Role of CTCF in HPV replication and viral DNA looping

 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。虽然已经开发了预防HPV感染的预防性疫苗，但对现有的HPV病变没有有效的治疗方法。因此，了解高危HPV的生产性生命周期是如何调节的，以确定潜在的新治疗靶点至关重要。HPV感染复层鳞状上皮细胞，并将其生产生命周期与受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存在于最上皮层中的高度分化细胞的细胞核。当HPV感染的细胞从基底层迁移时，它们被阻滞在G1期，并被诱导在最分化的层中重新进入S/G2期，以允许在称为扩增的过程中进行营养期病毒DNA复制。我的实验室最近证明，粘附蛋白，SMC 1和绝缘子转录因子，CTCF，作为HPV生命周期的关键调节因子。粘附蛋白SMC 1是有丝分裂前姐妹染色单体联合所必需的。此外，SMC 1的磷酸化形式在ATM应答的NBS 1/BRCA 1依赖性臂中起关键作用。在HPV阳性细胞中，SMC 1被组成性磷酸化，并定位于与γ-H2 AX以及CHK 2复合的不同核灶，并与HPV DNA结合。重要的是，SMC 1的敲低阻断了分化依赖性基因组扩增。pSMC 1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晚期区域的保守序列基序结合。在大多数HPV类型中发现了类似的基序。用shRNA敲低CTCF阻断基因组扩增，并且L2 ORF中CTCF结合基序的突变抑制未分化细胞中病毒附加体的稳定维持以及分化后基因组的扩增。这些发现表明了一种模型，其中SMC 1因子在HPV阳性细胞中组成性激活，并通过与CTCF形成复合物来募集病毒基因组，调节病毒复制。CTCF复合物通过阻断相邻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，其保护隐蔽病毒启动子免于被URR中的HPV增强子元件激活。CTCF的作用是组织染色质环，调节转录、剪接、复制和重组。本申请的重点是了解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研究的一个新领域，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？

 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?