Date of Award
Doctor of Philosophy
Biochemistry and Cellular and Molecular Biology
Bruce McKee, Rachael P. McCord, Jae Park, Keerthi Krishnan, Tarek Hewezi
The ability of a cell to sense and respond to various forms of stress is essential to maintain integrity of the genome. Numerous pathways have been implicated in cellular responses to environmental and genotoxic stresses, often involving proteins and complexes that bind DNA directly to orchestrate changes in transcription and genome organization. Chromatin insulators describe a class of protein complex that bind specific sequences in the genome and work through two classically described functions: to restrict communication between enhancers and promoters through physical separation into different genomic domains and to prevent the spread of heterochromatin into euchromatic regions of the genome. Insulator sites also demarcate the boundaries between topologically associated domain (TAD) boundaries. Drosophila melanogaster has an array of different insulator complexes, with each complex being recruited to different sequences. Here, we demonstrate an interaction between proteins that associate with the gypsy insulator and the phosphorylated histone variant H2Av (γ[lowercase gamma]H2Av), a marker of DNA double strand breaks. The gypsy retrotransposon contains binding sites for Suppressor of Hair Wing (Su(Hw)), which interacts with and recruits Mod(mdg4)67.2 and CP190. Components of this insulator complex colocalize with γ[lowercase gamma]H2Av throughout the genome and at specific gypsy loci. Mutation of insulator components prevents stable H2Av phosphorylation in polytene chromatin. Deficiency for Su(Hw) in particular is also associated with chromosomal aberrations in actively dividing larval neuroblasts. PP2A phosphatase inhibition strengthens the association between insulator components and γ[lowercase gamma]H2Av and rescues γ[lowercase gamma]H2Av localization in insulator mutants. Likewise, mutation of His2Av affects binding of gypsy insulator components in polytene chromatin. We demonstrate that mutation of su(Hw) suppresses the melanotic tumor phenotype associated with the lack of H2Av. We also show that γ[lowercase gamma]H2Av is a component of insulator stress bodies, and that phosphatase activity is required for insulator body dissolution after recovery from stress. We propose a model in which H2Av and its phosphorylation are functionally linked with the activity of insulator binding proteins. Together, our results add to the growing body of evidence linking genome organization to genome stability and provide potential clues to one such mechanism in flies.
Simmons, James Ryan, "The Histone Variant H2Av Regulates Stress Responses and Tissue Development through Interactions with Chromatin Insulator Proteins in Drosophila melanogaster. " PhD diss., University of Tennessee, 2021.