Klf4的精氨酸甲基化修饰在细胞重编程中的作用

Klf4 is an important transcription factor for maintaining the pluripotency of embryonic stem cells (ESCs), which is capable of reprogramming somatic cells to embryonic stem cells together with other transcription factors Oct3/4, Sox2. Prmt1 is a predominant protein arginine methyltransferase, which is necessary for early embryonic development. Prmt1-/- mice die at E6.5. However, the role of Prmt1 in the early embryonic development is still unknown. To explore the function of Prmt1 in embryonic stem cells, we successful established Prmt1 knockout embryonic stem cell line using CRISPR-Cas9 system, which had been confirmed at protein, mRNA and genome levels. Prmt1 knockout ESCs lose a tightly packed colony morphology and exhibit characteristic epithelial morphology. Prmt1-/- mESCs show a significantly decreased growth rate. Prmt1 knockout ESCs showed a decrease in the percentage of S-phase cells and a lengthening of G2/M, which agreed with the cell cycle remodeling of differentiated mESCs. Next, we determined whether there was stem factors change and lineage-specific differentiation upon Prmt1 deletion. Quantitative PCR results show stem-related transcription factors Oct4、Sox2 and Klf4 remained the same except the reduction of Nanog expression. Surprisingly, the lineage-specific marker genes had a specific expression pattern. Gata4, Gata6, Sox17 and Foxa2, primitive endoderm (PrE) markers, have a remarkably increase, however the mesoectoderm markers (Brachyury, Id2, Mix1, Nestin, Fgf5) and trophectoderm markers (Cdx2, Ascl2, Hand1) didn’t change obviously. The immunostaining showed the expression of Gata4 and Gata6 in Prmt1 knockout E14 rather than control E14. The above evidences indicated that the deletion of Prmt1 result in the embryonic differentiate to primitive endoderm. .Previous study of our lab found Prmt1 could interact with Klf4, which depends on Klf4 zinc finger domain. Using methylation assays in vitro, we illustrated that Klf4 could be methylated by Prmt1 and the methylation site located the repression and activation domain of Klf4 not the interaction zinc finger domain. The expression of Klf4 is not influenced by the Prmt1 deletion. .In conclusion, the deletion of Prmt1 could weaken the pluripotency of ESCs and promote ESCs differentiate to primitive endoderm. What’s more, the differentiation is connected with arginine methyltransferase activity of Prmt1. In addition, the methylation of Klf4 mediated by Prmt1 imply us whether this lineage-specific differentiation due to the loss of Klf4 methylation. We need more evidences to support our assumption..Based on the above observations, we aim to interpretation the regulatory mechanism of arginine methylated Klf4 by Prmt1 onto the PrE differentiation and reveal the role of arginine methylated Klf4 in cellular programming.

Klf4作为细胞重编程中一个重要转录因子，和Oct3/4、Sox2、c-Myc和Nanog等关键转录因子构成精细的调控网络，将已经分化的细胞重新诱导成具有胚胎干细胞特性的多能干性细胞（iPS）。蛋白质精氨酸甲基转移酶Prmt1介导的组蛋白和非组蛋白底物甲基化修饰参与许多重要的生命过程。Prmt1敲除的小鼠同样在胚胎发育到E6.5天发生胚胎致死，但其在胚胎发育过程中的作用知之甚少。本实验室前期研究发现， Prmt1可以特异性甲基化修饰Klf4；敲除Prmt1促使胚胎干细胞（ES）细胞向原始内胚层（primitive endoderm，PrE）分化。本集成项目拟在确定Klf4精氨酸甲基化修饰的位点以及Prmt1修饰Klf4对胚胎干细胞向PrE分化的影响的基础上，进一步阐释精氨酸甲基化修饰的Klf4对iPS诱导效率以及iPS再分化谱系潜能的影响。

小鼠早期胚胎的发育伴随着由一个受精卵细胞向多种类型细胞的分化。不同细胞的特异基因在时间和空间上受到精确控制。蛋白质精氨酸甲基转移酶（PRMT）催化蛋白质精氨酸残基的甲基添加，可以调节小鼠胚胎的早期发育。PRMT1基因敲除小鼠导致胚胎发育失败，死于6.5 天，但其调控机制未知。.小鼠胚胎干细胞（ES细胞）是一组来自小鼠胚胎内细胞团细胞（ICM）。ES细胞也可以有效地分化成不同胚系的细胞。PRMT1基因敲除的ES细胞是可以存活的，因此，我们利用mES基因敲除策略拟研究PRMT1在胚胎发育早期的功能及其调控机制。首先，我们利用CRISPR-Cas9技术在mES细胞中特异敲除PRMT1基因。发现PRMT1可以调节胚外内胚层（PrE）发育的关键调节基因Sox17，GATA4表达GATA6等。分子机制研究表明这个调节过程是通过多能性的维持核心成员KLF4的精氨酸甲基化来实现的。研究结果揭示PRMT1功能介导的Klf4甲基化在ES细胞向PrE分化过程中起重要作用。