CTCF维持基因组稳定性研究；HSV-1基因组复制过程中的动态变化研究

	CTCF维持基因组稳定性研究；HSV-1基因组复制过程中的动态变化研究
其他题名	CTCF maintenances genome stability；Danymic changes of HSV-1 genome during replication
	李卓然
学位类型	博士
导师	周巨民
	2016-07
学位授予单位	中国科学院研究生院
学位授予地点	北京
关键词	Ctcf 常见易断裂位点 Dna损伤应答人类i型单纯疱疹病毒复制受激发射损耗显微技术
其他摘要	CTCF通过保护易断裂位点完整性维持基因组稳定性。维持基因组稳定性和传递完整的基因组对细胞、器官乃至一个物种的生存都至关重要，但是，基因组中本身就存在一种被称为常见易断裂位点(CFSs)的座位，该位点常引起特定基因的缺失、异位或染色体重排。大多数的CFSs都能够响应阿非迪霉素(APH)的诱导。利用免疫荧光(IF)、流式细胞术(FCM)、实时定量反转录聚合酶链式反应(RT-qPCR)和蛋白印记(WB)等技术进行分析，发现敲低CTCF引起细胞CFSs断裂频率上升、基因组不稳定性加剧、γH2A.X和53BP1富集增加、细胞周期停滞和DNA修复相关蛋白表达水平变化。经APH处理并采用免疫荧光-荧光原位杂交(IF-FISH)和免疫共沉淀定量聚合酶链式反应(ChIP-qPCR)技术检测，发现CTCF能够在53BP1所标记的CFSs，或已知的CFSs处富集，说明CTCF可以通过保护CFSs来维持基因组稳定性。通过STED超分辨显微镜观察复制中的HSV-1基因组。人类I型单纯疱疹病毒(HSV-1)是一种重要的、以人类为宿主而且广泛存在的病原体。为了深入研究HSV-1的复制过程，将荧光原位杂交、免疫荧光与受激发射损耗显微技术(STED)相结合对复制中的病毒基因组和相关蛋白进行观察。利用定位于HSV-1基因组相同区域但以不同标记物标记的探针进行原位杂交，无论是前复制期还是复制后期，探针间的相关性和共定位频率都较高；而以定位于HSV-1基因组不同区域并以不同标记物标记的探针进行杂交时，探针间的平均距离随着病毒复制的进行而不断增大，说明，随着HSV-1进入复制阶段，其基因组逐渐从压缩状态转变为松散状态。HSV-1单链结合蛋白ICP8与病毒基因组间的平均距离比其与RNA复制酶II(RNA Pol II)间的距离小，说明，虽然HSV-1的转录和复制都在病毒复制区内进行，但是这两个生物学过程被分隔在不同亚结构内。HSV-1裂解感染是一个动态变化的过程，而STED显微镜能够被用于研究病毒的复制过程。; CTCF maintenances genome stability through protecting integrity of CFSs. Maintaining genome stability and transmission of intact genomes is critical for survival of cells, organisms, and species. However, the genome is not uniformly stable and contains common fragile sites (CFSs), which are specific genomic loci prone to deletions, translocations and other rearrangements in metaphase chromosomes. Most of CFSs are induced by treatment with aphidicolin (APH), an inhibitor of replicative polymerase. Here we studied the role of CTCF in genome stability, and found that CTCF knockdown augmented the instability of genome, increased CFSs expression. By IF, FCM, RT-qPCR and WB, we found CTCF knock down promoted CFSs expression, increased γH2A.X and 53BP1 foci numbers, caused cell cycle arrested, and changed expression levels of DNA damage related proteins. When cells were treated with APH and detected through IF-FISH or ChIP-qPCR methods, increased binding of CTCF to 53BP1 labeled CFSs, or known CFSs. These analyses suggest a role of CTCF in maintenance of genome stability through responding to expression of CFSs.Visualizing the replicating HSV-1 virus using STED super-resolution microscopy. HSV-1 is a ubiquitous and important human pathogen. To gain new insight into HSV-1 replication, we used a combination of STED microscopy and FISH or IF to observe the replication process. Using two different colored probes labeling the same region of viral genome, the two highly correlated in both pre-replication and replicating genomes. In comparison, when probes from different regions were used, the average distance between the two probes increased as virus enters replication, suggesting that the viral genome undergoes dynamic structure changes from compact to a relaxed formation and occupies larger space as it enters replication. Viral single strand binding protein ICP8 was seen closely positioned with viral genome, but distant from RNA Pol II, especially the RNA Pol II Ser2P modified form, suggesting that ICP8 marked regions of DNA replication is spatially separate from regions of active transcription represented by the elongating form of RNA Pol II within the viral replication compartments. These results shed interesting light to the dynamic process of HSV-1 lytic infection and suggests that stimulated emission depletion microscopy is capable of investigating events during HSV-1 replication.
学科领域	细胞生物学
语种	中文
文献类型	学位论文
条目标识符	http://ir.kiz.ac.cn/handle/152453/11994
专题	科研部门_基因调控与表达遗传（周巨民）
作者单位	中国科学院昆明动物研究所
推荐引用方式 GB/T 7714	李卓然. CTCF维持基因组稳定性研究；HSV-1基因组复制过程中的动态变化研究[D]. 北京. 中国科学院研究生院,2016.

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