SAHF核心元件BRG1-HP1-H3K9me复合物的鉴定与功能研究

Oncogene-induced senescence (OIS) is one of the most important topics in today's aging and cancer research areas. Senescence-associated heterochromatic foci (SAHF) play an essential role in the initiation and maintenance of OIS. The mechanism of SAHF formation and the specific gene targets suppressed by SAHF are the two most appealing aspects of OIS study. A major difference between SAHF and common heterochromatin is that the folding of the tightly packed structures of SAHF is irreversible. In addition, increasing evidence supports that SAHF are specific for their gene targets, and this selective suppression of gene transcription is crucial to the regulation of OIS process. Basing on our previous studies and latest results, we hypothesize that BRG1-HP1-H3K9me protein complexes do exist in senescent cells, and these complexes are the core structures for SAHF formation. In this project, we propose to overexpress H-RasG12V to induce cellular senescence and use a variety of methods to prove the existence of BRG1-HP1-H3K9me complexes in senescent cells. Then we plan to use a series of methods to regulate the formation of BRG1-HP1-H3K9me complexes and study their effects on SAHF formation. Additionally, we will search for other proteins which might also interact with BRG1-HP1-H3K9me complexes. We also will investigate whether the activation of other oncogenes will induce BRG1-HP1-H3K9me complexes, and whether these complexes exist in other cell lines after Ras activation. After we have a comprehensive understanding of BRG1-HP1-H3K9me complexes, we will search for the bound DNA of these complexes genome-wide, and verify these gene candidates. This study is expected to have great contribution to the mechanism of SAHF formation, and help researchers to profoundly understand complex oncogene-induced senescence. By searching for preferential targets of SAHF, we may nominate novel targets for cancer treatment and provide better theoretical foundation for the development of diagnostic and therapeutic reagents to treat cancers.

癌基因诱导的细胞衰老是当今衰老和癌症研究领域的一大热点。衰老相关的异染色质位点（SAHF）在癌基因诱导细胞衰老的发生和维持中极为重要。SAHF形成的分子机理及其对靶基因的选择性是当前癌基因诱导细胞衰老研究领域的两个重要问题。基于前期研究和最新的结果，我们提出：衰老细胞中存在BRG1-HP1-H3K9me复合物，它们是SAHF形成的核心元件。在本项目中，我们将验证BRG1-HP1-H3K9me复合物在衰老细胞中的存在，研究复合物的变化对SAHF形成的影响，搜寻其他可能与复合物结合的蛋白，并检验其他癌基因激活的衰老细胞中是否有相同复合物；同时以该复合物为导向，筛选和验证SAHF在全基因组范围内的抑制靶点。通过以上研究，有望对SAHF的形成机理进行深刻阐述，加深对癌基因诱导细胞衰老这一复杂有趣的生物学现象的理解；同时，为发现新的肿瘤治疗靶蛋白，开发新型癌症诊断和治疗试剂提供理论指导。

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。.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激活的细胞中，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。.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。