A stable dicentric chromosome: Both centromeres develop kinetochores and attach to the spindle in monocentric and dicentric configuration

A stable, dicentric human chromosome, which is known from light microscopy to show a 50:50 distribution between monocentric/dicentric appearance, was examined by conventional electron microscopy and after labelling the centromere with anticentromere antibodies from CREST serum. Both centromeres of the chromosome developed kinetochores whether in monocentric or dicentric configuration. The eight monocentrics observed had all developed kinetochores at the centromere outside the constriction; at least six of them also had kinetochores at the centromere in the constriction. The dicentrics from glutaraldehyde fixed cells had spindle microtubules attached to both kinetochore sets irrespective of monocentric/dicentric configuration. The chromosome thus appeared to use both centromeres, either equally or with one serving a chromatid adhesion function while the second was used for transport along the spindle.     

Behavior of dicentric chromosomes in budding yeast

DNA double-strand breaks arise in vivo when a dicentric chromosome (two centromeres on one chromosome) goes through mitosis with the two centromeres attached to opposite spindle pole bodies. Repair of the DSBs generates phenotypic diversity due to the range of monocentric derivative chromosomes that arise. To explore whether DSBs may be differentially repaired as a function of their spatial position in the chromosome, we have examined the structure of monocentric derivative chromosomes from cells containing a suite of dicentric chromosomes in which the distance between the two centromeres ranges from 6.5 kb to 57.7 kb. Two major classes of repair products, homology-based (homologous recombination (HR) and single-strand annealing (SSA)) and end-joining (non-homologous (NHEJ) and micro-homology mediated (MMEJ)) were identified. The distribution of repair products varies as a function of distance between the two centromeres. Genetic dependencies on double strand break repair (Rad52), DNA ligase (Lif1), and S phase checkpoint (Mrc1) are indicative of distinct repair pathway choices for DNA breaks in the pericentromeric chromatin versus the arms.     

The same molecular mechanism at the maternal meiosis I produces mono- and dicentric 8p duplications.

We studied 16 cases of 8p duplications, with a karyotype 46,XX or XY,dup(8p), associated with mental retardation, facial dysmorphisms, and brain defects. We demonstrate that these 8p rearrangements can be either dicentric (6 cases) with the second centromere at the tip of the short arm or monocentric (10 cases). The distal 8p23 region, from D8S349 to the telomere, including the defensin 1 locus, is deleted in all the cases. The region spanning from D8S252 to D8S265, at the proximal 8p23 region, is present in single copy, and the remaining part of the abnormal 8 short arm is duplicated in the dicentric cases and partially duplicated in the monocentric ones. The distal edge of the duplication always spans up to D8S552 (8p23.1), while its proximal edge includes the centromere in the dicentric cases and varies from case to case in the monocentric ones. The analysis of DNA polymorphisms indicates that the rearrangement is consistently of maternal origin. In the deleted region, only paternal alleles were present in the patient. In the duplicated region, besides one paternal allele, some loci showed two different maternal alleles, while others, which were duplicated by FISH analysis, showed only one maternal allele. We hypothesize that, at maternal meiosis I, there was abnormal pairing of chromosomes 8 followed by anomalous crossover at the regions delimited by D8S552 and D8S35 and by D8S252 and D8S349, which presumably contain inverted repeated sequences. The resulting dicentric chromosome, 8qter-8p23.1(D8S552)::8p23.1-(D8S35)-8q ter, due to the presence of two centromeres, breaks at anaphase I, generating an inverted duplicated 8p, dicentric if the breakage occurs at the centromere or monocentric if it occurs between centromeres.     

De novo generation of plant centromeres at tandem repeats

Artificial minichromosomes are highly desirable tools for basic research, breeding, and biotechnology purposes. We present an option to generate plant artificial minichromosomes via de novo engineering of plant centromeres in Arabidopsis thaliana by targeting kinetochore proteins to tandem repeat arrays at non-centromeric positions. We employed the bacterial lactose repressor/lactose operator system to guide derivatives of the centromeric histone H3 variant cenH3 to LacO operator sequences. Tethering of cenH3 to non-centromeric loci led to de novo assembly of kinetochore proteins and to dicentric carrier chromosomes which potentially form anaphase bridges. This approach will be further developed and may contribute to generating minichromosomes from preselected genomic regions, potentially even in a diploid background.     

Mutations in Cen3 Cause Aberrant Chromosome Segregation during Meiosis in Saccharomyces Cerevisiae

We investigated the structural requirements of the centromere from chromosome III (CEN3) of Saccharomyces cerevisiae by analyzing the ability of chromosomes with CEN3 mutations to segregate properly during meiosis. We analyzed diploid cells in which one or both copies of chromosome III carry a mutant centromere in place of the wild-type centromere and found that some alterations in the length, base composition and primary sequence characteristics of the central A+T-rich region (CDE II) of the centromere had a significant effect on the ability of the chromosome to segregate properly through meiosis. Chromosomes containing mutations which delete a portion of CDE II showed a high rate of premature disjunction at meiosis I. Chromosomes containing point mutations in CDE I or lacking CDE I appeared to segregate properly through meiosis; however, plasmids carrying centromeres with CDE I completely deleted showed an increased frequency of segregation to nonsister spores.     

Y isochromosome associated with a mosaic karyotype and inactivation of the centromere

Summary A patient with azoospermia and a Y isochromosome is described. The breakpoint producing this i(Y) was within the terminal short arm of the Y chromosome. Lymphocyte cultures from peripheral blood contained a high proportion of 45,X cells and cells with different Y-chromosome rearrangements. The i(Y) had either a monocentric or dicentric appearance. In dicentrics, anti-kinetochore immunofluorescence was present at both centromeres. However, this was also true for most of the functional monocentrics (pseudodicentrics). Kinetochore staining was generally positive at the site of the inactive centromeres; only a minority of the suppressed centromeres had lost their antigenic properties. Permanently growing lymphoblasts consistently showed a monocentric i(Y) with only one fluorescing kinetochore; the immunonegative Y centromere did not recover antigenicity.     

Alterations in the adenine-plus-thymine-rich region of CEN3 affect centromere function in Saccharomyces cerevisiae.

Centromere DNA from 11 of the 16 chromosomes of the yeast Saccharomyces cerevisiae have been analyzed and reveal three sequence elements common to each centromere, referred to as conserved centromere DNA elements (CDE). The adenine-plus-thymine (A + T)-rich central core element, CDE II, is flanked by two short conserved sequences, CDE I (8 base pairs [bp]) and CDE III (25 bp). Although no consensus sequence exists among the different CDE II regions, they do have three common features of sequence organization. First, the CDE II regions are similar in length, ranging from 78 to 86 bp measured from CDE I to the left boundary of CDE III. Second, the base composition is always greater than 90% A + T. Finally, the A and T residues in these segments are often arranged in runs of A and runs of T residues, sometimes with six or seven bases in a stretch. We constructed insertion, deletion, and replacement mutations in the CDE II region of the centromere from chromosome III, CEN3, designed to investigate the length and sequence requirements for function of the CDE II region of the centromere. We analyzed the effect of these altered centromeres on plasmid and chromosome segregation in S. cerevisiae. Our results show that increasing the length of CDE II from 84 to 154 bp causes a 100-fold increase in chromosome nondisjunction. Deletion mutations removing segments of the A + T-rich CDE II DNA also cause aberrant segregation. In some cases partial function could be restored by replacing the deleted DNA with fragments whose primary sequence or base composition is very different from that of the wild-type CDE II DNA. In addition, we found that identical mutations introduced into different positions in CDE II have very similar effects.     

Budding yeast Rad50, Mre11, Xrs2, and Hdf1, but not Rad52, are involved in the formation of deletions on a dicentric plasmid

We have previously shown that the RAD50, RAD52, MRE11, XRS2, and HDF1 genes of Saccharomyces cervisiae are involved in the formation of deletions by illegitimate recombination on a monocentric plasmid. In this study, we investigated the effects of mutations of these genes on formation of deletions of a dicentric plasmid, in which DNA double-strand breaks are expected to occur frequently because the two centromeres are pulled to opposite poles in mitosis. We transformed yeast cells with a dicentric plasmid, and after incubation for a few division cycles, cells carrying deleted plasmids were detected using negative selection markers. Deletions occurred at a higher frequency than on the monocentric plasmid and there were short regions of homology at the recombination junctions as observed on the monocentric plasmid. In rad50, mre11, xrs2, and hdf1 mutants, the frequency of occurrence of deletions was reduced by about 50-fold, while in the rad52 mutant, it was comparable to that in the wild-type strain. The end-joining functions of Rad50, Mre11, Xrs2, and Hdf1, suggest that these proteins play important roles in the joining of DNA ends produced on the dicentric plasmid during mitosis. Received: 30 October 1996 / Accepted: 28 February 1997     

Dicentric chromosomes and the inactivation of the centromere

Summary The origin and behavior of human dicentric chromosomes are reviewed. Most dicentrics between two non-homologous or two homologous chromosomes (isodicentrics), which are permanent members of a chromosome complement, probably originate from segregation of an adjacent quadriradial; such configurations are the result of a chromatid translocation between two nonhomologous chromosomes, or they represent an adjacent counterpart of a mitotic chiasma. The segregation of such a quadriradial may also give rise to a cell line monosomic for the chromosome concerned (e.g., a 45,X line). Contrary to the generally held opinion, isodicentrics rarely result from an isolocal break in two chromatids followed by rejoining of sister chromatids. In this case the daughter centromeres go to opposite poles in the next anaphase, and the resulting bridge breaks at a random point. This mechanism, therefore, leads to the formation of an isodicentric chromosome only if the two centromeres are close together, or if one centromere is immediately inactivated. Observations on the origin of dicentrics in Bloom syndrome support these conclusions. One centromere is permanently inactivated in most dicentric chromosomes, and even when the dicentric breaks into two chromosomes, the centromere is not reactivated. The appearance and behavior of the acentric X chromosomes show that their centromeres are similarly inactivated and not prematurely divided. Two Bloom syndrome lymphocytes, one with an extra chromosome 2 and the other with an extra chromosome 7, each having an inactivated centromere, show that this can also happen in monocentric autosomes.     

Reactivation of an Inactive Centromere Reveals Epigenetic and Structural Components for Centromere Specification in Maize

Stable maize (Zea mays) chromosomes were recovered from an unstable dicentric containing large and small versions of the B chromosome centromere. In the stable chromosome, the smaller centromere had become inactivated. This inactive centromere can be inherited from one generation to the next attached to the active version and loses all known cytological and molecular properties of active centromeres. When separated from the active centromere by intrachromosomal recombination, the inactive centromere can be reactivated. The reactivated centromere regains the molecular attributes of activity in anaphase I of meiosis. When two copies of the dicentric chromosome with one active and one inactive centromere are present, homologous chromosome pairing reduces the frequency of intrachromosomal recombination and thus decreases, but does not eliminate, the reactivation of inactive centromeres. These findings indicate an epigenetic component to centromere specification in that centromere inactivation can be directed by joining two centromeres in opposition. These findings also indicate a structural aspect to centromere specification revealed by the gain of activity at the site of the previously inactive sequences.     

Structure and mitotic stability of minichromosomes originating in yeast cells transformed with tandem dimers of CEN11 plasmids

Summary Large (10.5–13.5 kbp) circular minichromosomes containing the centromere of chromosome 11 (CEN11) and the MET14 gene of Saccharomyces cerevisiae in the YRp7 vector are considerably more stable during mitosis than smaller ones containing only the 1.6 kbp CEN11 SalI-fragment. Yeast transformants obtained with a tandem dimeric and thus dicentric form derived from this DNA varied in the mitotic stability of the TRP1 marker of the vector. The largest group of transformants contained minichromosomes which carried deletions located quite specifically at one of the two centromeres in the dimer, eliminating its function in mitosis. This group included also some minichromosomes which had been modified by intramolecular tandem amplification of the subunit carrying the deletion without losing the centromere within the unmodified subunit. The second major group carried minichromosomes which had been monomerized. Monomerized minichromosomes showed the relative low degree of mitotic stability typical for the original minichromosomes containing the 1.6 kbp CEN11 SalI-fragment. Increasing numbers of additional subunits carrying the TRP1-ARS1 sequences but lacking additional centromeres improved the mitotic stability considerably.     

CSE4 genetically interacts with the Saccharomyces cerevisiae centromere DNA elements CDE I and CDE II but not CDE III. Implications for the path of the centromere dna around a cse4p variant nucleosome

Each Saccharomyces cerevisiae chromosome contains a single centromere composed of three conserved DNA elements, CDE I, II, and III. The histone H3 variant, Cse4p, is an essential component of the S. cerevisiae centromere and is thought to replace H3 in specialized nucleosomes at the yeast centromere. To investigate the genetic interactions between Cse4p and centromere DNA, we measured the chromosome loss rates exhibited by cse4 cen3 double-mutant cells that express mutant Cse4 proteins and carry chromosomes containing mutant centromere DNA (cen3). When compared to loss rates for cells carrying the same cen3 DNA mutants but expressing wild-type Cse4p, we found that mutations throughout the Cse4p histone-fold domain caused surprisingly large increases in the loss of chromosomes carrying CDE I or CDE II mutant centromeres, but had no effect on chromosomes with CDE III mutant centromeres. Our genetic evidence is consistent with direct interactions between Cse4p and the CDE I-CDE II region of the centromere DNA. On the basis of these and other results from genetic, biochemical, and structural studies, we propose a model that best describes the path of the centromere DNA around a specialized Cse4p-nucleosome.     

Molecular definition of breakpoints associated with human Xq isochromosomes: implications for mechanisms of formation.

To test the centromere misdivision model of isochromosome formation, we have defined the breakpoints of cytogenetically monocentric and dicentric Xq isochromosomes (i(Xq)) from Turner syndrome probands, using FISH with cosmids and YACs derived from a contig spanning proximal Xp. Seven different pericentromeric breakpoints were identified, with 10 of 11 of the i(Xq)s containing varying amounts of material from Xp. Only one of the eight cytogenetically monocentric i(Xq)s demonstrated a single alpha-satellite (DXZ1) signal, consistent with classical models involving centromere misdivision. The remaining seven were inconsistent with such a model and had breakpoints that spanned proximal Xp11.21: one was between DXZ1 and the most proximal marker, ZXDA; one occurred between the duplicated genes, ZXDA and ZXDB; two were approximately 2 Mb from DXZ1; two were adjacent to ALAS2 located 3.5 Mb from DXZ1; and the largest had a breakpoint just distal to DXS1013E, indicating the inclusion of 8 Mb of Xp DNA between centromeres. The three cytologically dicentric i(Xq)s had breakpoints distal to DXS423E in Xp11.22 and therefore contained > or = 12 Mb of DNA between centromeres. These data demonstrate that the majority of breakpoints resulting in i(Xq) formation are in band Xp11.2 and not in the centromere itself. Therefore, we hypothesize that the predominant mechanism of i(Xq) formation involves sequences in the proximal short arm that are prone to breakage and reunion events between sister chromatids or homologous X chromosomes.     

Two-color hybridization with high complexity chromosome-specific probes and a degenerate alpha satellite probe DNA allows unambiguous discrimination between symmetrical and asymmetrical translocations

This report describes a fluorescence in situ hybridization approach to chromosome staining that facilitates detection of structural aberrations and allows discrimination between dicentric chromosomes and symmetrically translocated chromosomes. In this approach, selected whole chromosomes are stained in one color by hybridization with composite probes whose elements have DNA sequence homology along the length of the target chromosomes. In addition, all chromosomes are counterstained with a DNA specific dye so that structural aberrations between target and non-target chromosomes are clearly visible. Discrimination between dicentric chromosomes and symmetrical translocations is accomplished by hybridization with a second probe that is homologous to DNA sequences found in the centromeric region of all chromosomes. The centromeric marker is visualized in a different color, so that the number of centromeres per aberrant chromosome can be rapidly determined in the microscope by changing excitation and fluorescence filters.by H.F. Willard     

Nucleosome depletion alters the chromatin structure of Saccharomyces cerevisiae centromeres.

Saccharomyces cerevisiae centromeric DNA is packaged into a highly nuclease-resistant chromatin core of approximately 200 base pairs of DNA. The structure of the centromere in chromosome III is somewhat larger than a 160-base-pair nucleosomal core and encompasses the conserved centromere DNA elements (CDE I, II, and III). Extensive mutational analysis has revealed the sequence requirements for centromere function. Mutations affecting the segregation properties of centromeres also exhibit altered chromatin structures in vivo. Thus the structure, as delineated by nuclease digestion, correlated with functional centromeres. We have determined the contribution of histone proteins to this unique structural organization. Nucleosome depletion by repression of either histone H2B or H4 rendered the cell incapable of chromosome segregation. Histone repression resulted in increased nuclease sensitivity of centromere DNA, with up to 40% of CEN3 DNA molecules becoming accessible to nucleolytic attack. Nucleosome depletion also resulted in an alteration in the distribution of nuclease cutting sites in the DNA surrounding CEN3. These data provide the first indication that authentic nucleosomal subunits flank the centromere and suggest that nucleosomes may be the central core of the centromere itself.     

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.     

Construction of midget chromosomes in wheat.

To test the usefulness of breakage-fusion-bridge (BFB) cycles in generating new chromosome aberrations in bread wheat (Triticum aestivum L.) and to extend the range of aberrations available, a series of midget chromosomes was produced from the long arm of chromosome 1B. Using a reverse tandem duplication initiated chromatid type BFB cycle, the 1BL arm was broken and fused with centromeres of either chromosome 5BL or 1RS to form dicentric chromosomes. The 1R and 5B centromeres were broken by centric misdivision. Among the progenies of plants with dicentric chromosomes, two classes of monocentric chromosomes were selected: deficient chromosomes 1B and chromosomes that had 1RS or 5BL for one arm and various fragments of 1BL for the other arm. Following centric misdivision of these monocentrics, midget chromosomes 1BL were isolated: deficient and deletion telocentrics and telocentrics derived from interstitial regions of 1BL. By chance, one deficient chromosome 1BS and one deletion chromosome 1BS were identified in unrelated lines of the same wheat. Following centric misdivision of these chromosomes, two midget chromosomes covering the whole of 1BS were added to the set.     

Holocentric plant meiosis: first sisters,then homologues

Meiosis is a crucial process of sexual reproduction by forming haploid gametes from diploid precursor cells. It involves 2 subsequent divisions (meiosis I and meiosis II) after one initial round of DNA replication. Homologous monocentric chromosomes are separated during the first and sister chromatids during the second meiotic division. The faithful segregation of monocentric chromosomes is realized by mono-orientation of fused sister kinetochores at metaphase I and by bi-orientation of sister kinetochores at metaphase II. Conventionally this depends on a 2-step loss of cohesion, along chromosome arms during meiosis I and at sister centromeres during meiosis II.     

Alteration of Terminal Heterochromatin and Chromosome Rearrangements in Derivatives of Wheat-Rye Hybrids

Wheat-rye addition and substitution lines and their self progenies revealed variations in telomeric heterochromatin and centromeres.Furthermore,a mitotically unstable dicentric chromosome and stable mu...     

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.