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The physical locations of citrus centromere are revealed by combining genetic and immunological assays for the first time and nine citrus centromere-specific markers for cytogenetics are mined. Centromere localization is challenging, because highly redundant repetitive sequences in centromeric regions make sequence assembly difficult. Although several citrus genomes have been released, the centromeric regions and their characteristics remain to be elucidated. Here, we mapped citrus centromeres through half-tetrad analysis (HTA) that included the genotyping of 54 tetraploid hybrids derived from 2n megagametophytes of Nadorcott tangor with 212 single nucleotide polymorphism (SNP) markers. The sizes of centromeric regions, which estimated based on the heterozygosity restitution rate pattern along the chromosomes, ranged from 1.12 to 18.19 Mb. We also profiled the binding sequences with the centromere-specific histone variant CenH3 by chromatin immunoprecipitation sequencing (ChIP-seq). Based on the positions of the top ten CenH3-enriched contigs, the sizes of centromeric regions were estimated to range from 0.01 to 7.60 Mb and were either adjacent to or included in the centromeric regions identified by HTA. We used DNA probes from two repeats selected from the centromeric regions and seven CenH3-binding centromeric repeats to verify centromeric locations by fluorescence in situ hybridization (FISH). Centromere localization in citrus will contribute to the mining of centromeric/pericentromeric markers, thus to facilitate the rapid identification of mechanisms underlying 2n gamete formation and serve the polyploidy breeding.

Centromere structure and function are defined by the epigenetic modification of histones at centromeric and pericentromeric chromatin. The constitutive heterochromatin found at pericentromeric regions is highly enriched for H3K9me3 and H4K20me3. Although mis-expression of the methyltransferase enzymes that regulate these marks, Suv39 and Suv420, is common in disease, the consequences of such changes are not well understood. Our data show that increased centromere localization of Suv39 and Suv420 suppresses centromere transcription and compromises localization of the mitotic kinase Aurora B, decreasing microtubule dynamics and compromising chromosome alignment and segregation. We find that inhibition of Suv420 methyltransferase activity partially restores Aurora B localization to centromeres and that restoration of the Aurora B-containing chromosomal passenger complex to the centromere is sufficient to suppress mitotic errors that result when Suv420 and H4K20me3 is enriched at centromeres. Consistent with a role for Suv39 and Suv420 in negatively regulating Aurora B, high expression of these enzymes corresponds with increased sensitivity to Aurora kinase inhibition in human cancer cells, suggesting that increased H3K9 and H4K20 methylation may be an underappreciated source of chromosome mis-segregation in cancer. This article has an associated First Person interview with the first author of the paper.

Kinetoplastids are evolutionarily divergent eukaryotes that have an unconventional set of kinetochore proteins. Marcianò et al. show that KKT2 and KKT3 play important roles in kinetochore assembly in Trypanosoma brucei. Centromere localization of these proteins is mediated by the unique centromere localization domain., The kinetochore is the macromolecular protein complex that assembles onto centromeric DNA and binds spindle microtubules. Evolutionarily divergent kinetoplastids have an unconventional set of kinetochore proteins. It remains unknown how kinetochores assemble at centromeres in these organisms. Here, we characterize KKT2 and KKT3 in the kinetoplastid parasite Trypanosoma brucei. In addition to the N-terminal kinase domain and C-terminal divergent polo boxes, these proteins have a central domain of unknown function. We show that KKT2 and KKT3 are important for the localization of several kinetochore proteins and that their central domains are sufficient for centromere localization. Crystal structures of the KKT2 central domain from two divergent kinetoplastids reveal a unique zinc-binding domain (termed the CL domain for centromere localization), which promotes its kinetochore localization in T. brucei. Mutations in the equivalent domain in KKT3 abolish its kinetochore localization and function. Our work shows that the unique central domains play a critical role in mediating the centromere localization of KKT2 and KKT3.

Summary: Bub1 is required for the kinetochore/centromere localization of two essential mitotic kinases Plk1 and Aurora B. Surprisingly, stable depletion of Bub1 by ∼95% in human cells marginally affects whole chromosome segregation fidelity. We show that CENP-U, which is recruited to kinetochores by the CENP-P and CENP-Q subunits of the CENP-O complex, is required to prevent chromosome mis-segregation in Bub1-depleted cells. Mechanistically, Bub1 and CENP-U redundantly recruit Plk1 to kinetochores to stabilize kinetochore-microtubule attachments, thereby ensuring accurate chromosome segregation. Furthermore, unlike its budding yeast homolog, the CENP-O complex does not regulate centromeric localization of Aurora B. Consistently, depletion of Bub1 or CENP-U sensitizes cells to the inhibition of Plk1 but not Aurora B kinase activity. Taken together, our findings provide mechanistic insight into the regulation of kinetochore function, which may have implications for targeted treatment of cancer cells with mutations perturbing kinetochore recruitment of Plk1 by Bub1 or the CENP-O complex.

Chromosome biorientation is established by the four-member chromosomal passenger complex (CPC) through phosphorylation of incorrect kinetochore-microtubule attachments. During chromosome alignment, the CPC localizes to the inner centromere, the inner kinetochore and spindle microtubules. Here we show that a small region of the CPC subunit INCENP/Sli15 is required to target the complex to all three of these locations in budding yeast. This region, the SAH, is essential for phosphorylation of outer kinetochore substrates, chromosome segregation, and viability. By restoring the CPC to each of these three locations individually, we found that inner centromere localization is sufficient to establish chromosome biorientation and viability independently of the other two targeting mechanisms. Remarkably, although neither the inner kinetochore nor microtubule binding activities was able to rescue viability individually, they were able to do so when combined. We have therefore identified two parallel pathways by which the CPC can promote chromosome biorientation and proper completion of mitosis.

Significance Meiosis is the specialized cell division that generates haploid sperm and eggs. Proper segregation of chromosomes in meiosis is required to prevent pregnancy loss and birth defects. This requires that the replicated copies of each chromosome remain attached as the homologous copies of each chromosome segregate in the first meiotic division. The replicated copies of each chromosome then segregate in the second meiotic division. The Shugoshin proteins protect attachments between the replicated chromosome copies in meiosis I. This paper shows that, in Drosophila meiosis, a phosphatase and the Shugoshin MEI-S332 reciprocally regulate each other’s localization to centromeres and together they thus function to ensure accurate segregation., Proper segregation of chromosomes in meiosis is essential to prevent miscarriages and birth defects. This requires that sister chromatids maintain cohesion at the centromere as cohesion is released on the chromatid arms when the homologs segregate at anaphase I. The Shugoshin proteins preserve centromere cohesion by protecting the cohesin complex from cleavage, and this has been shown in yeasts to be mediated by recruitment of the protein phosphatase 2A B′ (PP2A B′). In metazoans, delineation of the role of PP2A B′ in meiosis has been hindered by its myriad of other essential roles. The Drosophila Shugoshin MEI-S332 can bind directly to both of the B′ regulatory subunits of PP2A, Wdb and Wrd, in yeast two-hybrid experiments. Exploiting experimental advantages of Drosophila spermatogenesis, we found that the Wdb subunit localizes first along chromosomes in meiosis I, becoming restricted to the centromere region as MEI-S332 binds. Wdb and MEI-S332 show colocalization at the centromere region until release of sister-chromatid cohesion at the metaphase II/anaphase II transition. MEI-S332 is necessary for Wdb localization, but, additionally, both Wdb and Wrd are required for MEI-S332 localization. Thus, rather than MEI-S332 being hierarchical to PP2A B′, these proteins reciprocally ensure centromere localization of the complex. We analyzed functional relationships between MEI-S332 and the two forms of PP2A by quantifying meiotic chromosome segregation defects in double or triple mutants. These studies revealed that both Wdb and Wrd contribute to MEI-S332’s ability to ensure accurate segregation of sister chromatids, but, as in centromere localization, they do not act solely downstream of MEI-S332.

Gene-centromere (G-C) mapping provides insight into vertebrate genome composition, structure and evolution. Although medaka fish are important experimental animals, no genome-wide G-C map of medaka has been constructed. In this study, we used 112 interspecific triploid hybrids and 152 DNA markers to make G-C maps of all 24 linkage groups (LGs). Under the assumption of 50% interference, 24 centromeres were localized onto all corresponding medaka LGs. Comparison with 21 centromere positions deduced from putative centromeric repeats revealed that 19 were localized inside the centromeric regions of the G-C maps, whereas two were not. Based on the centromere positions indicated in the G-C maps and those of centromeric repeats on each LG, we classified chromosomes as either biarmed or monoarmed; n = 24 = 10 metacentrics/submetacentrics + 14 subtelocentrics/acrocentrics, which is consistent with the results of previous karyological reports. This study helps to elucidate genome evolution mechanisms, and integrates physical and genetic maps with karyological information of medaka.

Chromosome segregation requires the centromere, the site on chromosomes where kinetochores assemble in mitosis to attach chromosomes to the mitotic spindle. Centromere identity is defined epigenetically by the presence of nucleosomes containing the histone H3 variant CENP-A. New CENP-A nucleosome assembly occurs at the centromere every cell cycle during G1, but how CENP-A nucleosome assembly is spatially and temporally restricted remains poorly understood. Centromere recruitment of factors required for CENP-A assembly is mediated in part by the three-protein Mis18 complex (Mis18α, Mis18β, M18BP1). Here we show that Xenopus M18BP1 localizes to centromeres during metaphase - prior to CENP-A assembly - by binding to CENP-C using a highly conserved SANTA domain. We find that Cdk phosphorylation of M18BP1 is necessary for M18BP1 to bind CENP-C and localize to centromeres in metaphase. Surprisingly, mutations which disrupt the metaphase M18BP1/CENP-C interaction cause defective nuclear localization of M18BP1 in interphase, resulting in defective CENP-A nucleosome assembly. We propose that M18BP1 may identify centromeric sites in metaphase for subsequent CENP-A nucleosome assembly in interphase.

CENP-A is a centromere-specific histone H3 variant that epigenetically determines centromere identity, but how CENP-A is deposited at the centromere remains obscure. We previously reported that CENP-A K124 ubiquitylation, mediated by the CUL4A-RBX1-COPS8 complex, is essential for CENP-A deposition at the centromere. However, a recent report stated that CENP-A K124R mutants show no defects in centromere localization and cell viability. In the present study, we found that EYFP tagging induces additional ubiquitylation of EYFP-CENP-A K124R, which allows the mutant protein to bind to HJURP. Using a previously developed conditional CENP-A knockout system and our CENP-A K124R knock-in mutant created by the CRISPR-Cas9 system, we show that the Flag-tagged or untagged CENP-A K124R mutant is lethal. This lethality is rescued by monoubiquitin fusion, indicating that CENP-A ubiquitylation is essential for viability.

Dynamic changes in CCAN organization during progression of the cell cycle are examined in chicken DT40 cells. CENP-C166-324 is sufficient for interphase centromere localization through association with CENP-L-N, and CENP-C643-864 is essential for mitotic centromere localization through binding to CENP-A nucleosomes., The kinetochore is a crucial structure for faithful chromosome segregation during mitosis and is formed in the centromeric region of each chromosome. The 16-subunit protein complex known as the constitutive centromere-associated network (CCAN) forms the foundation for kinetochore assembly on the centromeric chromatin. Although the CCAN can be divided into several subcomplexes, it remains unclear how CCAN proteins are organized to form the functional kinetochore. In particular, this organization may vary as the cell cycle progresses. To address this, we analyzed the relationship of centromeric protein (CENP)-C with the CENP-H complex during progression of the cell cycle. We find that the middle portion of chicken CENP-C (CENP-C166–324) is sufficient for centromere localization during interphase, potentially through association with the CENP-L-N complex. The C-terminus of CENP-C (CENP-C601–864) is essential for centromere localization during mitosis, through binding to CENP-A nucleosomes, independent of the CENP-H complex. On the basis of these results, we propose that CCAN organization changes dynamically during progression of the cell cycle.

Despite their essential role in the process of chromosome segregation in eukaryotes, kinetochore proteins are highly diverse across species, being lost, duplicated, created, or diversified during evolution. Based on comparative genomics, the duplication of the inner kinetochore proteins CenH3 and Cenp-C, which are interdependent in their roles of establishing centromere identity and function, can be said to be rare in animals. Surprisingly, the Drosophila CenH3 homolog Cid underwent four independent duplication events during evolution. Particularly interesting are the highly diverged Cid1 and Cid5 paralogs of the Drosophila subgenus, which are probably present in over one thousand species. Given that CenH3 and Cenp-C likely co-evolve as a functional unit, we investigated the molecular evolution of Cenp-C in species of Drosophila. We report yet another Cid duplication (leading to Cid6) within the Drosophila subgenus and show that not only Cid, but also Cenp-C is duplicated in the entire subgenus. The Cenp-C paralogs, which we named Cenp-C1 and Cenp-C2, are highly divergent. Both Cenp-C1 and Cenp-C2 retain key motifs involved in centromere localization and function, while some functional motifs are conserved in an alternate manner between the paralogs. Interestingly, both Cid5 and Cenp-C2 are male germline-biased and evolved adaptively. However, it is currently unclear if the paralogs subfunctionalized or if the new copies acquired a new function. Our findings point towards a specific inner kinetochore composition in a specific context (i.e., spermatogenesis), which could prove valuable for the understanding of how the extensive kinetochore diversity is related to essential cellular functions.

Mis18α, a component of Mis18 complex comprising of Mis18α, Mis18β, and M18BP1, is known to localize at the centromere from late telophase to early G1 phase and plays a priming role in CENP-A deposition. Although its role in CENP-A deposition is well established, the other function of Mis18α remains unknown. Here, we elucidate a new function of Mis18α that is critical for the proper progression of cell cycle independent of its role in CENP-A deposition. We find that Aurora B kinase phosphorylates Mis18α during mitosis not affecting neither centromere localization of Mis18 complex nor centromere loading of CENP-A. However, the replacement of endogenous Mis18α by phosphorylation-defective mutant causes mitotic defects including micronuclei formation, chromosome misalignment, and chromosomal bridges. Together, our data demonstrate that Aurora B kinase-mediated mitotic phosphorylation of Mis18α is a crucial event for faithful cell cycle progression through the enhanced recruitment of polo-like kinase 1 to the kinetochore.

Despite their essential role in the process of chromosome segregation in eukaryotes, kinetochore proteins are highly diverse across species, being lost, duplicated, created, or diversified during evolution. Based on comparative genomics, the duplication of the inner kinetochore proteins CenH3 and Cenp-C, which are interdependent in their roles of stablishing centromere identity and function, can be said to be rare in animals. Surprisingly, the Drosophila CenH3 homolog Cid underwent four independent duplication events during evolution. Particularly interesting are the highly diverged and subfunctionalized Cid1 and Cid5 paralogs of the Drosophila subgenus, which show that over one thousand Drosophila species may encode two Cid genes, making those with a single copy a minority. Given that CenH3 and Cenp-C likely co-evolve as a functional unit, we investigated the molecular evolution of Cenp-C in species of Drosophila. We report yet another Cid duplication within the Drosophila subgenus and show that not only Cid, but also Cenp-C is duplicated in the entire subgenus. The Cenp-C paralogs, which we named Cenp-C1 and Cenp-C2, are highly divergent. The retention of key motifs involved in centromere localization and function by both Cenp-C1 and Cenp-C2 makes neofunctionalization unlikely. In contrast, the alternate conservation of some functional motifs between the proteins is indicative of subfunctionalization. Interestingly, both Cid5 and Cenp-C2 are male germline-biased and evolved adaptively. Our findings point towards a specific inner kinetochore composition in a specific context (i.e., spermatogenesis), which could prove valuable for the understanding of how the extensive kinetochore diversity is related to essential cellular functions.

Most human cancers show chromosomal instability (CIN), but the precise mechanisms remain uncertain. Annexin A2 is frequently overexpressed in human cancers, and its relationship to tumorigenesis is poorly understood. We found that annexin A2 is overexpressed in the nuclei of CIN cells compared with cells with microsatellite instability (MIN). Ectopic annexin A2 expression in MIN cells results in a high level of aneuploidy and induces lagging chromosomes; suppression of annexin A2 in CIN cells reduces such CIN signatures with apoptosis of highly aneuploid cells. Ectopic expression of annexin A2 in MIN cells reduces the expression of centromere proteins. Conversely, annexin A2-knockdown in CIN cells increases the expression of centromere proteins. Moreover, the endogenous expression levels of centromere proteins in CIN cells were greatly reduced compared with MIN cell lines. The reduced expression of centromere proteins likely occurred due to aberrant centromere localization of coilin, a major component of the Cajal bodies. These results suggest that nuclear accumulation of annexin A2 has a crucial role in CIN by disrupting centromere function.

Essential Novelty The evolution of essential function for newly originated genes presents a conundrum, in that prior to the gene's origin either the essential function was absent or else performed by another gene or set of genes. In order to better understand how new genes acquire essential function, Ross et al. (p. 1211 ) investigated the origin of the Drosophila gene Umbrea. Umbrea became an essential protein in certain Drosophila species through the gain of localization at the centromere and a role in chromosome segregation.

Although we have long known the repetitive nature of the DNA in plant and animal centromeres of plant and animals and have identified hundreds of kinetochore proteins, closing the mechanistic gap between the centromere DNA and the kinetochore protein complex has remained a challenge. Much research

Aurora-A kinase functions mainly in centrosome maturation, separation and spindle formation. It has also been found to be amplified or overexpressed in a range of solid tumors, which is linked with tumor progression and poor prognosis. Importantly, Aurora-A inhibitors are being studied in a number of ongoing clinical trials. However, whether and how Aurora-A has a role in the regulation of the mitotic checkpoint is controversial. Additionally, the function of nuclear-accumulated Aurora-A in late G2 phase is not clear. Here we show that knockout, inhibition or blockade of the nuclear entry of Aurora-A severely decreased the centromere localization of Aurora-B and the phosphorylation of histone H3 threonine 3 (H3T3-ph) mediated by the kinase Haspin in late G2 phase. We further reveal that nuclear-accumulated Aurora-A phosphorylates Haspin at multiple sites at its N-terminus and that this promotes H3T3-ph and the rapid recruitment to the centromere of the chromosomal passenger complex. In addition, Aurora-A facilitates the association of Aurora-B with their common substrates: Haspin and Plk1. Notably, these functions of Aurora-A are mostly independent of Plk1. Thus we demonstrate that, in late G2 and prophase, Aurora-A phosphorylates Haspin to trigger the Haspin-H3T3-ph-Aurora-B positive feedback loop that supports the timely establishment of the chromosomal passenger complex and the mitotic checkpoint before spindle assembly.

Faithful chromosome segregation during mitosis requires that the kinetochores of all sister chromatids become stably connected to microtubules derived from opposite spindle poles. How stable chromosome bi-orientation is accomplished and coordinated with anaphase onset remains incompletely understood. Here we show that stable chromosome bi-orientation requires inner centromere localization of the non-enzymatic subunits of the chromosomal passenger complex (CPC) to maintain centromeric cohesion. Precise inner centromere localization of the CPC appears less relevant for Aurora B-dependent resolution of erroneous kinetochore–microtubule (KT–MT) attachments and for the stabilization of bi-oriented KT–MT attachments once sister chromatid cohesion is preserved via knock-down of WAPL. However, Aurora B inner centromere localization is essential for mitotic checkpoint silencing to allow spatial separation from its kinetochore substrate KNL1. Our data infer that the CPC is localized at the inner centromere to sustain centromere cohesion on bi-oriented chromosomes and to coordinate mitotic checkpoint silencing with chromosome bi-orientation., Precise chromosome segregation during mitosis requires coordination of stable chromosome bi-orientation with anaphase onset, however the underlying mechanism is not clear. Here the authors show that inner centromere localization of the chromosomal passenger complex maintains centromeric cohesion on bi-oriented chromosomes and allows mitotic checkpoint silencing.

The chromosomal passenger complex (CPC), consisting of the serine/threonine kinase Aurora B, the inner centromere protein INCENP, Survivin, and Borealin/DasraB, has essential functions at the centromere in ensuring correct chromosome alignment and segregation. Despite observations that small interfering RNA-mediated knockdown of any one member of the CPC abolishes localization of the other subunits, it remains unclear how the complex is targeted to the centromere. We have now identified a ternary subcomplex of the CPC comprising Survivin, Borealin, and the N-terminal 58 amino acids of INCENP in vitro and in vivo. This subcomplex was found to be essential and sufficient for targeting to the centromere. Notably, Aurora B kinase, the enzymatic core of the CPC, was not required for centromere localization of the subcomplex. We demonstrate that CPC targeting to the centromere does not depend on CENP-A and hMis12, two core components for kinetochore/centromere assembly, and provide evidence that the CPC may be directed to centromeric DNA directly via the Borealin subunit. Our findings thus establish a functional module within the CPC that assembles on the N terminus of INCENP and controls centromere recruitment.

Analyzing the features of the chromosomes can be very useful for diagnosis of many genetic disorders or prediction of possible abnormalities that may occur in the future generations. For this purpose, karyotype is often used, for which to be made, it is necessary to identify each one of the 24 chromosomes from the microscopic images. Definition and extraction of the morphological and band pattern-based features for each chromosome is the first step to identify them. Centromere location is an important morphological feature. In this paper, a novel algorithm for centromere localization is presented. The procedure is based on the calculation and analyzing the concavity degree of the chromosome's boundary pixels. In this method, the centerline of the chromosome is computed and the score of each pixel on the centerline is considered as the sum of the concavity degree of two pixels on the chromosome's boundary that are perpendicular to it. Finally, location of the centromere is estimated as one pixel on the centerline which is corresponding to the maximum score. When applied the proposed algorithm on 50 images, an average error of 2.25 pixels for centromere localization is achieved.

Centromeres form the site of chromosome attachment to microtubules during mitosis. Identity of these loci is maintained epigenetically by nucleosomes containing the histone H3 variant CENP-A. Propagation of CENP-A chromatin is uncoupled from DNA replication initiating only during mitotic exit. We now demonstrate that inhibition of Cdk1 and Cdk2 activities is sufficient to trigger CENP-A assembly throughout the cell cycle in a manner dependent on the canonical CENP-A assembly machinery. We further show that the key CENP-A assembly factor Mis18BP1(HsKNL2) is phosphorylated in a cell cycle-dependent manner that controls its centromere localization during mitotic exit. These results strongly support a model in which the CENP-A assembly machinery is poised for activation throughout the cell cycle but kept in an inactive noncentromeric state by Cdk activity during S, G2, and M phases. Alleviation of this inhibition in G1 phase ensures tight coupling between DNA replication, cell division, and subsequent centromere maturation. FCT doctoral fellowship: (SFRH/BD/33219/2007); FCT grant: (BIA-BCM/100557/2008); Fundação Calouste Gulbenkian; European Commission FP7 programme; EMBO installation grant.

The attachment of sister kinetochores to microtubules from opposite spindle poles is essential for faithful chromosome segregation. Kinetochore assembly requires centromere‐specific nucleosomes containing the histone H3 variant CenH3. However, the functional roles of the canonical histones (H2A, H2B, H3, and H4) in chromosome segregation remain elusive. Using a library of histone point mutants in Saccharomyces cerevisiae , 24 histone residues that conferred sensitivity to the microtubule‐depolymerizing drugs thiabendazole (TBZ) and benomyl were identified. Twenty‐three of these mutations were clustered at three spatially separated nucleosomal regions designated TBS‐I, ‐II, and ‐III ( T BZ/ b enomyl‐ s ensitive regions I–III). Elevation of mono‐polar attachment induced by prior nocodazole treatment was observed in H2A‐I112A (TBS‐I), H2A‐E57A (TBS‐II), and H4‐L97A (TBS‐III) cells. Severe impairment of the centromere localization of Sgo1, a key modulator of chromosome bi‐orientation, occurred in H2A‐I112A and H2A‐E57A cells. In addition, the pericentromeric localization of Htz1, the histone H2A variant, was impaired in H4‐L97A cells. These results suggest that the spatially separated nucleosomal regions, TBS‐I and ‐II, are necessary for Sgo1‐mediated chromosome bi‐orientation and that TBS‐III is required for Htz1 function.

The chromosomal passenger complex consisting of Borealin, INCENP, Survivin, and Aurora B follows a dynamic pattern of localization to perform its role as a regulator of chromosome alignment, aspects of the spindle assembly checkpoint, and cytokinesis. Post-translational modifications of chromosomal passenger proteins play an important role in regulating the localization and function of the complex. Borealin displays a slower electrophoretic mobility during mitosis as a result of phosphorylation. Here we show that phosphorylation at S219 is responsible for this mobility shift. An S219A mutant of Borealin that cannot be phosphorylated at this site displays a defect in centromere localization that is evident in cells arrested in mitosis with nocodazole. Further, the S219A form of Borealin is unable to efficiently rescue mitotic defects that occur upon knock-down of the endogenous protein. These defects are correlated with a reduction in the intensity of Mad2 staining at kinetochores in cells expressing the S219A form of Borealin. These results highlight an important role for phosphorylation of Borealin at S219 in the proper progression through mitosis.

Location, Location, Location Cell division is orchestrated by a complex signaling pathway that ensures the correct segregation of newly replicated chromosomes to the two daughter cells. The pathway is controlled in part by restricting the activity of critical regulators to specific subcellular locations. For example, the chromosomal passenger complex (CPC) is recruited to chromosomes during mitosis where it oversees kinetochore activity and cytokinesis (see Perspective by Musacchio ). Wang et al. (p. 231 , published online 12 August), Kelly et al. (p. 235 , published online 12 August), and Yamagishi et al. (p. 239 ) now show that the phosphorylation of the chromatin protein, histone H3, acts to bring the CPC to chromosomes, thereby activating its aurora B kinase subunit. The Survivin subunit of CPC binds specifically to phosphorylated H3, with the phosphorylation at centromeres being carried out by the mitosis-specific kinase, haspin. Furthermore, Bub1 phosphorylation of histone H2A recruits shugoshin, a centromeric CPC adapter. Thus, these two histone marks in combination define the inner centromere.

Rapid evolution is a hallmark of centromeric DNA in eukaryotic genomes. Yet, the centromere itself has a conserved functional role that is mediated by the kinetochore protein complex. To broaden our understanding about both the DNA and proteins that interact at the functional centromere, we sought to gain a detailed view of the evolutionary events that have shaped the primate kinetochore. Specifically, we performed comparative mapping and sequencing of the genomic regions encompassing the genes encoding three foundation kinetochore proteins: Centromere Proteins A, B, and C (CENP-A, CENP-B, and CENP-C). A histone H3 variant, CENP-A provides the foundation of the centromere-specific nucleosome. Comparative sequence analyses of the CENP-A gene in 14 primate species revealed encoded amino-acid residues within both the histone-fold domain and the N-terminal tail that are under strong positive selection. Similar comparative analyses of CENP-C, another foundation protein essential for centromere function, identified amino-acid residues throughout the protein under positive selection in the primate lineage, including several in the centromere localization and DNA-binding regions. Perhaps surprisingly, the gene encoding CENP-B, a kinetochore protein that binds specifically to alpha-satellite DNA, was not found to be associated with signatures of positive selection. These findings point to important and distinct evolutionary forces operating on the DNA and proteins of the primate centromere.

SummaryThe centromere is responsible for accurate chromosome segregation. Mammalian centromeres are specified epigenetically, with all active centromeres containing centromere-specific chromatin in which CENP-A replaces histone H3 within the nucleosome. The proteins responsible for assembly of human CENP-A into centromeric nucleosomes during the G1 phase of the cell cycle are shown here to be distinct from the chromatin assembly factors previously shown to load other histone H3 variants. Here we demonstrate that prenucleosomal CENP-A is complexed with histone H4, nucleophosmin 1, and HJURP. Recruitment of new CENP-A into nucleosomes at replicated centromeres is dependent on HJURP. Recognition by HJURP is mediated through the centromere targeting domain (CATD) of CENP-A, a region that we demonstrated previously to induce a unique conformational rigidity to both the subnucleosomal CENP-A heterotetramer and the corresponding assembled nucleosome. We propose HJURP to be a cell-cycle-regulated CENP-A-specific histone chaperone required for centromeric chromatin assembly.

The mechanisms ensuring specific incorporation of CENP-A at centromeres are poorly understood. Mis16 and Mis18 are required for CENP-A localization at centromeres and form a complex that is conserved from fission yeast to human. Fission yeast sim1 mutants that alleviate kinetochore domain silencing are defective in Scm3(Sp), the ortholog of budding yeast Scm3(Sc). Scm3(Sp) depends on Mis16/18 for its centromere localization and like them is recruited to centromeres in late anaphase. Importantly, Scm3(Sp) coaffinity purifies with CENP-A(Cnp1) and associates with CENP-A(Cnp1) in vitro, yet localizes independently of intact CENP-A(Cnp1) chromatin and is differentially released from chromatin. While Scm3(Sc) has been proposed to form a unique hexameric nucleosome with CENP-A(Cse4) and histone H4 at budding yeast point centromeres, we favor a model in which Scm3(Sp) acts as a CENP-A(Cnp1) receptor/assembly factor, cooperating with Mis16 and Mis18 to receive CENP-A(Cnp1) from the Sim3 escort and mediate assembly of CENP-A(Cnp1) into subkinetochore chromatin.

Centromeres are the structural and functional foundation for kinetochore formation, spindle attachment, and chromosome segregation. In this study, we isolated factors required for centromere propagation using genome-wide RNA interference screening for defects in centromere protein A (CENP-A; centromere identifier [CID]) localization in Drosophila melanogaster. We identified the proteins CAL1 and CENP-C as essential factors for CID assembly at the centromere. CID, CAL1, and CENP-C coimmunoprecipitate and are mutually dependent for centromere localization and function. We also identified the mitotic cyclin A (CYCA) and the anaphase-promoting complex (APC) inhibitor RCA1/Emi1 as regulators of centromere propagation. We show that CYCA is centromere localized and that CYCA and RCA1/Emi1 couple centromere assembly to the cell cycle through regulation of the fizzy-related/CDH1 subunit of the APC. Our findings identify essential components of the epigenetic machinery that ensures proper specification and propagation of the centromere and suggest a mechanism for coordinating centromere inheritance with cell division.

A universal mark of centromeric chromatin is its packaging by a variant of histone H3 known as centromeric H3 (CenH3). The mechanism by which CenH3s are incorporated specifically into centromere DNA or the specialized function they serve there is not known. In a genetic approach to identify factors involved in CenH3 deposition, we screened for dosage suppressors of a temperature-sensitive cse4 allele in Saccharomyces cerevisiae (Cse4 is the S. cerevisiae CenH3). Independent screens yielded ORF YDL139C, which we named SCM3 . Dosage suppression by SCM3 was specific for alleles affecting the histone fold domain of Cse4. Copurification and two-hybrid studies showed that Scm3 and Cse4 interact in vivo , and chromatin immunoprecipitation revealed that Scm3, like Cse4, is found associated with centromere DNA. Scm3 contains two essential protein domains, a Leu-rich nuclear export signal and a heptad repeat domain that is widely conserved in fungi. A conditional scm3 allele was generated to allow us to deplete Scm3. Upon Scm3 depletion, cells undergo a Mad2-dependent G 2 /M arrest, and centromere localization of Cse4 is perturbed. We suggest that S. cerevisiae Scm3 defines a previously undescribed family of fungal kinetochore proteins important for CenH3 localization.

Centromeres direct chromosomal inheritance by nucleating assembly of the kinetochore, a large multiprotein complex required for microtubule attachment during mitosis. Centromere identity in humans is epigenetically determined, with no DNA sequence either necessary or sufficient. A prime candidate for the epigenetic mark is assembly into centromeric chromatin of centromere protein A (CENP-A), a histone H3 variant found only at functional centromeres. A new covalent fluorescent pulse-chase labeling approach using SNAP tagging has now been developed and is used to demonstrate that CENP-A bound to a mature centromere is quantitatively and equally partitioned to sister centromeres generated during S phase, thereby remaining stably associated through multiple cell divisions. Loading of nascent CENP-A on the megabase domains of replicated centromere DNA is shown to require passage through mitosis but not microtubule attachment. Very surprisingly, assembly and stabilization of new CENP-A–containing nucleosomes is restricted exclusively to the subsequent G1 phase, demonstrating direct coupling between progression through mitosis and assembly/maturation of the next generation of centromeres.

The centromere is the chromosomal site that joins to microtubules during mitosis for proper segregation. Determining the location of a centromere-specific histone H3 called CENP-A at the centromere is vital for understanding centromere structure and function. Here, we report the identification of three human proteins essential for centromere/kinetochore structure and function, hMis18alpha, hMis18beta, and M18BP1, the complex of which is accumulated specifically at the telophase-G1 centromere. We provide evidence that such centromeric localization of hMis18 is essential for the subsequent recruitment of de novo-synthesized CENP-A. If any of the three is knocked down by RNAi, centromere recruitment of newly synthesized CENP-A is rapidly abolished, followed by defects such as misaligned chromosomes, anaphase missegregation, and interphase micronuclei. Tricostatin A, an inhibitor to histone deacetylase, suppresses the loss of CENP-A recruitment to centromeres in hMis18alpha RNAi cells. Telophase centromere chromatin may be primed or licensed by the hMis18 complex and RbAp46/48 to recruit CENP-A through regulating the acetylation status in the centromere.

Nucleosomes containing the centromere-specific histone H3 variant centromere protein A (CENP-A) create the chromatin foundation for kinetochore assembly. To understand the mechanisms that selectively target CENP-A to centromeres, we took a functional genomics approach in the nematode Caenorhabditis elegans, in which failure to load CENP-A results in a signature kinetochore-null (KNL) phenotype. We identified a single protein, KNL-2, that is specifically required for CENP-A incorporation into chromatin. KNL-2 and CENP-A localize to centromeres throughout the cell cycle in an interdependent manner and coordinately direct chromosome condensation, kinetochore assembly, and chromosome segregation. The isolation of KNL-2–associated chromatin coenriched CENP-A, indicating their close proximity on DNA. KNL-2 defines a new conserved family of Myb DNA-binding domain–containing proteins. The human homologue of KNL-2 is also specifically required for CENP-A loading and kinetochore assembly but is only transiently present at centromeres after mitotic exit. These results implicate a new protein class in the assembly of centromeric chromatin and suggest that holocentric and monocentric chromosomes share a common mechanism for CENP-A loading.

Accurate detection of the human metaphase chromosome centromere is an critical element of cytogenetic diagnostic techniques, including chromosome enumeration, karyotyping and radiation biodosimetry. Existing image processing methods can perform poorly in the presence of irregular boundaries, shape variations and premature sister chromatid separation, which can adversely affect centromere localization. We present a centromere detection algorithm that uses a novel profile thickness measurement technique on irregular chromosome structures defined by contour partitioning. Our algorithm generates a set of centromere candidates which are then evaluated based on a set of features derived from images of chromosomes. Our method also partitions the chromosome contour to isolate its telomere regions and then detects and corrects for sister chromatid separation. When tested with a chromosome database consisting of 1400 chromosomes collected from 40 metaphase cell images, the candidate based centromere detection algorithm was able to accurately localize 1220 centromere locations yielding a detection accuracy of 87%. We also introduce a Candidate Based Centromere Confidence (CBCC) metric which indicates an approximate confidence value of a given centromere detection and can be readily extended into other candidate related detection problems.

Menopause results from loss of ovarian function and marks the end of a woman's reproductive life. Alleles of the human SYCP2L locus are associated with age at natural menopause (ANM). SYCP2L is a paralogue of the synaptonemal complex protein SYCP2 and is expressed exclusively in oocytes. Here we report that SYCP2L localizes to centromeres of dictyate stage oocytes, which represent the limited pool of primordial oocytes that are formed perinatally and remain arrested till ovulation. Centromere localization of SYCP2L requires its C-terminal portion, which is missing in truncated variants resulting from low-frequency nonsense mutations identified in humans. Female mice lacking SYCP2L undergo a significantly higher progressive loss of oocytes with age compared with wild-type females and are less fertile. Specifically, the pool of primordial oocytes becomes more rapidly depleted in SYCP2L-deficient than in wild-type females, such that with aging, fewer oocytes undergo maturation in developing follicles. We find that a human SYCP2L intronic single nucleotide polymorphism (SNP) rs2153157, which is associated with ANM, changes the splicing efficiency of U12-type minor introns and may therefore regulate the steady-state amount of SYCP2L transcript. Furthermore, the more efficiently spliced allele of this intronic SNP in SYCP2L is associated with increased ANM. Our results suggest that SYCP2L promotes the survival of primordial oocytes and thus provide functional evidence for its association with ANM in humans.

Using immunostaining with antibodies against cohesin subunits, we show here that cohesin-enriched structures analogous to the so-called centromere protein bodies (PB) are the characteristic of galliform lampbrush chromosomes. Their centromeric location was verified by FISH with certain DNA probes. PB-like structures were used as markers for centromere localization in chicken lampbrush chromosomes. The gap predicted to be centromeric in current chicken chromosome 3 sequence assembly was found to correspond to the non-centromeric cluster of CNM repeat on the q-arm of chromosome 3; the centromere is proposed to be placed at another position. The majority of chicken microchromosomes were found to be acrocentric, in contrast to Japanese quail microchromosomes which are biarmed. Centromere cohesin-enriched structures on chicken and quail lampbrush microchromosomes co-localize with pericentromeric CNM and BglII- repeats respectively. FISH to the nascent transcripts on chicken lampbrush chromosomes revealed numerous non-centromeric CNM clusters in addition to pericentromeric arrays. Complementary CNM transcripts from both C- and G-rich DNA strands were revealed during the lampbrush stage.

The centromere is a chromatin structure essential for correct segregation of sister chromatids, and defects in this region often lead to aneuploidy and cancer. We have previously reported purification of the interphase centromere complex (ICEN) from HeLa cells, and have demonstrated the presence of 40 proteins (ICEN1-40), along with CENP-A, -B, -C, -H and hMis6, by proteomic analysis. Here we report analysis of seven ICEN components with unknown function. Centromere localization of EGFP-tagged ICEN22, 24, 32, 33, 36, 37 and 39 was observed in transformant cells. Depletion of each of these proteins by short RNA interference produced abnormal metaphase cells carrying misaligned chromosomes and also produced cells containing aneuploid chromosomes, implying that these ICEN proteins take part in kinetochore functions. Interestingly, in the ICEN22, 32, 33, 37 or 39 siRNA-transfected cells, CENP-H and hMis6 signals disappeared from all the centromeres in abnormal mitotic cells containing misaligned chromosomes. These results suggest that the seven components of the ICEN complex are predominantly localized at the centromeres and are required for kinetochore function perhaps through or not through loading of CENP-H and hMis6 onto the centromere.

In vertebrates, centromeres lack defined sequences and are thought to be propagated by epigenetic mechanisms involving the incorporation of specialized nucleosomes containing the histone H3 variant centromere protein (CENP)-A. However, the precise mechanisms that target CENP-A to centromeres remain poorly understood. Here, we isolated a multi-subunit complex, which includes the established inner kinetochore components CENP-H and CENP-I, and nine other proteins, from both human and chicken cells. Our analysis of these proteins demonstrates that the CENP-H-I complex can be divided into three functional sub-complexes, each of which is required for faithful chromosome segregation. Interestingly, newly expressed CENP-A is not efficiently incorporated into centromeres in knockout mutants of a subclass of CENP-H-I complex proteins, indicating that the CENP-H-I complex may function, in part, as a marker directing CENP-A deposition to centromeres.

Summary CENP-A is a centromere-specific histone H3 variant that epigenetically determines centromere identity to ensure kinetochore assembly and proper chromosome segregation, but the precise mechanism of its specific localization within centromeric heterochromatin remains obscure. We have discovered that CUL4A-RBX1-COPS8 E3 ligase activity is required for CENP-A ubiquitylation on lysine 124 (K124) and CENP-A centromere localization. A mutation of CENP-A, K124R, reduces interaction with HJURP (a CENP-A-specific histone chaperone) and abrogates localization of CENP-A to the centromere. Addition of monoubiquitin is sufficient to restore CENP-A K124R to centromeres and the interaction with HJURP, indicating that "signaling" ubiquitylation is required for CENP-A loading at centromeres. The CUL4A-RBX1 complex is required for loading newly synthesized CENP-A and maintaining preassembled CENP-A at centromeres. Thus, CENP-A K124R ubiquitylation, mediated by the CUL4A-RBX1-COPS8 complex, is essential for CENP-A deposition at the centromere.

Protein sumoylation by small ubiquitin-like modifier (SUMO) proteins is an important post-translational regulatory modification. A role in the control of chromosome dynamics was first suggested when SUMO was identified as high-copy suppressor of the centromere protein CENP-C mutants. CENP-C itself contains a consensus sumoylation sequence motif that partially overlaps with its DNA binding and centromere localization domain. To ascertain whether CENP-C can be sumoylated, tandem mass spectrometry (MS) based strategy was developed for high sensitivity identification and sequencing of sumoylated isopeptides present among in-gel-digested tryptic peptides of SDS-PAGE fractionated target proteins. Without a predisposition to searching for the expected isopeptides based on calculated molecular mass and relying instead on the characteristic MS/MS fragmentation pattern to identify sumolylation, we demonstrate that several other lysine residues located not within the perfect consensus sumoylation motif psiKXE/D, where psi represents a large hydrophobic amino acid, and X represents any amino acid, can be sumolylated with a reconstituted in vitro system containing only the SUMO proteins, E1-activating enzyme and E2-conjugating enzyme (Ubc9). In all cases, target sites that can be sumoylated by SUMO-2 were shown to be equally susceptible to SUMO-1 attachments which include specific sites on SUMO-2 itself, Ubc9, and the recombinant CENP-C fragments. Two non-consensus sites on one of the CENP-C fragments were found to be sumoylated in addition to the predicted site on the other fragment. The developed methodologies should facilitate future studies in delineating the dynamics and substrate specificities of SUMO-1/2/3 modifications and the respective roles of E3 ligases in the process.

The mitotic and meiotic chromosomes of Mus macedonicus from Israel are described. The autosomal complement of M. macedonicus (2n = NF = 40) does not differ from M. musculus and allied species. Useful cytogenetic markers for M. macedonicus include the small Y-chromosome, the enlarged centromeric block of heterochromatin (consisting of several sub-blocks) in the X-chromosomes, the relatively high number of chromosomes (6 pairs) bearing nucleolar organizing regions (NORs), and the centromere localization of NORs.

During folliculogenesis, the oocyte undergoes changes in chromatin organization that are functional to the acquisition of the developmental competence. In the antral compartment of the mouse ovary, two types of oocytes have been described on the basis of the presence or absence of a ring of Hoechst-bright fluorescence around the nucleolus. It is not known whether these changes of chromatin organization entail changes in the localization of chromosomal domains. Making use of CREST serum against centromeric proteins and confocal microscopy, here we describe the three-dimensional localization of the centromeres within the nuclear volume of mouse antral oocytes. Our data reveal that centromeres change localization during oocyte maturation showing a dramatic coalescence around the nucleolus surface at the stages preceding ovulation. These results further our knowledge on the nuclear organization of the mouse oocyte and represent a first and necessary contribution for future studies on the role that nuclear architecture may exert as an epigenetic regulator of genome functioning.

Kinetochores are the chromosomal sites for spindle interaction and play a vital role for chromosome segregation. The composition of kinetochore proteins and their cellular roles are, however, poorly understood in higher eukaryotes. We identified a novel kinetochore protein family conserved from yeast to human that is essential for equal chromosome segregation. The human homologue hMis12 of yeast spMis12/scMtw1 retains conserved sequence features and locates at the kinetochore region indistinguishable from CENP-A, a centromeric histone variant. RNA interference (RNAi) analysis of HeLa cells shows that the reduced hMis12 results in misaligned metaphase chromosomes, lagging anaphase chromosomes, and interphase micronuclei without mitotic delay, while CENP-A is located at kinetochores. Further, the metaphase spindle length is abnormally extended. Spindle checkpoint protein hMad2 temporally localizes at kinetochores at early mitotic stages after RNAi. The RNAi deficiency of CENP-A leads to a similar mitotic phenotype, but the kinetochore signals of other kinetochore proteins, hMis6 and CENP-C, are greatly diminished. RNAi for hMis6, like that of a kinetochore kinesin CENP-E, induces mitotic arrest. Kinetochore localization of hMis12 is unaffected by CENP-A RNAi, demonstrating an independent pathway of CENP-A in human kinetochores.

Xenopus kinesin catastrophe modulator-1 (XKCM1) is a Kin I kinesin family member that uses the energy of ATP hydrolysis to depolymerize microtubules [1–3]. We demonstrated previously that XKCM1 is essential for mitotic-spindle assembly in vitro and acts by regulating microtubule dynamics as a pure protein, in extracts and in cells [2, 4, 5]. A portion of the XKCM1 pool is specifically localized to centromeres during mitosis and may be important in chromosome movement. To selectively analyze the function of centromere-bound XKCM1, we generated glutathione-S-transferase (GST) fusion proteins containing the N-terminal globular domain (GST-NT), the centrally located catalytic domain (GST-CD), and the C-terminal α-helical tail (GST-CT) of XKCM1. The GST-NT protein targeted to centromeres during spindle assembly, suggesting that the N-terminal domain of XKCM1 is sufficient for centromere localization. Addition of GST-NT prior to or after spindle assembly replaced endogenous XKCM1, indicating that centromere targeting is a dynamic process. Loss of endogenous XKCM1 from centromeres caused a misalignment of chromosomes on the metaphase plate without affecting global spindle structure. These results suggest that centromere bound XKCM1 has an important role in chromosome positioning on the spindle.

Background The pericentromeric heterochromatin is an important element for the regulation of gene silencing. Its spatial distribution during interphase appears to be cell-type specific. This study analyzes three-dimensional (3D) centromere distribution patterns during cellular differentiation along the neutrophil pathway. Methods Differentiation of the promyelocytic leukemia cell line NB4 was induced by retinoic acid. Centromeres in interphase nuclei were visualized by immunofluorescence staining of centromere-associated proteins with CREST serum. 3D images of nuclei were obtained by confocal microscopy. Automated methods for the segmentation of point-like objects in 3D images were implemented to detect the position of centromeres. Features of centromere localization patterns were determined by constructing the minimal spanning tree of the centromere distribution. Results In differentiated NB4 cells, the number of centromere conglomerates (chromocenters) was decreased and the distance between chromocenters was increased as compared with untreated controls. The nuclear volume did not differ between the two groups. Conclusions The measured rearrangement of centromeres indicates a progressive clustering of heterochromatin and a global remodeling of interphase chromosome territories during differentiation of NB4 cells. The developed methods for the analysis of 3D centromere distribution patterns provide the opportunity for a fast and objective analysis of heterochromatin remodeling. Cytometry 47:217–225, 2002. © 2002 Wiley-Liss, Inc.

Summary Centromeres are specified and maintained by sequence-independent epigenetic mechanisms through the incorporation of CENP-A into centromeres. Given that CENP-A incorporation requires the Mis18 complex to be in the centromere region, it is necessary to precisely understand how the Mis18 complex localizes to the centromere region. Here, we showed that centromere localization of the Mis18 complex depends on CENP-A, but not CENP-C or CENP-T, in chicken DT40 cells. Furthermore, we demonstrated that M18BP1/KNL2, a member of the Mis18 complex, contained the CENP-C-like motif in chicken and other vertebrates, which is essential for centromere localization and M18BP1/KNL2 function in DT40 cells. We also showed that in vitro reconstituted CENP-A nucleosome, but not H3 nucleosome, bound to the CENP-C-like motif containing M18BP1/KNL2. Based on these results, we conclude that M18BP1/KNL2 is essential for centromere formation through direct binding to CENP-A nucleosome in non-mammalian vertebrates. This explains how new CENP-A recognizes the centromere position.

Accurate mitosis requires the chromosomal passenger protein complex (CPC) containing Aurora B kinase, borealin, INCENP, and survivin, which orchestrates chromosome dynamics. However, the chromatin factors that specify the CPC to the centromere remain elusive. Here we show that borealin interacts directly with heterochromatin protein 1α (HP1α) and that this interaction is mediated by an evolutionarily conserved PXVXL motif in the C-terminal borealin with the chromo shadow domain of HP1α. This borealin-HP1α interaction recruits the CPC to the centromere and governs an activation of Aurora B kinase judged by phosphorylation of Ser-7 in CENP-A, a substrate of Aurora B. Consistently, modulation of the motif PXVXL leads to defects in CPC centromere targeting and aberrant Aurora B activity. On the other hand, the localization of the CPC in the midzone is independent of the borealin-HP1α interaction, demonstrating the spatial requirement of HP1α in CPC localization to the centromere. These findings reveal a previously unrecognized but direct link between HP1α and CPC localization in the centromere and illustrate the critical role of borealin-HP1α interaction in orchestrating an accurate cell division.

The Shugoshin (Sgo) protein family helps to ensure proper chromosome segregation by protecting cohesion at the centromere by preventing cleavage of the cohesin complex. Some Sgo proteins also influence other aspects of kinetochore-microtubule attachments. Although many Sgo members require Aurora B kinase to localize to the centromere, factors controlling delocalization are poorly understood and diverse. Moreover, it is not clear how Sgo function is inactivated and whether this is distinct from delocalization. We investigated these questions in Drosophila melanogaster, an organism with superb chromosome cytology to monitor Sgo localization and quantitative assays to test its function in sister-chromatid segregation in meiosis. Previous research showed that in mitosis in cell culture, phosphorylation of the Drosophila Sgo, MEI-S332, by Aurora B promotes centromere localization, whereas Polo phosphorylation promotes delocalization. These studies also suggested that MEI-S332 can be inactivated independently of delocalization, a conclusion supported here by localization and function studies in meiosis. Phosphoresistant and phosphomimetic mutants for the Aurora B and Polo phosphorylation sites were examined for effects on MEI-S332 localization and chromosome segregation in meiosis. Strikingly, MEI-S332 with a phosphomimetic mutation in the Aurora B phosphorylation site prematurely dissociates from the centromeres in meiosis I. Despite the absence of MEI-S332 on meiosis II centromeres in male meiosis, sister chromatids segregate normally, demonstrating that detectable levels of this Sgo are not essential for chromosome congression, kinetochore biorientation, or spindle assembly., David H. Koch Institute for Integrative Cancer Research at MIT (Fellowship), National Science Foundation (U.S.) (Grant MCB-0646593)

application/pdf, Article, Cell Structure and Functions. 26, p.705-718

Centromeres of most organisms are embedded within constitutive heterochromatin, the condensed regions of chromosomes that account for a large fraction of complex genomes. The functional significance of this centromere–heterochromatin relationship, if any, is unknown. One possibility is that heterochromatin provides a suitable environment for assembly of centromere components, such as special centromeric nucleosomes that contain distinctive histone H3-like proteins. We describe a Drosophila H3-like protein, Cid (for centromere identifier) that localizes exclusively to fly centromeres. When the cid upstream region drives expression of H3 and H2B histone–green fluorescent protein fusion genes in Drosophila cells, euchromatin-specific deposition results. Remarkably, when the cid upstream region drives expression of yeast, worm, and human centromeric histone–green fluorescent protein fusion proteins, localization is preferentially within Drosophila pericentric heterochromatin. Heterochromatin-specific localization also was seen for yeast and worm centromeric proteins constitutively expressed in human cells. Preferential localization to heterochromatin in heterologous systems is unexpected if centromere-specific or site-specific factors determine H3-like protein localization to centromeres. Rather, the heterochromatic state itself may help localize centromeric components.

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.