TY - JOUR
T1 - Development of an intracellular, DNA methyltransferase-specific, and gene-specific assay for studying dynamic DNA methylation
AU - Gonzalez-Bosquet, Jesus
AU - Chu, Yongli
AU - Chen, Hai Bin
AU - Dowdy, Sean C.
AU - Podratz, Karl C.
AU - Li, Jinping
AU - Jiang, Shi Wen
PY - 2014
Y1 - 2014
N2 - 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.
AB - 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.
KW - Dna methylation
KW - Dna methyltransferase
KW - Epigenetic regulation
KW - Methylation assay
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U2 - 10.2174/13816128113199990539
DO - 10.2174/13816128113199990539
M3 - Article
C2 - 23888947
AN - SCOPUS:84903715575
SN - 1381-6128
VL - 20
SP - 1664
EP - 1673
JO - Current pharmaceutical design
JF - Current pharmaceutical design
IS - 11
ER -