Hemidemethylation is sufficient for chromatin relaxation and transcriptional activation of methylated aprt gene in mouse P19 embryonal carcinoma cell line

Somat Cell Mol Genet. 1993 May;19(3):221-9. doi: 10.1007/BF01233070.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Adenine Phosphoribosyltransferase / genetics*
  • Adenine Phosphoribosyltransferase / metabolism
  • Animals
  • Antineoplastic Agents / pharmacology
  • Azacitidine / analogs & derivatives
  • Azacitidine / pharmacology
  • Chromatin / metabolism*
  • Decitabine
  • Enzyme Activation
  • Gene Expression Regulation, Enzymologic
  • Methylation
  • Mice
  • Teratoma
  • Transcription, Genetic*
  • Tumor Cells, Cultured

Substances

  • Antineoplastic Agents
  • Chromatin
  • Decitabine
  • Adenine Phosphoribosyltransferase
  • Azacitidine