Engineering Cell Lines to Investigate the Function of Mammalian SWI/SNF Complexes

Student thesis: Doctoral ThesisDoctor of Philosophy

Abstract

Mammalian SWI/SNF complexes belong to the class of chromatin remodelling complexes, and consist of approximately 15 subunits. Bioinformatics analyses of tumour sample sequencing studies have shown that members of the mSWI/SNF complexes are mutated in almost 20% of all cases, suggesting many subunits have tumour suppressor functions. The residual complex that remains upon inactivation of a subunit is thought to have tumorigenic effects, but how loss of different subunits affects the complex and chromatin architecture is still under investigation. Furthermore, most studies into the biological role of SWI/SNF in cells rely on knock-down or knock-out models, which potentially give rise to substantial indirect effects following cellular adaptation to subunit loss. Consequently, it is of great interest to generate targeted approaches to study SWI/SNF loss of function over short time scales. With this aim, we have developed a conditional protein degradation approach in mouse embryonic stem cells. This enables substantial depletion of the target subunit within two hours. Immunoprecipitation of complexes depleted for SWI/SNF components showed specific degradation of the targeted subunit. The effect of these residual complexes on chromatin architecture was probed using ATAC-seq. This showed that chromatin accessibility was lost at many sites following depletion of SWI/SNF components. These areas of decreased accessibility frequently occurred at enhancers, and overlapped with the binding sites for pluripotency factors OCT4, SOX2 and NANOG. The development of this system for acute depletion provides a means to assess the order in which subsequent changes to chromatin modifications and gene expression occur following loss of different subunits.
Date of Award2018
Original languageEnglish
SponsorsCancer Research UK
SupervisorTom Owen-Hughes (Supervisor)

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