Histone-Histone Interactions and Centromere Function

Abstract

Cse4p is a structural component of the core centromere of Saccharomyces cerevisiae and is a member of the conserved CENP-A family of specialized histone H3 variants. The histone H4 allele hhf1-20 confers defects in core centromere chromatin structure and mitotic chromosome transmission. We have proposed that Cse4p and histone H4 interact through their respective histone fold domains to assemble a nucleosome-like structure at centromeric DNA. To test this model, we targeted random mutations to the Cse4p histone fold domain and isolated three temperature-sensitive cse4 alleles in an unbiased genetic screen. Two of the cse4alleles contain mutations at the Cse4p-H4 interface. One of these requires two widely separated mutations demonstrating long-range cooperative interactions in the structure. The third cse4allele is mutated at its helix 2-helix 3 interface, a region required for homotypic H3 fold dimerization. Overexpression of wild-type Cse4p and histone H4 confer reciprocal allele-specific suppression of cse4 and hhf1 mutations, providing strong evidence for Cse4p-H4 protein interaction. Overexpression of histone H3 is dosage lethal in cse4 mutants, suggesting that histone H3 competes with Cse4p for histone H4 binding. However, the relative resistance of the Cse4p-H4 pathway to H3 interference argues that centromere chromatin assembly must be highly regulated.

ACKNOWLEDGMENTS

We thank Forrest Spencer and Daniel Burke for plasmids and strains; Pamela Meluh, David Auble, Michael Christman, David Allis, Daniel Burke, and Van Moudrianakis for helpful discussions; and William Ross for expert technical assistance with the flow cytometry.

This work was supported by NIH grant GM28920 to M.M.S.