Type
Text
Type
Thesis
Advisor
Luk, Ed | Hollingsworth, Nancy.
Date
2013-12-01
Keywords
Biochemistry
Department
Department of Biochemistry and Cell Biology.
Language
en_US
Source
This work is sponsored by the Stony Brook University Graduate School in compliance with the requirements for completion of degree.
Identifier
http://hdl.handle.net/11401/76907
Publisher
The Graduate School, Stony Brook University: Stony Brook, NY.
Format
application/pdf
Abstract
In this study, I investigated how the well-conserved histone variant H2A.Z is removed from promoters to prepare genes for transcription. H2A.Z is found at the majority of nucleosomes near promoters in place of the canonical histone H2A. Why H2A.Z is localized to gene promoters in all eukaryotes is unclear. One hypothesis is that H2A.Z marks nucleosomes for disassembly, thereby opening up promoters to give room for the transcription machinery to assemble. How H2A.Z-containing nucleosomes are removed from promoter sites is also unknown, but the Luk laboratory has recently found that ATP is necessary for this process. We propose that there may be an evictor enzyme that specifically recognizes H2A.Z and removes it from promoters. Although H2A and H2A.Z can assemble together with H2B, H3, H4 and DNA into nucleosomes that are structurally similar, 40% of the amino acids in the polypeptides of H2A and H2A.Z differ. This thesis tests the hypothesis that the putative evictor may recognize some of the unique residues of H2A.Z to mediate nucleosome disassembly. The genes HTZ1 and HTA1 encode H2A.Z and H2A in yeast, respectively. Using a collection of yeast expression vectors bearing various domain swap genes of HTZ1 and HTA1, the unique amino acid residues of Htz1 that are important for histone eviction were looked for. Substitution of Htz1 sequences required for eviction with the corresponding region of Hta1 should result in a loss of function and failure to complement the phenotype of htz1 depleted yeast. Nine domain swap (Htz1/Hta1) proteins that failed to fully complement the htz1 depletion phenotype in yeast were identified. The Htz1/Hta1 protein-containing strains that failed to complement were then assayed for their enrichment at promoters by chromatin immunoprecipitation (ChIP) and qPCR assays. Our model predicts that proteins defective for recognition by the evictor protein will accumulate at promoters. However, none of these chimeric proteins accumulate at promoters. Instead, all of these Htz1/Hta1 proteins are depleted at promoters relative to wild-type Htz1 and five of them have lost some or all of their ability to accumulate at promoters. The data suggest that these proteins lack the Htz1-specific residues important for promoter specific deposition. Furthermore, the data support the idea that the specific residues important in promoter specific deposition of Htz1 are located in the alpha1 helix, loop1 and alpha2 helix regions of Htz1. | 37 pages
Recommended Citation
D'Orazio, Karole Nicole, "Identification of specific residues necessary for the eviction of H2A.Z-containing nucleosomes" (2013). Stony Brook Theses and Dissertations Collection, 2006-2020 (closed to submissions). 2781.
https://commons.library.stonybrook.edu/stony-brook-theses-and-dissertations-collection/2781