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. 1996 May;16(5):2065–2073. doi: 10.1128/mcb.16.5.2065

Characterization of the cooperative function of inhibitory sequences in Ets-1.

M D Jonsen 1, J M Petersen 1, Q P Xu 1, B J Graves 1
PMCID: PMC231193  PMID: 8628272

Abstract

DNA binding by the eukaryotic transcription factor Ets-1 is negatively regulated by an intramolecular mechanism. Quantitative binding assays compared the DNA-binding activities of native Ets-1, three deletion mutants, and three tryptic fragments. Ets-1 and activated Ets-1 polypeptides differed in DNA-binding affinity as much as 23-fold. Inhibition was mediated by two regions flanking the minimal DNA-binding domain. Both regions regulated affinity by enhancing dissociation of the protein-DNA complex. Three lines of evidence indicated that inhibition requires cooperative interaction between the two regions: first, the two inhibitory regions acted through a common mechanism; second, neither region functioned independently of the other; finally, mutation of the C-terminal inhibitory region altered the conformation of the N-terminal inhibitory region. In addition, partial proteolysis detected an identical altered conformation in the N-terminal inhibitory region of Ets-1 bound to DNA. This finding suggested that repression is transiently disrupted during DNA binding. These results provide evidence that the two inhibitory regions of Ets-1 are structurally, as well as functionally, coupled. In addition, conformational change is shown to be a critical component of the inhibition mechanism. A cooperative, allosteric model of autoinhibition is described. Autoinhibition of Ets-1 could be relieved by either protein partner(s) or posttranslational modifications.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Carey J. Gel retardation. Methods Enzymol. 1991;208:103–117. doi: 10.1016/0076-6879(91)08010-f. [DOI] [PubMed] [Google Scholar]
  2. Dalton S., Treisman R. Characterization of SAP-1, a protein recruited by serum response factor to the c-fos serum response element. Cell. 1992 Feb 7;68(3):597–612. doi: 10.1016/0092-8674(92)90194-h. [DOI] [PubMed] [Google Scholar]
  3. Donaldson L. W., Petersen J. M., Graves B. J., McIntosh L. P. Secondary structure of the ETS domain places murine Ets-1 in the superfamily of winged helix-turn-helix DNA-binding proteins. Biochemistry. 1994 Nov 22;33(46):13509–13516. doi: 10.1021/bi00250a001. [DOI] [PubMed] [Google Scholar]
  4. Donaldson L. W., Petersen J. M., Graves B. J., McIntosh L. P. Solution structure of the ETS domain from murine Ets-1: a winged helix-turn-helix DNA binding motif. EMBO J. 1996 Jan 2;15(1):125–134. [PMC free article] [PubMed] [Google Scholar]
  5. Dudek H., Tantravahi R. V., Rao V. N., Reddy E. S., Reddy E. P. Myb and Ets proteins cooperate in transcriptional activation of the mim-1 promoter. Proc Natl Acad Sci U S A. 1992 Feb 15;89(4):1291–1295. doi: 10.1073/pnas.89.4.1291. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Erpel T., Superti-Furga G., Courtneidge S. A. Mutational analysis of the Src SH3 domain: the same residues of the ligand binding surface are important for intra- and intermolecular interactions. EMBO J. 1995 Mar 1;14(5):963–975. doi: 10.1002/j.1460-2075.1995.tb07077.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Fisher R. J., Fivash M., Casas-Finet J., Erickson J. W., Kondoh A., Bladen S. V., Fisher C., Watson D. K., Papas T. Real-time DNA binding measurements of the ETS1 recombinant oncoproteins reveal significant kinetic differences between the p42 and p51 isoforms. Protein Sci. 1994 Feb;3(2):257–266. doi: 10.1002/pro.5560030210. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Fisher R. J., Mavrothalassitis G., Kondoh A., Papas T. S. High-affinity DNA-protein interactions of the cellular ETS1 protein: the determination of the ETS binding motif. Oncogene. 1991 Dec;6(12):2249–2254. [PubMed] [Google Scholar]
  9. Fleischman L. F., Pilaro A. M., Murakami K., Kondoh A., Fisher R. J., Papas T. S. c-Ets-1 protein is hyperphosphorylated during mitosis. Oncogene. 1993 Mar;8(3):771–780. [PubMed] [Google Scholar]
  10. Frankel A. D., Kim P. S. Modular structure of transcription factors: implications for gene regulation. Cell. 1991 May 31;65(5):717–719. doi: 10.1016/0092-8674(91)90378-c. [DOI] [PubMed] [Google Scholar]
  11. Fried M. G. Measurement of protein-DNA interaction parameters by electrophoresis mobility shift assay. Electrophoresis. 1989 May-Jun;10(5-6):366–376. doi: 10.1002/elps.1150100515. [DOI] [PubMed] [Google Scholar]
  12. Giese K., Kingsley C., Kirshner J. R., Grosschedl R. Assembly and function of a TCR alpha enhancer complex is dependent on LEF-1-induced DNA bending and multiple protein-protein interactions. Genes Dev. 1995 Apr 15;9(8):995–1008. doi: 10.1101/gad.9.8.995. [DOI] [PubMed] [Google Scholar]
  13. Gill S. C., von Hippel P. H. Calculation of protein extinction coefficients from amino acid sequence data. Anal Biochem. 1989 Nov 1;182(2):319–326. doi: 10.1016/0003-2697(89)90602-7. [DOI] [PubMed] [Google Scholar]
  14. Giovane A., Pintzas A., Maira S. M., Sobieszczuk P., Wasylyk B. Net, a new ets transcription factor that is activated by Ras. Genes Dev. 1994 Jul 1;8(13):1502–1513. doi: 10.1101/gad.8.13.1502. [DOI] [PubMed] [Google Scholar]
  15. Golay J., Introna M., Graf T. A single point mutation in the v-ets oncogene affects both erythroid and myelomonocytic cell differentiation. Cell. 1988 Dec 23;55(6):1147–1158. doi: 10.1016/0092-8674(88)90259-0. [DOI] [PubMed] [Google Scholar]
  16. Green M., Schuetz T. J., Sullivan E. K., Kingston R. E. A heat shock-responsive domain of human HSF1 that regulates transcription activation domain function. Mol Cell Biol. 1995 Jun;15(6):3354–3362. doi: 10.1128/mcb.15.6.3354. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Grimm S., Baeuerle P. A. The inducible transcription factor NF-kappa B: structure-function relationship of its protein subunits. Biochem J. 1993 Mar 1;290(Pt 2):297–308. doi: 10.1042/bj2900297. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Gunther C. V., Graves B. J. Identification of ETS domain proteins in murine T lymphocytes that interact with the Moloney murine leukemia virus enhancer. Mol Cell Biol. 1994 Nov;14(11):7569–7580. doi: 10.1128/mcb.14.11.7569. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Gégonne A., Bosselut R., Bailly R. A., Ghysdael J. Synergistic activation of the HTLV1 LTR Ets-responsive region by transcription factors Ets1 and Sp1. EMBO J. 1993 Mar;12(3):1169–1178. doi: 10.1002/j.1460-2075.1993.tb05758.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Hager D. A., Burgess R. R. Elution of proteins from sodium dodecyl sulfate-polyacrylamide gels, removal of sodium dodecyl sulfate, and renaturation of enzymatic activity: results with sigma subunit of Escherichia coli RNA polymerase, wheat germ DNA topoisomerase, and other enzymes. Anal Biochem. 1980 Nov 15;109(1):76–86. doi: 10.1016/0003-2697(80)90013-5. [DOI] [PubMed] [Google Scholar]
  21. Hagman J., Grosschedl R. An inhibitory carboxyl-terminal domain in Ets-1 and Ets-2 mediates differential binding of ETS family factors to promoter sequences of the mb-1 gene. Proc Natl Acad Sci U S A. 1992 Oct 1;89(19):8889–8893. doi: 10.1073/pnas.89.19.8889. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Hahn S. L., Wasylyk B. The oncoprotein v-Ets is less selective in DNA binding than c-Ets-1 due to the C-terminal sequence change. Oncogene. 1994 Sep;9(9):2499–2512. [PubMed] [Google Scholar]
  23. Hupp T. R., Meek D. W., Midgley C. A., Lane D. P. Regulation of the specific DNA binding function of p53. Cell. 1992 Nov 27;71(5):875–886. doi: 10.1016/0092-8674(92)90562-q. [DOI] [PubMed] [Google Scholar]
  24. Hupp T. R., Sparks A., Lane D. P. Small peptides activate the latent sequence-specific DNA binding function of p53. Cell. 1995 Oct 20;83(2):237–245. doi: 10.1016/0092-8674(95)90165-5. [DOI] [PubMed] [Google Scholar]
  25. Kemp B. E., Pearson R. B. Intrasteric regulation of protein kinases and phosphatases. Biochim Biophys Acta. 1991 Aug 13;1094(1):67–76. doi: 10.1016/0167-4889(91)90027-u. [DOI] [PubMed] [Google Scholar]
  26. Kowenz-Leutz E., Twamley G., Ansieau S., Leutz A. Novel mechanism of C/EBP beta (NF-M) transcriptional control: activation through derepression. Genes Dev. 1994 Nov 15;8(22):2781–2791. doi: 10.1101/gad.8.22.2781. [DOI] [PubMed] [Google Scholar]
  27. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  28. Leprince D., Gegonne A., Coll J., de Taisne C., Schneeberger A., Lagrou C., Stehelin D. A putative second cell-derived oncogene of the avian leukaemia retrovirus E26. Nature. 1983 Nov 24;306(5941):395–397. doi: 10.1038/306395a0. [DOI] [PubMed] [Google Scholar]
  29. Liang H., Mao X., Olejniczak E. T., Nettesheim D. G., Yu L., Meadows R. P., Thompson C. B., Fesik S. W. Solution structure of the ets domain of Fli-1 when bound to DNA. Nat Struct Biol. 1994 Dec;1(12):871–875. doi: 10.1038/nsb1294-871. [DOI] [PubMed] [Google Scholar]
  30. Lieberman P. M., Schmidt M. C., Kao C. C., Berk A. J. Two distinct domains in the yeast transcription factor IID and evidence for a TATA box-induced conformational change. Mol Cell Biol. 1991 Jan;11(1):63–74. doi: 10.1128/mcb.11.1.63. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Lim F., Kraut N., Framptom J., Graf T. DNA binding by c-Ets-1, but not v-Ets, is repressed by an intramolecular mechanism. EMBO J. 1992 Feb;11(2):643–652. doi: 10.1002/j.1460-2075.1992.tb05096.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Nelsen B., Tian G., Erman B., Gregoire J., Maki R., Graves B., Sen R. Regulation of lymphoid-specific immunoglobulin mu heavy chain gene enhancer by ETS-domain proteins. Science. 1993 Jul 2;261(5117):82–86. doi: 10.1126/science.8316859. [DOI] [PubMed] [Google Scholar]
  33. Nunn M. F., Seeburg P. H., Moscovici C., Duesberg P. H. Tripartite structure of the avian erythroblastosis virus E26 transforming gene. Nature. 1983 Nov 24;306(5941):391–395. doi: 10.1038/306391a0. [DOI] [PubMed] [Google Scholar]
  34. Nye J. A., Petersen J. M., Gunther C. V., Jonsen M. D., Graves B. J. Interaction of murine ets-1 with GGA-binding sites establishes the ETS domain as a new DNA-binding motif. Genes Dev. 1992 Jun;6(6):975–990. doi: 10.1101/gad.6.6.975. [DOI] [PubMed] [Google Scholar]
  35. Petersen J. M., Skalicky J. J., Donaldson L. W., McIntosh L. P., Alber T., Graves B. J. Modulation of transcription factor Ets-1 DNA binding: DNA-induced unfolding of an alpha helix. Science. 1995 Sep 29;269(5232):1866–1869. doi: 10.1126/science.7569926. [DOI] [PubMed] [Google Scholar]
  36. Pognonec P., Boulukos K. E., Gesquière J. C., Stéhelin D., Ghysdael J. Mitogenic stimulation of thymocytes results in the calcium-dependent phosphorylation of c-ets-1 proteins. EMBO J. 1988 Apr;7(4):977–983. doi: 10.1002/j.1460-2075.1988.tb02904.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Rabault B., Ghysdael J. Calcium-induced phosphorylation of ETS1 inhibits its specific DNA binding activity. J Biol Chem. 1994 Nov 11;269(45):28143–28151. [PubMed] [Google Scholar]
  38. Rabindran S. K., Haroun R. I., Clos J., Wisniewski J., Wu C. Regulation of heat shock factor trimer formation: role of a conserved leucine zipper. Science. 1993 Jan 8;259(5092):230–234. doi: 10.1126/science.8421783. [DOI] [PubMed] [Google Scholar]
  39. Shapiro L. H. Myb and Ets proteins cooperate to transactivate an early myeloid gene. J Biol Chem. 1995 Apr 14;270(15):8763–8771. doi: 10.1074/jbc.270.15.8763. [DOI] [PubMed] [Google Scholar]
  40. Shi Y., Kroeger P. E., Morimoto R. I. The carboxyl-terminal transactivation domain of heat shock factor 1 is negatively regulated and stress responsive. Mol Cell Biol. 1995 Aug;15(8):4309–4318. doi: 10.1128/mcb.15.8.4309. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Skalicky J. J., Donaldson L. W., Petersen J. M., Graves B. J., McIntosh L. P. Structural coupling of the inhibitory regions flanking the ETS domain of murine Ets-1. Protein Sci. 1996 Feb;5(2):296–309. doi: 10.1002/pro.5560050214. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Soderling T. R. Protein kinases. Regulation by autoinhibitory domains. J Biol Chem. 1990 Feb 5;265(4):1823–1826. [PubMed] [Google Scholar]
  43. Soudant N., Albagli O., Dhordain P., Flourens A., Stéhelin D., Leprince D. A residue of the ETS domain mutated in the v-ets oncogene is essential for the DNA-binding and transactivating properties of the ETS-1 and ETS-2 proteins. Nucleic Acids Res. 1994 Sep 25;22(19):3871–3879. doi: 10.1093/nar/22.19.3871. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Sun W., Graves B. J., Speck N. A. Transactivation of the Moloney murine leukemia virus and T-cell receptor beta-chain enhancers by cbf and ets requires intact binding sites for both proteins. J Virol. 1995 Aug;69(8):4941–4949. doi: 10.1128/jvi.69.8.4941-4949.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Thompson C. C., Brown T. A., McKnight S. L. Convergence of Ets- and notch-related structural motifs in a heteromeric DNA binding complex. Science. 1991 Aug 16;253(5021):762–768. doi: 10.1126/science.1876833. [DOI] [PubMed] [Google Scholar]
  46. Wasylyk C., Flores P., Gutman A., Wasylyk B. PEA3 is a nuclear target for transcription activation by non-nuclear oncogenes. EMBO J. 1989 Nov;8(11):3371–3378. doi: 10.1002/j.1460-2075.1989.tb08500.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Wasylyk C., Kerckaert J. P., Wasylyk B. A novel modulator domain of Ets transcription factors. Genes Dev. 1992 Jun;6(6):965–974. doi: 10.1101/gad.6.6.965. [DOI] [PubMed] [Google Scholar]
  48. Wasylyk C., Wasylyk B. Oncogenic conversion of Ets affects redox regulation in-vivo and in-vitro. Nucleic Acids Res. 1993 Feb 11;21(3):523–529. doi: 10.1093/nar/21.3.523. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Werner M. H., Clore M., Fisher C. L., Fisher R. J., Trinh L., Shiloach J., Gronenborn A. M. The solution structure of the human ETS1-DNA complex reveals a novel mode of binding and true side chain intercalation. Cell. 1995 Dec 1;83(5):761–771. doi: 10.1016/0092-8674(95)90189-2. [DOI] [PubMed] [Google Scholar]
  50. Williams S. C., Baer M., Dillner A. J., Johnson P. F. CRP2 (C/EBP beta) contains a bipartite regulatory domain that controls transcriptional activation, DNA binding and cell specificity. EMBO J. 1995 Jul 3;14(13):3170–3183. doi: 10.1002/j.1460-2075.1995.tb07319.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Woods D. B., Ghysdael J., Owen M. J. Identification of nucleotide preferences in DNA sequences recognised specifically by c-Ets-1 protein. Nucleic Acids Res. 1992 Feb 25;20(4):699–704. doi: 10.1093/nar/20.4.699. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Wotton D., Ghysdael J., Wang S., Speck N. A., Owen M. J. Cooperative binding of Ets-1 and core binding factor to DNA. Mol Cell Biol. 1994 Jan;14(1):840–850. doi: 10.1128/mcb.14.1.840. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Zuo J., Rungger D., Voellmy R. Multiple layers of regulation of human heat shock transcription factor 1. Mol Cell Biol. 1995 Aug;15(8):4319–4330. doi: 10.1128/mcb.15.8.4319. [DOI] [PMC free article] [PubMed] [Google Scholar]

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