Resolving genome-wide views of chromatin structure to identify fundamental principles of genome organization
Rizzo, Jason Michael
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Chromatin, the collection of DNA and proteins found in eukaryotic nuclei, is organized into a distinct structure to facilitate the compaction of genomic DNA. Chromatin structure is highly conserved across eukaryotic species and the organization of chromatin is known to have far-reaching implications on the accessibility and functionality of genetic information stored in nucleotide sequences. The purpose of this research is to improve our understanding of the global principles controlling chromatin structure. Work in this thesis utilizes the genetic model of Saccharomyces cerevisiae because of its high experimental tractability, strong conservation of eukaryotic transcription machinery, and extensive genome annotation. This thesis begins with a study defining the technical biases present in genome-wide chromatin mapping experiments and outlining the development and validation of an improved methodology that standardizes the collection and analysis of genome-wide maps of chromatin structure. This standardized collection and analysis of chromatin structures ultimately enables unbiased comparisons between genome-wide chromatin states, which help to expand our understanding of chromatin biology. Accordingly, global principles of genome organization are explored by using this improved methodology to map and compare genome-wide chromatin structures in various experimentally perturbed chromatin states. First, genome-wide chromatin structures are compared in the presence and absence of a conserved transcriptional repressor protein that is known to act through chromatin, Tup1p. Analysis of wild-type and tup1Δ ; chromatin structures enabled identification of a dominant role for Tup1p in establishing nucleosome positioning and occupancy at -1 and -2 promoter nucleosomes directly upstream of transcription start sites as part of is repressive mechanism. Additionally, analysis of chromatin organization at Tup1-regulated genes suggests a link between Tup1 regulation and transcriptional plasticity, whereby more open promoter structures are targeted by a larger number of regulatory factors with distinct patterns, to enable more variable gene expression across different environments. Finally, genome-wide chromatin structures are mapped and compared following an experimental depletion of nucleosomes to explore the role of histone dosage in chromatin organization. Structural responses to nucleosome depletion suggest alternative mechanisms for the establishment of the 5' barrier against which nucleosomes are packed and the spacing between 5' nucleosomes. Packing barriers at the 5' ends of genes are established through a mechanism that is sensitive to histone dosage, whereas spacing between 5' nucleosomes is maintained more resiliently in response to nucleosome depletion, suggesting a histone dose-independent mechanism. Loss of nucleosomes also occurs in a non-random fashion and with distinct structural changes that appear to relate to differences in transcriptional machinery and promoter elements at gene classes. Overall, this behavior is consistent with the active placement of nucleosomes surrounding transcription start sites and suggests that statistical packing principles, once thought to organize a large portion of the genome, may not be as prominent.