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     

Sequence of centromere separation: orderly separation of multicentric chromosomes in mouse L cells

Mouse L cells have many dicentric chromosomes and one with eight centromeres. All eight centromeres behave similarly until midmetaphase when most centromeres split into two units each in apparently quick succession but out-of-phase. This premature separation leaves one or perhaps two closely located centromeres intact, which separate at late metaphase-anaphase, drawing the two chromatids to opposite poles. Such dominance of one centromere over all others, though unexplained, ensures the lack of any mitotic abnormality such as bridges or fragments. These observations show that all the centromeres are retained as functional primary constrictions except for a change in functional regulation when more than one centromere are located on a chromosome.     

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.     

Sequence of Centromere Separation: Role of Centromeric Heterochromatin

The late metaphase-early anaphase cells from various tissues of male Mus musculus, M. poschiavinus, M. spretus, M. castaneus, female and male Bos taurus (cattle) and female Myopus schisticolor (wood lemming) were analyzed for centromeres that showed separation into two daughter centromeres and those that did not show such separation. In all strains and species of mouse the Y chromosome is the first one to separate, as is the X or Y in the cattle. These sex chromosomes are devoid of constitutive heterochromatin, whereas all autosomes in these species carry detectable quantities. In cattle, the late replicating X chromosome appears to separate later than the active X. In the wood lemming the three pairs of autosomes with the least amount of centromeric constitutive heterochromatin separate first. These are followed by the separation of seven pairs of autosomes carrying medium amounts of constitutive heterochromatin. Five pairs of autosomes with the largest amounts of constitutive heterochromatin are the last in the sequence of separation. The sex chromosomes with medium amounts of constitutive heterochromatin around the centromere, and a very large amount of distal heterochromatin, separate among the very late ones but are not the last. These observations assign a specific role to centromeric constitutive heterochromatin and also indicate that nonproximal heterochromatin does not exert control over the sequence in which the centromeres in the genome separate. It appears that qualitative differences among various types of constitutive heterochromatin are as important as quantitative differences in controlling the separation of centromeres.     

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.     

Sequence of centromere separation: Separation in a quasi-stable mouse-human somatic cell hybrid

A quasi-stable mouse-human hybrid cell line, HR61, containing between one and ten human chromosomes was analyzed for the sequence of centromere separation. The purpose was to determine which genome of the two initiates centromere separation first. The data clearly indicate that the separation of centromeres of the human genome is not only initiated but is completed before any centromeres from the mouse chromosomes start splitting into daughter units. The information on whether uniparental chromosome loss results from a lack of deposition of kinetochore proteins was equivocal. The human genome also completes its DNA replication before the mouse genome does. Our studies, therefore, show that the timing of centromere separation is tightly linked to the completion of replication of DNA. At least in this cell line the segregant genome is not the one which exhibits delayed DNA replication.     

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.     

Identification of a subdomain of CENP-B that is necessary and sufficient for localization to the human centromere

We have combined in vivo and in vitro approaches to investigate the function of CENP-B, a major protein of human centromeric heterochromatin. Expression of epitope-tagged deletion derivatives of CENP-B in HeLa cells revealed that a single domain less than 158 residues from the amino terminus of the protein is sufficient to localize CENP-B to centromeres. Centromere localization was abolished if as few as 28 amino acids were removed from the amino terminus of CENP-B. The centromere localization signal of CENP-B can function in an autonomous fashion, relocating a fused bacterial enzyme to centromeres. The centromere localization domain of CENP-B specifically binds in vitro to a subset of alpha-satellite DNA monomers. These results suggest that the primary mechanism for localization of CENP-B to centromeres involves the recognition of a DNA sequence found at centromeres. Analysis of the distribution of this sequence in alpha-satellite DNA suggests that CENP-B binding may have profound effects on chromatin structure at centromeres.     

Is the time dimension of the cell cycle re-entry in AD regulated by centromere cohesion dynamics?

Chromosomal involvement is a legitimate, yet not well understood, feature of Alzheimer disease (AD). Firstly, AD affects more women than men. Secondly, the amyloid-β protein precursor genetic mutations, responsible for a cohort of familial AD cases, reside on chromosome 21, the same chromosome responsible for the developmental disorder Down's syndrome. Thirdly, lymphocytes from AD patients display a novel chromosomal phenotype, namely premature centromere separation (PCS). Other documented morphological phenomena associated with AD include the occurrence of micronuclei, aneuploidy, binucleation, telomere instability, and cell cycle re-entry protein expression. Based on these events, here we present a novel hypothesis that the time dimension of cell cycle re-entry in AD is highly regulated by centromere cohesion dynamics. In view of the fact that neurons can re-enter the cell division cycle, our hypothesis predicts that alterations in the signaling pathway leading to premature cell death in neurons is a consequence of altered regulation of the separation of centromeres as a function of time. It is well known that centromeres in the metaphase anaphase transition separate in a non-random, sequential order. This sequence has been shown to be deregulated in aging cells, various tumors, syndromes of chromosome instability, following certain chemical inductions, as well as in AD. Over time, premature chromosome separation is both a result of, and a driving force behind, further cohesion impairment, activation of cyclin dependent kinases, and mitotic catastrophe–a vicious circle resulting in cellular degeneration and death.     

A satellite-like sequence,representing a “clone gap” in the human genome,was likely involved in the seeding of a novel centromere in macaque

Although the human genome sequence is generally considered “finished”, the latest assembly (NCBI Build 36.1) still presents a number of gaps. Some of them are defined as “clone gaps” because they separate neighboring contigs. Evolutionary new centromeres are centromeres that repositioned along the chromosome, without marker order variation, during evolution. We have found that one human “clone gap” at 18q21.2 corresponds to an evolutionary new centromere in Old World Monkeys (OWM). The partially sequenced gap revealed a satellite-like structure. DNA stretches of the same satellite were found in the macaque (flanking the chromosome 18 centromere) and in the marmoset (New World Monkey), which was used as an outgroup. These findings strongly suggested that the repeat was present at the time of novel centromere seeding in OWM ancestor. We have provided, therefore, the first instance of a specific sequence hypothesized to have played a role in triggering the emergence of an evolutionary new centromere. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Diversity in requirement of genetic and epigenetic factors for centromere function in fungi

A centromere is a chromosomal region on which several proteins assemble to form the kinetochore. The centromere-kinetochore complex helps in the attachment of chromosomes to spindle microtubules to mediate segregation of chromosomes to daughter cells during mitosis and meiosis. In several budding yeast species, the centromere forms in a DNA sequence-dependent manner, whereas in most other fungi, factors other than the DNA sequence also determine the centromere location, as centromeres were able to form on nonnative sequences (neocentromeres) when native centromeres were deleted in engineered strains. Thus, in the absence of a common DNA sequence, the cues that have facilitated centromere formation on a specific DNA sequence for millions of years remain a mystery. Kinetochore formation is facilitated by binding of a centromere-specific histone protein member of the centromeric protein A (CENP-A) family that replaces a canonical histone H3 to form a specialized centromeric chromatin structure. However, the process of kinetochore formation on the rapidly evolving and seemingly diverse centromere DNAs in different fungal species is largely unknown. More interestingly, studies in various yeasts suggest that the factors required for de novo centromere formation (establishment) may be different from those required for maintenance (propagation) of an already established centromere. Apart from the DNA sequence and CENP-A, many other factors, such as posttranslational modification (PTM) of histones at centric and pericentric chromatin, RNA interference, and DNA methylation, are also involved in centromere formation, albeit in a species-specific manner. In this review, we discuss how several genetic and epigenetic factors influence the evolution of structure and function of centromeres in fungal species.     

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.     

Displacement and re-accumulation of centromeric cohesin during transient pre-anaphase centromere splitting

The ring-shaped cohesin complex links sister chromatids until their timely segregation during mitosis. Cohesin is enriched at centromeres where it provides the cohesive counterforce to bipolar tension produced by the mitotic spindle. As a consequence of spindle tension, centromeric sequences transiently split in pre-anaphase cells, in some organisms up to several micrometers. This ‘centromere breathing’ presents a paradox, how sister sequences separate where cohesin is most enriched. We now show that in the budding yeast Saccharomyces cerevisiae, cohesin binding diminishes over centromeric sequences that split during breathing. We see no evidence for cohesin translocation to surrounding sequences, suggesting that cohesin is removed from centromeres during breathing. Two pools of cohesin can be distinguished. Cohesin loaded before DNA replication, which has established sister chromatid cohesion, disappears during breathing. In contrast, cohesin loaded after DNA replication is partly retained. As sister centromeres re-associate after transient separation, cohesin is reloaded in a manner independent of the canonical cohesin loader Scc2/Scc4. Efficient centromere re-association requires the cohesion establishment factor Eco1, suggesting that re-establishment of sister chromatid cohesion contributes to the dynamic behaviour of centromeres in mitosis. These findings provide new insights into cohesin behaviour at centromeres. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

A tale of two centromeres—diversity of structure but conservation of function in plants and animals

The structural and functional aspects of two specific centromeres, one drawn from the animal kingdom (Drosophila) and the other from the plant kingdom (maize), are compared. Both cases illustrate an epigenetic component to centromere specification. The observations of neocentromeres in Drosophila and inactive centromeres in maize constitute one line of evidence for this hypothesis. Another common feature is the divisibility of centromere function with reduced stability as the size decreases. The systems differ in that Drosophila has no common sequence repeat at all centromeres, whereas maize has a 150-bp unit present in tandem arrays together with a centromere-specific transposon, centromere retrotransposon maize, present at all primary constrictions. Aspects of centromere structure known only from one or the other system might be common to both, namely, the presence of centromere RNAs in the kinetochore as found in maize and the organization of the centromeric histone 3 in tetrameric nucleosomes.     

Vertebrate shugoshin links sister centromere cohesion and kinetochore microtubule stability in mitosis

Drosophila MEI-S332 and fungal Sgo1 genes are essential for sister centromere cohesion in meiosis I. We demonstrate that the related vertebrate Sgo localizes to kinetochores and is required to prevent premature sister centromere separation in mitosis, thus providing an explanation for the differential cohesion observed between the arms and the centromeres of mitotic sister chromatids. Sgo is degraded by the anaphase-promoting complex, allowing the separation of sister centromeres in anaphase. Intriguingly, we show that Sgo interacts strongly with microtubules in vitro and that it regulates kinetochore microtubule stability in vivo, consistent with a direct microtubule interaction. Sgo is thus critical for mitotic progression and chromosome segregation and provides an unexpected link between sister centromere cohesion and microtubule interactions at kinetochores.     

Plant centromeres: structure and control

Recent work has led to a better understanding of the molecular components of plant centromeres. Conservation of at least some centromere protein constituents between plant and non-plant systems has been demonstrated. The identity and organization of plant centromeric DNA sequences are also beginning to yield to analysis. While there is little primary DNA sequence conservation among the characterized plant centromeres and their non-plant counterparts, some parallels in centromere genomic organisation can be seen across species. Finally, the emerging idea that centromere activity is controlled epigenetically finds support in an examination of the plant centromere literature.     

Budding yeast chromosome structure and dynamics during mitosis

Using green fluorescent protein probes and rapid acquisition of high-resolution fluorescence images, sister centromeres in budding yeast are found to be separated and oscillate between spindle poles before anaphase B spindle elongation. The rates of movement during these oscillations are similar to those of microtubule plus end dynamics. The degree of preanaphase separation varies widely, with infrequent centromere reassociations observed before anaphase. Centromeres are in a metaphase-like conformation, whereas chromosome arms are neither aligned nor separated before anaphase. Upon spindle elongation, centromere to pole movement (anaphase A) was synchronous for all centromeres and occurred coincident with or immediately after spindle pole separation (anaphase B). Chromatin proximal to the centromere is stretched poleward before and during anaphase onset. The stretched chromatin was observed to segregate to the spindle pole bodies at rates greater than centromere to pole movement, indicative of rapid elastic recoil between the chromosome arm and the centromere. These results indicate that the elastic properties of DNA play an as of yet undiscovered role in the poleward movement of chromosome arms.     

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.     

Genetic and genomic analysis of the AT-rich centromere DNA element II of Saccharomyces cerevisiae

Centromere DNA element II (CDEII) of budding yeast centromeres is an AT-rich sequence essential for centromere (CEN) function. Sequence analysis of Saccharomyces cerevisiae CDEIIs revealed that A(5-7)/T(5-7) tracts are statistically overrepresented at the expense of AA/TT and alternating AT. To test the hypothesis that this nonrandom sequence organization is functionally important, a CEN library in which the CDEII sequences were randomized was generated. The library was screened for functional and nonfunctional members following centromere replacement in vivo. Functional CENs contained CDEIIs with the highly biased A(n)/T(n) run distribution of native centromeres, while nonfunctional CDEIIs resembled those picked from the library at random. Run content, defined as the fraction of residues present in runs of four or more nucleotides, of the functional and nonfunctional CDEII populations differed significantly (P < 0.001). Computer searches of the genome for regions with an A + T content comparable to CDEIIs revealed that such loci are not unique to centromeres, but for 14 of the 16 chromosomes the AT-rich locus with the highest A(n > or =4) + T(n > or =4) run content was the centromere. Thus, the distinctive and nonrandom sequence organization of CDEII is important for centromere function and possesses informational content that could contribute to the determination of centromere identity.