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

Gregory E. Cooper, Peggy L. Bishop, Mitchell Turker

Research output: Contribution to journalArticle

10 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)221-229
Number of pages9
JournalSomatic Cell and Molecular Genetics
Volume19
Issue number3
DOIs
StatePublished - May 1993
Externally publishedYes

Fingerprint

Adenine Phosphoribosyltransferase
Embryonal Carcinoma Stem Cells
Transcriptional Activation
Chromatin
Cell Line
Genes
DNA Methylation
Clone Cells
decitabine
Nucleic Acid Conformation
Genetic Promoter Regions
Alleles
RNA
Phenotype
Messenger RNA

ASJC Scopus subject areas

  • Genetics
  • Cell Biology

Cite this

@article{77ea4b63751e48338ff47e2a94d806cf,
title = "Hemidemethylation is sufficient for chromatin relaxation and transcriptional activation of methylated aprt gene in mouse P19 embryonal carcinoma cell line",
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.",
author = "Cooper, {Gregory E.} and Bishop, {Peggy L.} and Mitchell Turker",
year = "1993",
month = "5",
doi = "10.1007/BF01233070",
language = "English (US)",
volume = "19",
pages = "221--229",
journal = "Somatic Cell and Molecular Genetics",
issn = "0740-7750",
publisher = "Springer GmbH & Co, Auslieferungs-Gesellschaf",
number = "3",

}

TY - JOUR

T1 - Hemidemethylation is sufficient for chromatin relaxation and transcriptional activation of methylated aprt gene in mouse P19 embryonal carcinoma cell line

AU - Cooper, Gregory E.

AU - Bishop, Peggy L.

AU - Turker, Mitchell

PY - 1993/5

Y1 - 1993/5

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=0027226371&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0027226371&partnerID=8YFLogxK

U2 - 10.1007/BF01233070

DO - 10.1007/BF01233070

M3 - Article

C2 - 7687384

AN - SCOPUS:0027226371

VL - 19

SP - 221

EP - 229

JO - Somatic Cell and Molecular Genetics

JF - Somatic Cell and Molecular Genetics

SN - 0740-7750

IS - 3

ER -