ABSTRACT
Allele-specific epigenetic events regulate the expression of specific genes such as tumor suppressor genes. Methods to biochemically identify epigenetic regulators remain limited. Here, we used insertional chromatin immunoprecipitation (iChIP) to address this issue. iChIP combined with quantitative mass spectrometry identified DNA methyltransferase 1 (DNMT1) and epigenetic regulators as proteins that potentially interact with a region of the p16INK4A gene that is CpG-methylated in one allele in HCT116 cells. Some of the identified proteins are involved in the CpG methylation of this region, and of these, DEAD-box helicase 24 (DDX24) contributes to CpG methylation by regulating the protein levels of DNMT1. Thus, iChIP is a useful method to identify proteins which bind to a target locus of interest.
Subject(s)
CpG Islands , Cyclin-Dependent Kinase Inhibitor p16 , DNA Methylation , Epigenesis, Genetic , Humans , Chromatin Immunoprecipitation , Cyclin-Dependent Kinase Inhibitor p16/genetics , Cyclin-Dependent Kinase Inhibitor p16/metabolism , DEAD-box RNA Helicases/metabolism , DEAD-box RNA Helicases/genetics , DNA (Cytosine-5-)-Methyltransferases/metabolism , DNA (Cytosine-5-)-Methyltransferases/genetics , HCT116 CellsABSTRACT
BACKGROUND: Analysis of CpG methylation is informative for cancer diagnosis. Previously, we developed a novel method to discriminate CpG methylation status in target DNA by blocking recombinase polymerase amplification (RPA), an isothermal DNA amplification technique, using methyl-CpG binding domain (MBD) protein 2 (MBD2). The method was named MBD protein interference-RPA (MBDi-RPA). In this study, MBDi-RPA was performed using methyl-CpG binding protein 2 (MeCP2), another MBD family protein, as the blocking agent. RESULTS: MBDi-RPA using MeCP2 detected low levels of CpG methylation, showing that it had higher sensitivity than MBDi-RPA using MBD2. We also developed real-time RPA, which enabled rapid analysis of DNA amplification without the need for laborious agarose gel electrophoresis and used it in combination with MBDi-RPA. We termed this method real-time MBDi-RPA. The method using MeCP2 could determine the abundance ratio of CpG-methylated target DNA simply and rapidly, although highly sensitive detection was challenging. SIGNIFICANCE AND NOVELTY: Real-time MBDi-RPA using MeCP2 could be potentially useful for estimating CpG methylation status in target DNA prior to more detailed analyses.