PDF(Pubmed)
Abstract：
Inhibition of protein kinase C (PKC) efficiently promoted the self-renewal of embryonic stem cells (ESCs). However, information about the function of PKC inhibition remains lacking. Here, RNA-sequencing showed that the addition of Go6983 significantly inhibited the expression of de novo methyltransferases (Dnmt3a and Dnmt3b) and their regulator Dnmt3l, resulting in global hypomethylation of DNA in mouse ESCs. Mechanistically, PR domain-containing 14 (Prdm14), a site-specific transcriptional activator, partially contributed to Go6983-mediated repression of Dnmt3 genes. Administration of Go6983 increased Prdm14 expression mainly through the inhibition of PKCδ. High constitutive expression of Prdm14 phenocopied the ability of Go6983 to maintain` mouse ESC stemness in the absence of self-renewal-promoting cytokines. In contrast, the knockdown of Prdm14 eliminated the response to PKC inhibition and substantially impaired the Go6983-induced resistance of mouse ESCs to differentiation. Furthermore, liquid chromatography-mass spectrometry profiling and Western blotting revealed low levels of Suv39h1 and Suv39h2 in Go6983-treated mouse ESCs. Suv39h enzymes are histone methyltransferases that recognize dimethylated and trimethylated histone H3K9 specifically and usually function as transcriptional repressors. Consistently, the inhibition of Suv39h1 by RNA interference or the addition of the selective inhibitor chaetocin increased Prdm14 expression. Moreover, chromatin immunoprecipitation assay showed that Go6983 treatment led to decreased enrichment of dimethylation and trimethylation of H3K9 at the Prdm14 promoter but increased RNA polymerase Ⅱ binding affinity. Together, our results provide novel insights into the pivotal association between PKC inhibition-mediated self-renewal and epigenetic changes, which will help us better understand the regulatory network of stem cell pluripotency.
摘要：
抑制蛋白激酶C(PKC)可有效促进胚胎干细胞(ESC)的自我更新。然而,关于PKC抑制功能的信息仍然缺乏。这里,RNA-测序显示，添加Go6983显著抑制了从头甲基转移酶(Dnmt3a和Dnmt3b)及其调节因子Dnmt3l的表达，导致小鼠ESC中DNA的全局低甲基化。机械上,含PR结构域14(Prdm14)，一个位点特异性转录激活因子，部分有助于Go6983介导的Dnmt3基因抑制。施用Go6983主要通过抑制PKCδ来增加Prdm14表达。Prdm14的高组成型表达表型证实了Go6983在不存在促进自我更新的细胞因子的情况下维持小鼠ESC干性的能力。相比之下,Prdm14的敲低消除了对PKC抑制的反应,并显著损害了Go6983诱导的小鼠ESC对分化的抗性.此外，LC-MS分析和蛋白质印迹显示，在Go6983处理的小鼠ESC中，Suv39h1和Suv39h2水平较低。Suv39h酶是组蛋白甲基转移酶,其特异性地识别二甲基化和三甲基化组蛋白H3K9并且通常作为转录阻遏物起作用。始终如一，通过RNA干扰或添加选择性抑制剂chaetocin对Suv39h1的抑制增加了Prdm14的表达。此外，ChIP实验表明，Go6983处理导致Prdm14启动子处H3K9的二甲基化和三甲基化富集减少，但增加了RNA聚合酶Ⅱ的结合亲和力。一起,我们的研究结果为PKC抑制介导的自我更新和表观遗传变化之间的关键关联提供了新的见解，这将有助于我们更好地理解干细胞多能性的调节网络。
