RA-Induced Transcriptional Silencing of Checkpoint Kinase-2 through Promoter Methylation by Dnmt3b Is Required for Neuronal Differentiation of P19 Cells.

In a previous study, we identified several novel targets of Dnmt3b using a chromatin library from retinoic acid (RA)-treated P19 cells. The present study describes the regulation of expression and function of checkpoint kinase (Chk2), which was one of the target genes of Dnmt3b. Chromatin immunoprecipitation followed by quantitative PCR analysis showed that recruitment of Dnmt3b on Chk2 promoter is induced following RA treatment of P19 cells. Both bisulfite genomic sequence and COBRA analyses showed that the methylation level of Chk2 promoter is progressively increased during RA-induced neuronal differentiation of P19 cells. Concomitantly, both mRNA and protein expression of Chk2 are reduced as determined by real-time PCR and Western blot analysis, respectively. Suppression of Dnmt3b expression by lentiviral-mediated shRNA resulted in increased expression and reduced methylation of Chk2, which clearly showed that Dnmt3b is responsible for transcriptional silencing of Chk2 gene in RA-treated P19 cells. Neuronal differentiation of P19 cells was inhibited upon enforced Chk2 expression in P19 cells, which showed that the decrease in endogenous expression of Chk2 is essential for normal differentiation. Ectopic Chk2 expression also negatively regulated cell cycle arrest and apoptosis following RA treatment, which could also contribute to impaired neuronal differentiation. Together, this study described the regulation of Chk2 expression through promoter methylation and also presented a novel role of Chk2 during neuronal differentiation, which is independent of its previously known function in DNA damage response.