; 22: 2663-2672Crossref PubMed Scopus (73) Google Scholar). Likewise, microinjection of E2F-3A antibodies demonstrates that E2F-3A is the only member of the E2F family essential for S phase induction under physiological expression levels (10Leone G. DeGregori J. Yan Z. Jakoi L. Ishida S. Williams R.S. Nevins J.R. Genes Dev. 1998; 12: 2120-2130Crossref PubMed Scopus (308) Google Scholar). Because E2F-3A and -3B share an identical DNA binding domain, it is likely that they bind and regulate the same subset of E2F-regulated promoters that are not efficiently recognized by other members of the E2F family. Recent studies contribute to a model for E2F activity in which E2F·Rb complexes in G0 serve to actively repress transcription of the promoters to which they are bound (2Harbour J.W. Dean D.C. Genes Dev. 2000; 14: 2393-2409Crossref PubMed Scopus (955) Google Scholar, 3Dyson N. Genes Dev. 1998; 12: 2245-2262Crossref PubMed Scopus (1962) Google Scholar). In late G1, the E2F·Rb complexes dissociate (due to Cdk phosphorylation), and E2F-regulated promoters are then bound by activating members of the E2F family. Within this general model it is clear that different members of the E2F family have unique roles (3Dyson N. Genes Dev. 1998; 12: 2245-2262Crossref PubMed Scopus (1962) Google Scholar,51Lavia P. Jansen-Durr P. Bioessays. 1999; 21: 221-230Crossref PubMed Scopus (143) Google Scholar). Promoter binding analysis has shown that distinct complexes of E2F and pRb family members mediate activation or repression throughout the cell cycle (52Takahashi Y. Rayman J.B. Dynlacht B.D. Genes Dev. 2000; 14: 804-816PubMed Google Scholar).Based upon its G0 expression pattern and association with pRb, E2F-3B likely fits into this model primarily as a transcriptional repressor but could also contribute in the activation of genes, including E2F-1, -2, and -3A, in late G1. In contrast, E2F-3A likely serves exclusively as a potent transcriptional activator of genes essential for S phase entry during its brief appearance late in G1. Because E2F-3B has a DNA binding domain that is identical to E2F-3A, its major function may be to make sure that specific E2F-3A-responsive genes will not be active in G0by bringing pRb to the promoter. If E2F-3B is a repressor in G0, inhibition of E2F-3B at G1/S transition will be as important as activation of E2F-3A for G1/S transition. Because both forms are transcriptional activators when not bound by pRb (see Fig. 4), they must be destabilized and removed during S phase lest they induce apoptosis. Finally, once S phase is completed and cyclin A levels drop, E2F-3B levels rebound due to its constitutive synthesis (note the 27- and 30-h time points of Fig. 5). This rebounding of E2F-3B activity is likely necessary to serve as a tether for pRb as cells enter the next G1. The promoter of E2F-3A is regulated by Myc and E2F, and is activated in late G1 when cells start to progress into S-phase. E2F-3B, however, is transcribed from an alternative promoter, which is equally active throughout the cell cycle (49Adams M.R. Sears R. Nuckolls F. Leone G. Nevins J.R. Mol. Cell. Biol. 2000; 20: 3633-3639Crossref PubMed Scopus (111) Google Scholar). Thus, the observed regulation of E2F-3B is apparently at the post-transcriptional level. E2F-3B protein level is the highest in G0, and it rapidly decreases as cells enter S phase. E2F-3A, however, is expressed only at the G1/S transition, and its level goes down after cells progress into S phase. We demonstrate here that cyclin A likely accounts for the observed down-regulation of E2F-3B during S phase. We note that the down-regulation of E2F-3A in S phase appears to lag behind the down-regulation of E2F-3B. We explain this observation in terms of competing synthesis and degradation steps. E2F-3B is synthesized constitutively throughout the cell cycle. Thus, its down-regulation is apparent immediately following up-regulation of cyclin A. In contrast, E2F-3A synthesis occurs in a surge only at the G1/S boundary. Thus, early in S phase, transcriptional up-regulation of E2F-3A temporarily overcomes post-transcriptional down-regulation by cyclin A. Later in S phase, when the cyclin A protein level is high and E2F-3A expression is no longer activated, two levels of down-regulation take over and a rapid decrease in E2F-3A activity is observed. E2F-3A and -3B have particularly important roles in the regulation of the G1/S transition. For example, E2F-3 null mice are the only E2F knockout animals in which defects in cellular proliferation are observed (8Humbert P.O. Verona R. Trimarchi J.M. Rogers C. Dandapani S. Lees J.A. Genes Dev. 2000; 14: 690-703PubMed Google Scholar, 50Cloud J.E. Rogers C. Reza T.L. Ziebold U. Stone J.R. Picard M.H. Caron A.M. Bronson R.T. Lees J.A. Mol. Cell. Biol. 2002; 22: 2663-2672Crossref PubMed Scopus (73) Google Scholar). Likewise, microinjection of E2F-3A antibodies demonstrates that E2F-3A is the only member of the E2F family essential for S phase induction under physiological expression levels (10Leone G. DeGregori J. Yan Z. Jakoi L. Ishida S. Williams R.S. Nevins J.R. Genes Dev. 1998; 12: 2120-2130Crossref PubMed Scopus (308) Google Scholar). Because E2F-3A and -3B share an identical DNA binding domain, it is likely that they bind and regulate the same subset of E2F-regulated promoters that are not efficiently recognized by other members of the E2F family. Recent studies contribute to a model for E2F activity in which E2F·Rb complexes in G0 serve to actively repress transcription of the promoters to which they are bound (2Harbour J.W. Dean D.C. Genes Dev. 2000; 14: 2393-2409Crossref PubMed Scopus (955) Google Scholar, 3Dyson N. Genes Dev. 1998; 12: 2245-2262Crossref PubMed Scopus (1962) Google Scholar). In late G1, the E2F·Rb complexes dissociate (due to Cdk phosphorylation), and E2F-regulated promoters are then bound by activating members of the E2F family. Within this general model it is clear that different members of the E2F family have unique roles (3Dyson N. Genes Dev. 1998; 12: 2245-2262Crossref PubMed Scopus (1962) Google Scholar,51Lavia P. Jansen-Durr P. Bioessays. 1999; 21: 221-230Crossref PubMed Scopus (143) Google Scholar). Promoter binding analysis has shown that distinct complexes of E2F and pRb family members mediate activation or repression throughout the cell cycle (52Takahashi Y. Rayman J.B. Dynlacht B.D. Genes Dev. 2000; 14: 804-816PubMed Google Scholar). Based upon its G0 expression pattern and association with pRb, E2F-3B likely fits into this model primarily as a transcriptional repressor but could also contribute in the activation of genes, including E2F-1, -2, and -3A, in late G1. In contrast, E2F-3A likely serves exclusively as a potent transcriptional activator of genes essential for S phase entry during its brief appearance late in G1. Because E2F-3B has a DNA binding domain that is identical to E2F-3A, its major function may be to make sure that specific E2F-3A-responsive genes will not be active in G0by bringing pRb to the promoter. If E2F-3B is a repressor in G0, inhibition of E2F-3B at G1/S transition will be as important as activation of E2F-3A for G1/S transition. Because both forms are transcriptional activators when not bound by pRb (see Fig. 4), they must be destabilized and removed during S phase lest they induce apoptosis. Finally, once S phase is completed and cyclin A levels drop, E2F-3B levels rebound due to its constitutive synthesis (note the 27- and 30-h time points of Fig. 5). This rebounding of E2F-3B activity is likely necessary to serve as a tether for pRb as cells enter the next G1. We thank Drs. Nancy Olashaw, Rhonda Croxton, Eric Haura, and Yihong Ma for critically evaluating our work. We also thank Drs. Gustavo Leone, Patrick Hearing, Edward Leof, and Jack Pledger for reagents critical to this work. We acknowledge Dr. Lanming Zhang and Dr. Marybeth Colter and Jodi Kroeger of Moffitt Core Facilities who performed DNA sequencing and flow cytometry experiments, respectively.

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