Biochemical studies of aberrant chromatin regulation in cancer

项目概要/摘要 真核细胞需要严格调控整体基因表达以维持稳态 并对环境刺激做出反应。DNA缠绕在组蛋白周围， 结构，染色质，并提供了一个平台，复杂的基因表达的调整 通过物理和化学调节。不出所料，这些染色质的破坏 调节机制作为疾病的驱动因素在癌症中特别普遍。完成 拟议的项目将阐明健康染色质调节的机制及其 疾病的中断，提供必要的洞察力，以开发改进的治疗方法， 各种癌症的干预措施。 在F99阶段，我研究了B型肝炎对染色质信号传导的破坏 乙型肝炎病毒（HBV）是世界范围内肝细胞癌的主要原因。HBV维持慢性 通过建立一个独立的微型染色体，称为共价 闭合环状DNA（cccDNA），在很大程度上逃避免疫检测和常规染色质 监管机制。进一步促成这种逃避的是病毒蛋白HBx，其具有 记录的角色重定向许多染色质效应子，包括转录因子， 降解机制和表观基因组修饰剂。到目前为止，在我的论文工作中，我已经开发了一个 用于生物化学和生物物理研究的体外重构cccDNA的平台，确定 HBx表达需要cccDNA中的组蛋白占据，并显示HBx直接结合 到核小体我论文的其余部分将用于测试 其他已知的相互作用对cccDNA压缩和基因表达的影响， 使用基因座特异性蛋白质组学和表观基因组学研究的细胞中cccDNA景观。 K 00相移聚焦于ATP依赖性染色质重塑酶，一类 这些蛋白质在超过20%的癌症中被突变或过度表达。我特别 我打算研究冠心病家族的重塑，这已被牵连的驱动程序的各种 癌症类型。我将运用我在染色质生物化学方面的专业知识， 包括冷冻电子显微镜作为研究结构和功能的手段， CHD染色质重塑。与此同时，我将发展基因编辑技术的技能， 野生型酶，并将临床相关的CHD突变体引入细胞中进行表观基因组分析。 分析疾病中的重塑功能障碍。将这些新方法与我的 生物化学，化学生物学和生物物理学的背景将使我能够解决紧迫的问题 在我的职业生涯中，我一直在研究染色质和癌症生物学方面的问题， 一个独立的专注于癌症的教职

Project Summary/Abstract Eukaryotic cells require the tight regulation of global gene expression to maintain homeostasis and respond to environmental stimuli. DNA spools around histone proteins form this vital structure, chromatin, and provide a platform for the sophisticated tuning of gene expression through physical and chemical regulation. Unsurprisingly, the disruption of these chromatin regulatory mechanisms is particularly prevalent in cancers as a driver of disease. Completion of the proposed projects will shed light on the mechanisms of healthy chromatin regulation and its disruption in disease, providing the insight necessary to develop improved therapeutic interventions in a variety of cancers. In the F99 phase of this proposal, I study disrupted chromatin signaling by Hepatitis B Virus (HBV), a leading cause of hepatocellular carcinoma worldwide. HBV maintains chronic infections within hepatocytes by establishing an independent minichromosome, termed covalently closed circular DNA (cccDNA), that largely evades immune detection and conventional chromatin regulatory mechanisms. Further contributing to this evasion is the viral protein HBx, which has documented roles redirecting numerous chromatin effectors, including transcription factors, degradation machinery, and epigenome modifiers. So far in my thesis work, I have developed a platform to reconstitute cccDNA in vitro for biochemical and biophysical studies, determined that histone occupancy in cccDNA is required for HBx expression, and shown that HBx binds directly to nucleosomes. The remainder of my thesis work will be spent testing the biochemical effects of other known interactors on cccDNA compaction and gene expression and illuminating the cccDNA landscape in cells using locus-specific proteomic and epigenomic studies. The K00 phase shifts focus to ATP-dependent chromatin remodeling enzymes, a class of proteins shown to be mutated or overexpressed in more than 20% of cancers. In particular, I intend to study the CHD family of remodelers, which have been implicated as drivers of a variety of cancer types. I will apply my expertise in chromatin biochemistry and expand my technical repertoire to include cryo-electron microscopy as a means to study the structure and function of CHD chromatin remodelers. In parallel, I will develop skills in gene editing techniques to knockout wild-type enzyme and introduce clinically-relevant CHD mutants into cells for epigenomic analyses of remodeler dysfunction in disease. Combining these new approaches with my background in biochemistry, chemical biology, and biophysics will position me to address pressing questions in chromatin and cancer biology throughout the rest of my career as I pursue an independent, cancer-focused faculty position.