GSBS Dissertations and Theses

Approval Date

August 2000

Document Type

Doctoral Dissertation

Department

Graduate School of Biomedical Sciences, Biochemistry

Subjects

Chromatin; Enzymes; Academic Dissertations; Dissertations, UMMS

Abstract

Chromatin structure plays an essential role in the regulation of many nuclear processes such as transcription, replication, recombination, and repair. It is generally accepted that chromatin remodeling is a prerequisite step in gene activation. Over recent years, large multisubunit enzymes that regulate the accessibility of nucleosomal DNA have emerged as key regulators of eukaryotic transcription. It seems likely that similar enzymes contribute to the efficiency of DNA replication, recombination, and repair. These chromatin remodeling complexes can be classified into two broad groups: (1) the ATP-dependent enzymes, which utilize the energy of ATP hydrolysis to increase the accessibility of nucleosomal DNA; and (2) histone modifying enzymes that phosphorylate, acetylate, methylate, ubiquitinate, or ADP-ribosylate the nucleosomal histones (for review see Kingston and Narlikar, 1999; Muchardt and Yaniv, 1999; Brown et al., 2000; Vignali et al., 2000; Strahl and Allis, 2000).

The mechanism by which these two groups of large, multi-subunit enzymes function to alter chromatin structure is enigmatic. Studies suggest that ATP-dependent and histone acetyltransferase chromatin remodeling enzymes have widespread roles in gene expression and perform both independent and overlapping functions. Interestingly, although both groups of enzymes appear to be distinct, several features of these enzymes have been conserved from yeast to man. Thus, understanding the role of these similar features will be essential in order to elucidate the function of remodeling enzymes, their functional interrelationships, and may uncover the fundamental principals of chromatin remodeling. In this study, we use a combination of yeast molecular genetics and biochemistry to dissect out the function of individual parts of these chromatin remodeling machines and to understand how these large macromolecular assemblies are put together. In addition, we also investigate the mechanism by which the ATP-dependent enzymes exert their regulatory effects on chromatin structure.

Structure/function analysis of Saccharomyces cerevisiae Swi3p (conserved in SWI/SNF complexes across all eukaryotic phyla) reveals a unique scaffolding role for this protein as it is essential for assembly of SWI/SNF subunits. We have also characterized a novel motif that has homology to the Myb DNA binding domain, the SANT domain, and that is shared among transcriptional regulatory proteins implicated in chromatin remodeling. Mutational analysis of this domain in yeast Swi3p (SWI/SNF), Rsc8/Swh3p (RSC), and Ada2p (GCN5 HATs) reveals an essential function for the SANT domain in chromatin remodeling. Moreover, our studies suggest that this novel motif may be directly involved in mediating a functional interaction with chromatin components (i.e. histone amino terminal domains).

We have also directly compared the activities of several members of the ATP-dependent chromatin remodeling enzymes. Surprisingly, we find that these enzymes utilize similar amounts of ATP to increase nucleosomal DNA accessibility. In as much, we show that changes in histone octamer comformation or composition is not a requirement or consequence of chromatin remodeling by SWI/SNF. Taken together, these data suggest a similar mechanism for ATP-utilizing chromatin remodeling enzymes in which disruption of histone-DNA contacts occur without consequence to the structure of the histone octamer. These data have striking implications for how we view the mechanism of chromatin remodeling.

Comments

Some images did not scan well. Please consult original document. Pages 117-118 flipped in document but corrected in electronic file.

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Copyright is held by the author, with all rights reserved.

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