Logo of molcellbPermissionsJournals.ASM.orgJournalMCB ArticleJournal InfoAuthorsReviewers
Mol Cell Biol. 1996 Apr; 16(4): 1659–1667.
PMCID: PMC231152

Interaction of Sp1 with the growth- and cell cycle-regulated transcription factor E2F.


Within the region around 150 bp upstream of the initiation codon, which was previously shown to suffice for growth-regulated expression, the murine thymidine kinase gene carries a single binding site for transcription factor Sp1; about 10 bp downstream of this site, there is a binding motif for transcription factor E2F. The latter protein appears to be responsible for growth regulation of the promoter. Mutational inactivation of either the Sp1 or the E2F site almost completely abolishes promoter activity, suggesting that the two transcription factors interact directly in delivering an activation signal to the basic transcription machinery. This was verified by demonstrating with the use of glutathione S-transferase fusion proteins that E2F and Sp1 bind to each other in vitro. For this interaction, the C-terminal part of Sp1 and the N terminus of E2F1, a domain also present in E2F2 and E2F3 but absent in E2F4 and E2F5, were essential. Accordingly, E2F1 to E2F3 but not E2F4 and E2F5 were found to bind sp1 in vitro. Coimmunoprecipitation experiments showed that complexes exist in vivo, and it was estabilished that the distance between the binding sites for the two transcription factors was critical for optimal promoter activity. Finally, in vivo footprinting experiments indicated that both the sp1 and E2F binding sites are occupied throughout the cell cycle. Mutation of either binding motif abolished binding of both transcription factors in vivo, which may indicate cooperative binding of the two proteins to chromatin-organized DNA. Our data are in line with the hypothesis that E2F functions as a growth- and cell cycle regulated tethering factor between Sp1 and the basic transcription machinery.

Full Text

The Full Text of this article is available as a PDF (450K).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Adams CC, Workman JL. Binding of disparate transcriptional activators to nucleosomal DNA is inherently cooperative. Mol Cell Biol. 1995 Mar;15(3):1405–1421. [PMC free article] [PubMed]
  • Aubin RJ, Weinfeld M, Paterson MC. Factors influencing efficiency and reproducibility of polybrene-assisted gene transfer. Somat Cell Mol Genet. 1988 Mar;14(2):155–167. [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, Lam EW, Sørensen TS, Zamanian M, Girling R, La Thangue NB. DP-1: a cell cycle-regulated and phosphorylated component of transcription factor DRTF1/E2F which is functionally important for recognition by pRb and the adenovirus E4 orf 6/7 protein. EMBO J. 1994 Jul 1;13(13):3104–3114. [PMC free article] [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]
  • 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]
  • Blake MC, Jambou RC, Swick AG, Kahn JW, Azizkhan JC. Transcriptional initiation is controlled by upstream GC-box interactions in a TATAA-less promoter. Mol Cell Biol. 1990 Dec;10(12):6632–6641. [PMC free article] [PubMed]
  • Dou QP, Zhao S, Levin AH, Wang J, Helin K, Pardee AB. G1/S-regulated E2F-containing protein complexes bind to the mouse thymidine kinase gene promoter. J Biol Chem. 1994 Jan 14;269(2):1306–1313. [PubMed]
  • Dyson N, Dembski M, Fattaey A, Ngwu C, Ewen M, Helin K. Analysis of p107-associated proteins: p107 associates with a form of E2F that differs from pRB-associated E2F-1. J Virol. 1993 Dec;67(12):7641–7647. [PMC free article] [PubMed]
  • Fetzer J, Michael M, Bohner T, Hofbauer R, Folkers G. A fast method for obtaining highly pure recombinant herpes simplex virus type 1 thymidine kinase. Protein Expr Purif. 1994 Oct;5(5):432–441. [PubMed]
  • Fridovich-Keil JL, Markell PJ, Gudas JM, Pardee AB. DNA sequences required for serum-responsive regulation of expression from the mouse thymidine kinase promoter. Cell Growth Differ. 1993 Aug;4(8):679–687. [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]
  • Girling R, Partridge JF, Bandara LR, Burden N, Totty NF, Hsuan JJ, La Thangue NB. A new component of the transcription factor DRTF1/E2F. Nature. 1993 Mar 4;362(6415):83–87. [PubMed]
  • Gudas JM, Fridovich-Keil JL, Datta MW, Bryan J, Pardee AB. Characterization of the murine thymidine kinase-encoding gene and analysis of transcription start point heterogeneity. Gene. 1992 Sep 10;118(2):205–216. [PubMed]
  • Hagemeier C, Cook A, Kouzarides T. The retinoblastoma protein binds E2F residues required for activation in vivo and TBP binding in vitro. Nucleic Acids Res. 1993 Nov 11;21(22):4998–5004. [PMC free article] [PubMed]
  • Hamel PA, Gallie BL, Phillips RA. The retinoblastoma protein and cell cycle regulation. Trends Genet. 1992 May;8(5):180–185. [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]
  • Hiebert SW, Blake M, Azizkhan J, Nevins JR. Role of E2F transcription factor in E1A-mediated trans activation of cellular genes. J Virol. 1991 Jul;65(7):3547–3552. [PMC free article] [PubMed]
  • Hiebert SW, Chellappan SP, Horowitz JM, Nevins JR. The interaction of RB with E2F coincides with an inhibition of the transcriptional activity of E2F. Genes Dev. 1992 Feb;6(2):177–185. [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]
  • Hoey T, Weinzierl RO, Gill G, Chen JL, Dynlacht BD, Tjian R. Molecular cloning and functional analysis of Drosophila TAF110 reveal properties expected of coactivators. Cell. 1993 Jan 29;72(2):247–260. [PubMed]
  • Huber HE, Edwards G, Goodhart PJ, Patrick DR, Huang PS, Ivey-Hoyle M, Barnett SF, Oliff A, Heimbrook DC. Transcription factor E2F binds DNA as a heterodimer. Proc Natl Acad Sci U S A. 1993 Apr 15;90(8):3525–3529. [PMC free article] [PubMed]
  • Imataka H, Sogawa K, Yasumoto K, Kikuchi Y, Sasano K, Kobayashi A, Hayami M, Fujii-Kuriyama Y. Two regulatory proteins that bind to the basic transcription element (BTE), a GC box sequence in the promoter region of the rat P-4501A1 gene. EMBO J. 1992 Oct;11(10):3663–3671. [PMC free article] [PubMed]
  • Ivey-Hoyle M, Conroy R, Huber HE, Goodhart PJ, Oliff A, Heimbrook DC. Cloning and characterization of E2F-2, a novel protein with the biochemical properties of transcription factor E2F. Mol Cell Biol. 1993 Dec;13(12):7802–7812. [PMC free article] [PubMed]
  • Janson L, Pettersson U. Cooperative interactions between transcription factors Sp1 and OTF-1. Proc Natl Acad Sci U S A. 1990 Jun;87(12):4732–4736. [PMC free article] [PubMed]
  • Jordan KL, Haas AR, Logan TJ, Hall DJ. Detailed analysis of the basic domain of the E2F1 transcription factor indicates that it is unique among bHLH proteins. Oncogene. 1994 Apr;9(4):1177–1185. [PubMed]
  • Kadonaga JT, Carner KR, Masiarz FR, Tjian R. Isolation of cDNA encoding transcription factor Sp1 and functional analysis of the DNA binding domain. Cell. 1987 Dec 24;51(6):1079–1090. [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]
  • Kaelin WG, Jr, Pallas DC, DeCaprio JA, Kaye FJ, Livingston DM. Identification of cellular proteins that can interact specifically with the T/E1A-binding region of the retinoblastoma gene product. Cell. 1991 Feb 8;64(3):521–532. [PubMed]
  • Kitagawa M, Higashi H, Suzuki-Takahashi I, Segawa K, Hanks SK, Taya Y, Nishimura S, Okuyama A. Phosphorylation of E2F-1 by cyclin A-cdk2. Oncogene. 1995 Jan 19;10(2):229–236. [PubMed]
  • Kouzarides T. Transcriptional regulation by the retinoblastoma protein. Trends Cell Biol. 1993 Jul;3(7):211–213. [PubMed]
  • Krek W, Ewen ME, Shirodkar S, Arany Z, Kaelin WG, Jr, Livingston DM. Negative regulation of the growth-promoting transcription factor E2F-1 by a stably bound cyclin A-dependent protein kinase. Cell. 1994 Jul 15;78(1):161–172. [PubMed]
  • Lai JS, Herr W. Ethidium bromide provides a simple tool for identifying genuine DNA-independent protein associations. Proc Natl Acad Sci U S A. 1992 Aug 1;89(15):6958–6962. [PMC free article] [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]
  • La Thangue NB. DRTF1/E2F: an expanding family of heterodimeric transcription factors implicated in cell-cycle control. Trends Biochem Sci. 1994 Mar;19(3):108–114. [PubMed]
  • Lees JA, Saito M, Vidal M, Valentine M, Look T, Harlow E, Dyson N, Helin K. The retinoblastoma protein binds to a family of E2F transcription factors. Mol Cell Biol. 1993 Dec;13(12):7813–7825. [PMC free article] [PubMed]
  • Li R, Knight JD, Jackson SP, Tjian R, Botchan MR. Direct interaction between Sp1 and the BPV enhancer E2 protein mediates synergistic activation of transcription. Cell. 1991 May 3;65(3):493–505. [PubMed]
  • Li Y, Slansky JE, Myers DJ, Drinkwater NR, Kaelin WG, Farnham PJ. Cloning, chromosomal location, and characterization of mouse E2F1. Mol Cell Biol. 1994 Mar;14(3):1861–1869. [PMC free article] [PubMed]
  • Lieberman HB, Lin PF, Yeh DB, Ruddle FH. Transcriptional and posttranscriptional mechanisms regulate murine thymidine kinase gene expression in serum-stimulated cells. Mol Cell Biol. 1988 Dec;8(12):5280–5291. [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]
  • Merika M, Orkin SH. Functional synergy and physical interactions of the erythroid transcription factor GATA-1 with the Krüppel family proteins Sp1 and EKLF. Mol Cell Biol. 1995 May;15(5):2437–2447. [PMC free article] [PubMed]
  • Moran E. DNA tumor virus transforming proteins and the cell cycle. Curr Opin Genet Dev. 1993 Feb;3(1):63–70. [PubMed]
  • Mueller PR, Salser SJ, Wold B. Constitutive and metal-inducible protein:DNA interactions at the mouse metallothionein I promoter examined by in vivo and in vitro footprinting. Genes Dev. 1988 Apr;2(4):412–427. [PubMed]
  • Mueller PR, Wold B. In vivo footprinting of a muscle specific enhancer by ligation mediated PCR. Science. 1989 Nov 10;246(4931):780–786. [PubMed]
  • Murata Y, Kim HG, Rogers KT, Udvadia AJ, Horowitz JM. Negative regulation of Sp1 trans-activation is correlated with the binding of cellular proteins to the amino terminus of the Sp1 trans-activation domain. J Biol Chem. 1994 Aug 12;269(32):20674–20681. [PubMed]
  • Naeve GS, Sharma A, Lee AS. Temporal events regulating the early phases of the mammalian cell cycle. Curr Opin Cell Biol. 1991 Apr;3(2):261–268. [PubMed]
  • Nevins JR. E2F: a link between the Rb tumor suppressor protein and viral oncoproteins. Science. 1992 Oct 16;258(5081):424–429. [PubMed]
  • Nevins JR. Cell cycle targets of the DNA tumor viruses. Curr Opin Genet Dev. 1994 Feb;4(1):130–134. [PubMed]
  • Ogris E, Rotheneder H, Mudrak I, Pichler A, Wintersberger E. A binding site for transcription factor E2F is a target for trans activation of murine thymidine kinase by polyomavirus large T antigen and plays an important role in growth regulation of the gene. J Virol. 1993 Apr;67(4):1765–1771. [PMC free article] [PubMed]
  • Pagano M, Draetta G, Jansen-Dürr P. Association of cdk2 kinase with the transcription factor E2F during S phase. Science. 1992 Feb 28;255(5048):1144–1147. [PubMed]
  • Pardee AB. G1 events and regulation of cell proliferation. Science. 1989 Nov 3;246(4930):603–608. [PubMed]
  • Pascal E, Tjian R. Different activation domains of Sp1 govern formation of multimers and mediate transcriptional synergism. Genes Dev. 1991 Sep;5(9):1646–1656. [PubMed]
  • Plet A, Tourkine N, Mechti N, Jeanteur P, Blanchard JM. In vivo footprints between the murine c-myc P1 and P2 promoters. Oncogene. 1992 Sep;7(9):1847–1851. [PubMed]
  • Pugh BF, Tjian R. Mechanism of transcriptional activation by Sp1: evidence for coactivators. Cell. 1990 Jun 29;61(7):1187–1197. [PubMed]
  • Pugh BF, Tjian R. Transcription from a TATA-less promoter requires a multisubunit TFIID complex. Genes Dev. 1991 Nov;5(11):1935–1945. [PubMed]
  • Rotheneder H, Grabner M, Wintersberger E. Presence of regulatory sequences within intron 2 of the mouse thymidine kinase gene. Nucleic Acids Res. 1991 Dec 25;19(24):6805–6809. [PMC free article] [PubMed]
  • Sardet C, Vidal M, Cobrinik D, Geng Y, Onufryk C, Chen A, Weinberg RA. E2F-4 and E2F-5, two members of the E2F family, are expressed in the early phases of the cell cycle. Proc Natl Acad Sci U S A. 1995 Mar 14;92(6):2403–2407. [PMC free article] [PubMed]
  • Seiser C, Knöfler M, Rudelstorfer I, Haas R, Wintersberger E. Mouse thymidine kinase: the promoter sequence and the gene and pseudogene structures in normal cells and in thymidine kinase deficient mutants. Nucleic Acids Res. 1989 Jan 11;17(1):185–195. [PMC free article] [PubMed]
  • Seto E, Lewis B, Shenk T. Interaction between transcription factors Sp1 and YY1. Nature. 1993 Sep 30;365(6445):462–464. [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]
  • Sherr CJ. The ins and outs of RB: coupling gene expression to the cell cycle clock. Trends Cell Biol. 1994 Jan;4(1):15–18. [PubMed]
  • Slansky JE, Li Y, Kaelin WG, Farnham PJ. A protein synthesis-dependent increase in E2F1 mRNA correlates with growth regulation of the dihydrofolate reductase promoter. Mol Cell Biol. 1993 Mar;13(3):1610–1618. [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]
  • Weichselbraun I, Ogris E, Wintersberger E. Bidirectional promoter activity of the 5' flanking region of the mouse thymidine kinase gene. FEBS Lett. 1990 Nov 26;275(1-2):49–52. [PubMed]
  • Weinberg RA. The retinoblastoma protein and cell cycle control. Cell. 1995 May 5;81(3):323–330. [PubMed]
  • Weinzierl RO, Dynlacht BD, Tjian R. Largest subunit of Drosophila transcription factor IID directs assembly of a complex containing TBP and a coactivator. Nature. 1993 Apr 8;362(6420):511–517. [PubMed]
  • Wintersberger E. Biochemical events controlling initiation and propagation of the S phase of the cell cycle. Rev Physiol Biochem Pharmacol. 1991;118:49–95. [PubMed]
  • Wintersberger E, Rotheneder H, Grabner M, Beck G, Seiser C. Regulation of thymidine kinase during growth, cell cycle and differentiation. Adv Enzyme Regul. 1992;32:241–254. [PubMed]
  • Wu CL, Zukerberg LR, Ngwu C, Harlow E, Lees JA. In vivo association of E2F and DP family proteins. Mol Cell Biol. 1995 May;15(5):2536–2546. [PMC free article] [PubMed]
  • Xu M, Sheppard KA, Peng CY, Yee AS, Piwnica-Worms H. Cyclin A/CDK2 binds directly to E2F-1 and inhibits the DNA-binding activity of E2F-1/DP-1 by phosphorylation. Mol Cell Biol. 1994 Dec;14(12):8420–8431. [PMC free article] [PubMed]
  • Zhang Y, Chellappan SP. Cloning and characterization of human DP2, a novel dimerization partner of E2F. Oncogene. 1995 Jun 1;10(11):2085–2093. [PubMed]

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


Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...


  • Gene
    Gene links
  • Gene (nucleotide)
    Gene (nucleotide)
    Records in Gene identified from shared sequence links
  • GEO Profiles
    GEO Profiles
    Related GEO records
  • HomoloGene
    HomoloGene links
  • MedGen
    Related information in MedGen
  • Nucleotide
    Published Nucleotide sequences
  • Pathways + GO
    Pathways + GO
    Pathways, annotations and biological systems (BioSystems) that cite the current article.
  • Protein
    Published protein sequences
  • PubMed
    PubMed citations for these articles
  • Substance
    PubChem Substance links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...