MECHANISM OF HP1-MEDIATED HETEROCHROMATIN ASSEMBLY AND DURABILITY IN LIVE CELLS

HP1 介导的异染色质组装机制及其在活细胞中的耐久性

• 批准号: 10197949
• 负责人: Nathaniel A. Hathaway
• 金额: $ 31万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-07-01 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10197949
• 关键词: Alleles; Biological Assay; Biological Models; Cell Differentiation process; Cell Line; Cell division; Cells; Chemicals; Chromatin; Chromatin Modeling; Chromatin Structure; Complex; DNA; DNA Binding Domain; DNA Methylation; DNA Modification Process; DNA Sequence; Data; Dependence; Deposition; Development; Disease; Engineering; Enzymes; Euchromatin; Event; Excision; Fibroblasts; Future; Gene Expression; Gene Expression Regulation; Generations; Genes; Genetic Structures; Genetic Transcription; Goals; Heterochromatin; Histone Acetylation; Histone Deacetylase; Histone H3; Histones; Hour; Human; Human Development; Immune System Diseases; Individual; Kinetics; Laboratory Study; Lead; Lysine; Maintenance; Malignant Neoplasms; Malignant neoplasm of prostate; Mammalian Cell; Measures; Mechanics; Mediating; Memory; Methylation; Modeling; Modification; Mus; Mutate; Nuclear Lamina; Nucleosomes; Nucleotides; Organism; Pathology; Pathway interactions; Patients; Physiological; Post-Translational Protein Processing; Process; Promoter Regions; Proteins; Regulation; Regulatory Pathway; Reporter; Repression; Role; Series; Signal Transduction; Site; Stimulus; Structure; Support System; System; Tail; Testing; Therapeutic; Tissues; Tretinoin; Work; chromatin modification; density; developmental disease; embryonic stem cell; enzyme activity; gene repression; genetic information; heterochromatin-specific nonhistone chromosomal protein HP-1; high throughput screening; histone acetyltransferase; histone methyltransferase; histone modification; human disease; imaging modality; improved; in vivo; inhibitor/antagonist; malignant breast neoplasm; molecular mechanics; nervous system disorder; novel; overexpression; preservation; promoter; recruit; scaffold; small molecule; small molecule inhibitor; tool

Project Summary/Abstract My laboratory studies gene regulation dynamics in mouse embryonic stem cells and fibroblast tissues. We use these model systems to investigate the mechanics of chromatin regulatory pathways that provide stability to gene expression profiles through cell division allowing for mammalian tissue identity. Preservation of heterochromatin-mediated gene repression is critical for development and it is dysregulated in a number of human diseases. Yet, the molecular mechanics of formation and memory of heterochromatin repression domains are poorly understood. To understand how these regulatory processes function in vivo, we developed a novel platform that allows individual chromatin modifying activities such as Heterochromatin Protein-1 (HP1) to be recruited with high temporal control to native chromatin substrates. We have recently improved this system to allow us to rapidly change the structure of the endogenous chromatin substrate in order to explore the regulation of a diverse range of promoter and gene structures. The long-term goal of this project is to understand the mechanism of HP1-mediated gene repression and to determine the key features that provide heterochromatin stability through successive cell generations. Specifically, in this work we examine: (1) the influence on heterochromatin assembly of chromatin structural features such as: promoter transcriptional activity, DNA methylation, and histone posttranslational modifications, (2) the influence of these same chromatin features on the durability of heterochromatin gene repression. Additionally, using high throughput screening we have discovered novel inhibitors that disrupt HP1 mediated gene repression. We will develop these small molecule probes and use these compounds to define the role of individual enzymatic activities in heterochromatin assembly and durable gene repression. At the conclusion of these studies, using a combination of chemical approaches and novel in vivo tools, we will provide a new generalizable model for how HP1-mediated heterochromatin is assembled and maintained in living cells.

项目概要/摘要 我的实验室研究小鼠胚胎干细胞和成纤维细胞组织中的基因调控动态。我们使用 这些模型系统研究染色质调控途径的机制，为染色质提供稳定性 通过细胞分裂的基因表达谱允许哺乳动物组织身份。保存 异染色质介导的基因抑制对于发育至关重要，并且在许多方面都失调 人类疾病。然而，异染色质抑制的形成和记忆的分子机制 人们对这些领域知之甚少。为了了解这些调节过程在体内如何发挥作用，我们开发了 一个允许个体染色质修饰活动的新平台，例如异染色质蛋白-1 (HP1) 通过对天然染色质底物的高度时间控制来招募。我们最近对此进行了改进 系统使我们能够快速改变内源染色质底物的结构，以便探索 多种启动子和基因结构的调节。该项目的长期目标是 了解 HP1 介导的基因抑制机制并确定提供的关键特征 异染色质在连续细胞世代中的稳定性。具体来说，在这项工作中，我们研究：（1） 染色质结构特征对异染色质组装的影响，例如：启动子转录 活性、DNA 甲基化和组蛋白翻译后修饰，(2) 这些因素的影响 染色质特征对异染色质基因抑制的持久性的影响。此外，利用高通量 通过筛选，我们发现了能够破坏 HP1 介导的基因抑制的新型抑制剂。我们将开发 这些小分子探针并使用这些化合物来定义单个酶活性在 异染色质组装和持久的基因抑制。在这些研究的结论中，使用 结合化学方法和新颖的体内工具，我们将为 HP1 介导的异染色质如何在活细胞中组装和维持。