Cell cycle dependent methylation of Dam1 contributes to kinetochore integrity and faithful chromosome segregation

TL;DR

This study shows that cell cycle-dependent methylation of the Dam1 protein is crucial for proper kinetochore function and accurate chromosome segregation in yeast.

Contribution

The study reveals a novel role for cell cycle-regulated lysine methylation of Dam1 in kinetochore assembly and chromosomal stability.

Findings

Dam1 methylation peaks in metaphase and is mediated by Set1.

Jhd2 interacts with Dam1 and reduces its methylation, leading to kinetochore defects.

Altered Dam1 methylation causes chromosome missegregation and growth defects.

Abstract

The kinetochore, a megadalton structure composed of centromeric (CEN) DNA and protein complexes, is required for faithful chromosome segregation in eukaryotes. The evolutionarily conserved Dam1/DASH complex (Ska1 in metazoans) is one of the essential protein sub-complexes of the budding yeast kinetochore. Previous studies showed that methylation of lysine residue 233 in Dam1 by Set1 is important for haploid growth as mutation of lysine 233 to alanine results in lethality. In this study, we report that Set1-mediated cell cycle dependent Dam1 lysine methylation contributes to kinetochore assembly and chromosomal stability. Our results show that Dam1 methylation is cell cycle regulated with the highest levels of methylation in metaphase. Consistent with these results, co-immunoprecipitation experiments revealed an interaction between Dam1 with Set1 in metaphase cells. Set1 has been shown to colocalize with Jhd2, a histone lysine demethylase which demethylates Set1-methylated histones. Affinity purification-based mass spectroscopy of Jhd2 associated proteins identified seven of the ten subunits of the Dam1 complex; an association of Jhd2 with non-histone proteins, such as Dam1 has not been previously reported. We confirmed the interaction of Jhd2 with Dam1 and showed that cells overexpressing JHD2 exhibit reduced levels of methylated lysine in Dam1 in wild type and UBP8 deletion strains, growth defects in kinetochore mutants, reduced levels of kinetochore proteins at CEN chromatin, defects in kinetochore biorientation and chromosome missegregation. In summary, we have shown that cell cycle dependent methylation of Dam1 plays a crucial role in the maintenance of kinetochore assembly for faithful chromosome segregation.

Faithful chromosome segregation in eukaryotes is regulated by the kinetochore, a megadalton structure composed of centromeric DNA and associated proteins. Errors in chromosome segregation during the cell cycle result in chromosomal instability that is associated with human diseases, such as cancer and birth defects. Hence, the mechanisms and processes underlying kinetochore function and high-fidelity chromosome segregation are of utmost importance. The epigenetic modifications of evolutionarily conserved kinetochore protein Dam1 play an important role in kinetochore function and chromosome segregation. Phosphorylation of Dam1 by Aurora kinase Ipl1 modulates kinetochore-microtubule interaction, chromosome biorientation and segregation. However, the role of cell cycle mediated lysine methylation of Dam1 is not known. In this study, we showed that lysine methylation of Dam1 is cell cycle regulated with maximum enrichment in mitotic cells. We established an interaction of the histone lysine demethylase, Jhd2 with Dam1 and showed that cells with inducible expression of Jhd2 show defects in Dam1 methylation and kinetochore integrity with chromosome missegregation. In summary, we have defined a novel role for cell cycle dependent methylation of Dam1 in ensuring proper kinetochore assembly and chromosome segregation.

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