Structural and biochemical characterisation of human SWI/SNF-related chromatin remodelling complexes

Student thesis: Doctoral ThesisDoctor of Philosophy

Abstract

The genomes of eukaryotes exist as chromatin. The organisation of chromatin provides a means of regulating access to the underlying genetic information. One way in which eukaryotes regulate chromatin is through the action of chromatin remodelling motor proteins. Human forms of the SWI/SNF remodelling complex are a series of chromatin remodelling ATPases comprised of some 15 partially overlapping subunits. These complexes function at enhancers and other regulatory elements. They have been observed to be mutated at high rates in a range of human cancers. Characterisation of the structure and biochemical activities of these complexes is limited. We aimed to address this by developing a system for the expression of human SWI/SNF complex subunits in insect cells as recombinant proteins. A complex of the core proteins was successfully expressed and purified. This complex was found to have robust biochemical activity, and enabled the investigation of the effects on remodelling resulting from small molecule inhibitors targeting SWI/SNF domains, and the effects of introduced subunit mutations. Complexes bearing a tumour-associated mutation in the SMARCA4 ATPase were purified and their enzymatic activity on nucleosomes was characterised. The presence of mutant complexes was found to reduce the activity of wild-type complexes, consistent with their dominant negative phenotype in vivo. A sub-complex of human PBAF was purified by the incorporation of PBAF-specific subunits PBRM1 and BRD7 into the core complex. The biochemical activity of both complexes on nucleosomes was tested. Both complexes were then investigated for their remodelling activity on nucleosomes bearing acetylated H3 histones, which are known to stimulate binding to and remodelling of nucleosomes by yeast SWI/SNF complexes. The PBRM1 sub-complex was found to remodel acetylated nucleosomes at a reduced rate compared to the core complex. Structural analysis by cryo-EM was undertaken for both of the complexes, however only low resolution structures were obtained.
Date of Award2022
Original languageEnglish
SupervisorTom Owen-Hughes (Supervisor) & Victoria Cowling (Supervisor)

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