Duplication of centromeric histone H3 (HTR12) gene in Arabidopsis halleri and A. lyrata, plant species with multiple centromeric satellite sequences

Arabidopsis halleri and lyrata have three different major centromeric satellite sequences, a unique finding for a diploid Arabidopsis species. Since centromeric histones coevolve with centromeric satellites, these proteins would be predicted to show signs of selection when new centromere satellites have recently arisen. We isolated centromeric protein genes from A. halleri and lyrata and found that one of them, HTR12 (CENP-A), is duplicated, while CENP-C is not. Phylogenetic analysis indicates that the HTR12 duplication occurred after these species diverged from A. thaliana. Genetic mapping shows that HTR12 copy B has the same genomic location as the A. thaliana gene; the other copy (A, at the other end of the same chromosome) is probably the new copy. To test for selection since the duplication, we surveyed diversity at both HTR12 loci within A. lyrata. Overall, there is no strong evidence for an "evolutionary arms race" causing multiple replacement substitutions. The A. lyrata HTR12B sequences fall into three classes of haplotypes, apparently maintained for a long time, but they all encode the same amino acid sequence. In contrast, HTR12A has low diversity, but many variants are amino acid replacements, possibly due to independent selective sweeps within populations of the species.     

Centromeric localization and adaptive evolution of an Arabidopsis histone H3 variant

Centromeric H3-like histones, which replace histone H3 in the centromeric chromatin of animals and fungi, have not been reported in plants. We identified a histone H3 variant from Arabidopsis thaliana that encodes a centromere-identifying protein designated HTR12. By immunological detection, HTR12 localized at centromeres in both mitotic and meiotic cells. HTR12 signal revealed tissue- and stage-specific differences in centromere morphology, including a distended bead-like structure in interphase root tip cells. The anti-HTR12 antibody also detected spherical organelles in meiotic cells. Although the antibody does not label centromeres in the closely related species Arabidopsis arenosa, HTR12 signal was found on all centromeres in allopolyploids of these two species. Comparison of the HTR12 genes of A. thaliana and A. arenosa revealed striking adaptive evolution in the N-terminal tail of the protein, similar to the pattern seen in its counterpart in Drosophila. This finding suggests that the same evolutionary forces shape centromeric chromatin in both animals and plants.     

Polymorphic Chromosomal Specificity of Centromere Satellite Families in Arabidopsis halleri ssp. gemmifera

The chromosomal localizations of repetitive DNA clusters (ribosomal DNA and centromere satellites) were analyzed by fluorescent in situ hybridization in five strains of Arabidopsis halleri ssp. gemmifera. All five A. gemmifera strains have three chromosome pairs with 45S (5.8S-16S-26S) rDNA loci, and one pair with both 5S and 45S rDNA loci. These localizations are different from that of A. thaliana. Very unusually, there are three families of centromeric satellite DNAs (pAa, pAge1, and pAge2), and they showed polymorphism among the five strains studied. Overall, we found four different centromere satellite compositions. A plant from Fumuro was heterozygous for the chromosome specificities of centromere satellite families, possibly due to a reciprocal translocation involving centromere regions. Changes of centromeric satellite repeats appear to be rapid and frequent events in the history of A. gemmifera, and seem to occur by exchanging clusters as units.     

Chromatin immunoprecipitation reveals that the 180-bp satellite repeat is the key functional DNA element of Arabidopsis thaliana centromeres

The centromeres of Arabidopsis thaliana chromosomes contain megabases of complex DNA consisting of numerous types of repetitive DNA elements. We developed a chromatin immunoprecipitation (ChIP) technique using an antibody against the centromeric H3 histone, HTR12, in Arabidopsis. ChIP assays showed that the 180-bp centromeric satellite repeat was precipitated with the antibody, suggesting that this repeat is the key component of the centromere/kinetochore complex in Arabidopsis.     

A lineage‐specific centromere retrotransposon in Oryza brachyantha

Most eukaryotic centromeres contain large quantities of repetitive DNA, such as satellite repeats and retrotransposons. Unlike most transposons in plant genomes, the centromeric retrotransposon (CR) family is conserved over long evolutionary periods among a majority of the grass species. CR elements are highly concentrated in centromeres, and are likely to play a role in centromere function. In order to study centromere evolution in the Oryza (rice) genus, we sequenced the orthologous region to centromere 8 of Oryza sativa from a related species, Oryza brachyantha. We found that O. brachyantha does not have the canonical CRR (CR of rice) found in the centromeres of all other Oryza species. Instead, a new Ty3‐gypsy (Metaviridae) retroelement (FRetro3) was found to colonize the centromeres of this species. This retroelement is found in high copy numbers in the O. brachyantha genome, but not in other Oryza genomes, and based on the dating of long terminal repeats (LTRs) of FRetro3 it was amplified in the genome in the last few million years. Interestingly, there is a high level of removal of FRetro3 based on solo‐LTRs to full‐length elements, and this rapid turnover may have played a role in the replacement of the canonical CRR with the new element by active deletion. Comparison with previously described ChIP cloning data revealed that FRetro3 is found in CENH3‐associated chromatin sequences. Thus, within a single lineage of the Oryza genus, the canonical component of grass centromeres has been replaced with a new retrotransposon that has all the hallmarks of a centromeric retroelement.     

Absence of satellite III DNA in the centromere and the proximal long-arm region of human chromosome 14: analysis of a 14p- variant.

Cytogenetic methods and molecular probes derived from the centromere and short arm of chromosome 14 were used to investigate the structural properties of a chromosome 14 variant. Results of GTL, CBG, Ag-NOR, and non-banded Giemsa staining of the chromosomes suggested the complete absence of the short arm and possibly a large part of the centromere. Negative in situ hybridisation with an alpha satellite III probe confirmed the absence of the arm; the detection of normal amounts of alpha satellite DNA, however, indicated retention of the centromeric domain. The natural occurrence of a human acrocentric variant lacking a short arm was thus established. Within the detection limits of the methods used, the results demonstrate that satellite III DNA is not essential for normal centromeric activity and allow us to exclude the presence of this satellite DNA within the centromere and proximal long-arm region of human chromosome 14.     

The Evolutionary Origin of Man Can Be Traced in the Layers of Defunct Ancestral Alpha Satellites Flanking the Active Centromeres of Human Chromosomes

Alpha satellite domains that currently function as centromeres of human chromosomes are flanked by layers of older alpha satellite, thought to contain dead centromeres of primate progenitors, which lost their function and the ability to homogenize satellite repeats, upon appearance of a new centromere. Using cladistic analysis of alpha satellite monomers, we elucidated complete layer patterns on chromosomes 8, 17, and X and related them to each other and to primate alpha satellites. We show that discrete and chronologically ordered alpha satellite layers are partially symmetrical around an active centromere and their succession is partially shared in non-homologous chromosomes. The layer structure forms a visual representation of the human evolutionary lineage with layers corresponding to ancestors of living primates and to entirely fossil taxa. Surprisingly, phylogenetic comparisons suggest that alpha satellite arrays went through periods of unusual hypermutability after they became “dead” centromeres. The layer structure supports a model of centromere evolution where new variants of a satellite repeat expanded periodically in the genome by rounds of inter-chromosomal transfer/amplification. Each wave of expansion covered all or many chromosomes and corresponded to a new primate taxon. Complete elucidation of the alpha satellite phylogenetic record would give a unique opportunity to number and locate the positions of major extinct taxa in relation to human ancestors shared with extant primates. If applicable to other satellites in non-primate taxa, analysis of centromeric layers could become an invaluable tool for phylogenetic studies.     

Satellite DNA from the Y chromosome of the malaria vector Anopheles gambiae

Satellite DNA is an enigmatic component of genomic DNA with unclear function that has been regarded as "junk." Yet, persistence of these tandem highly repetitive sequences in heterochromatic regions of most eukaryotic chromosomes attests to their importance in the genome. We explored the Anopheles gambiae genome for the presence of satellite repeats and identified 12 novel satellite DNA families. Certain families were found in close juxtaposition within the genome. Six satellites, falling into two evolutionarily linked groups, were investigated in detail. Four of them were experimentally confirmed to be linked to the Y chromosome, whereas their relatives occupy centromeric regions of either the X chromosome or the autosomes. A complex evolutionary pattern was revealed among the AgY477-like satellites, suggesting their rapid turnover in the A. gambiae complex and, potentially, recombination between sex chromosomes. The substitution pattern suggested rolling circle replication as an array expansion mechanism in the Y-linked 53-bp satellite families. Despite residing in different portions of the genome, the 53-bp satellites share the same monomer lengths, apparently maintained by molecular drive or structural constraints. Potential functional centromeric DNA structures, consisting of twofold dyad symmetries flanked by a common sequence motif, have been identified in both satellite groups.     

Diversity of centromeric repeats in two closely related wild rice species, Oryza officinalis and Oryza rhizomatis

Oryza officinalis (CC, 2n=24) and Oryza rhizomatis (CC, 2n=24) belong to the Oryza genus, which contains more than 20 identified wild rice species. Although much has been known about the molecular composition and organization of centromeres in Oryza sativa, relatively little is known of its wild relatives. In the present study, we isolated and characterized a 126-bp centromeric satellite (CentO-C) from three bacterial artificial chromosomes of O. officinalis. In addition to CentO-C, low abundance of CentO satellites is also present in O. officinalis. In order to determine the chromosomal locations and distributions of CentO-C (126-bp), CentO (155 bp) and TrsC (366 bp) satellite within O. officinalis, fluorescence in situ hybridization examination was done on pachytene or metaphase I chromosomes. We found that only ten centromeres (excluding centromere 7 and 2) contain CentO-C arrays in O. officinalis, while centromere 7 comprises CentO satellites, and centromere 2 is devoid of any detectable satellites. For TrsC satellites, it was detected at multiple subtelomeric regions in O. officinalis, however, in O. rhizomatis, TrsC sequences were detected both in the four centromeric regions (CEN 3, 4, 10, 11) and the multiple subtelomeric regions. Therefore, these data reveal the evolutionary diversification pattern of centromere DNA within/or between close related species, and could provide an insight into the dynamic evolutionary processes of rice centromere.     

The formation and evolution of centromeric satellite repeats in Saccharum species

Centromeres in eukaryotes are composed of tandem DNAs and retrotransposons. However, centromeric repeats exhibit considerable diversity, even among closely related species, and their origin and evolution are largely unknown. We conducted a genome-wide characterization of the centromeric sequences in sugarcane (Saccharum officinarum). Four centromeric tandem repeat sequences, So1, So103, So137 and So119, were isolated. So1 has a monomeric length of 137 bp, typical of a centromeric satellite, and has evolved four variants. However, these So1 variants had distinct centromere distributions and some were unique to an individual centromere. The distributions of the So1 variants were unexpectedly consistent among the Saccharum species that had different basic chromosome numbers or ploidy levels, thus suggesting evolutionary stability for approximately 7 million years in sugarcane. So103, So137 and So119 had unusually longer monomeric lengths that ranged from 327 to 1371 bp and lacked translational phasing on the CENH3 nucleosomes. Moreover, So103, So137 and So119 seemed to be highly similar to retrotransposons, which suggests that they originated from these mobile elements. Notably, all three repeats were flanked by direct repeats, and formed extrachromosomal circular DNAs (eccDNAs). The presence of circular molecules for these retrotransposon-derived centromeric satellites suggests an eccDNA-mediated centromeric satellite formation pathway in sugarcane.     

Recognition of <Emphasis Type="Italic">A. thaliana</Emphasis> centromeres by heterologous CENH3 requires high similarity to the endogenous protein

The centromere is an essential chromosomal component assembling the kinetochore for chromosome attachment to the spindle microtubules and for directing the chromosome segregation during nuclear division. Kinetochore assembly requires deposition of the centromeric histone H3 variant (CENH3) into centromeric nucleosomes. CENH3 has a variable N-terminal and a more conserved C-terminal part, including the loop1 region of the histone fold domain, which is considered to be critical for centromere targeting. To investigate the structural requirements for centromere targeting, constructs for EYFP-tagged CENH3 of A. lyrata, A. arenosa, Capsella bursa-pastoris, Zea mays and Luzula nivea (the latter with holocentric chromosomes) were transformed into A. thaliana. Except for LnCENH3, all recombinant CENH3 proteins targeted A. thaliana centromeres, but the more distantly related the heterologous protein is, the lower is the efficiency of targeting. Alignment of CENH3 sequences revealed that the tested species share only three amino acids at loop1 region: threonine2, arginine12 and alanine15. These three amino acids were substituted by asparagine, proline and valine encoding sequences within a recombinant EYFP-AtCENH3 construct via PCR mutagenesis prior to transformation of A. thaliana. After transformation, immunostaining of root tip nuclei with anti-GFP antibodies yielded only diffuse signals, indicating that the original three amino acids are necessary but not sufficient for targeting A. thaliana centromeres.     

CENH3 interacts with the centromeric retrotransposon <Emphasis Type="Italic">cereba</Emphasis> and GC-rich satellites and locates to centromeric substructures in barley

The chromosomal location of centromere-specific histone H3 (CENH3) is the assembly site for the kinetochore complex of active centromeres. Chromatin immunoprecipitation data indicated that CENH3 interacts in barley with cereba, a centromeric retroelement (CR)-like element conserved among cereal centromeres and barley-specific GC-rich centromeric satellite sequences. Anti-CENH3 signals on extended chromatin fibers always colocalized with the centromeric sequences but did not encompass the entire area covered by such centromeric repeats. This indicates that the CENH3 protein is bound only to a fraction of the centromeric repeats. At mitotic metaphase, CENH3, histone H3, and serine 10 phosphorylated histone H3 predominated within distinct structural subdomains of the centromere, as demonstrated by immunogold labeling for high resolution scanning electron microscopy.     

Alpha satellite DNA in neotropical primates (Platyrrhini)

The alpha satellite DNA of Old World (catarhine) primates usually consists of similar, but not identical, ca. 170 bp sequences repeated tandemly hundreds to thousands of times. The 170 bp monomeric repeats are components of higher-order repeats, many of which are chromosome specific. Alpha satellites are found exclusively in centromeric regions where they appear to play a role in centromere function. We have found that alpha satellite DNA in neotropical (New World; platyrrhine) primates is very similar to its Old World counterpart: it consists of divergent ca. 170 bp subsequences that are arranged in tandem arrays with a ca. 340 bp periodicity. New and Old World alpha satellites share about 64% sequence identity overall, and contain several short sequence motifs that appear to be highly conserved. One exception to the tandemly arrayed 340 bp motif has been found: the major alpha satellite array in Chiropotes satanas (black bearded saki) has a 539 bp repeat unit that consists of a 338 bp dimer together with a duplication of 33 bp of the first monomeric unit and 168 bp of the second monomeric unit.     

The chromosomal distribution of the major and minor satellite is not conserved in the genusMus

The cytological distribution of the major and minor satellite first identified inMus musculus was studied in the karyotypes of three related subspecies and two other species of the genusMus. Both the major and minor satellite showed species dependent hybridization patterns. The major satellite is confined to the centromere region inM. musculus and related subspecies. However, inM spretus andM. caroli, the chromosomal arm regions contain this sequence class. In contrast the minor satellite is found at the kinetochore region inM. musculus and related subspecies but is distributed throughout the entire centromeric domain inM. spretus and appears to be excluded from the chromosomes ofM. caroli. There is an apparent correlation between the chromosomal location of these satellites and their phylogenetic relationship. Determination of the biological roles of the major and minor satellites fromM. musculus must take into account their differential chromosomal distribution in otherMus species.     

Searching for a Common Centromeric Structural Motif: Drosophila Centromeric Satellite DNAs Show Propensity to form Telomeric-Like Unusual DNA Structures

The molecular basis of centromere formation in a particular chromosomal region is not yet understood. In higher eukaryotes, no specific DNA sequence is required for the assembly of the kinetochore, but similar centromeric chromatins are formed on different centromere DNA sequences. Although epigenesis has been proposed as the main mechanism for centromere specification, DNA recognition must also play a role. Through the analysis of Drosophilacentromeric DNA sequences, we found that dodeca satellite and 18HT satellite are able to form unusual DNA structures similar to those formed by telomeric sequences. These findings suggest the existence of a common centromeric structural DNA motif which we feel merits further investigation. This revised version was published online in July 2006 with corrections to the Cover Date.     

Adaptive evolution of Cid, a centromere-specific histone in Drosophila

Centromeric DNA is generally composed of large blocks of tandem satellite repeats that change rapidly due to loss of old arrays and expansion of new repeat classes. This extreme heterogeneity of centromeric DNA is difficult to reconcile with the conservation of the eukaryotic chromosome segregation machinery. Histone H3-like proteins, including Cid in Drosophila melanogaster, are a unique chromatin component of centromeres. In comparisons between closely related species of Drosophila, we find an excess of replacement changes that have been fixed since the separation of D. melanogaster and D. simulans, suggesting adaptive evolution. The last adaptive changes appear to have occurred recently, as evident from a reduction in polymorphism in the melanogaster lineage. Adaptive evolution has occurred both in the long N-terminal tail as well as in the histone fold of Cid. In the histone fold, the replacement changes have occurred in the region proposed to mediate binding to DNA. We propose that this rapid evolution of Cid is driven by a response to the changing satellite repeats at centromeres. Thus, centromeric H3-like proteins may act as adaptors between evolutionarily labile centromeric DNA and the conserved kinetochore machinery.     

Isolation, cloning and characterization of two major satellite DNA families of rabbit (Oryctolagus cuniculus)

We report here the isolation, cloning and characterization of two abundant centromeric satellite sequences (Rsat I and Rsat II) what are not related to each other, and that of a divergent subfamily (Rsat IIE) of rabbit (Oryctolagus cuniculus). The Rsat I monomers had a 375 base pair (bp) average length, while repeat units Rsat II and Rsat IIE were approximately 585 bp long. Variable amounts of Rsat I were detected by FISH at the centromeric region of 11 chromosome pairs of the complement. Rsat II hybridized to the centromere of 12 different chromosomes, and two of these were labeled also with the Rsat IIE probe. Two-color in situ hybridizations with the satellite probes and rDNA revealed that the NOR chromosomes carried different satellites. Rsat I was abundant on chromosome 20 and 21, but it was undetectable on chromosomes 13 and 16. Large Rsat II arrays were found on chromosomes 16, 20 and 21, but reduced amount was detected on chromosome 13. The variant Rsat IIE was prominent on chromosome 16, but was absent from the other rDNA-bearing chromosomes. The rDNA signal on chromosome 21 was localized to the 21q(ter) region, what can be a useful cytological marker in comparative cytological studies. These data show that rabbit chromosomes form at least four distinct groups based on the satellite composition of their centromeres. The differences in the chromosomal distribution of satellite families will help easy FISH identification of individual chromosomes, as well as to unveil the evolutionary history of the Leporidae karyotype.     

An overview of plant centromeres

The centromere is a defining region that mediates chromosome attachment to kinetochore microtubules and proper segregation of the sister chromatids. Intriguingly, satellite DNA and centromeric retrotransposon as major DNA constituents of centromere showed baffling diversification and species-specific. However, the key kinetochore proteins are conserved in both plants and animals, particularly the centromere-specific histone H3-1ike protein （CENH3） in all functional centromeres. Recent studies have highlighted the importance of epigenetic mechanisms in the establishment and maintenance of centromere identity. Here, we review the progress and compendium of research on plant centromere in the light of recent data.