Date of Award

2022

Degree Type

Dissertation

Degree Name

Doctor of Philosophy in Biological and Environmental Sciences

Specialization

Cell & Molecular Biology

Department

Cell & Molecular Biology

First Advisor

Arnob Dutta

Abstract

The multi-subunit chromatin remodeler, SWI/SNF, is evolutionarily conserved from yeast to humans. The general function of this remodeler is in gene regulation and manipulation of chromatin architecture. In yeast, SWI/SNF acts upon only a subset of genes but under stress conditions, SWI/SNF is targeted to a greater set of genes. The human SWI/SNF has vital roles in cell differentiation, development, and cellular processes and variations of the complex can exist across tissues and cells. SWI/SNF is recruited to chromatin via two methods: covalent modifications of histones and interaction with transcription factors. Specific subunits of the complex contain domains that can identify modifications on histone tails as well as specific sequences on transcription factors. Environmental cues can change the landscape of chromatin, in which cellular responses are necessary to coordinate transcriptional changes. A review of SWI/SNF composition, function, and genomic instability is detailed in Chapter I.

In Chapter II, we identified that human SWI/SNF subcomplexes are not static as a response to hypoxic (1% O2) conditions. First, we probed for exclusive subunits elevated in this condition and identified the complex associated with the subunits. To verify our results, we knocked down the exclusive subunits and measured cell survivability and induction of SWI/SNF dependent genes. All three complexes were present in normoxic conditions, but do not remain fixed upon hypoxic induction, instead the balance of the complexes was shifted as a response to environmental cues, in this case, hypoxia.

In Chapter III, we hypothesized that there are novel interactors of yeast SWI/SNF due to aberrant complexes present when loss of subunits disrupts the integrity of the complex. Using transformation knockout experiments of different SWI/SNF subunits, we observed a synthetic genetic interaction occurring between altered SWI/SNF complexes and the 26S proteasome, specifically the 19S regulatory particle (19S RP) subcomplex. Our studies showed that defects in the 19S RP affected SWI/SNF growth, but not the 20S core particle, and the 26S proteasome regulated biochemical functions and genomic landscape of yeast SWI/SNF. This study provides evidence that the proteasome is regulating functions of yeast SWI/SNF through an association rather than a degradation process in which the 26S proteasome is known for, and shows another non-canonical role of the proteasome.

In Chapter IV, we examined another novel interactor of yeast SWI/SNF, Pyk1, a pyruvate kinase that has been shown to have protein kinase activity in humans, PKM2. Using post-translational modification mass spectrometric analysis, we determined phosphorylation sites on Snf2 and Swi3 that could be targeted by Pyk1 for its protein kinase activity. Pyk1 has also been found to phosphorylate histone 3 at threonine 11 (H3T11), a possible recruitment marker for SWI/SNF. We performed remodeling assays using phosphorylated histones but were unable to determine any changes in remodeling activity of SWI/SNF. Another complex able to phosphorylate H3T11 in yeast is CK2. We purified the complex and performed in vitro kinase activity and were able to detect phosphorylation on the histones. SWI/SNF remodeling activity was surprisingly decreased in the presence of phosphorylation of H3T11, suggesting that H3pT11 prevents SWI/SNF from remodeling nucleosomes.

Available for download on Wednesday, May 17, 2023

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