Institute of Genetics and Cytology, Northeast Normal University, Changchun, China.
PLoS One. 2013;8(2):e55826. doi: 10.1371/journal.pone.0055826. Epub 2013 Feb 7.
DNA methylation is an important mechanism of gene silencing in mammals catalyzed by a group of DNA methyltransferases including Dnmt1, Dnmt3a, and Dnmt3b which are required for the establishment of genomic methylation patterns during development and differentiation. In this report, we studied the role of DNA methyltransferases during retinoic acid induced neuronal differentiation of P19 cells. We observed an increase in the mRNA and protein level of Dnmt3b, whereas the expression of Dnmt1 and Dnmt3a was decreased after RA treatment of P19 cells which indicated that Dnmt3b is more important during neuronal differentiation of P19 cells. Dnmt3b enriched chromatin library from RA treated P19 cells identified dipeptidyl peptidase 6 (Dpp6) gene as a novel target of Dnmt3b. Further, quantitative ChIP analysis showed that the amount of Dnmt3b recruited on Dpp6 promoter was equal in both RA treated as well as untreated p19 cells. Bisulfite genomic sequencing, COBRA, and methylation specific PCR analysis revealed that Dpp6 promoter was heavily methylated in both RA treated and untreated P19 cells. Dnmt3b was responsible for transcriptional silencing of Dpp6 gene as depletion of Dnmt3b resulted in increased mRNA and protein expression of Dpp6. Consequently, the average methylation of Dpp6 gene promoter was reduced to half in Dnmt3b knockdown cells. In the absence of Dnmt3b, Dnmt3a was associated with Dpp6 gene promoter and regulated its expression and methylation in P19 cells. RA induced neuronal differentiation was inhibited upon ectopic expression of Dpp6 in P19 cells. Taken together, the present study described epigenetic silencing of Dpp6 expression by DNA methylation and established that its ectopic expression can act as negative signal during RA induced neuronal differentiation of P19 cells.
DNA 甲基化是哺乳动物中基因沉默的一种重要机制，由一组 DNA 甲基转移酶（DNMTs）催化，包括 Dnmt1、Dnmt3a 和 Dnmt3b，它们在发育和分化过程中建立基因组甲基化模式是必需的。在本报告中，我们研究了 DNA 甲基转移酶在视黄酸诱导 P19 细胞神经元分化中的作用。我们观察到 Dnmt3b 的 mRNA 和蛋白水平增加，而 Dnmt1 和 Dnmt3a 的表达在 P19 细胞用 RA 处理后降低，这表明 Dnmt3b 在 P19 细胞的神经元分化中更为重要。从用 RA 处理的 P19 细胞富集的 Dnmt3b 染色质文库中鉴定出二肽基肽酶 6（Dpp6）基因为 Dnmt3b 的新靶点。此外，定量 ChIP 分析表明，在 RA 处理和未处理的 p19 细胞中，Dnmt3b 募集到 Dpp6 启动子上的量相等。亚硫酸氢盐基因组测序、COBRA 和甲基化特异性 PCR 分析显示，Dpp6 启动子在 RA 处理和未处理的 P19 细胞中均高度甲基化。Dnmt3b 负责 Dpp6 基因的转录沉默，因为 Dnmt3b 的耗竭导致 Dpp6 的 mRNA 和蛋白表达增加。因此，Dnmt3b 敲低细胞中 Dpp6 基因启动子的平均甲基化降低到一半。在缺乏 Dnmt3b 的情况下，Dnmt3a 与 Dpp6 基因启动子结合，并调节其在 P19 细胞中的表达和甲基化。在 P19 细胞中异位表达 Dpp6 会抑制 RA 诱导的神经元分化。综上所述，本研究描述了 Dpp6 表达的表观遗传沉默通过 DNA 甲基化实现，并证实其异位表达可作为 RA 诱导 P19 细胞神经元分化的负信号。
