SAHF核心元件BRG1-HP1-H3K9me复合物的鉴定

发布时间：2018-09-17 14:10

【摘要】：研究背景和意义癌基因诱导的细胞衰老(Oncogene-induced senescence,OIS)是当今衰老和癌症研究领域的一个重要热点。SAHF(senescence-associated heterochromatic foci)在OIS的发生和维持中具有核心地位,因此,SAHF形成的机制是目前OIS研究中最受关注的问题之一。普通异染色质,其紧密折叠结构具有解散的潜力,而SAHF紧密折叠结构具有不可逆转性,导致这一现象的机理尚不明确。基于我们前期的研究和最新的结果,我们推测在衰老的细胞中存在BRG1-HP1-H3K9me复合物,而该复合物是SAHF形成的核心元件,能使SAHF紧密折叠不可逆转。研究方法与结果在本研究中,我们通过表达H-RAS~(G12V)诱导了细胞衰老,通过免疫荧光染色,我们发现在RAS过表达的细胞中BRG1、HP1和H3K9me共定位,且都存在于SAHF中。免疫共沉淀实验结果也显示,在RAS激活细胞中,BRG1、HP1和H3K9me之间的相互作用显著增强。此外,GST-Pull down的结果证实BRG1和HP1以及H3K9me之间存在相互作用。在此基础上,我们通过过表达BRG1(295-634AA)或者BRG1(1223-1420AA)竞争性地抑制BRG1-HP1-H3K9me复合物的形成,进而抑制了RAS诱导的SAHF的形成。证明了BRG1-HP1-H3K9me复合物是SAHF形成的核心结构。结论本研究证实了RAS诱导的衰老细胞中存在BRG1-HP1-H3K9me蛋白复合物,这些复合物是SAHF形成的核心元件。这些结果将有助于推进SAHF形成的机制研究,并帮助研究人员深入了解复杂的OIS过程。
[Abstract]:Background and significance of oncogene-induced cell senescence (Oncogene-induced senescence,OIS) is an important research hotspot in the field of aging and cancer. SAHF-SAHF (senescence-associated heterochromatic foci) plays a central role in the occurrence and maintenance of OIS). Therefore, the mechanism of formation of OIS is one of the most concerned issues. Common heterochromatin has the potential to dissolve its compact folding structure, while the SAHF compact folding structure has irreversibility, so the mechanism of this phenomenon is not clear. Based on our previous studies and recent results, we speculate that there is a BRG1-HP1-H3K9me complex in aging cells, which is the core component of SAHF formation and makes SAHF tightly folded irreversibly. Methods and results in this study, we induced cell senescence by expression of H-RAS-G12V. By immunofluorescence staining, we found that BRG1,HP1 and H3K9me were co-localized in the overexpression of RAS and both existed in SAHF. The results of co-immunoprecipitation also showed that the interaction between HP1 and H3K9me was significantly enhanced in RAS activated cells. In addition, the results of GST-Pull down confirm the interaction between BRG1, HP1 and H3K9me. On this basis, we competitively inhibit the formation of BRG1-HP1-H3K9me complexes by overexpression of BRG1 (295-634AA) or BRG1 (1223-1420AA), and further inhibit the formation of SAHF induced by RAS. It is proved that BRG1-HP1-H3K9me complex is the core structure of SAHF. Conclusion this study confirmed the existence of BRG1-HP1-H3K9me protein complexes in RAS-induced aging cells, which are the core components of SAHF formation. These results will help advance the mechanism of SAHF formation and help researchers understand complex OIS processes in depth.
【学位授予单位】：江苏大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R3416

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