Skip to main content
NCBI home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1987 Nov;7(11):4089–4099. doi: 10.1128/mcb.7.11.4089

Multiple 5'-flanking regions of the human alpha-skeletal actin gene synergistically modulate muscle-specific expression.

G E Muscat 1, L Kedes 1
PMCID: PMC368080  PMID: 2828926

Abstract

Transfection into myogenic and nonmyogenic cell lines was used to investigate the transcriptional regulation of the human alpha-skeletal actin gene. We demonstrated that 1,300 base pairs of the 5'-flanking region directed high-level transient expression of the bacterial chloramphenicol acetyltransferase gene in differentiated mouse C2C12 and rat L8 myotubes but not in mouse nonmuscle L.TK- and HuT-12 cells. Unidirectional 5' deletion analysis and heterologous promoter stimulation experiments demonstrated that at least three transcription-regulating subdomains lie in this 1,300-base-pair region. A proximal cis-acting transcriptional element located between positions -153 and -87 relative to the start of transcription at +1 was both sufficient and necessary for muscle-specific expression and developmental regulation during myogenesis in the two myogenic cell systems. The region 3' of position -87 interacted with factors present in both myogenic and fibroblastic cells and appeared to define, or to be a major component of, the basal promoter. In C2C12 myotubes, but not in L8 myotubes, a distal sequence domain between positions -1300 and -626 and the proximal sequence domain between positions -153 and -87 each induced transcription about 10-fold and synergistically increased CAT expression 100-fold over levels achieved by the sequences 3' of position -87. Furthermore, these cis-acting elements independently and synergistically modulated an enhancerless, heterologous simian virus 40 promoter in a tissue-specific manner. DNA fragments which included the proximal domain displayed classical enhancerlike properties. The central region between positions -626 and -153, although required in neither cell line, had a positive, two- to threefold, additive role in augmenting expression in L8 cells but not in C2C12 cells. This suggests that certain elements between positions -1300 and -153 appear to be differentially utilized for maximal expression in different myogenic cells and that the particular combination of domains used is dependent on the availability, in kind or amount, of trans-acting, transcription-modulating factors present in each cell type. Thus, multiple myogenic factors that vary qualitatively and quantitatively may be responsible for the different and complex modulatory programs of actin gene expression observed during in vivo muscle differentiation.

Full text

PDF
4089

Images in this article

4092Image
on p.4092
4093Image
on p.4093
4095Image
on p.4095

Selected References

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

  1. Alwine J. C. Transient gene expression control: effects of transfected DNA stability and trans-activation by viral early proteins. Mol Cell Biol. 1985 May;5(5):1034–1042. doi: 10.1128/mcb.5.5.1034. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Atchison M. L., Perry R. P. The role of the kappa enhancer and its binding factor NF-kappa B in the developmental regulation of kappa gene transcription. Cell. 1987 Jan 16;48(1):121–128. doi: 10.1016/0092-8674(87)90362-x. [DOI] [PubMed] [Google Scholar]
  3. Bains W., Ponte P., Blau H., Kedes L. Cardiac actin is the major actin gene product in skeletal muscle cell differentiation in vitro. Mol Cell Biol. 1984 Aug;4(8):1449–1453. doi: 10.1128/mcb.4.8.1449. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Benoist C., O'Hare K., Breathnach R., Chambon P. The ovalbumin gene-sequence of putative control regions. Nucleic Acids Res. 1980 Jan 11;8(1):127–142. doi: 10.1093/nar/8.1.127. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bergsma D. J., Grichnik J. M., Gossett L. M., Schwartz R. J. Delimitation and characterization of cis-acting DNA sequences required for the regulated expression and transcriptional control of the chicken skeletal alpha-actin gene. Mol Cell Biol. 1986 Jul;6(7):2462–2475. doi: 10.1128/mcb.6.7.2462. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bienz M., Pelham H. R. Heat shock regulatory elements function as an inducible enhancer in the Xenopus hsp70 gene and when linked to a heterologous promoter. Cell. 1986 Jun 6;45(5):753–760. doi: 10.1016/0092-8674(86)90789-0. [DOI] [PubMed] [Google Scholar]
  7. Breathnach R., Chambon P. Organization and expression of eucaryotic split genes coding for proteins. Annu Rev Biochem. 1981;50:349–383. doi: 10.1146/annurev.bi.50.070181.002025. [DOI] [PubMed] [Google Scholar]
  8. Carmon Y., Czosnek H., Nudel U., Shani M., Yaffe D. DNAase I sensitivity of genes expressed during myogenesis. Nucleic Acids Res. 1982 May 25;10(10):3085–3098. doi: 10.1093/nar/10.10.3085. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Chepelinsky A. B., King C. R., Zelenka P. S., Piatigorsky J. Lens-specific expression of the chloramphenicol acetyltransferase gene promoted by 5' flanking sequences of the murine alpha A-crystallin gene in explanted chicken lens epithelia. Proc Natl Acad Sci U S A. 1985 Apr;82(8):2334–2338. doi: 10.1073/pnas.82.8.2334. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Ciliberto G., Dente L., Cortese R. Cell-specific expression of a transfected human alpha 1-antitrypsin gene. Cell. 1985 Jun;41(2):531–540. doi: 10.1016/s0092-8674(85)80026-x. [DOI] [PubMed] [Google Scholar]
  11. D'Onofrio C., Colantuoni V., Cortese R. Structure and cell-specific expression of a cloned human retinol binding protein gene: the 5'-flanking region contains hepatoma specific transcriptional signals. EMBO J. 1985 Aug;4(8):1981–1989. doi: 10.1002/j.1460-2075.1985.tb03881.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Davidson I., Fromental C., Augereau P., Wildeman A., Zenke M., Chambon P. Cell-type specific protein binding to the enhancer of simian virus 40 in nuclear extracts. Nature. 1986 Oct 9;323(6088):544–548. doi: 10.1038/323544a0. [DOI] [PubMed] [Google Scholar]
  13. Devlin R. B., Emerson C. P., Jr Coordinate accumulation of contractile protein mRNAs during myoblast differentiation. Dev Biol. 1979 Mar;69(1):202–216. doi: 10.1016/0012-1606(79)90286-0. [DOI] [PubMed] [Google Scholar]
  14. Edlund T., Walker M. D., Barr P. J., Rutter W. J. Cell-specific expression of the rat insulin gene: evidence for role of two distinct 5' flanking elements. Science. 1985 Nov 22;230(4728):912–916. doi: 10.1126/science.3904002. [DOI] [PubMed] [Google Scholar]
  15. Efstratiadis A., Posakony J. W., Maniatis T., Lawn R. M., O'Connell C., Spritz R. A., DeRiel J. K., Forget B. G., Weissman S. M., Slightom J. L. The structure and evolution of the human beta-globin gene family. Cell. 1980 Oct;21(3):653–668. doi: 10.1016/0092-8674(80)90429-8. [DOI] [PubMed] [Google Scholar]
  16. Fujita T., Ohno S., Yasumitsu H., Taniguchi T. Delimitation and properties of DNA sequences required for the regulated expression of human interferon-beta gene. Cell. 1985 Jun;41(2):489–496. doi: 10.1016/s0092-8674(85)80022-2. [DOI] [PubMed] [Google Scholar]
  17. Godbout R., Ingram R., Tilghman S. M. Multiple regulatory elements in the intergenic region between the alpha-fetoprotein and albumin genes. Mol Cell Biol. 1986 Feb;6(2):477–487. doi: 10.1128/mcb.6.2.477. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Goodbourn S., Zinn K., Maniatis T. Human beta-interferon gene expression is regulated by an inducible enhancer element. Cell. 1985 Jun;41(2):509–520. doi: 10.1016/s0092-8674(85)80024-6. [DOI] [PubMed] [Google Scholar]
  19. Gopal T. V., Shimada T., Baur A. W., Nienhuis A. W. Contribution of promoter to tissue-specific expression of the mouse immunoglobulin kappa gene. Science. 1985 Sep 13;229(4718):1102–1104. doi: 10.1126/science.2994213. [DOI] [PubMed] [Google Scholar]
  20. 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]
  21. Graham F. L., van der Eb A. J. A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology. 1973 Apr;52(2):456–467. doi: 10.1016/0042-6822(73)90341-3. [DOI] [PubMed] [Google Scholar]
  22. Grichnik J. M., Bergsma D. J., Schwartz R. J. Tissue restricted and stage specific transcription is maintained within 411 nucleotides flanking the 5' end of the chicken alpha-skeletal actin gene. Nucleic Acids Res. 1986 Feb 25;14(4):1683–1701. doi: 10.1093/nar/14.4.1683. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Grosveld G. C., Shewmaker C. K., Jat P., Flavell R. A. Localization of DNA sequences necessary for transcription of the rabbit beta-globin gene in vitro. Cell. 1981 Jul;25(1):215–226. doi: 10.1016/0092-8674(81)90246-4. [DOI] [PubMed] [Google Scholar]
  24. Gunning P., Hardeman E., Wade R., Ponte P., Bains W., Blau H. M., Kedes L. Differential patterns of transcript accumulation during human myogenesis. Mol Cell Biol. 1987 Nov;7(11):4100–4114. doi: 10.1128/mcb.7.11.4100. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Gunning P., Leavitt J., Muscat G., Ng S. Y., Kedes L. A human beta-actin expression vector system directs high-level accumulation of antisense transcripts. Proc Natl Acad Sci U S A. 1987 Jul;84(14):4831–4835. doi: 10.1073/pnas.84.14.4831. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Gunning P., Ponte P., Blau H., Kedes L. alpha-skeletal and alpha-cardiac actin genes are coexpressed in adult human skeletal muscle and heart. Mol Cell Biol. 1983 Nov;3(11):1985–1995. doi: 10.1128/mcb.3.11.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Hammer R. E., Krumlauf R., Camper S. A., Brinster R. L., Tilghman S. M. Diversity of alpha-fetoprotein gene expression in mice is generated by a combination of separate enhancer elements. Science. 1987 Jan 2;235(4784):53–58. doi: 10.1126/science.2432657. [DOI] [PubMed] [Google Scholar]
  28. Hanauer A., Levin M., Heilig R., Daegelen D., Kahn A., Mandel J. L. Isolation and characterization of cDNA clones for human skeletal muscle alpha actin. Nucleic Acids Res. 1983 Jun 11;11(11):3503–3516. doi: 10.1093/nar/11.11.3503. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Herbomel P., Bourachot B., Yaniv M. Two distinct enhancers with different cell specificities coexist in the regulatory region of polyoma. Cell. 1984 Dec;39(3 Pt 2):653–662. doi: 10.1016/0092-8674(84)90472-0. [DOI] [PubMed] [Google Scholar]
  30. Hickey R., Skoultchi A., Gunning P., Kedes L. Regulation of a human cardiac actin gene introduced into rat L6 myoblasts suggests a defect in their myogenic program. Mol Cell Biol. 1986 Sep;6(9):3287–3290. doi: 10.1128/mcb.6.9.3287. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Knoll B. J., Zarucki-Schulz T., Dean D. C., O'Malley B. W. Definition of the ovalbumin gene promoter by transfer of an ovalglobin fusion gene into cultured cells. Nucleic Acids Res. 1983 Oct 11;11(19):6733–6754. doi: 10.1093/nar/11.19.6733. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Laimins L. A., Khoury G., Gorman C., Howard B., Gruss P. Host-specific activation of transcription by tandem repeats from simian virus 40 and Moloney murine sarcoma virus. Proc Natl Acad Sci U S A. 1982 Nov;79(21):6453–6457. doi: 10.1073/pnas.79.21.6453. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Laimins L., Holmgren-König M., Khoury G. Transcriptional "silencer" element in rat repetitive sequences associated with the rat insulin 1 gene locus. Proc Natl Acad Sci U S A. 1986 May;83(10):3151–3155. doi: 10.1073/pnas.83.10.3151. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Laimins L., Holmgren-König M., Khoury G. Transcriptional "silencer" element in rat repetitive sequences associated with the rat insulin 1 gene locus. Proc Natl Acad Sci U S A. 1986 May;83(10):3151–3155. doi: 10.1073/pnas.83.10.3151. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Luse D. S., Roeder R. G. Accurate transcription initiation on a purified mouse beta-globin DNA fragment in a cell-free system. Cell. 1980 Jul;20(3):691–699. doi: 10.1016/0092-8674(80)90315-3. [DOI] [PubMed] [Google Scholar]
  36. Manley J. L., Fire A., Cano A., Sharp P. A., Gefter M. L. DNA-dependent transcription of adenovirus genes in a soluble whole-cell extract. Proc Natl Acad Sci U S A. 1980 Jul;77(7):3855–3859. doi: 10.1073/pnas.77.7.3855. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Mayer Y., Czosnek H., Zeelon P. E., Yaffe D., Nudel U. Expression of the genes coding for the skeletal muscle and cardiac actions in the heart. Nucleic Acids Res. 1984 Jan 25;12(2):1087–1100. doi: 10.1093/nar/12.2.1087. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Melloul D., Aloni B., Calvo J., Yaffe D., Nudel U. Developmentally regulated expression of chimeric genes containing muscle actin DNA sequences in transfected myogenic cells. EMBO J. 1984 May;3(5):983–990. doi: 10.1002/j.1460-2075.1984.tb01917.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Minty A. J., Alonso S., Caravatti M., Buckingham M. E. A fetal skeletal muscle actin mRNA in the mouse and its identity with cardiac actin mRNA. Cell. 1982 Aug;30(1):185–192. doi: 10.1016/0092-8674(82)90024-1. [DOI] [PubMed] [Google Scholar]
  40. Minty A., Blau H., Kedes L. Two-level regulation of cardiac actin gene transcription: muscle-specific modulating factors can accumulate before gene activation. Mol Cell Biol. 1986 Jun;6(6):2137–2148. doi: 10.1128/mcb.6.6.2137. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Minty A., Kedes L. Upstream regions of the human cardiac actin gene that modulate its transcription in muscle cells: presence of an evolutionarily conserved repeated motif. Mol Cell Biol. 1986 Jun;6(6):2125–2136. doi: 10.1128/mcb.6.6.2125. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Miwa T., Kedes L. Duplicated CArG box domains have positive and mutually dependent regulatory roles in expression of the human alpha-cardiac actin gene. Mol Cell Biol. 1987 Aug;7(8):2803–2813. doi: 10.1128/mcb.7.8.2803. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Mizushima-Sugano J., Roeder R. G. Cell-type-specific transcription of an immunoglobulin kappa light chain gene in vitro. Proc Natl Acad Sci U S A. 1986 Nov;83(22):8511–8515. doi: 10.1073/pnas.83.22.8511. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Mohun T. J., Garrett N., Gurdon J. B. Upstream sequences required for tissue-specific activation of the cardiac actin gene in Xenopus laevis embryos. EMBO J. 1986 Dec 1;5(12):3185–3193. doi: 10.1002/j.1460-2075.1986.tb04628.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Nir U., Walker M. D., Rutter W. J. Regulation of rat insulin 1 gene expression: evidence for negative regulation in nonpancreatic cells. Proc Natl Acad Sci U S A. 1986 May;83(10):3180–3184. doi: 10.1073/pnas.83.10.3180. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Nudel U., Greenberg D., Ordahl C. P., Saxel O., Neuman S., Yaffe D. Developmentally regulated expression of a chicken muscle-specific gene in stably transfected rat myogenic cells. Proc Natl Acad Sci U S A. 1985 May;82(10):3106–3109. doi: 10.1073/pnas.82.10.3106. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Okazaki K., Yasuda K., Kondoh H., Okada T. S. DNA sequences responsible for tissue-specific expression of a chicken alpha-crystallin gene in mouse lens cells. EMBO J. 1985 Oct;4(10):2589–2595. doi: 10.1002/j.1460-2075.1985.tb03975.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Ordahl C. P., Cooper T. A. Strong homology in promoter and 3'-untranslated regions of chick and rat alpha-actin genes. Nature. 1983 May 26;303(5915):348–349. doi: 10.1038/303348a0. [DOI] [PubMed] [Google Scholar]
  49. Pastorcic M., Wang H., Elbrecht A., Tsai S. Y., Tsai M. J., O'Malley B. W. Control of transcription initiation in vitro requires binding of a transcription factor to the distal promoter of the ovalbumin gene. Mol Cell Biol. 1986 Aug;6(8):2784–2791. doi: 10.1128/mcb.6.8.2784. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Ryals J., Dierks P., Ragg H., Weissmann C. A 46-nucleotide promoter segment from an IFN-alpha gene renders an unrelated promoter inducible by virus. Cell. 1985 Jun;41(2):497–507. doi: 10.1016/s0092-8674(85)80023-4. [DOI] [PubMed] [Google Scholar]
  51. Schirm S., Jiricny J., Schaffner W. The SV40 enhancer can be dissected into multiple segments, each with a different cell type specificity. Genes Dev. 1987 Mar;1(1):65–74. doi: 10.1101/gad.1.1.65. [DOI] [PubMed] [Google Scholar]
  52. Schmidt A., Rossi P., de Crombrugghe B. Transcriptional control of the mouse alpha 2(I) collagen gene: functional deletion analysis of the promoter and evidence for cell-specific expression. Mol Cell Biol. 1986 Feb;6(2):347–354. doi: 10.1128/mcb.6.2.347. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Seiler-Tuyns A., Eldridge J. D., Paterson B. M. Expression and regulation of chicken actin genes introduced into mouse myogenic and nonmyogenic cells. Proc Natl Acad Sci U S A. 1984 May;81(10):2980–2984. doi: 10.1073/pnas.81.10.2980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Suzuki Y., Tsuda M., Takiya S., Hirose S., Suzuki E., Kameda M., Ninaki O. Tissue-specific transcription enhancement of the fibroin gene characterized by cell-free systems. Proc Natl Acad Sci U S A. 1986 Dec;83(24):9522–9526. doi: 10.1073/pnas.83.24.9522. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Talkington C. A., Nishioka Y., Leder P. In vitro transcription of normal, mutant, and truncated mouse alpha-globin genes. Proc Natl Acad Sci U S A. 1980 Dec;77(12):7132–7136. doi: 10.1073/pnas.77.12.7132. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Theisen M., Stief A., Sippel A. E. The lysozyme enhancer: cell-specific activation of the chicken lysozyme gene by a far-upstream DNA element. EMBO J. 1986 Apr;5(4):719–724. doi: 10.1002/j.1460-2075.1986.tb04273.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Tsai S. Y., Tsai M. J., O'Malley B. W. Specific 5' flanking sequences are required for faithful initiation of in vitro transcription of the ovalbumin gene. Proc Natl Acad Sci U S A. 1981 Feb;78(2):879–883. doi: 10.1073/pnas.78.2.879. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Yaffe D., Saxel O. Serial passaging and differentiation of myogenic cells isolated from dystrophic mouse muscle. Nature. 1977 Dec 22;270(5639):725–727. doi: 10.1038/270725a0. [DOI] [PubMed] [Google Scholar]
  59. Zarraga A. M., Danishefsky K., Deshpande A., Nicholson D., Mendola C., Siddiqui M. A. Characterization of 5'-flanking region of heart myosin light chain 2A gene. Structural and functional evidence for promoter activity. J Biol Chem. 1986 Oct 15;261(29):13852–13860. [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES