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Title: TAZ2 domain of CBP binds DNA to regulate histone acetylation
Author: Sheahan, Thomas William
ISNI:       0000 0004 9348 9465
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2020
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DNA sequences that regulate gene expression are marked by distinct chromatin architectures that segregate these functional elements from the surrounding genome. In mammals, DNA methylation is prevalent throughout the genome and is repressive to transcription. However, the majority of gene promoters are associated with regions of CG- and CpG-rich DNA called CpG islands (CGIs), which are unmethylated and permissive to transcription. Although previous studies have shown that CGIs act as platforms for the recruitment of both active and repressive chromatin-modifying activities, it remains only partially understood how mechanistically the chromatin environment at CGIs is established. To address this, an unbiased proteomics approach was adopted to generate an inventory of CGI-binding proteins with the aim of understanding how binding to CGIs influences promoter function. In parallel to this, a candidate approach was used to gain a greater understanding of how the chromatin landscape is established at distal regulatory regions called enhancers. Enhancers are typically associated with monomethylation of H3K4, placed by the histone methyltransferases (HMTs) MLL3 and MLL4, and with acetylation of H3K27, placed by the histone acetyltransferase (HAT) CBP and its paralogue p300. To understand how MLL3 and MLL4 contribute to enhancer function, the MLL3/MLL4 complexes were purified from mammalian cells and interaction partners were analysed by mass spectrometry to determine complex composition. To address how CBP contributes to the chromatin environment at enhancers, an in vitro domain mapping approach was developed using purified CBP and recombinant nucleosome templates. This showed that the CBP TAZ2 domain, located downstream of the catalytic HAT domain, is required for efficient acetylation of H3K27 in chromatin and that this is mediated through the TAZ2 domain driving association with nucleosomes via sequence-independent interaction with DNA. Further work showed that the TAZ2 domain is important for stable binding to chromatin in vivo and facilitates specific acetylation of H3K27 to activate transcription from regulatory elements. Together, this work elucidates a novel mechanism by which CBP HAT activity is selective for H3K27, forming the basis of a model in which mechanisms that determine HAT substrate specificity are vital to ensure robust regulation of gene expression.
Supervisor: Voigt, Philipp ; Bird, Adrian Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
Keywords: CpG islands ; CGIs ; H3K4 ; histone methyltransferases ; chromatin ; CBP TAZ2