Mechanism For Nucleosome Organization Reconstituted Across A Eukaryotic Genome
Open Access
- Author:
- Wal, Megha
- Graduate Program:
- Biochemistry, Microbiology, and Molecular Biology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- December 03, 2015
- Committee Members:
- Benjamin Franklin Pugh, Dissertation Advisor/Co-Advisor
Benjamin Franklin Pugh, Committee Chair/Co-Chair
Joseph C. Reese, Committee Member
Song Tan, Committee Member
Ross Cameron Hardison, Committee Member
Tae Hee Lee, Committee Member - Keywords:
- nucleosome
chromatin remodelers
in vitro reconstitution
yeast - Abstract:
- The eukaryotic genome is packaged into chromatin, which renders the DNA inaccessible to DNA-templated cellular processes. These include DNA replication, repair, recombination and transcription. Packaging of the genome into chromatin is achieved via nucleosome formation using histone proteins. Positions of these nucleosomes play a highly important role in determining access to DNA. Till date, several studies have focused into the principles determining nucleosome positioning in the cell. Such studies have revealed an integrative model of nucleosome organization. This model includes contribution from DNA sequence preferences, trans-acting factors, statistical positioning and process of transcription. While these studies have provided a basic framework to study the determinants of nucleosome positioning, a lot of questions still remain unanswered. We do not have a deep understanding about role of particular factors in different aspects of nucleosome positioning, and how this is brought about. With this objective in mind, we took a nucleosome reconstitution approach to determine the direct and sufficient roles of different factors in nucleosome positioning. We used high-resolution MNase-ChIP-sequencing strategy to determine nucleosome positions in the yeast genome. Interestingly we found that DNA sequence and transcription have very limited roles in nucleosome organization, while ATP-dependent chromatin remodelers play a major role in nucleosome organization. Specifically we show how nucleosome positioning is reconstituted in a step-wise manner using DNA, histones, chromatin remodelers and sequence-specific factors as minimal constituents. In stage 1, DNA sequence features determine NFR formation and positioning with the help of RSC remodeling complex, which directly reads the DNA sequence to catalyze nucleosome removal. In stage 2, +1 nucleosome is reconstituted using two alternative approaches. In the first approach, INO80 remodeling complex positions +1 nucleosome using DNA sequence and shape features while in the second approach ISW2 remodeler uses Abf1 and Reb1 sequence-specific binding proteins as barriers to reconstitute the +1 nucleosome. In stage 3 & 4, nucleosomal arrays are established with correct spacing using ISW1a remodeler. Taken together, the work in this thesis has provided advancement into the role of chromatin remodelers as major players in nucleosome organization. We provide some initial evidence for remodeler mechanisms in nucleosome restructuring and a minimal in-vitro system to reconstitute physiological nucleosome organization.