Hypermethylation of metallothionein-I promoter and suppression of its induction in cell lines overexpressing the large subunit of Ku protein.
We have shown previously that the heavy metal-induced metallothionein-I (MT-I) gene expression is specifically repressed in a rat fibroblast cell line (Ku-80) overexpressing the 80-kDa subunit of Ku autoantigen but not in cell lines overexpressing the 70-kDa subunit or Ku heterodimer. Here, we explored the molecular mechanism of silencing of MT-I gene in Ku-80 cells. Genomic footprinting analysis revealed both basal and heavy metal-inducible binding at specific cis elements in the parental cell line (Rat-1). By contrast, MT-I promoter in Ku-80 cells was refractory to any transactivating factors, implying alteration of chromatin structure. Treatment of two clonal lines of Ku-80 cells with 5-azacytidine, a potent DNA demethylating agent, rendered MT-I gene inducible by heavy metals, suggesting that the gene is methylated in these cells. Bisulfite genomic sequencing revealed that all 21 CpG dinucleotides in MT-I immediate promoter were methylated in Ku-80 cells, whereas only four CpG dinucleotides were methylated in Rat-1 cells. Almost all methylated CpG dinucleotides were demethylated in Ku-80 cells after 5-azacytidine treatment. To our knowledge, this is the first report that describes hypermethylation of a specific gene promoter and its resultant silencing in response to overexpression of a cellular protein.
Majumder
S
S
Department of Medical Biochemistry, College of Medicine, The Ohio State University, Columbus, Ohio 43210, USA.
Ghoshal
K
K
Li
Z
Z
Jacob
S T
ST
D000818:Animals; D034961:Antigens, Nuclear; D001374:Azacitidine / Q000494:pharmacology; D001483:Base Sequence; D002460:Cell Line; D018899:CpG Islands / Q000235:genetics; D004247:DNA; D018983:DNA Footprinting; D004265:DNA Helicases; D019175:DNA Methylation; D004268:DNA-Binding Proteins / Q000235:genetics*; D015870:Gene Expression / Q000187:drug effects; D020868:Gene Silencing; D000072200:Ku Autoantigen; D008668:Metallothionein / Q000235:genetics*; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000235:genetics*; D011401:Promoter Regions, Genetic; D051381:Rats
D034961:Antigens, Nuclear; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D004247:DNA; D008668:Metallothionein; D004265:DNA Helicases; C106893:XRCC5 protein, human; C000606509:Xrcc6 protein, human; C000606511:Xrcc6 protein, rat; D000072200:Ku Autoantigen; D001374:Azacitidine