Skip to main content
NCBI home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1995 Oct;15(10):5355–5362. doi: 10.1128/mcb.15.10.5355

A DNA methylation site in the male-specific P450 (Cyp 2d-9) promoter and binding of the heteromeric transcription factor GABP.

N Yokomori 1, R Kobayashi 1, R Moore 1, T Sueyoshi 1, M Negishi 1
PMCID: PMC230784  PMID: 7565685

Abstract

The Cyp 2d-9 gene encodes the male-specific steroid 16 alpha-hydroxylase in mouse liver and shares a conserved regulatory element (-100TTCCGGGC-93) with another male-specific Slp promoter. As shown with the Slp promoter (N. Yokomori, R. Moore, and M. Negishi, Proc. Natl. Acad. Sci. USA 92:1302-1306, 1995), the male-preferential demethylation also occurs at CpG/-97 in the Cyp 2d-9 promoter. The transcription factor which specifically binds to the demethylated element has been purified. The peptide sequences reveal that the factor consists of GABP alpha and GABP beta 1 with Ets and Notch motifs, respectively. Both DNase I footprinting and gel shift assays indicate that the bacterially expressed glutathione S-transferase-GABP fusion proteins bind to the regulatory element only when CpG/-97 is demethylated. Moreover, Cyp 2d-9 promoter is trans-activated by coexpression of GABP proteins in HepG2 cells. Given the additional results that CpG/-50 of the female-specific steroid 15 alpha-hydroxylase (Cyp 2a-4) promoter is preferentially demethylated in the females, the sex-specific expressions of the P450 genes correlate very well with DNA demethylation. We also conclude that GABP is a methylation-sensitive transcription factor and is a potential transcription activator of the male-specific Cyp 2d-9 promoter.

Full Text

The Full Text of this article is available as a PDF (572.2 KB).

Selected References

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

  1. Aguado F., Rodrigo J., Cacicedo L., Mellström B. Distribution of insulin-like growth factor-I receptor mRNA in rat brain. Regulation in the hypothalamo-neurohypophysial system. J Mol Endocrinol. 1993 Oct;11(2):231–239. doi: 10.1677/jme.0.0110231. [DOI] [PubMed] [Google Scholar]
  2. Bird A. The essentials of DNA methylation. Cell. 1992 Jul 10;70(1):5–8. doi: 10.1016/0092-8674(92)90526-i. [DOI] [PubMed] [Google Scholar]
  3. Bolwig G. M., Bruder J. T., Hearing P. Different binding site requirements for binding and activation for the bipartite enhancer factor EF-1A. Nucleic Acids Res. 1992 Dec 25;20(24):6555–6564. doi: 10.1093/nar/20.24.6555. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Brown T. A., McKnight S. L. Specificities of protein-protein and protein-DNA interaction of GABP alpha and two newly defined ets-related proteins. Genes Dev. 1992 Dec;6(12B):2502–2512. doi: 10.1101/gad.6.12b.2502. [DOI] [PubMed] [Google Scholar]
  5. Carter R. S., Avadhani N. G. Cooperative binding of GA-binding protein transcription factors to duplicated transcription initiation region repeats of the cytochrome c oxidase subunit IV gene. J Biol Chem. 1994 Feb 11;269(6):4381–4387. [PubMed] [Google Scholar]
  6. Comb M., Goodman H. M. CpG methylation inhibits proenkephalin gene expression and binding of the transcription factor AP-2. Nucleic Acids Res. 1990 Jul 11;18(13):3975–3982. doi: 10.1093/nar/18.13.3975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Dannan G. A., Guengerich F. P., Waxman D. J. Hormonal regulation of rat liver microsomal enzymes. Role of gonadal steroids in programming, maintenance, and suppression of delta 4-steroid 5 alpha-reductase, flavin-containing monooxygenase, and sex-specific cytochromes P-450. J Biol Chem. 1986 Aug 15;261(23):10728–10735. [PubMed] [Google Scholar]
  8. Davis R. L., Weintraub H., Lassar A. B. Expression of a single transfected cDNA converts fibroblasts to myoblasts. Cell. 1987 Dec 24;51(6):987–1000. doi: 10.1016/0092-8674(87)90585-x. [DOI] [PubMed] [Google Scholar]
  9. Frommer M., McDonald L. E., Millar D. S., Collis C. M., Watt F., Grigg G. W., Molloy P. L., Paul C. L. A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands. Proc Natl Acad Sci U S A. 1992 Mar 1;89(5):1827–1831. doi: 10.1073/pnas.89.5.1827. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Genuario R. R., Kelley D. E., Perry R. P. Comparative utilization of transcription factor GABP by the promoters of ribosomal protein genes rpL30 and rpL32. Gene Expr. 1993;3(3):279–288. [PMC free article] [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. Gorski K., Carneiro M., Schibler U. Tissue-specific in vitro transcription from the mouse albumin promoter. Cell. 1986 Dec 5;47(5):767–776. doi: 10.1016/0092-8674(86)90519-2. [DOI] [PubMed] [Google Scholar]
  13. Gustafsson J. A., Mode A., Norstedt G., Skett P. Sex steroid induced changes in hepatic enzymes. Annu Rev Physiol. 1983;45:51–60. doi: 10.1146/annurev.ph.45.030183.000411. [DOI] [PubMed] [Google Scholar]
  14. Gustafsson J. A., Stenberg A. On the obligatory role of the hypophysis in sexual differentiation hepatic metabolism in rats. Proc Natl Acad Sci U S A. 1976 May;73(5):1462–1465. doi: 10.1073/pnas.73.5.1462. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. 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]
  16. Klages S., Möllers B., Renkawitz R. The involvement of demethylation in the myeloid-specific function of the mouse M lysozyme gene downstream enhancer. Nucleic Acids Res. 1992 Apr 25;20(8):1925–1932. doi: 10.1093/nar/20.8.1925. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. LaMarco K., Thompson C. C., Byers B. P., Walton E. M., McKnight S. L. Identification of Ets- and notch-related subunits in GA binding protein. Science. 1991 Aug 16;253(5021):789–792. doi: 10.1126/science.1876836. [DOI] [PubMed] [Google Scholar]
  18. Li E., Beard C., Jaenisch R. Role for DNA methylation in genomic imprinting. Nature. 1993 Nov 25;366(6453):362–365. doi: 10.1038/366362a0. [DOI] [PubMed] [Google Scholar]
  19. Lindberg R., Burkhart B., Ichikawa T., Negishi M. The structure and characterization of type I P-450(15) alpha gene as major steroid 15 alpha-hydroxylase and its comparison with type II P-450(15) alpha gene. J Biol Chem. 1989 Apr 15;264(11):6465–6471. [PubMed] [Google Scholar]
  20. Maxam A. M., Gilbert W. A new method for sequencing DNA. Proc Natl Acad Sci U S A. 1977 Feb;74(2):560–564. doi: 10.1073/pnas.74.2.560. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Mode A., Norstedt G., Simic B., Eneroth P., Gustafsson J. A. Continuous infusion of growth hormone feminizes hepatic steroid metabolism in the rat. Endocrinology. 1981 Jun;108(6):2103–2108. doi: 10.1210/endo-108-6-2103. [DOI] [PubMed] [Google Scholar]
  22. Nonaka M., Kimura H., Yeul Y. D., Yokoyama S., Nakayama K., Takahashi M. Identification of the 5'-flanking regulatory region responsible for the difference in transcriptional control between mouse complement C4 and Slp genes. Proc Natl Acad Sci U S A. 1986 Oct;83(20):7883–7887. doi: 10.1073/pnas.83.20.7883. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Noshiro M., Negishi M. Pretranslational regulation of sex-dependent testosterone hydroxylases by growth hormone in mouse liver. J Biol Chem. 1986 Dec 5;261(34):15923–15927. [PubMed] [Google Scholar]
  24. Ström A., Eguchi H., Mode A., Legraverend C., Tollet P., Strömstedt P. E., Gustafsson J. A. Characterization of the proximal promoter and two silencer elements in the CYP2C11 gene expressed in rat liver. DNA Cell Biol. 1994 Aug;13(8):805–819. doi: 10.1089/dna.1994.13.805. [DOI] [PubMed] [Google Scholar]
  25. Sundseth S. S., Alberta J. A., Waxman D. J. Sex-specific, growth hormone-regulated transcription of the cytochrome P450 2C11 and 2C12 genes. J Biol Chem. 1992 Feb 25;267(6):3907–3914. [PubMed] [Google Scholar]
  26. Tate P. H., Bird A. P. Effects of DNA methylation on DNA-binding proteins and gene expression. Curr Opin Genet Dev. 1993 Apr;3(2):226–231. doi: 10.1016/0959-437x(93)90027-m. [DOI] [PubMed] [Google Scholar]
  27. Thompson C. C., Brown T. A., McKnight S. L. Convergence of Ets- and notch-related structural motifs in a heteromeric DNA binding complex. Science. 1991 Aug 16;253(5021):762–768. doi: 10.1126/science.1876833. [DOI] [PubMed] [Google Scholar]
  28. Tilghman S. M. DNA methylation: a phoenix rises. Proc Natl Acad Sci U S A. 1993 Oct 1;90(19):8761–8762. doi: 10.1073/pnas.90.19.8761. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Watanabe H., Sawada J., Yano K., Yamaguchi K., Goto M., Handa H. cDNA cloning of transcription factor E4TF1 subunits with Ets and notch motifs. Mol Cell Biol. 1993 Mar;13(3):1385–1391. doi: 10.1128/mcb.13.3.1385. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Watanabe H., Wada T., Handa H. Transcription factor E4TF1 contains two subunits with different functions. EMBO J. 1990 Mar;9(3):841–847. doi: 10.1002/j.1460-2075.1990.tb08181.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Wong G., Itakura T., Kawajiri K., Skow L., Negishi M. Gene family of male-specific testosterone 16 alpha-hydroxylase (C-P-450(16 alpha)) in mice. Organization, differential regulation, and chromosome localization. J Biol Chem. 1989 Feb 15;264(5):2920–2927. [PubMed] [Google Scholar]
  32. Wong G., Kawajiri K., Negishi M. Gene family of male-specific testosterone 16 alpha-hydroxylase (C-P-450(16) alpha) in mouse liver: cDNA sequences, neonatal imprinting, and reversible regulation by androgen. Biochemistry. 1987 Dec 29;26(26):8683–8690. doi: 10.1021/bi00400a029. [DOI] [PubMed] [Google Scholar]
  33. Wu J., Issa J. P., Herman J., Bassett D. E., Jr, Nelkin B. D., Baylin S. B. Expression of an exogenous eukaryotic DNA methyltransferase gene induces transformation of NIH 3T3 cells. Proc Natl Acad Sci U S A. 1993 Oct 1;90(19):8891–8895. doi: 10.1073/pnas.90.19.8891. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. YATES F. E., HERBST A. L., URQUHART J. Sex difference in rate of ring A reduction of delta 4-3-keto-steroids in vitro by rat liver. Endocrinology. 1958 Dec;63(6):887–902. doi: 10.1210/endo-63-6-887. [DOI] [PubMed] [Google Scholar]
  35. Yokomori N., Moore R., Negishi M. Sexually dimorphic DNA demethylation in the promoter of the Slp (sex-limited protein) gene in mouse liver. Proc Natl Acad Sci U S A. 1995 Feb 28;92(5):1302–1306. doi: 10.1073/pnas.92.5.1302. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Yoshioka H., Lang M., Wong G., Negishi M. A specific cis-acting element regulates in vitro transcription of sex-dependent mouse steroid 16 alpha-hydroxylase (C-P450(16 alpha)) gene. J Biol Chem. 1990 Aug 25;265(24):14612–14617. [PubMed] [Google Scholar]
  37. Zaphiropoulos P. G., Mode A., Norstedt G., Gustafsson J. A. Regulation of sexual differentiation in drug and steroid metabolism. Trends Pharmacol Sci. 1989 Apr;10(4):149–153. doi: 10.1016/0165-6147(89)90167-3. [DOI] [PubMed] [Google Scholar]

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

RESOURCES