Analysis of primary structural determinants that distinguish the centromere-specific function of histone variant Cse4p from histone H3.

The nucleosome core is composed of an evolutionarily conserved octamer of four core histone proteins, H2A, H2B, H3, and H4, and 145 to 147 bp of DNA (26). Histone-histone and histone-DNA interactions that form the core nucleosome are mediated through a globular, highly evolutionarily conserved histone-fold domain found in all four core histones. The histone-fold domain is composed of a long central α-helix (helix II) flanked by two short α-helices (helix I and helix III) (Fig. ​(Fig.1).1). The three α-helices are separated by two β-strand loops (loop I and loop II). H3-H4 and H2A-H2B dimers form in an antiparallel fashion such that the helix II α-helices cross each other and juxtapose loop I with loop II from the two different molecules (1, 11). This arrangement creates three regions in each dimer that bind the minor groove of DNA, two loop I-loop II regions and a region formed from the two adjacent helix I α-helices. In the case of H3, an additional N-terminal helix also makes DNA contacts (11). During nucleosome assembly, two H3-H4 heterodimers form a tetramer through an H3-H3′ interaction called the four-helix bundle (Fig. ​(Fig.1).1). To complete the octamer, two H2A-H2B heterodimers bind to the tetramer through a four-helix bundle interaction involving H4 and H2B (11). FIG. 1 (A) Alignment of the histone-fold domains of S. cerevisiae H3, Cse4p, and human CENP-A. Residues that are identical in two or three of the proteins are indicated in boldface italic type. The α-helices and β-loops in the histone-fold domain ... The kinetochore is a complex of specialized centromeric chromatin and proteins that mediates interactions between the chromosomal DNA and spindle fiber during mitosis and meiosis. Proper assembly of the kinetochore on centromeric chromatin and attachment of the kinetochore to the spindle fiber are essential for accurate chromosome segregation. Studies in a variety of organisms support the idea that epigenetic mechanisms, as well as primary DNA sequences, impart identity and function to centromeric DNA (10), indicating a critical role for histone proteins and other chromatin components in determining the functional state of a centromere. The organization of centromeric chromatin and the assembly of functional centromeres are specified at least in part by the H3-like histone variants Cse4p and CENP-A, discovered in Saccharomyces cerevisiae and mammals, respectively (16, 23, 27). Both proteins have uniquely different N termini and C-terminal histone-fold domains that are more than 60% identical to the histone-fold domain of H3 (Fig. ​(Fig.1)1) (23). In humans, tandem arrays of AT-rich α-satellite DNA are found in the inner kinetochore plate where CENP-A localizes. CENP-A copurifies with core histones in nucleosome-like structures (16) and binds predominantly to α-satellite DNA in phased nucleosomal arrays (25). Immunolocalization studies of CENP-A proteins expressed from transfected DNA in the presence of endogenous CENP-A show that the histone-fold domain mediates targeting of the protein to the mammalian centromere and that the unique N terminus is not required for centromere localization (24). Similar assays on mutant CENP-A proteins containing H3 substitutions in analogous sites in the histone-fold domain show that residues throughout the histone-fold domain of CENP-A cooperate to localize the protein to the mammalian centromere (19). The S. cerevisiae centromere contains three conserved DNA elements, CDE I, CDE II, and CDE III, which are sufficient and required for high-fidelity mitotic and meiotic chromosome segregation (4, 5). In vivo all three CDE elements are packaged into a 150- to 220-bp nuclease-resistant chromatin structure that is flanked by phased nucleosomal arrays (3, 6). Depletion of either histone H4 or H2B renders the CDE elements accessible to nucleases, showing that core histone proteins play a critical role in centromere structure (18). Cse4p is a component of yeast centromeric chromatin. Mutant cse4 alleles cause cell cycle arrest at G2-M, increased chromosome missegregation and disrupt centromere chromatin structure (23, 14). Cse4p associates with yeast centromere DNA in vivo and is an integral component of yeast chromatin with biochemical properties similar to H3 (14, 23). Genetic evidence supports direct interactions between Cse4p and H4. Overexpression of CSE4 suppresses the temperature-sensitive phenotype of a mutant H4 allele (hhf1-20) that causes a G2-M cell cycle arrest and increased chromosome missegregation (22). In addition, overexpression of CSE4 rescues the defective centromeric chromatin structure observed in hhf1-20 cells (14), further supporting a direct interaction between Cse4p and H4 at the centromere. Interestingly, the hhf1-20 mutations are located in a region of H4 which, according to the nucleosome crystal structure, would be adjacent to loop I of Cse4p, a region of Cse4p that diverges significantly from H3. By analogy to the structure of H3 in standard nucleosomes, the N terminus of Cse4p would be located outside the nucleosome core, while the histone-fold domain would be located in the core of the nucleosome. We reason that some regions of Cse4p have functions similar to those of histone H3 (e.g., nucleosome assembly), while other regions are involved in centromere specific functions (e.g., kinetochore assembly, sister chromatid cohesion). As a first step toward understanding how Cse4p functions in organizing centromeric chromatin, we conducted a systematic mutagenic analysis of the protein. Deletions were made in the Cse4p N terminus, and Cse4p histone-fold domain amino acids that differ between Cse4p and yeast H3 were changed to the H3 counterparts. We found that the N terminus of Cse4p, which differs significantly in length and primary amino acid sequence from H3, is required for cell viability. Analysis of the histone-fold domain revealed that extensive substitution by H3 amino acids is lethal but that any single amino acid in the Cse4p histone fold can be changed to the analogous H3 residue without disrupting essential Cse4p function. Many cse4 mutations, though viable, caused elevated rates of mitotic chromosome loss. The results are discussed in the context of the known three-dimensional structure of H3 in the nucleosome and compared with findings for CENP-A.