MECHANISM OF HP1-MEDIATED HETEROCHROMATIN ASSEMBLY AND DURABILITY IN LIVE 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力学。我们使用 这些模型系统研究染色质调节途径的机制， 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是至关重要的，并且它在许多发育中失调。 人类疾病。然而，异染色质抑制的形成和记忆的分子机制 域知之甚少。为了了解这些调节过程在体内的功能，我们开发了 一种新的平台，允许个体染色质修饰活性，如异染色质蛋白-1（HP 1） 以高时间控制募集到天然染色质底物。我们最近改进了这一点 系统，使我们能够快速改变内源性染色质底物的结构，以探索 多种启动子和基因结构的调节。该项目的长期目标是 了解HP 1介导的基因抑制机制，并确定提供 通过连续细胞世代的异染色质稳定性。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装配的影响，例如：启动子转录 活性，DNA甲基化和组蛋白翻译后修饰，（2）这些相同的影响 染色质特征对异染色质基因阻遏持久性的影响。此外，使用高吞吐量 通过筛选，我们发现了破坏HP 1介导的基因抑制的新抑制剂。我们将开发 这些小分子探针，并使用这些化合物来确定个别酶活性的作用， 异染色质装配和持久的基因阻遏。在这些研究的结论中，使用 结合化学方法和新的体内工具，我们将提供一个新的通用模型， HP 1介导的异染色质如何在活细胞中组装和维持。

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.