RBFOX2基因組范圍內(nèi)招募RPC2和調(diào)節(jié)H3K27me3修飾的分子機(jī)制研究
發(fā)布時(shí)間：2018-08-04 14:02
【摘要】：Polycomb Repressive Complex 2(PRC2)是具有組蛋白甲基轉(zhuǎn)移酶活性的復(fù)合物,可以通過催化H3K27me3修飾來抑制基因轉(zhuǎn)錄活性。關(guān)于PRC2如何被招募到染色質(zhì)特定區(qū)域的研究尚存在許多爭(zhēng)議,是目前表觀遺傳學(xué)研究領(lǐng)域的熱點(diǎn)和難點(diǎn)之一。最新的生化實(shí)驗(yàn)證明PRC2可以混亂結(jié)合RNA,另外細(xì)胞內(nèi)數(shù)據(jù)也表明PRC2亞基可以結(jié)合處于適量表達(dá)水平的mRNA,并且更傾向于結(jié)合5‘端區(qū)域。因此研究者提出PRC2可以通過結(jié)合新生RNA(Nascent RNA)被招募到染色質(zhì)基因啟動(dòng)子區(qū)域。RNA結(jié)合蛋白FOX2(RNA Binding Protein FOX2,RBFOX2)是廣泛存在于多種組織和細(xì)胞中的剪切因子,該蛋白對(duì)神經(jīng)、心肌和骨骼肌系統(tǒng)正常發(fā)育和功能維持具有十分重要的功能。本組之前研究表明RBFOX2除了具有剪切調(diào)節(jié)功能外,還具有抑制基因轉(zhuǎn)錄活性的作用。本文經(jīng)過一列實(shí)驗(yàn)研究,證明RBFOX2可以通過招募PRC2來發(fā)揮轉(zhuǎn)錄抑制作用,并且在胚胎干細(xì)胞(Embryonic Stem Cells,ESCs)中該招募過程受到ERK1/2通路的調(diào)節(jié)。本論文主要包括以下實(shí)驗(yàn)結(jié)果:(1)RBFOX2 ChIP-seq數(shù)據(jù)顯示RBFOX2主要結(jié)合基因轉(zhuǎn)錄起始位點(diǎn)(Transcription Start Sites,TSS);經(jīng)比對(duì)分析后發(fā)現(xiàn)RBFOX2和PRC2亞基SUZ12結(jié)合位點(diǎn)十分接近,并且RBFOX2信號(hào)聚集峰值和SUZ12峰值也高度重合。另一方面,通過特異基因誘導(dǎo)表達(dá)模型發(fā)現(xiàn)RBFXO2是通過轉(zhuǎn)錄激活產(chǎn)生的新生RNA招募到基因轉(zhuǎn)錄起始區(qū)域。(2)免疫共沉淀實(shí)驗(yàn)(Co-Immunoprecipitation,Co-IP)證明RBFOX2和PRC2可以不依賴RNA或DNA直接蛋白-蛋白相互作用。體外GST-Pulldown也同樣證明RBFOX2可以捕獲整個(gè)PRC2,并且RBFOX2的C端區(qū)域同PRC2相互作用。(3)ESCs在常規(guī)培養(yǎng)體系下,RBFox2基因沉默會(huì)引起整個(gè)基因組啟動(dòng)子區(qū)域H3K27me3修飾降低;但是在“2i”(ERK1/2通路抑制劑PD0325901和GSK3抑制劑CHIR99021)培養(yǎng)體系下,RBFox2沉默不會(huì)引起啟動(dòng)子區(qū)域H3K27me3修飾變化。(4)RBFOX2具有兩個(gè)主要的剪切變異體,分別命名為RBFOX2a和RBFOX2f,不同細(xì)胞中主要的剪切變異體不同。并且在干細(xì)胞分化過程中會(huì)出現(xiàn)主要剪切變異體從RBFOX2a向RBFOX2f轉(zhuǎn)變的過程。另外,ERK1/2信號(hào)通路激活可以磷酸化RBFOX2a,不能磷酸化RBFOX2f。(5)RBFOX2磷酸化修飾不會(huì)改變自身的亞細(xì)胞定位,也不能改變同PRC2的相互作用能力。但是RBFOX2磷酸化修飾會(huì)改變自身RNA結(jié)合能力和特異性剪切調(diào)節(jié)能力。通過以上結(jié)果,我們對(duì)新生RNA介導(dǎo)的PRC2招募方式進(jìn)行改進(jìn),得出新的PRC2招募模型:啟動(dòng)子區(qū)域新生RNA捕獲RBFOX2蛋白,RBFOX2通過蛋白-蛋白相互作用招募PRC2到基因啟動(dòng)子區(qū)域。當(dāng)該基因轉(zhuǎn)錄活性較高,轉(zhuǎn)錄激活標(biāo)記H3K4me3和H3K36me3會(huì)抑制PRC2染色質(zhì)結(jié)合及催化活性,PRC2無法催化H3K27me3修飾;當(dāng)基因轉(zhuǎn)錄活性適中或較低,PRC2活性不會(huì)被抑制,可以催化H3K27me修飾,防止這些基因過度激活。在這個(gè)模型中,編碼基因轉(zhuǎn)錄生成的mRNA除了翻譯蛋白發(fā)揮功能外,還可以作為自身轉(zhuǎn)錄活性的信號(hào),通過招募其他轉(zhuǎn)錄調(diào)節(jié)蛋白來維持局部轉(zhuǎn)錄活性穩(wěn)定。另一方面,ESCs在“2i”培養(yǎng)體系下會(huì)處于更加原始狀態(tài)(Naive)狀態(tài),并且基因組范圍內(nèi)啟動(dòng)子區(qū)域H3K27me3修飾水平更低。我們認(rèn)為ERK1/2通路抑制劑可以使RBFOX2去磷酸化改變其RNA結(jié)合能力,進(jìn)而降低PRC2招募能力,這一途徑是ESCs在原始狀態(tài)下具有非常低的H3K27me3修飾的原因之一。
[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.
【學(xué)位授予單位】：重慶大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：Q78
，
本文編號(hào)：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.
【學(xué)位授予單位】：重慶大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：Q78
，
本文編號(hào)：2164114
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