Biochemical studies of aberrant chromatin regulation in cancer

癌症中异常染色质调控的生化研究

• 批准号: 10305344
• 负责人: Nicholas Prescott
• 金额: $ 4.75万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-01 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10305344
• 关键词: ATP Hydrolysis; Address; Architecture; Binding; Biochemical; Biochemistry; Biology; Biophysics; Cancer Biology; Case Study; Cell Line; Cell physiology; Cells; Cellular biology; Chemicals; Chromatin; Chromatin Fiber; Chromatin Remodeling Factor; Chromatin Structure; Chronic; Chronic Hepatitis B; Circular DNA; Complex; Cryoelectron Microscopy; DNA; Data; Detection; Disease; Enzymes; Epigenetic Process; Eukaryotic Cell; Faculty; Family; Foundations; Functional disorder; Gatekeeping; Gene Expression; Gene Expression Profiling; Genes; Genetic Transcription; Genome; Goals; Hepatitis B Virus; Hepatocyte; Histones; Homeostasis; Host Defense; Immune; In Vitro; Individual; Investigation; Knock-out; Libraries; Light; Maintenance; Malignant Neoplasms; Malignant neoplasm of liver; Mass Spectrum Analysis; Molecular Biology; Molecular Conformation; Molecular and Cellular Biology; Mutate; Mutation; Nucleosome Core Particle; Nucleosomes; Oncogenic; Oncornaviruses; Output; Pathologic; Phase; Play; Population; Positioning Attribute; Post-Translational Protein Processing; Primary carcinoma of the liver cells; Proteins; Proteomics; Recombinants; Regulation; Regulatory Pathway; Research; Research Project Grants; Resolution; Rest; Role; Signal Transduction; Stimulus; Structure; System; Techniques; Testing; Therapeutic Intervention; Viral Proteins; Virus; Virus Replication; Work; biophysical analysis; cancer type; career; chromatin protein; chromatin remodeling; chronic infection; clinically relevant; crosslink; epigenome; epigenomics; established cell line; experience; improved; insight; member; mutant; novel strategies; novel therapeutics; overexpression; reconstitution; recruit; response; skills; structural biology; structural genomics; transcription factor; transcriptome; tumorigenesis

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.

项目摘要/摘要 真核细胞需要对全球基因表达进行严格调控才能维持体内平衡 并对环境刺激做出反应。围绕组蛋白蛋白的DNA卷轴形成了这一至关重要的 结构，染色质，并为基因表达的复杂调节提供平台 通过物理和化学调节。不出所料，这些染色质的破坏 调控机制作为疾病的驱动力在癌症中尤其普遍。完成 拟议的项目将阐明健康的染色质调节机制及其 疾病的中断，提供必要的洞察力来开发改进的治疗方法 对多种癌症的干预。 在这项提议的F99阶段，我研究了乙肝引起的染色质信号中断 病毒(乙肝病毒)，全球肝细胞癌的主要原因。乙肝病毒保持慢性 通过建立一个独立的微染色体在肝细胞内感染，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。进一步导致这种逃避的是病毒蛋白HBx，它具有 有文献记载的角色重定向了许多染色质效应器，包括转录因子， 降解机制和表观基因组修饰物。到目前为止，在我的论文工作中，我已经开发了一种 用于生化和生物物理研究的体外重建cccDNA的平台，确定 HBx的表达需要cccDNA中的组蛋白占位，并表明HBx直接结合 核小体。我剩下的论文工作将用来测试 其他已知的影响cccDNA紧凑和基因表达的相互作用因素 利用特定基因座蛋白质组学和表观基因组学研究细胞中的cccDNA景观。 K00将焦点转移到依赖于ATP的染色质重塑酶上，这是一类 在超过20%的癌症中，蛋白质显示出突变或过度表达。尤其是，我 打算研究CHD重建器家族，它们被认为是各种疾病的驱动因素 癌症类型。我将应用我在染色质生物化学方面的专业知识，并扩展我的技术 将冷冻电子显微镜作为研究细胞结构和功能的一种手段 先天性心脏病染色质重构体。与此同时，我将发展基因编辑技术的技能，以敲除 野生型酶并将临床相关的CHD突变体导入细胞进行表观基因组学研究 疾病中重塑功能障碍的分析。将这些新方法与我的 在生物化学、化学生物学和生物物理学方面的背景将使我能够解决紧迫的问题 在我余下的职业生涯中，我一直在研究染色质和癌症生物学方面的问题 独立的、专注于癌症的教师职位。