Investigating the role of histones in fission yeast centromere function

Mellone, Barbara (2002). Investigating the role of histones in fission yeast centromere function. PhD thesis The Open University.



The centromere is the chromosomal region which is responsible for the accurate segregation of chromosomes during mitosis and meiosis. Failure to properly segregate replicated chromosomes causes aneuploidy and contributes to cancer progression.
Fission Yeast centromeres display several features in common with the centromeres of higher eukaryotes. They are assembled in a specialised silent heterochromatin composed of underacetylated histones and methylated histone H3.
To investigate the role of histone H3 and H4 N-tails in centromere structure and function, conserved lysines of histone N-termini were mutated to mimic hyperacetylated or non-methylated states. Since the fission yeast haploid genome contains three copies of both histones H3 and H4, initially a strain harbouring a single H3 and H4 gene was generated and analysed. This phenotypically wild type strain provided a genetic background in which to perform site-directed mutagenesis of the histone tails. These mutants showed that the H4 tail is not critical for silencing, while the H3 tail plays an essential role and is required for centromere function.
Centromeric nucleosomes contain an essential histone H3-like protein, CENP-A. Antibodies were raised against the fission yeast CENP-A homologue Cnpl and were used to map Cnp1 association with the central domain of the centromere. Cnp1 appears to replace histone H3 in this domain and can coat a large fragment of non-centromeric DMA artificially inserted within this centromeric domain. Futhermore, strains expressing more histone H3 than H4 showed delocalisation of Cnp1, alleviation of centromeric silencing and missegregation of chromosomes in mitosis indicating that a fine balance between histone H3 and H4 is important for centromere function and that histone H3 can compete with Cnp1 in nucleosome assembly.

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