CENP-O class proteins form a stable complex and are required for proper kinetochore function

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.     

The functional region of CENP-H interacts with the Nuf2 complex that localizes to centromere during mitosis

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.     

CENP-H, a constitutive centromere component, is required for centromere targeting of CENP-C in vertebrate cells

CENP-H has recently been discovered as a constitutive component of the centromere that co-localizes with CENP-A and CENP-C throughout the cell cycle. The precise function, however, remains poorly understood. We examined the role of CENP-H in centromere function and assembly by generating a conditional loss-of-function mutant in the chicken DT40 cell line. In the absence of CENP-H, cell cycle arrest at metaphase, consistent with loss of centromere function, was observed. Immunocytochemical analysis of the CENP-H-deficient cells demonstrated that CENP-H is necessary for CENP-C, but not CENP-A, localization to the centromere. These findings indicate that centromere assembly in vertebrate cells proceeds in a hierarchical manner in which localization of the centromere-specific histone CENP-A is an early event that occurs independently of CENP-C and CENP-H.     

CENP-I is essential for centromere function in vertebrate cells

We identified a novel essential centromere protein, CENP-I, which shows sequence similarity with fission yeast Mis6 protein, and we showed that CENP-I is a constitutive component of the centromere that colocalizes with CENP-A, -C, and -H throughout the cell cycle in vertebrate cells. To determine the precise function of CENP-I, we examined its role in centromere function by generating a conditional loss-of-function mutant in the chicken DT40 cell line. In the absence of CENP-I, cells arrested at prometaphase with misaligned chromosomes for long periods of time. Eventually, cells exited mitosis without undergoing cytokinesis. Immunocytochemical analysis of CENP-I-deficient cells demonstrated that both CENP-I and CENP-H are necessary for localization of CENP-C but not CENP-A to the centromere.     

CENP-A is required for accurate chromosome segregation and sustained kinetochore association of BubR1

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.     

The human Mis12 complex is required for kinetochore assembly and proper chromosome segregation

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.     

Scc1/Rad21/Mcd1 is required for sister chromatid cohesion and kinetochore function in vertebrate cells.

Proteolytic cleavage of the cohesin subunit Scc1 is a consistent feature of anaphase onset, although temporal differences exist between eukaryotes in cohesin loss from chromosome arms, as distinct from centromeres. We describe the effects of genetic deletion of Scc1 in chicken DT40 cells. Scc1 loss caused premature sister chromatid separation but did not disrupt chromosome condensation. Scc1 mutants showed defective repair of spontaneous and induced DNA damage. Scc1-deficient cells frequently failed to complete metaphase chromosome alignment and showed chromosome segregation defects, suggesting aberrant kinetochore function. Notably, the chromosome passenger INCENP did not localize normally to centromeres, while the constitutive kinetochore proteins CENP-C and CENP-H behaved normally. These results suggest a role for Scc1 in mitotic regulation, along with cohesion.     

Creation and characterization of temperature-sensitive CENP-C mutants in vertebrate cells

CENP-C is an evolutionarily conserved centromere protein that is thought to be an important component in kinetochore assembly in vertebrate cells. However, the functional role of CENP-C in cell cycle progression remains unclear. To further understand CENP-C function, we developed a method incorporating the hyper-recombinogenic chicken B lymphocyte cell line DT40 to create several temperature-sensitive CENP-C mutants in DT40 cells. We found that, under restrictive conditions, one temperature-sensitive mutant, ts4-11, displayed metaphase delay and chromosome missegregation but proceeded through the cell cycle until arrest at G₁ phase. Furthermore, ts4-11 cells were transfected with a human HeLa cell cDNA library maintained in a retroviral vector, and genes that suppressed the temperature-sensitive phenotype were identified. One of these suppressor genes encodes SUMO-1, which is a ubiquitin-like protein. This finding suggests that SUMO-1 may be involved in centromere function in vertebrate cells. The novel strategy reported here will be useful and applicable to a wide range of proteins that have general cell-autonomous function in vertebrate cells.     

Vertebrate kinetochore protein architecture: protein copy number

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.     

Roles of vertebrate Smc5 in sister chromatid cohesion and homologous recombinational repair

The structural maintenance of chromosomes (Smc) family members Smc5 and Smc6 are both essential in budding and fission yeasts. Yeast smc5/6 mutants are hypersensitive to DNA damage, and Smc5/6 is recruited to HO-induced double-strand breaks (DSBs), facilitating intersister chromatid recombinational repair. To determine the role of the vertebrate Smc5/6 complex during the normal cell cycle, we generated an Smc5-deficient chicken DT40 cell line using gene targeting. Surprisingly, Smc5(-) cells were viable, although they proliferated more slowly than controls and showed mitotic abnormalities. Smc5-deficient cells were sensitive to methyl methanesulfonate and ionizing radiation (IR) and showed increased chromosome aberration levels upon irradiation. Formation and resolution of Rad51 and gamma-H2AX foci after irradiation were altered in Smc5 mutants, suggesting defects in homologous recombinational (HR) repair of DNA damage. Ku70(-/-) Smc5(-) cells were more sensitive to IR than either single mutant, with Rad54(-/-) Smc5(-) cells being no more sensitive than Rad54(-/-) cells, consistent with an HR function for the vertebrate Smc5/6 complex. Although gene targeting occurred at wild-type levels, recombinational repair of induced double-strand breaks was reduced in Smc5(-) cells. Smc5 loss increased sister chromatid exchanges and sister chromatid separation distances in mitotic chromosomes. We conclude that Smc5/6 regulates recombinational repair by ensuring appropriate sister chromatid cohesion.     

敲除 Sam68 基因导致白血病细胞系细胞生长迟缓和 G2/M 期延长

RNA 结合蛋白 Sam68 是细胞有丝分裂期 Src 酪氨酸磷酸化的靶蛋白 . 尽管确切机制尚不清楚,一些人还是认为 Sam68 可通过调控 RNA 的代谢参与细胞周期调控 . 利用基因打靶技术,在 DT40 细胞分离出 Sam68 基因缺失的细胞系 . 利用该细胞系,进行 Sam68 的功能解析 . 与野生型细胞系相比, Sam68 基因缺失细胞表现出明显的生长速度迟缓 . 通过细胞周期研究揭示 , 这些细胞生长速度延迟是由于细胞周期中的 G2/M 期延长 . 因为参与细胞周期 G2/M 期调控的周期因子 Cdc2 激酶的活性没有改变,所以提示 Sam68 不依赖于 Cdc2 激酶的活性参与细胞周期中 G2/M 期调控 .     

Disruption of a Conserved CAP-D3 Threonine Alters Condensin Loading on Mitotic Chromosomes Leading to Chromosome Hypercondensation

The condensin complex plays a key role in organizing mitotic chromosomes. In vertebrates, there are two condensin complexes that have independent and cooperative roles in folding mitotic chromosomes. In this study, we dissect the role of a putative Cdk1 site on the condensin II subunit CAP-D3 in chicken DT40 cells. This conserved site has been shown to activate condensin II during prophase in human cells, and facilitate further phosphorylation by polo-like kinase I. We examined the functional significance of this phosphorylation mark by mutating the orthologous site of CAP-D3 (CAP-D3^T1403A) in chicken DT40 cells. We show that this mutation is a gain of function mutant in chicken cells; it disrupts prophase, results in a dramatic shortening of the mitotic chromosome axis, and leads to abnormal INCENP localization. Our results imply phosphorylation of CAP-D3 acts to limit condensin II binding onto mitotic chromosomes. We present the first in vivo example that alters the ratio of condensin I:II on mitotic chromosomes. Our results demonstrate this ratio is a critical determinant in shaping mitotic chromosomes.     

RNAi knockdown of human kinetochore protein CENP-H

The inner kinetochore protein complex binds to centromeres during the whole cell cycle. It serves as the basis for the binding of further kinetochore proteins during mitosis. CENP-H is one of the inner kinetochore proteins which is conserved amongst many eukaryotes. By specific RNAi knockdown, we reduced the CENP-H protein level in human HEp-2 cells down to less than 5% of its normal value. In these CENP-H knocked-down cells, we observed severe mitotic phenotypes like misaligned chromosomes and multipolar spindles, however, no mitotic arrest. Strong reduction of CENP-H resulted in a slightly reduced CENP-C level at the kinetochores and normal localisation of hBubR1, indicating a functional mitotic checkpoint at the hBubR1 protein level. In CENP-H knocked-down human cells, the misaligned chromosomes contained only reduced levels of CENP-E. Our data clearly indicate that CENP-H has an important impact on the architecture and function of the human kinetochore complex.     

Trf1 Is Not Required for Proliferation or Functional Telomere Maintenance in Chicken DT40 Cells

The telomere end-protection complex prevents the ends of linear eukaryotic chromosomes from degradation or inappropriate DNA repair. The homodimeric double-stranded DNA-binding protein, Trf1, is a component of this complex and is essential for mouse embryonic development. To define the requirement for Trf1 in somatic cells, we deleted Trf1 in chicken DT40 cells by gene targeting. Trf1-deficient cells proliferated as rapidly as control cells and showed telomeric localization of Trf2, Rap1, and Pot1. Telomeric G-strand overhang lengths were increased in late-passage Trf1-deficient cells, although telomere lengths were unaffected by Trf1 deficiency, as determined by denaturing Southern and quantitative FISH analysis. Although we observed some clonal variation in terminal telomere fragment lengths, this did not correlate with cellular Trf1 levels. Trf1 was not required for telomere seeding, indicating that de novo telomere formation can proceed without Trf1. The Pin2 isoform and a novel exon 4, 5–deleted isoform localized to telomeres in Trf1-deficient cells. Trf1-deficient cells were sensitive to DNA damage induced by ionizing radiation. Our data demonstrate that chicken DT40 B cells do not require Trf1 for functional telomere structure and suggest that Trf1 may have additional, nontelomeric roles involved in maintaining genome stability.     

Role of inositol 1,4,5-trisphosphate receptors in apoptosis in DT40 lymphocytes

The role of inositol 1,4,5-trisphosphate receptors (IP(3)R) in caspase-3 activation and cell death was investigated in DT40 chicken B-lymphocytes stably expressing various IP(3)R constructs. Both full-length type-I IP(3)R and a truncated construct corresponding to the caspase-3 cleaved "channel-only" fragment were able to support staurosporine (STS)-induced caspase-3 activation and cell death even when the IP(3)R construct harbored a mutation that inactivates the pore of the Ca(2+) channel (D2550A). However, a full-length wild-type IP(3)R did not promote caspase-3 activation when the 159-amino acid cytosol-exposed C-terminal tail was deleted. STS caused an increase in cytosolic free Ca(2+) in DT40 cells expressing wild-type or pore-dead IP(3)R mutants. However, in the latter case all the Ca(2+) increase originated from Ca(2+) entry across the plasma membrane. Caspase-3 activation of pore-dead DT40 cells was also more sensitive to extracellular Ca(2+) chelation when compared with wild-type cells. STS-mediated release of cytochrome c into the cytosol and mitochondrial membrane potential depolarization could also be observed in DT40 cells lacking IP(3)Rs or containing the pore-dead mutant. We conclude that nonfunctional IP(3)Rs can sustain apoptosis in DT40 lymphocytes, because they facilitate Ca(2+) entry mechanisms across the plasma membrane. Although the intrinsic ion-channel function of IP(3)Rs is dispensable for apoptosis induced by STS, the C-terminal tail of IP(3)Rs appears to be essential, possibly reflecting key protein-protein interactions with this domain.     

The accuracy of segregation of human mini-chromosomes varies in different vertebrate cell lines, correlates with the extent of centromere formation and provides evidence for a trans-acting centromere maintenance activity

We show that the accuracy of mitotic segregation of three engineered, mapped human mini-chromosomes differs between human, mouse and chicken cell lines. We have studied the cause of these differences by analysing the extent of centromere formation on one mini-chromosome immunocytochemically. In human and chicken cell lines the mini-chromosomes segregate accurately and form centromeres but in one mouse cell line centromere formation is undetectable and mitotic segregation is inaccurate. These results indicate that the centromere is maintained by an activity that functions in trans and varies either in amount or specificity between different cells. Structurally defined mini-chromosomes may allow this activity to be studied.