Enzymatic methylation of DNA in cultured human cells studied by stable isotope incorporation and mass spectrometry

Jason L. Herring, Daniel K. Rogstad, Lawrence Sowers

Research output: Contribution to journalArticle

12 Citations (Scopus)

Abstract

Enzymatic methylation of cytosine residues in DNA, in conjunction with covalent histone modifications, establishes an epigenetic code essential for the proper control of gene expression in higher organisms. Once established during cellular differentiation, the epigenetic code must be faithfully transmitted to progeny cells. However, epigenetic perturbations can be found in most if not all cancer cells, and the mechanisms leading to these changes are not well understood. In this paper, we describe a series of experiments aimed at understanding the dynamic process of DNA methylation that follows DNA replication. Cells in culture can be propagated in the presence of 15N-enriched uridine, which labels the pyrimidine precursor pool as well as newly replicated DNA. Simultaneous culture in the presence of 2H-enriched methionine results in labeling of newly methylated cytosine residues. An ensemble of 5-methylcytosine residues differing in the degree of isotopic enrichment is generated, which can be examined by mass spectrometry. Using this method, we demonstrate that the kinetics of both DNA replication and methylation of newly replicated DNA are indistinguishable. The majority of methylation following DNA replication is shown to occur on the newly synthesized DNA. The method reported here does, however, suggest an unexpected methylation of parental DNA during DNA replication, which might indicate a previously undescribed chromatin remodeling process. The method presented here will be useful in monitoring the dynamic process of DNA methylation and will allow a more detailed understanding of the mechanisms of clinically used methylation inhibitors and environmental toxicants.

Original languageEnglish (US)
Pages (from-to)1060-1068
Number of pages9
JournalChemical Research in Toxicology
Volume22
Issue number6
DOIs
StatePublished - Jun 15 2009
Externally publishedYes

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Methylation
DNA Methylation
DNA Replication
Isotopes
Mass spectrometry
Cultured Cells
Mass Spectrometry
Cells
Epigenomics
Cytosine
DNA
Histone Code
5-Methylcytosine
Chromatin Assembly and Disassembly
Uridine
Methionine
Cell Culture Techniques
Gene Expression
Gene expression
Histones

ASJC Scopus subject areas

  • Toxicology

Cite this

Enzymatic methylation of DNA in cultured human cells studied by stable isotope incorporation and mass spectrometry. / Herring, Jason L.; Rogstad, Daniel K.; Sowers, Lawrence.

In: Chemical Research in Toxicology, Vol. 22, No. 6, 15.06.2009, p. 1060-1068.

Research output: Contribution to journalArticle

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