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Papers In Press, published online ahead of print August 12, 2002
J. Biol. Chem, 10.1074/jbc.C200408200
Submitted on July 15, 2002
Revised on August 12, 2002
Accepted on August 12, 2002
Pharmacology Dept., McGill University, Montreal, Quebec H3G 1Y6
Corresponding Author: mszyf{at}pharma.mcgill.ca
MBD2 is the only member of a family of methyl CpG binding proteins which has been reported to be both a transcriptional repressor and a DNA demethylase (dMTase). To understand the apparently contradictory function of MBD2/dMTase, we studied the effects of dMTase overexpression on the activity of various in vitro methylated promoters transiently transfected into HEK293 cells. We found that forced expression of a MBD2/dMTase expression vector (His-dMTase) differentially activated two methylated reporters, pSV40-CAT (the SV40 enhancerless promoter adjacent to the CAT reporter gene), and pGL2T+I4xTBRE (a region of the p21 promoter next to the luciferase reporter gene) in a time- and dose-dependent manner. His-dMTase increased pSV40-CAT expression by 3-10 fold after 96 hours, while pGL2T+I4xTBRE expression was increased by 2-3 fold after only 48 hours, and did not further increase at 96 hours. Gene activation was not universal, since no effect was seen with the p19-ARF promoter. We then assessed whether activation might be due to demethylation within the promoter region. Using bisulfite mapping, we found that exogenous expression of His-dMTase induced demethylation at 8 of the 10 CpG sites within the SV40 promoter. The observation that His-dMTase increases the demethylase activity in the cells was also confirmed using an in vitro CpG demethylase assay, with a mC32pG oligonucleotide substrate and purified Q-Sepharose fractions from HEK293 cells transfected with His-dMTase or empty pcDNA3.1His vector. We propose that a single protein possessing both repressor and demethylase functions has evolved to co-ordinate a program that requires suppression of some methylated genes and activation of others.
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