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. 1995 May;15(5):2429–2436. doi: 10.1128/mcb.15.5.2429

Negative regulation of the vascular smooth muscle alpha-actin gene in fibroblasts and myoblasts: disruption of enhancer function by sequence-specific single-stranded-DNA-binding proteins.

S Sun 1, E S Stoflet 1, J G Cogan 1, A R Strauch 1, M J Getz 1
PMCID: PMC230472  PMID: 7739527

Abstract

Transcriptional activation and repression of the vascular smooth muscle (VSM) alpha-actin gene in myoblasts and fibroblasts is mediated, in part, by positive and negative elements contained within an approximately 30-bp polypurine-polypyrimidine tract. This region contains binding sites for an essential transcription-activating protein, identified as transcriptional enhancer factor I (TEF-1), and two tissue-restrictive, sequence-specific, single-stranded-DNA-binding activities termed VACssBF1 and VACssBF2. TEF-1 has no detectable single-stranded-DNA-binding activity, while VACssBF1 and VACssBF2 have little, if any, affinity for double-stranded DNA. Site-specific mutagenesis experiments demonstrate that the determinants of VACssBF1 and VACssBF2 binding lie on opposite strands of the DNA helix and include the TEF-1 recognition sequence. Functional analysis of this region reveals that the CCAAT box-binding protein nuclear factor Y (NF-Y) can substitute for TEF-1 in activating VSM alpha-actin transcription but that the TEF-1-binding site is essential for the maintenance of full transcriptional repression. Importantly, replacement of the TEF-1-binding site with that for NF-Y diminishes the ability of VACssBF1 and VACssBF2 to bind to separated single strands. Additional activating mutations have been identified which lie outside of the TEF-1-binding site but which also impair single-stranded-DNA-binding activity. These data support a model in which VACssBF1 and VACssBF2 function as repressors of VSM alpha-actin transcription by stabilizing a local single-stranded-DNA conformation, thus precluding double-stranded-DNA binding by the essential transcriptional activator TEF-1.

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Selected References

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  1. Altiok S., Groner B. Interaction of two sequence-specific single-stranded DNA-binding proteins with an essential region of the beta-casein gene promoter is regulated by lactogenic hormones. Mol Cell Biol. 1993 Dec;13(12):7303–7310. doi: 10.1128/mcb.13.12.7303. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Blank R. S., McQuinn T. C., Yin K. C., Thompson M. M., Takeyasu K., Schwartz R. J., Owens G. K. Elements of the smooth muscle alpha-actin promoter required in cis for transcriptional activation in smooth muscle. Evidence for cell type-specific regulation. J Biol Chem. 1992 Jan 15;267(2):984–989. [PubMed] [Google Scholar]
  3. Cintorino M., Bellizzi de Marco E., Leoncini P., Tripodi S. A., Xu L. J., Sappino A. P., Schmitt-Gräff A., Gabbiani G. Expression of alpha-smooth-muscle actin in stromal cells of the uterine cervix during epithelial neoplastic changes. Int J Cancer. 1991 Apr 1;47(6):843–846. doi: 10.1002/ijc.2910470609. [DOI] [PubMed] [Google Scholar]
  4. Danilition S. L., Frederickson R. M., Taylor C. Y., Miyamoto N. G. Transcription factor binding and spacing constraints in the human beta-actin proximal promoter. Nucleic Acids Res. 1991 Dec 25;19(24):6913–6922. doi: 10.1093/nar/19.24.6913. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Darby I., Skalli O., Gabbiani G. Alpha-smooth muscle actin is transiently expressed by myofibroblasts during experimental wound healing. Lab Invest. 1990 Jul;63(1):21–29. [PubMed] [Google Scholar]
  6. Davidson I., Xiao J. H., Rosales R., Staub A., Chambon P. The HeLa cell protein TEF-1 binds specifically and cooperatively to two SV40 enhancer motifs of unrelated sequence. Cell. 1988 Sep 23;54(7):931–942. doi: 10.1016/0092-8674(88)90108-0. [DOI] [PubMed] [Google Scholar]
  7. Davis T. L., Firulli A. B., Kinniburgh A. J. Ribonucleoprotein and protein factors bind to an H-DNA-forming c-myc DNA element: possible regulators of the c-myc gene. Proc Natl Acad Sci U S A. 1989 Dec;86(24):9682–9686. doi: 10.1073/pnas.86.24.9682. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Desmoulière A., Geinoz A., Gabbiani F., Gabbiani G. Transforming growth factor-beta 1 induces alpha-smooth muscle actin expression in granulation tissue myofibroblasts and in quiescent and growing cultured fibroblasts. J Cell Biol. 1993 Jul;122(1):103–111. doi: 10.1083/jcb.122.1.103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Farrance I. K., Mar J. H., Ordahl C. P. M-CAT binding factor is related to the SV40 enhancer binding factor, TEF-1. J Biol Chem. 1992 Aug 25;267(24):17234–17240. [PubMed] [Google Scholar]
  10. Foster D. N., Min B., Foster L. K., Stoflet E. S., Sun S., Getz M. J., Strauch A. R. Positive and negative cis-acting regulatory elements mediate expression of the mouse vascular smooth muscle alpha-actin gene. J Biol Chem. 1992 Jun 15;267(17):11995–12003. [PubMed] [Google Scholar]
  11. Gorman C. M., Moffat L. F., Howard B. H. Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. Mol Cell Biol. 1982 Sep;2(9):1044–1051. doi: 10.1128/mcb.2.9.1044. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Ishiji T., Lace M. J., Parkkinen S., Anderson R. D., Haugen T. H., Cripe T. P., Xiao J. H., Davidson I., Chambon P., Turek L. P. Transcriptional enhancer factor (TEF)-1 and its cell-specific co-activator activate human papillomavirus-16 E6 and E7 oncogene transcription in keratinocytes and cervical carcinoma cells. EMBO J. 1992 Jun;11(6):2271–2281. doi: 10.1002/j.1460-2075.1992.tb05286.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Jansen-Dürr P., Boshart M., Lupp B., Bosserhoff A., Frank R. W., Schütz G. The rat poly pyrimidine tract binding protein (PTB) interacts with a single-stranded DNA motif in a liver-specific enhancer. Nucleic Acids Res. 1992 Mar 25;20(6):1243–1249. doi: 10.1093/nar/20.6.1243. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kariya K., Farrance I. K., Simpson P. C. Transcriptional enhancer factor-1 in cardiac myocytes interacts with an alpha 1-adrenergic- and beta-protein kinase C-inducible element in the rat beta-myosin heavy chain promoter. J Biol Chem. 1993 Dec 15;268(35):26658–26662. [PubMed] [Google Scholar]
  15. Larsen A., Weintraub H. An altered DNA conformation detected by S1 nuclease occurs at specific regions in active chick globin chromatin. Cell. 1982 Jun;29(2):609–622. doi: 10.1016/0092-8674(82)90177-5. [DOI] [PubMed] [Google Scholar]
  16. Lazard D., Sastre X., Frid M. G., Glukhova M. A., Thiery J. P., Koteliansky V. E. Expression of smooth muscle-specific proteins in myoepithelium and stromal myofibroblasts of normal and malignant human breast tissue. Proc Natl Acad Sci U S A. 1993 Feb 1;90(3):999–1003. doi: 10.1073/pnas.90.3.999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Luckow B., Schütz G. CAT constructions with multiple unique restriction sites for the functional analysis of eukaryotic promoters and regulatory elements. Nucleic Acids Res. 1987 Jul 10;15(13):5490–5490. doi: 10.1093/nar/15.13.5490. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Mar J. H., Ordahl C. P. A conserved CATTCCT motif is required for skeletal muscle-specific activity of the cardiac troponin T gene promoter. Proc Natl Acad Sci U S A. 1988 Sep;85(17):6404–6408. doi: 10.1073/pnas.85.17.6404. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Mar J. H., Ordahl C. P. M-CAT binding factor, a novel trans-acting factor governing muscle-specific transcription. Mol Cell Biol. 1990 Aug;10(8):4271–4283. doi: 10.1128/mcb.10.8.4271. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Min B. H., Foster D. N., Strauch A. R. The 5'-flanking region of the mouse vascular smooth muscle alpha-actin gene contains evolutionarily conserved sequence motifs within a functional promoter. J Biol Chem. 1990 Sep 25;265(27):16667–16675. [PubMed] [Google Scholar]
  21. O'Neill D., Bornschlegel K., Flamm M., Castle M., Bank A. A DNA-binding factor in adult hematopoietic cells interacts with a pyrimidine-rich domain upstream from the human delta-globin gene. Proc Natl Acad Sci U S A. 1991 Oct 15;88(20):8953–8957. doi: 10.1073/pnas.88.20.8953. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Pan W. T., Liu Q. R., Bancroft C. Identification of a growth hormone gene promoter repressor element and its cognate double- and single-stranded DNA-binding proteins. J Biol Chem. 1990 Apr 25;265(12):7022–7028. [PubMed] [Google Scholar]
  23. Patton J. G., Mayer S. A., Tempst P., Nadal-Ginard B. Characterization and molecular cloning of polypyrimidine tract-binding protein: a component of a complex necessary for pre-mRNA splicing. Genes Dev. 1991 Jul;5(7):1237–1251. doi: 10.1101/gad.5.7.1237. [DOI] [PubMed] [Google Scholar]
  24. Rønnov-Jessen L., Petersen O. W. Induction of alpha-smooth muscle actin by transforming growth factor-beta 1 in quiescent human breast gland fibroblasts. Implications for myofibroblast generation in breast neoplasia. Lab Invest. 1993 Jun;68(6):696–707. [PubMed] [Google Scholar]
  25. Santoro I. M., Yi T. M., Walsh K. Identification of single-stranded-DNA-binding proteins that interact with muscle gene elements. Mol Cell Biol. 1991 Apr;11(4):1944–1953. doi: 10.1128/mcb.11.4.1944. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Sappino A. P., Schürch W., Gabbiani G. Differentiation repertoire of fibroblastic cells: expression of cytoskeletal proteins as marker of phenotypic modulations. Lab Invest. 1990 Aug;63(2):144–161. [PubMed] [Google Scholar]
  27. Sappino A. P., Skalli O., Jackson B., Schürch W., Gabbiani G. Smooth-muscle differentiation in stromal cells of malignant and non-malignant breast tissues. Int J Cancer. 1988 May 15;41(5):707–712. doi: 10.1002/ijc.2910410512. [DOI] [PubMed] [Google Scholar]
  28. Schürch W., Seemayer T. A., Lagacé R. Stromal myofibroblasts in primary invasive and metastatic carcinomas. A combined immunological, light and electron microscopic study. Virchows Arch A Pathol Anat Histol. 1981;391(2):125–139. doi: 10.1007/BF00437591. [DOI] [PubMed] [Google Scholar]
  29. Stoflet E. S., Schmidt L. J., Elder P. K., Korf G. M., Foster D. N., Strauch A. R., Getz M. J. Activation of a muscle-specific actin gene promoter in serum-stimulated fibroblasts. Mol Biol Cell. 1992 Oct;3(10):1073–1083. doi: 10.1091/mbc.3.10.1073. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Treisman R., Ammerer G. The SRF and MCM1 transcription factors. Curr Opin Genet Dev. 1992 Apr;2(2):221–226. doi: 10.1016/s0959-437x(05)80277-1. [DOI] [PubMed] [Google Scholar]
  31. Wang Z. Y., Lin X. H., Nobuyoshi M., Deuel T. F. Identification of a single-stranded DNA-binding protein that interacts with an S1 nuclease-sensitive region in the platelet-derived growth factor A-chain gene promoter. J Biol Chem. 1993 May 15;268(14):10681–10685. [PubMed] [Google Scholar]
  32. Wilkison W. O., Min H. Y., Claffey K. P., Satterberg B. L., Spiegelman B. M. Control of the adipsin gene in adipocyte differentiation. Identification of distinct nuclear factors binding to single- and double-stranded DNA. J Biol Chem. 1990 Jan 5;265(1):477–482. [PubMed] [Google Scholar]
  33. Wolffe A. P. Structural and functional properties of the evolutionarily ancient Y-box family of nucleic acid binding proteins. Bioessays. 1994 Apr;16(4):245–251. doi: 10.1002/bies.950160407. [DOI] [PubMed] [Google Scholar]
  34. Yagil G. Paranemic structures of DNA and their role in DNA unwinding. Crit Rev Biochem Mol Biol. 1991;26(5-6):475–559. doi: 10.3109/10409239109086791. [DOI] [PubMed] [Google Scholar]

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