Structural Investigations of the NuRD Complex and Its Interaction with Chromatin

<p dir="ltr">Class I Histone deacetylase (HDAC) complexes are large molecular machines that act upon chromatin to maintain a cell’s ability to regulate gene expression. The Nucleosome Remodelling and Deacetylase complex (NuRD) is a part of a family of seven distinct class I HDAC complexes. Each member is unique with distinct components, stoichiometries, and functions. Where the activity and specificity of the Class I HDAC enzymes are determined by diverse complex recruitment in various cellular contexts. While parts of the NuRD complex are structurally well-characterised its assembly is less well understood. Additionally, its chromatin binding activity and mechanism of engaging chromatin are not completely elucidated. Chromatin recruitment of the HDAC1/2 enzyme by the NuRD complex deacetylase module is enabled by chromatinbinding domain-containing proteins MTA1/2/3, MBD2/3, and RBBP4/7. Using cryoelectron microscopy, the structure and topology of the MTA-BAH and ZnF domains, as well as the MBD-IDR with the core complex have been determined. This has led to a greater understanding of the assembly of the deacetylase module and how it interacts with chromatin.</p><p dir="ltr">To investigate how NuRD interacts with its chromatin substrate, electromobility shift assays (EMSAs) with NuRD sub-complexes and recombinant and endogenous chromatin were performed. Moreover, the endogenous NuRD complex was purified from CRISPR/Cas9 gene-edited HCT116 cell lines and co-purifies bound to nucleosomes. This discovery was investigated further using biochemical, proteomic and electron microscopy methods. The research described in this thesis, in combination with multi-genomic approaches, will contribute to the understanding of NuRD complex diversity, localisation and function in the regulation of gene expression, cell differentiation, organism development and disease.</p>