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Nucleic Acids Research, 1989, Vol. 17, No. 13 5135-5148
© 1989

MOLECULAR BIOLOGY

In vitro DNA cytosine roethytation of cis-regulatory elements modulates c-Ha-ras promoter activity in vivo

Mack J. Rachal, Heahyun Yoo, Frederick F. Becker and Jean-Numa Lapeyre*

Department of Molecular Pathology, Section of Experimental Pathology, The University of Texas M.D.Anderson Cancer Center 1515 Holcombe Boulevard, Houston, TX 77030, USA

*To whom corresspondance to be addressed

Received March 1, 1989. Revised June 4, 1989. Accepted June 1, 1989.

The effect of DNA cytosine methylation on promoter activity was assessed using a transient expression system employing pHrasCAT. This 551 bp Ha-ras-1 gene promoter region is enriched with 84 CpG dinucleotides, six functional GC boxes, and is prototypic of many genes possessing CpG islands in their promoter regions. Bacteria] modification enzymes Hhal methyl transferase (MTase) and Hpall MTase, alone or in combination with a human placenta] DNA methyltransferase (HP MTase) that methylates CpG sites in a generalized manner, including asymmetric elements such as GC box CpG's, were used to methylate at different types of sites in the promoter. Methylation of Hhal and Hpall sites reduced CAT expression by –70% –80%, whereas methylation at generalized CpG sites with HP MTase inactivated the promoter by <95%. The inhibition of H-ras promoter activity was not attributable to methylation-induced differences in DNA uptake or stability in the cell, topological form of the plasmid, or methylation effects in non-promoter regions.


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