Role of DNA sequence in CENP-ACnp1 assembly at fission yeast centromeres
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The centromere is the site of kinetochore assembly that ensures proper chromosome segregation. Active centromeres are formed at chromosomal locations that do not appear to share homology between different species; this and other analyses has lead to the conclusion that centromeres are epigenetically determined. In all organisms, centromere location is specified by the assembly of unusual nucleosomes containing the histone H3 variant CENP-A in place of H3. However, an apparent paradox is that CENP-A in most organisms generally occurs on certain preferred sequences. The analyses presented focuses on the influence of DNA sequence on the selection of the locus where CENP-A chromatin are formed and whether there are any particular DNA features that promote CENP-ACnp1 assembly at centromeres in Schizosaccharomyces pombe. S. pombe provides an excellent model to study the structure and function of a complex eukaryotic centromere as it possesses epigenetically regulated centromeres that are structurally related to those of metazoa. Furthermore, plasmid-based circular minichromosomes provide a useful tool for studying these centromeres and the inactivation of minichromosome centromeres does not affect cell viability. The main aim of this study was to identify those features that enable centromeric sequences to assemble CENP-A chromatin. Manipulated circular minichromosomes were utilised to investigate the minimal central core sequence requirement for establishment of CENP-ACnp1 chromatin. These analyses showed that a minimal 2kb region from the central core of cen2 could form a functional centromere. A second aim was to analyse the DNA sequence requirements for centromere function on this minimal 2 kb region. To facilitate this, the endogenous central core region of endogenous cen2 was replaced with the central core region of cen1. This modified strain allows the structural and biological properties associated with plasmid borne central core 2 sequences to be analysed. Transcription of central domain sequences has been proposed to play a role in CENP-A establishment and/or maintenance. To explore the contribution of transcription potential promoters were mapped within the minimal 2 kb sequence and their regulatory elements investigated. Mutation of the minimal DNA element impedes its ability to assemble CENP-A chromatin. Therefore the primary DNA sequence of fission yeast centromeres is important for establishing functional centromeres and thus centromere location not entirely epigenetically regulated. It remains to be determined if the characteristics associated with theses sequences, and their mode of action, are conserved at other centromeres.