RA-Induced Transcriptional Silencing of Checkpoint Kinase-2 through Promoter Methylation by Dnmt3b Is Required for Neuronal Differentiation of P19 Cells
RA-Induced Transcriptional Silencing of Checkpoint Kinase-2 through Promoter Methylation by Dnmt3b Is Required for Neuronal Differentiation of P19 Cells
复制标题
P19 细胞神经元分化需要 RA 通过 Dnmt3b 启动子甲基化诱导检查点激酶 2 转录沉默
DOI：
10.1016/j.jmb.2017.07.005
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发表时间：
2017
影响因子：
5.6
通讯作者：
Zhu Xiaojuan
中科院分区：
文献类型：
--
作者：
Sheikh Muhammad Abid;Malik Yousra Saeed;Zhu Xiaojuan
在之前的研究中，我们使用维甲酸(RA)处理的P19细胞的染色质文库鉴定了Dnmt3b的几个新靶点。本研究描述了作为Dnmt3b靶基因之一的检查点激酶(Chk2)的表达和功能的调节。染色质免疫沉淀和定量PCR分析表明，RA处理P19细胞后，Dnmt3b在Chk2启动子上的募集被诱导。亚硫酸氢盐基因组序列分析和COBRA分析均表明，在维甲酸诱导的P19细胞神经分化过程中，Chk2启动子甲基化水平逐渐升高。同时，实时荧光定量聚合酶链式反应和蛋白质印迹分析分别检测到Chk2的mRNA和蛋白表达均降低。慢病毒介导的shRNA抑制Dnmt3b的表达导致Chk2表达增加和甲基化减少，这清楚地表明Dnmt3b是导致RA处理的P19细胞Chk2基因转录沉默的原因。P19细胞中Chk2的表达增强抑制了P19细胞的神经分化，这表明内源性Chk2表达的降低是正常分化所必需的。在RA治疗后，异位Chk2的表达也负向调节细胞周期停滞和细胞凋亡，这也可能导致神经元分化受损。总之，这项研究描述了Chk2通过启动子甲基化对其表达的调节，并提出了Chk2在神经元分化过程中的一个新的作用，该作用独立于其在DNA损伤反应中的已知功能。
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.