Epigenetic regulation of Dpp6 expression by Dnmt3b and its novel role in the inhibition of RA induced neuronal differentiation of P19 cells.
Epigenetic regulation of Dpp6 expression by Dnmt3b and its novel role in the inhibition of RA induced neuronal differentiation of P19 cells.
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Dnmt3b 对 Dpp6 表达的表观遗传调控及其在抑制 RA 诱导的 P19 细胞神经元分化中的新作用
DOI：
10.1371/journal.pone.0055826
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发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Zhu X
中科院分区：
文献类型：
--
作者：
Sheikh MA;Malik YS;Yu H;Lai M;Wang X;Zhu X
DNA甲基化是哺乳动物基因沉默的一个重要机制，由一组DNA甲基转移酶催化，包括Dnmt 1，Dnmt 3a和Dnmt 3b，它们在发育和分化过程中建立基因组甲基化模式所必需。本文研究了DNA甲基转移酶在维甲酸诱导P19细胞神经元分化过程中的作用。我们观察到RA处理后P19细胞中Dnmt 3b的mRNA和蛋白水平增加，而Dnmt 1和Dnmt 3a的表达降低，这表明Dnmt 3b在P19细胞的神经元分化过程中更重要。从RA处理的P19细胞中富集Dnmt3b的染色质文库鉴定二肽基肽酶6（Dpp6）基因为Dnmt3b的新靶点。此外，定量ChIP分析显示，在RA处理的和未处理的p19细胞中，Dpp6启动子上募集的Dnmt3b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，在RA处理和未处理的P19细胞中，Dpp6启动子均高度甲基化。Dnmt3b的缺失导致Dpp6的mRNA和蛋白表达增加，因此Dnmt3b负责Dpp6基因的转录沉默。因此，Dpp6基因启动子的平均甲基化在Dnmt3b敲低细胞中降低至一半。在Dnmt3b缺失的情况下，Dnmt3a与Dpp6基因启动子相关，并调节P19细胞中Dpp6基因的表达和甲基化。维甲酸诱导的神经元分化抑制后，异位表达Dpp6在P19细胞。综上所述，本研究描述了Dpp6表达的DNA甲基化的表观遗传沉默，并建立了其异位表达可以作为负信号在RA诱导的P19细胞的神经元分化。
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.
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