本文選題：SUVH2 + SUVH9��； 參考：《中國(guó)農(nóng)業(yè)大學(xué)》2016年博士論文

【摘要】：RNA介導(dǎo)的基因沉默在真核生物的基因表達(dá)調(diào)控中發(fā)揮重要作用,而RNA介導(dǎo)的DNA甲基化(RdDM)途徑是植物介導(dǎo)DNA重復(fù)序列與轉(zhuǎn)座子沉默,維持基因組穩(wěn)定的重要機(jī)制。在擬南芥中,包含SET結(jié)構(gòu)域的SU(VAR)3-9同源蛋白SUVH2與SUVH9是RdDM途徑的重要組分,但具體的作用機(jī)制還不清楚。在利用質(zhì)譜分析SUVH2和SUVH9 (SUVH2/9)的互作蛋白時(shí),我們發(fā)現(xiàn)SUVH2/9與RdDM途徑中的DDR蛋白復(fù)合體(包括DRD1、DMS3與RDM1)相互作用。利用酵母雙雜交,以及體內(nèi)蛋白免疫共沉淀(co-IP)與凝膠阻滯實(shí)驗(yàn),我們進(jìn)一步證實(shí)了SUVH2/9與DDR蛋白復(fù)合體的相互作用。通過(guò)SUVH2/9結(jié)構(gòu)域截短片段與DMS3結(jié)合實(shí)驗(yàn)的分析,表明SET結(jié)構(gòu)域的缺失并不影響SUVH2/9與DDR蛋白復(fù)合體的互作。另外SET結(jié)構(gòu)域保守位點(diǎn)的定點(diǎn)突變,也并不影響SUVH2在RdDM途徑中的作用。這說(shuō)明SUVH2/9在體內(nèi)可能并不具備組蛋白甲基轉(zhuǎn)移酶活性。之前的研究表明,DDR蛋白復(fù)合體能夠在全基因組范圍內(nèi)介導(dǎo)RNA聚合酶pol V與染色質(zhì)的結(jié)合。通過(guò)染色質(zhì)免疫沉淀的方法,我們發(fā)現(xiàn)在suvh2suvh9突變體中,DDR蛋白復(fù)合體和pol V與染色質(zhì)的結(jié)合都有顯著的下降,這說(shuō)明SUVH2和SUVH9通過(guò)促進(jìn)DDR蛋白復(fù)合體與染色質(zhì)結(jié)合,介導(dǎo)pol V在染色體上的招募過(guò)程,進(jìn)而參與RdDM途徑。MORC家族蛋白中的MORC1與MORC6通過(guò)改變?nèi)旧|(zhì)的構(gòu)象介導(dǎo)轉(zhuǎn)錄水平的基因沉默,其功能不依賴于相關(guān)位點(diǎn)的DNA甲基化或組蛋白修飾的改變。通過(guò)蛋白復(fù)合體的質(zhì)譜分析、酵母雙雜交以及體內(nèi)蛋白免疫共沉淀實(shí)驗(yàn),我們發(fā)現(xiàn)MORC6與MORC1及MORC2形成異源二聚體,并且能夠與SUVH2及SUVH9相互作用。對(duì)morc6與suvh2suvh9進(jìn)行全基因組甲基化測(cè)序分析,結(jié)果表明MORC6參與了一小部分RdDM位點(diǎn)DNA甲基化的形成過(guò)程。針對(duì)morc6與suvh2suvh9的RNA深度測(cè)序,結(jié)果表明在suvh2suvh9中轉(zhuǎn)錄特異上調(diào)的大部分位點(diǎn),DNA甲基化水平相對(duì)于野生型有明顯下降,而在suvh2suvh9與morc6中轉(zhuǎn)錄共同上調(diào)的位點(diǎn),DNA甲基化水平都與野生型相當(dāng),這說(shuō)明SUVH2/9除了參與RdDM途徑,還通過(guò)與MORC家族蛋白以不改變DNA甲基化的其它途徑參與了轉(zhuǎn)錄水平的基因沉默。通過(guò)熒光素酶互補(bǔ)以及蛋白免疫共沉淀實(shí)驗(yàn),我們證明MORC6能與RdDM途徑中重要組分IDN2以及SWI/SNF類型染色質(zhì)重塑因子相互作用。此外,利用RT-PCR,我們發(fā)現(xiàn)IDN2與SW13D也參與了MORC6作用位點(diǎn)的基因沉默過(guò)程。這些結(jié)果暗示MORC6在調(diào)控染色質(zhì)結(jié)構(gòu)過(guò)程中需要SUVH2/9、1DN2和SWI/SNF類型染色質(zhì)重塑因子的參與。以上結(jié)果表明,不具備組蛋白甲基轉(zhuǎn)移酶活性的SUVH2/9,通過(guò)結(jié)合DDR蛋白復(fù)合體負(fù)責(zé)pol V在染色質(zhì)上的招募,從而參與了RdDM途徑和轉(zhuǎn)錄水平基因沉默。此外,SUVH2/9還通過(guò)與MORC蛋白家族的相互作用,參與了MORC1/6介導(dǎo)的依賴于染色質(zhì)結(jié)構(gòu)調(diào)控的轉(zhuǎn)錄水平基因沉默過(guò)程。
[Abstract]:RNA mediated gene silencing plays an important role in the regulation of gene expression in eukaryotes, while RNA mediated DNA methylation RdDMpathway is an important mechanism of plant mediated DNA repeats and transposons silencing and maintaining genomic stability. In Arabidopsis thaliana, SU(VAR)3-9 homologous protein SUVH2 and SUVH9, which contain SET domain, are important components of RdDM pathway, but the specific mechanism is unclear. When we analyzed the interaction proteins of SUVH2 and SUVH9 SUVH2 / 9 by mass spectrometry, we found that the DDR protein complex (including DRD1DMS3 and RDM1) in SUVH2/9 and RdDM pathway interact with each other. The interaction between SUVH2/9 and DDR protein complex was further confirmed by yeast two-hybrid and protein co-precipitation co-IPI in vivo and gel block assay. The analysis of SUVH2/9 domain truncation and DMS3 binding experiments showed that the absence of SET domain did not affect the interaction between SUVH2/9 and DDR protein complex. In addition, site-directed mutation of conserved loci in SET domain did not affect the role of SUVH2 in the RdDM pathway. This suggests that SUVH2/9 may not have histone methyltransferase activity in vivo. Previous studies have shown that the DDR protein complex can mediate the binding of RNA polymerase pol V to chromatin on a genome-wide basis. By the method of chromatin immunoprecipitation, we found that the binding of suvh2suvh9 protein complex and pol V to chromatin decreased significantly in suvh2suvh9 mutants, which indicated that SUVH2 and SUVH9 could promote the binding of DDR protein complex to chromatin. Mediating the recruitment of pol V on chromosomes, thus participating in the RdDM pathway. MORC1 and MORC6 in the MORC family proteins mediate transcriptional gene silencing by changing chromatin conformation. Its function is independent of DNA methylation or histone modification at the related sites. By mass spectrometry of protein complex, yeast two-hybrid and protein immunoprecipitation in vivo, we found that MORC6 formed heterodimer with MORC1 and MORC2, and could interact with SUVH2 and SUVH9. The whole genome methylation of morc6 and suvh2suvh9 was analyzed. The results showed that MORC6 was involved in the formation of DNA methylation at a small number of RdDM sites. The results of RNA deep sequencing of morc6 and suvh2suvh9 showed that the methylation level of most of the up-regulated sites in suvh2suvh9 was significantly lower than that of wild type. The level of SUVH2/9 methylation in suvh2suvh9 and morc6 is similar to that in wild type, which indicates that SUVH2/9 participates in the RdDM pathway and participates in gene silencing at the transcription level through other pathways that do not alter DNA methylation with MORC family proteins. By luciferase complementation and protein immunoprecipitation, we demonstrated that MORC6 interacts with IDN2, an important component of the RdDM pathway, and SWI/SNF type chromatin remodeling factors. In addition, using RT-PCR, we found that IDN2 and SW13D are also involved in the gene silencing process of MORC6 interaction sites. These results suggest that MORC6 requires the involvement of SUVH2 / 91DN2 and SWI/SNF type chromatin remodeling factors in the regulation of chromatin structure. These results suggest that SUVH _ 2 / 9, which does not have histone methyltransferase activity, is involved in RdDM pathway and transcription level gene silencing by binding DDR protein complex to pol V in chromatin recruitment. In addition, SUVH _ 2 / 9 participates in the transcription-level gene silencing mediated by MORC1/6 through interaction with the MORC protein family.
【學(xué)位授予單位】：中國(guó)農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：Q78

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