The RSC nucleosome-remodeling complex is required for Cohesin's association with chromosome arms

The fidelity of chromosome segregation requires that the cohesin protein complex bind together newly replicated sister chromatids both at centromeres and at discrete sites along chromosome arms. Segregation of the yeast 2 micro plasmid also requires cohesin, which is recruited to the plasmid partitioning locus. Here we report that the RSC chromatin-remodeling complex regulates the differential association of cohesin with centromeres and chromosome arms. RSC cycles on and off chromosomal arm and plasmid cohesin binding sites in a cell cycle-regulated manner 15 min preceding Mcd1p, the central cohesin subunit. We show that in rsc mutants Mcd1p fails to associate with chromosome arms but still binds to centromeres, and that consequently, the arm regions of mitotic sister chromosomes separate precociously while cohesion at centromeres is unaffected. Our data suggest a role for RSC in facilitating the loading of cohesin specifically onto chromosome arms, thereby ensuring sister chromatid cohesion and proper chromosome segregation.     

A novel member of the Swi6p family of fission yeast chromo domain-containing proteins associates with the centromere in vivo and affects chromosome segregation

We previously used a genetic approach to identify a new class of Schizosaccharomyces pombe genes (chromosome loss when overexpressed; clo genes) that, when present in elevated dosage, cause the loss of an otherwise stable cen1 linear minichromosome at high rates. Here we report the identities of two clo genes; one encodes histone H3.3 and the other, designated clo2, encodes a novel protein with significant homology to fission yeast Swi6p, human and Drosophila HP1 heterochromatin proteins, and other chromo domain-containing proteins. Members of this group have been shown to localize to heterochromatic DNA, including centromeres, and to play roles in chromatin formation and organization. The S. pombe Clo2 protein localizes to centromere DNA in vivo, and overexpression of clo2 leads to a dramatic increase in the rate of mitotic loss of an artificial chromosome. Clo2p is not essential for mitotic growth, however, even in cells that also lack Swi6p. Thus, fission yeast appears to utilize multiple, functionally redundant, HP1-related proteins for heterochromatin-associated activities at centromeres and perhaps elsewhere in the genome.     

Segregation after mitotic crossing-over in isodicentric X chromosomes

Segregation after mitotic crossing-over in an isodicentric (idic) X chromosome with one active and one inactive centromere has given rise to two new cell lines, one in which the idic(Xpter) chromosome has two active centromeres (most of these chromosomes also have an inversion) and another in which neither centromere is active. The two X chromosomes are attached at the telomeres of their short arms. Similar segregation has given rise to two other cell lines with idic(Xq-) chromosomes. Other observations on segregation after mitotic crossing-over are reviewed. Unequal crossing-over has apparently played a major role in the evolution of various genes and heterochromatin. Retinoblastoma and Wilms tumor are in some cases associated with homozygosity of a chromosome segment resulting from mitotic crossing-over. Similarly, the high incidence of cancer in Bloom syndrome may be caused by mitotic crossing-over leading to homozygosity or amplification of oncogenes.     

Genetic evidence for interactions between yeast importin α (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.     

Pat1: a topoisomerase II-associated protein required for faithful chromosome transmission in Saccharomyces cerevisiae.

Saccharomyces cerevisiae top2 mutants deficient in topoisomerase II activity are defective in chromosome segregation during both mitotic and meiotic cell divisions. To identify proteins that act in concert with topoisomerase II during chromosome segregation in S.cerevisiae, we have used a two-hybrid cloning approach. We report the isolation of the PAT1 gene (for protein associated with topoisomerase II), which encodes a novel 90 kDa proline- and glutamine-rich protein that interacts with a highly conserved, leucine-rich region of topoisomerase II in vivo. Strains lacking Pat1p exhibit a slow growth rate and a phenotype reminiscent of conditional top2 mutants grown at the semi-permissive temperature; most notably, a reduced fidelity of chromosome segregation during both mitosis and meiosis. These findings indicate that the PAT1 gene is necessary for accurate chromosome transmission during cell division in eukaryotic cells and suggest that the interaction of Pat1p and topoisomerase II is an important component of this function.     

EBNA1 partitions Epstein-Barr virus plasmids in yeast cells by attaching to human EBNA1-binding protein 2 on mitotic chromosomes

Epstein-Barr virus (EBV) episomal genomes are stably maintained in human cells and are partitioned during cell division by mitotic chromosome attachment. Partitioning is mediated by the viral EBNA1 protein, which binds both the EBV segregation element (FR) and a mitotic chromosomal component. We previously showed that the segregation of EBV-based plasmids can be reconstituted in Saccharomyces cerevisiae and is absolutely dependent on EBNA1, the EBV FR sequence, and the human EBNA1-binding protein 2 (EBP2). We have now used this yeast system to elucidate the functional contribution of human EBP2 to EBNA1-mediated plasmid partitioning. Human EBP2 was found to attach to yeast mitotic chromosomes in a cell cycle-dependent manner and cause EBNA1 to associate with the mitotic chromosomes. The domain of human EBP2 that binds both yeast and human chromosomes was mapped and shown to be functionally distinct from the EBNA1-binding domain. The functionality and localization of human EBP2 mutants and fusion proteins indicated that the attachment of EBNA1 to mitotic chromosomes is crucial for EBV plasmid segregation in S. cerevisiae, as it is in humans, and that this is the contribution of human EBP2. The results also indicate that plasmid segregation in S. cerevisiae can occur through chromosome attachment.     

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. Received: 18 November 1998 / Accepted: 17 March 1999     

Human artificial chromosomes with alpha satellite-based de novo centromeres show increased frequency of nondisjunction and anaphase lag

Human artificial chromosomes have been used to model requirements for human chromosome segregation and to explore the nature of sequences competent for centromere function. Normal human centromeres require specialized chromatin that consists of alpha satellite DNA complexed with epigenetically modified histones and centromere-specific proteins. While several types of alpha satellite DNA have been used to assemble de novo centromeres in artificial chromosome assays, the extent to which they fully recapitulate normal centromere function has not been explored. Here, we have used two kinds of alpha satellite DNA, DXZ1 (from the X chromosome) and D17Z1 (from chromosome 17), to generate human artificial chromosomes. Although artificial chromosomes are mitotically stable over many months in culture, when we examined their segregation in individual cell divisions using an anaphase assay, artificial chromosomes exhibited more segregation errors than natural human chromosomes (P < 0.001). Naturally occurring, but abnormal small ring chromosomes derived from chromosome 17 and the X chromosome also missegregate more than normal chromosomes, implicating overall chromosome size and/or structure in the fidelity of chromosome segregation. As different artificial chromosomes missegregate over a fivefold range, the data suggest that variable centromeric DNA content and/or epigenetic assembly can influence the mitotic behavior of artificial chromosomes.     

A role for histone H4K16 hypoacetylation in Saccharomyces cerevisiae kinetochore function

Hypoacetylated H4 is present at regional centromeres; however, its role in kinetochore function is poorly understood. We characterized H4 acetylation at point centromeres in Saccharomyces cerevisiae and determined the consequences of altered H4 acetylation on chromosome segregation. We observed low levels of tetra-acetylated and K16 acetylated histone H4 (H4K16Ac) at centromeres. Low levels of H4K16Ac were also observed at noncentromeric regions associated with Cse4p. Inhibition of histone deacetylases (HDAC) using nicotinamide (NAM) caused lethality in cse4 and hhf1-20 kinetochore mutants and increased centromeric H4K16Ac. Overexpression of Sas2-mediated H4K16 acetylation activity in wild-type cells led to increased rates of chromosome loss and synthetic dosage lethality in kinetochore mutants. Consistent with increased H4K16 acetylation as a cause of the phenotypes, deletion of the H4K16 deacetylase SIR2 or a sir2-H364Y catalytic mutant resulted in higher rates of chromosome loss compared to wild-type cells. Moreover, H4K16Q acetylmimic mutants displayed increased rates of chromosome loss compared to H4K16R nonacetylatable mutants and wild-type cells. Our work shows that hypoacetylated centromeric H4 is conserved across eukaryotic centromeres and hypoacetylation of H4K16 at centromeres plays an important role in accurate chromosome segregation.     

Components of the human spindle checkpoint control mechanism localize specifically to the active centromere on dicentric chromosomes

The spindle checkpoint control mechanism functions to ensure faithful chromosome segregation by delaying cell division until all chromosomes are correctly oriented on the mitotic spindle. Initially identified in budding yeast, several mammalian spindle checkpoint-associated proteins have recently been identified and partially characterized. These proteins associate with all active human centromeres, including neocentromeres, in the early stages of mitosis prior to the commencement of anaphase. We have examined the status of proteins associated with the checkpoint protein complex (BUB1, BUBR1, BUB3, MAD2), the anaphase-promoting complex (Tsg24, p55CDC), and other proteins associated with mitotic checkpoint control (ERK1, 3F3/2 epitope, hZW10), on a human dicentric chromosome. Each of these proteins was found to specifically associate with only the active centromere, suggesting that only active centromeres participate in the spindle checkpoint. This finding complements previous studies on multicentric chromosomes demonstrating specific association of structural and motor-related centromere proteins with active centromeres, and suggests that centromere inactivation is accompanied by loss of all functionally important centromere proteins.     

The Yin and Yang of centromeric cohesion of sister chromatids: mitotic kinases meet protein phosphatase 2A

Accurate chromosome segregation during meiosis and mitosis is essential for the maintenance of genomic stability. Defects in the regulation of chromosome segregation during division predispose cells to undergo mitotic catastrophe or neoplastic transformation. Cohesin, a molecular glue holding sister chromatids together, is removed from chromosomes in a stepwise fashion during mitosis and meiosis. Cohesin at centromeres but not on chromosome arm remains intact until anaphase onset during early mitosis and the initiation of anaphase II during meiosis. Several recent studies indicate that the activity of protein phosphatase 2A is essential for maintaining the integrity of centromeric cohesin. Shugoshin, a guardian for sister chromatid segregation, may cooperate with and/or mediate PP2A function by suppressing the phosphorylation status of centromeric proteins including cohesin.     

Phosphorylation of centromeric histone H3 variant regulates chromosome segregation in Saccharomyces cerevisiae

The centromeric histone H3 variant (CenH3) is essential for chromosome segregation in eukaryotes. We identify posttranslational modifications of Saccharomyces cerevisiae CenH3, Cse4. Functional characterization of cse4 phosphorylation mutants shows growth and chromosome segregation defects when combined with kinetochore mutants okp1 and ame1. Using a phosphoserine-specific antibody, we show that the association of phosphorylated Cse4 with centromeres increases in response to defective microtubule attachment or reduced cohesion. We determine that evolutionarily conserved Ipl1/Aurora B contributes to phosphorylation of Cse4, as levels of phosphorylated Cse4 are reduced at centromeres in ipl1 strains in vivo, and in vitro assays show phosphorylation of Cse4 by Ipl1. Consistent with these results, we observe that a phosphomimetic cse4-4SD mutant suppresses the temperature-sensitive growth of ipl1-2 and Ipl1 substrate mutants dam1 spc34 and ndc80, which are defective for chromosome biorientation. Furthermore, cell biology approaches using a green fluorescent protein–labeled chromosome show that cse4-4SD suppresses chromosome segregation defects in dam1 spc34 strains. On the basis of these results, we propose that phosphorylation of Cse4 destabilizes defective kinetochores to promote biorientation and ensure faithful chromosome segregation. Taken together, our results provide a detailed analysis, in vivo and in vitro, of Cse4 phosphorylation and its role in promoting faithful chromosome segregation.     

A conserved protein, Nuf2, is implicated in connecting the centromere to the spindle during chromosome segregation: a link between the kinetochore function and the spindle checkpoint

The centromere is crucial for the proper segregation of chromosomes in all eukaryotic cells. We identified a centromeric protein, Nuf2, which is conserved in fission yeast, human, nematode, and budding yeast. Gene disruption of nuf2+ in the fission yeast Schizosaccharomyces pombe caused defects in chromosome segregation and the spindle checkpoint: the mitotic spindle elongated without segregating the chromosomes, indicating that spindle function was compromised, but that this abnormality did not result in metaphase arrest. Certain nuf2 temperature-sensitive mutations, however, caused metaphase arrest with condensed chromosomes and a short spindle, indicating that, while these mutations caused abnormalities in spindle function, the spindle checkpoint pathway remained intact. Metaphase arrest in these cells was dependent on the spindle checkpoint component Mad2. Interestingly, Nuf2 disappeared from the centromere during meiotic prophase when centromeres lose their connection to the spindle pole body. We propose that Nuf2 acts at the centromere to establish a connection with the spindle for proper chromosome segregation, and that Nuf2 function is also required for the spindle checkpoint.     

PP2A is required for centromeric localization of Sgo1 and proper chromosome segregation

Loss of sister-chromatid cohesion triggers chromosome segregation in mitosis and occurs through two mechanisms in vertebrate cells: (1) phosphorylation and removal of cohesin from chromosome arms by mitotic kinases, including Plk1, during prophase, and (2) cleavage of centromeric cohesin by separase at the metaphase-anaphase transition. Bub1 and the MEI-S332/Shugoshin (Sgo1) family of proteins protect centromeric cohesin from mitotic kinases during prophase. We show that human Sgo1 binds to protein phosphatase 2A (PP2A). PP2A localizes to centromeres in a Bub1-dependent manner. The Sgo1-PP2A interaction is required for centromeric localization of Sgo1 and proper chromosome segregation in human cells. Depletion of Plk1 by RNA interference (RNAi) restores centromeric localization of Sgo1 and prevents chromosome missegregation in cells depleted of PP2A_Aalpha. Our findings suggest that Bub1 targets PP2A to centromeres, which in turn maintains Sgo1 at centromeres by counteracting Plk1-mediated chromosome removal of Sgo1.     

Differential kinetochore protein requirements for establishment versus propagation of centromere activity in Saccharomyces cerevisiae

Dicentric chromosomes undergo a breakage-fusion-bridge cycle as a consequence of having two centromeres on the same chromatid attach to opposite spindle poles in mitosis. Suppression of dicentric chromosome breakage reflects loss of kinetochore function at the kinetochore-microtubule or the kinetochore-DNA interface. Using a conditionally functional dicentric chromosome in vivo, we demonstrate that kinetochore mutants exhibit quantitative differences in their degree of chromosome breakage. Mutations in chl4/mcm17/ctf17 segregate dicentric chromosomes through successive cell divisions without breakage, indicating that only one of the two centromeres is functional. Centromere DNA introduced into the cell is unable to promote kinetochore assembly in the absence of CHL4. In contrast, established centromeres retain their segregation capacity for greater than 25 generations after depletion of Chl4p. The persistent mitotic stability of established centromeres reveals the presence of an epigenetic component in kinetochore segregation. Furthermore, this study identifies Chl4p in the initiation and specification of a heritable chromatin state.     

Role of chromosomal passenger complex in chromosome segregation and cytokinesis

Chromosomal passenger proteins associate with chromosomes early in mitosis and transfer to the spindle at ana/telophase. Recent results show that aurora B/AIM-1 (aurora and Ipl1-like midbody-associated protein kinase), which is responsible for mitotic histone H3 phosphorylation, INCENP (Inner Centromere protein) and Survivin/BIR are in a macromolecular complex as novel chromosomal passenger proteins. Aurora B/AIM-1 can bind to Survivin and the C-terminal region of INCENP, respectively, and colocalizes with both proteins to the centromeres, midzone and midbody. Disruption of either aurora B/AIM-1 or INCENP function leads to sever defects in chromosome segregation and cytokinesis. Moreover, the formation of the central spindle through anaphase to cytokinesis is also disrupted severely. These data suggest that chromosomal passenger complex is required for proper chromosome segregation by phosphorylating histone H3, and cytokinesis by ensuring the correct assembly of the midzone and midbody microtubule. Chromosomal passenger protein complex may couple chromosome segregation with cytokinesis.     

The Suv39h–HP1 histone methylation pathway is dispensable for enrichment and protection of cohesin at centromeres in mammalian cells

Sister chromatids are physically connected by cohesin complexes. This sister chromatid cohesion is essential for the biorientation of chromosomes on the mitotic and meiotic spindle. In many species, cohesion between chromosome arms is partly dissolved in prophase of mitosis, whereas cohesion is protected at centromeres until the onset of anaphase. In vertebrates, the protein Sgo1, protein phosphatase 2A, and several other proteins are required for protection of centromeric cohesin in early mitosis. In fission yeast, the recruitment of heterochromatin protein Swi6/HP1 to centromeres by the histone-methyltransferase Clr4/Suv39h is required for enrichment of cohesin at centromeres already in interphase. We have tested if the Suv39h–HP1 histone methylation pathway is also required for enrichment and mitotic protection of cohesin at centromeres in mammalian cells. We show that cohesin and HP1 proteins partially colocalize at mitotic centromeres but that cohesin localization is not detectably altered in mouse embryonic fibroblasts that lack Suv39h genes and in which HP1 proteins can, therefore, not be properly enriched in pericentric heterochromatin. Our data indicate that the Suv39h–HP1 pathway is not essential for enrichment and mitotic protection of cohesin at centromeres in mammalian cells.     

The Synaptonemal Complex Protein Zip1 Promotes Bi-Orientation of Centromeres at Meiosis I

In meiosis I, homologous chromosomes become paired and then separate from one another to opposite poles of the spindle. In humans, errors in this process are a leading cause of birth defects, mental retardation, and infertility. In most organisms, crossing-over, or exchange, between the homologous partners provides a link that promotes their proper, bipolar, attachment to the spindle. Attachment of both partners to the same pole can sometimes be corrected during a delay that is triggered by the spindle checkpoint. Studies of non-exchange chromosomes have shown that centromere pairing serves as an alternative to exchange by orienting the centromeres for proper microtubule attachment. Here, we demonstrate a new role for the synaptonemal complex protein Zip1. Zip1 localizes to the centromeres of non-exchange chromosomes in pachytene and mediates centromere pairing and segregation of the partners at meiosis I. Exchange chromosomes were also found to experience Zip1-dependent pairing at their centromeres. Zip1 was found to persist at centromeres, after synaptonemal complex disassembly, remaining there until microtubule attachment. Disruption of this centromere pairing, in spindle checkpoint mutants, randomized the segregation of exchange chromosomes. These results demonstrate that Zip1-mediated pairing of exchange chromosome centromeres promotes an initial, bipolar attachment of microtubules. This activity of Zip1 lessens the load on the spindle checkpoint, greatly reducing the chance that the cell will exit the checkpoint delay with an improperly oriented chromosome pair. Thus exchange, the spindle checkpoint, and centromere pairing are complementary mechanisms that ensure the proper segregation of homologous partners at meiosis I.