Recognition of the centromere-specific histone Cse4 by the chaperone Scm3

A specialized nucleosome is a component of all eukaryotic kinetochores. The core of this nucleosome contains a centromere-specific histone, CENP-A (the Cse4 gene product in budding yeast), instead of the usual H3. Assembly of a centromeric nucleosome depends on a specific chaperone, called Scm3 in yeast and HJURP in higher eukaryotes. We describe here the structure of a complex formed by an N-terminal fragment of Scm3 with the histone-fold domains of Cse4, and H4, all prepared as recombinant proteins derived from the budding yeast Kluyveromyces lactis. The contacts of Scm3 with Cse4 explain its selectivity for the centromere-specific histone; key residues at the interface are conserved in HJURP, indicating a common mechanism for centromeric-histone deposition. We also report the structure of a (Cse4 : H4)₂ heterotetramer; comparison with the structure of the Scm3:Cse4:H4 complex shows that tetramer formation and DNA-binding require displacement of Scm3 from the nucleosome core. The two structures together suggest that specific contacts between the chaperone and Cse4, rather than an altered overall structure of the nucleosome core, determine the selective presence of Cse4 at centromeres.Faithful transfer of genetic information from a mother cell is crucial for the survival of its daughters. During mitosis, an assembly of protein complexes, the kinetochore, connects each centromere with spindle microtubules and monitors bipolar attachment (1). A hallmark of kinetochores in all eukaryotes is a centromere-specific nucleosome, in which a centromere-specific H3 variant, CENP-A (sometimes designated CenH3 and known as Cse4 in budding yeast), replaces the canonical H3 (2–5). CENP-A/Cse4 is very well conserved, despite the divergence of centromeric DNA from budding yeast (which have short “point centromeres,” approximately 150–220 bp in length) to higher eukaryotes (with much longer, “regional centromeres”) (6).In point-centromere yeasts, a kinetochore-associated protein, Scm3, targets Cse4 nucleosomes to centromeres (7–9). Scm3, which associates specifically with Cse4 and not with H3, has orthologs in fission-yeast (Scm3^SP) and in higher eukaryotes (HJURP) (10–15). Centromeric localization of Scm3 is determined by Ndc10, a component of centromere-binding-factor 3 (CBF3) (8); elimination of CBF3 blocks deposition of the centromeric nucleosome (16). In organisms with regional centromeres, CENP-A deposition appears to be epigenetically directed. Localization of the Scm3 homolog depends on a set of proteins known as the Mis16–Mis18 complex (13, 17), as well as on the presence of CENP-A in neighboring nucleosomes and on defined H3 modifications in the interspersed chromatin (18).The so-called “CENP-A targeting domain” (CATD)—loop 1 and helix II of the CENP-A/Cse4 histone-fold domain (HFD)—is crucial for centromeric-histone function (19). Substitution of several CATD residues with their H3 counterparts disrupts CENP-A localization (20), and a chimeric H3-CATD histone functionally replaces CENP-A in vivo (19). Evidence that Scm3 and HJURP are assembly chaperones for Cse4 and CENP-A, respectively, therefore suggests that these proteins recognize features of the CATD (7–14).We report here the crystal structure (at 2.3-Å resolution) of a complex containing Scm3, Cse4, and H4, all from the budding yeast Kluyveromyces lactis. The structure shows that Scm3 interacts with Cse4 helix II and that its contacts explain selectivity for Cse4. Comparison of this structure with those of a (Cse4 : H4)₂ heterotetramer, also reported here, and of a conventional nucleosome (21) shows that tetramer formation and DNA-binding will displace Scm3. Conservation in HJURP and Scm3^SP of key residues at the Scm3:Cse4 interface indicates a common mechanism by which these chaperones recognize CENP-A/Cse4 and deposit it at centromeres. Our structure thus suggests that the principal difference between point and regional centromeres is in recruitment of the centromeric-histone–chaperone and that the structure and higher-order interactions of the centromere-specific nucleosome itself are essentially the same in all eukaryotes.