Title:Development of an Intracellular, DNA Methyltransferase-Specific, and Gene-Specific Assay for Studying Dynamic DNA Methylation
Volume: 20
Issue: 11
Author(s): Jesus Gonzalez-Bosquet, Yongli Chu, Hai Bin Chen, Sean C. Dowdy, Karl C. Podratz, Jinping Li and Shi-Wen Jiang
Affiliation:
Keywords:
DNA methyltransferase, methylation assay, DNA methylation, epigenetic regulation.
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.
