Budding yeast CENP-A^Cse4interacts with the N-terminus of Sgo1 and regulates its association with centromeric chromatin

Abstract

Shugoshin is an evolutionarily conserved protein, which is involved in tension sensing on mitotic chromosomes, kinetochore biorientation, and protection of centromeric (CEN) cohesin for faithful chromosome segregation. Interaction of the C-terminus of Sgo1 with phosphorylated histone H2A regulates its association with CEN and pericentromeric (peri-CEN) chromatin, whereas mutations in histone H3 selectively compromise the association of Sgo1 with peri-CEN but not CEN chromatin. Given that histone H3 is absent from CEN and is replaced by a histone H3 variant CENP-A, we investigated if CENP-A interacts with Sgo1 and promotes its association with the CEN chromatin. In this study, we found that Sgo1 interacts with CENP-A in vivo and in vitro. The N-terminus coiled-coil domain of Sgo1 without the C-terminus (sgo1-NT) is sufficient for its interaction with CENP-A, association with CEN but not the peri-CEN, and this CEN association is cell cycle dependent with maximum enrichment in mitosis. In agreement with the role of CENP-A in CEN maintenance of Sgo1, depletion of CENP-A results in the loss of Sgo1 and sgo1-NT from the CEN chromatin. The N-terminus of Sgo1 is required for genome stability as a mutant lacking the N-terminus (sgo1-CT) exhibits increased chromosome missegregation when compared to a sgo1-NT mutant. In summary, our results define a novel role for the N-terminus of Sgo1 in CENP-A mediated recruitment of Sgo1 to CEN chromatin for faithful chromosome segregation.

A Failsafe for Sensing Chromatid Tension in Mitosis with the Histone H3 Tail inSaccharomyces cerevisiae

“…Figure 8A shows that recombinant Sgo1p specifically interacts with the tail domains of H3 and Cse4p, but not that of Cnn1p or the GST control. This Sgo1p-Cse4p interaction is consistent with a recent report by Basrai and colleagues (Mishra et al 2017). To test whether acetylation directly influences the H3 tail-Sgo1p interaction, we acetylated the H3 tail-GST fusion protein in vitro with Gcn5p (Figure 8B).…”

Tripartite Chromatin Localization of Budding Yeast Shugoshin Involves Higher-Ordered Architecture of Mitotic Chromosomes

“…Sgo1p also binds the N’ tail of the centromere-specific histone H3 variant, Cse4p (M ishra et al 2017). It is likely that phos.H2A and Cse4p provide the docking site for Sgo1p that nucleates outward spread toward the pericentric regions.…”

“…While a Gly-to-Ser mutation in the TSM has no effect on cohesin localization, both pericentric and centromeric (though to a lesser extent) enrichment of Sgo1p is compromised ((L uo et al 2010) and Figure 3C). The establishment of the centromeric and pericentric domain of Sgo1p likely follows a spillover model in that Sgo1p is first recruited to the centromeres via direct association with Cse4p (M ishra et al 2017) and histone H2A phosphorylated at Ser121 by kinase Bub1p (F ernius and H ardwick 2007; K awashima et al 2010). Congregation of Sgo1p molecules at centromeres permits its spread to the adjacent pericentric nucleosomes where cohesin has already been loaded.…”

“…This spread may result from the turnover of a transient complex involving Sgo1p and centromeric proteins. Alternatively, the homodimerization activity of Sgo1p, evidenced by yeast two-hybrid tests (M ishra et al 2017), may facilitate the growth of the Sgo1p domain from centromeres to pericentric regions where the cohesin complex resides. By binding to nucleosomes, cohesin may also help to make the tension sensing motif more accessible for Sgo1p before biorientation is established (F ernius and H ardwick 2007; K awashima et al 2010; L uo et al 2010; L uo et al 2016).…”

“…Shugoshin is recruited to centromeres by binding to histone H2A phosphorylated by the Bub1 kinase (K awashima et al 2010; L iu et al 2013a). The centromeric recruitment of budding yeast Sgo1p may also involve the interaction with the centromere-specific histone H3 variant Cse4p (M ishra et al 2017). In human mitotic cells, Sgo1 recruited to the outer kinetochore nucleosomes is then driven by RNA polymerase II to the inner centromere where it is retained by cohesin (L iu et al 2015).…”