The AD1 transactivation domain of E2A contains a highly conserved helix which is required for its activity in both Saccharomyces cerevisiae and mammalian cells (original) (raw)

Abstract

A conserved region, designated the AD1 domain, is present in a class of helix-loop-helix (HLH) proteins, E proteins, that includes E12, E47, HEB, E2-2, and a Xenopus laevis HLH protein closely related to E12. We demonstrate that the AD1 domain in E2A and the conserved region of E2-2 activate transcription in both yeast and mammalian cells. The AD1 domain contains a highly conserved putative helix that is crucial for its transactivation properties. Circular dichroism spectroscopy data show that AD1 is structured and contains distinctive helical properties. In addition, we show that a synthetic peptide corresponding to the conserved region is unstructured in aqueous solution at neutral pH but can adopt an alpha-helical conformation in the presence of the hydrophobic solvent trifluoroethanol. Amino acid substitutions that destabilize the helix abolish the transactivation ability of the AD1 domain. Both structural and functional analyses of AD1 reveal striking similarities to the acidic class of activators. Remarkably, when wild-type and mutant proteins are expressed in mammalian cells and Saccharomyces cerevisiae, identical patterns of transactivation are observed, suggesting that the target molecule is conserved between S. cerevisiae and mammals. These data show that transactivation by E proteins is mediated, in part, by a strikingly conserved peptide that has the ability to form a helix in a hydrophobic solvent. We propose that the unstructured domain may become helical upon interaction with its cellular target molecule.

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Selected References

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  1. Altschul S. F., Gish W., Miller W., Myers E. W., Lipman D. J. Basic local alignment search tool. J Mol Biol. 1990 Oct 5;215(3):403–410. doi: 10.1016/S0022-2836(05)80360-2. [DOI] [PubMed] [Google Scholar]
  2. Aronheim A., Shiran R., Rosen A., Walker M. D. The E2A gene product contains two separable and functionally distinct transcription activation domains. Proc Natl Acad Sci U S A. 1993 Sep 1;90(17):8063–8067. doi: 10.1073/pnas.90.17.8063. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bain G., Maandag E. C., Izon D. J., Amsen D., Kruisbeek A. M., Weintraub B. C., Krop I., Schlissel M. S., Feeney A. J., van Roon M. E2A proteins are required for proper B cell development and initiation of immunoglobulin gene rearrangements. Cell. 1994 Dec 2;79(5):885–892. doi: 10.1016/0092-8674(94)90077-9. [DOI] [PubMed] [Google Scholar]
  4. Caudy M., Grell E. H., Dambly-Chaudière C., Ghysen A., Jan L. Y., Jan Y. N. The maternal sex determination gene daughterless has zygotic activity necessary for the formation of peripheral neurons in Drosophila. Genes Dev. 1988 Jul;2(7):843–852. doi: 10.1101/gad.2.7.843. [DOI] [PubMed] [Google Scholar]
  5. Caudy M., Vässin H., Brand M., Tuma R., Jan L. Y., Jan Y. N. daughterless, a Drosophila gene essential for both neurogenesis and sex determination, has sequence similarities to myc and the achaete-scute complex. Cell. 1988 Dec 23;55(6):1061–1067. doi: 10.1016/0092-8674(88)90250-4. [DOI] [PubMed] [Google Scholar]
  6. Chou P. Y., Fasman G. D. Prediction of protein conformation. Biochemistry. 1974 Jan 15;13(2):222–245. doi: 10.1021/bi00699a002. [DOI] [PubMed] [Google Scholar]
  7. Cline T. W. The affairs of daughterless and the promiscuity of developmental regulators. Cell. 1989 Oct 20;59(2):231–234. doi: 10.1016/0092-8674(89)90280-8. [DOI] [PubMed] [Google Scholar]
  8. Cordle S. R., Henderson E., Masuoka H., Weil P. A., Stein R. Pancreatic beta-cell-type-specific transcription of the insulin gene is mediated by basic helix-loop-helix DNA-binding proteins. Mol Cell Biol. 1991 Mar;11(3):1734–1738. doi: 10.1128/mcb.11.3.1734. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Corneliussen B., Thornell A., Hallberg B., Grundström T. Helix-loop-helix transcriptional activators bind to a sequence in glucocorticoid response elements of retrovirus enhancers. J Virol. 1991 Nov;65(11):6084–6093. doi: 10.1128/jvi.65.11.6084-6093.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Cronmiller C., Schedl P., Cline T. W. Molecular characterization of daughterless, a Drosophila sex determination gene with multiple roles in development. Genes Dev. 1988 Dec;2(12A):1666–1676. doi: 10.1101/gad.2.12a.1666. [DOI] [PubMed] [Google Scholar]
  11. Dahlman-Wright K., Baumann H., McEwan I. J., Almlöf T., Wright A. P., Gustafsson J. A., Härd T. Structural characterization of a minimal functional transactivation domain from the human glucocorticoid receptor. Proc Natl Acad Sci U S A. 1995 Feb 28;92(5):1699–1703. doi: 10.1073/pnas.92.5.1699. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Donaldson L., Capone J. P. Purification and characterization of the carboxyl-terminal transactivation domain of Vmw65 from herpes simplex virus type 1. J Biol Chem. 1992 Jan 25;267(3):1411–1414. [PubMed] [Google Scholar]
  13. Durfee T., Becherer K., Chen P. L., Yeh S. H., Yang Y., Kilburn A. E., Lee W. H., Elledge S. J. The retinoblastoma protein associates with the protein phosphatase type 1 catalytic subunit. Genes Dev. 1993 Apr;7(4):555–569. doi: 10.1101/gad.7.4.555. [DOI] [PubMed] [Google Scholar]
  14. Garnier J., Osguthorpe D. J., Robson B. Analysis of the accuracy and implications of simple methods for predicting the secondary structure of globular proteins. J Mol Biol. 1978 Mar 25;120(1):97–120. doi: 10.1016/0022-2836(78)90297-8. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. Henthorn P., Kiledjian M., Kadesch T. Two distinct transcription factors that bind the immunoglobulin enhancer microE5/kappa 2 motif. Science. 1990 Jan 26;247(4941):467–470. doi: 10.1126/science.2105528. [DOI] [PubMed] [Google Scholar]
  17. Hu J. S., Olson E. N., Kingston R. E. HEB, a helix-loop-helix protein related to E2A and ITF2 that can modulate the DNA-binding ability of myogenic regulatory factors. Mol Cell Biol. 1992 Mar;12(3):1031–1042. doi: 10.1128/mcb.12.3.1031. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hunger S. P., Ohyashiki K., Toyama K., Cleary M. L. Hlf, a novel hepatic bZIP protein, shows altered DNA-binding properties following fusion to E2A in t(17;19) acute lymphoblastic leukemia. Genes Dev. 1992 Sep;6(9):1608–1620. doi: 10.1101/gad.6.9.1608. [DOI] [PubMed] [Google Scholar]
  19. Inaba T., Roberts W. M., Shapiro L. H., Jolly K. W., Raimondi S. C., Smith S. D., Look A. T. Fusion of the leucine zipper gene HLF to the E2A gene in human acute B-lineage leukemia. Science. 1992 Jul 24;257(5069):531–534. doi: 10.1126/science.1386162. [DOI] [PubMed] [Google Scholar]
  20. Ito H., Fukuda Y., Murata K., Kimura A. Transformation of intact yeast cells treated with alkali cations. J Bacteriol. 1983 Jan;153(1):163–168. doi: 10.1128/jb.153.1.163-168.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Johnson W. C., Jr Circular dichroism and its empirical application to biopolymers. Methods Biochem Anal. 1985;31:61–163. doi: 10.1002/9780470110522.ch2. [DOI] [PubMed] [Google Scholar]
  22. Johnson W. C., Jr Protein secondary structure and circular dichroism: a practical guide. Proteins. 1990;7(3):205–214. doi: 10.1002/prot.340070302. [DOI] [PubMed] [Google Scholar]
  23. Kamps M. P., Murre C., Sun X. H., Baltimore D. A new homeobox gene contributes the DNA binding domain of the t(1;19) translocation protein in pre-B ALL. Cell. 1990 Feb 23;60(4):547–555. doi: 10.1016/0092-8674(90)90658-2. [DOI] [PubMed] [Google Scholar]
  24. Lassar A. B., Davis R. L., Wright W. E., Kadesch T., Murre C., Voronova A., Baltimore D., Weintraub H. Functional activity of myogenic HLH proteins requires hetero-oligomerization with E12/E47-like proteins in vivo. Cell. 1991 Jul 26;66(2):305–315. doi: 10.1016/0092-8674(91)90620-e. [DOI] [PubMed] [Google Scholar]
  25. Ma J., Ptashne M. A new class of yeast transcriptional activators. Cell. 1987 Oct 9;51(1):113–119. doi: 10.1016/0092-8674(87)90015-8. [DOI] [PubMed] [Google Scholar]
  26. Mitchell P. J., Tjian R. Transcriptional regulation in mammalian cells by sequence-specific DNA binding proteins. Science. 1989 Jul 28;245(4916):371–378. doi: 10.1126/science.2667136. [DOI] [PubMed] [Google Scholar]
  27. Monica K., LeBrun D. P., Dedera D. A., Brown R., Cleary M. L. Transformation properties of the E2a-Pbx1 chimeric oncoprotein: fusion with E2a is essential, but the Pbx1 homeodomain is dispensable. Mol Cell Biol. 1994 Dec;14(12):8304–8314. doi: 10.1128/mcb.14.12.8304. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Murre C., Bain G., van Dijk M. A., Engel I., Furnari B. A., Massari M. E., Matthews J. R., Quong M. W., Rivera R. R., Stuiver M. H. Structure and function of helix-loop-helix proteins. Biochim Biophys Acta. 1994 Jun 21;1218(2):129–135. doi: 10.1016/0167-4781(94)90001-9. [DOI] [PubMed] [Google Scholar]
  29. Murre C., McCaw P. S., Baltimore D. A new DNA binding and dimerization motif in immunoglobulin enhancer binding, daughterless, MyoD, and myc proteins. Cell. 1989 Mar 10;56(5):777–783. doi: 10.1016/0092-8674(89)90682-x. [DOI] [PubMed] [Google Scholar]
  30. Murre C., McCaw P. S., Vaessin H., Caudy M., Jan L. Y., Jan Y. N., Cabrera C. V., Buskin J. N., Hauschka S. D., Lassar A. B. Interactions between heterologous helix-loop-helix proteins generate complexes that bind specifically to a common DNA sequence. Cell. 1989 Aug 11;58(3):537–544. doi: 10.1016/0092-8674(89)90434-0. [DOI] [PubMed] [Google Scholar]
  31. Murre C., Voronova A., Baltimore D. B-cell- and myocyte-specific E2-box-binding factors contain E12/E47-like subunits. Mol Cell Biol. 1991 Feb;11(2):1156–1160. doi: 10.1128/mcb.11.2.1156. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Nelson C., Shen L. P., Meister A., Fodor E., Rutter W. J. Pan: a transcriptional regulator that binds chymotrypsin, insulin, and AP-4 enhancer motifs. Genes Dev. 1990 Jun;4(6):1035–1043. doi: 10.1101/gad.4.6.1035. [DOI] [PubMed] [Google Scholar]
  33. Nourse J., Mellentin J. D., Galili N., Wilkinson J., Stanbridge E., Smith S. D., Cleary M. L. Chromosomal translocation t(1;19) results in synthesis of a homeobox fusion mRNA that codes for a potential chimeric transcription factor. Cell. 1990 Feb 23;60(4):535–545. doi: 10.1016/0092-8674(90)90657-z. [DOI] [PubMed] [Google Scholar]
  34. O'Hare P., Williams G. Structural studies of the acidic transactivation domain of the Vmw65 protein of herpes simplex virus using 1H NMR. Biochemistry. 1992 Apr 28;31(16):4150–4156. doi: 10.1021/bi00131a035. [DOI] [PubMed] [Google Scholar]
  35. Peverali F. A., Ramqvist T., Saffrich R., Pepperkok R., Barone M. V., Philipson L. Regulation of G1 progression by E2A and Id helix-loop-helix proteins. EMBO J. 1994 Sep 15;13(18):4291–4301. doi: 10.1002/j.1460-2075.1994.tb06749.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Quong M. W., Massari M. E., Zwart R., Murre C. A new transcriptional-activation motif restricted to a class of helix-loop-helix proteins is functionally conserved in both yeast and mammalian cells. Mol Cell Biol. 1993 Feb;13(2):792–800. doi: 10.1128/mcb.13.2.792. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Rashbass J., Taylor M. V., Gurdon J. B. The DNA-binding protein E12 co-operates with XMyoD in the activation of muscle-specific gene expression in Xenopus embryos. EMBO J. 1992 Aug;11(8):2981–2990. doi: 10.1002/j.1460-2075.1992.tb05368.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Sawada S., Littman D. R. A heterodimer of HEB and an E12-related protein interacts with the CD4 enhancer and regulates its activity in T-cell lines. Mol Cell Biol. 1993 Sep;13(9):5620–5628. doi: 10.1128/mcb.13.9.5620. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Schlissel M., Voronova A., Baltimore D. Helix-loop-helix transcription factor E47 activates germ-line immunoglobulin heavy-chain gene transcription and rearrangement in a pre-T-cell line. Genes Dev. 1991 Aug;5(8):1367–1376. doi: 10.1101/gad.5.8.1367. [DOI] [PubMed] [Google Scholar]
  40. Schmitz M. L., dos Santos Silva M. A., Altmann H., Czisch M., Holak T. A., Baeuerle P. A. Structural and functional analysis of the NF-kappa B p65 C terminus. An acidic and modular transactivation domain with the potential to adopt an alpha-helical conformation. J Biol Chem. 1994 Oct 14;269(41):25613–25620. [PubMed] [Google Scholar]
  41. Sun X. H., Baltimore D. An inhibitory domain of E12 transcription factor prevents DNA binding in E12 homodimers but not in E12 heterodimers. Cell. 1991 Jan 25;64(2):459–470. doi: 10.1016/0092-8674(91)90653-g. [DOI] [PubMed] [Google Scholar]
  42. Van Hoy M., Leuther K. K., Kodadek T., Johnston S. A. The acidic activation domains of the GCN4 and GAL4 proteins are not alpha helical but form beta sheets. Cell. 1993 Feb 26;72(4):587–594. doi: 10.1016/0092-8674(93)90077-4. [DOI] [PubMed] [Google Scholar]
  43. Wang M. M., Reed R. R. Molecular cloning of the olfactory neuronal transcription factor Olf-1 by genetic selection in yeast. Nature. 1993 Jul 8;364(6433):121–126. doi: 10.1038/364121a0. [DOI] [PubMed] [Google Scholar]
  44. Wülbeck C., Fromental-Ramain C., Campos-Ortega J. A. The HLH domain of a zebrafish HE12 homologue can partially substitute for functions of the HLH domain of Drosophila DAUGHTERLESS. Mech Dev. 1994 May;46(2):73–85. doi: 10.1016/0925-4773(94)90077-9. [DOI] [PubMed] [Google Scholar]
  45. Yaffe M. P., Schatz G. Two nuclear mutations that block mitochondrial protein import in yeast. Proc Natl Acad Sci U S A. 1984 Aug;81(15):4819–4823. doi: 10.1073/pnas.81.15.4819. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Yoshihara T., Inaba T., Shapiro L. H., Kato J. Y., Look A. T. E2A-HLF-mediated cell transformation requires both the trans-activation domains of E2A and the leucine zipper dimerization domain of HLF. Mol Cell Biol. 1995 Jun;15(6):3247–3255. doi: 10.1128/mcb.15.6.3247. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Zhuang Y., Soriano P., Weintraub H. The helix-loop-helix gene E2A is required for B cell formation. Cell. 1994 Dec 2;79(5):875–884. doi: 10.1016/0092-8674(94)90076-0. [DOI] [PubMed] [Google Scholar]