Lim, MWC; (2016) Biochemical Characterization of the CHD1, CHD5 and SMARCAD1 Chromatin Remodellers. Doctoral thesis , UCL (University College London).

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Abstract

By altering nucleosome positioning and composition, chromatin remodellers can modulate genomic accessibility, thereby regulating DNA metabolic processes. However, many remain poorly characterized, especially regarding the mechanisms underlying their regulation and genomic specificity. Some chromatin remodellers form multi-subunit complexes that change in subunit composition during development. It was reasoned that identifying tissue-specific subunits of chromatin remodellers would help elucidate their regulation. Thus, endogenous CHD1 and CHD5 were purified from mouse embryonic stem cells and brains respectively. However, detailed mass spectrometry analysis failed to reveal any novel tissue-specific complex subunits of either enzyme. The poorly characterized chromatin remodeller, SMARCAD1, is implicated in the maintenance of heterochromatin and double-stranded DNA break repair. SMARCAD1 constitutively forms a stoichiometric interaction with KAP1, a transcriptional co-repressor. The SMARCAD1-KAP1 complex was reconstituted in vitro with pure recombinant proteins. Although both SMARCAD1 and the SMARCAD1-KAP1 complex display nucleosome-stimulated ATPase activity, SMARCAD1 exhibits greater activity than the complex, suggesting that KAP1 is a negative regulator. Beyond a conserved SWI2/SNF2-like ATPase domain, SMARCAD1 features tandem CUE domains, which in other proteins, function as ubiquitin-binding domains. In cells, we find that point mutations affecting the SMARCAD1 CUE domains abrogate the SMARCAD1-KAP1 interaction. Significantly, using recombinant proteins that are not ubiquitylated, the SMARCAD1-KAP1 interaction was shown to be a direct, CUE domain-dependent, protein-protein interaction, involving the first SMARCAD1 CUE domain and the RBCC domain of KAP1. Unexpectedly, in silico approaches fail to identify a ubiquitin-homology domain within KAP1, suggesting either that KAP1 possesses an atypical ubiquitinhomology domain or that the first SMARCAD1 CUE domain uniquely recognizes a ligand structurally distinct from ubiquitin. An attempt to determine the co-crystal structure of the SMARCAD1-KAP1 interaction interface is described. Overall, the biochemical and structural characterization of the SMARCAD1-KAP1 complex offer mechanistic insight into the regulation of a chromatin remodeller with important roles in DNA metabolism.

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