Project description:Specialized chromatin exists at centromeres and must be precisely transmitted during DNA replication. The mechanisms involved in the propagation of these structures remain elusive. Fission yeast centromeres are composed of two chromatin domains: the central CENP-ACnp1 kinetochore domain and flanking heterochromatin domains. Here we show that fission yeast Mcl1, a DNA polymerase ? (Pol?) accessory protein, is critical for maintenance of centromeric chromatin. In a screen for mutants that alleviate both central domain and outer repeat silencing, we isolated several cos mutants, of which cos1 is allelic to mcl1. The mcl1-101 mutation causes reduced CENP-ACnp1 in the central domain and an aberrant increase in histone acetylation in both domains. These phenotypes are also observed in a mutant of swi7+, which encodes a catalytic subunit of Pol?. Mcl1 forms S-phase-specific nuclear foci, which colocalize with those of PCNA and Pol?. These results suggest that Mcl1 and Pol? are required for propagation of centromere chromatin structures during DNA replication. Keywords: ChIP-chip analysis, S. pombe Antibodies used were ?-acetyl-histone H4 antiserum (Upstate) and ?-histone H4 pan antibody (Upstate). ChIP-on-chip was carried out using IVT amplification method. ChIP-chip data were comfirmed by real-time PCR.
Project description:Specialized chromatin exists at centromeres and must be precisely transmitted during DNA replication. The mechanisms involved in the propagation of these structures remain elusive. Fission yeast centromeres are composed of two chromatin domains: the central CENP-ACnp1 kinetochore domain and flanking heterochromatin domains. Here we show that fission yeast Mcl1, a DNA polymerase α (Polα) accessory protein, is critical for maintenance of centromeric chromatin. In a screen for mutants that alleviate both central domain and outer repeat silencing, we isolated several cos mutants, of which cos1 is allelic to mcl1. The mcl1-101 mutation causes reduced CENP-ACnp1 in the central domain and an aberrant increase in histone acetylation in both domains. These phenotypes are also observed in a mutant of swi7+, which encodes a catalytic subunit of Polα. Mcl1 forms S-phase-specific nuclear foci, which colocalize with those of PCNA and Polα. These results suggest that Mcl1 and Polα are required for propagation of centromere chromatin structures during DNA replication. Keywords: ChIP-chip analysis, S. pombe
Project description:Meiosis is a specialized cell division that generates gametes, such as eggs and sperm. Errors in meiosis result in miscarriages and are the leading cause of birth defects, however the molecular origins of these defects remain unknown. Studies in model organisms are beginning to identify the genes and pathways important for meiosis, but the parts list is still poorly defined. Here we present a comprehensive catalogue of genes required for meiosis in the fission yeast, Schizosaccharomyces pombe. Our genome-wide functional screen surveyed all non-essential genes for roles in chromosome segregation and spore formation. Novel genes required at distinct stages of the meiotic chromosome segregation and differentiation programme were identified. Preliminary characterization implicated three of these genes in centrosome/spindle pole body function, centromere and cohesion function. Our findings represent a near-complete parts list of genes required for meiosis in fission yeast, providing a valuable resource to advance our molecular understanding of meiosis.