A study of dynamic changes to chromatin structure and composition

In addition to the plethora of post translational modifications (PTMs) to histone tails, an additional level of structural and regulatory complexity is conferred to the nucleosome by histone variant proteins. Histone variants are alternative forms of histone proteins which replace their canonical counterparts in the nucleosome thereby effecting different properties to the resultant nucleosome.;The primarily repressive H2A variant macroH2A was identified in 1992, but the factors that regulate macroH2A's deposition into the nucleosome are unknown. Via a large scale unbiased approach, I identified the SWI/SNF helicase, ATRX, as a novel, chromatin-free interactor of macroH2A. Using deletion constructs of ATRX, I found that the N' terminal region of ATRX suffices for this interaction to persist in a chromatin-free manner.;Upon depleting cells of ATRX I found increased levels of endogenous macroH2A in chromatin, while whole cell levels and mRNA transcripts were unaffected. After noting an increased deposition of macroH2A at telomeres in cells depleted of ATRX, I proceeded to delineate the relationship between ATRX loss and macroH2A gain in the alpha thalassemia disorder, characterized by mutations in ATRX which result in the loss of alpha globin gene expression. Reasoning that the increase to macroH2A chromatin deposition at telomeres likely also persists to the telomere proximal alpha globin genes in cells lacking ATRX contributing to the silencing of this locus, I proceeded to analyze macroH2A's genome wide deposition via ChIP-sequencing. Thereby I noted an increased presence of macroH2A at the alpha globin gene cluster in cells depleted of ATRX.;Mechanistically, it is unknown how the loss of functional ATRX results in the alpha thalassemia phenotype of the ATRX syndrome. My work hence contributes to the understanding of how the loss of ATRX, at least in part, results in the loss of alpha globin expression - via the aberrant deposition macroH2A.