Chmadrin: a novel Ki-67 antigen-related perichromosomal protein possibly implicated in higher order chromatin structure.
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A novel perichromosomal protein, which we have named chmadrin, was identified from rat kangaroo PtK2 cells. The deduced amino acid sequence revealed structural homologies in several limited regions to the Ki-67 antigen (pKi-67). The subcellular localization of chmadrin was found to be similar to that of pKi-67 throughout the cell cycle, that is, predominantly nucleolar during interphase and perichromosomal in the mitotic phase. In addition, a certain population of the protein was found to be localized in heterochromatic foci in interphase nuclei. Transient expression analysis of the truncated proteins corresponding to the conserved regions clearly demonstrated the structural basis for the characteristic cellular localization. Residues 494-778, which show extensive similarity to the corresponding region of pKi-67, were efficiently targeted to nucleoli, whereas a repetitive structure found at the C-terminal portion, whose similarity to pKi-67 is weak, was localized precisely to mitotic chromosomes. The C-terminal portion was designated the 'LR domain' since several LR (leucine and arginine) pairs commonly appear in chmadrin and pKi-67. When overproduced in the interphase nuclei, the LR domain induced the formation of aberrant heterochromatin as a structural constituent. These are the first empirical data suggesting the involvement of perichromosomal proteins in the organization of chromatin structure. (+info)
hSNF5/INI1 inactivation is mainly associated with homozygous deletions and mitotic recombinations in rhabdoid tumors.
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The chromatin-remodeling hSNF5/INI1 gene has recently been shown to act as a tumor suppressor gene in rhabdoid tumors (RTs). In an attempt to further characterize the main chromosomal mechanisms involved in hSNF5/INI1 inactivation in RTs, we report here the molecular cytogenetic data obtained in 12 cell lines harboring hSNF5/INI1 mutations and/or deletions in relation to the molecular genetic analysis using polymorphic markers extended to both extremities of chromosome 22q. On the whole, mitotic recombination occurring in the proximal part of chromosome 22q, as demonstrated in five cases, and nondisjunction/duplication, highly suspected in two cases (processes leading respectively to partial or complete isodisomy), appear to be major mechanisms associated with hSNF5/INI1 inactivation. Such isodisomy accompanies each of the RTs exhibiting two cytogenetically normal chromosomes 22. This results in homozygosity for the mutation at the hSNF5/INI1 locus. An alternate mechanism accounting for hSNF5/INI1 inactivation observed in these tumors is homozygous deletion in the rhabdoid consensus region. This was observed in each of the four tumors carrying a chromosome 22q abnormality and, in particular, in the three tumors with chromosomal translocations. Only one case of our series illustrates the mutation/deletion classical model proposed for the double-hit inactivation of a tumor suppressor gene. (+info)
A maize homolog of mammalian CENPC is a constitutive component of the inner kinetochore.
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Genes for three maize homologs (CenpcA, CenpcB, and CenpcC) of the conserved kinetochore assembly protein known as centromere protein C (CENPC) have been identified. The C-terminal portion of maize CENPC shares similarity with mammalian CENPC and its yeast homolog Mif2p over a 23-amino acid region known as region I. Immunolocalization experiments combined with three-dimensional light microscopy demonstrated that CENPC is a component of the kinetochore throughout interphase, mitosis, and meiosis. It is shown that sister kinetochore separation occurs in two discrete phases during meiosis. A partial separation of sister kinetochores occurs in prometaphase I, and a complete separation occurs in prometaphase II. CENPC is absent on structures known as neocentromeres that, in maize, demonstrate poleward movement but lack other important features of centromeres/kinetochores. CENPC and a previously identified centromeric DNA sequence interact closely but do not strictly colocalize on meiotic chromosomes. These and other data indicate that CENPC occupies an inner domain of the maize kinetochore. (+info)
Hec1p, an evolutionarily conserved coiled-coil protein, modulates chromosome segregation through interaction with SMC proteins.
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hsHec1p, a Homo sapiens coiled-coil-enriched protein, plays an important role in M-phase progression in mammalian cells. A Saccharomyces cerevisiae protein, identical to Tid3p/Ndc80p and here designated scHec1p, has similarities in structure and biological function to hsHec1p. Budding yeast cells deleted in the scHEC1/NDC80 allele are not viable, but this lethal phenotype can be rescued by hsHEC1 under control of the endogenous scHEC1 promoter. At the nonpermissive temperature, significant mitotic delay, chromosomal missegregation, and decreased viability were observed in yeast cells with temperature-sensitive (ts) alleles of hsHEC1. In the hshec1-113 ts mutant, we found a single-point mutation changing Trp395 to a stop codon, which resulted in the expression of a C-terminally truncated 45-kDa protein. The binding of this mutated protein, hshec1-113p, to five identified hsHec1p-associated proteins was unchanged, while its binding to human SMC1 protein and yeast Smc1p was ts. Hec1p also interacts with Smc2p, and the binding of the mutated hshec1-113p to Smc2p was not ts. Overexpression of either hsHEC1 or scHEC1 suppressed the lethal phenotype of smc1-2 and smc2-6 at nonpermissive temperatures, suggesting that the interactions between Hec1p and Smc1p and -2p are biologically significant. These results suggest that Hec1 proteins play a critical role in modulating chromosomal segregation, in part, through their interactions with SMC proteins. (+info)
The gene for the embryonic stem cell coactivator UTF1 carries a regulatory element which selectively interacts with a complex composed of Oct-3/4 and Sox-2.
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UTF1 is a transcriptional coactivator which has recently been isolated and found to be expressed mainly in pluripotent embryonic stem (ES) cells (A. Okuda, A. Fukushima, M. Nishimoto, et al., EMBO J. 17:2019-2032, 1998). To gain insight into the regulatory network of gene expression in ES cells, we have characterized the regulatory elements governing UTF1 gene expression. The results indicate that the UTF1 gene is one of the target genes of an embryonic octamer binding transcription factor, Oct-3/4. UTF1 expression is, like the FGF-4 gene, regulated by the synergistic action of Oct-3/4 and another embryonic factor, Sox-2, implying that the requirement for Sox-2 by Oct-3/4 is not limited to the FGF-4 enhancer but is rather a general mechanism of activation for Oct-3/4. Our biochemical analyses, however, also reveal one distinct difference between these two regulatory elements: unlike the FGF-4 enhancer, the UTF1 regulatory element can, by its one-base difference from the canonical octamer-binding sequence, selectively recruit the complex comprising Oct-3/4 and Sox-2 and preclude the binding of the transcriptionally inactive complex containing Oct-1 or Oct-6. Furthermore, our analyses reveal that these properties are dictated by the unique ability of the Oct-3/4 POU-homeodomain that recognizes a variant of the Octamer motif in the UTF1 regulatory element. (+info)
Stabilization of chromatin structure by PRC1, a Polycomb complex.
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The Polycomb group (PcG) genes are required for maintenance of homeotic gene repression during development. Mutations in these genes can be suppressed by mutations in genes of the SWI/SNF family. We have purified a complex, termed PRC1 (Polycomb repressive complex 1), that contains the products of the PcG genes Polycomb, Posterior sex combs, polyhomeotic, Sex combs on midleg, and several other proteins. Preincubation of PRC1 with nucleosomal arrays blocked the ability of these arrays to be remodeled by SWI/SNF. Addition of PRC1 to arrays at the same time as SWI/SNF did not block remodeling. Thus, PRC1 and SWI/SNF might compete with each other for the nucleosomal template. Several different types of repressive complexes, including deacetylases, interact with histone tails. In contrast, PRC1 was active on nucleosomal arrays formed with tailless histones. (+info)
A central role for cohesins in sister chromatid cohesion, formation of axial elements, and recombination during yeast meiosis.
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A multisubunit complex, called cohesin, containing Smc1p, Smc3p, Scc1p, and Scc3p, is required for sister chromatid cohesion in mitotic cells. We show here that Smc3p and a meiotic version of Scc1p called Rec8p are required for cohesion between sister chromatids, for formation of axial elements, for reciprocal recombination, and for preventing hyperresection of double-strand breaks during meiosis. Both Rec8p and Smc3p colocalize with chromosome cores independently of synapsis during prophase I and largely disappear from chromosome arms after pachytene but persist in the neighborhood of centromeres until the onset of anaphase II. The eukaryotic cell's cohesion apparatus is required both for the repair of recombinogenic lesions and for chromosome segregation and therefore appears to lie at the heart of the meiotic process. (+info)
Cohesins bind to preferential sites along yeast chromosome III, with differential regulation along arms versus the centric region.
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Sister chromatid cohesion is mediated by evolutionary conserved chromosomal proteins, termed "cohesins." Using an extension of chromatin immunoprecipitation, we have analyzed the distribution of cohesins Mcd1/ Sccl and Smc1 along yeast chromosome III. Both proteins occur preferentially at the same approximately 23 positions. Sites in a approximately 50 kb region around the centromere give especially intense signals. Prominent centric region binding appears to emerge from a more even distribution, probably by differential loss of cohesins along the chromosome arms. Cohesin binding peaks correspond closely to peaks of high local AT composition, a base composition periodicity of approximately 15 kb that is distinct from the approximately 50 kb periodicity of base composition isochores, consistent with axis association of cohesins. The methodology described can be used to analyze the distribution of any DNA-binding protein and, via microchips, along entire genomes. (+info)