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

Abstract

Post-translational 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 methylation and Cse4-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.

Keywords: CENP-A; Cse4; Dsn1; Ndc80; Set2; Spc24; Spc25.

Fig. 1.

CENP-A^Cse4 is methylated at arginine 143 and lysine 131. a) Overview of the amino acid sequence of Cse4. R143me and K131me sites are located in the α-N-helix and C-terminal to the Cenp-A^N helix, respectively. Cse4 is phosphorylated at S33 (S33ph), methylated at R37 (R37me), and acetylated at K49 (K49ac). b) R143 corresponds to R52 in canonical H3. Pairwise alignment of yeast H3 and Cse4 was generated with EMBOSS needle. c) Top, location of Cse4-R143 (red) and -K130 (magenta) in the context of CCAN-Cenp-A^Nuc (PDB 6QLD). K130 is shown, because K131 is not visible in the structure. Bottom, view of the α-N and Cenp-A^N helices and the location of R143 and K130 (as a proxy for K131) of CCAN-Cenp-A^Nuc. The CCAN subunits were omitted for clarity.

Fig. 2.

The absence of Cse4-R143 methylation enhances the centromeric defect of spc25-1 and dsn1-7. a) Mutation of Cse4-R143 to alanine caused slow growth in the spc25-1 background. Strains with the indicated genotypes were serially diluted and spotted on YPD medium, and plates were incubated for 3 days and the indicated temperatures. b) cse4-R143A enhanced the plasmid maintenance defect of spc25-1. Error bars give SD of three independent experiments. *Significant difference, P < 0.05. n.s., not significant. c) spc25-1 cse4-R143A cells arrested at the G2/M phase of the cell cycle at the restrictive temperature. Cells were grown to early logarithmic phase at 23°C and shifted to 30°C for 3 h. DNA content as measured by FACS analysis is shown. d) cse4-R143E and -R143Q enhanced the growth defect of spc25-1. Serial dilutions of cse4Δ strains with the cse4 alleles on a plasmid were spotted on YPD plates and incubated for 2 days at the indicated temperatures. e) cse4-R143A enhanced the growth defect of dsn1-7. Serial dilutions of the indicated strains were spotted on YPD plates and growth for 2 days at the indicated temperatures.

Fig. 3.

The deletion of SET2 partially suppressed the centromeric defect of cse4-R143A spc25-1. a) set2Δ suppressed the temperature-sensitive growth defect of spc25-1 cse4-R143A, but not spc25-1 alone. Cell growth was assessed as in Fig. 2a. b) set2Δ suppressed the cell-cycle defect of spc25-1 cse4-R143A. FACS was conducted as in Fig. 2c. The experiment was carried out simultaneously with that presented in Fig. 2c. For further controls, see Supplementary Fig. 3a in Supplementary File 1. c) cse4-K131A partially suppressed the growth defect of spc25-1 cse4-R143A. Analysis was performed with plasmid-borne cse4 alleles as in Fig. 2d.

Fig. 4.

Suppressor mutations of spc25-1 cse4-R143A are located in the tetramer junction and the Spc24-Ndc80 coiled coil of the NDC80 complex. a) Suppression of the growth defect of spc25-1 cse4-R143A by mutations in NDC80 and SPC24. Serial dilutions of the strains were spotted on full medium and grown for 3 days at the indicated temperatures. b) Schematic of the S. cerevisiae kinetochore. c) Schematic of NDC80c illustrating the subcomplexes of Nuf2 with Ndc80 and Spc24 with Spc25. d) Localization of residues mutated in second-site suppressors of the spc25-1 cse4-R143A temperature-sensitive growth defect in the structure of the NDC80c^e-dwarf structure (PDB 5TD8, (Valverde et al. 2016)). Color schematic of proteins as in b). The respective residues are shown as yellow spheres. The suppressor mutations localize to the tetramerization junction and the Spc24-Spc24 stalk. Image generated using PyMol.