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At least 19 recordsLinked to original sources
PubMed2003

Dynamic relocalization of the chromosomal passenger complex proteins inner centromere protein (INCENP) and aurora-B kinase during male mouse meiosis.

INCENP and aurora-B kinase are two chromosomal passenger proteins that are thought to play key roles in coordinating chromosome segregation with cytokinesis in somatic cells. Here we have analyzed their subcellular distribution, and that of phosphorylated histone H3, and the timing of their relative appearance in mouse spermatocytes during both meiotic divisions. Our results show that in mitotic spermatogonial cells, INCENP and aurora-B show the same pattern of distribution as they do in cultured somatic cells. INCENP labels the synaptonemal complex central element from zygotene up to late pachytene when it begins to relocalize to heterochromatic chromocentres. Aurora-B first appears at chromocentres in late diplotene before the initial phosphorylation of histone H3. INCENP and aurora-B concentrate at centromeres during diakinesis and appear during metaphase I as T-shaped signals at their inner domains, just below associated sister kinetochores. During late anaphase I both proteins relocalize to the spindle midzone. Both proteins colocalize at a connecting strand traversing the centromere region and joining sister kinetochores, in metaphase II centromeres. This strand disappears at the metaphase II/anaphase II transition and relocalizes to the spindle midzone. We discuss the complex dynamic relocalization of the chromosomal passenger complex during prophase I. Additionally, we suggest that this complex may regulate sister-chromatid centromere cohesion during both meiotic divisions.

3T3 Cells↗
PubMed1995

Evidence that the MIF2 gene of Saccharomyces cerevisiae encodes a centromere protein with homology to the mammalian centromere protein CENP-C.

The MIF2 gene of Saccharomyces cerevisiae has been implicated in mitosis. Here we provide genetic evidence that MIF2 encodes a centromere protein. Specifically, we found that mutations in MIF2 stabilize dicentric minichromosomes and confer high instability (i.e., a synthetic acentric phenotype) to chromosomes that bear a cis-acting mutation in element I of the yeast centromeric DNA (CDEI). Similarly, we observed synthetic phenotypes between mutations in MIF2 and trans-acting mutations in three known yeast centromere protein genes-CEP1/CBF1/CPF1, NDC10/CBF2, and CEP3/CBF3B. In addition, the mif2 temperature-sensitive phenotype can be partially rescued by increased dosage of CEP1. Synthetic lethal interactions between a cep1 null mutation and mutations in either NDC10 or CEP3 were also detected. Taken together, these data suggest that the Mif2 protein interacts with Cep1p at the centromere and that the yeast centromere indeed exists as a higher order protein-DNA complex. The Mif2 and Cep1 proteins contain motifs of known transcription factors, suggesting that assembly of the yeast centromere is analogous to that of eukaryotic enhancers and origins of replication. We also show that the predicted Mif2 protein shares two short regions of homology with the mammalian centromere Ag CENP-C and that two temperature-sensitive mutations in MIF2 lie within these regions. These results provide evidence for structural conservation between yeast and mammalian centromeres.

Amino Acid Sequence↗
PubMed1995

Analysis of protein-DNA and protein-protein interactions of centromere protein B (CENP-B) and properties of the DNA-CENP-B complex in the cell cycle.

We previously reported that centromere protein B (CENP-B) forms a stable complex (designated complex A) containing two alphoid DNAs in vitro. Domains in the CENP-B polypeptide involved in the formation of complex A were determined in the present study with truncated derivatives expressed in Escherichia coli and in rabbit reticulocyte lysates. It was revealed by gel mobility shift analyses that polypeptides containing the NH2-terminal DNA-binding domain bind a DNA molecule as a monomer, while dimerizing at a novel hydrophobic domain in the COOH-terminal region of 59 amino acid residues. This polypeptide dimerization activity at the COOH-terminal region was also confirmed with the two-hybrid system in Saccharomyces cerevisiae cells. The results thus proved that CENP-B polypeptides form a homodimer at the COOH-terminal hydrophobic domain, each binding a DNA strand at their NH2-terminal domains. The dimerization and DNA-binding domains fall into two of the three completely conserved sequences found in human and mouse CENP-B, and complex A-forming activity was also detected in nuclear extracts of mouse cells. Metaphase-specific phosphorylation of CENP-B was also detected, but this had no effect on its complex A-forming activity. On the basis of the present results, we propose that CENP-B plays an important role in the assembly of specific centromere structures by forming unique DNA-protein complexes at the sites of CENP-B boxes on the centromeric repetitive DNA both in interphase nuclei and on mitotic chromosomes.

Amino Acid Sequence↗
PubMed2000

Interaction of yeast kinetochore proteins with centromere-protein/transcription factor Cbf1.

The centromere-kinetochore complex of Saccharomyces cerevisiae is a specialized chromosomal substructure that mediates attachment of duplicated chromosomes to the mitotic spindle by a regulated network of protein-DNA and protein-protein interactions. We have used in vitro assays to analyze putative molecular interactions between components of the yeast centromerekinetochore complex. Glutathione S-transferase pull-down experiments showed the direct interaction of in vitro translated p110, p64, and p58 of the essential CBF3 kinetochore protein complex with Cbf1p, a basic region helix-loop-helix zipper protein (bHLHzip) that specifically binds to the CDEI region on the centromere DNA. Furthermore, recombinant p64 and p23 each stimulated the in vitro DNA binding activity of Cbf1p. The N-terminal 70 amino acids of p23 were sufficient to mediate this effect. P64 could also promote the multimerization activity of Cbf1p in the presence of centromere DNA in vitro. These results show the direct physical interaction of Cbf1p and CBF3 subunits and provide evidence that CBF3 components can promote the binding of Cbf1p to its binding site in the yeast kinetochore. A functional comparison of the centromere binding proteins with transcription factors binding at MET16 promoters reveals the strong analogy between centromeres and the MET16 promoter.

Basic Helix-Loop-Helix Leucine Zipper Transcriptio↗
PubMed1991

The centromere protein CENP-B behaves as a microtubule-associated protein.

The centromere of mitotic chromosomes organizes the kinetochore, a proteinaceous matrix that interfaces with spindle microtubules at one plane and with the centromeric DNA at the other. Antibodies present in the sera from patients with CREST scleroderma recognize several proteins localized to the centromere. We have studied the ability of the two main human centromere proteins CENP-A (18 kD) and CENP-B (80 kD) to bind tubulin, in order to correlate with one of the putative functional roles in spindle microtubule attachment. CENP-A was partially solubilized from nuclear extracts by high salt treatment and then purified by reverse phase HPLC. CENP-B was obtained by gel electrophoresis and electroelution from nuclear insoluble extracts. CENP-B binds to tubulin while no significant interaction was found for CENP-A. CENP-B binds to the C-terminal region of tubulin, a characteristic similar to that found for other better characterized microtubule-associated proteins.

Antibodies↗
PubMed1989

Centromere proteins. I. Mitosis specific centromere antigen recognized by anti-centromere autoantibodies.

Human anti-centromere sera from scleroderma patients were used to detect centromere antigens of mouse fibroblast cells. An Mr = 59,000 centromere protein was localized exclusively on mitotic chromosomes. The association of this protein with the mitotic chromosomes proved to be DNase I sensitive. In interphase nuclei, this centromere antigen was not detectable by immunoblot techniques. The results suggest that the Mr = 59,000 mitosis specific protein may be necessary for the structural stability of kinetochores during mitosis.

Animals↗
PubMed1995

Sequence similarities between the yeast chromosome segregation protein Mif2 and the mammalian centromere protein CENP-C.

A short stretch of strong homology between the Saccharomyces cerevisiae chromosome segregation protein Mif2 and the DNA-binding motifs of the Drosophila D1 and mammalian HMGI(Y) chromosomal proteins suggested that Mif2 may act directly on chromosomes. Because this conserved motif is involved in binding A.T DNA, it was proposed that Mif2 may interact with chromosomes at the highly A + T-rich DNA element found in yeast centromeres. Comparison of the Mif2 amino-acid sequence with sequence databases showed that Mif2 shares at least two regions of similarity with the mammalian centromere protein CENP-C, suggesting an evolutionary conservation of centromere protein function from yeast to mammals. The order, spacing and location of these regions are also similar in the two proteins. Sequence analysis of several conditional lethal alleles of MIF2 generated by random mutagenesis revealed mutations in regions homologous to CENP-C, as well as in the highly conserved A.T DNA-binding motif. A potential phosphorylation site for p34cdc2 kinase located adjacent to the A.T DNA-binding motif was also found to be mutated in one of the mutants, suggesting that phosphorylation at this site may be important for Mif2 function and possibly for DNA binding.

Amino Acid Sequence↗
PubMed1992

Centromere protein B assembles human centromeric alpha-satellite DNA at the 17-bp sequence, CENP-B box.

We purified 15,000-fold from HeLa cell nuclear extract the centromere antigen that reacts specifically with the 17-bp sequence, designated previously as CENP-B box, in human centromeric alpha-satellite (alphoid) DNA by a two-step procedure including an oligonucleotide affinity column. The purified protein was identified as the centromere protein B (CENP-B) by its mobility on SDS-PAGE (80 kD), and reactivities to a monoclonal antibody raised to CENP-B (bacterial fusion protein) and to anticentromere sera from patients with autoimmune diseases. Direct binding by CENP-B of the CENP-B box sequence in the alphoid DNA has been proved using the purified CENP-B by DNA mobility-shift assay, Southwestern blotting, and DNase I protection analysis. The binding constant of the antigen to the CENP-B box sequence is 6 x 10(8) M-1. DNA mobility-shift assays indicated that the major complex formed between the CENP-B and the DNA contains two DNA molecules, suggesting the importance of the CENP-B/CENP-B box interaction in organization of higher ordered chromatin structures in the centromere and/or kinetochore. Location of DNA binding and dimerization domains in CENP-B was discussed based on the DNA mobility-shift assays performed with a protein fraction containing intact and partial cleavage products of CENP-B.

Autoantigens↗
PubMed2000

Centromere/kinetochore localization of human centromere protein A (CENP-A) exogenously expressed as a fusion to green fluorescent protein.

Three human centromere proteins, CENP-A, CENP-B and CENP-C, are a set of autoantigens specifically recognized by anticentromere antibodies often produced by patients with scleroderma. Microscopic observation has indicated that CENP-A and CENP-C localize to the inner plate of metaphase kinetochore, while CENP-B localizes to the centromere heterochromatin beneath the kinetochore. The antigenic structure, called "prekinetochore", is also present in interphase nuclei, but little is known about its molecular organization and the relative position of these antigens. Here, to visualize prekinetochore in living cells, we first obtained a stable human cell line, MDA-AF8-A2, in which human CENP-A is exogenously expressed as a fusion to a green fluorescent protein of Aequorea victoria. Simultaneous staining with anti-CENP-B and anti-CENP-C antibodies showed that the recombinant CENP-A colocalized with the endogenous CENP-C and constituted small discrete dots attaching to larger amorphous mass of CENP-B heterochromatin. When the cell growth was arrested in G1/ S phase with hydroxyurea, CENP-B heterochromatin was sometimes highly extended, while the relative location between GFP-fused CENP-A and the endogenous CENP-C was not affected. These results indicated that the fluorescent CENP-A faithfully localizes to the centromere/kinetochore throughout the cell cycle. We then obtained several mammalian cell lines where the same GFP-fused human CENP-A construct was stably expressed and their centromere/kinetochore is fluorescent throughout the cell cycle. These cell lines will further be used for visualizing the prekinetochore locus in interphase nuclei as well as analyzing kinetochore dynamics in the living cells.

Animals↗
PubMed2001

Essential roles of Drosophila inner centromere protein (INCENP) and aurora B in histone H3 phosphorylation, metaphase chromosome alignment, kinetochore disjunction, and chromosome segregation.

We have performed a biochemical and double-stranded RNA-mediated interference (RNAi) analysis of the role of two chromosomal passenger proteins, inner centromere protein (INCENP) and aurora B kinase, in cultured cells of Drosophila melanogaster. INCENP and aurora B function is tightly interlinked. The two proteins bind to each other in vitro, and DmINCENP is required for DmAurora B to localize properly in mitosis and function as a histone H3 kinase. DmAurora B is required for DmINCENP accumulation at centromeres and transfer to the spindle at anaphase. RNAi for either protein dramatically inhibited the ability of cells to achieve a normal metaphase chromosome alignment. Cells were not blocked in mitosis, however, and entered an aberrant anaphase characterized by defects in sister kinetochore disjunction and the presence of large amounts of amorphous lagging chromatin. Anaphase A chromosome movement appeared to be normal, however cytokinesis often failed. DmINCENP and DmAurora B are not required for the correct localization of the kinesin-like protein Pavarotti (ZEN-4/CHO1/MKLP1) to the midbody at telophase. These experiments reveal that INCENP is required for aurora B kinase function and confirm that the chromosomal passengers have essential roles in mitosis.

Anaphase↗
PubMed2005

Cohesion proteins are present in centromere protein bodies associated with avian lampbrush chromosomes.

Proteins of sister chromatid cohesion are important for maintenance of meiotic chromosome structure and retention of homologous chromosomes in bivalents during diplotene. Localization of the cohesion proteins within nuclei of growing oocytes merits special attention, particularly in avian oocytes, in which diplotene chromosomes assume the form of lampbrush chromosomes (LBCs). We performed indirect immunostaining using antibodies against cohesins SMC1alpha, SMC1beta, SMC3, Rad21, and the SA/STAG family on chaffinch, pigeon and duck LBCs spreads, and frozen ovary sections. On LBCs spreads, antibodies to the majority of cohesins showed punctate staining on chromosome axes. LBC lateral loops, where sister chromatids are separated, did not show cohesin components. The spherical entities attached to the LBCs centromeres in avian germinal vesicles, the so-called protein bodies (PBs), were enriched in SMC1alpha, SMC3, Rad21, STAG1 and STAG2. The synaptonemal complex component SYCP3, which also participates in cohesion, was detected in the axes of avian lampbrush bivalents and, to a greater degree, in the PBs. In vitellogenic oocytes, cohesion proteins persist in the PBs associated with condensing bivalents when they concentrate into the karyosphere. These results indicate that cohesion proteins accumulate in centromere PBs in avian oocytes and are involved into structural maintenance of lampbrush chromosome axes.

Animals↗
PubMed2001

Determination of the binding constants of the centromere protein Cbf1 to all 16 centromere DNAs of Saccharomyces cerevisiae.

Cbf1p is a Saccharomyces cerevisiae chromatin protein belonging to the basic region helix-loop-helix leucine zipper (bHLHzip) family of DNA binding proteins. Cbf1p binds to a conserved element in the 5'-flanking region of methionine biosynthetic genes and to centromere DNA element I (CDEI) of S.cerevisiae centromeric DNA. We have determined the apparent equilibrium dissociation constants of Cbf1p binding to all 16 CDEI DNAs in gel retardation assays. Binding constants of full-length Cbf1p vary between 1.7 and 3.8 nM. However, the dissociation constants of a Cbf1p deletion variant that has been shown to be fully sufficient for Cbf1p function in vivo vary in a range between 3.2 and 12 nM. In addition, native polyacrylamide gel electrophoresis revealed distinct changes in the 3D structure of the Cbf1p/CEN complexes. We also show that the previously reported DNA binding stimulation activity of the centromere protein p64 functions on both the Cbf1 full-length protein and a deletion variant containing only the bHLHzip domain of Cbf1p. Our results suggest that centromeric DNA outside the consensus CDEI sequence and interaction of Cbf1p with adjacent centromere proteins contribute to the complex formation between Cbf1p and CEN DNA.

Basic Helix-Loop-Helix Leucine Zipper Transcriptio↗
PubMed1998

Genetic mapping of mouse centromere protein (Incenp and Cenpe) genes.

Inner centromere protein (INCENP) and centromere protein E (CENPE) are two functionally important proteins of the higher eukaryotic centromere. Using a mouse Incenp genomic DNA and a mouse Cenpe cDNA to analyze recombinant inbred mouse sets, as well as interspecific backcross panels, we have mapped these genes to the proximal regions of mouse Chromosomes 19 and 6, respectively. Comparison of Cenpe and human CENPE, which maps to chromosome region 4q24-->q25, has further identified a new region of homology between the two species.

Animals↗
PubMed1992

Anti-helix-loop-helix domain antibodies: discovery of autoantibodies that inhibit DNA binding activity of human centromere protein B (CENP-B).

Centromere protein B (CENP-B) is one of the centromere DNA binding proteins constituting centromeric heterochromatin of human chromosomes. This protein was originally identified as the target antigen in autoimmune disease patients (often with scleroderma). In this study, we cloned a human CENP-B cDNA which was longer than the previously isolated one and expressed functional recombinant CENP-B in Escherichia coli. The DNA binding domain was finely located within the N-terminal 134-amino-acid residues covering a predicted helix-loop-helix (HLH) structure, by using a set of recombinant products with stepwise deletions from the C-terminus. From the analysis of their reactivity to anti-centromere sera from autoimmune disease patients, four epitopes were mapped on CENP-B antigen. In addition to two epitopes at the C-terminus, two were found on the HLH region at the N-terminus. In the analysis of the interaction between the antigen and autoantibodies, we found that the DNA binding activity of CENP-B was distorted by the attack of the anti-HLH domain antibodies in in vitro binding reactions. Our results suggest that the direct inhibition of the DNA binding activity by the autoantibodies might be involved in patients' autoimmune reactions in vivo.

Amino Acid Sequence↗
PubMed2004

Adaptive evolution of centromere proteins in plants and animals.

BACKGROUND: Centromeres represent the last frontiers of plant and animal genomics. Although they perform a conserved function in chromosome segregation, centromeres are typically composed of repetitive satellite sequences that are rapidly evolving. The nucleosomes of centromeres are characterized by a special H3-like histone (CenH3), which evolves rapidly and adaptively in Drosophila and Arabidopsis. Most plant, animal and fungal centromeres also bind a large protein, centromere protein C (CENP-C), that is characterized by a single 24 amino-acid motif (CENPC motif). RESULTS: Whereas we find no evidence that mammalian CenH3 (CENP-A) has been evolving adaptively, mammalian CENP-C proteins contain adaptively evolving regions that overlap with regions of DNA-binding activity. In plants we find that CENP-C proteins have complex duplicated regions, with conserved amino and carboxyl termini that are dissimilar in sequence to their counterparts in animals and fungi. Comparisons of Cenpc genes from Arabidopsis species and from grasses revealed multiple regions that are under positive selection, including duplicated exons in some grasses. In contrast to plants and animals, yeast CENP-C (Mif2p) is under negative selection. CONCLUSIONS: CENP-Cs in all plant and animal lineages examined have regions that are rapidly and adaptively evolving. To explain these remarkable evolutionary features for a single-copy gene that is needed at every mitosis, we propose that CENP-Cs, like some CenH3s, suppress meiotic drive of centromeres during female meiosis. This process can account for the rapid evolution and the complexity of centromeric DNA in plants and animals as compared to fungi.

Adaptation, Physiological↗
PubMed1985

Three related centromere proteins are absent from the inactive centromere of a stable isodicentric chromosome.

We developed an aqueous spreading procedure that permits simultaneous analysis of human chromosomes by Q-banding and indirect immunofluorescence. Using this methodology and anticentromere antibodies from an autoimmune patient we compared the active and inactive centromeres of an isodicentric X chromosome. We show that a family of structurally related human centromere proteins (CENP-A, CENP-B, and CENP-C) is detectable only at the active centromere. These antigens therefore may be regarded both as morphological and functional markers for active centromeres.

Cell Transformation, Viral↗
PubMed1987

The inner centromere protein (INCENP) antigens: movement from inner centromere to midbody during mitosis.

We describe a novel set of polypeptide antigens that shows a dramatic change in structural localization during mitosis. Through metaphase these antigens define a new chromosomal substructure that is located between the sister chromatids. Because the antigens are concentrated in the pericentromeric region, we have provisionally termed them the INCENPs (inner centromere proteins). The INCENPs (two polypeptides of 155 and 135 kD) were identified with a monoclonal antibody that was raised against the bulk proteins of the mitotic chromosome scaffold fraction. These two polypeptides are the most tightly bound chromosomal proteins known. When scaffolds are prepared, 100% of the detectable INCENPs remain scaffold associated. We were therefore unprepared for the fate of the INCENPs at anaphase. As the sister chromatids separate, the INCENPs dissociate fully from them, remaining behind at the metaphase plate as the chromatids migrate to the spindle poles. During anaphase the INCENPs are found on coarse fibers in the central spindle, and also in close apposition to the cell membrane in the region of the forming contractile ring. During telophase, the INCENPs gradually become focused onto the forming midbody, together with which they are ultimately discarded. Several possible in vivo roles for the INCENPs are suggested by these data: regulation of sister chromatid pairing, stabilization of the plane of cleavage, and separation of spindle poles at anaphase.

Anaphase↗
PubMed2000

Early disruption of centromeric chromatin organization in centromere protein A (Cenpa) null mice.

Centromere protein A (Cenpa for mouse, CENP-A for other species) is a histone H3-like protein that is thought to be involved in the nucleosomal packaging of centromeric DNA. Using gene targeting, we have disrupted the mouse Cenpa gene and demonstrated that the gene is essential. Heterozygous mice are healthy and fertile whereas null mutants fail to survive beyond 6.5 days postconception. Affected embryos show severe mitotic problems, including micronuclei and macronuclei formation, nuclear bridging and blebbing, and chromatin fragmentation and hypercondensation. Immunofluorescence analysis of interphase cells at day 5.5 reveals complete Cenpa depletion, diffuse Cenpb foci, absence of discrete Cenpc signal on centromeres, and dispersion of Cenpb and Cenpc throughout the nucleus. These results suggest that Cenpa is essential for kinetochore targeting of Cenpc and plays an early role in organizing centromeric chromatin at interphase. The evidence is consistent with the proposal of a critical epigenetic function for CENP-A in marking a chromosomal region for centromere formation.

Animals↗