Development of an intracellular, DNA methyltransferase-specific, and gene-specific assay for studying dynamic DNA methylation

Jesus Gonzalez-Bosquet, Yongli Chu, Hai Bin Chen, Sean Christopher Dowdy, Karl C. Podratz, Jinping Li, Shi Wen Jiang

Research output: Contribution to journalArticle

1 Citation (Scopus)

Abstract

A growing body of evidence supports that DNA methylation-mediated silencing of tumor suppressor genes plays a significant role in cancer development. DNA methylatransferase (DNMT) is the enzyme catalyzing the methylation modification of cytosines in a CpG dinucleotide context. In humans, this reaction is highly selective for certain gene promoters and/or genomic DNA domains. Elucidation of the intracellular targeting mechanism by DNMT has become the key task for understanding epigenetic regulation in cancers. Unfortunately, no suitable method is available to explore this important cell function. This study focuses on the development of an efficient technique for measuring the intracellular, DNMT isoform-specific, and methylated gene-specific, DNA methylation alterations. The technique, designated IMA for Intracellular DNA Methylation Assay, takes advantage of covalent arresting of active DNMT molecules by aza-deoxycytidine (ADC), a modified cytosine homologue readily incorporated into genomic DNA at the cytosine position. The DNMT-DNA complex was isolated by a modified ETOH precipitation procedure to remove cellular proteins including free DNMT. Chromatin immunoprecipitation using DNMT isoform-specific antibody was subsequently performed to collect DNMT-bound DNA fragments. PCR amplification was used to detect and quantify the isolated gene fragment. Validation of the IMA was performed by manipulating the DNMT activity, by treating with antisense oligonucleotides against DNMT1 and DNMT3B, and repeating the IMA experiment. One of the main discoveries with this technique was the observation of DNMT3B maintenance methylation activity. This new technique can be applied to examine the dynamic DNMT-specific action on diversified methylation-silenced genes, in a variety of cell culture conditions.

Original languageEnglish (US)
Pages (from-to)1664-1673
Number of pages10
JournalCurrent Pharmaceutical Design
Volume20
Issue number11
DOIs
StatePublished - 2014

Fingerprint

Methyltransferases
DNA Methylation
DNA
Genes
Cytosine
Methylation
Protein Isoforms
Deoxycytidine
Antisense Oligonucleotides
Chromatin Immunoprecipitation
Tumor Suppressor Genes
Epigenomics
Neoplasms
Cell Culture Techniques

Keywords

  • Dna methylation
  • Dna methyltransferase
  • Epigenetic regulation
  • Methylation assay

ASJC Scopus subject areas

  • Drug Discovery
  • Pharmacology
  • Medicine(all)

Cite this

Development of an intracellular, DNA methyltransferase-specific, and gene-specific assay for studying dynamic DNA methylation. / Gonzalez-Bosquet, Jesus; Chu, Yongli; Chen, Hai Bin; Dowdy, Sean Christopher; Podratz, Karl C.; Li, Jinping; Jiang, Shi Wen.

In: Current Pharmaceutical Design, Vol. 20, No. 11, 2014, p. 1664-1673.

Research output: Contribution to journalArticle

Gonzalez-Bosquet, Jesus ; Chu, Yongli ; Chen, Hai Bin ; Dowdy, Sean Christopher ; Podratz, Karl C. ; Li, Jinping ; Jiang, Shi Wen. / Development of an intracellular, DNA methyltransferase-specific, and gene-specific assay for studying dynamic DNA methylation. In: Current Pharmaceutical Design. 2014 ; Vol. 20, No. 11. pp. 1664-1673.
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