Methylation of CpG Islands in the Rat 7-dehydrocholesterol Reductase Promoter Suppresses Transcriptional Activation

  • Kim, Jai-Hyun (Department of Biochemistry and Yonsei Proteome Research Center, Yonsei University) ;
  • Hwang, Eun-Ha (Department of Biochemistry and Yonsei Proteome Research Center, Yonsei University) ;
  • Park, Hye-Jung (Department of Biological Sciences and Bio/Molecular Informatics Center, Konkuk University) ;
  • Paik, Young-Ki (Department of Biochemistry and Yonsei Proteome Research Center, Yonsei University) ;
  • Shim, Yhong-Hee (Department of Biological Sciences and Bio/Molecular Informatics Center, Konkuk University)
  • Received : 2004.08.17
  • Accepted : 2005.01.13
  • Published : 2005.04.30

Abstract

In mammals, 7-dehydrocholesterol reductase (Dhcr7) is the terminal enzyme in cholesterol biosynthesis. We previously reported that the Dhcr7 proximal promoter (-179 to +1), which contains CpG islands, is responsible for sterol-mediated expression of the rat gene. In the present study, we examined whether methylation of this region affects the transcriptional activity of the Dhcr7 gene. In vitro DNA methylation of the Dhcr7 promoter and luciferase-reporter assays showed that DNA methylation of the CpG islands suppressed transcription. Furthermore, treatment of the methylated Dhcr7 promoter with the demethylating agent, 5-aza-2'-deoxycytidine (5-Aza-CdR), reversed the suppression of promoter activity. These results indicate that methylation of the CpG islands is an important transcriptional regulatory mechanism in the Dhcr7 promoter.

Keywords

7-Dehydrocholesterol Reductase;DNA Methylation;Transcriptional Activity

Acknowledgement

Supported by : Korea Science and Engineering Foundation

References

  1. Bae, S. H., Lee, J. N., Fitzky, B. U., Seong, J., and Paik, Y. K. (1999) Cholesterol biosynthesis from lanosterol: molecular cloning, tissue distribution, expression, chromosomal localization, and regulation of rat 7-dehydrocholesterol reductase, a Smith-Lemli-Opitz syndrome-related protein. J. Biol. Chem. 274, 14624-14631 https://doi.org/10.1074/jbc.274.21.14624
  2. Roder, K., Hung, M. S., Lee, T. L., Lin, T. Y., Xiao, H., et al. (2000) Transcriptional repression by Drosophila methyl- CpG-binding proteins. Mol. Cell. Biol. 20, 7401-7409 https://doi.org/10.1128/MCB.20.19.7401-7409.2000
  3. Smith, D. W., Lemli, L., and Opitz, J. M. (1964) A newly recognized syndrome of multiple congenital anomalies. J. Pedatt. 64, 210-217 https://doi.org/10.1016/S0022-3476(64)80264-X
  4. Kim, J. H., Lee, J. N., and Paik, Y. K. (2001) Cholesterol biosynthesis from lanosterol. A concerted role for Sp1 and NFY- binding sites for sterol-mediated regulation of rat 7- dehydrocholesterol reductase gene expression. J. Biol. Chem. 276, 18153-18160 https://doi.org/10.1074/jbc.M101661200
  5. Gaylor, J. L. (2002) Membrane-bound enzymes of cholesterol synthesis from lanosterol. Biochem. Biophys. Res. Commun. 292, 1139-1146 https://doi.org/10.1006/bbrc.2001.2008
  6. Lee, J. N., Bae, S. H., and Paik, Y. K. (2002) Structure and alternative splicing of the rat 7-dehydrocholesterol reductase gene. Biochim. Biophys. Acta 1576, 148-156 https://doi.org/10.1016/S0167-4781(02)00285-3
  7. Clark, S. J., Harrison, J., Paul, C. L., and Frommer, M. (1994) High sensitivity mapping of methylated cytosine. Nucleic Acids Res. 22, 2990-2997 https://doi.org/10.1093/nar/22.15.2990
  8. Ghoshal, K., Majumder, S., Dong, Z., Li, X., and Jacob, S. T. (2000) Suppression of metallothionein gene expression in a rat hepatoma because of promoter-specific DNA methylation. J. Biol. Chem. 275, 539-547 https://doi.org/10.1074/jbc.275.1.539
  9. Bae, S. H. and Paik, Y. K. (1997) Cholesterol biosynthesis from lanosterol: development of a novel assay method and characterization of rat liver microsomal lanosterol ${\Delta^{24}$-reductase. Biochem. J. 326, 609-616
  10. Bird, A. (1992) The essentials of DNA methylation. Cell 70, 5-8 https://doi.org/10.1016/0092-8674(92)90526-I
  11. Dehart, D. B., Lanoue, L., and Sulik, K. K. (1997) Pathogenesis of malformations in a rodent model for Smith-Lemli-Opitz syndrome. Am. J. Med. Genet. 68, 328-337 https://doi.org/10.1002/(SICI)1096-8628(19970131)68:3<328::AID-AJMG15>3.0.CO;2-V
  12. Irons, M., Elias, E. R., Tint, G. S., Salen, G., Frieden, R., et al. (1994) Abnormal cholesterol metabolism in the Smith-Lemli- Opitz syndrome: report of clinical and biochemical findings in four patients and treatment in one patient. Am. J. Med. Genet. 50, 347-352 https://doi.org/10.1002/ajmg.1320500409
  13. Cedar, H. (1988) DNA methylation and gene activity. Cell 53, 3-4 https://doi.org/10.1016/0092-8674(88)90479-5
  14. Harrington, M. A., Jones, P. A., Imagawa, M., and Karin, M. (1988) Cytosine methylation does not affect binding of transcription factor Sp1. Proc. Natl. Acad. Sci. USA 85, 2066- 2070
  15. Yoon, S. S., Kim H. J., Chung, D. H., and Kim, T. J. (2004) CD99 costimulation Up-Regulates T cell receptor-mediated activation of JNK and AP-1. Mol. Cells 18, 186-191
  16. Singal, R. and Ginder, G. D. (1999) DNA methylation. Blood 93, 4059-4070