RBFOX2基因组范围内招募RPC2和调节H3K27me3修饰的分子机制研究
发布时间：2018-08-04 14:02
【摘要】：Polycomb Repressive Complex 2(PRC2)是具有组蛋白甲基转移酶活性的复合物,可以通过催化H3K27me3修饰来抑制基因转录活性。关于PRC2如何被招募到染色质特定区域的研究尚存在许多争议,是目前表观遗传学研究领域的热点和难点之一。最新的生化实验证明PRC2可以混乱结合RNA,另外细胞内数据也表明PRC2亚基可以结合处于适量表达水平的mRNA,并且更倾向于结合5‘端区域。因此研究者提出PRC2可以通过结合新生RNA(Nascent RNA)被招募到染色质基因启动子区域。RNA结合蛋白FOX2(RNA Binding Protein FOX2,RBFOX2)是广泛存在于多种组织和细胞中的剪切因子,该蛋白对神经、心肌和骨骼肌系统正常发育和功能维持具有十分重要的功能。本组之前研究表明RBFOX2除了具有剪切调节功能外,还具有抑制基因转录活性的作用。本文经过一列实验研究,证明RBFOX2可以通过招募PRC2来发挥转录抑制作用,并且在胚胎干细胞(Embryonic Stem Cells,ESCs)中该招募过程受到ERK1/2通路的调节。本论文主要包括以下实验结果:(1)RBFOX2 ChIP-seq数据显示RBFOX2主要结合基因转录起始位点(Transcription Start Sites,TSS);经比对分析后发现RBFOX2和PRC2亚基SUZ12结合位点十分接近,并且RBFOX2信号聚集峰值和SUZ12峰值也高度重合。另一方面,通过特异基因诱导表达模型发现RBFXO2是通过转录激活产生的新生RNA招募到基因转录起始区域。(2)免疫共沉淀实验(Co-Immunoprecipitation,Co-IP)证明RBFOX2和PRC2可以不依赖RNA或DNA直接蛋白-蛋白相互作用。体外GST-Pulldown也同样证明RBFOX2可以捕获整个PRC2,并且RBFOX2的C端区域同PRC2相互作用。(3)ESCs在常规培养体系下,RBFox2基因沉默会引起整个基因组启动子区域H3K27me3修饰降低;但是在“2i”(ERK1/2通路抑制剂PD0325901和GSK3抑制剂CHIR99021)培养体系下,RBFox2沉默不会引起启动子区域H3K27me3修饰变化。(4)RBFOX2具有两个主要的剪切变异体,分别命名为RBFOX2a和RBFOX2f,不同细胞中主要的剪切变异体不同。并且在干细胞分化过程中会出现主要剪切变异体从RBFOX2a向RBFOX2f转变的过程。另外,ERK1/2信号通路激活可以磷酸化RBFOX2a,不能磷酸化RBFOX2f。(5)RBFOX2磷酸化修饰不会改变自身的亚细胞定位,也不能改变同PRC2的相互作用能力。但是RBFOX2磷酸化修饰会改变自身RNA结合能力和特异性剪切调节能力。通过以上结果,我们对新生RNA介导的PRC2招募方式进行改进,得出新的PRC2招募模型:启动子区域新生RNA捕获RBFOX2蛋白,RBFOX2通过蛋白-蛋白相互作用招募PRC2到基因启动子区域。当该基因转录活性较高,转录激活标记H3K4me3和H3K36me3会抑制PRC2染色质结合及催化活性,PRC2无法催化H3K27me3修饰;当基因转录活性适中或较低,PRC2活性不会被抑制,可以催化H3K27me修饰,防止这些基因过度激活。在这个模型中,编码基因转录生成的mRNA除了翻译蛋白发挥功能外,还可以作为自身转录活性的信号,通过招募其他转录调节蛋白来维持局部转录活性稳定。另一方面,ESCs在“2i”培养体系下会处于更加原始状态(Naive)状态,并且基因组范围内启动子区域H3K27me3修饰水平更低。我们认为ERK1/2通路抑制剂可以使RBFOX2去磷酸化改变其RNA结合能力,进而降低PRC2招募能力,这一途径是ESCs在原始状态下具有非常低的H3K27me3修饰的原因之一。
[Abstract]:Polycomb Repressive Complex 2 (PRC2) is a complex of histone methyltransferase activity, which can inhibit gene transcription activity by catalyzing H3K27me3 modification. There are many controversies about how PRC2 is recruited to specific chromatin areas. It is one of the hotspots and difficulties in the field of epigenetic studies. Biochemical experiments have shown that PRC2 can be confused with RNA, and the intracellular data also indicate that PRC2 subunits can bind to a moderate level of mRNA and tend to bind to the 5 'end region. Therefore, the researchers suggest that PRC2 can be recruited to the chromatic gene promoter region.RNA binding protein FOX2 (RNA B) by combining new RNA (Nascent RNA). Inding Protein FOX2, RBFOX2) is a shear factor widely distributed in a variety of tissues and cells. This protein has a very important function in the normal development and function maintenance of nerve, myocardium and skeletal muscle. Previous studies have shown that RBFOX2 has the effect of inhibiting gene transcriptional activity in addition to shear regulation. After a series of experimental studies, it has been shown that RBFOX2 can play a transcriptional inhibition by recruiting PRC2 and that the recruitment process is regulated by ERK1/2 pathway in Embryonic Stem Cells (ESCs). This paper mainly includes the following experimental results: (1) RBFOX2 ChIP-seq data shows that RBFOX2 mainly combines the gene transcription starting site (Trans) Cription Start Sites, TSS); after comparison analysis found that RBFOX2 and PRC2 subunit SUZ12 binding sites are very close, and RBFOX2 signal aggregation peak and SUZ12 peak are also highly recoincide. On the other hand, through specific gene induced expression model, RBFXO2 is found to be recruited to the gene transcriptional starting region through the activation of the birth of the newborn RNA. (2) Co-Immunoprecipitation (Co-IP) proved that RBFOX2 and PRC2 can not rely on RNA or DNA direct protein protein interaction. In vitro GST-Pulldown also proves that RBFOX2 can capture the whole PRC2 and the C end region of RBFOX2 interacts with PRC2. (3) under the regular culture system, the gene silencing will cause the whole base. H3K27me3 modification in the promoter region of the group is reduced; but under the culture of "2I" (ERK1/2 pathway inhibitor PD0325901 and GSK3 inhibitor CHIR99021), RBFox2 silence does not cause H3K27me3 modification in the promoter region. (4) RBFOX2 has two major shear variants, named RBFOX2a and RBFOX2f, and the main scissors in different cells. In the process of stem cell differentiation, the main shear variants change from RBFOX2a to RBFOX2f. In addition, the activation of ERK1/2 signal pathway can phosphorylate RBFOX2a, and the phosphorylation of RBFOX2f. (5) RBFOX2 can not change the subcellular localization of its own and can not change the interaction capacity with PRC2. But RBFOX2 phosphorylation modification could change the ability of RNA binding and specific shear regulation. Through the above results, we improved the recruitment of PRC2 by new RNA, and obtained a new PRC2 recruitment model: the newborn RNA in the promoter region captured RBFOX2 protein, RBFOX2 through protein protein interaction recruited PRC2 to gene initiation. When the transcriptional activity is high, transcriptional activation markers H3K4me3 and H3K36me3 inhibit PRC2 chromatin binding and catalytic activity, PRC2 can not catalyze H3K27me3 modification; when the gene transcriptional activity is moderate or low, the PRC2 activity is not suppressed, and the H3K27me modification can be catalyzed to prevent these genes from overactivating. In this model, the coding is encoded. In addition to the function of the translated protein, mRNA can also serve as a signal of its own transcriptional activity and maintain local transcriptional activity by recruiting other transcriptional regulatory proteins. On the other hand, ESCs will be in a more primitive state (Naive) state under the "2I" culture system, and the promoter region H3K2 is initiated within the genome range. The level of 7me3 modification is lower. We think that the ERK1/2 pathway inhibitor can cause RBFOX2 dephosphorylation to change its RNA binding ability and reduce PRC2 recruitment, which is one of the reasons that ESCs has very low H3K27me3 modification in the original state.
【学位授予单位】：重庆大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q78
，
本文编号：2164114
[Abstract]:Polycomb Repressive Complex 2 (PRC2) is a complex of histone methyltransferase activity, which can inhibit gene transcription activity by catalyzing H3K27me3 modification. There are many controversies about how PRC2 is recruited to specific chromatin areas. It is one of the hotspots and difficulties in the field of epigenetic studies. Biochemical experiments have shown that PRC2 can be confused with RNA, and the intracellular data also indicate that PRC2 subunits can bind to a moderate level of mRNA and tend to bind to the 5 'end region. Therefore, the researchers suggest that PRC2 can be recruited to the chromatic gene promoter region.RNA binding protein FOX2 (RNA B) by combining new RNA (Nascent RNA). Inding Protein FOX2, RBFOX2) is a shear factor widely distributed in a variety of tissues and cells. This protein has a very important function in the normal development and function maintenance of nerve, myocardium and skeletal muscle. Previous studies have shown that RBFOX2 has the effect of inhibiting gene transcriptional activity in addition to shear regulation. After a series of experimental studies, it has been shown that RBFOX2 can play a transcriptional inhibition by recruiting PRC2 and that the recruitment process is regulated by ERK1/2 pathway in Embryonic Stem Cells (ESCs). This paper mainly includes the following experimental results: (1) RBFOX2 ChIP-seq data shows that RBFOX2 mainly combines the gene transcription starting site (Trans) Cription Start Sites, TSS); after comparison analysis found that RBFOX2 and PRC2 subunit SUZ12 binding sites are very close, and RBFOX2 signal aggregation peak and SUZ12 peak are also highly recoincide. On the other hand, through specific gene induced expression model, RBFXO2 is found to be recruited to the gene transcriptional starting region through the activation of the birth of the newborn RNA. (2) Co-Immunoprecipitation (Co-IP) proved that RBFOX2 and PRC2 can not rely on RNA or DNA direct protein protein interaction. In vitro GST-Pulldown also proves that RBFOX2 can capture the whole PRC2 and the C end region of RBFOX2 interacts with PRC2. (3) under the regular culture system, the gene silencing will cause the whole base. H3K27me3 modification in the promoter region of the group is reduced; but under the culture of "2I" (ERK1/2 pathway inhibitor PD0325901 and GSK3 inhibitor CHIR99021), RBFox2 silence does not cause H3K27me3 modification in the promoter region. (4) RBFOX2 has two major shear variants, named RBFOX2a and RBFOX2f, and the main scissors in different cells. In the process of stem cell differentiation, the main shear variants change from RBFOX2a to RBFOX2f. In addition, the activation of ERK1/2 signal pathway can phosphorylate RBFOX2a, and the phosphorylation of RBFOX2f. (5) RBFOX2 can not change the subcellular localization of its own and can not change the interaction capacity with PRC2. But RBFOX2 phosphorylation modification could change the ability of RNA binding and specific shear regulation. Through the above results, we improved the recruitment of PRC2 by new RNA, and obtained a new PRC2 recruitment model: the newborn RNA in the promoter region captured RBFOX2 protein, RBFOX2 through protein protein interaction recruited PRC2 to gene initiation. When the transcriptional activity is high, transcriptional activation markers H3K4me3 and H3K36me3 inhibit PRC2 chromatin binding and catalytic activity, PRC2 can not catalyze H3K27me3 modification; when the gene transcriptional activity is moderate or low, the PRC2 activity is not suppressed, and the H3K27me modification can be catalyzed to prevent these genes from overactivating. In this model, the coding is encoded. In addition to the function of the translated protein, mRNA can also serve as a signal of its own transcriptional activity and maintain local transcriptional activity by recruiting other transcriptional regulatory proteins. On the other hand, ESCs will be in a more primitive state (Naive) state under the "2I" culture system, and the promoter region H3K2 is initiated within the genome range. The level of 7me3 modification is lower. We think that the ERK1/2 pathway inhibitor can cause RBFOX2 dephosphorylation to change its RNA binding ability and reduce PRC2 recruitment, which is one of the reasons that ESCs has very low H3K27me3 modification in the original state.
【学位授予单位】：重庆大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q78
，
本文编号：2164114
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