Methylation of CENP-A/Cse4 on arginine 143 and lysine 131 regulates kinetochore stability in yeast

Abstract

Posttranslational modifications on histones are well known to regulate chromatin structure and function, but much less information is available on modifications of the centromeric histone H3 variant and their effect at the kinetochore. Here, we report two modifications on the centromeric histone H3 variant CENP-A/Cse4 in the yeast Saccharomyces cerevisiae, methylation at arginine 143 (R143me) and lysine 131 (K131me), that affect centromere stability and kinetochore function. Both R143me and K131me lie in the core region of the centromeric nucleosome, near the entry/exit sites of the DNA from the nucleosome. Unexpectedly, mutation of Cse4-R143 (cse4-R143A) exacerbated the kinetochore defect of mutations in components of the NDC80 complex of the outer kinetochore (spc25-1) and the MIND complex (dsn1-7). The analysis of suppressor mutations of the spc25-1 cse4-R143A growth defect highlighted residues in Spc24, Ndc80 and Spc25 that localize to the tetramerization domain of the NDC80 complex and the Spc24-Spc25 stalk, suggesting that the mutations enhance interactions among NDC80 complex components and thus stabilize the complex. Furthermore, the Set2 histone methyltransferase inhibited kinetochore function in spc25-1 cse4-R143A cells, possibly by methylating Cse4-K131. Taken together, our data suggest that Cse4-R143 and -K131 methylation affect the stability of the centromeric nucleosome, which is detrimental in the context of defective NDC80 tetramerization and can be compensated for by strengthening interactions among NDC80 complex components.

Misregulation of cell cycle-dependent methylation of budding yeast CENP-A contributes to chromosomal instability

“…A recent study has identified methylation within the C-terminus of Cse4 (K131 and R143), which affects the stability of CEN nucleosomes in the context of defects in outer kinetochore proteins, such as those of the Ndc80 complex ( Nguyen et al. , 2023 ).…”

“…, 2019 ). The lack of any detectable phenotype in cse4-R37A could be due to compensatory effect produced by the methylation of R143 in the C-terminus of Cse4 ( Nguyen et al. , 2023 ) or other methylated arginine residues in Cse4 that are yet to be identified.…”

“…For example, phosphorylation of Cse4 by protein kinases Cdc7, Cdc5, and Ipl1 modulate kinetochore integrity, chromosome biorientation, and segregation (Boeckmann et al, 2013;Mishra et al, 2021). Various studies have previously identified methylation of Cse4 arginine 37 (R37) in the essential N-terminus domain (END) of Cse4 (Samel et al, 2012;Boeckmann et al, 2013;Anedchenko et al, 2019;Nguyen et al, 2023). Methylation of Cse4-R37 seems to be required in the context of a defective kinetochore as cse4-R37A strain exhibits synthetic genetic interactions with mutants corresponding to inner kinetochore, namely COMA (Ctf19, Okp1, Mcm21, and Ame1) complex (Samel et al, 2012).…”

“…It has been proposed that the essential components of the COMA complex namely, Okp1 and Ame1 serve as readers for Cse4-R37 methylation, and their association with methylated Cse4-R37 acts as a signal for the loading of other kinetochore components (Anedchenko et al, 2019). A recent study has shown that methylation of Cse4-lysine (K) 131 and Cse4-R143 affect the stability of CEN nucleosome, and their absence is detrimental when combined with the defects in the outer kinetochore proteins, such as the those of the Ndc80 complex (Nguyen et al, 2023).…”

“…, 2019 ). A recent study has shown that methylation of Cse4-lysine (K) 131 and Cse4-R143 affect the stability of CEN nucleosome, and their absence is detrimental when combined with the defects in the outer kinetochore proteins, such as the those of the Ndc80 complex ( Nguyen et al. , 2023 ).…”