Promoter-specific activation and demethylation by MBD2/demethylase.
MBD2 is the only member of a family of methyl-CpG-binding proteins that 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 chloramphenicol acetyltransferase (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 h, while pGL2T+I4xTBRE expression was increased by 2-3-fold after only 48 h and did not further increase at 96 h. Gene activation was not universal because 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 coordinate a program that requires suppression of some methylated genes and activation of others.
Detich
Nancy
N
Department of Pharmacology and Therapeutics, McGill University, Montreal, Quebec H3G 1Y6, Canada.
Theberge
Johanne
J
Szyf
Moshe
M
D001483:Base Sequence; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000378:metabolism; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins / Q000378:metabolism; D019175:DNA Methylation; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D017930:Genes, Reporter; D005822:Genetic Vectors; D006801:Humans; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000737:chemistry; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000378:metabolism; D013447:Sulfites / Q000494:pharmacology; D013997:Time Factors; D014158:Transcription, Genetic; D014162:Transfection
C493956:CDKN1A protein, human; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D004268:DNA-Binding Proteins; C115368:MBD2 protein; D009841:Oligonucleotides; D011994:Recombinant Proteins; D013447:Sulfites; D015500:Chloramphenicol O-Acetyltransferase