Structural Investigations of the NuRD Complex and Its Interaction with Chromatin

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.

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.