The Chromatin-Remodeling Factor FACT Contributes to Centromeric Heterochromatin Independently of RNAi

Centromeres exert vital cellular functions in mitosis and meiosis. A specialized histone and other chromatin-bound factors nucleate a dynamic protein assembly that is required for the proper segregation of sister chromatids. In several organisms including the fission yeast S. pombe, a RNAi-related pathway further strengthens the epigenetic maintenance of centromere location, primarily through repressing transcription near centromeres. Little is known how accessory factors such as histone chaperones and chromatin remodelling factors might be required to establish a functioning centromere. Here we show that the chaperone and remodelling complex FACT is required for transcriptional gene silencing at centromeres and for accurate chromosome segregation during mitosis. We show that Spt16 and Pob3 are two subunits of the S. pombe FACT complex. Surprisingly, yeast strains deleted for Pob3 are viable and show a marked loss of gene silencing at centromeric repeats and at the mating locus. Importantly, Pob3-null strains fail to undergo mitosis at high accuracy and phenocopy histone methylation and RNAi mutants. Processing of centromeric RNA transcripts into siRNAs is maintained in Pob3 mutants, but Swi6-association at the centromere is affected. Our studies provide the first experimental evidence for a role of the RNA polymerase II cofactor FACT in centromere function. Keywords: Expression profiling of pob3 vs wt cDNA expression profiling was carried out according to (Xue et al., 2004). We used the S. pombe ORF spotted microarrays (Eurogentec, Belgium custom DNA microarray services).GeneSpring software was used for all data analysis. Expression profiling mutant vs wt data sets were normalized using Lowess (per spot per chip) intensity-dependent normalization, which corrects nonlinear rates for dye incorporation. Cut off values of 2,0 were used to generate ‘high’ and ‘low’ gene lists for ORF and IGR regions.