Epigenetic Regulation Involved In Sporulation And Heterochromatin Silencing In Schizosaccharomyces Pombe

COMPASS is a highly conserved complex in eukaryotes which is responsible for methylation of histone H3K4 and regulates transcriptions of genes involved in multiple biological processes. Ash2 is an important subunit of COMPASS. Previously, we found that the deletion of ash2+ caused defect in expressions of the DNA replication and cell-cycle related genes. In this study, multiple alignments analysis revealed that Ash2 in pombe shares two conserved domain with other homologues. The deletion of ash2+ reduces the rate of growth and increases the cell’s sensibility to TBZ and Oxygen Stess. We indicate that Ash2 is localized in nucleus and contributes to methylation of H3K4.To further investment to the functions of Ash2, we used yeast two-hybrid screening to find proteins interacting with Ash2. Finally, we got eight proteins.We confirmed the interaction between Ash2 and Ste4 in vitro. Ste4 is a regulatory factor during sporulation. The result indicates Ash2 may play a role in sporulation.Schizosaccharomyces pombe, under conditions of nitrogen starvation and mating pheromones undergoes meiosis instead of mitosis, which results in conjugation and sporulation. During this progress, the pheromone-responsive MAPK pathway plays an important role in regulating the conjuation and the transcriptional activation of genes required for meiosis. Spkl, a key component of MAPK pathway, activates Stell through protein phosphorylation. As a transcriptional factor, Stell activates the transcriptions of several genes requied for meiosis, including mei2+, mam2+ and map3+. The role of methylation of histone H3K4 in the sporualtion of fission yeast is poorly understood. Deletion of ash2+ resulted in a delay of sporulation and a substantial drop of sporulation efficiency. ChIP and RT-PCR analysis showed that deletion of ash2+ caused a reduction of H3K4me2 level in the coding region of spk1+ as well as a reduction of its mRNA level. Although the mRNA level of stell+ kept unchanged, the levels of Stell-targetted genes, such as mei2+,mam2+ and map3+, all reduced in ash2△ cells. There results suggest that Ash2 regulates the transcriptional activation of spkl+ through H3K4 methylation, and that helps to increase the activity of Stell and promotes the transcriptional activations of target genes downstream. This study investigates a potential role of Ash2 in the sporulation, which might provide new clue to elucidate the link between meiosis and epigenetic regulation.Heterochromatin is a specialized chromatin structure that inhibits transcription and DNA recombination. In the fission yeast Schizosaccharomyces pombe, heterochromatin is associated with telomeres, the silent mating-type loci, and repetitive DNA elements surrounding centromeres. To identify novel factors that contribute to heterochromatin organization at pericentromeres, We first performed a global screen in fission yeast mutation library. We got 82 mutations which showed serious defect in gene silencing of heterochromatin. There are many mutations among the results have been reported involved in the function of heterochromatin previously, such as clr4△ and rdpl△. We first identified a novel factor yoxl+ which is essential for heterochromatic integrity. As a repressor of the MBF complex, Yoxl and the other transcriptional repressor Nrml are both involved in confining MBF-dependent transcription to the G1 phase of the cell cycle.We found the deletion of yox1+ decreased level of H3K9me2 but increased the expressions of the reported genes ade6+ and ura4+ in heterochromatin. Interestingly, we found Yox1 didn’t locate in the centromeric heterochromatin region. Deletion of yoxl+ didn’t show defect in heterochromatin at mating-type loci through Atfl/Pcrl pathway or RNAi pathway. Furthermore,we found the deletion of nrml+ also affected the organization of heterochromatin at pericentromeres. Yox1 and Nrm1 acted in the same pathway to regulate the gene silencing in the centromeric heterochromatin region. Together, these lines of evidence suggest that Yoxl may contribute to the gene silencing in the centromeric heterochromatin region by cell-cycle control. This study might provide a new clue to elucidate the link between cell cycle and heterochromatin organization.