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.

项目概要/摘要 真核细胞需要严格调控全局基因表达以维持体内平衡 并对环境刺激作出反应。 DNA 缠绕在组蛋白周围形成了这一重要的 结构、染色质，并为基因表达的复杂调整提供平台 通过物理和化学调节。毫不奇怪，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尤其普遍。完成 拟议的项目将揭示健康染色质调控的机制及其 破坏疾病，提供开发改进治疗所需的洞察力 对多种癌症进行干预。 在该提案的 F99 阶段，我研究了乙型肝炎破坏的染色质信号传导 病毒 (HBV)，全世界肝细胞癌的主要原因。乙肝病毒持续慢性化 通过建立独立的微型染色体（称为共价）在肝细胞内进行感染 闭环 DNA (cccDNA)，很大程度上逃避免疫检测和传统染色质 监管机制。病毒蛋白 HBx 进一步促成了这种逃避，它具有 记录的角色重定向许多染色质效应器，包括转录因子， 降解机制和表观基因组修饰剂。到目前为止，在我的论文工作中，我已经开发了一个 体外重建 cccDNA 用于生化和生物物理研究的平台，确定 cccDNA 中的组蛋白占据是 HBx 表达所必需的，并且表明 HBx 直接结合 到核小体。我论文的其余部分将用于测试其生化效应 其他已知的 cccDNA 压缩和基因表达相互作用因子，并阐明了 使用位点特异性蛋白质组学和表观基因组研究观察细胞中的 cccDNA 景观。 K00 阶段将焦点转向 ATP 依赖性染色质重塑酶，这是一类 超过 20% 的癌症中显示出突变或过度表达的蛋白质。特别是，我 打算研究 CHD 重塑者家族，这些家族被认为是多种疾病的驱动者 癌症类型。我将运用我在染色质生物化学方面的专业知识并扩展我的技术 曲目包括冷冻电子显微镜作为研究结构和功能的手段 CHD 染色质重塑者。与此同时，我将培养基因编辑技术的技能，以实现基因敲除 野生型酶并将临床相关的 CHD 突变体引入细胞进行表观基因组学研究 疾病中重塑者功能障碍的分析。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 在我余下的职业生涯中，当我追求染色质和癌症生物学的问题时， 独立的、以癌症为中心的教员职位。