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

项目摘要/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。我们用 这些模型系统用来研究染色质调节通路的机制，这些通路为 通过细胞分裂的基因表达谱，允许哺乳动物组织的同一性。保存 异染色质介导的基因抑制对发育至关重要，在许多 人类疾病。然而，异染色质抑制的形成和记忆的分子机制 人们对领域的了解很少。为了了解这些调节过程如何在体内发挥作用，我们开发了 一种允许单个染色质修饰活动的新平台，如异染色质蛋白-1(HP1) 对天然染色质底物有较高的时间控制力。我们最近改进了这一点 系统允许我们快速改变内源性染色质底物的结构，以便探索 对多种启动子和基因结构的调控。这个项目的长期目标是 了解HP1介导的基因抑制的机制，并确定提供 异染色质在连续几代细胞中的稳定性。具体地说，在这项工作中，我们检查：(1) 影响异染色质组装的染色质结构特征如：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 染色质的特性决定了异染色质基因抑制的持久性。此外，使用高吞吐量 通过筛选，我们发现了新的抑制剂，可以破坏HP1介导的基因抑制。我们将发展 这些小分子探针和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。在这些研究结束时，使用 结合化学方法和新的活体工具，我们将提供一个新的可推广的模型 HP1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.