Strong nucleosomes reside in meiotic centromeres of C. elegans

Recently discovered strong nucleosomes (SNs) are characterized by strongly periodical DNA sequence, with visible rather than hidden sequence periodicity. In a quest for possible functions of the SNs, it has been found that the SNs concentrate within centromere regions of A. thaliana chromosomes . They, however, have been detected in Caenorhabditis elegans as well, although the holocentric chromosomes of this species do not have centromeres. Scrutinizing the SNs of C. elegans and their distributions along the DNA sequences of the chromosomes, we have discovered that the SNs are located mainly at the ends of the chromosomes of C. elegans. This suggests that, perhaps, the ends of the chromosomes fulfill some function(s) of centromeres in this species, as also indicated by the cytogenetic studies on meiotic chromosomes in spermatocytes of C. elegans, where the end-to-end association is observed. The centromeric involvement of the SNs, also found in A. thaliana, opens new horizons for the chromosome and centromere structure studies.     

Strong nucleosomes of yeasts

Yeast genome lacks visibly periodic sequences characteristic of strong nucleosomes (SNs) originally discovered in A. thaliana, C. elegans, and H. sapiens. Yet, the sequences with good match to the (RRRRRYYYYY)_n consensus of the SNs do show preference to centromere regions of Schizosaccharomyces pombe, Saccharomyces cerevisiae, and Cryptococcus neoformans – property characteristic of SNs of higher eukaryotes. Candida albicans is the first exception detected so far, where their SNs do not have any affinity to the centromeres, nor pericentromeric regions. Three of the four yeast genomes analyzed possess unique repeating centromere-specific SN sequences (C. albicans, again, is an exception). The results firmly indicate that centromeres of plants, animals, and yeasts in general have special chromatin structure, favoring SNs.     

TA-periodic (“601”-like) centromeric nucleosomes of A. thaliana

The bulk of strong nucleosomes (SNs, with visibly periodic DNA sequences) is described by consensus pattern of 5 or 6 base runs of purines alternating with similar runs of pyrimidines – RR/YY SNs. Yet, the strongest known nucleosome positioning sequence, the 601 clone of Lowary and Widom, is rather periodic repetition of TA dinucleotides following one another every 10 bases. We located “601”-like TA-periodic sequences in the genome of A. thaliana. Several families of such sequences are discovered repeating almost exclusively in centromeres. Thus, while A. thaliana SNs of RR/YY type have strong affinity to pericentromeric regions, as it has been previously found, the SNs of TA periodic type concentrate rather in centromeres.     

Single base discrimination of CENP-B repeats on mouse and human Chromosomes with PNA-FISH

The satellite repeat structure of the mammalian centromere contains the CENP-B protein binding site. Using the peptide nucleic acid-fluorescence in situ hybridization (PNA-FISH), we show by direct PNA-DNA binding that all detectable CENP-B sites in a mammalian genome might have the same sequence. Two species-specific PNA 17-mers, pMm and pMc, were identified from CENP-B binding sites of Mus musculus and M. caroli, respectively. Fluorescence in situ hybridization confirmed that pMc hybridized to M. caroli centromeres only; however, pMm cross-hybridized to M. musculus and human centromeres. By using a series of CENP-B PNA 17-mers containing 1, 2, 3, 5, and 7 base-pair mismatches to their DNA counterparts, we further demonstrate that PNA-FISH can discriminate between two CENP-B DNA sequences that differ by a single base-pair in mouse and human centromeres, suggesting the degree of conservation of CENP-B sequences throughout the genome. In comparison with DNA oligonucleotides, PNA oligomers demonstrate the higher sequence specificity, improved stability, reproducibility, and lower background. Therefore, PNA oligomers have significant advantages over DNA oligonucleotide probes in analyzing microsatellites in a genome. Received: 16 June 1998 / Accepted: 3 September 1998     

Sequence structure of Lowary/Widom clones forming strong nucleosomes

Lowary and Widom selected from random sequences those which form exceptionally stable nucleosomes, including clone 601, the current champion of strong nucleosome (SN) sequences. This unique sequence database (LW sequences) carries sequence elements which confer stability on the nucleosomes formed on the sequences, and, thus, may serve as source of information on the structure of “ideal” or close to ideal nucleosome DNA sequence. An important clue is also provided by crystallographic study of Vasudevan and coauthors on clone 601 nucleosomes. It demonstrated that YR·YR dinucleotide stacks (primarily TA·TA) follow one another at distances 10 or 11 bases or multiples thereof, such that they all are located on the interface between DNA and histone octamer. Combining this important information with alignment of the YR-containing 10-mers and 11-mers from LW sequences, the bendability matrices of the stable nucleosome DNA are derived. The matrices suggest that the periodically repeated TA (YR), RR, and YY dinucleotides are the main sequence features of the SNs. This consensus coincides with the one for recently discovered SNs with visibly periodic DNA sequences. Thus, the experimentally observed stable LW nucleosomes and SNs derived computationally appear to represent the same entity – exceptionally stable SNs.     

JunctionViewer: customizable annotation software for repeat-rich genomic regions

Background  

Repeat-rich regions such as centromeres receive less attention than their gene-rich euchromatic counterparts because the former are difficult to assemble and analyze. Our objectives were to 1) map all ten centromeres onto the maize genetic map and 2) characterize the sequence features of maize centromeres, each of which spans several megabases of highly repetitive DNA. Repetitive sequences can be mapped using special molecular markers that are based on PCR with primers designed from two unique "repeat junctions". Efficient screening of large amounts of maize genome sequence data for repeat junctions, as well as key centromere sequence features required the development of specific annotation software.     

Mouse centromere mapping using oligonucleotide probes that detect variants of the minor satellite

Cytologically, the centromere is found at the very end of most Mus musculus chromosomes, co-localizing with an array of minor satellite sequences. It is separated from the euchromatin of the long arm by a large domain of heterochromatin, composed in part of arrays of major satellite sequences. We used oligonucleotide probes that specifically detect regions of sequence variation found in certain cloned minor satellite sequences. They detect a limited subset of the minor satellite arrays in the mouse genome, based on both pulsed-field gel electrophoresis and in situ hybridization data, and provide direct molecular genetic markers for individual centromeres in some inbred mouse strains. Array size polymorphisms detected by these probes map to positions consisten with the centromeres of chromosomes 1 and 14 in the BXD recombinant inbred (RI) strains. The genetic distances between these minor satellite arrays and loci on the long arms of chromosomes 1 and 14 are consistent with repression of meiotic recombination in the heterochromatic domains separating them. The existence of chromosome-specific minor satellite sequences implies that the rate of sequence exchange between non-homologous chromosomes relative to the rate between homologous chromosomes is much lower than has previously been postulated. We suggest that the high degree of sequence homogeneity of mouse satellite sequences may instead reflect recent common ancestry.     

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

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

Robertsonian Fusion and Centromere Repositioning Contributed to the Formation of Satellite-free Centromeres During the Evolution of Zebras

Centromeres are epigenetically specified by the histone H3 variant CENP-A and typically associated with highly repetitive satellite DNA. We previously discovered natural satellite-free neocentromeres in Equus caballus and Equus asinus. Here, through ChIP-seq with an anti-CENP-A antibody, we found an extraordinarily high number of centromeres lacking satellite DNA in the zebras Equus burchelli (15 of 22) and Equus grevyi (13 of 23), demonstrating that the absence of satellite DNA at the majority of centromeres is compatible with genome stability and species survival and challenging the role of satellite DNA in centromere function. Nine satellite-free centromeres are shared between the two species in agreement with their recent separation. We assembled all centromeric regions and improved the reference genome of E. burchelli. Sequence analysis of the CENP-A binding domains revealed that they are LINE-1 and AT-rich with four of them showing DNA amplification. In the two zebras, satellite-free centromeres emerged from centromere repositioning or following Robertsonian fusion. In five chromosomes, the centromeric function arose near the fusion points, which are located within regions marked by traces of ancestral pericentromeric sequences. Therefore, besides centromere repositioning, Robertsonian fusions are an important source of satellite-free centromeres during evolution. Finally, in one case, a satellite-free centromere was seeded on an inversion breakpoint. At 11 chromosomes, whose primary constrictions seemed to be associated with satellite repeats by cytogenetic analysis, satellite-free neocentromeres were instead located near the ancestral inactivated satellite-based centromeres; therefore, the centromeric function has shifted away from a satellite repeat containing locus to a satellite-free new position.     

Epigenetically-Inherited Centromere and Neocentromere DNA Replicates Earliest in S-Phase

Eukaryotic centromeres are maintained at specific chromosomal sites over many generations. In the budding yeast Saccharomyces cerevisiae, centromeres are genetic elements defined by a DNA sequence that is both necessary and sufficient for function; whereas, in most other eukaryotes, centromeres are maintained by poorly characterized epigenetic mechanisms in which DNA has a less definitive role. Here we use the pathogenic yeast Candida albicans as a model organism to study the DNA replication properties of centromeric DNA. By determining the genome-wide replication timing program of the C. albicans genome, we discovered that each centromere is associated with a replication origin that is the first to fire on its respective chromosome. Importantly, epigenetic formation of new ectopic centromeres (neocentromeres) was accompanied by shifts in replication timing, such that a neocentromere became the first to replicate and became associated with origin recognition complex (ORC) components. Furthermore, changing the level of the centromere-specific histone H3 isoform led to a concomitant change in levels of ORC association with centromere regions, further supporting the idea that centromere proteins determine origin activity. Finally, analysis of centromere-associated DNA revealed a replication-dependent sequence pattern characteristic of constitutively active replication origins. This strand-biased pattern is conserved, together with centromere position, among related strains and species, in a manner independent of primary DNA sequence. Thus, inheritance of centromere position is correlated with a constitutively active origin of replication that fires at a distinct early time. We suggest a model in which the distinct timing of DNA replication serves as an epigenetic mechanism for the inheritance of centromere position.     

Centromere structures highlighted by the 100%-complete Cyanidioschyzon merolae genome

Centromere dynamics are largely unknown in lower plants (algae). We have recently identified the centromere-specific histone H3 variant (CENH3) and clarified the dynamic centromere rearrangement at mitosis in the primitive red alga Cyanidioschyzon merolae. We also showed that the CENH3-containing nucleosomes constituted the kinetochore closely interacting with the nuclear envelope. CENH3 visualization during the whole cell cycle suggests that C. merolae centromeres are monocentric and confined to specific loci. We completed 100% no-gap telomereto-telomere sequencing of the C. merolae genome. Interestingly, a single A+T-rich region has been identified on each fully sequenced chromosome. No centromere-like A+T-rich repetitive sequence have been found within these regions, implying that the C. merolae centromeres may be ‘point’ centromeres, or be comprised of nonrepetitive heterogeneous DNA sequences.Key words: centromere, chromosome structure, complete nuclear genome, Cyanidioschyzon, repetitive DNACentromere function is evolutionarily conserved in almost all eukaryotes. It is known that centromeric DNAs undergo rapid evolution and have no obvious constraints on their sequence conservation. However, several centromere proteins are conserved at the domain and motif level, suggesting that key protein-protein interactions, retained through centromere evolution, might allow for the functional conservation and DNA sequence diversity of the centromere. Most prominent are centromere-specific histone H3 (CENH3) family proteins, because the histone fold domain of this family is well conserved among all the eukaryotic lineages to assemble the centromeric nucleosomes with other conventional histones.¹We previously clarified the centromere movement and reconstitution during the cell cycle by tracing the CENH3 in the ultrasmall unicellular red alga Cyanidioschyzon merolae. On the relationship between the kinetochore and the nuclear envelope (NE), which had been poorly understood in red algae, we demonstrated using electron microscopy that they are closely associated at mitosis. Given the cellular characteristics that the chromosomes barely condense and the NE remains intact throughout the cell cycle in C. merolae, we postulate that this kinetochore-NE interaction might produce a ‘signal’ until the uncondensed and lagged chromosome arm regions have been completely segregated and the tension on the NE has been attenuated. Visualization of CENH3 proved that the C. merolae chromosomes are not holocentric (entire chromosomes serve as centromeres) but are likely to be monocentric (one ‘regional’ or ‘point’ centromere occurs on each chromosome).²Previous C. merolae genome sequencing showed that several chromosomes possess single A+T-rich regions, which are annotated as putative centromeric regions. However, there were many gaps in the whole genome assembly that had not been fully sequenced, and a full picture of the putative centromeric regions was unclear.³ Recently, we finished the 100% complete genome sequencing and obtained all the 20 chromosome assemblies as full telomere-to-telomere sequences.⁴ Although generally the rDNA regions are highly repetitive and structurally unstable in most eukaryotes, the C. merolae complete genome sequence included the complete set of three ‘static’ singlet rDNA clusters scattered across different chromosomal loci, which is one of the most distinguishing structural characteristics.⁵Figure 1 shows the overall G+C content distribution on the fully sequenced chromosomes. It is interesting to note that a single A+T-rich region is present on each chromosome. We postulate that the single A+T-rich regions are something more than just stochastic distribution, and are likely to play some role in the maintenance of chromosome structure, since these regions show essentially a one-on-one relationship with chromosomes. Although less clear, the genome sequence of the unicellular green alga Ostreococcus tauri (Prasinophyceae) similarly shows a biased A+T distribution pattern.⁶ To determine whether the A+T-rich regions are associated with repetitive sequences like most centromeric regions in other species, we employed Tandem repeats finder, a program used to search for repeat sequences.⁷ However, we have not found any repeat sequence, any common pattern or any particular rule concerning the size, A+T% or sequence of these regions.Open in a separate windowFigure 1G+C content and distribution on the Cyanidioschyzon merolae chromosomes. Arrowheads indicate the positions of the putative centromeric regions.Several pioneering works provide useful information on the centromere structures in lower eukaryotes. The centromeres of the human malaria parasite Plasmodium falciparum are composed of extremely A+T-rich repetitive elements.⁸ In the trypanosome parasites, the Trypanosoma brucei chromosomes possess A+T-rich repeats within the centromeric region as identified by etoposide-mediated topoisomerase-II cleavage analysis, while these A+T elements are not found in T. cruzi.⁹ It is also important to note that centromeres in Candida albicans are all comprised of different and unique DNA sequences, and are maintained by an epigenetic mechanism.^10,11We presume that monocentric C. merolae chromosomes are unlikely to possess ‘regional’ centromeres composed of A+T-rich centromeric repeats, but rather the centromere structure is similar to ‘point’ (approximately 120 bp) centromeres in Saccharomyces cerevisiae.¹ Alternatively, the functional C. merolae centromeres might be non-repetitive, heterogeneous DNA elements, lacking in inter-chromosomal sequence similarities. With the increasing numbers of lower plant genome sequences that are available, comparative analysis of centromeric sequences will help to elucidate the evolution of centromere DNA-protein interactions in the plant kingdom.     

A novel repetitive sequence associated with the centromeric regions of Arabidopsis thaliana chromosomes

 The middle repetitive fraction of the Arabidopsis genome has been relatively poorly characterized. We describe here a novel repetitive sequence cloned in the plasmid mi167, and present in ∼90 copies in the genome of Arabidopsis thaliana ecotype Columbia. Hybridization analysis to physically mapped YAC clones representing Arabidopsis chromosome 4 revealed four mi167-hybridizing loci, all clustered near the centromere. No other loci were detected on YAC clones covering chromosome 4. In situ hybridisation experiments to Arabidopsis chromosome spreads showed that mi167-hybridizing sequences are clustered at the centromeric heterochromatin of all five chromosomes; on two chromosomes the hybridization appeared to be localised on one arm. Additional mi167-hybridizing loci were detected, one of which was adjacent to a non-centromeric, heterochromatic region. This work supports the idea that the majority of middle repetitive DNA in the Arabidopsis genome is clustered. It also adds to our understanding of the organization of the centromere of Arabidopsis chromosome 4. Received: 19 February 1996 / Accepted: 30 June 1996     

A tale of two centromeres—diversity of structure but conservation of function in plants and animals

The structural and functional aspects of two specific centromeres, one drawn from the animal kingdom (Drosophila) and the other from the plant kingdom (maize), are compared. Both cases illustrate an epigenetic component to centromere specification. The observations of neocentromeres in Drosophila and inactive centromeres in maize constitute one line of evidence for this hypothesis. Another common feature is the divisibility of centromere function with reduced stability as the size decreases. The systems differ in that Drosophila has no common sequence repeat at all centromeres, whereas maize has a 150-bp unit present in tandem arrays together with a centromere-specific transposon, centromere retrotransposon maize, present at all primary constrictions. Aspects of centromere structure known only from one or the other system might be common to both, namely, the presence of centromere RNAs in the kinetochore as found in maize and the organization of the centromeric histone 3 in tetrameric nucleosomes.     

Centromeric DNA characterization in the model grass Brachypodium distachyon provides insights on the evolution of the genus

Brachypodium distachyon is a well‐established model monocot plant, and its small and compact genome has been used as an accurate reference for the much larger and often polyploid genomes of cereals such as Avena sativa (oats), Hordeum vulgare (barley) and Triticum aestivum (wheat). Centromeres are indispensable functional units of chromosomes and they play a core role in genome polyploidization events during evolution. As the Brachypodium genus contains about 20 species that differ significantly in terms of their basic chromosome numbers, genome size, ploidy levels and life strategies, studying their centromeres may provide important insight into the structure and evolution of the genome in this interesting and important genus. In this study, we isolated the centromeric DNA of the B. distachyon reference line Bd21 and characterized its composition via the chromatin immunoprecipitation of the nucleosomes that contain the centromere‐specific histone CENH3. We revealed that the centromeres of Bd21 have the features of typical multicellular eukaryotic centromeres. Strikingly, these centromeres contain relatively few centromeric satellite DNAs; in particular, the centromere of chromosome 5 (Bd5) consists of only ~40 kb. Moreover, the centromeric retrotransposons in B. distachyon (CRBds) are evolutionarily young. These transposable elements are located both within and adjacent to the CENH3 binding domains, and have similar compositions. Moreover, based on the presence of CRBds in the centromeres, the species in this study can be grouped into two distinct lineages. This may provide new evidence regarding the phylogenetic relationships within the Brachypodium genus.