Project description:The meiotic cohesin Rec8 is required for the stepwise segregation of chromosomes during the two rounds of meiotic division. By directly measuring chromosome compaction in living cells of the fission yeast Schizosaccharomyces pombe, we found an additional role for the meiotic cohesin in the compaction of chromosomes during meiotic prophase. In the absence of Rec8, chromosomes were decompacted relative to those of wild-type cells. Conversely, loss of the cohesin-associated protein Pds5 resulted in hyper-compaction. While this hyper-compaction requires Rec8, binding of Rec8 to chromatin was reduced in the absence of Pds5, indicating that Pds5 promotes chromosome association of Rec8. To explain these observations, we propose that meiotic prophase chromosomes are organized as chromatin loops emanating from a Rec8-containing axis; the absence of Rec8 disrupts the axis, resulting in disorganized chromosomes, whereas reduced Rec8 loading results in a longitudinally compacted axis with fewer attachment points and longer chromatin loops. Keywords: ChIP-chip analysis ChIP analysis of Rec8: In all cases, hybridization data for ChIP fraction was compared with that of SUP (supernatant) fraction. Pombe chromosome II, III array was used.
Project description:The meiotic cohesin Rec8 is required for the stepwise segregation of chromosomes during the two rounds of meiotic division. By directly measuring chromosome compaction in living cells of the fission yeast Schizosaccharomyces pombe, we found an additional role for the meiotic cohesin in the compaction of chromosomes during meiotic prophase. In the absence of Rec8, chromosomes were decompacted relative to those of wild-type cells. Conversely, loss of the cohesin-associated protein Pds5 resulted in hyper-compaction. While this hyper-compaction requires Rec8, binding of Rec8 to chromatin was reduced in the absence of Pds5, indicating that Pds5 promotes chromosome association of Rec8. To explain these observations, we propose that meiotic prophase chromosomes are organized as chromatin loops emanating from a Rec8-containing axis; the absence of Rec8 disrupts the axis, resulting in disorganized chromosomes, whereas reduced Rec8 loading results in a longitudinally compacted axis with fewer attachment points and longer chromatin loops. Keywords: ChIP-chip analysis
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.
Project description:We used CLIP-Seq to determine the RNAs bound specifically to RNA binding protein Mei2 in early meiosis in fission yeast. We added a TAP tag to the C-terminal ends of two meiotic RNA binding proteins, Mei2 and Msa1. We used an untagged fission yeast strain as a negative control. These strains were nitrogen starved and allowed to progress into meiosis, after which they were harvested, lyzed and crosslinking immunoprecipitaion was performed. The RNAs purified after CLIP were sequenced
Project description:Meiotic recombination facilitates accurate pairing and faithful segregation of homologous chromosomes by forming physical connections (crossovers) between homologs. Developmentally programmed DNA double-strand breaks (DSBs) generated by Spo11 protein (Rec12 in fission yeast) initiate meiotic recombination. Until recently, attempts to address the basis of the highly non-random distribution of DSBs on a genome-wide scale have been limited to 0.1–1 kb resolution of DSB position. We have assessed individual DSB events across the Schizosaccharomyces pombe genome at near-nucleotide resolution by deep-sequencing the short oligonucleotides connected to Rec12 following DNA cleavage. The single oligonucleotide size-class generated by Rec12 allowed us to effectively analyze all break events. Our high-resolution DSB map shows that the influence of underlying nucleotide sequence and chromosomal architecture differs in multiple ways from that in budding yeast. Rec12 action is not strongly restricted to nucleosome-depleted regions but is nevertheless spatially biased with respect to chromatin structure. Furthermore, we find strong evidence across the genome for differential DSB repair previously predicted to account for crossover invariance (constant cM/kb in spite of DSB hotspots). Our genome-wide analyses demonstrate evolutionarily fluid factors contributing to crossover initiation and its regulation.