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. 1990 Jul 11;18(13):3975–3982. doi: 10.1093/nar/18.13.3975

CpG methylation inhibits proenkephalin gene expression and binding of the transcription factor AP-2.

M Comb 1, H M Goodman 1
PMCID: PMC331101  PMID: 1695733

Abstract

DNA methylation at HpaII (CmCGG) sites inhibits expression of a human proenkephalin-CAT fusion gene when it is transiently expressed in CV-1 cells or stably expressed in C6-glioma cells. The inhibitory effects of HpaII methylation have been mapped to a site within the human proenkephalin promoter located at position -72 relative to the start site of transcription. This region spans a cAMP and phorbol ester inducible enhancer and methylation at this position inhibits both basal transcription and transcription induced by either cAMP or TPA. The HpaII site is located within an element which binds the transcription factor AP-2. In vitro methylation at this HpaII site inhibits the binding of AP-2. These results suggest that CpG methylation inhibits proenkephalin gene expression by directly interfering with the binding of a positively acting transcription factor previously shown to be essential for maximal basal, cAMP, and TPA inducible transcription.

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Selected References

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

  1. Becker P. B., Ruppert S., Schütz G. Genomic footprinting reveals cell type-specific DNA binding of ubiquitous factors. Cell. 1987 Nov 6;51(3):435–443. doi: 10.1016/0092-8674(87)90639-8. [DOI] [PubMed] [Google Scholar]
  2. Ben-Hattar J., Beard P., Jiricny J. Cytosine methylation in CTF and Sp1 recognition sites of an HSV tk promoter: effects on transcription in vivo and on factor binding in vitro. Nucleic Acids Res. 1989 Dec 25;17(24):10179–10190. doi: 10.1093/nar/17.24.10179. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Ben-Hattar J., Jiricny J. Methylation of single CpG dinucleotides within a promoter element of the Herpes simplex virus tk gene reduces its transcription in vivo. Gene. 1988 May 30;65(2):219–227. doi: 10.1016/0378-1119(88)90458-1. [DOI] [PubMed] [Google Scholar]
  4. Bird A. P. CpG-rich islands and the function of DNA methylation. Nature. 1986 May 15;321(6067):209–213. doi: 10.1038/321209a0. [DOI] [PubMed] [Google Scholar]
  5. Bird A., Taggart M., Frommer M., Miller O. J., Macleod D. A fraction of the mouse genome that is derived from islands of nonmethylated, CpG-rich DNA. Cell. 1985 Jan;40(1):91–99. doi: 10.1016/0092-8674(85)90312-5. [DOI] [PubMed] [Google Scholar]
  6. Busslinger M., Hurst J., Flavell R. A. DNA methylation and the regulation of globin gene expression. Cell. 1983 Aug;34(1):197–206. doi: 10.1016/0092-8674(83)90150-2. [DOI] [PubMed] [Google Scholar]
  7. Cedar H. DNA methylation and gene activity. Cell. 1988 Apr 8;53(1):3–4. doi: 10.1016/0092-8674(88)90479-5. [DOI] [PubMed] [Google Scholar]
  8. Comb M., Birnberg N. C., Seasholtz A., Herbert E., Goodman H. M. A cyclic AMP- and phorbol ester-inducible DNA element. 1986 Sep 25-Oct 1Nature. 323(6086):353–356. doi: 10.1038/323353a0. [DOI] [PubMed] [Google Scholar]
  9. Comb M., Mermod N., Hyman S. E., Pearlberg J., Ross M. E., Goodman H. M. Proteins bound at adjacent DNA elements act synergistically to regulate human proenkephalin cAMP inducible transcription. EMBO J. 1988 Dec 1;7(12):3793–3805. doi: 10.1002/j.1460-2075.1988.tb03264.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Comb M., Rosen H., Seeburg P., Adelman J., Herbert E. Primary structure of the human proenkephalin gene. DNA. 1983;2(3):213–229. doi: 10.1089/dna.1983.2.213. [DOI] [PubMed] [Google Scholar]
  11. Dignam J. D., Lebovitz R. M., Roeder R. G. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res. 1983 Mar 11;11(5):1475–1489. doi: 10.1093/nar/11.5.1475. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. 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]
  13. Ehrlich M., Wang R. Y. 5-Methylcytosine in eukaryotic DNA. Science. 1981 Jun 19;212(4501):1350–1357. doi: 10.1126/science.6262918. [DOI] [PubMed] [Google Scholar]
  14. Gorman C., Padmanabhan R., Howard B. H. High efficiency DNA-mediated transformation of primate cells. Science. 1983 Aug 5;221(4610):551–553. doi: 10.1126/science.6306768. [DOI] [PubMed] [Google Scholar]
  15. Harrington M. A., Jones P. A., Imagawa M., Karin M. Cytosine methylation does not affect binding of transcription factor Sp1. Proc Natl Acad Sci U S A. 1988 Apr;85(7):2066–2070. doi: 10.1073/pnas.85.7.2066. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hyman S. E., Comb M., Lin Y. S., Pearlberg J., Green M. R., Goodman H. M. A common trans-acting factor is involved in transcriptional regulation of neurotransmitter genes by cyclic AMP. Mol Cell Biol. 1988 Oct;8(10):4225–4233. doi: 10.1128/mcb.8.10.4225. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hyman S. E., Comb M., Pearlberg J., Goodman H. M. An AP-2 element acts synergistically with the cyclic AMP- and phorbol ester-inducible enhancer of the human proenkephalin gene. Mol Cell Biol. 1989 Jan;9(1):321–324. doi: 10.1128/mcb.9.1.321. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Iguchi-Ariga S. M., Schaffner W. CpG methylation of the cAMP-responsive enhancer/promoter sequence TGACGTCA abolishes specific factor binding as well as transcriptional activation. Genes Dev. 1989 May;3(5):612–619. doi: 10.1101/gad.3.5.612. [DOI] [PubMed] [Google Scholar]
  19. Imagawa M., Chiu R., Karin M. Transcription factor AP-2 mediates induction by two different signal-transduction pathways: protein kinase C and cAMP. Cell. 1987 Oct 23;51(2):251–260. doi: 10.1016/0092-8674(87)90152-8. [DOI] [PubMed] [Google Scholar]
  20. Kadonaga J. T., Tjian R. Affinity purification of sequence-specific DNA binding proteins. Proc Natl Acad Sci U S A. 1986 Aug;83(16):5889–5893. doi: 10.1073/pnas.83.16.5889. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kelley D. E., Pollok B. A., Atchison M. L., Perry R. P. The coupling between enhancer activity and hypomethylation of kappa immunoglobulin genes is developmentally regulated. Mol Cell Biol. 1988 Feb;8(2):930–937. doi: 10.1128/mcb.8.2.930. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Keshet I., Lieman-Hurwitz J., Cedar H. DNA methylation affects the formation of active chromatin. Cell. 1986 Feb 28;44(4):535–543. doi: 10.1016/0092-8674(86)90263-1. [DOI] [PubMed] [Google Scholar]
  23. Kovesdi I., Reichel R., Nevins J. R. Role of an adenovirus E2 promoter binding factor in E1A-mediated coordinate gene control. Proc Natl Acad Sci U S A. 1987 Apr;84(8):2180–2184. doi: 10.1073/pnas.84.8.2180. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kruczek I., Doerfler W. Expression of the chloramphenicol acetyltransferase gene in mammalian cells under the control of adenovirus type 12 promoters: effect of promoter methylation on gene expression. Proc Natl Acad Sci U S A. 1983 Dec;80(24):7586–7590. doi: 10.1073/pnas.80.24.7586. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Langner K. D., Vardimon L., Renz D., Doerfler W. DNA methylation of three 5' C-C-G-G 3' sites in the promoter and 5' region inactivate the E2a gene of adenovirus type 2. Proc Natl Acad Sci U S A. 1984 May;81(10):2950–2954. doi: 10.1073/pnas.81.10.2950. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Langner K. D., Weyer U., Doerfler W. Trans effect of the E1 region of adenoviruses on the expression of a prokaryotic gene in mammalian cells: resistance to 5' -CCGG- 3' methylation. Proc Natl Acad Sci U S A. 1986 Mar;83(6):1598–1602. doi: 10.1073/pnas.83.6.1598. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Lee K. A., Hai T. Y., SivaRaman L., Thimmappaya B., Hurst H. C., Jones N. C., Green M. R. A cellular protein, activating transcription factor, activates transcription of multiple E1A-inducible adenovirus early promoters. Proc Natl Acad Sci U S A. 1987 Dec;84(23):8355–8359. doi: 10.1073/pnas.84.23.8355. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. 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]
  29. Mitchell P. J., Wang C., Tjian R. Positive and negative regulation of transcription in vitro: enhancer-binding protein AP-2 is inhibited by SV40 T antigen. Cell. 1987 Sep 11;50(6):847–861. doi: 10.1016/0092-8674(87)90512-5. [DOI] [PubMed] [Google Scholar]
  30. Murray E. J., Grosveld F. Site specific demethylation in the promoter of human gamma-globin gene does not alleviate methylation mediated suppression. EMBO J. 1987 Aug;6(8):2329–2335. doi: 10.1002/j.1460-2075.1987.tb02508.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Razin A., Riggs A. D. DNA methylation and gene function. Science. 1980 Nov 7;210(4470):604–610. doi: 10.1126/science.6254144. [DOI] [PubMed] [Google Scholar]
  32. Razin A., Szyf M., Kafri T., Roll M., Giloh H., Scarpa S., Carotti D., Cantoni G. L. Replacement of 5-methylcytosine by cytosine: a possible mechanism for transient DNA demethylation during differentiation. Proc Natl Acad Sci U S A. 1986 May;83(9):2827–2831. doi: 10.1073/pnas.83.9.2827. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Saluz H. P., Jiricny J., Jost J. P. Genomic sequencing reveals a positive correlation between the kinetics of strand-specific DNA demethylation of the overlapping estradiol/glucocorticoid-receptor binding sites and the rate of avian vitellogenin mRNA synthesis. Proc Natl Acad Sci U S A. 1986 Oct;83(19):7167–7171. doi: 10.1073/pnas.83.19.7167. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Seed B., Sheen J. Y. A simple phase-extraction assay for chloramphenicol acyltransferase activity. Gene. 1988 Jul 30;67(2):271–277. doi: 10.1016/0378-1119(88)90403-9. [DOI] [PubMed] [Google Scholar]
  35. Sen R., Baltimore D. Multiple nuclear factors interact with the immunoglobulin enhancer sequences. Cell. 1986 Aug 29;46(5):705–716. doi: 10.1016/0092-8674(86)90346-6. [DOI] [PubMed] [Google Scholar]
  36. Shimada T., Inokuchi K., Nienhuis A. W. Site-specific demethylation and normal chromatin structure of the human dihydrofolate reductase gene promoter after transfection into CHO cells. Mol Cell Biol. 1987 Aug;7(8):2830–2837. doi: 10.1128/mcb.7.8.2830. [DOI] [PMC free article] [PubMed] [Google Scholar]

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