Title

Functional implications of macrohistones in embryonic stem cells and differentiated progeny

Date of Completion

January 2011

Keywords

Biology, Genetics

Degree

Ph.D.

Abstract

Chromatin properties are increasingly gaining interest as important factors in the maintenance of the pluripotent nature of ESCs, as well as a determinant factor in lineage specification during differentiation. Histone variants, specialized isoforms of canonical core histones that confer distinct functional properties to the nucleosomes, are thought to have significant impact on these fundamental processes. Macrohistones (mH2As) belong to the family of closely related proteins that is comprised of three subtypes; mH2A1.1 and mH2A1.2 are splice variants of the gene H2afy, while mH2A2 is encoded by a separate gene (H2afy2), located on a different chromosome. Protein products of these two genes have been found enriched on the inactive X chromosome and other heterochromatic regions, findings implicating mH2As in gene repression. Microarray expression analysis in mH2A1.2-deficient ESCs demonstrated significant regulation for the subset of genes associated with early cell lineage decisions. Approximately 1/3 of the regulated genes were down-regulated, in contrast to the proposed repressive function for mH2As. Chromatin immunoprecipitation sequencing (ChIP-Seq) studies revealed the presence of mH2A1-nucleosomes in the chromatin of male ESCs on all autosomes, while the sex chromosomes were markedly devoid of mH2A1 signals across their entire lengths. RNAi-induced depletion of the mH2A-pool in male and female ESCs did not lead to defects in growth rates or developmental potential of these cells either in vitro or in vivo. No difference was observed for ESCs that retain mH2A1.1 in the differentiated state, compared to ESCs that are depleted for all mH2A isoforms. Imprinted expression of a selected subset of genes was unaffected in female knockdown ESC lines. Results the presented here imply an important function for mH2A1 in the control of developmentally-regulated genes in ESCs, but indicate the presence of multiple epigenetic mechanisms that the can compensate for the function of mH2As during in vitro development. ^^