Hemidemethylation is sufficient for chromatin relaxation and transcriptional activation of methylated aprt gene in mouse P19 embryonal carcinoma cell line.
A series of clones displaying a high-frequency "switching" phenotype for expression of the adenine phosphoribosyltransferase (aprt) gene was previously isolated from the P19 mouse embryonal carcinoma stem cell line. In a subset of these clones, loss of aprt expression was correlated with increased DNA methylation, a nuclease-resistant chromatin conformation, and loss of RNA transcription; reactivation was associated with a reversal of these parameters. In this report, the role of DNA methylation in transcriptional inactivation was studied in the H22D3 clone. The cells of this clone contain a single inactive aprt allele that is methylated. Mass cultures of H22D3 were treated with 2-deoxy-5'-azacytidine (5aCdr) and found to reactivate aprt at frequencies ranging from 60 to 90%. Treated cultures were then assayed over time for aprt mRNA, chromatin conformation, and DNA methylation of the aprt gene. These studies demonstrated that 5aCdr treatment resulted in promoter region-specific hemidemethylation and chromatin relaxation starting at 12 h. This was followed by the appearance of RNA transcripts at 18 h and increasing levels of APRT enzymatic activity at 36 h after treatment. Complete demethylation occurred significantly later. Experiments in which cells were treated with 5aCdr for varying periods of time demonstrated that a single round of analog incorporation was sufficient for transcriptional reactivation of aprt in H22D3.
Cooper
G E
GE
Department of Microbiology & Immunology, University of Kentucky College of Medicine, Lexington 40536.
Bishop
P L
PL
Turker
M S
MS
D000228:Adenine Phosphoribosyltransferase / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology; D001374:Azacitidine / Q000031:analogs & derivatives / Q000494:pharmacology; D002843:Chromatin / Q000378:metabolism*; D000077209:Decitabine; D004789:Enzyme Activation; D015971:Gene Expression Regulation, Enzymologic; D008745:Methylation; D051379:Mice; D013724:Teratoma; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
D000970:Antineoplastic Agents; D002843:Chromatin; D000077209:Decitabine; D000228:Adenine Phosphoribosyltransferase; D001374:Azacitidine