Centromere separation and association in the nuclei of an interspecific hybrid between Torenia fournieri and T. baillonii (Scrophulariaceae) during mitosis and meiosis

In the nuclei of some interspecific hybrid and allopolyploid plant species, each genome occupies a separate spatial domain. To analyze this phenomenon, we studied localization of the centromeres in the nuclei of a hybrid between Torenia fournieri and T. baillonii during mitosis and meiosis using three-dimensional fluorescence in situ hybridization (3D-FISH) probed with species-specific centromere repeats. Centromeres of each genome were located separately in undifferentiated cells but not differentiated cells, suggesting that cell division might be the possible force causing centromere separation. However, no remarkable difference of dividing distance was detected between chromatids with different centromeres in anaphase and telophase, indicating that tension of the spindle fiber attached to each chromatid is not the cause of centromere separation in Torenia. In differentiated cells, centromeres in both genomes were not often observed for the expected chromosome number, indicating centromere association. In addition, association of centromeres from the same genome was observed at a higher frequency than between different genomes. This finding suggests that centromeres within one genome are spatially separated from those within the other. This close position may increase possibility of association between centromeres of the same genome. In meiotic prophase, all centromeres irrespective of the genome were associated in a certain portion of the nucleus. Since centromere association in the interspecific hybrid and amphiploid was tighter than that in the diploid parents, it is possible that this phenomenon may be involved in sorting and pairing of homologous chromosomes.     

Decondensation of pericentric heterochromatin alters the sequence of centromere separation in mouse cells

The centromeres of a genome separate in a sequential, nonrandom manner that is apparently dependent upon the quantity and quality of pericentric heterochromatin. It is becoming increasingly clear that the biological properties of a centromere depend upon its physicochemical makeup, such as its tertiary structure, and not necessarily on its particular nucleotide sequence. To test this idea we altered the physical state of the AT-rich pericentric heterochromatin of mouse with Hoechst 33258 (bis-benzimidazole) and studied a biological parameter, viz., sequence of separation. We report that an alteration in the physical state of heterochromatin, i.e., decondensation, is accompanied by aberrations in the pattern of centromere separation. The most dramatic effect seems to be on chromosomes with large blocks of heterochromatin. Many chromosomes with large blocks of heterochromatin that, in untreated cells, separate late tend to separate early. Decondensation with Hoechst 33258 does not seem to alter the sequence of separation of inactive centromeres relative to that of active centromeres. These data indicate that alteration in the physical parameters of the pericentric heterochromatin might dispose the centromeres to errors. It is likely that this aberration results from early replication of the pericentric heterochromatin associated with active centromeres. Received: 24 August 1998; in revised form: 24 August 1998 / Accepted: 28 August 1998     

Strong nucleosomes of mouse genome including recovered centromeric sequences

Recently discovered strong nucleosomes (SNs) characterized by visibly periodical DNA sequences have been found to concentrate in centromeres of Arabidopsis thaliana and in transient meiotic centromeres of Caenorhabditis elegans. To find out whether such affiliation of SNs to centromeres is a more general phenomenon, we studied SNs of the Mus musculus. The publicly available genome sequences of mouse, as well as of practically all other eukaryotes do not include the centromere regions which are difficult to assemble because of a large amount of repeat sequences in the centromeres and pericentromeric regions. We recovered those missing sequences using the data from MNase-seq experiments in mouse embryonic stem cells, where the sequence of DNA inside nucleosomes, including missing regions, was determined by 100-bp paired-end sequencing. Those nucleosome sequences, which are not matching to the published genome sequence, would largely belong to the centromeres. By evaluating SN densities in centromeres and in non-centromeric regions, we conclude that mouse SNs concentrate in the centromeres of telocentric mouse chromosomes, with ~3.9 times excess compared to their density in the rest of the genome. The remaining non-centromeric SNs are harbored mainly by introns and intergenic regions, by retro-transposons, in particular. The centromeric involvement of the SNs opens new horizons for the chromosome and centromere structure studies.     

Telomere Disruption Results in Non-Random Formation of De Novo Dicentric Chromosomes Involving Acrocentric Human Chromosomes

Genome rearrangement often produces chromosomes with two centromeres (dicentrics) that are inherently unstable because of bridge formation and breakage during cell division. However, mammalian dicentrics, and particularly those in humans, can be quite stable, usually because one centromere is functionally silenced. Molecular mechanisms of centromere inactivation are poorly understood since there are few systems to experimentally create dicentric human chromosomes. Here, we describe a human cell culture model that enriches for de novo dicentrics. We demonstrate that transient disruption of human telomere structure non-randomly produces dicentric fusions involving acrocentric chromosomes. The induced dicentrics vary in structure near fusion breakpoints and like naturally-occurring dicentrics, exhibit various inter-centromeric distances. Many functional dicentrics persist for months after formation. Even those with distantly spaced centromeres remain functionally dicentric for 20 cell generations. Other dicentrics within the population reflect centromere inactivation. In some cases, centromere inactivation occurs by an apparently epigenetic mechanism. In other dicentrics, the size of the α-satellite DNA array associated with CENP-A is reduced compared to the same array before dicentric formation. Extra-chromosomal fragments that contained CENP-A often appear in the same cells as dicentrics. Some of these fragments are derived from the same α-satellite DNA array as inactivated centromeres. Our results indicate that dicentric human chromosomes undergo alternative fates after formation. Many retain two active centromeres and are stable through multiple cell divisions. Others undergo centromere inactivation. This event occurs within a broad temporal window and can involve deletion of chromatin that marks the locus as a site for CENP-A maintenance/replenishment.     

Sequence of centromere separation: influence of pericentromeric heterochromatin (repetitive DNA) inMus

A relationship between the sequence of centromere separation and quantity of pericentromeric constitutive heterochromatin was studied using bone marrow cells ofMus musculus molossinus and three cell lines, viz., SEWA-Rec 4, brain tumor and L-cells, ofM. m. domesticus origin. The timing of separation of a centromere into two daughter centromeres is related to the quantity of pericentromeric heterochromatin. In these genomes, having qualitatively uniform DNA in their heterochromatin fraction, the chromosomes with none or small quantities of heterochromatin separate first. These are followed by those chromosomes which have increasingly larger quantities of heterochromatin. It appears that one function of repetitive DNA (pericentromeric heterochromatin) is to regulate the timing of separation of centromeres.     

An SMC ATPase mutant disrupts chromosome segregation in Caulobacter

Accurate replication and segregation of the bacterial genome are essential for cell cycle progression. We have identified a single amino acid substitution in the Caulobacter structural maintenance of chromosomes (SMC) protein that disrupts chromosome segregation and cell division. The E1076Q point mutation in the SMC ATPase domain caused a dominant-negative phenotype in which DNA replication was able to proceed, but duplicated parS centromeres, normally found at opposite cell poles, remained at one pole. The cellular positions of other chromosomal loci were in the wild-type order relative to the parS centromere, but chromosomes remained unsegregated and appeared to be stacked upon one another. Purified SMC-E1076Q was deficient in ATP hydrolysis and exhibited abnormally stable binding to DNA. We propose that SMC spuriously links the duplicated chromosome immediately after passage of the replication fork. In wild-type cells, ATP hydrolysis opens the SMC dimer, freeing one chromosome to segregate to the opposite pole. The loss of ATP hydrolysis causes the SMC-E1076Q dimer to remain bound to both chromosomes, inhibiting segregation.     

CENP-B interacts with CENP-C domains containing Mif2 regions responsible for centromere localization

Recently, human artificial chromosomes featuring functional centromeres have been generated efficiently from naked synthetic alphoid DNA containing CENP-B boxes as a de novo mechanism in a human cultured cell line, but not from the synthetic alphoid DNA only containing mutations within CENP-B boxes, indicating that CENP-B has some functions in assembling centromere/kinetochore components on alphoid DNA. To investigate whether any interactions exist between CENP-B and the other centromere proteins, we screened a cDNA library by yeast two-hybrid analysis. An interaction between CENP-B and CENP-C was detected, and the CENP-C domains required were determined to overlap with three Mif2 homologous regions, which were also revealed to be involved in the CENP-C assembly of centromeres by expression of truncated polypeptides in cultured cells. Overproduction of truncated CENP-B containing no CENP-C interaction domains caused abnormal duplication of CENP-C domains at G2 and cell cycle delay at metaphase. These results suggest that the interaction between CENP-B and CENP-C may be involved in the correct assembly of CENP-C on alphoid DNA. In other words, a possible molecular linkage may exist between one of the kinetochore components and human centromere DNA through CENP-B/CENP-B box interaction.     

Sequence of centromere separation: differential replication of pericentric heterochromatin in multicentric chromosomes

The dicentric and multicentric chromosomes in L cells and a brain tumor cell line of mouse display only one site of kinetochore formation associated with the active centromere. The accessory or inactive centromeres show premature separation. These cell lines were treated with 10^–6 M 5-bromodeoxyuridine (BrdUrd) followed by anti-BrdUrd antibody to study the pattern of replication of pericentric heterochromatin flanking the active vs inactive centromeres. Regardless of its quantity, heterochromatin around the inactive centromere replicates earlier than that associated with the active centromere. There appears to be a relationship between the timing of separation of a centromere and the timing of replication of pericentric heterochromatin. The premature replication of heterochromatin associated with an inactive centromere may be responsible for its premature separation and, hence, inactivity.     

Cd bands and centromeric function in dicentric chromosomes

Summary The Cd technique was applied to two cases of dicentric attached X chromosomes (XpXp and XqXq) and to cells from an established cell line of tumor origin (MaNo9) in which dicentrics with two active centromeres were present and dicentrics with one active and one inactive centromere. It was confirmed that the Cd technique discriminates between active and latent centromeres, and it was demonstrated that true dicentrics and dicentrics with one latent centromere can co-exist in the same cells. This indicates that the mechanism of centromere inactivation is a phenomenon that is specific to each chromosome and not generalized at the level of the individual cell.     

Robertsonian Fusion and Centromere Repositioning Contributed to the Formation of Satellite-free Centromeres During the Evolution of Zebras

Centromeres are epigenetically specified by the histone H3 variant CENP-A and typically associated with highly repetitive satellite DNA. We previously discovered natural satellite-free neocentromeres in Equus caballus and Equus asinus. Here, through ChIP-seq with an anti-CENP-A antibody, we found an extraordinarily high number of centromeres lacking satellite DNA in the zebras Equus burchelli (15 of 22) and Equus grevyi (13 of 23), demonstrating that the absence of satellite DNA at the majority of centromeres is compatible with genome stability and species survival and challenging the role of satellite DNA in centromere function. Nine satellite-free centromeres are shared between the two species in agreement with their recent separation. We assembled all centromeric regions and improved the reference genome of E. burchelli. Sequence analysis of the CENP-A binding domains revealed that they are LINE-1 and AT-rich with four of them showing DNA amplification. In the two zebras, satellite-free centromeres emerged from centromere repositioning or following Robertsonian fusion. In five chromosomes, the centromeric function arose near the fusion points, which are located within regions marked by traces of ancestral pericentromeric sequences. Therefore, besides centromere repositioning, Robertsonian fusions are an important source of satellite-free centromeres during evolution. Finally, in one case, a satellite-free centromere was seeded on an inversion breakpoint. At 11 chromosomes, whose primary constrictions seemed to be associated with satellite repeats by cytogenetic analysis, satellite-free neocentromeres were instead located near the ancestral inactivated satellite-based centromeres; therefore, the centromeric function has shifted away from a satellite repeat containing locus to a satellite-free new position.     

Possible causes of variation in trivalent orientation frequencies in pearl millet and rye

In pearl millet, chain trivalents composed of two telocentric and one metacentric chromosomes, showed an excess of linear orientation over the 1/3 expected with random centromere activation and inactivity of a central centromere stretched between the two outer centromeres. Chain trivalents composed of two metacentrics and one telo or of three metacentrics behaved as predicted. The difference was explained by assuming precocious activation of completely terminal centromeres as opposed to median centromeres. This early activity was reflected in precocious separation at late metaphase. In rye, all trivalents composed of two telos and one metacentric showed alternate orientation and anaphase separation did not precede that of metacentric chromosomes. It is concluded that in rye terminal centromeres are not precocious and that the spindle at meiosis is not long enough to permit stretching of the central centromere, which consequently always has the opportunity to orient and to induce the other centromeres to choose the opposite pole either directly or after reorientation, accumulating the most stable (alternate) orientation type.     

Chromatin assembly at kinetochores is uncoupled from DNA replication

The specification of metazoan centromeres does not depend strictly on centromeric DNA sequences, but also requires epigenetic factors. The mechanistic basis for establishing a centromeric "state" on the DNA remains unclear. In this work, we have directly examined replication timing of the prekinetochore domain of human chromosomes. Kinetochores were labeled by expression of epitope-tagged CENP-A, which stably marks prekinetochore domains in human cells. By immunoprecipitating CENP-A mononucleosomes from synchronized cells pulsed with [(3)H]thymidine we demonstrate that CENP-A-associated DNA is replicated in mid-to-late S phase. Cytological analysis of DNA replication further demonstrated that centromeres replicate asynchronously in parallel with numerous other genomic regions. In contrast, quantitative Western blot analysis demonstrates that CENP-A protein synthesis occurs later, in G2. Quantitative fluorescence microscopy and transient transfection in the presence of aphidicolin, an inhibitor of DNA replication, show that CENP-A can assemble into centromeres in the absence of DNA replication. Thus, unlike most genomic chromatin, histone synthesis and assembly are uncoupled from DNA replication at the kinetochore. Uncoupling DNA replication from CENP-A synthesis suggests that regulated chromatin assembly or remodeling could play a role in epigenetic centromere propagation.     

Sequence of centromere separation: characterization of multicentric chromosomes in a rat cell line

The B₁ cell line of rat cerebral endothelium origin exhibits several dicentric and multicentric chromosomes. These chromosomes, unlike multicentrics in mouse (Vig and Zinkowski 1986) do not show premature centromere separation. All centromeres deposit kinetochore proteins and appear to be functional. Even the centromeres which fail to migrate to the poles during anaphase and make side arm bridges bind to spindle microtubules. Some multicentric chromosomes show kinetochores spaced apart with intervening stretches of euchromatin while others are located adjacent to each other thus exhibiting tandem repeats and forming a compound kinetochore (Brinkeley et al. 1984). Also, unlike mouse multicentric chromosomes in which different pericentric regions and the centromeres replicate at different times, the rat chromosomes appear to replicate all pericentric and centric regions in a given multicentric simultaneously. The present studies indicate that centromeres in rat and mouse replicate during the last part of the S-phase and in continuation with the pericentric heterochromatin.     

Formation of primary constriction and heterochromatin in mouse does not require minor satellite DNA.

Whereas the major satellite fraction in mouse extends its domain from the centromere to the distal end of the pericentric heterochromatin, the minor satellite DNA is present specifically in the centromere or primary constriction. We hybridized the biotinylated minor satellite sequence to L929 cells of mouse origin. The sequence hybridized to all chromosomes. Whereas hybridization was detected on all active centromeres, the inactive centromeres in certain dicentrics did not show any signal. This satellite, however, was detected in all inactive centromeres in a heptacentric chromosome. The intensity of fluorescence on the inactive centromeres of the heptacentric was similar to that present on the active centromeres. Several heterochromatin blocks, which were not associated with any centromere, were also found to lack hybridization with the minor satellite. The inactive centromeres, whether carrying the minor satellite DNA fraction or not, generally do not react with the antikinetochore antibodies present in the scleroderma serum. These studies are interpreted to show that (1) the primary constriction in mouse can be formed without the participation of minor satellite, (2) heterochromatin in mouse may constitute without this fraction, (3) the major and minor satellite may not be interspersed but are joined at some defined boundary, and (4) the binding of CENP-B does not depend upon the quantity of minor satellite or the number of CENP boxes present in the inactive centromeres.     

Unusual chromosome cleavage dynamic in rodent neonatal germ cells

At the metaphase/anaphase transition in the mouse and rat male germ lines during the perinatal period, sister centromeres separate before sister chromatids. This gives the chromosomes an unusual appearance that resembles the premature centromere division described in some human pathological conditions such as Roberts syndrome. At the same period, there is also an unusual pattern of DNA methylation, with strongly demethylated heterochromatin and methylated euchromatin. This suggests that chromosome DNA methylation may modulate chromatid and centromere splitting, without altering normal chromosome segregation.     

Effects of excess centromeres and excess telomeres on chromosome loss rates.

The linear chromosomes of eukaryotes contain specialized structures to ensure their faithful replication and segregation to daughter cells. Two of these structures, centromeres and telomeres, are limited, respectively, to one and two copies per chromosome. It is possible that the proteins that interact with centromere and telomere DNA sequences are present in limiting amounts and could be competed away from the chromosomal copies of these elements by additional copies introduced on plasmids. We have introduced excess centromeres and telomeres into Saccharomyces cerevisiae and quantitated their effects on the rates of loss of chromosome III and chromosome VII by fluctuation analysis. We show that (i) 600 new telomeres have no effect on chromosome loss; (ii) an average of 25 extra centromere DNA sequences increase the rate of chromosome III loss from 0.4 x 10(-4) events per cell division to 1.3 x 10(-3) events per cell division; (iii) centromere DNA (CEN) sequences on circular vectors destabilize chromosomes more effectively than do CEN sequences on 15-kb linear vectors, and transcribed CEN sequences have no effect on chromosome stability. We discuss the different effects of extra centromere and telomere DNA sequences on chromosome stability in terms of how the cell recognizes these two chromosomal structures.     

Epigenetically-Inherited Centromere and Neocentromere DNA Replicates Earliest in S-Phase

Eukaryotic centromeres are maintained at specific chromosomal sites over many generations. In the budding yeast Saccharomyces cerevisiae, centromeres are genetic elements defined by a DNA sequence that is both necessary and sufficient for function; whereas, in most other eukaryotes, centromeres are maintained by poorly characterized epigenetic mechanisms in which DNA has a less definitive role. Here we use the pathogenic yeast Candida albicans as a model organism to study the DNA replication properties of centromeric DNA. By determining the genome-wide replication timing program of the C. albicans genome, we discovered that each centromere is associated with a replication origin that is the first to fire on its respective chromosome. Importantly, epigenetic formation of new ectopic centromeres (neocentromeres) was accompanied by shifts in replication timing, such that a neocentromere became the first to replicate and became associated with origin recognition complex (ORC) components. Furthermore, changing the level of the centromere-specific histone H3 isoform led to a concomitant change in levels of ORC association with centromere regions, further supporting the idea that centromere proteins determine origin activity. Finally, analysis of centromere-associated DNA revealed a replication-dependent sequence pattern characteristic of constitutively active replication origins. This strand-biased pattern is conserved, together with centromere position, among related strains and species, in a manner independent of primary DNA sequence. Thus, inheritance of centromere position is correlated with a constitutively active origin of replication that fires at a distinct early time. We suggest a model in which the distinct timing of DNA replication serves as an epigenetic mechanism for the inheritance of centromere position.     

Sequence of centromere separation of mitotic chromosomes in Chinese hamster

Chromosome preparations in late metaphase cells from bone marrow of colcemid treated male Chinese hamsters were used to analyse the sequence of separation of sister centromeres. Chromatids of chromosomes 2 and 1 are the first ones to separate at centromeres, followed by members of group B, D and C. Some acrocentric chromosome is always the last one to separate at the centromere. The data point to a possible correlation between the position of a centromere in the separation sequence in the genome and the amount of centromeric heterochromatin as well as relation to the phenomenon of non-disjunction.     

CENP-B binds a novel centromeric sequence in the Asian mouse Mus caroli.

Minor satellite DNA, found at Mus musculus centromeres, is not present in the genome of the Asian mouse Mus caroli. This repetitive sequence family is speculated to have a role in centromere function by providing an array of binding sites for the centromere-associated protein CENP-B. The apparent absence of CENP-B binding sites in the M. caroli genome poses a major challenge to this hypothesis. Here we describe two abundant satellite DNA sequences present at M. caroli centromeres. These satellites are organized as tandem repeat arrays, over 1 Mb in size, of either 60- or 79-bp monomers. All autosomes carry both satellites and small amounts of a sequence related to the M. musculus major satellite. The Y chromosome contains small amounts of both major satellite and the 60-bp satellite, whereas the X chromosome carries only major satellite sequences. M. caroli chromosomes segregate in M. caroli x M. musculus interspecific hybrid cell lines, indicating that the two sets of chromosomes can interact with the same mitotic spindle. Using a polyclonal CENP-B antiserum, we demonstrate that M. caroli centromeres can bind murine CENP-B in such an interspecific cell line, despite the absence of canonical 17-bp CENP-B binding sites in the M. caroli genome. Sequence analysis of the 79-bp M. caroli satellite reveals a 17-bp motif that contains all nine bases previously shown to be necessary for in vitro binding of CENP-B. This M. caroli motif binds CENP-B from HeLa cell nuclear extract in vitro, as indicated by gel mobility shift analysis. We therefore suggest that this motif also causes CENP-B to associate with M. caroli centromeres in vivo. Despite the sequence differences, M. caroli presents a third, novel mammalian centromeric sequence producing an array of binding sites for CENP-B.     

Dicentric chromosome formation and epigenetics of centromere formation in plants

Plant centromeres are generally composed of tandem arrays of simple repeats that form a complex chromosome locus where the kinetochore forms and microtubules attach during mitosis and meiosis. Each chromosome has one centromere region, which is essential for accurate division of the genetic material. Recently, chromosomes containing two centromere regions (called dicentric chromosomes) have been found in maize and wheat. Interestingly, some dicentric chromosomes are stable because only one centromere is active and the other one is inactivated. Because such arrays maintain their typical structure for both active and inactive centromeres, the specification of centromere activity has an epigenetic component independent of the DNA sequence. Under some circumstances, the inactive centromeres may recover centromere function, which is called centromere reactivation. Recent studies have highlighted the important changes, such as DNA methylation and histone modification, that occur during centromere inactivation and reactivation.