Role of CTCF in HPV replication and viral DNA looping

CTCF 在 HPV 复制和病毒 DNA 循环中的作用

• 批准号: 9094415
• 负责人: Laimonis A. LAIMINS
• 金额: $ 22.4万
• 依托单位: NORTHWESTERN UNIVERSITY AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-07-01 至 2018-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9094415
• 关键词: Anus; Architecture; Area; BRCA1 gene; Binding; Binding Proteins; Binding Sites; Biological Assay; CCCTC-binding factor; CHEK2 gene; Calcium; Cell Cycle; Cell Nucleus; Cell division; Cells; Cervical; Chromatin; Chromatin Loop; Chromosomes; Complex; Conserved Sequence; DNA; DNA Damage; DNA biosynthesis; Digestion; Distal; Enhancers; Episome; Epithelial; Epithelial Cells; Epithelium; G2 Phase; Gene Expression; Genetic Enhancer Element; Genetic Recombination; Genetic Transcription; Genome; Genomics; H2AFX gene; HPV-High Risk; Health; Human; Human Papillomavirus; Human papilloma virus 31; Human papilloma virus infection; Insulator Elements; Laboratories; Lesion; Life Cycle Stages; Link; Maintenance; Malignant Neoplasms; Malignant neoplasm of cervix uteri; Mediating; Methods; Mitosis; Modeling; Molecular Conformation; Mutate; Mutation; NBS1 gene; Normal Cell; Nuclear; Open Reading Frames; Papillomavirus; Pathway interactions; Phosphotransferases; Play; Polymerase; Process; Proteins; RNA Splicing; Recruitment Activity; Role; Series; Sister Chromatid; Site; Spliced Genes; Stratified Squamous Epithelium; Stratum Basale; Structure; Undifferentiated; Vaccines; Viral; Viral Genome; Virion; Virus Replication; Woman; arm; ataxia telangiectasia mutated protein; chromatin immunoprecipitation; cohesin; crosslink; effective therapy; keratinocyte; knock-down; malignant mouth neoplasm; new therapeutic target; next generation sequencing; prevent; promoter; prophylactic; response; restriction enzyme; stable cell line; transcription factor; viral DNA; virus episome maintenance

 DESCRIPTION (provided by applicant): Human papillomaviruses (HPV) are the causative agents of cervical, anal and many oral cancers. While prophylactic vaccines to prevent HPV infections have been developed, there is no effective treatment for existing HPV lesions. It is therefore of critical importance to understand how the productive life cycle of high-risk HPVs is regulated to identify potential new therapeutic targets. HPVs infect stratified squamous epithelia and link their productive life cycles to the differentiation of the infected cell. Vegetative genom replication, late gene expression, and virion assembly are restricted to the nuclei of highly differentiated cells present in the uppermost epithelial layers. When HPV infected cells migrate from the basal layer, they become arrested in G1 and are induced to re-enter S/G2 phases in the most differentiated layers to allow for vegetative viral DNA replication in a process referred to as amplification. My laboratory recently demonstrated that the cohesin protein, SMC1 and the insulator transcription factor, CTCF, as critical regulators of the HPV life cycle. The cohesin protein, SMC1, is necessary for sister chromatid association prior to mitosis. In addition the phosphorylated form of SMC1 plays a critical role in the NBS1/BRCA1 dependent arm of the ATM response. In HPV positive cells SMC1 is constitutively phosphorylated and localized to distinct nuclear foci in complexes with γ-H2AX, as well as CHK2 and are bound to HPV DNA. Importantly, knockdown of SMC1 blocks differentiation-dependent genome amplification. pSMC1 also forms complexes with the insulator transcription factor CTCF and our studies show that it binds to conserved sequence motifs in the L2 late region of HPV 31. Similar motifs are found in most HPV types. Knockdown of CTCF with shRNAs blocks genome amplification and mutation of the CTCF binding motifs in the L2 ORF inhibits stable maintenance of viral episomes in undifferentiated cells as well as amplification of genomes upon differentiation. These findings suggest a model in which SMC1 factors are constitutively activated in HPV positive cells and recruited to viral genomes through complex formation with CTCF that regulate viral replication. CTCF complexes mediate insulator function by blocking adjacent enhancer function and CTCF complexes bound to HPV genomes also act as insulator elements that shield cryptic viral promoters from activation by the HPV enhancer element in the URR. CTCF acts to organize chromatin loops that regulate transcription, splicing, replication and recombination. This application focuses on understanding the role of CTCF in mediating stable maintenance of HPV episomes in undifferentiated cells, chromatin organization of viral DNAs and the inter- as well as intramolecular looping of viral DNAs. This is a new area of study for human papillomaviruses that I believe will help us understand the role of supramolecular structures of viral DNAs in the viral life cycle. Aim 1: What is the role of CTCF sites in HPV replication and gene expression? Aim 2: Does HPV form inter or intramolecular DNA loops through CTCF interactions?

 描述（由适用提供）：人乳头瘤病毒（HPV）是宫颈，肛门和许多口服癌的真正药物。尽管已经开发了预防HPV感染的预防性疫苗，但现有的HPV病变尚无有效的治疗方法。因此，了解高风险HPV的产品生命周期以确定潜在的新治疗靶点至关重要。 HPV感染了分层的鳞状上皮，并将其生产寿命与感染细胞的分化联系起来。营养基因组复制，晚期基因表达和病毒体组装仅限于上皮层中存在的高度分化细胞的核。当HPV感染的细胞从基本层迁移时，它们在G1中被捕，并被诱导在最分化的层中重新进入S/G2相，以允许在称为扩增的过程中进行营养病毒DNA复制。我的实验室最近证明，粘着蛋白SMC1和绝缘子转录因子CTCF是HPV生命周期的关键调节剂。在有丝分裂之前，粘合素蛋白SMC1是姐妹染色单体缔合所必需的。另外，SMC1的磷酸化形式在ATM响应的NBS1/BRCA1依赖臂中起关键作用。在HPV阳性细胞中，SMC1在具有γ-H2AX和CHK2的复合物中的组成型磷酸化并将其定位于不同的核灶，并与HPV DNA结合。重要的是，SMC1的敲低阻止了依赖分化的基因组扩增。 PSMC1还与绝缘子转录因子CTCF形成复合物，我们的研究表明，它与HPV 31的L2晚期区域中的配置序列基序结合。在大多数HPV类型中都发现了类似的基序。用shRNA敲低CTCF会阻止L2 ORF中CTCF结合基序的基因组扩增和突变，可抑制未分化细胞中病毒spipopomes的稳定维持以及分化后基因组的扩增。这些发现表明了一个模型，其中SMC1因子在HPV阳性细胞中始终激活，并通过与调节病毒复制的CTCF形成复合形成的病毒基因组。 CTCF复合物通过阻止与HPV基因组结合的相邻增强子功能和CTCF复合物通过阻止IRR中HPV增强子元素激活的绝缘体元件来通过阻断相邻的增强子功能和CTCF复合物来实现介质绝缘子功能。 CTCF的作用是组织调节转录，剪接，复制和重组的染色质环。该应用的重点是了解CTCF在介导未分化细胞中HPV促进稳定维持中的作用，病毒DNA的染色质组织以及病毒DNA的分子内和分子内环。这是人类乳头瘤病毒的新研究领域，我认为这将有助于我们了解病毒DNA在病毒生命周期中的超分子结构的作用。 AIM 1：CTCF位点在HPV复制和基因表达中的作用是什么？ AIM 2：HPV是否通过CTCF相互作用形成间分子内DNA环？