HIGH-RESOLUTION GENOME-WIDE ORGANIZATION OF THE YEAST TRANSCRIPTION MACHINERY
Open Access
- Author:
- Venters, Bryan Jacob
- Graduate Program:
- Biochemistry, Microbiology, and Molecular Biology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- June 05, 2008
- Committee Members:
- Benjamin Franklin Pugh, Committee Chair/Co-Chair
Joseph C. Reese, Committee Member
Ross Cameron Hardison, Committee Member
David Scott Gilmour, Committee Member
Naomi S Altman, Committee Member - Keywords:
- ChIP-chip
Tiling microarrays
Pre-initiation complex
Chromatin
Saccharomyces
Mot1 - Abstract:
- The yeast genome is packaged into chromatin, which regulates promoter access to the transcription apparatus. The current view of transcription initiation is that sequence-specific regulators interact with their cognate DNA motifs in response to cellular signals. The regulators recruit transcriptional coactivators or corepressors, which in turn alter the local chromatin environment to control the rate of assembly of the pre-initiation complex (PIC). The PIC is composed of the general transcription factors (GTFs) and Pol II. The TATA binding protein (TBP) is a central fixture in the transcription of nearly all genes in the cell, and consequently its distribution and activity is controlled by numerous regulatory proteins. One of them, Mot1, is an essential protein that regulates the genomic distribution of TBP. To delineate how different conserved regions of Mot1 regulate transcription, I conducted an in vivo structure/function study. Using genome-wide expression and location profiling of Mot1 mutants, I found that Mot1-regulated genes are equally dependent on all the tested regions of Mot1, except for the ATPase region, which indicates that Mot1 functions in the cell as an indivisible regulatory unit to facilitate the appropriate genomic distribution of TBP. Recent studies have illuminated the nucleosomal architecture in yeast with base pair precision, thus setting the stage for understanding how the transcription machinery is organized in its native chromatin context throughout the genome. To determine the genome-wide spatial distribution of the transcription apparatus at high-resolution, I mapped the locations of protein factors representing different stages of transcription using chromatin immunoprecipitation coupled to tiled DNA microarray detection (ChIP-chip). By mapping nearly 30 components of the yeast transcription machinery at ~40 bp resolution, I find that several organizational themes emerge. First, the -1 promoter nucleosome is the preferred target for all chromatin remodelers except SWR-C, suggesting that this nucleosome plays a major role in regulating promoter access. Second, GTFs are all found ~60bp upstream of the transcription start site (TSS) in the nucleosome-free region (NFR) irrespective of whether a TATA-box is present, which indicates that transcription tends to start at a fixed distance from the PIC. Third, TFIID displays a broad distribution across the NFR, whereas SAGA binding is confined to the upstream promoter region near the -1 nucleosome, indicating that these central complexes in the two coactivator pathways interact with promoters in distinct chromatin environments. Fourth, although Pol II is widely distributed throughout the genome as previously reported, the pattern of Pol II density across the coding region varies from gene to gene instead of a plateau-like distribution, indicating that Pol II is rate-limited at different stages of the transcription cycle in a gene dependent manner. Lastly, the location linkage analysis uses binding site information derived from ChIP-chip to define a network of connectivity between transcription factors and chromatin remodeling complexes, and generates testable hypotheses about genome-wide recruitment dependencies without a priori knowledge of protein-protein interactions. Taken together, the work presented in this thesis reveals the organization of the transcription machinery at promoters genome-wide, and provides a contextual framework for subsequent location profiling experiments.