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(申请人提供)：人乳头瘤病毒(HPV)是宫颈癌、肛门癌和许多口腔癌的病原体。虽然已经开发了预防HPV感染的预防性疫苗，但还没有针对现有HPV病变的有效治疗方法。因此，了解如何调节高危HPV的生产生命周期，以确定潜在的新治疗靶点是至关重要的。HPV感染复层鳞状上皮，并将其生产生命周期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粒子组装仅限于存在于最上层的高度分化的细胞的细胞核。当人乳头瘤病毒感染细胞从基底层迁移时，它们被阻止在G1期，并被诱导在最分化的层重新进入S/G2期，以便在一个称为扩增的过程中允许病毒的营养复制。我的实验室最近证明，粘附素蛋白SMC1和绝缘体转录因子CTCF是HPV生命周期的关键调节因子。粘附素蛋白SMC1是有丝分裂前姐妹染色单体联合所必需的。此外，SMC1的磷酸化形式在ATM反应的NBS1/BRCA1依赖臂中起着关键作用。在人乳头瘤病毒阳性细胞中，Smc1被结构性磷酸化，并定位于与γ-H_2AX和Chk2的复合体中的不同的核病灶，并与HPVDNA结合。重要的是，SMC1基因的敲除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HPV31 L2晚期区域的保守序列基序结合。在大多数HPV类型中都发现了类似的基序。用shRNAs敲除CTCF可阻断基因组扩增，L2ORF中CTCF结合基序的突变抑制未分化细胞中病毒表型的稳定维持以及基因组在分化时的扩增。这些发现提出了一种模型，在该模型中，SMC1因子在HPV阳性细胞中被结构性激活，并通过与调节病毒复制的CTCF形成复合体而被招募到病毒基因组中。CTCF复合体通过阻断邻近的增强子功能来调节绝缘体功能，与HPV基因组结合的CTCF复合体也作为绝缘体元件，保护隐蔽的病毒启动子不被URR中的HPV增强子元件激活。CTCF起着组织染色质环的作用，这些染色质环调控转录、剪接、复制和重组。这一应用侧重于了解CTCF在介导未分化细胞中HPV异构体的稳定维持、病毒DNA的染色质组织以及病毒DNA的分子间和分子内环中的作用。这是对人乳头瘤病毒的一个新的研究领域，我相信这将有助于我们了解病毒DNA的超分子结构在病毒生命周期中的作用。目的1：CTCF位点在HPV复制和基因表达中的作用是什么？目的2：HPV是否通过CTCF相互作用形成分子间或分子内DNA环？