Degree Type

Dissertation

Date of Award

2015

Degree Name

Doctor of Philosophy

Department

Biochemistry, Biophysics and Molecular Biology

First Advisor

Michael Shogren-Knaak

Abstract

The SAGA family of co-activators play a major role in regulating eukaryotic gene expression. These multiprotein complexes are highly conserved across all eukaryotic species, from budding yeast to humans. Transcription of inducible genes is facilitated by SAGA, by mediating acetylation of amino tails of the histone H3. The acetyltransferase activity of SAGA is harbored in the Gcn5 subunit of the complex. Previous studies characterized the enzymatic properties of the recombinantly expressed HAT domain of Gcn5 from Saccharomyces cerevisiae, on synthetic H3 peptides. We were interested in studying the properties of endogenous Gcn5, in the context of full SAGA complex. We also utilized nucleosomes as substrates, since the basic structural unit of chromatin is a nucleosome.

Towards this end, we first turned our efforts into developing a reliable, reproducible and sensitive assay for detecting acetylation on nucleosomes. Previous studies utilized filter binding assays, which work great for short peptides. Since these peptides are positively charged, they bind to the filter in an efficient and reproducible manner. Nucleosomes, being net negatively charged and structurally fragile, required an alternative approach. We developed bead based strategies which immobilize the nucleosome substrates, and thus circumvented the above-mentioned issues.

We utilized a trinucleosome system to study the pattern of acetylation established across the nucleosomal array under saturating and sub-saturating conditions. We observed a 2 fold preference for nucleosomes harboring longer linker lengths on one side. By further optimizing the bead based assay for performing initial rate, steady state kinetics, we designed several nucleosomes harboring different lengths of linkers on both sides. Our data suggest that linker DNA longer than 80 bp on one end stimulate the HAT activity of SAGA. We also show that a long linker in combination with linker on the other side of the nucleosome stimulates SAGA to the maximum potential.

Steady state data on different nucleosomes revealed that SAGA binds to all nucleosomes pretty tightly, but the turnover rates were lower than expected. Our bead based assay also allows us to perform single turnover experiments, which would give important information on the nature of chemical step. Preliminary data suggests that indeed, the first turnover is pretty fast, but subsequent cycles are much slower, indicating a step post catalysis, such as product release, to be limiting. We are currently working towards understanding this burst phase in more detail.

In vivo, different factors work in concert to regulate gene transcription. For example, DNA bound activators recruit SAGA to target genes, to initiate transcription. Previous studies have followed a combination of genetic and biochemical approaches to qualitatively assess this interaction. In order to investigate the effect of this interaction from a quantitative perspective, we incorporated activator binding site in the linker DNA of one of the nucleosomes. Our preliminary data suggests that activator modulates SAGA complex in potentially 2 different ways, even beyond the stimulation mediated by linker DNA. This might explain how the cell is able to quickly respond to stress environments and turn on genes to achieve homeostasis.

Overall, our results help us in gaining better insights into the mechanisms employed by the SAGA complex in maintaining basal levels of acetylation under normal conditions, and establishing hyperacetylated domains under inducing conditions.

DOI

https://doi.org/10.31274/etd-180810-4545

Copyright Owner

Chitvan Mittal

Language

en

File Format

application/pdf

File Size

130 pages

Included in

Biochemistry Commons

Share

COinS