Epigenetic Regulation During the HPV Life Cycle

HPV生命周期的生产阶段仅限于上皮的最上层，在通常已经退出细胞周期的细胞中。E7蛋白改变细胞周期调节，将分化的细胞推回到细胞周期，允许多产的复制和病毒粒子的产生。E7细胞周期失调导致基因组不稳定，这是癌症发展的驱动力。我们的长期目标是了解调节多产病毒复制的机制，这对了解HPV如何导致癌症很重要。HPV基因组与组蛋白相关，尽管组蛋白翻译后修饰对病毒生命周期的影响尚不清楚。该建议的重点是了解HPV如何利用SETD2甲基转移酶促进病毒复制。SETD2在转录延伸过程中将三甲基标记添加到赖氨酸36上的组蛋白H3 （H3K36me3）上，并通过向H3K36me3募集大量效应蛋白来调节多种细胞过程。我们将确定SETD2活性促进病毒复制的机制，并确定E7如何增加SETD2的稳定性。我们假设HPV通过setd2介导的H3K36me3在病毒和细胞染色质上的表观遗传调节病毒生命周期。具体目的是：(1)通过检测SETD2敲低是否影响病毒基因组上的转录、RNA加工和DNA修复，以及鉴定与HPV DNA结合的H3K36me3读取器，确定SETD2促进病毒复制的机制。(2)通过检测已知的SETD2稳定性调节剂，以及利用BirA标记的SETD2与质谱联用非偏倚方法鉴定SETD2的相互作用伙伴，确定E7增加SETD2蛋白稳定性的机制。由于SETD2对维持基因组稳定性至关重要，因此了解HPV如何利用这种表观遗传修饰因子促进病毒复制是很重要的。了解SETD2活性如何促进病毒生命周期将有助于深入了解病毒持久性和基因组不稳定性的机制。这些研究也可能确定治疗hpv相关疾病的治疗靶点。

The productive phase of the HPV life cycle is restricted to the uppermost layer of the epithelium, in cells that have normally exited the cell cycle. The E7 protein alters cell cycle regulation to push differentiating cells back into the cell cycle, allowing for productive replication and virion production. Cell cycle deregulation by E7 leads to genomic instability that is a driving force in cancer development. Our long-term goal is to understand mechanisms that regulate productive viral replication, which is important to understanding how HPV causes cancer. HPV genomes are histone-associated, though the impact of histone post-translational modifications on the viral life cycle is unclear. This proposal focuses on understanding how HPV utilizes the SETD2 methyltransferase to facilitate viral replication. SETD2 adds the trimethyl mark to histone H3 on lysine 36 (H3K36me3) during transcription elongation and regulates multiple cellular processes through the recruitment of numerous effector proteins to H3K36me3. We will identify the mechanisms by which SETD2 activity contributes to viral replication and determine how E7 increases the stability of SETD2. We hypothesize that HPV epigenetically regulates the viral life cycle through SETD2-mediated H3K36me3 on viral, as well as cellular chromatin. Specific Aims to test this are: (1) To determine the mechanism by which SETD2 facilitates viral replication by examining whether SETD2 knockdown affects transcription, RNA processing and DNA repair on viral genomes, as well as by identifying H3K36me3 readers bound to HPV DNA. (2) To determine the mechanism by which E7 increases the protein stability of SETD2 by examining known modulators of SETD2 stability, as well as by identifying SETD2 interacting partners though a non-biased approach using a BirA- tagged SETD2 coupled with mass spectrometry. Since SETD2 is essential to the maintenance of genomic stability, it is important to understand how HPV utilizes this epigenetic modifier to facilitate viral replication. Understanding how SETD2 activity promotes the viral life cycle will provide insight into mechanisms of viral persistence, as well as genomic instability. These studies may also identify therapeutic targets for the treatment of HPV-associated diseases.