Ma, Meiji Kit-Wan
Characterisation of histone modifications at insulator elements.
PhD thesis, University of Glasgow.
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The genomes of higher eukaryotes are marked by distinct chromatin domains, which allow for the control of different gene expression states. It is thought that the boundaries of chromatin domains could be formed by DNA sequence elements called insulators. The paradigm HS4 insulator element is located at a boundary between the β-globin gene cluster and an adjacent condensed chromatin domain. Proteins that bind to the HS4 sequence recruit enzymes that mediate a number of histone modifications generally associated with chromatin accessibility. Inspired by yeast genetic studies, we hypothesised that H2B ubiquitination might be a key regulator of these ‘active’ marks. It was found that HS4 and another chromatin boundary at the neighbouring FOLR1 gene locus, HSA/HSB, are sites of H2B ubiquitination. The ubiquitination E3 ligase RNF20 was found to be necessary for global H2B ubiquitination and for methylation of H3K4, in a trans-histone modification pathway that is conserved from yeast to man. RNAi-mediated knockdown of RNF20 not only resulting in the depletion of H2B ubiquitination normally found at chromatin boundaries, but also disrupted their H3K4 methylation and acetylation at multiple histones. H2B ubiquitination is a master controller of the active chromatin state at the HS4 and HSA/HSB chromatin boundaries. Long term depletion of RNF20 expression leads to a compromise of the boundaries, allowing the spreading of heterochromatin into the FOLR1 and β-globin gene loci, resulting in gene silencing. This study also looked at the recruitment of factors that mediate the incorporation of the histone variant H2A.Z at chromatin boundary elements in vertebrates. It was found that insulator binding proteins control H2A.Z incorporation and acetylation.
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