Active Centromeres Research Articles

CENP-C binds the alpha-satellite DNA in vivo at specific centromere domains.

CENP-C is a fundamental component of the centromere, highly conserved among species and necessary for the proper assembly of the kinetochore structure and for the metaphase-anaphase transition. Although CENP-C can bind DNA in vitro, the identification of the DNA sequences associated with it in vivo and the significance of such an interaction have been, until now, elusive. To address this problem we took advantage of a chromatin-immunoprecipitation procedure and applied this technique to human HeLa cells. Through this approach we could establish that: (1) CENP-C binds the alpha-satellite DNA selectively; (2) the CENP-C region between amino acids 410 and 537, previously supposed to contain a DNA-binding domain, is indeed required to perform such a function in vivo; and (3) the profile of the alpha-satellite DNA associated with CENP-C is essentially identical to that recognized by CENP-B. However, further biochemical and ultrastructural characterization of CENP-B/DNA and CENP-C/DNA complexes, relative to their DNA components and specific spatial distribution in interphase nuclei, surprisingly reveals that CENP-C and CENP-B associate with the same types of alpha-satellite arrays but in distinct non-overlapping centromere domains. Our results, besides extending previous observations on the role of CENP-C in the formation of active centromeres, show, for the first time, that CENP-C can associate with the centromeric DNA sequences in vivo and, together with CENP-B, defines a highly structured organization of the alpha-satellite DNA within the human centromere.

Construction of neocentromere-based human minichromosomes for gene delivery and centromere studies.

Human neocentromeres are fully functional centromeres that arise naturally in non-centromeric regions devoid of alpha-satellite DNA. We have successfully produced a series of minichromosomes by telomere-associated truncation of a marker chromosome mardel(10) containing a neocentromere. The resulting minichromosomes are either linear or circular in nature, and range in size from approximately 650 kb to 2 Mb. These minichromosomes exhibit full centromeric activity, bind to essential centromere proteins, and are mitotically stable over many generations. They provide a useful system for dissecting the functional domains of complex eukaryotic centromeres and as vectors for therapeutic gene delivery.

Molecular cloning and sequence analysis of hamster CENP-A cDNA.

BackgroundThe centromere is a specialized locus that mediates chromosome movement during mitosis and meiosis. This chromosomal domain comprises a uniquely packaged form of heterochromatin that acts as a nucleus for the assembly of the kinetochore a trilaminar proteinaceous structure on the surface of each chromatid at the primary constriction. Kinetochores mediate interactions with the spindle fibers of the mitotic apparatus. Centromere protein A (CENP-A) is a histone H3-like protein specifically located to the inner plate of kinetochore at active centromeres. CENP-A works as a component of specialized nucleosomes at centromeres bound to arrays of repeat satellite DNA.ResultsWe have cloned the hamster homologue of human and mouse CENP-A. The cDNA isolated was found to contain an open reading frame encoding a polypeptide consisting of 129 amino acid residues with a C-terminal histone fold domain highly homologous to those of CENP-A and H3 sequences previously released. However, significant sequence divergence was found at the N-terminal region of hamster CENP-A that is five and eleven residues shorter than those of mouse and human respectively. Further, a human serine 7 residue, a target site for Aurora B kinase phosphorylation involved in the mechanism of cytokinesis, was not found in the hamster protein. A human autoepitope at the N-terminal region of CENP-A described in autoinmune diseases is not conserved in the hamster protein.ConclusionsWe have cloned the hamster cDNA for the centromeric protein CENP-A. Significant differences on protein sequence were found at the N-terminal tail of hamster CENP-A in comparison with that of human and mouse. Our results show a high degree of evolutionary divergence of kinetochore CENP-A proteins in mammals. This is related to the high diverse nucleotide repeat sequences found at the centromere DNA among species and support a current centromere model for kinetochore function and structural plasticity.

CENP-A, -B, and -C chromatin complex that contains the I-type alpha-satellite array constitutes the prekinetochore in HeLa cells.

CENP-A is a component of centromeric chromatin and defines active centromere regions by forming centromere-specific nucleosomes. We have isolated centromeric chromatin containing the CENP-A nucleosome, CENP-B, and CENP-C from HeLa cells using anti-CENP-A and/or anti-CENP-C antibodies and shown that the CENP-A/B/C complex is predominantly formed on alpha-satellite DNA that contains the CENP-B box (alphaI-type array). Mapping of hypersensitive sites for micrococcal nuclease (MNase) digestion indicated that CENP-A nucleosomes were phased on the alphaI-type array as a result of interactions between CENP-B and CENP-B boxes, implying a repetitive configuration for the CENP-B/CENP-A nucleosome complex. Molecular mass analysis by glycerol gradient sedimentation showed that MNase digestion released a CENP-A/B/C chromatin complex of three to four nucleosomes into the soluble fraction, suggesting that CENP-C is a component of the repetitive CENP-B/CENP-A nucleosome complex. Quantitative analysis by immunodepletion of CENP-A nucleosomes showed that most of the CENP-C and approximately half the CENP-B took part in formation of the CENP-A/B/C chromatin complex. A kinetic study of the solubilization of CENPs showed that MNase digestion first released the CENP-A/B/C chromatin complex into the soluble fraction, and later removed CENP-B and CENP-C from the complex. This result suggests that CENP-A nucleosomes form a complex with CENP-B and CENP-C through interaction with DNA. On the basis of these results, we propose that the CENP-A/B/C chromatin complex is selectively formed on the I-type alpha-satellite array and constitutes the prekinetochore in HeLa cells.

Active, but not inactive, human centromeres display topoisomerase II activity in vivo.

Eukaryotic centromeres are composed of centromere DNA and the multiple proteins directly or indirectly associated with it. One important DNA-binding protein in the centromere is DNA topoisomerase II (topo II). In the genome in general, topo II has two functions, one structural and one enzymatic, the latter catalyzing DNA strand-passage reactions. It has been demonstrated that topo II accumulates at centromeres during the first part of mitosis, and disappears again at anaphase, but it has not been clear whether it serves a structural or an enzymatic function at the centromere. To investigate this issue, we developed the topo II-induced self-primed in situ assay (Topo-SPRINS). In this assay, DNA breaks created by topo II are stabilized with the topo II inhibitor VM-26 in vivo, and used as 'primers' for localized DNA synthesis in vitro. The assay revealed that topo II has enzymatic activity at mitotic centromeres and that the activity is relatively constant across centromeres. Furthermore, the activity was observed at a neocentromere, and, in multicentric chromosomes, the activity was restricted to the active centromere. The topo II activity is thus selectively present at functioning centromeres, indicating that it plays a role in mitotic centromere function.

Localization of human SMC1 protein at kinetochores.

Proper cohesion of sister chromatids is prerequisite for correct segregation of chromosomes during cell division. The cohesin multiprotein complex, conserved in eukaryotes, is required for sister chromatid cohesion. Human cohesion is composed of a stable heterodimer of the structural maintenance of chromosomes (SMC) family proteins, hSMC1 and hSMC3, and non-SMC components, hRAD21 and SA1 (or SA2). In yeast, cohesion associates with chromosomes from late G1 to metaphase and is required for the establishment and maintenance of both chromosome arm and centromeric cohesion. However, in human cells, the majority of cohesion dissociates from chromosomes before mitosis. Although it was recently shown that a small amount of hRAD21 localizes to the centromeres during metaphase, the presence of other cohesion components at the centromere has not been demonstrated in human cells. Here we report the mitosis-specific localization of hSMC1 to the kinetochores. hSMC1 is targeted to the kinetochore region during prophase concomitant with kinetochore assembly and remains through anaphase. Importantly, hSMC1 is targeted only to the active centromere on dicentric chromosomes. These results suggest that hSMC1 is an integral component of the functional kinetochore structure during mitosis.

Visualizing nuclear proteins together with transcribed and inactive genes in structurally preserved cells

Recent data support the idea that the mammalian nucleus is organized in a functionally significant way. Immunocytochemistry has revealed the existence of diverse nuclear “bodies” and compartments. Fluorescence in situ hybridization (FISH) has shown that chromosomes change their spatial relationships during dynamic cell cycle progression and that nuclear organization can change during development and differentiation when patterns of gene expression are established or modified. To determine the relationship between nuclear organization and genome function is an important goal for biology. This article describes an immunoFISH technique, which is a useful tool for investigating the functional organization of the nucleus. It combines immunocytochemistry with FISH to allow associations between proteins, DNA, and RNA to be visualized in a single-step analysis using confocal microscopy. Immunocytochemistry and FISH were thought incompatible since cell preparation flattens nuclei and the harsh DNA denaturation treatment required for FISH destroys proteins. The immunoFISH technique successfully overcomes these problems and can reveal interactions between nuclear components not readily detectable using other experimental approaches. The interactions of single-copy, endogenous loci with nuclear proteins or bodies can be seen, as can spatial compartmentalization of these loci, in cells preserved in three dimensions, representative of the situation in vivo. Allelic differences in transcription can be related to nuclear location and protein interactions of the individual alleles since genes, RNA and proteins can be visualized together. Chromosome behavior can be followed through mitosis to investigate centromere activity or vector segregation efficiency, for example. Visual data obtained using the immunoFISH technique have provided insight into the functional significance of nuclear organization and its role in cell biology.

The S. pombe aurora-related kinase Ark1 associates with mitotic structures in a stage dependent manner and is required for chromosome segregation.

Metazoans contain three aurora-related kinases. Aurora A is required for spindle formation while aurora B is required for chromosome condensation and cytokinesis. Less is known about the function of aurora C. S. pombe contains a single aurora-related kinase, Ark1. Although Ark1 protein levels remained constant as cells progressed through the mitotic cell cycle, its distribution altered during mitosis and meiosis. Throughout G2 Ark1 was concentrated in one to three nuclear foci that were not associated with the spindle pole body/centromere complex. Following commitment to mitosis Ark1 associated with chromatin and was particularly concentrated at several sites including kinetochores/centromeres. Kinetochore/centromere association diminished during anaphase A, after which it was distributed along the spindle. The protein became restricted to a small central zone that transiently enlarged as the spindle extended. As in many other systems mitotic fission yeast cells exhibit a much greater degree of phosphorylation of serine 10 of histone H3 than interphase cells. A number of studies have linked this modification with chromosome condensation. Ark1 immuno-precipitates phosphorylated serine 10 of histone H3 in vitro. This activity was highest in mitotic extracts. The absence of the histone H3 phospho-serine 10 epitope from mitotic cells in which the ark1(+) gene had been deleted (ark1.Delta1); the inability of these cells to resolve their chromosomes during anaphase and the co-localisation of this phospho-epitope with Ark1 early in mitosis, all suggest that Ark1 phosphorylates serine 10 of histone H3 in vivo. ark1.Delta1 cells also exhibited a reduction in kinetochore activity and a minor defect in spindle formation. Thus the enzyme activity, localisation and phenotype arising from our manipulations of this single fission yeast aurora kinase family member suggest that this single kinase is executing functions that are separately implemented by distinct aurora A and aurora B kinases in higher systems.

A well-differentiated liposarcoma with a new type of chromosome 12-derived markers

Well-differentiated liposarcomas (WDLPS) are cytogenetically characterized by the presence of supernumerary ring or giant rod marker chromosomes. These supernumerary chromosomes are composed of amplified sequences from chromosome 12 (12q14∼15) in association with amplified segments from various other chromosomes, and contain alterations of the alpha satellite sequences. We report a case of WDLPS of the lipoma-like and sclerosing subtype that contains a novel type of supernumerary marker chromosome. Instead of rings or giant rods, these cells had three apparently identical copies of a subtelocentric supernumerary marker with a size and shape similar to C-group chromosomes. Fluorescence in situ hybridization analysis revealed that the markers were composed of amplified material from 12q14∼15, including the genes MDM2 and CDK4. Similar to the rings and giant rods observed in other WDLPS cases, these unusual markers had no alpha satellite repeats at the primary constriction site, but centromeric activity could be demonstrated by using anti-centromere protein C antibodies. These findings show that the supernumerary markers of WDLPS may be variable in size and shape, but consistently share the same genomic structure, specifically 12q amplified sequences together with centromere alterations, and underline the importance of molecular methods in the diagnosis of adipose tissue tumors.

Specification of kinetochore-forming chromatin by the histone H3 variant CENP-A.

The mechanisms that specify precisely where mammalian kinetochores form within arrays of centromeric heterochromatin remain largely unknown. Localization of CENP-A exclusively beneath kinetochore plates suggests that this distinctive histone might direct kinetochore formation by altering the structure of heterochromatin within a sub-region of the centromere. To test this hypothesis, we experimentally mistargeted CENP-A to non-centromeric regions of chromatin and determined whether other centromere-kinetochore components were recruited. CENP-A-containing non-centromeric chromatin assembles a subset of centromere-kinetochore components, including CENP-C, hSMC1, and HZwint-1 by a mechanism that requires the unique CENP-A N-terminal tail. The sequence-specific DNA-binding protein CENP-B and the microtubule-associated proteins CENP-E and HZW10 were not recruited, and neocentromeric activity was not detected. Experimental mistargeting of CENP-A to inactive centromeres or to acentric double-minute chromosomes was also not sufficient to assemble complete kinetochore activity. The recruitment of centromere-kinetochore proteins to chromatin appears to be a unique function of CENP-A, as the mistargeting of other components was not sufficient for assembly of the same complex. Our results indicate at least two distinct steps in kinetochore assembly: (1) precise targeting of CENP-A, which is sufficient to assemble components of a centromere-prekinetochore scaffold; and (2) targeting of kinetochore microtubule-associated proteins by an additional mechanism present only at active centromeres.

The chromokinesin Kid is necessary for chromosome arm orientation and oscillation, but not congression, on mitotic spindles.

Chromokinesins have been postulated to provide the polar ejection force needed for chromosome congression during mitosis. We have evaluated that possibility by monitoring chromosome movement in vertebrate-cultured cells using time-lapse differential interference contrast microscopy after microinjection with antibodies specific for the chromokinesin Kid. 17.5% of cells injected with Kid-specific antibodies have one or more chromosomes that remain closely opposed to a spindle pole and fail to enter anaphase. In contrast, 82.5% of injected cells align chromosomes in metaphase, progress to anaphase, and display chromosome velocities not significantly different from control cells. However, injected cells lack chromosome oscillations, and chromosome orientation is atypical because chromosome arms extend toward spindle poles during both congression and metaphase. Furthermore, chromosomes cluster into a mass and fail to oscillate when Kid is perturbed in cells containing monopolar spindles. These data indicate that Kid generates the polar ejection force that pushes chromosome arms away from spindle poles in vertebrate-cultured cells. This force increases the efficiency with which chromosomes make bipolar spindle attachments and regulates kinetochore activities necessary for chromosome oscillation, but is not essential for chromosome congression.

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.

The activation of a neocentromere in Drosophila requires proximity to an endogenous centromere.

The centromere is essential for proper segregation and inheritance of genetic information. Centromeres are generally regulated to occur exactly once per chromosome; failure to do so leads to chromosome loss or damage and loss of linked genetic material. The mechanism for faithful regulation of centromere activity and number is unknown. The presence of ectopic centromeres (neocentromeres) has allowed us to probe the requirements and characteristics of centromere activation, maintenance, and structure. We utilized chromosome derivatives that placed a 290-kilobase "test segment" in three different contexts within the Drosophila melanogaster genome--immediately adjacent to (1) centromeric chromatin, (2) centric heterochromatin, or (3) euchromatin. Using irradiation mutagenesis, we freed this test segment from the source chromosome and genetically assayed whether the liberated "test fragment" exhibited centromere activity. We observed that this test fragment behaved differently with respect to centromere activity when liberated from different chromosomal contexts, despite an apparent sequence identity. Test segments juxtaposed to an active centromere produced fragments with neocentromere activity, whereas test segments far from centromeres did not. Once established, neocentromere activity was stable. The imposition of neocentromere activity on juxtaposed DNA supports the hypothesis that centromere activity and identity is capable of spreading and is regulated epigenetically.

Centromeres

The centromere is the chromosomal site which associates with spindle microtubules and is responsible for equal division of the chromosomes to both daughter cells. Centromeres can often be recognised cytologically as a distinctive narrow region on metaphase chromosomes known as the primary constriction. The terms ‘centromere’ and ‘kinetochore’ are often used synonymously but it is best to consider the centromere as the DNA and chromatin on which the kinetochore is assembled and the kinetochore as the protein machine, including centromeric chromatin, which mediates attachment to the spindle. Centromeres are found near the centre of the chromosome on metacentric chromosomes, but they can also be submetacentric, between the middle and an end; acrocentric, close to an end; and telocentric, at an end. Some organisms such as Caenorhabditis elegans are holocentric and assemble centromeric chromatin and kinetochores along the entire chromosome. Delving further you discover that centromeres are eccentric! The centromere region is also a site of strong cohesion; in higher eukaryotes it is the last point of contact between sister-chromatids at metaphase prior to their separation at anaphase. Cohesion between sister centromeres is intimately associated with kinetochore function as it counteracts forces of microtubule–kinetochore attachment and possibly aids orientation and attachment of sister kinetochores to opposite poles. Centromeres are frequently associated with heterochromatin. Genes are silenced if placed in these regions by accident or design. It is not known why these regions are transcriptionally silent and whether silencing itself is important for centromere function. In fission yeast, at least, it appears that the silent state is a by-product of full kinetochore assembly. Centromeres range in size and complexity from a relatively minute 125 bp in budding yeast, through more complex structures of 40–120 kb in fission yeast, to enormous arrays of relatively simple repeats extending for 250 kb in flies and 5000 kb in humans. Regardless of size, they all assemble kinetochores that segregate chromosomes with awe-inspiring fidelity. The number of microtubules per metaphase kinetochore varies from one in budding yeast, 2–4 in fission yeast and from 4 to over 40 in mammals. DNA sequence is not conserved between centromeres of different organisms. However, all active centromeres have a histone H3-like protein, such as CENP-A, which must contribute to the assembly of a specialised centromere specific chromatin structure. Yes and no. Yes for yeast. But it's more complicated for mammalian centromeres. In humans vast arrays of so-called alphoid satellite repeats are found at centromeres, and mammalian artificial chromosomes have been created that contain alphoid DNA. So, you think, alphoid DNA is the determinant of centromeric activity! But in some dicentric chromosomes, both centromeres contain alphoid DNA, but only one is active, as assayed by the presence of important kinetochore components such as CENP-A and CENP-C. And in certain rare cases centromere activity has been gained by regions which have never before acted as centromeres. These neocentromeres contain no alphoid DNA but have been propagated through up to three human generations. These strange goings-on indicate that centromeres are epigenetically regulated. Presumably there is a meta-stable mark that normally ensures that centromeres and kinetochores do assemble at the same place division after division. Epigenetic phenomena also contribute to centromere function in fission yeast and Drosophila, and probably other organisms. Where can I find out more?

The spindle checkpoint of the yeast Saccharomyces cerevisiae requires kinetochore function and maps to the CBF3 domain.

We have measured the activity of the spindle checkpoint in null mutants lacking kinetochore activity in the yeast Saccharomyces cerevisiae. We constructed deletion mutants for nonessential genes by one-step gene replacements. We constructed heterozygous deletions of one copy of essential genes in diploid cells and purified spores containing the deletion allele. In addition, we made gene fusions for three essential genes to target the encoded proteins for proteolysis (degron alleles). We determined that Ndc10p, Ctf13p, and Cep3p are required for checkpoint activity. In contrast, cells lacking Cbf1p, Ctf19p, Mcm21p, Slk19p, Cse4p, Mif2p, Mck1p, and Kar3p are checkpoint proficient. We conclude that the kinetochore plays a critical role in checkpoint signaling in S. cerevisiae. Spindle checkpoint activity maps to a discreet domain within the kinetochore and depends on the CBF3 protein complex.

A Novel Chromatin Immunoprecipitation and Array (CIA) Analysis Identifies a 460-kb CENP-A-Binding Neocentromere DNA

Centromere protein A (CENP-A) is an essential histone H3-related protein that constitutes the specialized chromatin of an active centromere. It has been suggested that this protein plays a key role in the epigenetic marking and transformation of noncentromeric genomic DNA into functional neocentromeres. Neocentromeres have been identified on more than two-thirds of the human chromosomes, presumably involving different noncentromeric DNA sequences, but it is unclear whether some generalized sequence properties account for these neocentromeric sites. Using a novel method combining chromatin immunoprecipitation and genomic array hybridization, we have identified a 460-kb CENP-A-binding DNA domain of a neocentromere derived from the 20p12 region of an invdup (20p) human marker chromosome. Detailed sequence analysis indicates that this domain contains no centromeric α-satellite, classical satellites, or other known pericentric repetitive sequence motifs. Putative gene loci are detected, suggesting that their presence does not preclude neocentromere formation. The sequence is not significantly different from surrounding non-CENP-A-binding DNA in terms of the prevalence of various interspersed repeats and binding sites for DNA-interacting proteins (Topoisomerase II and High-Mobility-Group protein I). Notable variations include a higher AT content similar to that seen in human α-satellite DNA and a reduced prevalence of long terminal repeats (LTRs), short interspersed repeats (SINEs), and Alus. The significance of these features in neocentromerization is discussed.

A novel chromatin immunoprecipitation and array (CIA) analysis identifies a 460-kb CENP-A-binding neocentromere DNA.

Centromere protein A (CENP-A) is an essential histone H3-related protein that constitutes the specialized chromatin of an active centromere. It has been suggested that this protein plays a key role in the epigenetic marking and transformation of noncentromeric genomic DNA into functional neocentromeres. Neocentromeres have been identified on more than two-thirds of the human chromosomes, presumably involving different noncentromeric DNA sequences, but it is unclear whether some generalized sequence properties account for these neocentromeric sites. Using a novel method combining chromatin immunoprecipitation and genomic array hybridization, we have identified a 460-kb CENP-A-binding DNA domain of a neocentromere derived from the 20p12 region of an invdup (20p) human marker chromosome. Detailed sequence analysis indicates that this domain contains no centromeric alpha-satellite, classical satellites, or other known pericentric repetitive sequence motifs. Putative gene loci are detected, suggesting that their presence does not preclude neocentromere formation. The sequence is not significantly different from surrounding non-CENP-A-binding DNA in terms of the prevalence of various interspersed repeats and binding sites for DNA-interacting proteins (Topoisomerase II and High-Mobility-Group protein I). Notable variations include a higher AT content similar to that seen in human alpha-satellite DNA and a reduced prevalence of long terminal repeats (LTRs), short interspersed repeats (SINEs), and Alus. The significance of these features in neocentromerization is discussed.

Efficient male and female germline transmission of a human chromosomal vector in mice.

A small accessory chromosome that was mitotically stable in human fibroblasts was transferred into the hprt(-) hamster cell line CH and developed as a human chromosomal vector (HCV) by the introduction of a selectable marker and the 3' end of an HPRT minigene preceded by a loxP sequence. This HCV is stably maintained in the hamster cell line. It consists mainly of alphoid sequences of human chromosome 20 and a fragment of human chromosome region 1p22, containing the tissue factor gene F3. The vector has an active centromere, and telomere sequences are lacking. By transfecting a plasmid containing the 5' end of HPRT and a Cre-encoding plasmid into the HCV(+) hamster cell line, the HPRT minigene was reconstituted by Cre-mediated recombination and expressed by the cells. The HCV was then transferred to male mouse R1-ES cells and it did segregate properly. Chimeras were generated containing the HCV as an independent chromosome in a proportion of the cells. Part of the male and female offspring of the chimeras did contain the HCV. The HCV(+) F1 animals harbored the extra chromosome in >80% of the cells. The HCV was present as an independent chromosome with an active centromere and the human F3 gene was expressed from the HCV in a human-tissue-specific manner. Both male and female F1 mice did transmit the HCV to F2 offspring as an independent chromosome with properties similar to the original vector. This modified small accessory chromosome, thus, shows the properties of a useful chromosomal vector: It segregates stably as an independent chromosome, sequences can be inserted in a controlled way and are expressed from the vector, and the HCV is transmitted through the male and female germline in mice.

Histone H3 phosphorylation of mammalian chromosomes.

Inferences about the role and location of phosphorylated histone H3 are derived primarily from biochemical studies. A few direct observations at chromosome level have shown that phosphorylation begins at the site of heterochromatin and spreads throughout the chromosome. However, a comparative study of chromosomes of mouse (L929 cells), Chinese hamster (CHO 9 cells) and the Indian muntjac (male cells) reveals some distinguishable details among mammalian species. Whereas the L929 cells exhibit the typical pattern of phosphorylation at the region of centromeric heterochromatin associated with the active centromere, the heterochromatin blocks associated with the inactive centromeres also show label of about equivalent intensity. Throughout the cell cycle, heterochromatin exhibits sharper (denser) and better defined label than does euchromatin which expresses somewhat diffuse label. The centromere constriction on biarmed chromosomes, originating in Robertsonian translocations, appears phosphorylated in some, if not all chromosomes. A similar situation was found for the CHO 9 cells indicating that phosphorylation does include the region in which H3 is supposedly replaced by CENP-A. An interesting feature of the CHO cell line was the dense label at and near the telomeres; this feature was not observed in either the mouse or the Indian muntjac. The centromere regions of the Indian muntjac chromosomes showed three sites of label in the multicentric X chromosome and two each on chromosome pair number 1 and Y2; the sites coinciding with the reaction sites of antikinetochore antibodies. Also, the X and Y, chromosomes of Indian muntjac show intense phosphorylation at the sites of secondary constrictions. The chromosomes of all three species were phosphorylated throughout the cell cycle. As the chromosomes started to decondense during anaphase, heavy phosphorylation was observed in the form of discontinuous beaded structures indicating partial despiralization of the chromosome. Interestingly, when cells had completed karyokinesis and resolved into two independent nuclei, the phosphorylation was observed at the midbody. At this stage, the cytoplasm appeared to be again phosphorylated.

Only Centromeres Can Supply the Partition System Required for ARS Function in the Yeast Yarrowia lipolytica

Autonomously replicating sequences ( ARSs) in the yeast Yarrowia lipolytica require two components: an origin of replication ( ORI) and centromere ( CEN) DNA, both of which are necessary for extrachromosomal maintenance. To investigate this cooperation in more detail, we performed a screen for genomic sequences able to confer high frequency of transformation to a plasmid-borne ORI. Our results confirm a cooperation between ORI and CEN sequences to form an ARS, since all sequences identified in this screen displayed features of centromeric DNA and included the previously characterized CEN1-1, CEN3-1 and CEN5-1 fragments. Two new centromeric DNAs were identified as they are unique, map to different chromosomes (II and IV) and induce chromosome breakage after genomic integration. A third sequence, which is adjacent to, but distinct from the previously characterized CEN1-1 region was isolated from chromosome I. Although these CEN sequences do not share significant sequence similarities, they display a complex pattern of short repeats, including conserved blocks of 9 to 14 bp and regions of dyad symmetry. Consistent with their A+T-richness and strong negative roll angle, Y. lipolytica CEN -derived sequences, but not ORIs, were capable of binding isolated Drosophila nuclear scaffolds. However, a Drosophila scaffold attachment region that functions as an ARS in other yeasts was unable to confer autonomous replication to an ORI-containing plasmid. Deletion analysis of CEN1-1 showed that the sequences responsible for the induction of chromosome breakage could be eliminated without compromising extrachromosomal maintenance. We propose that, while Y. lipolytica CEN DNA is essential for plasmid maintenance, this function can be supplied by several sub-fragments which, together, form the active chromosomal centromere. This complex organization of Y. lipolytica centromeres is reminiscent of the regional structures described in the yeast Schizosaccharomyces pombe or in multicellular eukaryotes.