Dicentric chromosomes: unique models to study centromere function and inactivation

Dicentric chromosomes are products of genome rearrangement that place two centromeres on the same chromosome. Depending on the organism, dicentric stability varies after formation. In humans, dicentrics occur naturally in a substantial portion of the population and usually segregate successfully in mitosis and meiosis. Their stability has been attributed to inactivation of one of the two centromeres, creating a functionally monocentric chromosome that can segregate normally during cell division. The molecular basis for centromere inactivation is not well understood, although studies in model organisms and in humans suggest that genomic and epigenetic mechanisms can be involved. Furthermore, constitutional dicentric chromosomes ascertained in patients presumably represent the most stable chromosomes, so the spectrum of dicentric fates, if it exists, is not entirely clear. Studies of engineered or induced dicentrics in budding yeast and plants have provided significant insight into the fate of dicentric chromosomes. And, more recently, studies have shown that dicentrics in humans can also undergo multiple fates after formation. Here, we discuss current experimental evidence from various organisms that has deepened our understanding of dicentric behavior and the intriguingly complex process of centromere inactivation.     

Chromosome stability is maintained by short intercentromeric distance in functionally dicentric human Robertsonian translocations

While the formation of a dicentric chromosome often leads to chromosome instability, human dicentric Robertsonian translocations usually remain stable. To investigate the basis for this stability, we have examined the centromeres of 15 structurally dicentric rob(13q14q) Robertsonian translocations using immunofluorescence and fluorescence in situ hybridization (FISH). The immunofluorescence detection of centromere protein C (CENP-C) was used as a marker for centromere function as CENP-C seems to play an essential role in kinetochore structure and stability and was previously shown to be absent from inactive centromeres. In all 15 translocation-containing cell lines, CENP-C was confined to only one of the centromeres of the translocation in a fraction of the cells analyzed. This suggests that centromere inactivation commonly occurs on dicentric Robertsonian translocations and may serve as one mechanism allowing for their stability. However, in the majority of the translocations (12 out of 15), a portion of the cells analyzed displayed CENP-C immunofluorescence at both centromeres, suggesting that both centromeres were active and that the translocation was functionally dicentric. The percentage of cells with CENP-C at both centromeres ranged from 2% to 82%. These results support the hypothesis that the close proximity of two functional centromeres on Robertsonian translocations allows them to remain stable.     

Evolution of compound centromeres. A new phenomenon

A new type of centromere aberration in a transformed cell line of rat cerebral endothelial origin is described. These cells exhibit normal monocentric, dicentric, and multicentric chromosomes. The centromeres in dicentrics and multicentrics express variable locations along the chromosome. The centromeres in some of the multicentrics are located next to each other, with small intervening noncentromeric chromatin. In others, the centromeres appear to be in the immediate vicinity of each other with no evidence of intervening chromatin. This organization of the centromeres results in what appears to be a compound centromere composed of some multiples of single centromeres. All centromeres deposit kinetochore proteins that respond to kinetochore antibody. This evidence and that obtained from electron microscopy permits the conclusion that various centromeres/kinetochores in the compound structure are functional. The study presented here points to the existence of compound large centromeres--a novel phenomenon in cytogenetics--that may be prevalent in cancer cells. In the present cell line these regions appear as long, neck-like structures in some chromosomes and may be similar to some in vivo situations such as the X in Indian muntjac.     

Centromere activity in dicentric small supernumerary marker chromosomes

Twenty-five dicentric small supernumerary marker chromosomes (sSMC) derived from #13/21, #14, #15, #18, and #22 were studied by immunohistochemistry for their centromeric activity. Centromere protein (CENP)-B was applied as marker for all centromeres and CENP-C to label the active ones. Three different ‘predominant’ activation patterns could be observed, i.e., centric fusion or either only one or all two centromeres were active. In one inherited case, the same activation pattern was found in mother and son. In acrocentric-derived sSMC, all three activation patterns could be present. In contrary, in chromosome 18-derived sSMC, only the fusion type was observed. In concordance with previous studies a certain centromeric plasticity was observed in up to 13% of the cells of an individual case. Surprisingly, the obtained data suggests a possible influence of the sSMC carrier’s gender on the implementation of the predominant activation pattern; especially, only one active centromere was found more frequently in female than in male carriers. Also, it might be suggested that dicentric sSMC with one active centromere could be less stable than such with two active ones—centromeric plasticity might have an influence here, as well. Also, centromere activity in acrocentric-derived dicentrics could be influenced by heteromorphisms of the corresponding short arms. Finally, evidence is provided that the closer the centromeres of a dicentric are and if they are not fused, the more likely it was that both of them became active. In concordance and refinement with previous studies, a distance of 1.4 Mb up to about 13 Mb the two active centromere state was favored, while centromeric distance of over ∼15 Mb lead to inactivation of one centromere. Overall, here, the first and largest ever undertaken study in dicentric sSMC is presented, providing evidence that the centromeric activation pattern is, and parental origin may be of interest for their biology. Influence of mechanisms similar or identical to meiotic imprinting in the centromeric regions of human chromosomes might be present. Furthermore, centromeric activation pattern could be at least in parts meaningful for the clinical outcome of dicentric sSMC, as sSMC stability and mosaicism can make the difference between clinically normal and abnormal phenotypes.     

Sequence of centromere separation: a mechanism for orderly separation of dicentrics

Stable dicentric chromosomes from three mouse cell lines (viz., SEWA Rec4, brain tumor, and L-cells), as well as a human t(9;11) line were analyzed for the sequence in which the two centromeres separate. At prometaphase, as well as in many cells at midmetaphase, the dicentrics express the two centromeres in the form of two primary constrictions. As the cell advances to late metaphase, one of the constrictions loosens the two chromatids so that eventually there is no connection between them. The other centromere stays intact during this period and separates into two units at the metaanaphase junction along with the rest of the genome. The centromere that separates prematurely (out-of-phase) usually is the same in a given dicentric. It is proposed that such a prematurely separating centromere does not function as active element during chromatid migration. Apparently, in dicentrics some sort of control is exerted to eliminate the functioning of one centromere. The nature of such control is not understood at this time. The mouse dicentrics "synthesize" only one kinetochore as definable by antikinetochore antibody studies.     

Making CENs of mammalian artificial chromosomes.

Mammalian artificial chromosomes (MACs) hold the promise of providing autonomous vectors for gene therapy in dividing cells. They would not require insertion into the genome and could include sufficient genomic sequences that surround the therapeutic gene to ensure proper tissue-specific and temporal regulation. Several groups have reported successful formation of MACs in human cells using transfection strategies that included alpha satellite DNA, the primary DNA found at normal human centromeres. These results, although extremely encouraging, have limitations such as unpredictable chromosome formation and success thus far in only one transformed human cell line. Examination of other cells where alpha satellite DNA has integrated into ectopic chromosomal locations, as well as naturally occurring dicentric and neocentromere-containing cell lines, suggests that alpha satellite DNA may not be necessary or sufficient for centromere formation. Overall, these results suggest that epigenetic modifications of centromeric DNA are required for efficient centromere formation. Models for this centromere-specific epigenetic modification include a specialized chromatin structure and differential replication timing of centromeric DNA. Thus, further investigation of these centromere-specific epigenetic modifications may suggest strategies for increasing the efficiency of generating human artificial chromosomes for use as gene therapy vectors.     

A new stable human dicentric chromosome, tdic(4;21)(p16;q22), in a woman with first trimester abortion.

A woman with first trimester abortion and a dicentric chromosome formed from a 4 and a 21 is described. The dicentric chromosome was stable and in the majority of cells the 21 centromere was active, while in a minority the chromosome 4 centromere was active. This shows that both centromeres were functional, but that only one functioned in any given cell. Suppression of the activity of one centromere might be the mechanism by which this dicentric chromosome achieved its stability. A dicentric formed from a chromosome 4 and a 21 has not apparently been previously reported.     

Further evidence that CENP-C is a necessary component of active centromeres: studies of a dic(X; 15) with simultaneous immunofluorescence and FISH

The stability of certain dicentric chromosomes in humans seemsto result from inactivation of one centromere, yielding a functionallymonocentric chromosome. Centromere protein C (CENP-C) was previouslyshown to be present at active centromeres but absent from theinactive centromere of one homologous dicentric rearrangement.We have combined indirect immunofluorescence detection of CENP-Cand fluorescence in situ hybridization with chromo-some-specific     

Stable dicentric autosome, tdic (8:22)(p23:p13), in a mentally retarded girl.

A dicentric autosome, tdic(8:22)(p23:p13), was found in all metaphase cells examined from the peripheral blood of a mentally retarded girl. It is suggested that the centromere of chromosome 22 was inactive, allowing the dicentric to behave as a monocentric element. The involvement of acrocentric chromosomes in the stable dicentric autosomes of man is discussed.     

Inactivation of a centromere during the formation of a translocation in maize

Fluorescence in situ hybridization analysis of a reciprocal translocation in maize between chromosomes 1 and 5 that has been used extensively in maize genetics revealed the presence of an inactive centromere at or near the breakpoints of the two chromosomes. This centromere contains both the satellite repeat, CentC, and the centromeric retrotransposon family, CRM, that are typical of centromere regions in maize. This site does not exhibit any of the tested biochemical features of active centromeres such as association with CENP-C and phosphorylation of serine-10 on histone H3. The most likely scenario for this chromosome arrangement is that a centromere was included in the repair process that formed the translocation but became inactive and has been inherited in this state for several decades. The documentation of an inactive A chromosome centromere in maize extends the evidence for an epigenetic component to centromere function in plants. This case provides an experimental example of how karyotype evolution might proceed via changes in centromere inactivation.     

Centromeres of filamentous fungi

How centromeres are assembled and maintained remains one of the fundamental questions in cell biology. Over the past 20?years, the idea of centromeres as precise genetic loci has been replaced by the realization that it is predominantly the protein complement that defines centromere localization and function. Thus, placement and maintenance of centromeres are excellent examples of epigenetic phenomena in the strict sense. In contrast, the highly derived "point centromeres" of the budding yeast Saccharomyces cerevisiae and its close relatives are counterexamples for this general principle of centromere maintenance. While we have learned much in the past decade, it remains unclear if mechanisms for epigenetic centromere placement and maintenance are shared among various groups of organisms. For that reason, it seems prudent to examine species from many different phylogenetic groups with the aim to extract comparative information that will yield a more complete picture of cell division in all eukaryotes. This review addresses what has been learned by studying the centromeres of filamentous fungi, a large, heterogeneous group of organisms that includes important plant, animal and human pathogens, saprobes, and symbionts that fulfill essential roles in the biosphere, as well as a growing number of taxa that have become indispensable for industrial use.     

Dicentric X isochromosomes in man.

Four cases of Turner's syndrome are presented in which an apparent X isochromosome i(Xq) has been found to possess two regions of centromeric heterochromatin. It is suggested that these chromosomes were isodicentric structures capable of functioning as monocentric elements as a result of the inactivation of one centromere. The prevalence of mosaicism is believed to be a consequence of the dicentric nature of these chromosomes, and it is considered possible that a high proportion of X isochromosmes are structurally dicentric. Banding patterns showed that the exchange site involved in the formation of the dicentric chromosome was different in at least three of the cases.     

Distinct DNA methylation patterns associated with active and inactive centromeres of the maize B chromosome

Centromeres are determined by poorly understood epigenetic mechanisms. Centromeres can be activated or inactivated without changing the underlying DNA sequences. However, virtually nothing is known about the epigenetic transition of a centromere from an active to an inactive state because of the lack of examples of the same centromere exhibiting alternative forms and being distinguishable from other centromeres. The centromere of the supernumerary B chromosome of maize provides such an opportunity because its functional core can be cytologically tracked, and an inactive version of the centromere is available. We developed a DNA fiber-based technique that can be used to assess the levels of cytosine methylation associated with repetitive DNA sequences. We report that DNA sequences in the normal B centromere exhibit hypomethylation. This methylation pattern is not affected by the genetic background or structural rearrangement of the B chromosome, but is slightly changed when the B chromosome is transferred to oat as an addition chromosome. In contrast, an inactive version of this same centromere exhibits hypermethylation, indicating that the inactive centromere was modified into a different epigenetic state at the DNA level.     

Identification of centromeric antigens in dicentric Robertsonian translocations: CENP-C and CENP-E are necessary components of functional centromeres

Robertsonian translocations are the most common structural dicentricrearrangements in humans. The stability of these dicentricsis attributed to the inactivation of one centromere by mechanismswhich are currently unknown. The presence and amounts of centromericproteins (CENPs) differ between the centromeres of the few dicentricswhich have been studied, providing a limited understanding ofthe protein components necessary for centromeric function. However,CENP-C previously has been observed only at the active centromeresin two dicentric chromosomes. In the present investigation,the presence and localizations of several centromeric antigens,CENP-B, -C and -E, have been determined in 12 dicentric Roberisoniantranslocations. Each translocation was studied initially usingin situ hybridization with     

Characterization by fluorescence and electron microscopy in situ hybridization of a double Y isochromosome

A patient with mixed gonadal dysgenesis and Y isochromosomes i(Y) is described. Lymphocyte cultures from peripheral blood contained a high proportion of 45,X cells and several other cell lines with two different marker chromosomes (mars). These markers had either a monocentric (mar1) or a dicentric appearance (mar2). Following high-resolution GTG, RBG, QFQ, and CBG bandings, five cell lines were identified; 45,X/46,X, + mar1/46,X, + mar2/47,X, + mar1x2/47,X, + mar2x2. The percentages were 66/6/26/1/1%, respectively. Chromosome banding analyses were insufficient for characterization of the markers. In situ hybridization of specific probes for the Y centromere and its short arm showed, both in fluorescence and electron microscopy (EM), two different Y rearrangements. Mar1 is an isochromosome for the short arm i(Yp) and mar2 is a dicentric which was shown by EM to be a double isochromosome Yp, inv dup i(Yp). The breakpoint producing mar1 is within the centromere and the one producing mar2 is within one of the short arms of the Y isochromosome. The findings of different cell populations in peripheral blood lymphocytes indicate the postzygotic instability of this i(Yp). © 1995 Wiley-Liss, Inc.     

Stability of monocentric and dicentric ring minichromosomes in <Emphasis Type="Italic">Arabidopsis</Emphasis>

A dicentric ring minichromosome (miniδ) was identified in transgenic Arabidopsis thaliana and added to a wild type as a supernumerary chromosome. This line is relatively stable and has been maintained for generations, notwithstanding its ring and dicentric structure. To determine the mechanism for stable transmission of miniδ, the structure and behavior of two new types of ring minichromosomes (miniδ1 and miniδ1-1) derived from miniδ were investigated. Fluorescence in situ hybridization analysis revealed that miniδ1 is dicentric just like miniδ, whereas miniδ1-1 is monocentric. The estimated sizes of miniδ1 and miniδ1-1 were 3.8~5.0 and 1.7 Mb, respectively. The sizes of the two centromeres on miniδ1 were identical (ca. 270 kb) and similar to that of miniδ1-1 (ca. 250 kb). Miniδ1 was relatively stable during mitosis and meiosis, as is miniδ, whereas miniδ1-1 was unstable during mitosis, and the number of minichromosomes per cell varied. This possibly resulted from misdivision caused by a short centromere on monocentric miniδ1-1. Transmission through the female was quite limited for all three ring minichromosomes (0–3.2%), whereas that through the male was relatively high (15.4–27.3%) compared with that of other supernumerary chromosomes in Arabidopsis. Ring structure without telomeres itself seems not to limit the female transmission.     

Localization of human SMC1 protein at kinetochores

Proper cohesion of sister chromatids is prerequisite for correct segregation of chromosomes during cell division. The cohesin multiprotein complex, conserved in eukaryotes, is required for sister chromatid cohesion. Human cohesin is composed of a stable heterodimer of the structural maintenance of chromosomes (SMC) family proteins, hSMC1 and hSMC3, and non-SMC components, hRAD21 and SA1 (or SA2). In yeast, cohesin associates with chromosomes from late G1 to metaphase and is required for the establishment and maintenance of both chromosome arm and centromeric cohesion. However, in human cells, the majority of cohesin dissociates from chromosomes before mitosis. Although it was recently shown that a small amount of hRAD21 localizes to the centromeres during metaphase, the presence of other cohesin components at the centromere has not been demonstrated in human cells. Here we report the mitosis-specific localization of hSMC1 to the kinetochores. hSMC1 is targeted to the kinetochore region during prophase concomitant with kinetochore assembly and remains through anaphase. Importantly, hSMC1 is targeted only to the active centromere on dicentric chromosomes. These results suggest that hSMC1 is an integral component of the functional kinetochore structure during mitosis. This revised version was published online in July 2006 with corrections to the Cover Date.     

Effect of colchicine and telocentric chromosome conformation on centromere and telomere dynamics at meiotic prophase I in wheat–rye additions

Association of telomeres in a bouquet and clustering of centromere regions have been proposed to be involved in the search and recognition of homologous partners. We have analysed the role of these structures in meiotic chromosome pairing in wheat–rye addition lines by applying colchicine for disturbing presynaptic telomere movements and by modifying the centromere position from submetacentric to telocentric for studying centromere effects. Rye chromosomes, wheat and rye centromeres, and telomeres were identified by fluorescence in-situ hybridization. Presynaptic association of centromeres in pairs or in more complex structures involved mainly non-homologous chromosomes as deduced from the behaviour of rye centromeres. While centromere association was not affected by colchicine, colchicine inhibited bouquet formation, which caused failure of homologous synapsis. Homologous centromeres of rye telocentrics associated earlier than those of rye submetacentric chromosomes, indicating that migration of the telocentrics’ centromeres to the telomere pole during bouquet formation facilitated their association. Homologous chromosomes associated in premeiotic interphase can recognize each other and initiate synapsis at zygotene. However, telomere convergence is needed for bringing together the majority of homologous pairs that normally occupy separate territories in premeiotic nuclei.