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。虽然已经开发出预防 HPV 感染的预防性疫苗，但目前还没有针对现有 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至关重要。 HPV 感染复层鳞状上皮，并将其生产生命周期与受感染细胞的分化联系起来。营养体基因组复制、晚期基因表达和病毒体组装仅限于最上上皮层中高度分化细胞的细胞核。当 HPV 感染的细胞从基底层迁移时，它们会停滞在 G1 期，并被诱导重新进入分化程度最高的层中的 S/G2 期，从而在称为扩增的过程中进行病毒 DNA 营养复制。我的实验室最近证明，粘连蛋白 SMC1 和绝缘体转录因子 CTCF 是 HPV 生命周期的关键调节因子。粘连蛋白 SMC1 对于有丝分裂前的姐妹染色单体关联是必需的。此外，SMC1 的磷酸化形式在 ATM 反应的 NBS1/BRCA1 依赖性臂中发挥着关键作用。在 HPV 阳性细胞中，SMC1 被组成型磷酸化，并定位于与 γ-H2AX 以及 CHK2 形成复合物的不同核灶，并与 HPV DNA 结合。重要的是，SMC1 的敲除可阻止分化依赖性基因组扩增。 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 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 shRNA 敲低 CTCF 会阻止基因组扩增，L2 ORF 中 CTCF 结合基序的突变会抑制未分化细胞中病毒附加体的稳定维持以及分化时基因组的扩增。这些发现提出了一种模型，其中 SMC1 因子在 HPV 阳性细胞中被组成型激活，并通过与调节病毒复制的 CTCF 形成复合物而被招募到病毒基因组中。 CTCF 复合物通过阻断邻近的增强子功能来介导绝缘子功能，并且与 HPV 基因组结合的 CTCF 复合物还充当绝缘子元件，保护隐性病毒启动子免受 URR 中 HPV 增强子元件的激活。 CTCF 负责组织染色质环，调节转录、剪接、复制和重组。该应用的重点是了解 CTCF 在介导未分化细胞中 HPV 附加体的稳定维持、病毒 DNA 的染色质组织以及病毒 DNA 的分子间和分子内循环中的作用。这是人类乳头瘤病毒的一个新研究领域，我相信它将帮助我们了解病毒 DNA 的超分子结构在病毒生命周期中的作用。目标 1：CTCF 位点在 HPV 复制和基因表达中起什么作用？目标 2：HPV 是否通过 CTCF 相互作用形成分子间或分子内 DNA 环？