Aberrant promoter methylation of the ABCG2 gene in renal carcinoma.
ABCG2 is a ubiquitous ATP-binding cassette transmembrane protein that is important in clinical drug resistance. Little is known about the mechanism(s) regulating the expression of ABCG2. We hypothesized that DNA methylation could play a role in the epigenetic regulation of ABCG2 gene expression. The promoter methylation status of three renal carcinoma cell lines was assessed with restriction enzyme digestion-coupled PCR and bisulfite genomic sequencing. Both UOK121 and UOK143, with known methylation of the VHL promoter, showed induction of ABCG2 expression after 5-aza-2'-deoxycytidine (5-aza-dC) treatment, suggesting that aberrant methylation of the ABCG2 gene was associated with gene silencing. In vitro methylation of the ABCG2 promoter-driven luciferase reporter vector resulted in a significant inhibition of transcription. Our data suggested that the ABCG2 gene is regulated coordinately at both histone and DNA levels. A chromatin immunoprecipitation assay demonstrated that the methylated promoter in UOK121 and UOK143, but not the unmethylated one in UOK181, is associated with the methyl CpG binding domain proteins (MBDs), MBD2 and MeCP2. Histone deacetylase 1 and a corepressor, mSin3A, were identified binding to the promoter region containing the CpG island, thereby suppressing ABCG2 transcription. Reactivation of ABCG2 was achieved by treatment with 5-aza-dC, a demethylating agent, concomitant with the release of MBDs from the promoter. Furthermore, the association of methylated lysine 9 on histone H3, a hallmark of promoter methylation, with the promoter was reduced following 5-aza-dC treatment. These data suggest that DNA methylation-dependent formation of a repressor complex in the CpG island contributes to inactivation of ABCG2.
To
Kenneth K W
KK
Experimental Therapeutics Section, Medical Oncology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892-4255, USA. tok@mail.nih.gov
Zhan
Z
Z
Bates
Susan E
SE
D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters / Q000378:metabolism*; D001374:Azacitidine / Q000494:pharmacology; D001483:Base Sequence; D002292:Carcinoma, Renal Cell / Q000378:metabolism*; D045744:Cell Line, Tumor; D018899:CpG Islands; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000235:genetics / Q000378:metabolism; D019175:DNA Methylation; D000090205:DNA Methyltransferase 3A; D044127:Epigenesis, Genetic; D006657:Histones / Q000378:metabolism; D006801:Humans; D007680:Kidney Neoplasms / Q000378:metabolism*; D008239:Lysine; D009363:Neoplasm Proteins / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D014158:Transcription, Genetic; D051794:Von Hippel-Lindau Tumor Suppressor Protein / Q000378:metabolism
C116614:ABCG2 protein, human; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters; C000717967:DNMT3A protein, human; D006657:Histones; D009363:Neoplasm Proteins; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases; D000090205:DNA Methyltransferase 3A; D051794:Von Hippel-Lindau Tumor Suppressor Protein; C497446:VHL protein, human; D008239:Lysine; D001374:Azacitidine