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

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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.