组蛋白精氨酸甲基转移酶CARM1促进基因转录机制的研究

发布时间：2018-03-02 12:35

本文选题：CARM1　切入点：H3精氨酸甲基化　出处：《华东师范大学》2010年硕士论文　论文类型：学位论文

【摘要】：精氨酸甲基转移酶4(Protein Arginine Methyltransferase4, PRMT4)又称为共激活因子相关的精氨酸甲基转移酶1(Coactivator-Associated Arginine Methyltransferase1,CARM1),它催化组蛋白H3氮端的2、17和26号位,碳端的128、129、131和134号位以及H2A上的精氨酸甲基化。其中精氨酸17和26号位是该酶的主要催化位点,并且其甲基化会影响基因的转录。CARM1在细胞核激素受体(Nuclear Receptor, NR)介导的转录激活中起转录共激活因子的作用。在激素的刺激下,初级共激活因子p160首先与NR结合,同时招募次级共激活因子PRMT1、CBP/p300和CARM1等结合到p160上。其中,PRMT1对H4R3位点的甲基化修饰促进了CBP/p300对H3K14位点的乙酰化,反过来乙酰化又促进了CARM1催化的H3R17位点的甲基化修饰,而这一位点的甲基化与基因的转录激活有关。此外CARM1还能结合染色质转录重塑复合物,增强其ATP酶的活性,最终激活基因的转录。目前,CARM1甲基化H3R17和H3R26位点后如何促进基因转录的具体机制还尚不清楚。本论文则以此为出发点,研究CARM1促进基因转录的可能机制。为了寻找能够与H3R17和H3R26位点甲基化的多肽特异性结合的效应蛋白,我们首先在体外做了Pull-down实验。我们发现当H3多肽的R17和R26两个位点的精氨酸甲基化后,多肽结合核蛋白的能力显著降低。经质谱分析,结合降低的蛋白有MTA1、TIFlα和TIF1γ等转录共抑制因子,此结果通过放射自显影和蛋白质免疫印记得到验证。根据这些结果我们推测CARM1促进基因转录的分子机制之一是降低转录共抑制因子与组蛋白的结合。然后我们利用CARM1敲除型小鼠胚胎成纤维细胞检测此修饰是否也影响转录共抑制因子与染色质的结合。提取野生型小鼠胚胎成纤维细胞和敲除型细胞的组蛋白和染色质,通过蛋白质免疫印记检测转录共抑制因子的结合状态。我们发现CARM1敲除型细胞的组蛋白和染色质同野生型相比结合了更多的转录共抑制因子。另一方面,我们又检测了CARM1的过量表达是否能反过来降低转录共抑制因子与染色质的结合。我们成功建立了可诱导表达CARM1的稳定细胞系。该细胞经盐酸多西环素诱导6个小时后,CARM1蛋白过量表达,提取诱导和未诱导细胞的染色质,通过蛋白质免疫印记检测其结合的转录共抑制因子。我们发现CARM1过量表达的细胞的染色质结合转录共抑制因子的能力确实要比未诱导的低。综上所述,我们得出CARM1促进基因转录的分子机制可能是通过R17和R26的甲基化来降低转录共抑制因子与染色质的结合来实现的。
[Abstract]:Arginine methyltransferase 4 protein Arginine Methyltransferase4 (PRMT4), also known as the arginine methyltransferase associated with coactivator, Coactivator-Associated Arginine methyltransferase1 (CARM1), catalyzes the N-terminal of histone H3 at 2G17 and 26. Arginine methylation at the carbon terminal sites 128,129,131 and 134 and the arginine methylation on the H2A site. The arginine 17 and 26 sites are the main catalytic sites of the enzyme. And its methylation may affect the transcription of genes. CARM1 acts as a transcription co-activator in nuclear receptor nuclear receptor (NR1) -mediated transcriptional activation. Under hormone stimulation, primary co-activator p160 binds to NR first. At the same time, the secondary co-activator PRMT1CBP / p300 and CARM1 were recruited to bind to p160. the methylation of H4R3 site by PRMT1 promoted the acetylation of H3K14 site by CBP/p300, which in turn promoted the methylation of H3R17 site catalyzed by CARM1. The methylation of this site is related to the transcriptional activation of the gene. In addition, CARM1 can bind to the chromatin transcriptional remodeling complex, enhance the activity of its ATP enzyme, and finally activate the transcription of the gene. At present, it is not clear how to promote gene transcription after methylation of CARM1 at H3R17 and H3R26 sites. This paper studies the possible mechanism of CARM1 promoting gene transcription. In order to find the effector proteins that can specifically bind to the methylated peptides at H3R17 and H3R26 sites, we first performed Pull-down experiments in vitro. We found that the arginine methylation of the R17 and R26 sites of H3 peptides was performed in vitro. The ability of peptide binding nucleoprotein was significantly decreased. By mass spectrometric analysis, the reduced proteins included transcriptional co-inhibitory factors such as MTA1TIFl 伪 and TIF1 纬. The results were verified by autoradiography and protein-immunological imprinting. Based on these results, we speculate that one of the molecular mechanisms of CARM1 promoting gene transcription is to reduce the binding of transcription co-suppressor to histone. Then we used CARM1 knockout mouse embryonic fibroblasts to detect whether the modification also affected the binding of transcription co-suppressor to chromatin. We extracted histone and chromatin from wild mouse embryonic fibroblasts and knockout cells. We found that the histone and chromatin of CARM1 knockout cells combined more transcription co-suppressive factors than wild-type cells. On the other hand, We also examined whether overexpression of CARM1 could in turn reduce the binding of transcription co-suppressor to chromatin. We successfully established a stable cell line that could induce the expression of CARM1. The cell line was induced by doxycycline hydrochloride for 6 small cells. Overexpression of CARM1 protein, The chromatin binding ability of the cells with CARM1 overexpression was lower than that of the uninduced cells. In conclusion, we conclude that the molecular mechanism of CARM1 promoting gene transcription may be through the methylation of R17 and R26 to reduce the binding of transcription co-suppressor and chromatin.
【学位授予单位】：华东师范大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R3411

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