Type
Text
Type
Dissertation
Advisor
Luk, Ed | Hollingsworth, Nancy | Sternglanz, Rolf | Futcher, Bruce | Outten, Caryn E.
Date
2016-12-01
Keywords
Chromatin, Epigenetics, H2A.Z, Nucleosome, Transcription | Molecular biology -- Biochemistry -- Cellular biology
Department
Department of Molecular and Cellular 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/11401/76474
Publisher
The Graduate School, Stony Brook University: Stony Brook, NY.
Format
application/pdf
Abstract
Approximately 147 base pairs of DNA wraps almost two turns around a protein core comprised of two copies of each H2A, H2B, H3 and H4 histones to form a nucleosome—the fundamental packaging unit of chromatin. In Saccharomyces cerevisiae, nucleosomes are organized along the genome in a closely spaced and non-random manner. This compact structure blocks assembly of the transcription machinery and thus is refractory to gene expression. Surrounding the promoters of most genes, a wider nucleosome depleted region (NDR), maintained in part by the activities of chromatin remodelers, serves as the platform for assembly of the transcription preinitiation complex (PIC). The nucleosome immediately downstream of the NDR, termed +1, covers the transcription start site (TSS) and is frequently assembled with the histone variant H2A.Z instead of H2A. The histones at the +1 nucleosome position turnover rapidly at both actively and infrequently transcribed genes, suggesting constitutive chromatin remodeling allows the PIC to engage the TSS. We hypothesize that the H2A.Z nucleosome is recognized for disassembly to facilitate a rapid transcriptional response. The mechanism by which the +1 H2A.Z nucleosomes are removed is the central focus of my thesis. Previous studies suggested that the PIC assembles immediately upstream of the +1 H2A.Z nucleosome. This idea led to the hypothesis that the transcription machinery itself functions as a chromatin remodeler that is responsible for ejecting the +1 nucleosome. To understand the contribution of the transcription machinery in the disassembly of the +1 H2A.Z nucleosome, I used conditional mutants to block PIC assembly. A quantitative ChIP-seq approach was developed that allows for detection of changes in global histone occupancy in order to measure H2A.Z levels genome-wide. Blocking PIC assembly resulted in the promoter-specific accumulation of H2A.Z at both active and infrequently transcribed genes, indicating the PIC is required for H2A.Z eviction. Previous reports have suggested that the ATP-dependent remodeling complex INO80 could play a role in H2A.Z eviction. However, I observed no change in global H2A.Z levels upon depletion of INO80. These findings suggest that the assembly of the PIC and/or its activity is required for the constitutive turnover of +1 H2A.Z nucleosomes. | Approximately 147 base pairs of DNA wraps almost two turns around a protein core comprised of two copies of each H2A, H2B, H3 and H4 histones to form a nucleosome—the fundamental packaging unit of chromatin. In Saccharomyces cerevisiae, nucleosomes are organized along the genome in a closely spaced and non-random manner. This compact structure blocks assembly of the transcription machinery and thus is refractory to gene expression. Surrounding the promoters of most genes, a wider nucleosome depleted region (NDR), maintained in part by the activities of chromatin remodelers, serves as the platform for assembly of the transcription preinitiation complex (PIC). The nucleosome immediately downstream of the NDR, termed +1, covers the transcription start site (TSS) and is frequently assembled with the histone variant H2A.Z instead of H2A. The histones at the +1 nucleosome position turnover rapidly at both actively and infrequently transcribed genes, suggesting constitutive chromatin remodeling allows the PIC to engage the TSS. We hypothesize that the H2A.Z nucleosome is recognized for disassembly to facilitate a rapid transcriptional response. The mechanism by which the +1 H2A.Z nucleosomes are removed is the central focus of my thesis. Previous studies suggested that the PIC assembles immediately upstream of the +1 H2A.Z nucleosome. This idea led to the hypothesis that the transcription machinery itself functions as a chromatin remodeler that is responsible for ejecting the +1 nucleosome. To understand the contribution of the transcription machinery in the disassembly of the +1 H2A.Z nucleosome, I used conditional mutants to block PIC assembly. A quantitative ChIP-seq approach was developed that allows for detection of changes in global histone occupancy in order to measure H2A.Z levels genome-wide. Blocking PIC assembly resulted in the promoter-specific accumulation of H2A.Z at both active and infrequently transcribed genes, indicating the PIC is required for H2A.Z eviction. Previous reports have suggested that the ATP-dependent remodeling complex INO80 could play a role in H2A.Z eviction. However, I observed no change in global H2A.Z levels upon depletion of INO80. These findings suggest that the assembly of the PIC and/or its activity is required for the constitutive turnover of +1 H2A.Z nucleosomes. | 172 pages
Recommended Citation
Tramantano, Michael Charles, "The role of the transcription machinery in the remodeling of the promoter-proximal nucleosomes in Saccharomyces cerevisiae" (2016). Stony Brook Theses and Dissertations Collection, 2006-2020 (closed to submissions). 2391.
https://commons.library.stonybrook.edu/stony-brook-theses-and-dissertations-collection/2391