Engineered kinetochores reveal distinct functions of the CCAN in recruiting CENP-A to the centromere and acting as a structural core to directly recruit kinetochore proteins.
CENP-A acts as an important epigenetic marker for kinetochore specification. However, the mechanisms by which CENP-A is incorporated into centromeres and the structural basis for kinetochore formation downstream of CENP-A remain unclear. Here, we used a unique chromosome-engineering system in which kinetochore proteins are targeted to a noncentromeric site after the endogenous centromere is conditionally removed. Using this system, we created two distinct types of engineered kinetochores, both of which were stably maintained in chicken DT40 cells. Ectopic targeting of full-length HJURP, CENP-C, CENP-I, or the CENP-C C terminus generated engineered kinetochores containing major kinetochore components, including CENP-A. In contrast, ectopic targeting of the CENP-T or CENP-C N terminus generated functional kinetochores that recruit the microtubule-binding Ndc80 complex and chromosome passenger complex (CPC), but lack CENP-A and most constitutive centromere-associated network (CCAN) proteins. Based on the analysis of these different engineered kinetochores, we conclude that the CCAN has two distinct roles: recruiting CENP-A to establish the kinetochore and serving as a structural core to directly recruit kinetochore proteins.
The multi-protein kinetochore complex must assemble at a specific site on each chromosome to achieve accurate chromosome segregation. Defining the nature of the DNA-protein interactions that specify the position of the kinetochore and provide a scaffold for kinetochore formation remain key goals. Here, we demonstrate that the centromeric histone-fold containing CENP-T-W and CENP-S-X complexes co-assemble to form a stable CENP-T-W-S-X heterotetramer. High-resolution structural analysis of the individual complexes and the heterotetramer reveals similarity to other histone fold-containing complexes including canonical histones within a nucleosome. The CENP-T-W-S-X heterotetramer binds to and supercoils DNA. Mutants designed to compromise heterotetramerization or the DNA-protein contacts around the heterotetramer strongly reduce the DNA binding and supercoiling activities in vitro and compromise kinetochore assembly in vivo. These data suggest that the CENP-T-W-S-X complex forms a unique nucleosome-like structure to generate contacts with DNA, extending the “histone code” beyond canonical nucleosome proteins.
Kinetochore; CENP-T-W; CENP-S-X; The X-ray Structure; DNA binding
Accurate chromosome segregation requires assembly of the multi-protein kinetochore complex at centromeres. Although prior work identified the centromeric histone H3-variant CENP-A as the important upstream factor necessary for centromere specification, in human cells CENP-A is not sufficient for kinetochore assembly. Here, we demonstrate that two constitutive DNA-binding kinetochore components, CENP-C and CENP-T, function to direct kinetochore formation. Replacing the DNA-binding regions of CENP-C and CENP-T with alternate chromosome-targeting domains recruits these proteins to ectopic loci, resulting in CENP-A-independent kinetochore assembly. These ectopic kinetochore-like foci are functional based on the stoichiometric assembly of multiple kinetochore components including the microtubule-binding KMN network, the presence of microtubule attachments, the microtubule-sensitive recruitment of the spindle checkpoint protein Mad2, and the segregation behavior of foci-containing chromosomes. We additionally find that CENP-T phosphorylation regulates the mitotic assembly of both endogenous and ectopic kinetochores. Thus, CENP-C and CENP-T form a critical regulated platform for vertebrate kinetochore assembly.
Mitosis; Centromere; Kinetochore; Chromosome Segregation
The N and C termini of CENP-T undergo tension-dependent separation, suggesting that CENP-T elongation is responsible for changes in the shape of the inner kinetochore.
The kinetochore forms a dynamic interface with microtubules from the mitotic spindle. Live-cell light microscopy–based observations on the dynamic structural changes within the kinetochore suggest that molecular rearrangements within the kinetochore occur upon microtubule interaction. However, the source of these rearrangements is still unclear. In this paper, we analyze vertebrate kinetochore ultrastructure by immunoelectron microscopy (EM) in the presence or absence of tension from spindle microtubules. We found that the inner kinetochore region defined by CENP-A, CENP-C, CENP-R, and the C-terminal domain of CENP-T is deformed in the presence of tension, whereas the outer kinetochore region defined by Ndc80, Mis12, and CENP-E is not stretched even under tension. Importantly, based on EM, fluorescence microscopy, and in vitro analyses, we demonstrated that the N and C termini of CENP-T undergo a tension-dependent separation, suggesting that CENP-T elongation is at least partly responsible for changes in the shape of the inner kinetochore.
Accurate chromosome segregation requires carefully regulated interactions between kinetochores and microtubules, but how plasticity is achieved to correct diverse attachment defects remains unclear. Here, we demonstrate that Aurora B kinase phosphorylates three spatially distinct targets within the conserved outer kinetochore KNL1/Mis12 complex/Ndc80 complex (KMN) network, the key player in kinetochore-microtubule attachments. The combinatorial phosphorylation of the KMN network generates graded levels of microtubule binding activity, with full phosphorylation severely compromising microtubule binding. Altering the phosphorylation state of each protein causes corresponding chromosome segregation defects. Importantly, the spatial distribution of these targets along the kinetochore axis leads to their differential phosphorylation in response to changes in tension and attachment state. In total, rather than generating exclusively binary changes in microtubule binding, our results suggest a mechanism for the tension-dependent fine tuning of kinetochore-microtubule interactions.
Mitosis; Kinetochore; Microtubule; Phosphorylation; Chromosome Segregation
Localization of the spindle and kinetochore proteins Astrin, SKAP, and LC8 is antagonized by Aurora B so that they target exclusively to bioriented kinetochores.
During mitosis, kinetochores play multiple roles to generate interactions with microtubules, and direct chromosome congression, biorientation, error correction, and anaphase segregation. However, it is unclear what changes at the kinetochore facilitate these distinct activities. Here, we describe a complex of the spindle- and kinetochore-associated protein Astrin, the small kinetochore-associated protein (SKAP), and the dynein light chain LC8. Although most dynein-associated proteins localize to unaligned kinetochores in an Aurora B–dependent manner, Astrin, SKAP, and LC8 localization is antagonized by Aurora B such that they target exclusively to bioriented kinetochores. Astrin–SKAP-depleted cells fail to maintain proper chromosome alignment, resulting in a spindle assembly checkpoint–dependent mitotic delay. Consistent with a role in stabilizing bioriented attachments, Astrin and SKAP bind directly to microtubules and are required for CLASP localization to kinetochores. In total, our results suggest that tension-dependent Aurora B phosphorylation can act to control outer kinetochore composition to provide distinct activities to prometaphase and metaphase kinetochores.
The stoichiometry of kinetochore components is determined, suggesting conservation between multiple microtubule-binding vertebrate and single microtubule-binding yeast kinetochores.
To define the molecular architecture of the kinetochore in vertebrate cells, we measured the copy number of eight kinetochore proteins that link kinetochore microtubules (MTs [kMTs]) to centromeric DNA. We used a fluorescence ratio method and chicken DT40 cell lines in which endogenous loci encoding the analyzed proteins were deleted and complemented using integrated green fluorescent protein fusion transgenes. For a mean of 4.3 kMTs at metaphase, the protein copy number per kMT is between seven and nine for members of the MT-binding KNL-1/Mis12 complex/Ndc80 complex network. It was between six and nine for four members of the constitutive centromere-associated network: centromere protein C (CENP-C), CENP-H, CENP-I, and CENP-T. The similarity in copy number per kMT for all of these proteins suggests that each MT end is linked to DNA by six to nine fibrous unit attachment modules in vertebrate cells, a conclusion that indicates architectural conservation between multiple MT-binding vertebrate and single MT-binding budding yeast kinetochores.
KNL targets PP1 to kinetochores, where it antagonizes Aurora B activity.
Regulated interactions between kinetochores and spindle microtubules are essential to maintain genomic stability during chromosome segregation. The Aurora B kinase phosphorylates kinetochore substrates to destabilize kinetochore–microtubule interactions and eliminate incorrect attachments. These substrates must be dephosphorylated to stabilize correct attachments, but how opposing kinase and phosphatase activities are coordinated at the kinetochore is unknown. Here, we demonstrate that a conserved motif in the kinetochore protein KNL1 directly interacts with and targets protein phosphatase 1 (PP1) to the outer kinetochore. PP1 recruitment by KNL1 is required to dephosphorylate Aurora B substrates at kinetochores and stabilize microtubule attachments. PP1 levels at kinetochores are regulated and inversely proportional to local Aurora B activity. Indeed, we demonstrate that phosphorylation of KNL1 by Aurora B disrupts the KNL1–PP1 interaction. In total, our results support a positive feedback mechanism by which Aurora B activity at kinetochores not only targets substrates directly, but also prevents localization of the opposing phosphatase.
Despite many decades of study, mitotic chromosome structure and composition remain poorly characterized. Here, we have integrated quantitative proteomics with bioinformatic analysis to generate a series of independent classifiers that describe the ∼4,000 proteins identified in isolated mitotic chromosomes. Integrating these classifiers by machine learning uncovers functional relationships between protein complexes in the context of intact chromosomes and reveals which of the ∼560 uncharacterized proteins identified here merits further study. Indeed, of 34 GFP-tagged predicted chromosomal proteins, 30 were chromosomal, including 13 with centromere-association. Of 16 GFP-tagged predicted nonchromosomal proteins, 14 were confirmed to be nonchromosomal. An unbiased analysis of the whole chromosome proteome from genetic knockouts of kinetochore protein Ska3/Rama1 revealed that the APC/C and RanBP2/RanGAP1 complexes depend on the Ska complex for stable association with chromosomes. Our integrated analysis predicts that up to 97 new centromere-associated proteins remain to be discovered in our data set.
► Method to define the protein composition of a complex nonpurifiable organelle ► Combines genetics and proteomics to study complexes in whole chromosomes ► Use of machine learning to uncover functional relationships between proteins ► Comprehensive list of >4000 mitotic chromosome-associated proteins
CELLBIO; PROTEINS; SYSBIO
INCENP fine tunes the level of aurora B kinase activity, which in turn correlates with different functional states of the chromosome passenger complex.
Dynamic localization of the chromosomal passenger complex (CPC) during mitosis is essential for its diverse functions. CPC targeting to centromeres involves interactions between Survivin, Borealin, and the inner centromere protein (CENP [INCENP]) N terminus. In this study, we investigate how interactions between the INCENP C terminus and aurora B set the level of kinase activity. Low levels of kinase activity, seen in INCENP-depleted cells or in cells expressing a mutant INCENP that cannot bind aurora B, are sufficient for a spindle checkpoint response when microtubules are absent but not against low dose taxol. Intermediate kinase activity levels obtained with an INCENP mutant that binds aurora B but cannot fully activate it are sufficient for a robust response against taxol, but cannot trigger CPC transfer from the chromosomes to the anaphase spindle midzone. This transfer requires significantly higher levels of aurora B activity. These experiments reveal that INCENP interactions with aurora B in vivo modulate the level of kinase activity, thus regulating CPC localization and functions during mitosis.
Histone variants play important roles in the epigenetic regulation of genome function. The histone variant H2A.Z is evolutionarily conserved from yeast to vertebrates, and it has been reported to have multiple effects upon gene expression and insulation, and chromosome segregation. Recently two genes encoding H2A.Z were identified in the vertebrate genome. However, it is not yet clear whether the proteins transcribed from these genes are functionally distinct. To address this issue, we knocked out each gene individually in chicken DT40 cells. We found that two distinct proteins, H2A.Z-1 and H2A.Z-2, were produced from these genes, and that these proteins could be separated on a long SDS–PAGE gel. The two isoforms were deposited to a similar extent by the SRCAP chromatin-remodeling complex, suggesting redundancy to their function. However, cells lacking either one of the two isoforms exhibited distinct alterations in cell growth and gene expression, suggesting that the two isoforms have differential effects upon nucleosome stability and chromatin structure. These findings provide insight into the molecular basis of the multiple functions of the H2A.Z gene products.
The constitutive centromere-associated network (CCAN) proteins are central to kinetochore assembly. To define the molecular architecture of this critical kinetochore network, we sought to determine the full complement of CCAN components and to define their relationships. This work identified a centromere protein S (CENP-S)–containing subcomplex that includes the new constitutive kinetochore protein CENP-X. Both CENP-S– and CENP-X–deficient chicken DT40 cells are viable but show abnormal mitotic behavior based on live cell analysis. Human HeLa cells depleted for CENP-X also showed mitotic errors. The kinetochore localization of CENP-S and -X is abolished in CENP-T– or CENP-K–deficient cells, but reciprocal experiments using CENP-S–deficient cells did not reveal defects in the localization of CCAN components. However, CENP-S– and CENP-X–deficient cells show a significant reduction in the size of the kinetochore outer plate. In addition, we found that intrakinetochore distance was increased in CENP-S– and CENP-X–deficient cells. These results suggest that the CENP-S complex is essential for the stable assembly of the outer kinetochore.
Centromere identity is thought to be determined by epigenetic mechanisms. The centromere-specific histone H3 variant CENP-A plays a central role in specifying the locus where the centromere is constructed. However, the precise mechanisms that target CENP-A to centromeric chromatin are poorly understood. Here, we show that facilitates chromatin transcription (FACT) localizes to centromeres in a CENP-H–containing complex-dependent manner. In conditional mutant cell lines for SSRP1, a subunit of FACT, centromere targeting of newly synthesized CENP-A is severely inhibited. The chromatin remodeling factor CHD1 binds to SSRP1 both in vivo and in vitro and associates with centromeres. The centromeric localization of CHD1 is lost in SSRP1-depleted cells. RNA interference knockdown of CHD1 leads to a decrease in the amount of centromere localized CENP-A. These findings indicate that the CENP-H–containing complex facilitates deposition of newly synthesized CENP-A into centromeric chromatin in cooperation with FACT and CHD1.
In addition to their pleiotropic functions under physiological conditions, transcription factors STAT3 and STAT5 also have oncogenic activities, but how activated STATs are transported to the nucleus has not been fully understood. Here we show that an MgcRacGAP mutant lacking its nuclear localizing signal (NLS) blocks nuclear translocation of p-STATs both in vitro and in vivo. Unlike wild-type MgcRacGAP, this mutant did not promote complex formation of phosphorylated STATs (p-STATs) with importin α in the presence of GTP-bound Rac1, suggesting that MgcRacGAP functions as an NLS-containing nuclear chaperone. We also demonstrate that mutants of STATs lacking the MgcRacGAP binding site (the strand βb) are hardly tyrosine phosphorylated after cytokine stimulation. Intriguingly, mutants harboring small deletions in the C′-adjacent region (βb-βc loop region) of the strand βb became constitutively active with the enhanced binding to MgcRacGAP. The molecular basis of this phenomenon will be discussed, based on the computer-assisted tertiary structure models of STAT3. Thus, MgcRacGAP functions as both a critical mediator of STAT's tyrosine phosphorylation and an NLS-containing nuclear chaperone of p-STATs.
Cohesin is an essential protein complex required for sister chromatid cohesion. Cohesin associates with chromosomes and establishes sister chromatid cohesion during interphase. During metaphase, a small amount of cohesin remains at the chromosome-pairing domain, mainly at the centromeres, whereas the majority of cohesin resides in the cytoplasm, where its functions remain unclear. We describe the mitosis-specific recruitment of cohesin to the spindle poles through its association with centrosomes and interaction with nuclear mitotic apparatus protein (NuMA). Overexpression of NuMA enhances cohesin accumulation at spindle poles. Although transient cohesin depletion does not lead to visible impairment of normal spindle formation, recovery from nocodazole-induced spindle disruption was significantly impaired. Importantly, selective blocking of cohesin localization to centromeres, which disrupts centromeric sister chromatid cohesion, had no effect on this spindle reassembly process, clearly separating the roles of cohesin at kinetochores and spindle poles. In vitro, chromosome-independent spindle assembly using mitotic extracts was compromised by cohesin depletion, and it was rescued by addition of cohesin that was isolated from mitotic, but not S phase, cells. The combined results identify a novel spindle-associated role for human cohesin during mitosis, in addition to its function at the centromere/kinetochore regions.
We previously identified a multisubunit complex (CENP-H/I complex) in kinetochores from human and chicken cells. We showed that the CENP-H/I complex is divided into three functional classes. In the present study, we investigated CENP-O class proteins, which include CENP-O, -P, -Q, -R, and -50 (U). We created chicken DT40 cell knockouts of each of these proteins, and we found that all knockout lines were viable, but that they showed slow proliferation and mitotic defects. Kinetochore localization of CENP-O, -P, -Q, and -50 was interdependent, but kinetochore localization of these proteins was observed in CENP-R–deficient cells. A coexpression assay in bacteria showed that CENP-O, -P, -Q, and -50 proteins form a stable complex that can associate with CENP-R. Phenotype analysis of knockout cells showed that all proteins except for CENP-R were required for recovery from spindle damage, and phosphorylation of CENP-50 was essential for recovery from spindle damage. We also found that treatment with the proteasome inhibitor MG132 partially rescued the severe mitotic phenotype observed in response to release from nocodazole block in CENP-50–deficient cells. This suggests that CENP-O class proteins are involved in the prevention of premature sister chromatid separation during recovery from spindle damage.
Chromosome segregation during mitosis requires the assembly of a large proteinaceous structure termed the kinetochore. In Caenorhabditis elegans, KNL-1 is required to target multiple outer kinetochore proteins. Here, we demonstrate that the vertebrate KNL1 counterpart is essential for chromosome segregation and is required to localize a subset of outer kinetochore proteins. However, unlike in C. elegans, depletion of vertebrate KNL1 does not abolish kinetochore localization of the microtubule-binding Ndc80 complex. Instead, we show that KNL1 and CENP-K, a subunit of a constitutively centromere-associated complex that is missing from C. elegans, coordinately direct Ndc80 complex localization. Simultaneously reducing both hKNL1 and CENP-K function abolishes all aspects of kinetochore assembly downstream of centromeric chromatin and causes catastrophic chromosome segregation defects. These findings explain discrepancies in kinetochore assembly pathways between different organisms and reveal a surprising plasticity in the assembly mechanism of an essential cell division organelle.
CENP-C is a conserved inner kinetochore component. To understand the precise roles of CENP-C in the kinetochore, we created a cell line with a conditional knockout of CENP-C with the tetracycline-inducible system in which the target protein is inactivated at the level of transcription. We found that CENP-C inactivation causes mitotic delay. However, observations of living cells showed that CENP-C-knockout cells progressed to the next cell cycle without normal cell division after mitotic delay. Interphase cells with two nuclei before subsequent cell death were sometimes observed. We also found that ∼60% of CENP-C–deficient cells had no Mad2 signals even after treatment with nocodazole, suggesting that lack of CENP-C impairs the Mad2 spindle checkpoint pathway. We also observed significant reductions in the signal intensities of Mis12 complex proteins at centromeres in CENP-C–deficient cells. CENP-C signals were also weak in interphase nuclei but not in mitotic chromosomes of cells with a knockout of CENP-K, a member of CENP-H complex proteins. These results suggest that centromere localization of CENP-C in interphase nuclei occurs upstream of localization of the Mis12 complex and downstream of localization of the CENP-H complex.
In eukaryotic nuclei, DNA is wrapped around a protein octamer composed of the core histones H2A, H2B, H3, and H4, forming nucleosomes as the fundamental units of chromatin. The modification and deposition of specific histone variants play key roles in chromatin function. In this study, we established an in vitro system based on permeabilized cells that allows the assembly and exchange of histones in situ. H2A and H2B, each tagged with green fluorescent protein (GFP), are incorporated into euchromatin by exchange independently of DNA replication, and H3.1-GFP is assembled into replicated chromatin, as found in living cells. By purifying the cellular factors that assist in the incorporation of H2A–H2B, we identified protein phosphatase (PP) 2C γ subtype (PP2Cγ/PPM1G) as a histone chaperone that binds to and dephosphorylates H2A–H2B. The disruption of PP2Cγ in chicken DT40 cells increased the sensitivity to caffeine, a reagent that disturbs DNA replication and damage checkpoints, suggesting the involvement of PP2Cγ-mediated histone dephosphorylation and exchange in damage response or checkpoint recovery in higher eukaryotes.
Chromatin assembly factor-1 (CAF-1), a complex consisting of p150, p60, and p48 subunits, is highly conserved from yeast to humans and facilitates nucleosome assembly of newly replicated DNA in vitro. To investigate roles of CAF-1 in vertebrates, we generated two conditional DT40 mutants, respectively, devoid of CAF-1p150 and p60. Depletion of each of these CAF-1 subunits led to delayed S-phase progression concomitant with slow DNA synthesis, followed by accumulation in late S/G2 phase and aberrant mitosis associated with extra centrosomes, and then the final consequence was cell death. We demonstrated that CAF-1 is necessary for rapid nucleosome formation during DNA replication in vivo as well as in vitro. Loss of CAF-1 was not associated with the apparent induction of phosphorylations of S-checkpoint kinases Chk1 and Chk2. To elucidate the precise role of domain(s) in CAF-1p150, functional dissection analyses including rescue assays were preformed. Results showed that the binding abilities of CAF-1p150 with CAF-1p60 and DNA polymerase sliding clamp proliferating cell nuclear antigen (PCNA) but not with heterochromatin protein HP1-γ are required for cell viability. These observations highlighted the essential role of CAF-1–dependent nucleosome assembly in DNA replication and cell proliferation through its interaction with PCNA.
During cell division, kinetochores form the primary chromosomal attachment sites for spindle microtubules. We previously identified a network of 10 interacting kinetochore proteins conserved between Caenorhabditis elegans and humans. In this study, we investigate three proteins in the human network (hDsn1Q9H410, hNnf1PMF1, and hNsl1DC31). Using coexpression in bacteria and fractionation of mitotic extracts, we demonstrate that these proteins form a stable complex with the conserved kinetochore component hMis12. Human or chicken cells depleted of Mis12 complex subunits are delayed in mitosis with misaligned chromosomes and defects in chromosome biorientation. Aligned chromosomes exhibited reduced centromere stretch and diminished kinetochore microtubule bundles. Consistent with this, localization of the outer plate constituent Ndc80HEC1 was severely reduced. The checkpoint protein BubR1, the fibrous corona component centromere protein (CENP) E, and the inner kinetochore proteins CENP-A and CENP-H also failed to accumulate to wild-type levels in depleted cells. These results indicate that a four-subunit Mis12 complex plays an essential role in chromosome segregation in vertebrates and contributes to mitotic kinetochore assembly.
We identified CENP-50 as a novel kinetochore component. We found that CENP-50 is a constitutive component of the centromere that colocalizes with CENP-A and CENP-H throughout the cell cycle in vertebrate cells. To determine the precise role of CENP-50, we examined its role in centromere function by generating a loss-of-function mutant in the chicken DT40 cell line. The CENP-50 knockout was not lethal; however, the growth rate of cells with this mutation was slower than that of wild-type cells. We observed that the time for CENP-50-deficient cells to complete mitosis was longer than that for wild-type cells. Centromeric localization of CENP-50 was abolished in both CENP-H- and CENP-I-deficient cells. Coimmunoprecipitation experiments revealed that CENP-50 interacted with the CENP-H/CENP-I complex in chicken DT40 cells. We also observed severe mitotic defects in CENP-50-deficient cells with apparent premature sister chromatid separation when the mitotic checkpoint was activated, indicating that CENP-50 is required for recovery from spindle damage.
CENP-A is an evolutionarily conserved, centromere-specific variant of histone H3 that is thought to play a central role in directing kinetochore assembly and in centromere function. Here, we have analyzed the consequences of disrupting the CENP-A gene in the chicken DT40 cell line. In CENP-A-depleted cells, kinetochore protein assembly is impaired, as indicated by mislocalization of the inner kinetochore proteins CENP-I, CENP-H, and CENP-C as well as the outer components Nuf2/Hec1, Mad2, and CENP-E. However, BubR1 and the inner centromere protein INCENP are efficiently recruited to kinetochores. Following CENP-A depletion, chromosomes are deficient in proper congression on the mitotic spindle and there is a transient delay in prometaphase. CENP-A-depleted cells further proceed through anaphase and cytokinesis with unequal chromosome segregation, suggesting that some kinetochore function remains following substantial depletion of CENP-A. We furthermore demonstrate that CENP-A-depleted cells exhibit a specific defect in maintaining kinetochore localization of the checkpoint protein BubR1 under conditions of checkpoint activation. Our data thus point to a specific role for CENP-A in assembly of kinetochores competent in the maintenance of mitotic checkpoint signaling.
CENP-H is a constitutive centromere component that localizes to the centromere throughout the cell cycle. Because CENP-H is colocalized with CENP-A and CENP-C, it is thought to be an inner centromere protein. We previously generated a conditional loss-of-function mutant of CENP-H and showed that CENP-H is required for targeting of CENP-C to the centromere in chicken DT40 cells. In the present study, we used this mutant to identify the functional region of chicken CENP-H necessary for centromere targeting and cell viability. This region was found by yeast two-hybrid analysis to interact with Hec1, which is a member of the Nuf2 complex that transiently localizes to the centromere during mitosis. Coimmunoprecipitation experiments revealed that CENP-H interacts with the Nuf2 complex in chicken DT40 cells. Photobleaching experiments showed that both Hec1 and CENP-H form stable associations with the centromeres during mitosis, suggesting that Hec1 acts as a structural component of centromeres during mitosis. On the basis of these results and previously published data, we propose that the Nuf2 complex functions as a connector between the inner and outer kinetochores.