Author: Ozgen Deniz

Publisher:

Published: 2014

Total Pages: 249

ISBN-13:

The nucleosome is the fundamental structural unit of DNA compaction in eukaryotic cells and is formed by the wrapping of 147 bp double stranded DNA around a histone octamer. Nucleosome organization plays a major role in controlling DNA accessibility to regulatory proteins, hence affecting cellular processes such as transcription, DNA replication and repair. Our study focuses on genome-wide nucleosome positioning in S. cerevisiae to explore nucleosome determinants and plasticity throughout the cell cycle and their interplay with gene expression based on cell mRNA abundance. We pursued the contribution of DNA physical properties on nucleosome organization around key regulatory regions such as TSSs and TTSs by analyzing genome-wide MNase-digestion profile of genomic DNA. We also implemented a systematic approach to standardize MNase-Seq experiments by minimizing the noise generated by extrinsic factors to enable an accurate analysis of the underlying principles of nucleosome positioning and dynamics. Moreover, we carried out a large-scale study of nucleosome plasticity throughout the cell cycle and its interplay with transcription based on a comparative analysis among nucleosome maps, gene expression data and MNase sensitivity assays. We then focused on nucleosome organization around DNA replication origins and its possible effect on origin activation. Finally, we sought to characterize centromeric nucleosome composition and its oscillation along cell cycle. During the course of these studies, we found that key regulatory regions such as 5' and 3' nucleosome free regions (NFRs) contain unusual physical properties that are intrinsic to genomic DNA. We further demonstrated that DNA physical properties and transcription factors act synergistically to define NFRs, especially in genes with an open promoter structure. Once NFR is defined, the nucleosome positioning around TSSs can be predicted by a simple statistical model, supporting the energy barrier model for nucleosome positioning determination. However, we also observed that nucleosomes are quite dynamic at distal 5' NFRs and do have distinct regulatory mechanisms. Our comparative analysis of nucleosome organization along cell cycle revealed that chromatin exhibits a distinct configuration due to DNA replication-dependent organization at S phase, showing higher sensitivity to MNase and displaying fuzzier nucleosomes along the genome. Moreover, we observed different features at M phase, where chromatin compaction is the highest and displays a slightly different pattern than in G1 and G2 phases. Interestingly, these changes in chromatin organization are sudden and acute and only affect some regions of the genome, whereas the majority of genes present conserved nucleosome patterns along cell cycle. Our individual gene analysis disclosed that the largest changes take place in cell cycle-dependent genes, indicating the interplay between chromatin and transcription. Moreover, a distinct nucleosome organization at high and low transcription rates further supports this relationship. The detailed analysis around replication origins shows that they display slightly wider NFRs at G1 phase due to pre-Replication complex binding. Once the replication origins are active, nucleosomes partially occupy NFRs up to a certain extent due to constitutive binding of ORC. Moreover, we provided further evidence that early firing origins tend to have more ordered nucleosome organization than late firing origins. Finally we illustrated that centromeric nucleosomes display a perfect positioning, confirming their strong centromeric sequence-dependent recruitment to DNA. The characterization of histone composition under physiological cell conditions suggested that the octameric nucleosome assembly model is favored in centromeres. Yet, our analysis along cell cycle showed centromeric nucleosome dynamics, proposing that its composition might oscillate along cell cycle. Taken together, our accurate study provides a dynamic picture of nucleosome positioning and its determinants; new insights into cell cycle-dependent chromatin organization on key regulatory regions and its interplay with gene expression; and adds a new dimension to the characterization of centromeric nucleosomes.