The centric H3 variants Cse4p and CENP-A have histone-fold domains that are more than 60% identical to the histone-fold domain of H3. The striking sequence homology and predicted structural similarities among these proteins, taken together with biochemical and genetic evidence (15
), strongly supports the idea that Cse4p and CENP-A replace H3 in centromere-specific nucleosomes in S. cerevisiae
and mammalian cells, respectively. The similarities among Cse4p, CENP-A, and H3 imply that these proteins carry out common functions, such as interacting with other core histones and nucleosome assembly. In the various Cse4p swap mutants, we incorporated all of the H3 residues that differ between the two proteins into mutant cse4
alleles that proved to be viable. Apparently, no single or small group of contiguous amino acids in the histone-fold domain of Cse4p acts alone to specify Cse4p function at the centromere. Rather, the results suggest that amino acids distributed throughout the histone-fold domain interact in combination to impart centromere function. A similar conclusion was reached for CENP-A (19
). Furthermore, since all regions of the Cse4p histone-fold domain can be individually changed to H3 residues, it is probable that Cse4p assembles into the nucleosome core by a mechanism similar to that of H3.
Judged simply by the number of Cse4p residues that could be altered without abolishing function, certain regions of the Cse4p histone-fold domain are more tolerant of H3 substitutions, while other regions are more sensitive to substitutions. In general, Cse4p can accommodate fairly extensive substitutions of H3 residues in the N helix, N loop, helix I, and loop I. The N helix, N loop, helix I, and loop I in H3 make extensive interactions with the DNA. Although we did not design the Cse4p-H3 swap mutants to directly test Cse4p-DNA interactions and the mutations that alter DNA contacts also change noncontact sites, we did observe a correlation between the severity of the cse4 phenotypes and the number of putative DNA contact sites altered by the N helix, N loop, helix I, and loop I mutations. H3 substitutions in Cse4p that change four of the putative DNA contact sites in these regions are lethal (cse4-290). However, the cse4 allele that changes three possible contact sites is viable but exhibits a severe chromosome loss phenotype (23.5 fold; cse4-279); those changing only two sites show modest increases in chromosome loss rates (8.6- and 9.4-fold; cse4-260 and cse4-278), while those changing one putative contact site have little or no phenotype (1.1-, 4.8-, and 5.4-fold; cse4-270, cse4-259, and cse4-262, respectively). The N helix, N loop, helix I, and loop II may be critical for providing specificity between Cse4p and the centromere DNA.
Helix II and especially helix III of Cse4p appeared to be very sensitive to substitution with H3 amino acids. In H3-H4 heterodimers, the long central helix II in H3 crosses over helix II in H4, positioning loop I of each molecule with loop II of the other molecule. Two H3-H4 dimers interact through the H3-H3′ four-helix bundle to form a tetramer, which then initiates binding to the DNA helix (1
). Replacing most of the Cse4p helix II residues with H3 residues is lethal, but smaller helix II substitutions are viable, suggesting that the smaller mutations do not prevent Cse4p-H4 dimer formation. However, the binding affinities between the mutant Cse4p protein and H4 might be altered by these mutations, thereby affecting the efficiency of Cse4p-H4 dimer and (Cse4p-H4)2
tetramer assembly and possibly interactions with centromeric DNA. Such destabilizing changes could explain the lethal and high chromosome loss phenotypes observed for the helix II cse4
The extreme C-terminal tail of Cse4p (ERS) is an important structural determinant of helix III. Substitution of H3 amino acids for the Cse4p C-terminal tail moderately increases the rate of mitotic chromosome loss (cse4-261) and can be lethal when combined with changes elsewhere in Cse4p. This is most dramatic in the case of the Q219-to-K mutation in helix III (cse4-101), which is viable on its own but lethal when combined with the H3 C-terminal tail (cse4-369). The C-terminal halves of the H3-H3′ helix III’s contact each other across the dyad axis of the nucleosome. By analogy, helix III of Cse4p would be expected to be critical for stabilizing the (Cse4p-H4)2 tetramer. Substitution of H3 residues in helix II and helix III in Cse4p might interfere with interactions between Cse4p-H4 dimers required to assemble (Cse4p-H4)2 tetramers. Substitution mutations that make helix II and helix III in Cse4p more like H3, such as lethal alleles cse4-286 or cse4-369, would be expected to disrupt Cse4p-Cse4p′ four-helix bundle interactions and reduce the number of (Cse4p-H4)2 tetramers available for centromere recognition.
Immunolocalization studies of CENP-A mutants containing H3 amino acid substitutions in the histone-fold domain have identified regions of CENP-A that are involved in centromere localization (19
). These data, taken together with results from this study, indicate that Cse4p and CENP-A may impart centromere function by similar mechanisms involving multiple sites within their histone-fold domains. The N helix, loop I and both ends of helix II of CENP-A were found to be critical for localization to the centromere (19
). The same regions of Cse4p were found to be important, since substituting them with H3 amino acids either was lethal (helix II substitution) or caused increased chromosome loss rates (N helix, N loop substitution, loop I substitution, and partial helix II substitutions). An obvious caveat to direct comparisons between the two studies is that the CENP-A experiments directly tested localization but did not analyze other possible functions of the protein. In addition, the CENP-A mutant proteins were studied in the presence of wild-type CENP-A. In contrast, the study reported here analyzed the viability and centromere function of mutant Cse4p but did not directly measure centromere localization. We infer that mutant Cse4p proteins are properly localized if they rescue the cse4
null mutation and confer wild-type or near-wild-type rates of mitotic chromosome loss. However, mutant phenotypes detected by our in vivo assays may reflect defects other than mislocalization.
Replacing the histone-fold domain of Cse4p with that of CENP-A cannot rescue the cse4
null phenotype, indicating that specific regions within the histone-fold domain probably provide specificity to the different centromeres. Interestingly, regions in the histone-fold domain of Cse4p that differ from H3 also differ between Cse4p and CENP-A (Fig. ). These divergent regions may provide each protein with the specificity required for the distinctly different yeast and mammalian centromeres. Our results did reveal potential differences between the CENP-A and Cse4p histone-fold domains. We clearly show that helix III of Cse4p is essential in determining centromere-specific function. The sequence of helix III in CENP-A varies only minimally from human H3, so a helix III replacement mutant of CENP-A was not tested (19
). The extreme C terminus of Cse4p is also an important determinant of Cse4p function. Exchanging this region caused increased rates of chromosome loss and, when combined with other viable alleles, caused synthetic lethality. Substituting the CENP-A C terminus with that of H3 did not affect centromere localization (19
); however, combinatorial mutations, including the C-terminal substitution, were not tested. Altering the single helix III amino acid that differs between human H3 and CENP-A in combination with the C-terminus substitution might inactivate CENP-A, as we observed for Cse4p when the helix III Q219-to-K change was combined with the QFI-to-ERS C-terminus substitution. Finally, our results show that helix I is involved in Cse4p function, whereas this region of CENP-A is not required for centromere localization.
Another feature of the Cse4p primary structure that is decidedly different from CENP-A is the unique N terminus. The Cse4p N terminus has no amino acid homology with the N termini of either H3 or CENP-A and, while the N termini of H3 and CENP-A are both about 40 amino acids in length, the N terminus of Cse4p is much longer (135 amino acids). These facts suggest that the N terminus of Cse4p functions differently from the N termini of H3 and CENP-A. Indeed, we identified at least one region of the Cse4p N terminus that is essential for Cse4p function. In contrast, the CENP-A N terminus is not required for localizing CENP-A to the centromere, although it might be involved in kinetochore functions not revealed by the localization assay. The entire N terminus of yeast H3 can be deleted without affecting viability, although cells exhibit slow growth and defects in transcription. The H3 N terminus extends outside the nucleosome core, where it interacts with proteins involved in silencing and transcriptional regulation (11
). The N terminus of Cse4p may also extend outside the nucleosome core, making it accessible for interactions with proteins involved in a variety of centromere functions, including sister chromatid cohesion, chromosome separation, kinetochore assembly, and chromatin formation or modification. Although we have not yet fully characterized the essential region in the Cse4p N terminus, the spacing between the essential region and the histone-fold domain must be somewhat flexible, since insertion of the triple HA epitope tag (37 amino acids) in frame between amino acids 79 and 80 (Fig. ) does not detectably affect Cse4p function.
Replacement of H3 with Cse4p or CENP-A in the nucleosome core confers two distinct advantages for packaging centromere DNA. First, since homotypic interactions within the octamer particle are restricted to the two H3 molecules, substitution of H3 with Cse4p would permit the formation of homotypic (Cse4p-H4)2
tetramers but not heterotypic tetramers containing one molecule each of Cse4p and H3. We propose that (Cse4p-H4)2
tetramers function to recognize the centromeric DNA and initiate assembly of a unique Cse4p nucleosome. Heterotypic (H4-Cse4p–H3-H4) tetramers might be defective in assembly or function at the centromere because of mislocalization of the tetramer to noncentromere sites. The second advantage is that homotypic (Cse4p-H4)2
tetramers would maximize the Cse4p-CEN DNA contact sites. Histone H3 is unique among the core histones in that it contacts the nucleosomal DNA at several distinct points along the 146 bp of DNA associated with the core octamer. The position of the (Cse4p-H4)2
tetramer determines the central dyad axis of the nucleosomal DNA, as well as the entry and exit points of the DNA helix on the histone octamer (11
). By making multiple DNA-protein contacts along the centromere DNA, the two Cse4p proteins in the proposed homotypic Cse4p nucleosome are positioned to recognize the centromere, identify the central dyad axis, and initiate assembly.