Accurate distribution of genetic material is critical for the formation of functional cells and their proliferation. During cell division, sister chromatids separate from each other and segregate to opposite poles. To ensure accurate chromosome segregation all sister chromatids should be attached to microtubules from opposite spindle poles, known as bi-orientation. Cohesin is a protein complex that holds sister chromatids together from the time of its replication in S phase until anaphase onset, and it is required for proper chromosome segregation both in mitosis and in meiosis. It is distributed intermittently along the full length of chromosomes with significant enrichment in the region surrounding the centromere, known as the pericentromere. This chromosome domain was shown to be crucial for chromosome bi-orientation. In my PhD I studied how the establishment of tension between sister chromatids in the process of bi-orientation affects the distribution of different pericentromeric proteins on budding yeast chromosomes. It was known that levels of cohesin at the pericentromere are decreased in response to the establishment of tension. I demonstrate that other proteins, such as subunits of condensin, members of the Chromosome Passenger Complex (CPC) and others, exhibit similar dynamics, and suggest a model to explain this phenomenon. Out of all studied proteins, Shugoshin (Sgo1) was the only one that was completely removed from the pericentromere in response to spindle tension establishment. There is evidence that Sgo1 plays a role in sensing spindle tension and halting the cell cycle until this has been achieved but how it does so is not known. Therefore, removal of Shugoshin from the pericentromere might be a signal for the cell that bi-orientation occurred. I then found that spindle tension itself is not sufficient for Sgo1 re-localization from the pericentromere, and there are other factors that affect it. I showed that deletion of RTS1, a highly conserved regulatory subunit of Protein Phosphatase 2A (PP2A), results in substantial enrichment of Shugoshin at the pericentromere in the situation when spindle tension is absent. In addition, Bub1 kinase, a protein that is required for Sgo1 localization, was found to be removed from the centromere in response to spindle tension as well as Sgo1. The role of Bub1 the in localization of Shugoshin is to phosphorylate histone H2A, which then becomes a mark for Sgo1 loading. Therefore, we assume that Sgo1 dynamics and, potentially, its role in sensing bi-orientation, are regulated through the array of phosphorylation and de-phosphorylation events at the pericentromeric area. Finally, I have also found that budding yeast Sgo1 undergoes the posttranslational modification as sumoylation. I showed that sumoylation of Shugoshin is not required for its removal from the pericentromere during biorientation. However, it might be important for the regulation of Sgo1 degradation and its role in the metaphase to anaphase transition in mitosis.

Barley (Hordeum vulgare) is an economically important crop species with a large diploid genome. Around a half of the barley genome and a fifth of the genes are constrained within a low-recombining pericentromeric (LR-PC) region. I explored the LR-PC gene component with a genomic investigation of gene expression, diversity and evolution. Chromatin environments were also explored in the LR and high recombining (HR) regions by surveying the genic and genomic distributions of nine histone modifications. Firstly, regions of HR and LR were identified and compared for gene evolution, expression and diversity. LR regions of the barley genome were found to be restrictive for gene evolution and diversity, but not gene expression. I employed a bioinformatics approach to identify ancient gene pairs in barley to determine the long-term effects of residency in those regions upon gene evolution. Gene pair loss in LR regions was found to be elevated relative to the HR regions. Applying the same method to rice and Brachypodium distachyon revealed the same situation, suggesting a universal process in the grasses for loss of gene pairs in LR regions. The chromosomal distributions of transposable elements (TEs) were also explored and examined for correlations with recombination rate. Secondly, I developed a chromatin immunoprecipitation followed by Next Generation Sequencing (ChIP-seq) protocol for the investigation of histone modifications in barley seedlings. A protocol was optimised for the fixation, extraction and sonication of barley chromatin. The protocol was applied using antibodies against 13 different histone modifications. Following DNA library construction and Illumina sequencing, a bioinformatics pipeline was devised to analyse the sequence data. NGS reads were mapped to a custom assembly of the barley cultivar Morex reference genome sequence before peak calling. Genomic and genic locations were determined for the covalently modified histones. Four modifications were discarded from further study on the basis of low peak numbers or unexpected chromosomal locations. The remaining nine modifications were classified into four groups based on chromosomal distributions. Groupings were closely mirrored by peak sharing relationships between the modifications except histone H3 lysine-27 tri-methylation (H3K27me3). In addition, chromatin states representing local chromatin environments were defined in the barley genome using the peak sharing data. Mapping the states onto the genome revealed a striking chromatin structure of the gene-rich chromosome arms. A telomere-proximal region bearing high levels of H3K27me3-containing states was found adjacent to an interior gene-rich region characterised by active chromatin states lacking H3K27me3. The LTR retroelement-rich interior was found to be associated with repressive chromatin states. The histone modification status of TE classes were also probed revealing unexpected differences relating to the genomic and genic distributions of these elements. Finally, a genome browser was created to host the information publicly.

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