SET结构域蛋白SUVH2与SUVH9在转录水平基因沉默中的功能研究

发布时间：2018-06-06 03:28

本文选题：SUVH2 + SUVH9　；参考：《中国农业大学》2016年博士论文

【摘要】：RNA介导的基因沉默在真核生物的基因表达调控中发挥重要作用,而RNA介导的DNA甲基化(RdDM)途径是植物介导DNA重复序列与转座子沉默,维持基因组稳定的重要机制。在拟南芥中,包含SET结构域的SU(VAR)3-9同源蛋白SUVH2与SUVH9是RdDM途径的重要组分,但具体的作用机制还不清楚。在利用质谱分析SUVH2和SUVH9 (SUVH2/9)的互作蛋白时,我们发现SUVH2/9与RdDM途径中的DDR蛋白复合体(包括DRD1、DMS3与RDM1)相互作用。利用酵母双杂交,以及体内蛋白免疫共沉淀(co-IP)与凝胶阻滞实验,我们进一步证实了SUVH2/9与DDR蛋白复合体的相互作用。通过SUVH2/9结构域截短片段与DMS3结合实验的分析,表明SET结构域的缺失并不影响SUVH2/9与DDR蛋白复合体的互作。另外SET结构域保守位点的定点突变,也并不影响SUVH2在RdDM途径中的作用。这说明SUVH2/9在体内可能并不具备组蛋白甲基转移酶活性。之前的研究表明,DDR蛋白复合体能够在全基因组范围内介导RNA聚合酶pol V与染色质的结合。通过染色质免疫沉淀的方法,我们发现在suvh2suvh9突变体中,DDR蛋白复合体和pol V与染色质的结合都有显著的下降,这说明SUVH2和SUVH9通过促进DDR蛋白复合体与染色质结合,介导pol V在染色体上的招募过程,进而参与RdDM途径。MORC家族蛋白中的MORC1与MORC6通过改变染色质的构象介导转录水平的基因沉默,其功能不依赖于相关位点的DNA甲基化或组蛋白修饰的改变。通过蛋白复合体的质谱分析、酵母双杂交以及体内蛋白免疫共沉淀实验,我们发现MORC6与MORC1及MORC2形成异源二聚体,并且能够与SUVH2及SUVH9相互作用。对morc6与suvh2suvh9进行全基因组甲基化测序分析,结果表明MORC6参与了一小部分RdDM位点DNA甲基化的形成过程。针对morc6与suvh2suvh9的RNA深度测序,结果表明在suvh2suvh9中转录特异上调的大部分位点,DNA甲基化水平相对于野生型有明显下降,而在suvh2suvh9与morc6中转录共同上调的位点,DNA甲基化水平都与野生型相当,这说明SUVH2/9除了参与RdDM途径,还通过与MORC家族蛋白以不改变DNA甲基化的其它途径参与了转录水平的基因沉默。通过荧光素酶互补以及蛋白免疫共沉淀实验,我们证明MORC6能与RdDM途径中重要组分IDN2以及SWI/SNF类型染色质重塑因子相互作用。此外,利用RT-PCR,我们发现IDN2与SW13D也参与了MORC6作用位点的基因沉默过程。这些结果暗示MORC6在调控染色质结构过程中需要SUVH2/9、1DN2和SWI/SNF类型染色质重塑因子的参与。以上结果表明,不具备组蛋白甲基转移酶活性的SUVH2/9,通过结合DDR蛋白复合体负责pol V在染色质上的招募,从而参与了RdDM途径和转录水平基因沉默。此外,SUVH2/9还通过与MORC蛋白家族的相互作用,参与了MORC1/6介导的依赖于染色质结构调控的转录水平基因沉默过程。
[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.
【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q78

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