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Doctoral thesis
Open access
English

Chemical and Genetic Tools to Study Nucleosomes and Chromatin Remodelers in Two Distantly Related Yeast Species

ContributorsCerato, Luca
Number of pages203
Imprimatur date2023-06-22
Defense date2021-09-29
Abstract

The first level of DNA compaction in eukaryotic chromosomes is the nucleosome, in which ~147 bp of DNA are wrapped in ~1.75 negative helical turns around an octamer comprised of histones H2A, H2B, H3 and H4. These basic subunits of chromatin can block access of the RNA polymerase II machinery to promoter regions. As a result, gene expression often requires the combined action of transcription factors (TFs) and chromatin remodelers (CRs) to generate what is referred to as a nucleosome-depleted region (NDR) that renders promoters accessible for pre-initiation complex (PIC) assembly and polymerase binding. The precise nature of NDRs is still unclear, and previous studies in S. cerevisiae from our lab and others provided evidence for a class of NDRs that contain MNase-sensitive particles dubbed “fragile nucleosomes” (FNs), in which the underlying DNA is proposed to be in a partially unwrapped nucleosomal state. The existence of FNs has been challenged by other groups, who proposed instead that these signals are due to non-nucleosomal particles based upon their failure to detect histones at these genomic regions by chromatin immunoprecipitation (ChIP). Although several arguments were raised in opposition to this claim, it motivated us to find new experimental approaches to further characterize FN particles, test their histone content and probe for their presence in a distantly related yeast, S. pombe. Additionally, as S. pombe CRs and TFs have not been studied as extensively as in S. Cerevisiae, it created an opportunity to compare their essential RSC complex which operates mainly at gene promoters.

This thesis presents new evidence for the existence of fragile nucleosomes in both the S. cerevisiae and S. pombe yeast species. It also highlights a significant difference in the activity and location of their RSC complex as well as a radically different system of DNA motifs in their protein-coding gene promoters. In the studies described here, we used various tools, sometimes in conjunction with MNase digestion and always together with high throughput DNA sequencing as a read-out.

eng
Keywords
  • Nucleosomes
  • Chromatin
  • RSC
  • Nucleosome-depleted region
  • Gene promoters
  • ChIP
  • S. cerevisiae
  • S. pombe
  • DNA motifs
  • Chromatin remodelers
  • Transcription initiation
Research group
Citation (ISO format)
CERATO, Luca. Chemical and Genetic Tools to Study Nucleosomes and Chromatin Remodelers in Two Distantly Related Yeast Species. 2023. doi: 10.13097/archive-ouverte/unige:171168
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Creation08/29/2023 2:41:38 PM
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