组蛋白去甲基化酶KDM5B是调节基因组稳定性的关键因子

发布时间：2018-03-28 16:05

本文选题：KDM5B　切入点：PARP1　出处：《天津医科大学》2017年博士论文

【摘要】：目的:维持基因组的稳定性是正常机体发育所必需的,并且对于预防癌症等疾病至关重要。基因的信息被包装进了染色质中,越来越多的证据表明染色质的环境在DNA损伤应答中起了重要作用。但是,目前尚不完全清楚DNA修复是如何被表观机制所调控的。本论文旨在研究赖氨酸特异的组蛋白去甲基化酶KDM5B在DNA双链损伤应答的各个方面所发挥的作用。方法与结果:1)通过细胞核内蛋白质组分分离实验,发现在DNA损伤应答时,KDM5B可以更多的与染色质结合;2)在细胞内经稳定转染引入核酸内切酶I-Sce1/AsiSI的识别序列,过表达I-Sce1/AsiSI核酸内切酶质粒诱导DNA双链损伤,通过染色质免疫共沉淀(Ch IP)实验,发现KDM5B可以被招募到DNA双链损伤位点附近的染色质区域;3)通过免疫共沉淀实验,发现KDM5B与PARP1有相互作用,并且此作用在DNA损伤时增强,与之相对应的,损伤时KDM5B的多聚腺苷二磷酸核糖化水平增高;4)分离与染色质相结合的细胞核蛋白及全细胞裂解液发现,敲低PARP1会影响DNA损伤时KDM5B与染色质的结合,若加入PJ-34(PARP1的酶活性抑制剂),也会影响KDM5B在染色质上的富集。通过定量染色质免疫共沉淀(qCh IP)实验,发现无论是PARP1的酶活性还是其与KDM5B的结合都影响KDM5B富集在DNA损伤区域的染色质上;5)利用NHEJ-GFP系统以及HR-DRGFP系统,通过FACS发现DNA双链损伤时,KDM5B能影响NHEJ及HR的修复效率;6)引入KDM5B无酶活性的突变体,利用NHEJ-GFP系统以及HR-DRGFP系统,通过流式细胞仪,发现KDM5B在DNA损伤中的作用依赖其去甲基化酶活性;进一步通过染色质免疫共沉淀(ChIP)实验,发现KDM5B能改变DNA损伤区域染色质的H3K4me3水平;7)通过免疫荧光实验以及peptide pull-down实验发现,DNA发生损伤时,KDM5B能招募BRCA1至损伤区域,影响其在损伤位点形成foci,且此作用依赖于KDM5B的去甲基化酶活性。但KDM5B对MDC1及gH2AX的IR后foci形成无影响;通过激光微束照射及免疫荧光实验,发现在DNA发生损伤时,KDM5B还能招募Ku70至损伤区域,此过程同样依赖于KDM5B的去甲基化酶活性;8)IR照射细胞或者加入NCS造成DNA双链损伤,发现KDM5B能影响损伤后检测点蛋白磷酸化水平;9)通过流式细胞术检测细胞周期,敲低KDM5B或者ATM并对细胞进行IR照射或者NCS处理,发现敲低KDM5B能增强细胞对基因毒性物质的敏感性;10)通过流式细胞术检测细胞周期,给予细胞IR照射或者转染siRNA等不同处理,发现KDM5B能影响细胞周期进程,敲低KDM5B能使细胞G1/S期过渡延迟,尤其是IR造成DNA损伤时,敲低KDM5B造成的对细胞周期的影响更加明显;11)通过在p53+/+细胞及p53-/-细胞内敲低KDM5B,Western blotting检测p53通路相关蛋白表达水平,发现KDM5B能影响p53+/+细胞中p53通路相关蛋白表达水平,但对于p53-/-细胞中p53通路蛋白无影响;而用流式细胞术检测细胞周期,发现KDM5B对细胞周期的影响只有在表达p53的细胞中存在,KDM5B对细胞周期的影响依赖于p53;结论:细胞经电离或内切酶处理后,KDM5B能富集在DNA损伤区域,这个过程是依赖于PARP1的。KDM5B是有效修复DNA双链损伤及招募Ku70与BRCA1的不可缺少的组分。KDM5B缺失能激活p53信号通路并使细胞对基因毒性损害更加敏感。此论文的结果证明KDM5B确实是一个DNA损伤反应蛋白,指出KDM5B是一个重要的基因守护者,是基因组稳定性的调节者。此论文使人们深入理解表观遗传调控对维持基因稳定的重要作用。
[Abstract]:Objective: normal body development required for maintaining genome stability, and for the prevention of cancer and other diseases is essential. Gene information is packaged into chromatin, more and more evidence that chromatin environment plays an important role in the DNA damage response. However, it is not entirely clear how DNA repair the apparent mechanism of regulation. This paper aims at demethylase KDM5B play in the various aspects of DNA double strand damage response of lysine specific histone function. Methods and results: 1) the nucleus protein separation experiments, found in response to DNA damage, KDM5B can be more binding with chromatin; 2) recognition sequence in cells by endonuclease I-Sce1/AsiSI after stable transfection, I-Sce1/AsiSI overexpression plasmid endonuclease induced DNA double strand damage, by chromatin immunoprecipitation ( Ch IP) experiment, found that KDM5B can be recruited to DNA double strand damage chromatin regions near the site; 3) by CO immunoprecipitation experiments showed that KDM5B interacted with PARP1, and this effect increased in response to DNA damage, and the corresponding damage, KDM5B poly adenosine ribose phosphate of two the increased level of nuclear protein; 4) separation and combination of chromatin and whole cell lysates showed that knockdown of PARP1 may affect the binding of KDM5B with chromatin during DNA damage, adding PJ-34 (enzyme inhibitor PARP1), will also affect the quality of the enrichment of KDM5B in dyeing. Co precipitation by quantitative chromatin immune (qCh IP) experiment, found that both the PARP1 activity or the combination of KDM5B and KDM5B are enriched in the influence of chromatin DNA damage area; 5) using NHEJ-GFP system and HR-DRGFP system, the FACS found that DNA double strand damage, KDM5B can affect the NHEJ The repair efficiency and HR; 6) the introduction of KDM5B enzyme activity of the mutant, using NHEJ-GFP system and HR-DRGFP system, by flow cytometry, found that the role of KDM5B in DNA damage dependent demethylase activity; further by chromatin immunoprecipitation (ChIP) experiments found that KDM5B could change DNA damage area the H3K4me3 level of chromatin; 7) by immunofluorescence assay and peptide pull-down assay showed that DNA damage, KDM5B recruits BRCA1 to the damaged area, affect the formation of foci in the injury site, and this effect depends on the KDM5B demethylase activity. But KDM5B MDC1 and gH2AX IR foci after the formation of no effect; by laser microbeam irradiation and immunofluorescence experiments, found that the injury occurred in the DNA, KDM5B can recruit Ku70 to the injured area, this process also depends on the KDM5B demethylase activity; 8) IR irradiated cells or add NC S caused DNA damage, KDM5B can detect the point of protein phosphorylation injury; 9) by flow cytometry, knockdown of KDM5B or ATM and IR or NCS irradiation treatment on the cell, found that knockdown of KDM5B can enhance the sensitivity of cells to genotoxic substances; 10) by cell cycle was measured by flow cytometry, IR cells transfected with siRNA irradiation or given different treatment, found that KDM5B can influence the cell cycle progression, knockdown of KDM5B can make the cell G1/S phase delay, especially IR caused DNA damage, caused by KDM5B knockdown on cell cycle effect is more obvious; 11) in p53+/+ cells knockdown of KDM5B and p53-/- cells, the expression level of Western blotting detection of p53 pathway related proteins and found that KDM5B can influence the p53+/+ cells in p53 pathway related protein expression level of p53-/- in p53 cells, but the protein has no effect and path; By flow cytometry, found that the influence of KDM5B on the cell cycle of p53 cells only in the expression, the effect of KDM5B on cell cycle dependent on p53; conclusion: cells by ionizing or enzyme treatment, KDM5B can be enriched in the DNA region of the injury, this process is dependent on the PARP1.KDM5B is effective in repairing DNA double strand breaks and the recruitment of Ku70 and BRCA1 is an indispensable component of the.KDM5B deletion could activate p53 signal pathway and make the cells more sensitive to genotoxic damage. The results indicate that KDM5B is indeed a DNA damage response protein, and points out that KDM5B is an important gene guardian, are regulators of genomic stability. This paper makes an understanding of epigenetic regulation plays an important role in maintaining genetic stability.

【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R3411

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