Faithful segregation of chromosomes to progeny cells is essential for species perpetuation. Eukaryotic cells use the spindle assembly checkpoint (SAC) to correct mistakes and prevent chromosome missegregation. SAC ensures that sister chromosomes are bioriented before segregation starts. Chromosome biorientation is manifested by tension between the two sister chromatids that are held together by the cohesin complex. The centromeric protein Shugoshin (Sgo1p in Saccharomyces cerevisiae) is an integral part of the tension sensing function of SAC. The underlying mechanism of how Shugoshin responds to tension, and relays this signal to the appropriate players in SAC remains largely unresolved. Here I use S. cerevisiae as a model to understand this tension-sensing mechanism by studying the recruitment of Sgo1p. Our lab has discovered that residues Lys42, Pro43, Gly44, and Thr45 of histone H3 form the core of a tension sensing motif, TSM, in S. cerevisiae. TSM functions by physically recruiting and retaining Sgo1p at pericentromere. I have identified a pair of antagonistic chromatin modifiers, Gcn5p and Rpd3p/Hos2p, that are critical for regulation of the H3-Sgo1p interaction in vivo. Deletion of GCN5 or overexpressing catalytically-dead mutants of Gcn5p suppresses the mitotic defects of tsm- mutants. In contrast, deleting RPD3 causes prominent chromosome instability phenotype in tsm- mutant G44S. In addition, both Gcn5p and Rpd3p are enriched at centromere and pericentromere. These results suggest the opposing enzymes Gcn5p and Rpd3p are parts of the mitotic checkpoint through regulating TSM/Sgo1p function. Genome-wide ChIP-seq assay reveals a unique TSM-dependent, tripartite domain of Sgo1p in each mitotic chromosome. This domain consists of one centromeric and two flanking peaks 3 – 4 kb away. Strikingly, this trident motif coincides with cohesin localization at centromeres and pericentromeres. The trident localization of Sgo1p requires both TSM and cohesin. Chromosome conformation capture assays reveal apparent looping at the centromeric and pericentric regions. The TSM-Sgo1p-cohesin triad is therefore at the center stage of higher-ordered chromatin architecture for error-free segregation.
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