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.     

Maize Centromere Structure and Evolution: Sequence Analysis of Centromeres 2 and 5 Reveals Dynamic Loci Shaped Primarily by Retrotransposons

We describe a comprehensive and general approach for mapping centromeres and present a detailed characterization of two maize centromeres. Centromeres are difficult to map and analyze because they consist primarily of repetitive DNA sequences, which in maize are the tandem satellite repeat CentC and interspersed centromeric retrotransposons of maize (CRM). Centromeres are defined epigenetically by the centromeric histone H3 variant, CENH3. Using novel markers derived from centromere repeats, we have mapped all ten centromeres onto the physical and genetic maps of maize. We were able to completely traverse centromeres 2 and 5, confirm physical maps by fluorescence in situ hybridization (FISH), and delineate their functional regions by chromatin immunoprecipitation (ChIP) with anti-CENH3 antibody followed by pyrosequencing. These two centromeres differ substantially in size, apparent CENH3 density, and arrangement of centromeric repeats; and they are larger than the rice centromeres characterized to date. Furthermore, centromere 5 consists of two distinct CENH3 domains that are separated by several megabases. Succession of centromere repeat classes is evidenced by the fact that elements belonging to the recently active recombinant subgroups of CRM1 colonize the present day centromeres, while elements of the ancestral subgroups are also found in the flanking regions. Using abundant CRM and non-CRM retrotransposons that inserted in and near these two centromeres to create a historical record of centromere location, we show that maize centromeres are fluid genomic regions whose borders are heavily influenced by the interplay of retrotransposons and epigenetic marks. Furthermore, we propose that CRMs may be involved in removal of centromeric DNA (specifically CentC), invasion of centromeres by non-CRM retrotransposons, and local repositioning of the CENH3.     

Centromere inactivation and epigenetic modifications of a plant chromosome with three functional centromeres

A chromosome with two functional centromeres is cytologically unstable and can only be stabilized when one of the two centromeres becomes inactivated via poorly understood mechanisms. Here, we report a transmissible chromosome with multiple centromeres in wheat. This chromosome encompassed one large and two small domains containing the centromeric histone CENH3. The two small centromeres are in a close vicinity and often fused as a single centromere on metaphase chromosomes. This fused centromere contained approximately 30% of the CENH3 compared to the large centromere. An intact tricentric chromosome was transmitted to about 70% of the progenies, which was likely a consequence of the dominating pulling capacity of the large centromere during anaphases of meiosis. The tricentric chromosome showed characteristics typical to dicentric chromosomes, including chromosome breaks and centromere inactivation. Remarkably, inactivation was always associated with the small centromeres, indicating that small centromeres are less likely to survive than large ones in dicentric chromosomes. The inactivation of the small centromeres also coincided with changes of specific histone modifications, including H3K27me2 and H3K27me3, of the pericentromeric chromatin.     

Immunocytochemical visualization of the centromeres during male and female meiosis in Lilium longiflorum

Immunofluorescence staining with an antiserum raised against a presumptive meiotic histone, which has been shown to appear prior to male meiosis in liliaceous plants, preferentially stained the centromere (kinetochore) region of meiotic chromosomes in microsporocytes and megasporocytes. Using this antiserum, we were able clearly to visualize the centromeres at all important meiotic stages in microsporocytes, namely, the association and fusion of centromeres of homologous chromosomes at zygotene-pachytene in prophase I, the disjunction of the homologous centromeres at diplotene, the doubling of each centromere at metaphase I and nonseparation of the sister centromeres at anaphase I, by confocal laser scanning microscopy. Thus, this report provides a complete picture of the behavior of centromeres during meiosis in a eukaryote for the first time. This antiserum also decorated centromeres during female meiosis in cryo-sectioned megasporocytes, but did not stain the centromeres of mitotic chromosomes in root-tip meristem. From these observations, it is suggested that a meiosis-specific centromere protein is required for the meiosis-specific behavior of the centromere. Received: 12 May 1997; in revised form: 20 August 1997 / Accepted: 25 August 1997     

Synaptonemal complex components persist at centromeres and are required for homologous centromere pairing in mouse spermatocytes

Recent studies in simple model organisms have shown that centromere pairing is important for ensuring high-fidelity meiotic chromosome segregation. However, this process and the mechanisms regulating it in higher eukaryotes are unknown. Here we present the first detailed study of meiotic centromere pairing in mouse spermatogenesis and link it with key events of the G2/metaphase I transition. In mouse we observed no evidence of the persistent coupling of centromeres that has been observed in several model organisms. We do however find that telomeres associate in non-homologous pairs or small groups in B type spermatogonia and pre-leptotene spermatocytes, and this association is disrupted by deletion of the synaptonemal complex component SYCP3. Intriguingly, we found that, in mid prophase, chromosome synapsis is not initiated at centromeres, and centromeric regions are the last to pair in the zygotene-pachytene transition. In late prophase, we first identified the proteins that reside at paired centromeres. We found that components of the central and lateral element and transverse filaments of the synaptonemal complex are retained at paired centromeres after disassembly of the synaptonemal complex along diplotene chromosome arms. The absence of SYCP1 prevents centromere pairing in knockout mouse spermatocytes. The localization dynamics of SYCP1 and SYCP3 suggest that they play different roles in promoting homologous centromere pairing. SYCP1 remains only at paired centromeres coincident with the time at which some kinetochore proteins begin loading at centromeres, consistent with a role in assembly of meiosis-specific kinetochores. After removal of SYCP1 from centromeres, SYCP3 then accumulates at paired centromeres where it may promote bi-orientation of homologous centromeres. We propose that, in addition to their roles as synaptonemal complex components, SYCP1 and SYCP3 act at the centromeres to promote the establishment and/or maintenance of centromere pairing and, by doing so, improve the segregation fidelity of mammalian meiotic chromosomes.     

Repeatless and Repeat-Based Centromeres in Potato: Implications for Centromere Evolution

Centromeres in most higher eukaryotes are composed of long arrays of satellite repeats. By contrast, most newly formed centromeres (neocentromeres) do not contain satellite repeats and instead include DNA sequences representative of the genome. An unknown question in centromere evolution is how satellite repeat-based centromeres evolve from neocentromeres. We conducted a genome-wide characterization of sequences associated with CENH3 nucleosomes in potato (Solanum tuberosum). Five potato centromeres (Cen4, Cen6, Cen10, Cen11, and Cen12) consisted primarily of single- or low-copy DNA sequences. No satellite repeats were identified in these five centromeres. At least one transcribed gene was associated with CENH3 nucleosomes. Thus, these five centromeres structurally resemble neocentromeres. By contrast, six potato centromeres (Cen1, Cen2, Cen3, Cen5, Cen7, and Cen8) contained megabase-sized satellite repeat arrays that are unique to individual centromeres. The satellite repeat arrays likely span the entire functional cores of these six centromeres. At least four of the centromeric repeats were amplified from retrotransposon-related sequences and were not detected in Solanum species closely related to potato. The presence of two distinct types of centromeres, coupled with the boom-and-bust cycles of centromeric satellite repeats in Solanum species, suggests that repeat-based centromeres can rapidly evolve from neocentromeres by de novo amplification and insertion of satellite repeats in the CENH3 domains.     

Centromere autoantigens are associated with the nucleolus

Because of their importance as target antigens in scleroderma and since all other major autoantigens in scleroderma can be localized to the interphase nucleolus, we were interested in a further investigation of the potential relationship between interphase centromeres and the nucleolus. Using human anticentromere autoantibodies (ACA) from patients with the CREST form of scleroderma as probes in indirect immunofluorescence microscopy, we observed nonrandom interphase "clumping" of centromeres in a distribution suggestive of nucleoli. By double-label immunofluorescence comparing the localization of centromeres to nucleolar proteins Ki-67, fibrillarin, or protein B23 (nucleophosmin), interphase centromeres appeared to be localized around and within nucleoli. A number of different ACA sera were tested on HEp-2, HeLa, PtK2, Indian muntjac, 3T3, and NRK cells, all with identical results indicating colocalization between centromeres and nucleoli. Immunoelectron microscopy revealed that interphase centromeres were distributed free in the nucleoplasm, in contact with the nuclear envelope, in contact with and on the periphery of nucleoli, and totally embedded within the confines of the nucleolus itself. Interestingly, actinomycin D treatment dissociated centromeres from localization within the segregated nucleolus. To determine if interphase centromeres were integral components of nucleoli, nucleoli were isolated according to classical methods. By double-label immunofluorescence, immunoelectron microscopy, and Western blotting, it was demonstrated that centromere autoantigens copurified with isolated nucleoli. These studies offer proof that some interphase centromeres can be associated with, and may even be considered part of, the interphase nucleolus. Furthermore, all of the major autoantigens in scleroderma can now be localized to the nucleolus.     

POLO kinase regulates the Drosophila centromere cohesion protein MEI-S332

Accurate segregation of chromosomes is critical to ensure that each daughter cell receives the full genetic complement. Maintenance of cohesion between sister chromatids, especially at centromeres, is required to segregate chromosomes precisely during mitosis and meiosis. The Drosophila protein MEI-S332, the founding member of a conserved protein family, is essential in meiosis for maintaining cohesion at centromeres until sister chromatids separate at the metaphase II/anaphase II transition. MEI-S332 localizes onto centromeres in prometaphase of mitosis or meiosis I, remaining until sister chromatids segregate. We elucidated a mechanism for controlling release of MEI-S332 from centromeres via phosphorylation by POLO kinase. We demonstrate that POLO antagonizes MEI-S332 cohesive function and that full POLO activity is needed to remove MEI-S332 from centromeres, yet this delocalization is not required for sister chromatid separation. POLO phosphorylates MEI-S332 in vitro, POLO and MEI-S332 bind each other, and mutation of POLO binding sites prevents MEI-S332 dissociation from centromeres.     

Strong epigenetic similarity between maize centromeric and pericentromeric regions at the level of small RNAs, DNA methylation and H3 chromatin modifications

Both kinetochore function and sister chromatid cohesion can depend upon pericentromere chromatin structure, and factors associated with heterochromatin have been proposed to have general, conserved roles in distinguishing centromeres and pericentromeres and in conferring pericentromere-intrinsic functions. We applied genome-wide sequencing approaches to quantify RNA expression, DNA methylation and histone modification distributions in maize (Zea mays), focusing on two maize chromosomes with nearly fully sequenced centromeres and pericentromeres. Aside from the presence of the Histone H3 variant common to all centromeres, Centromeric Histone H3 (CENH3), we found no RNA expression or chromatin modifications that clearly differentiate pericentromeres from either centromeres or from chromosome arms, nor did we identify an epigenetic signature that accurately predicts CENH3 location. RNA expression and chromatin modification frequencies were broadly associated with distance from centromeres, gradually peaking or dipping toward arms. When interpreted in the context of experimental data from other systems, our results suggest that centromeres may confer essential functions (such as cohesion retention) to flanking sequence regardless of the local heterochromatin profile.     

Centromeres are specialized replication domains in heterochromatin

The properties that define centromeres in complex eukaryotes are poorly understood because the underlying DNA is normally repetitive and indistinguishable from surrounding noncentromeric sequences. However, centromeric chromatin contains variant H3-like histones that may specify centromeric regions. Nucleosomes are normally assembled during DNA replication; therefore, we examined replication and chromatin assembly at centromeres in Drosophila cells. DNA in pericentric heterochromatin replicates late in S phase, and so centromeres are also thought to replicate late. In contrast to expectation, we show that centromeres replicate as isolated domains early in S phase. These domains do not appear to assemble conventional H3-containing nucleosomes, and deposition of the Cid centromeric H3-like variant proceeds by a replication-independent pathway. We suggest that late-replicating pericentric heterochromatin helps to maintain embedded centromeres by blocking conventional nucleosome assembly early in S phase, thereby allowing the deposition of centromeric histones.     

Dynamic elastic behavior of alpha-satellite DNA domains visualized in situ in living human cells

We have constructed a fluorescent alpha-satellite DNA-binding protein to explore the motile and mechanical properties of human centromeres. A fusion protein consisting of human CENP-B coupled to the green fluorescent protein (GFP) of A. victoria specifically targets to centromeres when expressed in human cells. Morphometric analysis revealed that the alpha-satellite DNA domain bound by CENPB-GFP becomes elongated in mitosis in a microtubule-dependent fashion. Time lapse confocal microscopy in live mitotic cells revealed apparent elastic deformations of the central domain of the centromere that occurred during metaphase chromosome oscillations. These observations demonstrate that the interior region of the centromere behaves as an elastic element that could play a role in the mechanoregulatory mechanisms recently identified at centromeres. Fluorescent labeling of centromeres revealed that they disperse throughout the nucleus in a nearly isometric expansion during chromosome decondensation in telophase and early G1. During interphase, centromeres were primarily stationary, although motility of individual or small groups of centromeres was occasionally observed at very slow rates of 7-10 microns/h.     

Maize centromeres: organization and functional adaptation in the genetic background of oat

Centromeric DNA sequences in multicellular eukaryotes are often highly repetitive and are not unique to a specific centromere or to centromeres at all. Thus, it is a major challenge to study the fine structure of individual plant centromeres. We used a DNA fiber-fluorescence in situ hybridization approach to study individual maize (Zea mays) centromeres using oat (Avena sativa)-maize chromosome addition lines. The maize centromere-specific satellite repeat CentC in the addition lines allowed us to delineate the size and organization of centromeric DNA of individual maize chromosomes. We demonstrate that the cores of maize centromeres contain mainly CentC arrays and clusters of a centromere-specific retrotransposon, CRM. CentC and CRM sequences are highly intermingled. The amount of CentC/CRM sequence varies from approximately 300 to >2800 kb among different centromeres. The association of CentC and CRM with centromeric histone H3 (CENH3) was visualized by a sequential detection procedure on stretched centromeres. The analysis revealed that CENH3 is always associated with CentC and CRM but that not all CentC or CRM sequences are associated with CENH3. We further demonstrate that in the chromosomal addition lines in which two CenH3 genes were present, one from oat and one from maize, the oat CENH3 was consistently incorporated by the maize centromeres.     

Meiotic nuclear reorganization: switching the position of centromeres and telomeres in the fission yeast Schizosaccharomyces pombe.

In fission yeast meiotic prophase, telomeres are clustered near the spindle pole body (SPB; a centrosome-equivalent structure in fungi) and take the leading position in chromosome movement, while centromeres are separated from the SPB. This telomere position contrasts with mitotic nuclear organization, in which centromeres remain clustered near the SPB and lead chromosome movement. Thus, nuclear reorganization switching the position of centromeres and telomeres must take place upon entering meiosis. In this report, we analyze the nuclear location of centromeres and telomeres in genetically well-characterized meiotic mutant strains. An intermediate structure for telomere-centromere switching was observed in haploid cells induced to undergo meiosis by synthetic mating pheromone; fluorescence in situ hybridization revealed that in these cells, both telomeres and centromeres were clustered near the SPB. Further analyses in a series of mutants showed that telomere-centromere switching takes place in two steps; first, association of telomeres with the SPB and, second, dissociation of centromeres from the SPB. The first step can take place in the haploid state in response to mating pheromone, but the second step does not take place in haploid cells and probably depends on conjugation-related events. In addition, a linear minichromosome was also co-localized with authentic telomeres instead of centromeres, suggesting that telomere clustering plays a role in organizing chromosomes within a meiotic prophase nucleus.     

The role of centromere alignment in meiosis I segregation of homologous chromosomes in Saccharomyces cerevisiae

During meiosis, homologous chromosomes pair and then segregate from each other at the first meiotic division. Homologous centromeres appear to be aligned when chromosomes are paired. The role of centromere alignment in meiotic chromosome segregation was investigated in Saccharomyces cerevisiae diploids that contained one intact copy of chromosome I and one copy bisected into two functional centromere-containing fragments. The centromere on one fragment was aligned with the centromere on the intact chromosome while the centromere on the other fragment was either aligned or misaligned. Fragments containing aligned centromeres segregated efficiently from the intact chromosome, while fragments containing misaligned centromeres segregated much less efficiently from the intact chromosome. Less efficient segregation was correlated with crossing over in the region between the misaligned centromeres. Models that suggest that these crossovers impede proper segregation by preventing either a segregation-promoting chromosome alignment on the meiotic spindle or some physical interaction between homologous centromeres are proposed.     

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.     

Analysis of three whole-arm translocations in a mouse sarcoma cell line

Three apparently whole-arm translocation chromosomes in the mouse sarcoma 180 ascites cell line have been studied by G- and C-banding and by Hoechst staining. All three chromosomes appear to have two centromeres. Both centromeres of one, which are very close together, are likely to be active and to produce parallel separation of the chromatids. The centromeres of the other two chromosomes are well separated. One of these two centromeres may be inactive either because the kinetochore organizer has been inactivated or because the kinetochore plate has been deleted, leaving the AT-rich centromeric constituative heterochromatin intact. The possibility that these whole arm translocations arose by telomeric fusion and the molecular basis of such fusions are discussed.     

Intergenic Locations of Rice Centromeric Chromatin

Centromeres are sites for assembly of the chromosomal structures that mediate faithful segregation at mitosis and meiosis. Plant and animal centromeres are typically located in megabase-sized arrays of tandem satellite repeats, making their precise mapping difficult. However, some rice centromeres are largely embedded in nonsatellite DNA, providing an excellent model to study centromere structure and evolution. We used chromatin immunoprecipitation and 454 sequencing to define the boundaries of nine of the 12 centromeres of rice. Centromere regions from chromosomes 8 and 9 were found to share synteny, most likely reflecting an ancient genome duplication. For four centromeres, we mapped discrete subdomains of binding by the centromeric histone variant CENH3. These subdomains were depleted in both intact and nonfunctional genes relative to interspersed subdomains lacking CENH3. The intergenic location of rice centromeric chromatin resembles the situation for human neocentromeres and supports a model of the evolution of centromeres from gene-poor regions.     

DNA and proteins of plant centromeres

In plants, as in all eukaryotes, centromeres are chromatin domains that govern the transmission of nuclear chromosomes to the next generation of cells/individuals. The DNA composition and sequence organization of centromeres has recently been elucidated for a few plant species. Although there is little sequence conservation among centromeres, they usually contain tandem repeats and retroelements. The occurrence of neocentromeres reinforces the idea that the positions of centromeres are determined epigenetically. In contrast to centromeric DNA, structural and transient kinetochoric proteins are highly conserved among eukaryotes. Candidate sequences have been identified for a dozen putative kinetochore protein homologues, and some have been localized to plant centromeres. The kinetochore protein CENH3, which substitutes histone H3 within centromeric nucleosomes, co-immunoprecipitates preferentially with centromeric sequences. The mechanism(s) of centromere assembly and the functional implication of (peri-)centromeric modifications of chromatin remain to be elucidated.     

Acrocentric centromere organization within the chromocenter of the human sperm nucleus.

It has recently been reported that in human sperm cells, the centromeres are clustered in a chromocenter in the interior region of the nucleus. The aim of the present study was to determine the intra-chromocenter organization of the five centromeres of the acrocentric chromosomes responsible for the biosynthesis of rRNA. The acrocentric centromeres were labeled by fluorescence in situ hybridization (FISH) after mild decondensation of the sperm nuclei to preserve the tail structure. The tail was used as a topographical marker for the orientation of the nucleus. The following results were obtained: (a) the association among the five centromeres was higher than expected from random distribution; (b) all the centromeres observed were randomly located within the chromocenter, occupying about 87% of the total area of the internal nucleus; (c) a major subpopulation of centromeres was located in a preferred area occupying 8.3% of the total nuclear area, with a peak 0.6 microm on the lateral axis and 1.0 microm on the apical side of the longitudinal axis; and (d) The dispersion of the centromeres was not influenced by the degree of the nuclear decondensation. We conclude that in human sperm nuclei, the acrocentric centromeres are organized within a nonlocalized structural element in the chromocenter. The chromocenter can range from an expanded size of 87% of the whole nucleus to a preferred size of 8.3% independent of the degree of nuclear decondensation. These findings have important implications for nuclear function (rRNA) that is not directly related to sperm cell function or early embryo development.     

Deposition,turnover, and release of CENH3 at <Emphasis Type="Italic">Arabidopsis</Emphasis> centromeres

The kinetochore is a complex multiprotein structure located at centromeres and required for the proper segregation of chromosomes during mitosis and meiosis. An important role in kinetochore assembly and function plays the centromeric histone H3 variant (CENH3). Cell cycle stage of CENH3 deposition to centromeres varies between different organisms. We confirmed by in vivo studies that deposition of Arabidopsis CENH3 takes place at centromeres during G2 and demonstrated that additionally a low turnover of CENH3 occurs along the cell cycle, apparently for replacement of damaged protein. Furthermore, enhanced yellow fluorescent protein (EYFP)-CENH3 of photobleached chromocenters is not replaced by EYFP-CENH3 molecules from unbleached centromeres of the same nucleus, indicating a stable incorporation of CENH3 into centromeric nucleosomes. In differentiated endopolyploid nuclei however, the amount of CENH3 at centromeres declines with age.