Dicentric chromosome formation and epigenetics of centromere formation in plants

Plant centromeres are generally composed of tandem arrays of simple repeats that form a complex chromosome locus where the kinetochore forms and microtubules attach during mitosis and meiosis. Each chromosome has one centromere region, which is essential for accurate division of the genetic material. Recently, chromosomes containing two centromere regions (called dicentric chromosomes) have been found in maize and wheat. Interestingly, some dicentric chromosomes are stable because only one centromere is active and the other one is inactivated. Because such arrays maintain their typical structure for both active and inactive centromeres, the specification of centromere activity has an epigenetic component independent of the DNA sequence. Under some circumstances, the inactive centromeres may recover centromere function, which is called centromere reactivation. Recent studies have highlighted the important changes, such as DNA methylation and histone modification, that occur during centromere inactivation and reactivation.     

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.     

Centromeres put epigenetics in the driver's seat

A defining feature of chromosomes is the centromere, the site for spindle attachment at mitosis and meiosis. Intriguingly, centromeres of plants and animals are maintained by both sequence-specific and sequence-independent (epigenetic) processes. Epigenetic inheritance might enable kinetochores (the structures that attach centromeres to spindles) to maintain an optimal size. However, centromeres are susceptible to the evolution of "selfish" DNA repeats that bind to kinetochore proteins. We argue that such sequence-specific interactions are evolutionarily unstable because they enable repeat arrays to influence kinetochore size. Changes in kinetochore size could affect the interaction of kinetochores with the spindle and, in principle, skew Mendelian segregation. We propose that key kinetochore proteins have adapted to disrupt such sequence-specific interactions and restore epigenetic inheritance.     

Centromeres: getting a grip of chromosomes

On monocentric chromosomes the centromere is the chromosomal site at which the kinetochore complex is assembled. This complex mediates the attachment and movement of chromosomes along spindle microtubules. The centromere is usually the last site to retain cohesion between sister centromeres. The location of the main sensor for defective spindle assembly at the kinetochore allows the release of this cohesion, and thus progression through mitosis, to be held in check until key events have been completed. The intricate nature of the centromere-kinetochore complexes and the events they co-ordinate and react to is presently being dissected by studies in several organisms. In particular, several new kinetochore proteins have been identified in many organisms over the last year.     

Characterization of kinetochores in multicentric chromosomes

Long-term cultures of certain rat and mouse cell lines carry several dicentric and some multicentric chromosomes. Using antikinetochore antibodies obtainable from serum of scleroderma (var. CREST) patients we studied the number of kinetochores formed along the length of these chromosomes. The rat cells displayed as many kinetochores as there were centromeres. However, mouse cells showed the synthesis of only one kinetochore in dicentric and multicentric chromosomes which had been in the culture for a period of 1 year or more. When translocations were induced by bleomycin in mouse L cells, the newly formed dicentric chromosomes showed the formation of two kinetochores. It is not known when the accessory centromeres lose their capacity to assemble kinetochore proteins. Possibly, in the rat the latent kinetochores lack a specific component which renders them ineffective for microtubule binding. The reason for the formation of only one kinetochore in mouse multicentric chromosomes is not clear. It may be due to the accumulation of mutations, modification of the kinetochore protein so that it lacks the antibody binding component, or a more effective regulatory gene than in the rat.     

The requirement for the Dam1 complex is dependent upon the number of kinetochore proteins and microtubules

The Dam1 complex attaches the kinetochore to spindle microtubules and is a processivity factor in vitro. In Saccharomyces cerevisiae, which has point centromeres that attach to a single microtubule, deletion of any Dam1 complex member results in chromosome segregation failures and cell death. In Schizosaccharomyces pombe, which has epigenetically defined regional centromeres that each attach to 3-5 kinetochore microtubules, Dam1 complex homologs are not essential. To determine why the complex is essential in some organisms and not in others, we used Candida albicans, a multimorphic yeast with regional centromeres that attach to a single microtubule. Interestingly, the Dam1 complex was essential in C. albicans, suggesting that the number of microtubules per centromere is critical for its requirement. Importantly, by increasing CENP-A expression levels, more kinetochore proteins and microtubules were recruited to the centromeres, which remained fully functional. Furthermore, Dam1 complex members became less crucial for growth in cells with extra kinetochore proteins and microtubules. Thus, the requirement for the Dam1 complex is not due to the DNA-specific nature of point centromeres. Rather, the Dam1 complex is less critical when chromosomes have multiple kinetochore complexes and microtubules per centromere, implying that it functions as a processivity factor in vivo as well as in vitro.     

Knockdown of CENH3 in Arabidopsis reduces mitotic divisions and causes sterility by disturbed meiotic chromosome segregation

The histone H3 variant (CENH3) of centromeric nucleosomes is essential for kinetochore assembly and thus for chromosome segregation in eukaryotes. The mechanism(s) that determine centromere identity, assembly and maintenance of kinetochores are still poorly understood. Although the role of CENH3 during mitosis has been studied in several organisms, little is known about its meiotic function. We show that RNAi-mediated CENH3 knockdown in Arabidopsis thaliana caused dwarfism as the result of a reduced number of mitotic divisions. The remaining mitotic divisions appeared to be error-free. CENH3 RNAi transformants had reduced fertility because of frequently disturbed meiotic chromosome segregation. N-terminally truncated EYFP-CENH3(C) is deposited to and functional within Arabidopsis centromeres of mitotic chromosomes, but cannot be loaded onto centromeres of meiotic nuclei. Thus the N-terminal part is apparently required for CENH3 loading during meiosis. EYFP-CENH3(C) expression reduces the amount of endogenous CENH3, thus mimicking the effect of RNAi. The consequences of reduced endogenous CENH3 and lack of meiotic incorporation of EYFP-CENH3(C) are reduced fertility caused by insufficient CENH3 loading to the centromeres of meiotic chromosomes, subsequent lagging of chromosomes and formation of micronuclei.     

Differential kinetochore protein requirements for establishment versus propagation of centromere activity in Saccharomyces cerevisiae

Dicentric chromosomes undergo a breakage-fusion-bridge cycle as a consequence of having two centromeres on the same chromatid attach to opposite spindle poles in mitosis. Suppression of dicentric chromosome breakage reflects loss of kinetochore function at the kinetochore-microtubule or the kinetochore-DNA interface. Using a conditionally functional dicentric chromosome in vivo, we demonstrate that kinetochore mutants exhibit quantitative differences in their degree of chromosome breakage. Mutations in chl4/mcm17/ctf17 segregate dicentric chromosomes through successive cell divisions without breakage, indicating that only one of the two centromeres is functional. Centromere DNA introduced into the cell is unable to promote kinetochore assembly in the absence of CHL4. In contrast, established centromeres retain their segregation capacity for greater than 25 generations after depletion of Chl4p. The persistent mitotic stability of established centromeres reveals the presence of an epigenetic component in kinetochore segregation. Furthermore, this study identifies Chl4p in the initiation and specification of a heritable chromatin state.     

Incorporation of Drosophila CID/CENP-A and CENP-C into centromeres during early embryonic anaphase

The centromere/kinetochore complex is indispensable for accurate segregation of chromosomes during cell divisions when it serves as the attachment site for spindle microtubules. Centromere identity in metazoans is believed to be governed by epigenetic mechanisms, because the highly repetitive centromeric DNA is neither sufficient nor required for specifying the assembly site of the kinetochore. A candidate for an epigenetic mark is the centromere-specific histone H3 variant CENP-A that replaces H3 in alternating blocks of chromatin exclusively in active centromeres. CENP-A acts as an initiator of kinetochore assembly, but the detailed dynamics of the deposition of metazoan CENP-A and of other constitutive kinetochore components are largely unknown. Here we show by quantitative fluorescence measurements in living early embryos that functional fluorescent fusion proteins of the Drosophila CENP-A and CENP-C homologs are rapidly incorporated into centromeres during anaphase. This incorporation is independent of ongoing DNA synthesis and pulling forces generated by the mitotic spindle, but strictly coupled to mitotic progression. Thus, our findings uncover a strikingly dynamic behavior of centromere components in anaphase.     

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     

A Highlights from MBoC Selection: Ipl1/Aurora-B is necessary for kinetochore restructuring in meiosis I in Saccharomyces cerevisiae

In mitosis, the centromeres of sister chromosomes are pulled toward opposite poles of the spindle. In meiosis I, the opposite is true: the sister centromeres move together to the same pole, and the homologous chromosomes are pulled apart. This change in segregation patterns demands that between the final mitosis preceding meiosis and the first meiotic division, the kinetochores must be restructured. In budding yeast, unlike mammals, kinetochores are largely stable throughout the mitotic cycle. In contrast, previous work with budding and fission yeast showed that some outer kinetochore proteins are lost in early meiosis. We use quantitative mass spectrometry methods and imaging approaches to explore the kinetochore restructuring process that occurs in meiosis I in budding yeast. The Ndc80 outer kinetochore complex, but not other subcomplexes, is shed upon meiotic entry. This shedding is regulated by the conserved protein kinase Ipl1/Aurora-B and promotes the subsequent assembly of a kinetochore that will confer meiosis-specific segregation patterns on the chromosome.     

Sequences associated with A chromosome centromeres are present throughout the maize B chromosome

Maize chromosome spreads containing the supernumerary B chromosome were hybridized with probes from various repetitive elements including CentC, CRM, and CentA, which have been localized to centromeric regions on the A chromosomes. Repetitive elements that are enriched or found exclusively near the centromeres of A chromosomes hybridized to many sites distinct from the centromere on the B chromosome. To examine whether these elements recruit kinetochore proteins at locations other than the canonical B centromere, cells were labeled with antibodies against CENH3, a key kinetochore protein. No labeling was detected outside the normal centromere and no evidence of B chromosome holocentromeric activity was observed. This finding suggests that, as in other higher eukaryotes, DNA sequence alone is insufficient to dictate kinetochore location in plants. Additionally, examination of the B centromere region in pachytene chromosomes revealed that the B-specific element ZmBs hybridizes to a much larger region than the site of hybridization of CentC, CRM, and CentA and the labeling by anti-CENH3 antibodies.This revised version was published online in December 2004 with corrections to Table 1.     

Centromere organization in chromosomes of the mouse

The ultrastructure of the centromere region of chromosomes from mouse L929 cells treated with agents that affect centromere condensation have been examined using light, transmission electron, and scanning electron microscopic techniques. Micrographs of expanded centromeres from treated chromosomes illustrate that both the biarmed chromosomes that were generated by Robertsonian fusion during the past history of the strain and the functional centromere of the multicentromeric marker chromosomes display a prominent gap. This gap probably represents the original site of association of the acrocentric chromosomes and is also the site of the kinetochore. Despite the multicentromeric nature of the marker chromosome a single pair of kinetochores were found only at the central heterochromatic region. The functional implications of these structural findings are discussed.     

Tethering Sister Centromeres to Each Other Suggests the Spindle Checkpoint Detects Stretch within the Kinetochore

The spindle checkpoint ensures that newly born cells receive one copy of each chromosome by preventing chromosomes from segregating until they are all correctly attached to the spindle. The checkpoint monitors tension to distinguish between correctly aligned chromosomes and those with both sisters attached to the same spindle pole. Tension arises when sister kinetochores attach to and are pulled toward opposite poles, stretching the chromatin around centromeres and elongating kinetochores. We distinguished between two hypotheses for where the checkpoint monitors tension: between the kinetochores, by detecting alterations in the distance between them, or by responding to changes in the structure of the kinetochore itself. To distinguish these models, we inhibited chromatin stretch by tethering sister chromatids together by binding a tetrameric form of the Lac repressor to arrays of the Lac operator located on either side of a centromere. Inhibiting chromatin stretch did not activate the spindle checkpoint; these cells entered anaphase at the same time as control cells that express a dimeric version of the Lac repressor, which cannot cross link chromatids, and cells whose checkpoint has been inactivated. There is no dominant checkpoint inhibition when sister kinetochores are held together: cells expressing the tetrameric Lac repressor still arrest in response to microtubule-depolymerizing drugs. Tethering chromatids together does not disrupt kinetochore function; chromosomes are successfully segregated to opposite poles of the spindle. Our results indicate that the spindle checkpoint does not monitor inter-kinetochore separation, thus supporting the hypothesis that tension is measured within the kinetochore.     

A coordinated interdependent protein circuitry stabilizes the kinetochore ensemble to protect CENP-A in the human pathogenic yeast Candida albicans

Unlike most eukaryotes, a kinetochore is fully assembled early in the cell cycle in budding yeasts Saccharomyces cerevisiae and Candida albicans. These kinetochores are clustered together throughout the cell cycle. Kinetochore assembly on point centromeres of S. cerevisiae is considered to be a step-wise process that initiates with binding of inner kinetochore proteins on specific centromere DNA sequence motifs. In contrast, kinetochore formation in C. albicans, that carries regional centromeres of 3-5 kb long, has been shown to be a sequence independent but an epigenetically regulated event. In this study, we investigated the process of kinetochore assembly/disassembly in C. albicans. Localization dependence of various kinetochore proteins studied by confocal microscopy and chromatin immunoprecipitation (ChIP) assays revealed that assembly of a kinetochore is a highly coordinated and interdependent event. Partial depletion of an essential kinetochore protein affects integrity of the kinetochore cluster. Further protein depletion results in complete collapse of the kinetochore architecture. In addition, GFP-tagged kinetochore proteins confirmed similar time-dependent disintegration upon gradual depletion of an outer kinetochore protein (Dam1). The loss of integrity of a kinetochore formed on centromeric chromatin was demonstrated by reduced binding of CENP-A and CENP-C at the centromeres. Most strikingly, Western blot analysis revealed that gradual depletion of any of these essential kinetochore proteins results in concomitant reduction in cellular protein levels of CENP-A. We further demonstrated that centromere bound CENP-A is protected from the proteosomal mediated degradation. Based on these results, we propose that a coordinated interdependent circuitry of several evolutionarily conserved essential kinetochore proteins ensures integrity of a kinetochore formed on the foundation of CENP-A containing centromeric chromatin.     

The CCAN complex: linking centromere specification to control of kinetochore-microtubule dynamics

For over 70 years, chromosomes have been known to oscillate back-and-forth on the metaphase plate. These movements are directed by kinetochores, the microtubule-attachment complexes on centromeres that regulate the dynamics of bound spindle microtubules. Recent evidence shows that the CCAN (Constitutive Centromere Associated Network) kinetochore network, which directly binds centromeric nucleosomes, plays a crucial role in the control of kinetochore microtubule dynamics. Here we review how this 15-subunit protein network functions within the kinetochore machinery, how it may adapt dynamically both in time and in space to the functional requirements necessary for controlled and faithful chromosome movements during cell division, and how this conserved protein network may have evolved in organisms with different cell division machineries.     

HCP-4/CENP-C promotes the prophase timing of centromere resolution by enabling the centromere association of HCP-6 in Caenorhabditis elegans

Prior to microtubule capture, sister centromeres resolve from one another, coming to rest on opposite surfaces of the condensing chromosome. Subsequent assembly of sister kinetochores at each sister centromere generates a geometry favorable for equal levels of segregation of chromatids. The holocentric chromosomes of Caenorhabditis elegans are uniquely suited for the study of centromere resolution and subsequent kinetochore assembly. In C. elegans, only two proteins have been identified as being necessary for centromere resolution, the kinase AIR-2 (prophase only) and the centromere protein HCP-4/CENP-C. Here we found that the loss of proteins involved in chromosome cohesion bypassed the requirement for HCP-4/CENP-C but not for AIR-2. Interestingly, the loss of cohesin proteins also restored the localization of HCP-6 to the kinetochore. The loss of the condensin II protein HCP-6 or MIX-1/SMC2 impaired centromere resolution. Furthermore, the loss of HCP-6 or MIX-1/SMC2 resulted in no centromere resolution when either nocodazole or RNA interference (RNAi) of the kinetochore protein KNL-1 perturbed spindle-kinetochore interactions. This result suggests that normal prophase centromere resolution is mediated by condensin II proteins, which are actively recruited to sister centromeres to mediate the process of resolution.     

The Rabl chromosome configuration masks a kinetochore reassembly mechanism in yeast mitosis

During cell cycle progression in metazoans, the kinetochore is assembled at mitotic onset and disassembled during mitotic exit. Once assembled, the kinetochore complex attached to centromeres interacts directly with the spindle microtubules, the vehicle of chromosome segregation. This reassembly program is assumed to be absent in budding and fission yeast, because most kinetochore proteins are stably maintained at the centromeres throughout the entire cell cycle. Here, we show that the reassembly program of the outer kinetochore at mitotic onset is unexpectedly conserved in the fission yeast Schizosaccharomyces pombe. We identified this behavior by removing the Rabl chromosome configuration, in which centromeres are permanently associated with the nuclear envelope beneath the spindle pole body during interphase. In addition to having evolutionary implications for kinetochore reassembly, our results aid the understanding of the molecular processes responsible for kinetochore disassembly and assembly during mitotic entry.     

Sequence of centromere separation: characterization of multicentric chromosomes in a rat cell line

The B₁ cell line of rat cerebral endothelium origin exhibits several dicentric and multicentric chromosomes. These chromosomes, unlike multicentrics in mouse (Vig and Zinkowski 1986) do not show premature centromere separation. All centromeres deposit kinetochore proteins and appear to be functional. Even the centromeres which fail to migrate to the poles during anaphase and make side arm bridges bind to spindle microtubules. Some multicentric chromosomes show kinetochores spaced apart with intervening stretches of euchromatin while others are located adjacent to each other thus exhibiting tandem repeats and forming a compound kinetochore (Brinkeley et al. 1984). Also, unlike mouse multicentric chromosomes in which different pericentric regions and the centromeres replicate at different times, the rat chromosomes appear to replicate all pericentric and centric regions in a given multicentric simultaneously. The present studies indicate that centromeres in rat and mouse replicate during the last part of the S-phase and in continuation with the pericentric heterochromatin.     

Neocentromeres: role in human disease,evolution, and centromere study

The centromere is essential for the proper segregation and inheritance of genetic information. Neocentromeres are ectopic centromeres that originate occasionally from noncentromeric regions of chromosomes. Despite the complete absence of normal centromeric alpha-satellite DNA, human neocentromeres are able to form a primary constriction and assemble a functional kinetochore. Since the discovery and characterization of the first case of a human neocentromere in our laboratory a decade ago, 60 examples of constitutional human neocentromeres distributed widely across the genome have been described. Typically, these are located on marker chromosomes that have been detected in children with developmental delay or congenital abnormalities. Neocentromeres have also been detected in at least two types of human cancer and have been experimentally induced in Drosophila. Current evidence from human and fly studies indicates that neocentromere activity is acquired epigenetically rather than by any alteration to the DNA sequence. Since human neocentromere formation is generally detrimental to the individual, its biological value must lie beyond the individual level, such as in karyotype evolution and speciation.