CTCF在HSV-1转录复制中的作用和CTCF参与DNA损伤修复的研究
文献类型：学位论文
| 作者 | 郎丰超 |
| 学位类别 | 博士 |
| 答辩日期 | 2015-04 |
| 授予单位 | 中国科学院研究生院 |
| 授予地点 | 北京 |
| 导师 | 周巨民 |
| 关键词 | I型人类疱疹病毒 宿主蛋白 CTCF 转录复制 DNA损伤修复 |
| 其他题名 | The role of CTCF in HSV-1 transcription and replication & CTCF participation in DNA damage repair |
| 中文摘要 | HSV-1复制区与宿主蛋白相互作用HSV-1是一类DNA病毒，感染上皮细胞后进入裂解感染，在裂解感染过程中HSV-1控制了细胞核，转录表达病毒RNA和蛋白，进行复制和病毒体组装。转录和复制过程中的HSV-1形成结构性的区域，其转录复制区域被高度有序的组织。HSV-1利用细胞内的转录复合体和染色质蛋白调控自身基因表达，对抗宿主细胞对病毒的抑制。HSV-1转录复制区域对宿主蛋白招募或排斥。我们发现HSV-1对组蛋白H3K4me3，H3K27me3，H3K9me3，H3S10p和γH2A.X有排斥作用，对没有修饰的总的组蛋白影响不大。RNA聚合酶II（RNA Pol II）在病毒DNA结合是动态的。RNA Pol II，RNA Pol II ser2p和RNA Pol II ser5p在病毒感染初期都被招募到HSV-1转录区域；随后，在转录区域的RNA Pol II和RNA Pol II ser2减少，而RNA Pol II ser5一直存在。复制中心还会招募DNA损伤修复（DNA damage repair，DDR）相关的蛋白ATM，53BP1，BRCA1和降解后的抗病毒组分SP100；降解泛素化酶RNF8和RNF168。HSV-1复制中心需要宿主染色质结构调控蛋白CTCF和cohesin的大量参与。 宿主蛋白CTCF与HSV-1裂解感染 染色质结构组织蛋白CTCF参与染色质界限，绝缘子和DNA环这样的高级结构形成和维持。我们之前的ChIP-seq数据表明CTCF可以结合在裂解期感染的HSV-1基因组上，结合位置包括病毒基因内部和启动子。我们用超分辨显微镜技术发现CTCF位于HSV-1复制区区域。DDR组蛋白γH2AX位于复制区外围，形成笼子一样的结构包裹在病毒外面。敲低CTCF后，HSV-1转录，复制和病毒产量下降，病毒基因和启动子上的异染色质组蛋白H3K27me3结合增加，RNA Pol II结合减少；CTCF敲低后，γH2AX渗透进入复制区。 CTCF在DNA损伤修复中的作用CTCF突变会引起人类小头畸形症（microcephaly）和智力低下，这些症状和许多DNA损伤修复蛋白基因突变引起的疾病非常相似；条件敲除CTCF，能够引发小鼠神经元缺失并阻断淋巴细胞的增殖和分化，增加肿瘤发生的机会；CTCF还可以帮助细胞抵抗凋亡和UV带来的伤害；CTCF的结合位点很多与三核苷酸串联重复（Trinucleotide repeat）邻近，并且与其稳定性密切相关。然而现在已报道的CTCF的功能都不能清楚地解释这些现象。 我们研究了CTCF在DNA损伤修复中的可能发挥的作用。我们发现CTCF在APH诱导下结合基因组fragile位点，敲低CTCF诱导细胞发生双链DNA断裂，激活细胞DNA损伤修复应答反应，并降低DSB修复效率。CTCF依赖ATM和NBS途径及其锌指结构被DNA损伤区域招募。 |
| 英文摘要 | Interaction between HSV-1 replication centers and host factors Herpes Simplex viruses type 1 (HSV-1) is a DNA virus that enters productive infection when they infect epithelial cells, where they take control of the host nucleus to make viral proteins, start viral DNA synthesis and assemble infectious virion, Replicating viral genomes formhighly organized replication centers to facilitate viral growth. In this process,HSV-1 is known to use host factors, including host chromatin and host transcription regulators to transcribe its genes, however, the invading virus also encounters host defense and stress responses to inhibit viral growth.HSV-1 replication centers selectively recruit cellular factors needed for viral growth, while excluding host factors that are deleterious for viral transcription or replication. Here we report that the viral replication centers selectively exclude modified histone H3, including heterochromatin mark H3K9me3 and active chromatin mark H3K4me3 and H3S10P, but not unmodified H3. We found that that there is a dynamic association between the viral replication centers and the host RNA Polymerase II. These centers also recruit components of the DNA Damage Response (DDR) pathway including 53BP1, BRCA1 and the host antiviral protein SP100. Importantly, we found that the ATM kinase is needed for the recruitment of CTCF to the viral centers. These results suggest that the HSV-1 replication centers take advantage the host signaling pathways to actively recruit or exclude host factors to benefit viral growth. Host factor CTCF and HSV-1 lytic infection CTCF is a host chromatin-organizing factor implicated in higher-order chromosome structures that include boundaries, insulators, and DNA-loops. Our previous ChIP-seq revealed that CTCF bound to numerous sites along the HSV-1 genome, including many lytic cycle promoter regulatory regions. Using super resolution microscopy we found that CTCF is recruited toHSV-1 replication centers and forms a web-like structure withinthese centers. In contrast, the DNA-damage associated histone γH2A.X forms a cage-like structure that appears excluded from the interior of viral replication centers.Knocking down CTCF resulted in the collapse of the γH2AX-cage-likestructures. CTCF knock-down also led to the attenuationof viral gene transcription, reduced viral genome copy number and viral yield. This further correlated with anincrease of H3K27me3 and a reduction of RNA pol II recruitment to viral genes. These results provide direct evidencethat CTCF coordinates viral transcription withthe structural organization of viralgenome during lytic infection. Role of CTCF in DNA damage repair. CTCF mutations in human and CTCF knock out in mice point to a defect in cell proliferation, phenotypes that could not be easily explained by the reported activities of CTCF. We studied the role of CTCF in genotoxic stress and found that CTCF is recruited to genomic fragile sites after APH treatment. Knocking down of CTCF leads to increased DNA damage and the induction of phosphorylated ATM and H2A.X. These effects were aggravated by UV irradiation or chemotherapy drugs. Using immunofluorescence, we demonstrated CTCF recruitment to ETO-induced DNA damage foci and UV laser-induced DNA damage areas. The recruitment of CTCF depends on ATM, NBS and the zinc finger region of CTCF, but is independent of RNF8 or 53BP1. By ChIP, we showed that CTCF is recruited to regions near the DSB breaks. Finally, we showed that CTCF knock down resulted in the reduction in HR type of repair, G1 arrest. These lines of evidence strongly suggest a function of CTCF in DNA damage repair, and that CTCF is essential for maintaining genome stability. |
| 语种 | 英语 |
| 源URL | [http://159.226.149.26:8080/handle/152453/10177]  |
| 专题 | 昆明动物研究所_基因调控与表达遗传 |
| 推荐引用方式 GB/T 7714 | 郎丰超. CTCF在HSV-1转录复制中的作用和CTCF参与DNA损伤修复的研究[D]. 北京. 中国科学院研究生院. 2015. |
入库方式： OAI收割
来源：昆明动物研究所
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