Esperanto for histones: CENP-A, not CenH3, is the centromeric histone H3 variant.

The first centromeric protein identified in any species was CENP-A, a divergent member of the histone H3 family that was recognised by autoantibodies from patients with scleroderma-spectrum disease. It has recently been suggested to rename this protein CenH3. Here, we argue that the original name should be maintained both because it is the basis of a long established nomenclature for centromere proteins and because it avoids confusion due to the presence of canonical histone H3 at centromeres.

CSE4 genetically interacts with the Saccharomyces cerevisiae centromere DNA elements CDE I and CDE II but not CDE III. Implications for the path of the centromere dna around a cse4p variant nucleosome.

Each Saccharomyces cerevisiae chromosome contains a single centromere composed of three conserved DNA elements, CDE I, II, and III. The histone H3 variant, Cse4p, is an essential component of the S. cerevisiae centromere and is thought to replace H3 in specialized nucleosomes at the yeast centromere. To investigate the genetic interactions between Cse4p and centromere DNA, we measured the chromosome loss rates exhibited by cse4 cen3 double-mutant cells that express mutant Cse4 proteins and carry chromosomes containing mutant centromere DNA (cen3). When compared to loss rates for cells carrying the same cen3 DNA mutants but expressing wild-type Cse4p, we found that mutations throughout the Cse4p histone-fold domain caused surprisingly large increases in the loss of chromosomes carrying CDE I or CDE II mutant centromeres, but had no effect on chromosomes with CDE III mutant centromeres. Our genetic evidence is consistent with direct interactions between Cse4p and the CDE I-CDE II region of the centromere DNA. On the basis of these and other results from genetic, biochemical, and structural studies, we propose a model that best describes the path of the centromere DNA around a specialized Cse4p-nucleosome.

The N terminus of the centromere H3-like protein Cse4p performs an essential function distinct from that of the histone fold domain.

Cse4p is an evolutionarily conserved histone H3-like protein that is thought to replace H3 in a specialized nucleosome at the yeast (Saccharomyces cerevisiae) centromere. All known yeast, worm, fly, and human centromere H3-like proteins have highly conserved C-terminal histone fold domains (HFD) but very different N termini. We have carried out a comprehensive and systematic mutagenesis of the Cse4p N terminus to analyze its function. Surprisingly, only a 33-amino-acid domain within the 130-amino-acid-long N terminus is required for Cse4p N-terminal function. The spacing of the essential N-terminal domain (END) relative to the HFD can be changed significantly without an apparent effect on Cse4p function. The END appears to be important for interactions between Cse4p and known kinetochore components, including the Ctf19p/Mcm21p/Okp1p complex. Genetic and biochemical evidence shows that Cse4p proteins interact with each other in vivo and that nonfunctional cse4 END and HFD mutant proteins can form functional mixed complexes. These results support different roles for the Cse4p N terminus and the HFD in centromere function and are consistent with the proposed Cse4p nucleosome model. The structure-function characteristics of the Cse4p N terminus are relevant to understanding how other H3-like proteins, such as the human homolog CENP-A, function in kinetochore assembly and chromosome segregation.

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

Cse4p is a variant of histone H3 that has an essential role in chromosome segregation and centromere chromatin structure in budding yeast. Cse4p has a unique 135-amino-acid N terminus and a C-terminal histone-fold domain that is more than 60% identical to histone H3 and the mammalian centromere protein CENP-A. Cse4p and CENP-A have biochemical properties similar to H3 and probably replace H3 in centromere-specific nucleosomes in yeasts and mammals, respectively. In order to identify regions of Cse4p that distinguish it from H3 and confer centromere function, a systematic site-directed mutational analysis was performed. Nested deletions of the Cse4p N terminus showed that this region of the protein contains at least one essential domain. The C-terminal histone-fold domain of Cse4p was analyzed by changing Cse4p amino acids that differ between Cse4p and H3 to the analogous H3 residues. Extensive substitution of contiguous Cse4p residues with H3 counterparts resulted in cell lethality. However, all large lethal substitution alleles could be subdivided into smaller viable alleles, many of which caused elevated rates of mitotic chromosome loss. The results indicate that residues critical for wild-type Cse4p function and high-fidelity chromosome transmission are distributed across the entire histone-fold domain. Our findings are discussed in the context of the known structure of H3 within the nucleosome and compared with previous results reported for CENP-A.

Genetic evidence for interactions between yeast importin alpha (Srp1p) and its nuclear export receptor, Cse1p.

The yeast Srp1p protein functions as an import receptor for proteins bearing basic nuclear localization signals. Cse1p, the yeast homolog of mammalian CAS, recycles Srp1p back to the cytoplasm after import substrates have been released into the nucleoplasm. In this report we describe genetic interactions between SRP1 and CSE1. Results from genetic suppression and synthetic lethality studies demonstrate that these gene products interact to ensure accurate chromosome segregation. We also describe new mutant alleles of CSE1 and analyze a new temperature-sensitive allele of CSE1, cse1-2. This allele causes high levels of chromosome missegregation and cell cycle arrest during mitosis at the nonpermissive temperature.

Functional interaction between the CSE2 gene product and centromeres in Saccharomyces cerevisiae.

The cse2-1 allele was identified through a genetic screen for mutations affecting chromosome segregation in Saccharomyces cerevisiae. This mutation confers cold and temperature sensitivity and causes increases in mitotic chromosome non-disjunction and loss. The CSE2 gene encodes a 17 kDa protein with a basic region-leucine zipper motif. Disruption of CSE2 is not lethal but results in the accumulation of large-budded cells. Here, we report that disruption of CSE2 results in a significant increase in chromosome missegregation, slower growth and defective meiosis. The combination of the CSE2 disruption and a mutant centromere results in a synergistic effect on both cell growth and cell viability. These data suggest a functional interaction between the CSE2 protein and the yeast centromere.

A mutation in CSE4, an essential gene encoding a novel chromatin-associated protein in yeast, causes chromosome nondisjunction and cell cycle arrest at mitosis.

The centromere, a differentiated region of the eukaryotic chromosome, mediates the segregation of sister chromatids at mitosis. In this study, a Saccharomyces cerevisiae chromosome mis-segregation mutant, cse4-1, has been isolated and shown to increase the nondisjunction frequency of a chromosome bearing a mutant centromere DNA sequence. In addition, at elevated temperatures the cse4-1 allele causes a mitosis-specific arrest with a predominance of large budded cells containing single G2 nuclei and short bipolar mitotic spindles. The wild-type gene, CSE4, is essential for cell division and encodes a protein containing a domain that is 64% identical to the highly conserved chromatin protein, histone H3. Biochemical experiments demonstrate that CSE4p has similar DNA-binding characteristics as those of histone H3 and might form a specialized nucleosome structure in vivo. Interestingly, the human centromere protein, CENP-A, also contains this H3-like domain. Data presented here indicate that CSE4p is required for proper kinetochore function in yeast and may represent an evolutionarily conserved protein necessary for assembly of the unique chromatin structure associated with the eukaryotic centromere.

SCM2, a tryptophan permease in Saccharomyces cerevisiae, is important for cell growth.

SCM2, a novel gene encoding a yeast tryptophan permease, was cloned as a high-copy-number suppressor of cse2-1. The cse2-1 mutation causes cold sensitivity, temperature sensitivity and chromosome missegregation. However, only the cold-sensitive phenotype of cse2-1 cells is suppressed by SCM2 at high copy. SCM2 is located on the left arm of yeast chromosome XV, adjacent to SUP3 and encodes a 65 kDa protein that is highly homologous to known amino acid permeases. Four out of five disrupted scm2 alleles (scm2 delta 1-delta 4) cause slow growth, whereas one disrupted allele (scm2 delta 5) is lethal. Cells with both the scm2 delta 1 and trp1-delta 101 mutations exhibit a synthetic cold-sensitive phenotype and grow much more slowly at the permissive temperature than cells with a single scm2 delta 1 or trp1-delta 101 mutation. A region of the predicted SCM2 protein is identical to the partial sequence recently reported for the yeast tryptophan permease TAP2, indicating that SCM2 and TAP2 probably encode the same protein.

CSE1 and CSE2, two new genes required for accurate mitotic chromosome segregation in Saccharomyces cerevisiae.

By monitoring the mitotic transmission of a marked chromosome bearing a defective centromere, we have identified conditional alleles of two genes involved in chromosome segregation (cse). Mutations in CSE1 and CSE2 have a greater effect on the segregation of chromosomes carrying mutant centromeres than on the segregation of chromosomes with wild-type centromeres. In addition, the cse mutations cause predominantly nondisjunction rather than loss events but do not cause a detectable increase in mitotic recombination. At the restrictive temperature, cse1 and cse2 mutants accumulate large-budded cells, with a significant fraction exhibiting aberrant binucleate morphologies. We cloned the CSE1 and CSE2 genes by complementation of the cold-sensitive phenotypes. Physical and genetic mapping data indicate that CSE1 is linked to HAP2 on the left arm of chromosome VII and CSE2 is adjacent to PRP2 on chromosome XIV. CSE1 is essential and encodes a novel 109-kDa protein. CSE2 encodes a 17-kDa protein with a putative basic-region leucine zipper motif. Disruption of CSE2 causes chromosome missegregation, conditional lethality, and slow growth at the permissive temperature.

A mutation in PLC1, a candidate phosphoinositide-specific phospholipase C gene from Saccharomyces cerevisiae, causes aberrant mitotic chromosome segregation.

We identified a putative Saccharomyces cerevisiae homolog of a phosphoinositide-specific phospholipase C (PI-PLC) gene, PLC1, which encodes a protein most similar to the delta class of PI-PLC enzymes. The PLC1 gene was isolated during a study of yeast strains that exhibit defects in chromosome segregation. plc1-1 cells showed a 10-fold increase in aberrant chromosome segregation compared with the wild type. Molecular analysis revealed that PLC1 encodes a predicted protein of 101 kDa with approximately 50 and 26% identity to the highly conserved X and Y domains of PI-PLC isozymes from humans, bovines, rats, and Drosophila melanogaster. The putative yeast protein also contains a consensus EF-hand domain that is predicted to bind calcium. Interestingly, the temperature-sensitive and chromosome missegregation phenotypes exhibited by plc1-1 cells were partially suppressed by exogenous calcium.

Analysis of centromere function in Saccharomyces cerevisiae using synthetic centromere mutants.

We constructed Saccharomyces cerevisiae centromere DNA mutants by annealing and ligating synthetic oligonucleotides, a novel approach to centromere DNA mutagenesis that allowed us to change only one structural parameter at a time. Using this method, we confirmed that CDE I, II, and III alone are sufficient for centromere function and that A + T-rich sequences in CDE II play important roles in mitosis and meiosis. Analysis of mutants also showed that a bend in the centromere DNA could be important for proper mitotic and meiotic chromosome segregation. In addition we demonstrated that the wild-type orientation of the CDE III sequence, but not the CDE I sequence, is critical for wild-type mitotic segregation. Surprisingly, we found that one mutant centromere affected the segregation of plasmids and chromosomes differently. The implications of these results for centromere function and chromosome structure are discussed.

In vivo genomic footprint of a yeast centromere.

We have used in vivo genomic footprinting to investigate the protein-DNA interactions within the conserved DNA elements (CDEI, CDEII, and CDEIII) in the centromere from chromosome III of the yeast Saccharomyces cerevisiae. The in vivo footprint pattern obtained from wild-type cells shows that some guanines within the centromere DNA are protected from methylation by dimethyl sulfate. These results are consistent with studies demonstrating that yeast cells contain sequence-specific centromere DNA-binding proteins. Our in vivo experiments on chromosomes with mutant centromeres show that some mutations which affect chromosome segregation also alter the footprint pattern caused by proteins bound to the centromere DNA. The results of this study provide the first fine-structure map of proteins bound to centromere DNA in living yeast cells and suggest a direct correlation between these protein-DNA interactions and centromere function.

Cis- and trans-acting factors involved in centromere function in Saccharomyces cerevisiae.

The function of centromeric DNA in the yeast Saccharomyces cerevisiae has been studied in detail. Twelve of the sixteen S. cerevisiae centromeres have been sequenced to date, and a consensus sequence has been identified. This sequence consists of a central region 78 to 86bp in length which is greater than 90% A + T, usually in runs of As and runs of Ts. The central region is flanked on one side by a highly conserved 8bp sequence and on the other side by a highly conserved 25bp sequence which contains partial dyad symmetry around a central C/G base pair. Mutational analyses have been used to determine the importance of each subset of the consensus sequence to centromere function. A protein which binds to the 8bp sequence and at least one that binds to the 25bp sequence have been identified. The roles of these proteins in centromere function in mitosis and meiosis are currently under investigation.

Saccharomyces cerevisiae mutants defective in chromosome segregation.

We have devised a genetic screen to identify trans-acting factors involved in chromosome transmission in yeast. This approach was designed to potentially identify a subset of genes encoding proteins that interact with centromere DNA. It has been shown that mutations in yeast centromere DNA cause aberrant chromosome segregation during mitosis and meiosis. We reasoned that the function of an altered centromere should be particularly sensitive to changes in factors with which it interacts. We constructed a disomic strain containing one copy of chromosome III with a wild-type centromere and one copy of chromosome III bearing the SUP11 gene and a mutant CEN3. This strain forms white colonies with red sectors due to nondisjunction of the chromosome bearing the mutant centromere. After mutagenesis we picked colonies that exhibited increased nondisjunction of the mutant chromosome as evidenced by increased red-white sectoring. Using this approach, we have isolated three trans-acting chromosome nondisjunction (cnd) mutants that are defective in maintaining chromosomes during mitosis in yeast.

Purification of the yeast centromere binding protein CP1 and a mutational analysis of its binding site.

CP1 is a yeast protein which binds to the highly conserved DNA element I (CDEI) of yeast centromeres. We have purified CP1 to near homogeneity; it is comprised of a single polypeptide of molecular weight 58,400. When bound to yeast CEN3 DNA, CP1 protects a 12-15-base pair region centered over CDEI. Methylation interference experiments show that methylations of residues located outside of the 8-base pair CDEI sequence have no detectable effect on CP1 binding, suggesting that the DNA sequences important for CP1 recognition are confined to the CDEI octanucleotide. The equilibrium constant for CP1 binding to CEN3 DNA is relatively low, 3 x 10(8) M-1. Using a novel method to determine relative DNA binding constants, we analyzed the effect of CDEI mutations on CP1 binding. A C to T point mutation at position 5 (CO1) reduces the equilibrium constant about 35-fold, while the insertion of an additional T at this position (CAT) reduces the equilibrium constant 1,400-fold. The effect of these mutations on mitotic centromere function in vivo was assessed using a plasmid stability assay. While the CO1 mutation had a slight effect, the CAT mutation significantly impaired function, implying that CP1 binding is required for the optimal mitotic function of yeast centromeres.

Mutations in CEN3 cause aberrant chromosome segregation during meiosis in Saccharomyces cerevisiae.

We investigated the structural requirements of the centromere from chromosome III (CEN3) of Saccharomyces cerevisiae by analyzing the ability of chromosomes with CEN3 mutations to segregate properly during meiosis. We analyzed diploid cells in which one or both copies of chromosome III carry a mutant centromere in place of the wild-type centromere and found that some alterations in the length, base composition and primary sequence characteristics of the central A+T-rich region (CDE II) of the centromere had a significant effect on the ability of the chromosome to segregate properly through meiosis. Chromosomes containing mutations which delete a portion of CDE II showed a high rate of premature disjunction at meiosis I. Chromosomes containing point mutations in CDE I or lacking CDE I appeared to segregate properly through meiosis; however, plasmids carrying centromeres with CDE I completely deleted showed an increased frequency of segregation to nonsister spores.

Chromatin structure of altered yeast centromeres.

We have investigated the chromatin structure of wild-type and mutationally altered centromere sequences in the yeast Saccharomyces cerevisiae by using an indirect end-labeling mapping strategy. Wild-type centromere DNA from chromosome III (CEN3) exhibits a nuclease-resistant chromatin structure 220-250 base pairs long, centered around the conserved centromere DNA element (CDE) III. A point mutation in CDE III that changes a central cytidine to a thymidine and completely disrupts centromere function has lost the chromatin conformation typically associated with the wild-type centromere. A second conserved DNA element, CDE I, is spatially separated from CDE III by 78-86 A + T-rich base pairs, which is termed CDE II. The sequence and spatial requirements for CDE II are less stringent; alterations in CDE II length and sequence can be tolerated to a limited extent. Nuclease-resistant cores are altered in dimension in two CDE II CEN3 mutations. Two CDE I deletion mutations that retain partial centromere function also show nuclease-resistant regions of reduced size and intensity. The results from a number of such altered centromeres indicate a correlation between the presence of a protected core and centromere function.

Yeast centromeres.

Significant progress has been made toward understanding the roles played by conserved centromere DNA sequences in both mitotic and meiotic chromosome segregation. We are just beginning to formulate a picture of what a yeast kinetochore actually looks like and what components other than CEN DNA are necessary for function. In the next few years some of the genes encoding structural components of the kinetochore, and perhaps some involved in regulation of kinetochore function, will be cloned. Work is already in progress to isolate and characterize the proteins necessary for the assembly, maintenance and function of this amazing biological structure.

A genetic analysis of dicentric minichromosomes in Saccharomyces cerevisiae.

We have developed an assay in S. cerevisiae in which clones of cells that contain intact dicentric minichromosomes are visually distinct from those that have rearranged to monocentric minichromosomes. We find that the instability of dicentric minichromosomes is apparently due to mitotic nondisjunction accompanied by occasional structural rearrangements. Monocentric minichromosomes arising by rearrangement of the plasmid are rapidly selected in the population since dicentric minichromosomes depress the rate of cell division. We show that the ability of one centromere to compete with another in dicentric minichromosomes requires the presence of both of the conserved structural elements, CDE II and CDE III. Dicentric minichromosomes can be stabilized if one of the centromeres on the molecule is functionally hypomorphic because of mutations in CDE II even though these mutant centromeres are highly efficient in monocentric molecules. Stable dicentric molecules can also be produced by decreasing the space between two wild-type centromeres on the same molecule. These results suggest plausible pathways for changes in chromosome number that accompany evolution.

Alterations in the adenine-plus-thymine-rich region of CEN3 affect centromere function in Saccharomyces cerevisiae.

Centromere DNA from 11 of the 16 chromosomes of the yeast Saccharomyces cerevisiae have been analyzed and reveal three sequence elements common to each centromere, referred to as conserved centromere DNA elements (CDE). The adenine-plus-thymine (A + T)-rich central core element, CDE II, is flanked by two short conserved sequences, CDE I (8 base pairs [bp]) and CDE III (25 bp). Although no consensus sequence exists among the different CDE II regions, they do have three common features of sequence organization. First, the CDE II regions are similar in length, ranging from 78 to 86 bp measured from CDE I to the left boundary of CDE III. Second, the base composition is always greater than 90% A + T. Finally, the A and T residues in these segments are often arranged in runs of A and runs of T residues, sometimes with six or seven bases in a stretch. We constructed insertion, deletion, and replacement mutations in the CDE II region of the centromere from chromosome III, CEN3, designed to investigate the length and sequence requirements for function of the CDE II region of the centromere. We analyzed the effect of these altered centromeres on plasmid and chromosome segregation in S. cerevisiae. Our results show that increasing the length of CDE II from 84 to 154 bp causes a 100-fold increase in chromosome nondisjunction. Deletion mutations removing segments of the A + T-rich CDE II DNA also cause aberrant segregation. In some cases partial function could be restored by replacing the deleted DNA with fragments whose primary sequence or base composition is very different from that of the wild-type CDE II DNA. In addition, we found that identical mutations introduced into different positions in CDE II have very similar effects.