Date of Award

8-1-2012

Degree Name

Doctor of Philosophy

Department

Molecular Biology, Microbiology and Biochemistry

First Advisor

Bartholomew, Blaine

Abstract

ISW1 was initially identified as a single complex. Four years after its identification it was reported to form two separate complexes, namely ISW1a and ISW1b. ISW1a is composed of Isw1 and Ioc3; ISW1b consist of Isw1, Ioc2 and Ioc4. While these complexes share the same catalytic subunit, these complexes differ in their catalytic properties and function in vivo. ISW1a was shown to be localized at the promoter region of genes, functioning in transcription repression. True to this function, it exhibits directional remodeling activity and the ability to space nucleosome arrays at ~175bp increments, promoting the formation of a repressive chromatin structure. ISW1b lacks both of these biochemical properties and was found localized within the coding regions of genes functioning in the regulation of transcription elongation and termination. These differences in activity are attributed to the differences in subunit composition and the domain organization of these two complexes. Our work has identified and characterized novel domains within the Ioc2 and Ioc3 subunits that modulate the distinct biochemical properties of these complexes. We found that the IRD (ISW1a Regulator of Directionality) domain of the Ioc3 subunit, functions as a regulatory motif that dictates the directionality preferences and spacing activity exhibited by ISW1a during remodeling. Deletion of this domain resulted in an unregulated ISW1a complex in vivo which severely altered growth under stress conditions, gene expression and nucleosome organization genome-wide. Our work also characterized two domains within the Ioc2 component of the ISW1b complex, namely Ioc2 458-551 and a PHD domain. We find that these domains contribute significantly to the affinity of ISW1b for nucleosome substrates, as well as the ability of this complex to stimulate ATPase activity and remodel. Strikingly we find that the PHD domain functions as a distinguishing feature that differentiates ISW1a from ISW1b. Loss of this domain resulted in a mutant complex that remodels similarly to ISW1a. In vivo we also find that both of these domains contribute to nucleosome positioning and the modulation of gene expression of ISW1b regulated genes genome-wide. Overall this study provides an examination of the distinguishing features of the ISW1a and ISW1b complexes both in vitro and in vivo.

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