Association of Chromosome Loss with Centromere-Adjacent Mitotic Recombination in a Yeast Disomic Haploid

Experiments designed to characterize the association between disomic chromosome loss and centromere-adjacent mitotic recombination were performed. Mitotic gene convertants were selected at two heteroallelic sites on the left arm of disomic chromosome III and tested for coincident chromosome loss. The principal results are: (1) Disomic chromosome loss is markedly enhanced (nearly 40-fold) over basal levels among mitotic gene convertants selected to arise close to the centromere; no such enhancement is observed among convertants selected to arise relatively far from the centromere. (2) Chromosome loss is primarily associated with proximal allele conversion at the centromere-adjacent site, and many of these convertants are reciprocally recombined in the adjacent proximal interval. (3) Partial aneuploid exceptions provisionally identified as carrying left arm telocentrics have been found. A testable model is proposed suggesting that centromere involvement in genetic recombination may precipitate segregational disfunction leading to mitotic chromosome loss.     

Centromere formation: from epigenetics to self-assembly

This review is part of the Chromosome segregation and Aneuploidy series that focuses on the importance of chromosome segregation mechanisms in maintaining genome stability. Centromeres are specialized chromosomal domains that serve as the foundation for the mitotic kinetochore, the interaction site between the chromosome and the mitotic spindle. The chromatin of centromeres is distinguished from other chromosomal loci by the unique incorporation of the centromeric histone H3 variant, centromere protein A. Here, we review the genetic and epigenetic factors that control the formation and maintenance of centromeric chromatin and propose a chromatin self-assembly model for organizing the higher-order structure of the centromere.     

Trisomy 21 mosaicism in two subjects from two generations.

In the course of a chromosome fragility investigation on the cancer prone hereditary disorder xeroderma pigmentosum, a low proportion of cells with a 47,XY,+21 karyotype was found in lymphocyte cultures of a patient not showing any Down syndrome symptom. The presence of trisomy 21 mosaicism was demonstrated also in peripheral blood of the healthy father and confirmed by "chromosome painting" that allowed a rapid detection of chromosomes 21 on metaphase cells and interphase nuclei. The trisomic cell line was not detected in fibroblast cultures. The analysis of chromosome 21 heteromorphism indicated that in both subjects the mosaic could result from either a diploid or an aneuploid zygote. Since in the trisomic cell line of the father and the son the extra chromosome 21 seems to be the same, a predisposition toward mitotic errors (non-disjunction or anaphase lagging) may be postulated, leading to the recurrent gain or loss of a specific chromosome 21. In order to test the hypothesis of an abnormal mitotic behaviour of the chromosome 21, we investigated the centromere separation index and the DNA restriction pattern in Southern blots probed with satellite DNA sequences specific for chromosome 21 centromere. Both the approaches did not reveal any peculiar feature that may account for the genetically determined proneness to mitotic error observed in the family.     

Molecular evidence for true isochromosome 21q

Summary In one family a duplicated 21q was shown to be a true isochromosome, which segregates from mosaic mother to non-mosaic child with full Down syndrome phenotype. Densitometric analysis of Southern blots, using probe pPW228C for the distal long arm of chromosome 21, indicated that the 21q duplication contains two copies of the allele detected by the probe. Maternal mosaic karyotype of 45,XX,-21/46,XX/46, XX,-21,+21i(21q) also suggested transverse mitotic centromere division as the origin of the 21q isochromosomes. Morphologic analysis of chromosome heteromorphisms strengthened this interpretation because the free 21 missing in the cell line with 45 chromosomes was also missing in cells with the isochromosome. In a second family the cytogenetic data also suggested transmission of an i(21q) from mosaic mother to nonmosaic Down syndrome child but molecular evidence did not prove identity of alleles in the duplicated chromosome 21.     

Centromere mitotic recombination in mammalian cells

Centromeres are special structures of eukaryotic chromosomes that hold sister chromatid together and ensure proper chromosome segregation during cell division. Centromeres consist of repeated sequences, which have hindered the study of centromere mitotic recombination and its consequences for centromeric function. We use a chromosome orientation fluorescence in situ hybridization technique to visualize and quantify recombination events at mouse centromeres. We show that centromere mitotic recombination occurs in normal cells to a higher frequency than telomere recombination and to a much higher frequency than chromosome-arm recombination. Furthermore, we show that centromere mitotic recombination is increased in cells lacking the Dnmt3a and Dnmt3b DNA methyltransferases, suggesting that the epigenetic state of centromeric heterochromatin controls recombination events at these regions. Increased centromere recombination in Dnmt3a,3b-deficient cells is accompanied by changes in the length of centromere repeats, suggesting that prevention of illicit centromere recombination is important to maintain centromere integrity in the mouse.     

Analysis of centromere function in Saccharomyces cerevisiae using synthetic centromere mutants

We constructed Saccharomyces cerevisiae centromere DNA mutants by annealing and ligating synthetic oligonucleotides, a novel approach to centromere DNA mutagenesis that allowed us to change only one structural parameter at a time. Using this method, we confirmed that CDE I, II, and III alone are sufficient for centromere function and that A+T-rich sequences in CDE II play important roles in mitosis and meiosis. Analysis of mutants also showed that a bend in the centromere DNA could be important for proper mitotic and meiotic chromosome segregation. In addition we demonstrated that the wild-type orientation of the CDE III sequence, but not the CDE I sequence, is critical for wild-type mitotic segregation. Surprisingly, we found that one mutant centromere affected the segregation of plasmids and chromosomes differently. The implications of these results for centromere function and chromosome structure are discussed.by M. Yanagida     

Genetic and physical mapping of two centromere-proximal regions of chromosome IV in Aspergillus nidulans

Chromosome IV is the smallest chromosome of Aspergillus nidulans. The centromere-proximal portion of the chromosome was mapped physically using overlapping clones of a cosmid genomic library. Two contiguous segments of a physical map, based on restriction mapping of cosmid clones, were generated, together covering more than 0.4 Mb DNA. A reverse genetic mapping approach was used to establish a correlation between physical and genetic maps; i.e., marker genes were integrated into physically mapped segments and subsequently mapped by mitotic and meiotic recombination. The resulting data, together with additional classical genetic mapping, lead to a substantial revision of the genetic map of the chromosome, including the position of the centromere. Comparison of physical and genetic maps indicates that meiotic recombination is low in subcentromeric DNA, its frequency being reduced from 1 crossover per 0.8 Mb to approximately 1 crossover per 5 Mb per meiosis. The portion of the chromosome containing the functional centromere was not mapped because repeat-rich regions hindered further chromosome walking. The size of the missing segment was estimated to be between 70 and 400 kb.     

The centromere of budding yeast

Stable maintenance of genetic information during meiosis and mitosis is dependent on accurate chromosome transmission. The centromere is a key component of the segregational machinery that couples chromosomes with the spindle apparatus. Most of what is known about the structure and function of the centromeres has been derived from studies on yeast cells. In Saccharomyces cerevisiae, the centromere DNA requirements for mitotic centromere function have been defined and some of the proteins required for an active complex have been identified. Centromere DNA and the centromere proteins form a complex that has been studied extensively at the chromatin level. Finally, recent findings suggest that assembly and activation of the centromere are integrated in tethe cell cycle.     

Mammalian Centromeres: DNA Sequence, Protein Composition, and Role in Cell Cycle Progression

The centromere is a specialized region of the eukaryotic chromosome that is responsible for directing chromosome movements in mitosis and for coordinating the progression of mitotic events at the crucial transition between metaphase and anaphase. In this review, we will focus on recent advances in the understanding of centromere composition at the protein and DNA level and of the role of centromeres in sister-chromatid cohesion and mitotic checkpoint control.     

Mitotic recombination and uniparental disomy in Beckwith-Wiedemann syndrome

Beckwith-Wiedemann syndrome (BWS) is a model human imprinting disorder resulting from altered activity of one or more genes in the 11p15.5 imprinted gene cluster. Approximately 20% of BWS cases have uniparental disomy (UPD) of chromosome 11. Such cases appear to result from mitotic recombination occurring in early embryogenesis and offer a rare opportunity to study mitotic recombination in nonneoplastic cells. We analyzed a cohort of 52 children with BWS and UPD using a panel of microsatellite markers for chromosome 11. All cases demonstrated mosaic paternal isodisomy, and IGF2 and H19 were included in the segment of UPD in all cases. However, the extent of segmental disomy was variable, with no evidence of clustering of the proximal UPD breakpoint. In most cases (92% of those informative) UPD did not involve 11q, but 4 patients demonstrated UPD for the whole of chromosome 11. In contrast to meiotic recombination, the mitotic recombination frequency did not decline near the centromere.     

Structural analysis of a yeast centromere

The most striking region of structural differentiation of a eukaryotic chromosome is the kinetochore. This chromosomal domain plays an integral role in the stability and propagation of genetic material to the progeny cells during cell division. The DNA component of this structure, which we refer to as the centromere, has been localized to a small region of 220–250 base pairs within the chromosomes from the yeast Saccharomyces cerevisiae. The centromere DNA (CEN) is organized in a unique structure in the cell nucleus and is required for chromosome stability during both mitotic and meiotic cell cycles. The centromeres from one chromosome can stabilize small circular minichromosomes or other yeast chromosomes. The centromeres may therefore interact with the same components of the segregation apparatus regardless of the chromosome in which they reside. The CEN DNA does not encode any regulatory RNAs or proteins, but rather is a cis-acting element that provides genetic stability to adjacent DNA sequences.     

Dissection of CENP-C–directed Centromere and Kinetochore Assembly

Eukaryotic cells ensure accurate chromosome segregation in mitosis by assembling a microtubule-binding site on each chromosome called the kinetochore that attaches to the mitotic spindle. The kinetochore is assembled specifically during mitosis on a specialized region of each chromosome called the centromere, which is constitutively bound by >15 centromere-specific proteins. These proteins, including centromere proteins A and C (CENP-A and -C), are essential for kinetochore assembly and proper chromosome segregation. How the centromere is assembled and how the centromere promotes mitotic kinetochore formation are poorly understood. We have used Xenopus egg extracts as an in vitro system to study the role of CENP-C in centromere and kinetochore assembly. We show that, unlike the histone variant CENP-A, CENP-C is not maintained at centromeres through spermatogenesis but is assembled at the sperm centromere from the egg cytoplasm. Immunodepletion of CENP-C from metaphase egg extract prevents kinetochore formation on sperm chromatin, and depleted extracts can be complemented with in vitro–translated CENP-C. Using this complementation assay, we have identified CENP-C mutants that localized to centromeres but failed to support kinetochore assembly. We find that the amino terminus of CENP-C promotes kinetochore assembly by ensuring proper targeting of the Mis12/MIND complex and CENP-K.     

Crossing over in chicken oogenesis: cytological and chiasma-based genetic maps of the chicken lampbrush chromosome 1

Chiasmata in diplotene bivalents are located at the points of physical exchange (crossing-over) between homologous chromosomes. We have studied chiasma distribution within chicken lampbrush chromosome 1 to estimate the crossing-over frequency between chromosome landmarks. The position of the centromere and chromosome region 1q3.3-1q3.6 on lampbrush chromosome 1 were determined by comparative physical mapping of the TTAGGG repeats in the chicken mitotic and lampbrush chromosomes. The comparison of the chiasma (=crossing over)-based genetic distances on chicken chromosome 1 with the genetic linkage map obtained in genetic experiments showed that current genetic distances estimated by the high-resolution genetic mapping of the East Lansing, Compton, and Wageningen chicken reference populations are 1.2-1.9 times longer than those based on chiasma counts. Conceivable reasons for this discrepancy are discussed.     

Identification of novel centromere/kinetochore-associated proteins using monoclonal antibodies generated against human mitotic chromosome scaffolds

We describe the generation of 11 monoclonal antibodies that bind to the centromere/kinetochore region of human mitotic chromosomes. These antibodies were raised against mitotic chromosome scaffolds and screened for centromere/kinetochore binding by indirect immunofluorescence against purified chromosomes. Immunoblot analyses with these antibodies revealed that all of the antigens are greater than 200 kD and are components of nuclei, chromosomes, and/or chromosome scaffolds. Comparison of the immunolocalization of the antigens with that observed for the centromere-associated protein CENP-B revealed that each of these centromere/kinetochore proteins lies more peripherally to the DNA than does CENP-B. In cells normally progressing through the cell cycle, these antigens displayed four distinct patterns of centromere/kinetochore association, corresponding to a minimum of four novel centromere/kinetochore-associated proteins.     

Gcn5p plays an important role in centromere kinetochore function in budding yeast

We report that the histone acetyltransferase Gcn5p is involved in cell cycle progression, whereas its absence induces several mitotic defects, including inefficient nuclear division, chromosome loss, delayed G₂ progression, and spindle elongation. The fidelity of chromosome segregation is finely regulated by the close interplay between the centromere and the kinetochore, a protein complex hierarchically assembled in the centromeric DNA region, while disruption of GCN5 in mutants of inner components results in sick phenotype. These synthetic interactions involving the ADA complex lay the genetic basis for the critical role of Gcn5p in kinetochore assembly and function. We found that Gcn5p is, in fact, physically linked to the centromere, where it affects the structure of the variant centromeric nucleosome. Our findings offer a key insight into a Gcn5p-dependent epigenetic regulation at centromere/kinetochore in mitosis.     

Molecular analysis of an idic(Y)(qter -->p11.32::p11.32-->qter) chromosome from a female patient with a complex karyotype

A female patient with a structurally abnormal idic(Y) (p11.32) chromosome was studied using fluorescence in situ hybridization and PCR to define the precise position of the breakpoint. The patient had a complex mosaic karyotype with eight cell lines and at least two morphologically distinct derivatives from the Y chromosome. The rearrangement was a result of a meiosis I exchange between sister chromatids at the pseudoautosomal region, followed by centromere misdivision at meiosis II. Due to instability of the dicentric Y chromosome, new cell lines later arose because of mitotic errors occurring during embryonic development. Physical examination revealed a normal female phenotype without genital ambiguity, a normal uterus and rudimentary gonads which were surgically removed.     

A genetic method for generating Drosophila eyes composed exclusively of mitotic clones of a single genotype.

The genetic analysis of a gene at a late developmental stage can be impeded if the gene is required at an earlier developmental stage. The construction of mosaic animals, particularly in Drosophila, has been a means to overcome this obstacle. However, the phenotypic analysis of mitotic clones is often complicated because standard methods for generating mitotic clones render mosaic tissues that are a composite of both mutant and phenotypically normal cells. We describe here a genetic method (called EGUF/hid) that uses both the GAL4/UAS and FLP/FRT systems to overcome this limitation for the Drosophila eye by producing genetically mosaic flies that are otherwise heterozygous but in which the eye is composed exclusively of cells homozygous for one of the five major chromosome arms. These eyes are nearly wild type in size, morphology, and physiology. Applications of this genetic method include phenotypic analysis of existing mutations and F(1) genetic screens to identify as yet unknown genes involved in the biology of the fly eye. We illustrate the utility of the method by applying it to lethal mutations in the synaptic transmission genes synaptotagmin and syntaxin.     

Chromatin conformation of yeast centromeres

The centromere region of Saccharomyces cerevisiae chromosome III has been replaced by various DNA fragments from the centromere regions of yeast chromosomes III and XI. A 289-base pair centromere (CEN3) sequence can stabilize yeast chromosome III through mitosis and meiosis. The orientation of the centromeric fragments within chromosome III has no effect on the normal mitotic or meiotic behavior of the chromosome. The structural integrity of the centromere region in these genomic substitution strains was examined by mapping nucleolytic cleavage sites within the chromatin DNA. A nuclease-protected centromere core of 220-250 base pairs was evident in all of the genomic substitution strains. The position of the protected region is determined strictly by the centromere DNA sequence. These results indicate that the functional centromere core is contained within 220- 250 base pairs of the chromatin DNA that is structurally distinct from the flanking nucleosomal chromatin.     

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.     

Segregation after mitotic crossing-over in isodicentric X chromosomes

Segregation after mitotic crossing-over in an isodicentric (idic) X chromosome with one active and one inactive centromere has given rise to two new cell lines, one in which the idic(Xpter) chromosome has two active centromeres (most of these chromosomes also have an inversion) and another in which neither centromere is active. The two X chromosomes are attached at the telomeres of their short arms. Similar segregation has given rise to two other cell lines with idic(Xq-) chromosomes. Other observations on segregation after mitotic crossing-over are reviewed. Unequal crossing-over has apparently played a major role in the evolution of various genes and heterochromatin. Retinoblastoma and Wilms tumor are in some cases associated with homozygosity of a chromosome segment resulting from mitotic crossing-over. Similarly, the high incidence of cancer in Bloom syndrome may be caused by mitotic crossing-over leading to homozygosity or amplification of oncogenes.