Network of phosphatases and HDAC complexes at repressed chromatin.

Tight regulation of gene expression is achieved by a variety of protein complexes that selectively bind chromatin, modify it and change its transcription competency. Histone acetylases (HATs) and deacetylases (HDACs) play an important role in this process. They can generate transcriptionally active or inactive chromatin through the addition (HATs) or removal (HDACs) of acetyl groups on histones, respectively. Repo-Man is a Protein Phosphatase 1 targeting subunit that accumulates on chromosomes during mitotic exit and mediates the removal of mitotic histone H3 phosphorylations. It was shown recently that Repo-Man also regulates heterochromatin formation in interphase and that its depletion favours the switch between transcriptionally inactive and active chromatin, demonstrating that its role goes well beyond mitosis. Here, we provide the first link between a phosphatase and HDAC complexes. We show that genome-wide Repo-Man binding sites overlap with chromatin regions bound by members of the three HDAC complexes (Sin3a, NuRD and CoREST). We establish that members of the NuRD and Sin3a HDAC complexes interact with Repo-Man by mass spectrometry and that Repo-Man is in close proximity to SAP18 (Sin3a) in interphase as observed by the Proximity Ligation Assay. Altogether, these data suggest a mechanism by which Repo-Man/PP1 complex, via interactions with HDACs, could stabilise gene repression.

Centromeres: old tales and new tools.

The centromere is a specialised region of the eukaryotic chromosome that directs the equal segregation of sister chromatids into two daughter cells during mitosis. In mitosis, the kinetochores mediate (1) microtubule capture and chromosome alignment at a metaphase plate; (2) the correction of improper microtubule attachments; (3) the maintenance of an active checkpoint until bi-orientation is achieved by the whole complement of chromosomes; (4) the establishment of tension within the centromere which, in turn, contributes to silencing of the spindle checkpoint and triggers the onset of anaphase. In this review, we will analyse how centromeres are organised with respect to chromatin types and arrangements.

Sister chromatid cohesion and genome stability in vertebrate cells.

For successful eukaryotic mitosis, sister chromatid pairs remain linked after replication until their kinetochores have been attached to opposite spindle poles by microtubules. This linkage is broken at the metaphase-anaphase transition and the sisters separate. In budding yeast, this sister chromatid cohesion requires a multi-protein complex called cohesin. A key component of cohesin is Scc1/Mcd1 (Rad21 in fission yeast). Disruption of the chicken orthologue of Scc1 by gene targeting in DT40 cells causes premature sister chromatid separation. Cohesion between sister chromatids is likely to provide a substrate for post-replicative DNA repair by homologous recombination. In keeping with this role of cohesion, Scc1 mutants also show defects in the repair of spontaneous and induced DNA damage. Scc1-deficient cells frequently fail to complete metaphase chromosome alignment and show chromosome segregation defects, suggesting aberrant kinetochore function. Consistent with this, the chromosomal passenger protein, INCENP (inner centromere protein) fails to localize to centromeres. Survivin, another passenger protein and one which interacts with INCENP, also fails to localize to centromeres in Scc1-deficient cells. These results show that cohesin maintains genomic stability by ensuring appropriate DNA repair and equal chromosome segregation at mitosis.

INCENP loss from an inactive centromere correlates with the loss of sister chromatid cohesion.

Inactive centromeres of stable dicentric chromosomes provide a unique opportunity to examine the resolution of sister chromatid cohesion in mitosis. Here we show for the first time that inactive centromeres are composed of heterochromatin, as defined by the presence of heterochromatin protein HP1(Hs alpha). We then show that both the inner centromere protein (INCENP) and its binding partner Aurora-B/AIM-1 kinase can also be detected at the inactive centromere. Thus, targeting of the chromosomal passengers is not dependent upon the presence of an active centromere/kinetochore. Furthermore, we show that the association of INCENP with the inactive centromere correlates strictly with the state of cohesion between sister chromatids: loss of cohesion is accompanied by loss of detectable INCENP. These results are consistent with recent suggestions that one function of the chromosomal passenger proteins may be to regulate sister chromatid separation in mitosis.

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.

INCENP is required for proper targeting of Survivin to the centromeres and the anaphase spindle during mitosis.

Three lines of investigation have suggested that interactions between Survivin and the chromosomal passenger proteins INCENP and Aurora-B kinase may be important for mitotic progression. First, interference with the function of Survivin/BIR1, INCENP, or Aurora-B kinase leads to similar defects in mitosis and cytokinesis [1-7] (see [8] for review). Second, INCENP and Aurora-B exist in a complex in Xenopus eggs [9] and in mammalian cultured cells [7]. Third, interference with Survivin or INCENP function causes Aurora-B kinase to be mislocalized in mitosis in both C. elegans and vertebrates [5, 7, 9]. Here, we provide evidence that Survivin, Aurora-B, and INCENP interact physically and functionally. Direct visualization of Survivin-GFP in mitotic cells reveals that it localizes identically to INCENP and Aurora-B. Survivin binds directly to both Aurora-B and INCENP in yeast two-hybrid and in vitro pull-down assays. The in vitro interaction between Survivin and Aurora-B is extraordinarily stable in that it resists 3 M NaCl. Finally, Survivin and INCENP interact functionally in vivo; in cells in which INCENP localization is disrupted, Survivin adheres to the chromosomes and no longer concentrates at the centromeres or transfers to the anaphase spindle midzone. Our data provide the first biochemical evidence that Survivin can interact directly with members of the chromosomal passenger complex.

Human INCENP colocalizes with the Aurora-B/AIRK2 kinase on chromosomes and is overexpressed in tumour cells.

The inner centromere protein (INCENP), which has previously been described in chicken, frog and mouse, is required for correct chromosome segregation and cytokinesis. We have identified the human INCENP gene by library screening and reverse transcription-polymerase chain reaction (RT-PCR) and localized it to chromosomal region 11q12. HsINCENP is a single-copy gene that consists of 17 exons and covers 25 kb of genomic DNA. The gene is expressed at highest levels in the colon, testis and prostate, consistent with its likely role in cell proliferation. HsINCENP encodes a highly basic protein of 915 amino acids that localizes to metaphase chromosomes and to the mitotic spindle and equatorial cortex at anaphase. Recently we showed that INCENP is stockpiled in a complex with the Aurora-B/XAIRK2 kinase in Xenopus eggs. Here we demonstrate that, consistent with such an interaction, the two proteins colocalize on human metaphase chromosomes. Levels of Aurora-B are increased in several human cancers, and we show here that HsINCENP protein levels are also significantly increased in several colorectal cancer cell lines.

Insertion of a loxP site in a size-reduced human accessory chromosome.

The generation in vitro of mammalian artificial chromosomes, in view of the possibility of developing new technologies for gene therapy, is still an ambitious goal. Mammalian artificial chromosomes, to be used as cloning and expression vectors, have been constructed either by de novo synthesis or by reduction of pre-existing chromosomes. In the work here reported, we introduced a loxP sequence into the pericentromeric region of a chromosome 9-derived X-ray-reduced minichromosome, with the purpose of generating a human chromosome vector (HCV). The modified accessory chromosome is linear and mitotically stable, has lost at least 1400 kb of alpha satellite DNA and normally binds CENP-B, CENP-C and CENP-E. The efficiency of gene targeting via loxP mediated homologous recombination was tested using the histone H2B-Green Fluorescent Protein chimaeric gene as a reporter. The frequency of site-specific insertion of the exogenous sequence was found to be about 50% and to occur in a controlled way with regard to the number of copies. The expression level of the fusion protein was stable over prolonged time in culture.

Mammalian centromeres: DNA sequence, protein composition, and role in cell cycle progression.

The centromere is a specialized region of the eukaryotic chromosome that is responsible for directing chromosome movements in mitosis and for coordinating the progression of mitotic events at the crucial transition between metaphase and anaphase. In this review, we will focus on recent advances in the understanding of centromere composition at the protein and DNA level and of the role of centromeres in sister-chromatid cohesion and mitotic checkpoint control.

Gene targeting to the centromeric DNA of a human minichromosome.

A human supernumerary minichromosome (MC), previously identified as a derivative of chromosome 9, has been introduced into Chinese hamster ovary (CHO) cells by means of cell fusion. A hybrid clone containing the MC as the only free human chromosome was isolated. A selectable marker gene (neo) inserted into a yeast artificial chromosome (YAC) has been successfully targeted to the MC centromeric DNA via co-transfection with chromosome-9-specific alpha satellite DNA. In situ hybridization and Southern blotting experiments demonstrated that the intact neo gene was integrated into the MC centromeric DNA. Studies on the clonal distribution and on the stability of the MC either in the presence or in the absence of the selective agent have been carried out. The MC is susceptible to further manipulations and may thus represent a model for the construction of a large-capacity vector for somatic gene therapy.

DNA fingerprint analysis for the detection of induced mutations in mammalian cells in culture.

A mutation assay in cultured mammalian cells based on the direct analysis of minisatellite DNA was developed. Band pattern variations reflect DNA alterations ranging from single base changes to complex rearrangements. By DNA fingerprinting a large number of autosomal loci throughout the human genome can be simultaneously checked, therefore minimizing the size of the samples of cell colonies to be scored in the absence of phenotypic selection. For the mutation assay chinese hamster cells (V79) were treated with Nitrosoguanidine and 14 independent colonies were isolated and expanded. DNA fingerprints were obtained after digestion of the DNA extracted from each clone with bothHinfI andHae III, and hybridisation with both 33.15 and 33.6 probes. Twelve colonies from untreated cells were also analysed. Several differences in the band pattern of treated colonies were observed when compared with untreated cells; digestion withHae III and hybridisation with 33.15 probe allowed the detection of the highest frequency of induced variants. The results suggest that minisatellite sequences are hypermutable sites that can be used to monitor the mutagenic potential of chemical agents directly at the DNA level, without phenotypic selection. Moreover, with the method herein decribed, it is possible to distinguish between true mutations and epimutations, such as those caused by changes in DNA methylation.

Variation of minisatellites in chemically induced mutagenesis and in gene amplification.

A mutation assay in cultured mammalian cells was developed based on direct analysis of minisatellite DNA. Chinese hamster cells (V79) were mutagenized with nitrosoguanidine and independent colonies were isolated and expanded. DNA fingerprints were then obtained after digestion with HinfI or HaeIII and hybridization with 33.15 and 33.6 probes (Jeffreys et al., 1985). 12 colonies from untreated cells were also analyzed. Digestion with HaeIII and hybridization with 33.15 probe detected the highest frequency of induced variants. The results suggest that minisatellite sequences are hypermutable sites that can be used to monitor the mutagenic effect of chemical agents. We have also analyzed the DNA fingerprints of 17 independent Chinese hamster (CHO) cell lines carrying amplification of the CAD gene. The DNA fingerprint analysis showed a variation in minisatellite regions in 3 lines while no variation was observed in independent colonies from the CHO parental cell line. The results suggest that these sequences may be hot spots for recombination during gene amplification.

Trisomy 21 mosaicism in two subjects from two generations.

In the course of a chromosome fragility investigation on the cancer prone hereditary disorder xeroderma pigmentosum, a low proportion of cells with a 47,XY,+21 karyotype was found in lymphocyte cultures of a patient not showing any Down syndrome symptom. The presence of trisomy 21 mosaicism was demonstrated also in peripheral blood of the healthy father and confirmed by "chromosome painting" that allowed a rapid detection of chromosomes 21 on metaphase cells and interphase nuclei. The trisomic cell line was not detected in fibroblast cultures. The analysis of chromosome 21 heteromorphism indicated that in both subjects the mosaic could result from either a diploid or an aneuploid zygote. Since in the trisomic cell line of the father and the son the extra chromosome 21 seems to be the same, a predisposition toward mitotic errors (non-disjunction or anaphase lagging) may be postulated, leading to the recurrent gain or loss of a specific chromosome 21. In order to test the hypothesis of an abnormal mitotic behaviour of the chromosome 21, we investigated the centromere separation index and the DNA restriction pattern in Southern blots probed with satellite DNA sequences specific for chromosome 21 centromere. Both the approaches did not reveal any peculiar feature that may account for the genetically determined proneness to mitotic error observed in the family.

Assignment of the human urokinase receptor gene (PLAUR) to 19q13.

Through in situ hybridization of a cDNA probe to metaphase chromosomes, we localized the gene for the human urokinase receptor (PLAUR) on chromosome 19. RBG-banding permitted subchromosomal localization of the PLAUR gene to 19q13.

Aneuploidy induced by chloral hydrate detected in human lymphocytes with the Y97 probe.

Chloral hydrate (CH) has been assayed for its ability to induce chromosome number variation in human lymphocytes in culture. Aneuploidy induction has been detected by means of in situ hybridization on interphase nuclei with a chromosome Y-specific DNA probe. A dose-dependent increase in the number of hyperdiploid nuclei was found at CH concentrations ranging from 250 to 750 micrograms/ml. These results represent a further contribution to the validation of the method which gave a positive response with drugs characterized by different mechanisms of action and therefore may be of general use in detecting aneuploidy in mammalian cells.

Aneuploidy assay on diethylstilbestrol by means of in situ hybridization of radioactive and biotinylated DNA probes on interphase nuclei.

Aneuploidy tests by means of in situ hybridization with chromosome-specific DNA probes on interphase nuclei have been carried out on human lymphocytes treated with diethylstilbestrol (DES). A DNA probe specific for chromosome Y (Y97), either radioactive or biotinylated, was used for the assays. Autoradiography or FITC-conjugated antibiotin antibodies were employed to visualize the hybridization sites. A significant increase of hyperdiploid nuclei was obtained with both procedures and a dose-related effect was revealed using the biotinylated probe. The results obtained, while giving further support to the evidence that DES is able to induce aneuploidy in cultured human cells, also indicate that the sensitivity of the assay can be improved by using biotinylated probes coupled with fluorescent antibodies.

Characterization of an X;X translocation by cytological analysis and in situ hybridization.

An X;X chromosomal translocation was ascertained by conventional cytogenetic analysis in a phenotypically normal woman with secondary amenorrhea. In situ hybridization was performed with previously mapped X-specific DNA sequences to study the rearrangement at the molecular level. The results allowed us to demonstrate that the subject is monosomic for the distal region of the short arm of the X and trisomic for the distal region of the long arm.

Detection of chromosome variation in interphase by in situ hybridization with repetitive DNA probes: potential applications to cytogenetic analysis and mutagenicity testing.

Individual chromosomes can be identified by means of in situ hybridization with DNA probes for chromosome-specific repetitive sequences. The efficiency and sensitivity of the method are strictly dependent on the characteristics of the probes and the experimental conditions. Using three probes with different copy numbers, we demonstrated that the target chromosomes can be visualized in interphase when the homologous sequences are repeated at least 50 times.Possible applications of interphase analysis to clinical cytogenetics and mutagenicity testing are discussed.