Degree Type

Dissertation

Date of Award

2010

Degree Name

Doctor of Philosophy

Department

Biochemistry, Biophysics and Molecular Biology

First Advisor

Michael A. Shogren-knaak

Abstract

The SAGA family of coactivators is a group of highly conserved histone acetyltransferases that is essential for transcription of genes participating in stress adaptation and developemental processes. Misregulation of SAGA function can lead to developmental diseases and cancer. One important function of SAGA in regulating gene expression is to acetylate nucleosomes at both gene promoters and coding regions. To understand the molecular mechanism of nucleosome acetylation, much work has been done using recombinant Gcn5, the catalytic subunit of SAGA, and histone proteins or peptides. However, little is known about how histones are acetylated by full SAGA complex in the context of chromatin and how this relates to expression of SAGA-dependent genes. In this study, we performed histone acetyltransferase (HAT) kinetic assay using purified SAGA complex and reconstituted nucleosomal arrays. We found that SAGA complex acetylates nucleosomes in a highly cooperative manner with a cooperativity coefficiency of 1.97 y 0.15, suggesting that its maximal acetylation activity can be augmented by binding to multiple nucleosomes. Next, we explored the requirement for cooperative nucleosome acetylation with respect to substrates and SAGA enzyme complex, respectively. To determine the substrate requirement for cooperativity, various novel nucleosomes were generated and investigated through HAT kinetic assay. We found that SAGA-mediated cooperativity occurs only when both histone H3 tails are present and properly oriented in the nucleosomes. Furthermore, the four acetylation lysine residues on both H3 tails must be present and unacetylated. To determine factors in SAGA complex required for cooperative nucleosome acetylation, we performed HAT kinetic assay using a SAGA trimeric subcomplex composed of Ada2, Ada3, and Gcn5 and our studies showed that these three subunits are the minimal requirement to recapitulate SAGA-mediated cooperativity. Additionally, we discovered a novel regulatory mechanism of SAGA that enhances its activity when cells were grown under stressful conditions. Our results showed that SAGA acetylates one of its subunit Ada3 and undergoes acetylation-dependent dimerization when cells sense stress signals in the environment. Most importantly, this acetylation-dependent dimerization of SAGA contributes to cooperative nucleosome acetylation and facilitates cell growth under stress conditions. To probe which protein domains within these three subunits are required for cooperativity, we generated a series of subcomplex mutations and discovered that Gcn5 bromodomain is necessary for cooperativity. To further study the functions of Gcn5 bromodomain in SAGA-mediated nucleosome acetylation, we performed HAT kinetic assay on nucleosomes containing either pre-acetylated single H3 tail or pre-acetylated histone H4K16. We found that the Gcn5 bromodomain facilitates cross-tail H3 acetylation in pre-acetylated nucleosomes. Collectively, our studies revealed a novel mechanism which SAGA modulates its activity and nucleosome acetylation. Furthermore, this regulatory mechanism has important implications in inducible gene transctiption and co-regulation of functionally related genes.

DOI

https://doi.org/10.31274/etd-180810-1446

Copyright Owner

Shanshan Li

Language

en

Date Available

2012-04-30

File Format

application/pdf

File Size

137 pages

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