Skip to main content
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1989 Jan 11;17(1):253–270. doi: 10.1093/nar/17.1.253

5' flanking and first intron sequences of the human beta-actin gene required for efficient promoter activity.

R M Frederickson 1, M R Micheau 1, A Iwamoto 1, N G Miyamoto 1
PMCID: PMC331549  PMID: 2911466

Abstract

We have identified a CCAAT box element that is required for the efficient transcription of the human beta-actin gene. Both in vivo transient transfection assays in cultured HeLa cells and in vitro run-off transcription assays in HeLa whole cell extracts demonstrated the requirement of this element for efficient promoter activity. A gel mobility shift assay revealed a Hela nuclear factor that specifically interacted with the beta-actin CCAAT element in vitro; mutation of the first three base pairs of the CCAAT pentanucleotide abolished binding of this factor. Competition gel shift experiments revealed that three sequence elements located within the beta-actin promoter, each containing a CC(A/T)6GG motif similar to that contained within the c-fos serum response element, were able to bind a different nuclear factor, serum response factor (SRF). One of these CC(A/T)6GG motifs is contained within a first intron fragment that enhanced transcription from a heterologous promoter in vivo.

Full text

PDF
253

Images in this article

Selected References

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

  1. Banerji J., Rusconi S., Schaffner W. Expression of a beta-globin gene is enhanced by remote SV40 DNA sequences. Cell. 1981 Dec;27(2 Pt 1):299–308. doi: 10.1016/0092-8674(81)90413-x. [DOI] [PubMed] [Google Scholar]
  2. Chirgwin J. M., Przybyla A. E., MacDonald R. J., Rutter W. J. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry. 1979 Nov 27;18(24):5294–5299. doi: 10.1021/bi00591a005. [DOI] [PubMed] [Google Scholar]
  3. Chodosh L. A., Baldwin A. S., Carthew R. W., Sharp P. A. Human CCAAT-binding proteins have heterologous subunits. Cell. 1988 Apr 8;53(1):11–24. doi: 10.1016/0092-8674(88)90483-7. [DOI] [PubMed] [Google Scholar]
  4. Cohen R. B., Sheffery M., Kim C. G. Partial purification of a nuclear protein that binds to the CCAAT box of the mouse alpha 1-globin gene. Mol Cell Biol. 1986 Mar;6(3):821–832. doi: 10.1128/mcb.6.3.821. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. DePonti-Zilli L., Seiler-Tuyns A., Paterson B. M. A 40-base-pair sequence in the 3' end of the beta-actin gene regulates beta-actin mRNA transcription during myogenesis. Proc Natl Acad Sci U S A. 1988 Mar;85(5):1389–1393. doi: 10.1073/pnas.85.5.1389. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Deschamps J., Meijlink F., Verma I. M. Identification of a transcriptional enhancer element upstream from the proto-oncogene fos. Science. 1985 Dec 6;230(4730):1174–1177. doi: 10.1126/science.3865371. [DOI] [PubMed] [Google Scholar]
  7. Dorn A., Bollekens J., Staub A., Benoist C., Mathis D. A multiplicity of CCAAT box-binding proteins. Cell. 1987 Sep 11;50(6):863–872. doi: 10.1016/0092-8674(87)90513-7. [DOI] [PubMed] [Google Scholar]
  8. Elder P. K., Schmidt L. J., Ono T., Getz M. J. Specific stimulation of actin gene transcription by epidermal growth factor and cycloheximide. Proc Natl Acad Sci U S A. 1984 Dec;81(23):7476–7480. doi: 10.1073/pnas.81.23.7476. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Erba H. P., Eddy R., Shows T., Kedes L., Gunning P. Structure, chromosome location, and expression of the human gamma-actin gene: differential evolution, location, and expression of the cytoskeletal beta- and gamma-actin genes. Mol Cell Biol. 1988 Apr;8(4):1775–1789. doi: 10.1128/mcb.8.4.1775. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Fried M., Crothers D. M. Equilibria and kinetics of lac repressor-operator interactions by polyacrylamide gel electrophoresis. Nucleic Acids Res. 1981 Dec 11;9(23):6505–6525. doi: 10.1093/nar/9.23.6505. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Garner M. M., Revzin A. A gel electrophoresis method for quantifying the binding of proteins to specific DNA regions: application to components of the Escherichia coli lactose operon regulatory system. Nucleic Acids Res. 1981 Jul 10;9(13):3047–3060. doi: 10.1093/nar/9.13.3047. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Graves B. J., Johnson P. F., McKnight S. L. Homologous recognition of a promoter domain common to the MSV LTR and the HSV tk gene. Cell. 1986 Feb 28;44(4):565–576. doi: 10.1016/0092-8674(86)90266-7. [DOI] [PubMed] [Google Scholar]
  13. Greenberg M. E., Greene L. A., Ziff E. B. Nerve growth factor and epidermal growth factor induce rapid transient changes in proto-oncogene transcription in PC12 cells. J Biol Chem. 1985 Nov 15;260(26):14101–14110. [PubMed] [Google Scholar]
  14. Greenberg M. E., Siegfried Z., Ziff E. B. Mutation of the c-fos gene dyad symmetry element inhibits serum inducibility of transcription in vivo and the nuclear regulatory factor binding in vitro. Mol Cell Biol. 1987 Mar;7(3):1217–1225. doi: 10.1128/mcb.7.3.1217. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Greenberg M. E., Ziff E. B. Stimulation of 3T3 cells induces transcription of the c-fos proto-oncogene. Nature. 1984 Oct 4;311(5985):433–438. doi: 10.1038/311433a0. [DOI] [PubMed] [Google Scholar]
  16. 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]
  17. Gronostajski R. M. Analysis of nuclear factor I binding to DNA using degenerate oligonucleotides. Nucleic Acids Res. 1986 Nov 25;14(22):9117–9132. doi: 10.1093/nar/14.22.9117. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Gronostajski R. M., Knox J., Berry D., Miyamoto N. G. Stimulation of transcription in vitro by binding sites for nuclear factor I. Nucleic Acids Res. 1988 Mar 25;16(5):2087–2098. doi: 10.1093/nar/16.5.2087. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. 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]
  20. Jorgensen R. A., Rothstein S. J., Reznikoff W. S. A restriction enzyme cleavage map of Tn5 and location of a region encoding neomycin resistance. Mol Gen Genet. 1979;177(1):65–72. doi: 10.1007/BF00267254. [DOI] [PubMed] [Google Scholar]
  21. Kawamoto T., Makino K., Niwa H., Sugiyama H., Kimura S., Amemura M., Nakata A., Kakunaga T. Identification of the human beta-actin enhancer and its binding factor. Mol Cell Biol. 1988 Jan;8(1):267–272. doi: 10.1128/mcb.8.1.267. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kost T. A., Theodorakis N., Hughes S. H. The nucleotide sequence of the chick cytoplasmic beta-actin gene. Nucleic Acids Res. 1983 Dec 10;11(23):8287–8301. doi: 10.1093/nar/11.23.8287. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Kost T. A., Theodorakis N., Hughes S. H. The nucleotide sequence of the chick cytoplasmic beta-actin gene. Nucleic Acids Res. 1983 Dec 10;11(23):8287–8301. doi: 10.1093/nar/11.23.8287. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Lazarides E., Revel J. P. The molecular basis of cell movement. Sci Am. 1979 May;240(5):100–113. doi: 10.1038/scientificamerican0579-100. [DOI] [PubMed] [Google Scholar]
  25. Lennard A. C., Egly J. M. The bidirectional upstream element of the adenovirus-2 major late promoter binds a single monomeric molecule of the upstream factor. EMBO J. 1987 Oct;6(10):3027–3034. doi: 10.1002/j.1460-2075.1987.tb02608.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Lohse P., Arnold H. H. The down-regulation of the chicken cytoplasmic beta actin during myogenic differentiation does not require the gene promoter but involves the 3' end of the gene. Nucleic Acids Res. 1988 Apr 11;16(7):2787–2803. doi: 10.1093/nar/16.7.2787. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Maniatis T., Goodbourn S., Fischer J. A. Regulation of inducible and tissue-specific gene expression. Science. 1987 Jun 5;236(4806):1237–1245. doi: 10.1126/science.3296191. [DOI] [PubMed] [Google Scholar]
  28. 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]
  29. 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]
  30. Miyamoto N. G., Moncollin V., Wintzerith M., Hen R., Egly J. M., Chambon P. Stimulation of in vitro transcription by the upstream element of the adenovirus-2 major late promoter involves a specific factor. Nucleic Acids Res. 1984 Dec 11;12(23):8779–8799. doi: 10.1093/nar/12.23.8779. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Miyamoto N. G. Nucleotide sequence of the human beta-actin promoter 5' flanking region. Nucleic Acids Res. 1987 Nov 11;15(21):9095–9095. doi: 10.1093/nar/15.21.9095. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Mohun T., Garrett N., Treisman R. Xenopus cytoskeletal actin and human c-fos gene promoters share a conserved protein-binding site. EMBO J. 1987 Mar;6(3):667–673. doi: 10.1002/j.1460-2075.1987.tb04806.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Nagata K., Guggenheimer R. A., Enomoto T., Lichy J. H., Hurwitz J. Adenovirus DNA replication in vitro: identification of a host factor that stimulates synthesis of the preterminal protein-dCMP complex. Proc Natl Acad Sci U S A. 1982 Nov;79(21):6438–6442. doi: 10.1073/pnas.79.21.6438. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Nakajima-Iijima S., Hamada H., Reddy P., Kakunaga T. Molecular structure of the human cytoplasmic beta-actin gene: interspecies homology of sequences in the introns. Proc Natl Acad Sci U S A. 1985 Sep;82(18):6133–6137. doi: 10.1073/pnas.82.18.6133. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Ng S. Y., Gunning P., Eddy R., Ponte P., Leavitt J., Shows T., Kedes L. Evolution of the functional human beta-actin gene and its multi-pseudogene family: conservation of noncoding regions and chromosomal dispersion of pseudogenes. Mol Cell Biol. 1985 Oct;5(10):2720–2732. doi: 10.1128/mcb.5.10.2720. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Nudel U., Zakut R., Shani M., Neuman S., Levy Z., Yaffe D. The nucleotide sequence of the rat cytoplasmic beta-actin gene. Nucleic Acids Res. 1983 Mar 25;11(6):1759–1771. doi: 10.1093/nar/11.6.1759. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Otey C. A., Kalnoski M. H., Bulinski J. C. Identification and quantification of actin isoforms in vertebrate cells and tissues. J Cell Biochem. 1987 Jun;34(2):113–124. doi: 10.1002/jcb.240340205. [DOI] [PubMed] [Google Scholar]
  38. Prywes R., Roeder R. G. Purification of the c-fos enhancer-binding protein. Mol Cell Biol. 1987 Oct;7(10):3482–3489. doi: 10.1128/mcb.7.10.3482. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Santoro C., Mermod N., Andrews P. C., Tjian R. A family of human CCAAT-box-binding proteins active in transcription and DNA replication: cloning and expression of multiple cDNAs. Nature. 1988 Jul 21;334(6179):218–224. doi: 10.1038/334218a0. [DOI] [PubMed] [Google Scholar]
  40. 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]
  41. Shani M., Zevin-Sonkin D., Saxel O., Carmon Y., Katcoff D., Nudel U., Yaffe D. The correlation between the synthesis of skeletal muscle actin, myosin heavy chain, and myosin light chain and the accumulation of corresponding mRNA sequences during myogenesis. Dev Biol. 1981 Sep;86(2):483–492. doi: 10.1016/0012-1606(81)90206-2. [DOI] [PubMed] [Google Scholar]
  42. Treisman R. Identification and purification of a polypeptide that binds to the c-fos serum response element. EMBO J. 1987 Sep;6(9):2711–2717. doi: 10.1002/j.1460-2075.1987.tb02564.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Treisman R. Identification of a protein-binding site that mediates transcriptional response of the c-fos gene to serum factors. Cell. 1986 Aug 15;46(4):567–574. doi: 10.1016/0092-8674(86)90882-2. [DOI] [PubMed] [Google Scholar]
  44. Treisman R. Transient accumulation of c-fos RNA following serum stimulation requires a conserved 5' element and c-fos 3' sequences. Cell. 1985 Oct;42(3):889–902. doi: 10.1016/0092-8674(85)90285-5. [DOI] [PubMed] [Google Scholar]
  45. Vandekerckhove J., Weber K. Actin typing on total cellular extracts: a highly sensitive protein-chemical procedure able to distinguish different actins. Eur J Biochem. 1981 Jan;113(3):595–603. doi: 10.1111/j.1432-1033.1981.tb05104.x. [DOI] [PubMed] [Google Scholar]
  46. Wasylyk B., Wasylyk C., Chambon P. Short and long range activation by the SV40 enhancer. Nucleic Acids Res. 1984 Jul 25;12(14):5589–5608. doi: 10.1093/nar/12.14.5589. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Wildeman A. G., Sassone-Corsi P., Grundström T., Zenke M., Chambon P. Stimulation of in vitro transcription from the SV40 early promoter by the enhancer involves a specific trans-acting factor. EMBO J. 1984 Dec 20;3(13):3129–3133. doi: 10.1002/j.1460-2075.1984.tb02269.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]
  49. Zenke M., Grundström T., Matthes H., Wintzerith M., Schatz C., Wildeman A., Chambon P. Multiple sequence motifs are involved in SV40 enhancer function. EMBO J. 1986 Feb;5(2):387–397. doi: 10.1002/j.1460-2075.1986.tb04224.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. van Ooyen A., van den Berg J., Mantei N., Weissmann C. Comparison of total sequence of a cloned rabbit beta-globin gene and its flanking regions with a homologous mouse sequence. Science. 1979 Oct 19;206(4416):337–344. doi: 10.1126/science.482942. [DOI] [PubMed] [Google Scholar]
  51. van Straaten F., Müller R., Curran T., Van Beveren C., Verma I. M. Complete nucleotide sequence of a human c-onc gene: deduced amino acid sequence of the human c-fos protein. Proc Natl Acad Sci U S A. 1983 Jun;80(11):3183–3187. doi: 10.1073/pnas.80.11.3183. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES