title: Development of an intracellular, DNA methyltransferase-specific, and gene-specific assay for studying dynamic DNA methylation.
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.
authors.forename: Jesus
authors.initials: J
authors.lastname: Gonzalez-Bosquet
authors.forename: Yongli
authors.initials: Y
authors.lastname: Chu
authors.forename: Hai Bin
authors.initials: HB
authors.lastname: Chen
authors.forename: Sean C
authors.initials: SC
authors.lastname: Dowdy
authors.forename: Karl C
authors.initials: KC
authors.lastname: Podratz
authors.forename: Jinping
authors.initials: J
authors.lastname: Li
authors.affiliation: Department of Biomedical Science, Mercer University School of Medicine, Savannah Campus, 4700 Waters Ave, Savannah, GA 31404-3089, USA. Jiang_s@mercer.edu.
authors.forename: Shi-Wen
authors.initials: SW
authors.lastname: Jiang
mesh_terms: D045744:Cell Line, Tumor; D047369:Chromatin Immunoprecipitation; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000235:genetics*; D053938:DNA Fragmentation; D019175:DNA Methylation; D044127:Epigenesis, Genetic; D020868:Gene Silencing; D006801:Humans; D016376:Oligonucleotides, Antisense / Q000008:administration & dosage; D016133:Polymerase Chain Reaction; D020033:Protein Isoforms; D000097584:DNA Methyltransferase 3B
chemical_list: D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases; D016376:Oligonucleotides, Antisense; D020033:Protein Isoforms; D000097584:DNA Methyltransferase 3B; C000619380:DNMT1 protein, human