Date of Award

12-1-2010

Degree Name

Master of Science

Department

Molecular Biology Microbiology and Biochemistry

First Advisor

Bartholomew, Blaine

Abstract

The compact structure of the eukaryotic genome dictates the accessibility to genes, and therefore adds an additional layer of regulation for gene expression. A specialized class of proteins called "chromatin remodelers" facilitates this process in the cell. The imitation switch (ISWI) subfamily of chromatin remodelers is a well studied class of proteins affecting gene expression. Its member ISW2 was recently shown to behave differently from other chromatin remodeling proteins. For instance, the ISW2 complex has been shown to be stimulated by ~5-6 fold in its ATPase activity when bound to a nucleosome rather than to a DNA molecule. Nucleosome remodeling by ISW2 has even been shown to depend on the N-terminal tail of histone H4 and therefore, the octamer of a nucleosome might be playing a significant role in nucleosome remodeling by the ISW2 complex. The aim in this investigation was to delineate the protein-protein interactions that the ISW2 complex establishes with the octamer upon binding to a nucleosome. Several histones with unique cysteines engineered at specific positions were refolded with other wild type histones to produce histone octamers with a single cysteine in one of the four histones. Based on previous reports from site-specific DNA photoaffinity cross-linking and hydroxyl-radical footprinting experiments, it was inferred that the SHL2, entry-exit position and the extranucleosomal linker DNA were contacted by the ISW2 complex on a nucleosome1. Considering these critical regions and taking into account the accessibility of residues in close proximity to these regions, five discrete positions were selected on the octamer surface for scanning the face of the nucleosome. The five sites were residues 19, 89 and 113 of histone H2A (H2A-19, H2A-89, H2A-113), residue 109 of histone H2B (H2B-109), and residue 80 of histone H3 (H3-80). Initially, octamers with cysteine at one position in one of the four histone proteins were reconstituted on a 0N70 DNA (where `N' represents the 147bp 601 DNA sequence, and the lengths of the linker DNA is represented by the numbers 0 and 70). Nucleosomes were modified with the protein-protein cross-linker- MAB (methanethiosulfonate-tetrafluorophenylazide-biotin) reagent. This reagent makes a disulfide bond with the cysteines in the octamer of a nucleosome. The MAB reagent had a distance of ~11.1Aº between its photoreactive tetrafluorophenylazide group and the disulfide forming methanethiosulfonate group. The ISW2 complex was bound to the modified mononucleosomes and cross-linked by irradiating with UV-light. Under reducing conditions the biotin moiety was transferred from the nucleosome to the ISW2 complex. The subunit of the remodeler that was photocross-linked at these positions on the nucleosome was blotted onto nitrocellulose and detected with streptavidin conjugated to horseradish peroxidase (HRP). The catalytic subunit-Isw2 of the ISW2 complex was cross-linked at all five positions but with the following order of intensity from most to least- H2A-89, H3-80, H2B-109, H2A-19, and H2A113. Mass spectrometry was used to decipher these residues, motifs or domains of the catalytic subunit- Isw2 that interacted with the octamer at each position. The ISW2 complex was digested with trypsin, and the biotinylated peptides were enriched using monomeric avidin affinity chromatography. The largest subunit of the ISW2 complex, Itc1, did not get cross-linked at any of the positions.

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