Identification Of Novel Targets Of DNA Methyltransferase3b, Their Regulation Of Expression And Function In Neuronal Differentiation

DNA methylation is an important mechanism of gene silencing in mammalscatalyzed by a group of DNA methyltransferases including Dnmt1, Dnmt3a, andDnmt3b which are required for the establishment of genomic methylation patternsduring development and differentiation. In this report, we studied the role of DNAmethyltransferases during retinoic acid induced neuronal differentiation of P19cells.We observed an increase in the mRNA and protein level of Dnmt3b, whereas theexpression of Dnmt1and Dnmt3a was decreased after RA treatment of P19cellswhich indicated that Dnmt3b is more important during neuronal differentiation of P19cells. Next, Dnmt3b enriched chromatin library from RA treated P19cells identifiedseveral novel target genes. We selected dipeptidyl peptidase6(Dpp6) and checkpointkinase2(Chk2) for further study. Dpp6is a member of dipeptidyl peptidase IVfamily of proteins which regulate diverse biological functions including celldifferentiation, apoptosis, proliferation, and carcinogenesis. Quantitative ChIPanalysis showed that the amount of Dnmt3b recruited on Dpp6promoter was equal inboth RA treated as well as untreated p19cells. Bisulfite genomic sequencing,COBRA, and methylation specific PCR analysis revealed that Dpp6promoter washeavily methylated in both RA treated and untreated P19cells. Dnmt3b wasresponsible for transcriptional silencing of Dpp6gene as depletion of Dnmt3bresulted in increased mRNA and protein expression of Dpp6. Consequently, theaverage methylation of Dpp6gene promoter was reduced to half in Dnmt3bknockdown cells. In the absence of Dnmt3b, Dnmt3a was associated with Dpp6genepromoter and regulated its expression and methylation in P19cells. RA inducedneuronal differentiation was inhibited upon ectopic expression of Dpp6in P19cells.Taken together, the present study described epigenetic silencing of Dpp6expressionby DNA methylation and established that its ectopic expression can act as negativesignal during RA induced neuronal differentiation of P19cells.We also selected Chk2for further study based on its important role in ensuringgenomic integrity, cell cycle progression, differentiation, and apoptosis. ChIP assaysshowed that recruitment of Dnmt3b on Chk2promoter is induced by RA treatment.The methylation level of Chk2promoter region increased steadily following RA treatment which is the result of Dnmt3b recruitment on its promoter. Both mRNA andprotein level of Chk2is decreased as the methylation increases during neuronaldifferentiation of P19cells. Dnmt3b knockdown resulted in decreased methylationand increased expression of Chk2in RA treated P19cells. A negative effect ofectopic Chk2expression was observed on neuronal differentiation of P19cells whichalso showed that the decrease in endogenous expression of Chk2is essential fornormal differentiation. Enforced Chk2expression negatively regulate cell cycle exitby keeping the cells in proliferating stage which ultimately results in impairedneuronal differentiation. Our study also established Chk2as a negative regulator ofapoptosis. The negative effect of forced Chk2expression on apoptosis might alsocontribute to impaired neuronal differentiation of P19cells observed in our study.Overall, we showed the regulation of Chk2expression through DNA methylation andreported its novel role during differentiation which is independent of its previouslyknown function in DNA damage response. Finally, we provided epigenetic regulationof two genes by Dnmt3b which could greatly strengthen our understanding aboutepigenetic regulation of gene expression during complex mechanisms of neuronaldifferentiation.