Exploration of the global manipulation of transcriptional networks by oncogenic

dhpv每年导致61万例肛门生殖器和口咽道癌症。HPV生命周期的完成依赖于感染上皮细胞的分化。感染发生在未分化的基底角化细胞中，染色质化的病毒DNA作为一个持续的片段被建立，编码E6和E7癌蛋白的病毒转录物被表达。细胞向上上皮的分化和迁移与晚期病毒启动子的激活和晚期衣壳蛋白的表达一致。这种依赖于分化的HPV生命周期模型被广泛接受，但控制基因表达的分子机制仍然很大程度上未知。为了协调这些复杂的转录事件，HPV利用了大量的宿主转录因子。例如，我们证明了宿主染色质调节剂CTCF （ccctc结合因子）被招募到致癌的HPV基因组中，以协调病毒癌基因的分化依赖性抑制。除了控制自身基因的表达外，病毒还创造了一个支持感染和复制的宿主环境。研究表明，分离的HPV蛋白表达改变细胞基因表达，逃避免疫激活，增加细胞生长和运动。然而，这些研究尚未扩展到检查感染期间发生的变化，转录重编程的机制尚不清楚。假设hpv在表观遗传学上对宿主进行重编程，以创造一个支持病毒持续存在的细胞环境，这些变化有助于hpv驱动的癌变。目的学生将使用最先进的HPV感染组织模型和先进的技术方法来探索宿主全基因组操作的复杂性以及由此导致的HPV诱导疾病的转录变化。目标1。分析HPV建立后宿主转录变化：为了获得高质量的转录组分析，将对HPV16或HPV18发作前后从不同疾病相关部位采集的等基因原代角质形成细胞进行RNA-Seq分析。分化上皮中HPV感染后的差异基因表达模式也将通过RNA-Seq来确定。显著改变的宿主途径将通过基因本体分析（www.broadinstitute.org）识别，感兴趣的途径将通过qRT-PCR、western blotting和表型研究进一步验证。2. 分析hpv介导的宿主转录重编程的机制基础。初步数据表明，HPV通过重新分配重要的转录调控因子（如CTCF）来诱导宿主的转录重编程。学生将通过ChIP-Seq分析这些因子和活性转录染色质（H3K4Me3）和抑制染色质（H3K27Me3）的表观遗传标记进一步探索宿主细胞重编程。宿主内转录中心的已鉴定变化将被映射到RNA-Seq实验中鉴定的基因转录变化上，从而对HPV对宿主细胞重编程的表观遗传进行全面概述。这些变化也将在器官型筏培养的分化上皮中进行评估，并将通过ATAC-Seq分析宿主细胞增强子的特异性转录因子募集来补充。3. 分析HPV对宿主细胞染色质结构和拓扑相关结构域（TADs）的整体重塑。染色质被精细的三维折叠分隔开来，使遥远的功能元素在物理上接近。为了分析hpv建立前后的染色体相互作用，我们将与新加坡国立大学S. Jha教授合作，使用染色体构象捕获结合高通量测序（Hi-C）技术。

BackgroundHPV causes 610,000 cancers per year of the anogenital and oropharyngeal tracts. HPV life cycle completion is dependent on the differentiation of infected epithelial cells. Infection is established in the undifferentiated basal keratinocytes where the chromatinised viral DNA is established as a persistent episome and viral transcripts that encode the E6 and E7 oncoproteins are expressed. Differentiation and migration of cells to the upper epithelium coincides with activation of the late virus promoter and expression of late capsid proteins. This differentiation-dependent model of the HPV life cycle is well accepted but the molecular mechanisms controlling gene expression remain largely unknown. To co-ordinate these complex transcriptional events, HPV utilises a plethora of host transcription factors. For example, we demonstrated that the host chromatin regulator CTCF (CCCTC-binding factor) is recruited to oncogenic HPV genomes to co-ordinate differentiation-dependent repression of viral oncogenes. As well as controlling expression of their own genes, viruses create a host environment that supports infection and replication. Studies have shown that expression of isolated HPV proteins alters cellular gene expression to evade immune activation and increase cellular growth and motility. However, these studies have not been extended to examine changes that occur during infection and the mechanisms of transcriptional reprogramming are not understood. HypothesisHPV epigenetically reprograms the host to create a cellular milieu supportive of viral persistence and these changes contribute to HPV-driven carcinogenesis. AimThe student will use state-of-the-art models of HPV infected tissue and advanced technological methods to explore the complexity of genome-wide manipulation of the host and resulting transcriptional changes that contribute to HPV-induced disease. Objectives 1. Analyse host transcription changes following HPV establishment: To produce high-quality transcriptome analysis pre- and post-HPV establishment RNA-Seq will be carried out on isogenic primary keratinocytes harvested from different disease-relevant body sites before and after establishment of HPV16 or HPV18 episomes. Differential gene expression patterns following HPV infection in differentiating epithelia will also be determined by RNA-Seq. Significantly altered host pathways will be identified by Gene Ontology analysis (www.broadinstitute.org) and pathways of interest will be further validated by qRT-PCR, western blotting and phenotypic studies. 2. Analyse the mechanistic underpinnings of HPV-mediated host transcriptional reprogramming. Preliminary data suggests that HPV induces transcriptional reprogramming of the host by redistributing important transcriptional regulators, such as CTCF. The student will explore host cell reprogramming further by ChIP-Seq analysis of these factors and epigenetic marks of actively transcribed chromatin (H3K4Me3) and repressed chromatin (H3K27Me3). Identified changes in transcriptional hubs within the host will be mapped onto the changes in gene transcription identified in the RNA-Seq experiments to give a complete overview of epigenetic host cell reprogramming by HPV. These changes will also be assessed in differentiating epithelia in organotypic raft cultures and will be complemented by the analysis of specific transcription factor recruitment to host cell enhancers by ATAC-Seq. 3. Analyse the global remodelling of host cell chromatin structure and topologically associating domains (TADs) by HPV. Chromatin is compartmentalised by elaborate three-dimensional folding that brings distant functional elements in close physical proximity. To analyse these chromosomal interactions pre- and post-HPV establishment, we will use chromosome conformation capture coupled to high-throughput sequencing (Hi-C) in collaboration with Prof. S. Jha, National University of Singapore.