Title: Roles for histone H2A variants in gene regulation
Description: Genomic packaging of DNA into nucleosomes makes DNA refractory to transcription. Therefore much of gene regulation occurs at the level of the nucleosome. Canonical histones are responsible for bulk packaging of DNA into nucleosomes but are often replaced at sites of gene regulation by non-allelic histone variants. The mechanism of how histone variants impact gene regulation is not well understood. This dissertation studies the biology of histone variants H2A.Z and H2A.Bbd, both which are hypothesized to be involved in gene regulation. The primary method to learn more about the biological function of these variants was analysis of proteins that interact with variant nucleosomes. A class of proteins, called ATP-dependent chromatin remodelers, was analyzed for interaction with H2A.Z since remodelers are also localized to gene control loci and display similar phenotypes. The relative association of chromatin remodelers with H2A.Z chromatin was determined and H2A.Z was found to be associated in vivo with remodeling complexes involved in transcription. Activity of remodeling enzymes on H2A.Z nucleosomes was further analyzed in vitro . Inclusion of H2A.Z stimulated activity of only the ISWI family of chromatin remodelers although all families remodeled H2A.Z nucleosomes. Essential amino acid residues on H2A.Z are required for increasing ISWI activity suggesting that stimulation may be important biologically.

Genomic loci containing the H2A.Bbd variant were determined by high-throughput sequencing of DNA from purified H2A.Bbd chromatin. Contrary to H2A.Z, H2A.Bbd was depleted from promoters but enriched in gene 'bodies' of expressed genes. Furthermore, chromatin containing H2A.Bbd was associated with both elongating RNA Polymerase II and multiple members of the spliceosome. We hypothesize that H2A.Bbd serves as a link between the concurrent processes of transcription and splicing. These studies highlight different ways histone H2A variants can function in gene expression. The H2A.Z variant which is mainly localized to promoters stimulates activity of proteins important in early stages of transcription and the H2A.Bbd variant functions further downstream during transcription elongation possibly linking elongation with processing of mRNA.