Functional characterisation of variant dMi-2 nucleosome remodeling complexes in epigenome regulation

多亚单位染色质调节复合体(CRCs)在正常情况下和疾病中对表观基因组的调节起着关键作用。大多数CRC含有修饰或重塑核小体的酶。许多经典的CRC，如PRC1、PRC2或NuRD，最初被认为是单一的复合体，现在被证明存在于不同的亚基组成和辅助蛋白的不同版本中。非酶亚基和辅助蛋白被认为是靶向或调节CRCs中所含的酶，并赋予CRCs特定的功能。然而，CRC专门化的分子机制还不是很清楚，因此，CRC的异质性是目前表观遗传学研究的一个热点。核小体重塑和去乙酰化(NuRD)复合体是一种丰富的CRC，在所有后生动物中都是保守的。它在干细胞生物学和发育中发挥重要作用，其中NuRD调节染色质结构、基因转录和细胞周期。此外，NuRD对于DNA修复是必不可少的，其亚单位在癌症中经常错误表达或突变。顾名思义，NuRD结合了核小体重塑和组蛋白脱乙酰酶活性。由于其丰富性、进化保守性、广泛的生物学功能以及其依赖于ATP的核小体重塑和组蛋白修饰活性的整合，NuRD被认为是研究CRC功能基本原理的首选范例。最近的工作表明，NuRD的依赖于ATP的重塑亚单位Mi-2/CHD4存在于几个特征不佳的替代组件中。不同的Mi-2/CHD4复合体在形成表观基因组方面的功能差异尚不清楚。在这个项目中，我们将使用CRISPR/CA、可诱导靶向蛋白耗竭和表观基因组分析相结合的方法来确定两个可供选择的包含Mi-2/CHD4的复合体dNuRD和dMec的功能。在全基因组水平上，我们将确定dNuRD和dMec(及其非酶亚基)对启动子和增强子的转录和染色质结构的调节作用。此外，我们将以生物学上重要的蜕皮激素系统为模型，定义dNuRD和dMec在激素诱导的基因激活中的作用。这些方法将定义替代的含Mi-2/CHD4的复合体用来塑造表观基因组的分子机制。在更一般的术语中，我们将学习不同CRC复合体的非酶亚基如何赋予酶亚基特定的功能。

Multisubunit chromatin regulating complexes (CRCs) play pivotal roles in epigenome regulation in normal settings and in disease. Most CRCs contain enzymes that modify or remodel nucleosomes. Many classic CRCs, such as PRC1, PRC2 or NuRD, that were originally thought of as single complexes have now been demonstrated to exist in different versions that vary in subunit composition and accessory proteins. Non-enzymatic subunits and accessory proteins are believed to target or regulate the enzymes contained within CRCs and to endow CRCs with specific functions. However, the molecular mechanisms underlying CRC specialisation are not well understood and, consequently, the CRC heterogeneity is currently a hot topic in epigenetic research.The Nucleosome Remodeling and Deacetylation (NuRD) complex is an abundant CRC that is conserved in all metazoans. It plays important roles in stem cell biology and development where NuRD regulates chromatin structure, gene transcription and the cell cycle. Moreover, NuRD is essential for DNA repair and its subunits are frequently misexpressed or mutated in cancer. As the name implies, NuRD combines nucleosome remodeling and histone deacetylase activities. Due to its abundance, evolutionary conservation, its wide ranging biological functions and its integration of ATP-dependent nucleosome remodeling and histone modification activities, NuRD serves as a premier paradigm to study fundamental principles of CRC function.Recent work suggests that Mi-2/CHD4, the ATP-dependent remodeling subunit of NuRD, exists in several, poorly characterised alternative assemblies. The functional differences between variant Mi-2/CHD4 complexes in shaping the epigenome are unknown. In this project, we will use a combination of CRISPR/Cas, inducible targeted protein depletion and epigenome analyses to identify the functions of two alternative Mi-2/CHD4-containing complexes, dNuRD and dMec. At a genomewide level, we will determine the contributions of dNuRD and dMec (and their non-enzymatic subunits) to the regulation of transcription and chromatin structure at promoters and enhancers. In addition, we will define the roles of dNuRD and dMec in hormone-induced gene activation, using the biologically important ecdysone system as a model.These approaches will define the molecular mechanisms that alternative Mi-2/CHD4-containing complexes use to shape the epigenome. In more general terms, we will learn how non-enzymatic subunits of variant CRC complexes endow the enzymatic subunit with specific functions.