• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of molcellbPermissionsJournals.ASM.orgJournalMCB ArticleJournal InfoAuthorsReviewers
Mol Cell Biol. Dec 1997; 17(12): 6994–7007.
PMCID: PMC232556

Subunit composition determines E2F DNA-binding site specificity.

Abstract

The product of the retinoblastoma (Rb) susceptibility gene, Rb-1, regulates the activity of a wide variety of transcription factors, such as E2F, in a cell cycle-dependent fashion. E2F is a heterodimeric transcription factor composed of two subunits each encoded by one of two related gene families, denoted E2F and DP. Five E2F genes, E2F-1 through E2F-5, and two DP genes, DP-1 and DP-2, have been isolated from mammals, and heterodimeric complexes of these proteins are expressed in most, if not all, vertebrate cells. It is not yet clear whether E2F/DP complexes regulate overlapping and/or specific cellular genes. Moreover, little is known about whether Rb regulates all or a subset of E2F-dependent genes. Using recombinant E2F, DP, and Rb proteins prepared in baculovirus-infected cells and a repetitive immunoprecipitation-PCR procedure (CASTing), we have identified consensus DNA-binding sites for E2F-1/DP-1, E2F-1/DP-2, E2F-4/DP-1, and E2F-4/DP-2 complexes as well as an Rb/E2F-1/DP-1 trimeric complex. Our data indicate that (i) E2F, DP, and Rb proteins each influence the selection of E2F-binding sites; (ii) E2F sites differ with respect to their intrinsic DNA-bending properties; (iii) E2F/DP complexes induce distinct degrees of DNA bending; and (iv) complex-specific E2F sites selected in vitro function distinctly as regulators of cell cycle-dependent transcription in vivo. These data indicate that the specific sequence of an E2F site may determine its role in transcriptional regulation and suggest that Rb/E2F complexes may regulate subsets of E2F-dependent cellular genes.

Full Text

The Full Text of this article is available as a PDF (1.1M).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Adnane J, Shao Z, Robbins PD. The retinoblastoma susceptibility gene product represses transcription when directly bound to the promoter. J Biol Chem. 1995 Apr 14;270(15):8837–8843. [PubMed]
  • Bagchi S, Raychaudhuri P, Nevins JR. Adenovirus E1A proteins can dissociate heteromeric complexes involving the E2F transcription factor: a novel mechanism for E1A trans-activation. Cell. 1990 Aug 24;62(4):659–669. [PubMed]
  • Bandara LR, Buck VM, Zamanian M, Johnston LH, La Thangue NB. Functional synergy between DP-1 and E2F-1 in the cell cycle-regulating transcription factor DRTF1/E2F. EMBO J. 1993 Nov;12(11):4317–4324. [PMC free article] [PubMed]
  • Bandara LR, La Thangue NB. Adenovirus E1a prevents the retinoblastoma gene product from complexing with a cellular transcription factor. Nature. 1991 Jun 6;351(6326):494–497. [PubMed]
  • Beijersbergen RL, Kerkhoven RM, Zhu L, Carlée L, Voorhoeve PM, Bernards R. E2F-4, a new member of the E2F gene family, has oncogenic activity and associates with p107 in vivo. Genes Dev. 1994 Nov 15;8(22):2680–2690. [PubMed]
  • Bennett JD, Farlie PG, Watson RJ. E2F binding is required but not sufficient for repression of B-myb transcription in quiescent fibroblasts. Oncogene. 1996 Sep 5;13(5):1073–1082. [PubMed]
  • Blake MC, Azizkhan JC. Transcription factor E2F is required for efficient expression of the hamster dihydrofolate reductase gene in vitro and in vivo. Mol Cell Biol. 1989 Nov;9(11):4994–5002. [PMC free article] [PubMed]
  • Buchkovich K, Duffy LA, Harlow E. The retinoblastoma protein is phosphorylated during specific phases of the cell cycle. Cell. 1989 Sep 22;58(6):1097–1105. [PubMed]
  • Cao L, Faha B, Dembski M, Tsai LH, Harlow E, Dyson N. Independent binding of the retinoblastoma protein and p107 to the transcription factor E2F. Nature. 1992 Jan 9;355(6356):176–179. [PubMed]
  • Chellappan SP, Hiebert S, Mudryj M, Horowitz JM, Nevins JR. The E2F transcription factor is a cellular target for the RB protein. Cell. 1991 Jun 14;65(6):1053–1061. [PubMed]
  • Chen PL, Scully P, Shew JY, Wang JY, Lee WH. Phosphorylation of the retinoblastoma gene product is modulated during the cell cycle and cellular differentiation. Cell. 1989 Sep 22;58(6):1193–1198. [PubMed]
  • Chittenden T, Livingston DM, Kaelin WG., Jr The T/E1A-binding domain of the retinoblastoma product can interact selectively with a sequence-specific DNA-binding protein. Cell. 1991 Jun 14;65(6):1073–1082. [PubMed]
  • Claudio PP, Howard CM, Baldi A, De Luca A, Fu Y, Condorelli G, Sun Y, Colburn N, Calabretta B, Giordano A. p130/pRb2 has growth suppressive properties similar to yet distinctive from those of retinoblastoma family members pRb and p107. Cancer Res. 1994 Nov 1;54(21):5556–5560. [PubMed]
  • Cobrinik D, Whyte P, Peeper DS, Jacks T, Weinberg RA. Cell cycle-specific association of E2F with the p130 E1A-binding protein. Genes Dev. 1993 Dec;7(12A):2392–2404. [PubMed]
  • Cress WD, Nevins JR. Interacting domains of E2F1, DP1, and the adenovirus E4 protein. J Virol. 1994 Jul;68(7):4213–4219. [PMC free article] [PubMed]
  • Cress WD, Nevins JR. A role for a bent DNA structure in E2F-mediated transcription activation. Mol Cell Biol. 1996 May;16(5):2119–2127. [PMC free article] [PubMed]
  • DeCaprio JA, Ludlow JW, Figge J, Shew JY, Huang CM, Lee WH, Marsilio E, Paucha E, Livingston DM. SV40 large tumor antigen forms a specific complex with the product of the retinoblastoma susceptibility gene. Cell. 1988 Jul 15;54(2):275–283. [PubMed]
  • DeGregori J, Kowalik T, Nevins JR. Cellular targets for activation by the E2F1 transcription factor include DNA synthesis- and G1/S-regulatory genes. Mol Cell Biol. 1995 Aug;15(8):4215–4224. [PMC free article] [PubMed]
  • Dyson N, Howley PM, Münger K, Harlow E. The human papilloma virus-16 E7 oncoprotein is able to bind to the retinoblastoma gene product. Science. 1989 Feb 17;243(4893):934–937. [PubMed]
  • Field SJ, Tsai FY, Kuo F, Zubiaga AM, Kaelin WG, Jr, Livingston DM, Orkin SH, Greenberg ME. E2F-1 functions in mice to promote apoptosis and suppress proliferation. Cell. 1996 May 17;85(4):549–561. [PubMed]
  • Fry CJ, Slansky JE, Farnham PJ. Position-dependent transcriptional regulation of the murine dihydrofolate reductase promoter by the E2F transactivation domain. Mol Cell Biol. 1997 Apr;17(4):1966–1976. [PMC free article] [PubMed]
  • Geng Y, Eaton EN, Picón M, Roberts JM, Lundberg AS, Gifford A, Sardet C, Weinberg RA. Regulation of cyclin E transcription by E2Fs and retinoblastoma protein. Oncogene. 1996 Mar 21;12(6):1173–1180. [PubMed]
  • Ginsberg D, Vairo G, Chittenden T, Xiao ZX, Xu G, Wydner KL, DeCaprio JA, Lawrence JB, Livingston DM. E2F-4, a new member of the E2F transcription factor family, interacts with p107. Genes Dev. 1994 Nov 15;8(22):2665–2679. [PubMed]
  • Helin K, Lees JA, Vidal M, Dyson N, Harlow E, Fattaey A. A cDNA encoding a pRB-binding protein with properties of the transcription factor E2F. Cell. 1992 Jul 24;70(2):337–350. [PubMed]
  • Helin K, Wu CL, Fattaey AR, Lees JA, Dynlacht BD, Ngwu C, Harlow E. Heterodimerization of the transcription factors E2F-1 and DP-1 leads to cooperative trans-activation. Genes Dev. 1993 Oct;7(10):1850–1861. [PubMed]
  • Hijmans EM, Voorhoeve PM, Beijersbergen RL, van 't Veer LJ, Bernards R. E2F-5, a new E2F family member that interacts with p130 in vivo. Mol Cell Biol. 1995 Jun;15(6):3082–3089. [PMC free article] [PubMed]
  • Horowitz JM, Yandell DW, Park SH, Canning S, Whyte P, Buchkovich K, Harlow E, Weinberg RA, Dryja TP. Point mutational inactivation of the retinoblastoma antioncogene. Science. 1989 Feb 17;243(4893):937–940. [PubMed]
  • Hsiao KM, McMahon SL, Farnham PJ. Multiple DNA elements are required for the growth regulation of the mouse E2F1 promoter. Genes Dev. 1994 Jul 1;8(13):1526–1537. [PubMed]
  • Hu QJ, Dyson N, Harlow E. The regions of the retinoblastoma protein needed for binding to adenovirus E1A or SV40 large T antigen are common sites for mutations. EMBO J. 1990 Apr;9(4):1147–1155. [PMC free article] [PubMed]
  • Huber HE, Goodhart PJ, Huang PS. Retinoblastoma protein reverses DNA bending by transcription factor E2F. J Biol Chem. 1994 Mar 4;269(9):6999–7005. [PubMed]
  • Johnson DG, Ohtani K, Nevins JR. Autoregulatory control of E2F1 expression in response to positive and negative regulators of cell cycle progression. Genes Dev. 1994 Jul 1;8(13):1514–1525. [PubMed]
  • Kaelin WG, Jr, Krek W, Sellers WR, DeCaprio JA, Ajchenbaum F, Fuchs CS, Chittenden T, Li Y, Farnham PJ, Blanar MA, et al. Expression cloning of a cDNA encoding a retinoblastoma-binding protein with E2F-like properties. Cell. 1992 Jul 24;70(2):351–364. [PubMed]
  • Karlseder J, Rotheneder H, Wintersberger E. Interaction of Sp1 with the growth- and cell cycle-regulated transcription factor E2F. Mol Cell Biol. 1996 Apr;16(4):1659–1667. [PMC free article] [PubMed]
  • Kerppola TK, Curran T. DNA bending by Fos and Jun: the flexible hinge model. Science. 1991 Nov 22;254(5035):1210–1214. [PubMed]
  • Kerppola TK, Curran T. Fos-Jun heterodimers and Jun homodimers bend DNA in opposite orientations: implications for transcription factor cooperativity. Cell. 1991 Jul 26;66(2):317–326. [PubMed]
  • Kim J, Zwieb C, Wu C, Adhya S. Bending of DNA by gene-regulatory proteins: construction and use of a DNA bending vector. Gene. 1989 Dec 21;85(1):15–23. [PubMed]
  • Kim SJ, Onwuta US, Lee YI, Li R, Botchan MR, Robbins PD. The retinoblastoma gene product regulates Sp1-mediated transcription. Mol Cell Biol. 1992 Jun;12(6):2455–2463. [PMC free article] [PubMed]
  • Knudson AG. Antioncogenes and human cancer. Proc Natl Acad Sci U S A. 1993 Dec 1;90(23):10914–10921. [PMC free article] [PubMed]
  • Koo HS, Wu HM, Crothers DM. DNA bending at adenine . thymine tracts. Nature. 1986 Apr 10;320(6062):501–506. [PubMed]
  • Kovesdi I, Reichel R, Nevins JR. Identification of a cellular transcription factor involved in E1A trans-activation. Cell. 1986 Apr 25;45(2):219–228. [PubMed]
  • Krek W, Livingston DM, Shirodkar S. Binding to DNA and the retinoblastoma gene product promoted by complex formation of different E2F family members. Science. 1993 Dec 3;262(5139):1557–1560. [PubMed]
  • Lam EW, La Thangue NB. DP and E2F proteins: coordinating transcription with cell cycle progression. Curr Opin Cell Biol. 1994 Dec;6(6):859–866. [PubMed]
  • Lam EW, Watson RJ. An E2F-binding site mediates cell-cycle regulated repression of mouse B-myb transcription. EMBO J. 1993 Jul;12(7):2705–2713. [PMC free article] [PubMed]
  • Lees JA, Buchkovich KJ, Marshak DR, Anderson CW, Harlow E. The retinoblastoma protein is phosphorylated on multiple sites by human cdc2. EMBO J. 1991 Dec;10(13):4279–4290. [PMC free article] [PubMed]
  • Lin SY, Black AR, Kostic D, Pajovic S, Hoover CN, Azizkhan JC. Cell cycle-regulated association of E2F1 and Sp1 is related to their functional interaction. Mol Cell Biol. 1996 Apr;16(4):1668–1675. [PMC free article] [PubMed]
  • Ludlow JW, Shon J, Pipas JM, Livingston DM, DeCaprio JA. The retinoblastoma susceptibility gene product undergoes cell cycle-dependent dephosphorylation and binding to and release from SV40 large T. Cell. 1990 Feb 9;60(3):387–396. [PubMed]
  • Magae J, Wu CL, Illenye S, Harlow E, Heintz NH. Nuclear localization of DP and E2F transcription factors by heterodimeric partners and retinoblastoma protein family members. J Cell Sci. 1996 Jul;109(Pt 7):1717–1726. [PubMed]
  • Mihara K, Cao XR, Yen A, Chandler S, Driscoll B, Murphree AL, T'Ang A, Fung YK. Cell cycle-dependent regulation of phosphorylation of the human retinoblastoma gene product. Science. 1989 Dec 8;246(4935):1300–1303. [PubMed]
  • Moberg K, Starz MA, Lees JA. E2F-4 switches from p130 to p107 and pRB in response to cell cycle reentry. Mol Cell Biol. 1996 Apr;16(4):1436–1449. [PMC free article] [PubMed]
  • Nevins JR. E2F: a link between the Rb tumor suppressor protein and viral oncoproteins. Science. 1992 Oct 16;258(5081):424–429. [PubMed]
  • Ohtani K, DeGregori J, Leone G, Herendeen DR, Kelly TJ, Nevins JR. Expression of the HsOrc1 gene, a human ORC1 homolog, is regulated by cell proliferation via the E2F transcription factor. Mol Cell Biol. 1996 Dec;16(12):6977–6984. [PMC free article] [PubMed]
  • Ohtani K, DeGregori J, Nevins JR. Regulation of the cyclin E gene by transcription factor E2F1. Proc Natl Acad Sci U S A. 1995 Dec 19;92(26):12146–12150. [PMC free article] [PubMed]
  • Ormondroyd E, de la Luna S, La Thangue NB. A new member of the DP family, DP-3, with distinct protein products suggests a regulatory role for alternative splicing in the cell cycle transcription factor DRTF1/E2F. Oncogene. 1995 Oct 19;11(8):1437–1446. [PubMed]
  • Ouellette MM, Chen J, Wright WE, Shay JW. Complexes containing the retinoblastoma gene product recognize different DNA motifs related to the E2F binding site. Oncogene. 1992 Jun;7(6):1075–1081. [PubMed]
  • Reichel R, Kovesdi I, Nevins JR. Activation of a preexisting cellular factor as a basis for adenovirus E1A-mediated transcription control. Proc Natl Acad Sci U S A. 1988 Jan;85(2):387–390. [PMC free article] [PubMed]
  • Rogers KT, Higgins PD, Milla MM, Phillips RS, Horowitz JM. DP-2, a heterodimeric partner of E2F: identification and characterization of DP-2 proteins expressed in vivo. Proc Natl Acad Sci U S A. 1996 Jul 23;93(15):7594–7599. [PMC free article] [PubMed]
  • Schulze A, Zerfass K, Spitkovsky D, Middendorp S, Bergès J, Helin K, Jansen-Dürr P, Henglein B. Cell cycle regulation of the cyclin A gene promoter is mediated by a variant E2F site. Proc Natl Acad Sci U S A. 1995 Nov 21;92(24):11264–11268. [PMC free article] [PubMed]
  • Schwarz JK, Devoto SH, Smith EJ, Chellappan SP, Jakoi L, Nevins JR. Interactions of the p107 and Rb proteins with E2F during the cell proliferation response. EMBO J. 1993 Mar;12(3):1013–1020. [PMC free article] [PubMed]
  • Sellers WR, Rodgers JW, Kaelin WG., Jr A potent transrepression domain in the retinoblastoma protein induces a cell cycle arrest when bound to E2F sites. Proc Natl Acad Sci U S A. 1995 Dec 5;92(25):11544–11548. [PMC free article] [PubMed]
  • Shan B, Zhu X, Chen PL, Durfee T, Yang Y, Sharp D, Lee WH. Molecular cloning of cellular genes encoding retinoblastoma-associated proteins: identification of a gene with properties of the transcription factor E2F. Mol Cell Biol. 1992 Dec;12(12):5620–5631. [PMC free article] [PubMed]
  • Shirodkar S, Ewen M, DeCaprio JA, Morgan J, Livingston DM, Chittenden T. The transcription factor E2F interacts with the retinoblastoma product and a p107-cyclin A complex in a cell cycle-regulated manner. Cell. 1992 Jan 10;68(1):157–166. [PubMed]
  • Slansky JE, Farnham PJ. Introduction to the E2F family: protein structure and gene regulation. Curr Top Microbiol Immunol. 1996;208:1–30. [PubMed]
  • Sterner JM, Murata Y, Kim HG, Kennett SB, Templeton DJ, Horowitz JM. Detection of a novel cell cycle-regulated kinase activity that associates with the amino terminus of the retinoblastoma protein in G2/M phases. J Biol Chem. 1995 Apr 21;270(16):9281–9288. [PubMed]
  • Strauss JK, Maher LJ., 3rd DNA bending by asymmetric phosphate neutralization. Science. 1994 Dec 16;266(5192):1829–1834. [PubMed]
  • Taya Y, Yasuda H, Kamijo M, Nakaya K, Nakamura Y, Ohba Y, Nishimura S. In vitro phosphorylation of the tumor suppressor gene RB protein by mitosis-specific histone H1 kinase. Biochem Biophys Res Commun. 1989 Oct 16;164(1):580–586. [PubMed]
  • Thompson JF, Landy A. Empirical estimation of protein-induced DNA bending angles: applications to lambda site-specific recombination complexes. Nucleic Acids Res. 1988 Oct 25;16(20):9687–9705. [PMC free article] [PubMed]
  • Tommasi S, Pfeifer GP. In vivo structure of the human cdc2 promoter: release of a p130-E2F-4 complex from sequences immediately upstream of the transcription initiation site coincides with induction of cdc2 expression. Mol Cell Biol. 1995 Dec;15(12):6901–6913. [PMC free article] [PubMed]
  • Udvadia AJ, Rogers KT, Higgins PD, Murata Y, Martin KH, Humphrey PA, Horowitz JM. Sp-1 binds promoter elements regulated by the RB protein and Sp-1-mediated transcription is stimulated by RB coexpression. Proc Natl Acad Sci U S A. 1993 Apr 15;90(8):3265–3269. [PMC free article] [PubMed]
  • Vairo G, Livingston DM, Ginsberg D. Functional interaction between E2F-4 and p130: evidence for distinct mechanisms underlying growth suppression by different retinoblastoma protein family members. Genes Dev. 1995 Apr 1;9(7):869–881. [PubMed]
  • Wade M, Blake MC, Jambou RC, Helin K, Harlow E, Azizkhan JC. An inverted repeat motif stabilizes binding of E2F and enhances transcription of the dihydrofolate reductase gene. J Biol Chem. 1995 Apr 28;270(17):9783–9791. [PubMed]
  • Weinberg RA. The retinoblastoma protein and cell cycle control. Cell. 1995 May 5;81(3):323–330. [PubMed]
  • Weinberg RA. E2F and cell proliferation: a world turned upside down. Cell. 1996 May 17;85(4):457–459. [PubMed]
  • Weintraub SJ, Chow KN, Luo RX, Zhang SH, He S, Dean DC. Mechanism of active transcriptional repression by the retinoblastoma protein. Nature. 1995 Jun 29;375(6534):812–815. [PubMed]
  • Weintraub SJ, Prater CA, Dean DC. Retinoblastoma protein switches the E2F site from positive to negative element. Nature. 1992 Jul 16;358(6383):259–261. [PubMed]
  • Wells J, Held P, Illenye S, Heintz NH. Protein-DNA interactions at the major and minor promoters of the divergently transcribed dhfr and rep3 genes during the Chinese hamster ovary cell cycle. Mol Cell Biol. 1996 Feb;16(2):634–647. [PMC free article] [PubMed]
  • Wells JM, Illenye S, Magae J, Wu CL, Heintz NH. Accumulation of E2F-4.DP-1 DNA binding complexes correlates with induction of dhfr gene expression during the G1 to S phase transition. J Biol Chem. 1997 Feb 14;272(7):4483–4492. [PubMed]
  • Whyte P, Buchkovich KJ, Horowitz JM, Friend SH, Raybuck M, Weinberg RA, Harlow E. Association between an oncogene and an anti-oncogene: the adenovirus E1A proteins bind to the retinoblastoma gene product. Nature. 1988 Jul 14;334(6178):124–129. [PubMed]
  • Yamasaki L, Jacks T, Bronson R, Goillot E, Harlow E, Dyson NJ. Tumor induction and tissue atrophy in mice lacking E2F-1. Cell. 1996 May 17;85(4):537–548. [PubMed]
  • Yee AS, Raychaudhuri P, Jakoi L, Nevins JR. The adenovirus-inducible factor E2F stimulates transcription after specific DNA binding. Mol Cell Biol. 1989 Feb;9(2):578–585. [PMC free article] [PubMed]
  • Zamanian M, La Thangue NB. Transcriptional repression by the Rb-related protein p107. Mol Biol Cell. 1993 Apr;4(4):389–396. [PMC free article] [PubMed]
  • Zhu L, van den Heuvel S, Helin K, Fattaey A, Ewen M, Livingston D, Dyson N, Harlow E. Inhibition of cell proliferation by p107, a relative of the retinoblastoma protein. Genes Dev. 1993 Jul;7(7A):1111–1125. [PubMed]
  • Zhu L, Zhu L, Xie E, Chang LS. Differential roles of two tandem E2F sites in repression of the human p107 promoter by retinoblastoma and p107 proteins. Mol Cell Biol. 1995 Jul;15(7):3552–3562. [PMC free article] [PubMed]
  • Zwicker J, Liu N, Engeland K, Lucibello FC, Müller R. Cell cycle regulation of E2F site occupation in vivo. Science. 1996 Mar 15;271(5255):1595–1597. [PubMed]

Articles from Molecular and Cellular Biology are provided here courtesy of American Society for Microbiology (ASM)

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

  • Compound
    Compound
    PubChem Compound links
  • MedGen
    MedGen
    Related information in MedGen
  • PubMed
    PubMed
    PubMed citations for these articles
  • Substance
    Substance
    PubChem Substance links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...