Retrotransposon-related DNA sequences in the centromeres of grass chromosomes.

Several distinct DNA fragments were subcloned from a sorghum (Sorghum bicolor) bacterial artificial chromosome clone 13I16 that was derived from a centromere. Three fragments showed significant sequence identity to either Ty3/gypsy- or Ty1/copia-like retrotransposons. Fluorescence in situ hybridization (FISH) analysis revealed that the Ty1/copia-related DNA sequences are not specific to the centromeric regions. However, the Ty3/gypsy-related sequences were present exclusively in the centromeres of all sorghum chromosomes. FISH and gel-blot hybridization showed that these sequences are also conserved in the centromeric regions of all species within Gramineae. Thus, we report a new retrotransposon that is conserved in specific chromosomal regions of distantly related eukaryotic species. We propose that the Ty3/gypsy-like retrotransposons in the grass centromeres may be ancient insertions and are likely to have been amplified during centromere evolution. The possible role of centromeric retrotransposons in plant centromere function is discussed.     

Intergenic Locations of Rice Centromeric Chromatin

Centromeres are sites for assembly of the chromosomal structures that mediate faithful segregation at mitosis and meiosis. Plant and animal centromeres are typically located in megabase-sized arrays of tandem satellite repeats, making their precise mapping difficult. However, some rice centromeres are largely embedded in nonsatellite DNA, providing an excellent model to study centromere structure and evolution. We used chromatin immunoprecipitation and 454 sequencing to define the boundaries of nine of the 12 centromeres of rice. Centromere regions from chromosomes 8 and 9 were found to share synteny, most likely reflecting an ancient genome duplication. For four centromeres, we mapped discrete subdomains of binding by the centromeric histone variant CENH3. These subdomains were depleted in both intact and nonfunctional genes relative to interspersed subdomains lacking CENH3. The intergenic location of rice centromeric chromatin resembles the situation for human neocentromeres and supports a model of the evolution of centromeres from gene-poor regions.     

Maize centromeric chromatin scales with changes in genome size

Centromeres are defined by the location of Centromeric Histone H3 (CENP-A/CENH3) which interacts with DNA to define the locations and sizes of functional centromeres. An analysis of 26 maize genomes including 110 fully assembled centromeric regions revealed positive relationships between centromere size and genome size. These effects are independent of variation in the amounts of the major centromeric satellite sequence CentC. We also backcrossed known centromeres into two different lines with larger genomes and observed consistent increases in functional centromere sizes for multiple centromeres. Although changes in centromere size involve changes in bound CENH3, we could not mimic the effect by overexpressing CENH3 by threefold. Literature from other fields demonstrate that changes in genome size affect protein levels, organelle size and cell size. Our data demonstrate that centromere size is among these scalable features, and that multiple limiting factors together contribute to a stable centromere size equilibrium.     

The Birth of the Centromere

The centromere is the region of the eukaryotic chromosome that determines kinetochore formation and sister chromatid cohesion. Centromeres interact with spindle microtubules to ensure chromatid segregation during mitosis and homologous chromosome segregation during meiosis I. In recent years, the overall organization of centromeres in several eukaryotic species has been described, yet the mechanisms of centromere definition remain elusive. Understanding the evolutionary origin of the centromere may well elucidate aspects of its function. With such intention, we hypothesize that centromeres were derived from telomeres during the evolution of the eukaryotic chromosome. We propose that the proto-eukaryotic cell could not have evolved a nucleus without concurrently evolving a new tubulin-based cytoskeleton, the microtubules, and a specific chromosomal region that enabled the chromosome-microtubule interaction, the centromere. The repetitive nature of the subtelomeric regions that gave rise to the centromeres forced the concerted evolution of the centromeres. Although this implies the absence of a conserved primary sequence, a conserved centromere-specific structural motif could still exist and determine where in the chromosome the centromere is to be formed.To support the “centromeres-from-telomeres” hypothesis, we discuss several situations, in meiosis and mitosis, where telomeric regions took over centromeric roles. The recently discovered phenomenon of centromere repositioning is also discussed because it has revealed new insights into how neocentromeres evolve.     

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.     

Maize Centromere Structure and Evolution: Sequence Analysis of Centromeres 2 and 5 Reveals Dynamic Loci Shaped Primarily by Retrotransposons

We describe a comprehensive and general approach for mapping centromeres and present a detailed characterization of two maize centromeres. Centromeres are difficult to map and analyze because they consist primarily of repetitive DNA sequences, which in maize are the tandem satellite repeat CentC and interspersed centromeric retrotransposons of maize (CRM). Centromeres are defined epigenetically by the centromeric histone H3 variant, CENH3. Using novel markers derived from centromere repeats, we have mapped all ten centromeres onto the physical and genetic maps of maize. We were able to completely traverse centromeres 2 and 5, confirm physical maps by fluorescence in situ hybridization (FISH), and delineate their functional regions by chromatin immunoprecipitation (ChIP) with anti-CENH3 antibody followed by pyrosequencing. These two centromeres differ substantially in size, apparent CENH3 density, and arrangement of centromeric repeats; and they are larger than the rice centromeres characterized to date. Furthermore, centromere 5 consists of two distinct CENH3 domains that are separated by several megabases. Succession of centromere repeat classes is evidenced by the fact that elements belonging to the recently active recombinant subgroups of CRM1 colonize the present day centromeres, while elements of the ancestral subgroups are also found in the flanking regions. Using abundant CRM and non-CRM retrotransposons that inserted in and near these two centromeres to create a historical record of centromere location, we show that maize centromeres are fluid genomic regions whose borders are heavily influenced by the interplay of retrotransposons and epigenetic marks. Furthermore, we propose that CRMs may be involved in removal of centromeric DNA (specifically CentC), invasion of centromeres by non-CRM retrotransposons, and local repositioning of the CENH3.     

植物着丝粒结构和功能的研究进展

着丝粒是真核生物有丝分裂和减数分裂染色体正确分离和传递所必需的染色体区域。十多年来, 已对包括拟南芥、水稻、玉米在内的一些植物的着丝粒进行了较深入的分子生物学研究。在不同的植物间, 着丝粒DNA的保守性很低, 呈现快速进化, 但着丝粒的DNA序列类型和组织方式基本相似, 一般是由夹杂排列着的卫星DNA串联重复阵列和着丝粒专一的反转录转座子构成。与着丝粒DNA相反, 着丝粒/着丝点的结构性和瞬时蛋白质在包括植物在内的真核生物中保守。与其他真核生物的情况一样, 拥有含着丝粒组蛋白H3(CENH3)的核小体是植物功能着丝粒染色质最基本的特征, CENH3在着丝粒染色质的识别和保持中起着关键作用。     

Plant centromeres: structure and control

Recent work has led to a better understanding of the molecular components of plant centromeres. Conservation of at least some centromere protein constituents between plant and non-plant systems has been demonstrated. The identity and organization of plant centromeric DNA sequences are also beginning to yield to analysis. While there is little primary DNA sequence conservation among the characterized plant centromeres and their non-plant counterparts, some parallels in centromere genomic organisation can be seen across species. Finally, the emerging idea that centromere activity is controlled epigenetically finds support in an examination of the plant centromere literature.     

The compact Brachypodium genome conserves centromeric regions of a common ancestor with wheat and rice

The evolution of five chromosomes of Brachypodium distachyon from a 12-chromosome ancestor of all grasses by dysploidy raises an interesting question about the fate of redundant centromeres. Three independent but complementary approaches were pursued to study centromeric region homologies among the chromosomes of Brachypodium, wheat, and rice. The genes present in pericentromeres of the basic set of seven chromosomes of wheat and the Triticeae, and the 80 rice centromeric genes spanning the CENH3 binding domain of centromeres 3, 4, 5, 7, and 8 were used as “anchor” markers to identify centromere locations in the B. distachyon chromosomes. A total of 53 B. distachyon bacterial artificial chromosome (BAC) clones anchored by wheat pericentromeric expressed sequence tags (ESTs) were used as probes for BAC-fluorescence in situ hybridization (FISH) analysis of B. distachyon mitotic chromosomes. Integrated sequence alignment and BAC-FISH data were used to determine the approximate positions of active and inactive centromeres in the five B. distachyon chromosomes. The following syntenic relationships of the centromeres for Brachypodium (Bd), rice (R), and wheat (W) were evident: Bd1-R6, Bd2-R5-W1, Bd3-R10, Bd4-R11-W4, and Bd5-R4. Six rice centromeres syntenic to five wheat centromeres were inactive in Brachypodium chromosomes. The conservation of centromere gene synteny among several sets of homologous centromeres of three species indicates that active genes can persist in ancient centromeres with more than 40 million years of shared evolutionary history. Annotation of a BAC contig spanning an inactive centromere in chromosome Bd3 which is syntenic to rice Cen8 and W7 pericentromeres, along with BAC FISH data from inactive centromeres revealed that the centromere inactivation was accompanied by the loss of centromeric retrotransposons and turnover of centromere-specific satellites during Bd chromosome evolution.     

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.     

Euchromatic subdomains in rice centromeres are associated with genes and transcription

The presence of the centromere-specific histone H3 variant, CENH3, defines centromeric (CEN) chromatin, but poorly understood epigenetic mechanisms determine its establishment and maintenance. CEN chromatin is embedded within pericentromeric heterochromatin in most higher eukaryotes, but, interestingly, it can show euchromatic characteristics; for example, the euchromatic histone modification mark dimethylated H3 Lys 4 (H3K4me2) is uniquely associated with animal centromeres. To examine the histone marks and chromatin properties of plant centromeres, we developed a genomic tiling array for four fully sequenced rice (Oryza sativa) centromeres and used chromatin immunoprecipitation-chip to study the patterns of four euchromatic histone modification marks: H3K4me2, trimethylated H3 Lys 4, trimethylated H3 Lys 36, and acetylated H3 Lys 4, 9. The vast majority of the four histone marks were associated with genes located in the H3 subdomains within the centromere cores. We demonstrate that H3K4me2 is not a ubiquitous component of rice CEN chromatin, and the euchromatic characteristics of rice CEN chromatin are hallmarks of the transcribed sequences embedded in the centromeric H3 subdomains. We propose that the transcribed sequences located in rice centromeres may provide a barrier preventing loading of CENH3 into the H3 subdomains. The separation of CENH3 and H3 subdomains in the centromere core may be favorable for the formation of three-dimensional centromere structure and for rice centromere function.     

Diversity in requirement of genetic and epigenetic factors for centromere function in fungi

A centromere is a chromosomal region on which several proteins assemble to form the kinetochore. The centromere-kinetochore complex helps in the attachment of chromosomes to spindle microtubules to mediate segregation of chromosomes to daughter cells during mitosis and meiosis. In several budding yeast species, the centromere forms in a DNA sequence-dependent manner, whereas in most other fungi, factors other than the DNA sequence also determine the centromere location, as centromeres were able to form on nonnative sequences (neocentromeres) when native centromeres were deleted in engineered strains. Thus, in the absence of a common DNA sequence, the cues that have facilitated centromere formation on a specific DNA sequence for millions of years remain a mystery. Kinetochore formation is facilitated by binding of a centromere-specific histone protein member of the centromeric protein A (CENP-A) family that replaces a canonical histone H3 to form a specialized centromeric chromatin structure. However, the process of kinetochore formation on the rapidly evolving and seemingly diverse centromere DNAs in different fungal species is largely unknown. More interestingly, studies in various yeasts suggest that the factors required for de novo centromere formation (establishment) may be different from those required for maintenance (propagation) of an already established centromere. Apart from the DNA sequence and CENP-A, many other factors, such as posttranslational modification (PTM) of histones at centric and pericentric chromatin, RNA interference, and DNA methylation, are also involved in centromere formation, albeit in a species-specific manner. In this review, we discuss how several genetic and epigenetic factors influence the evolution of structure and function of centromeres in fungal species.     

植物着丝粒结构及进化的研究进展

植物着丝粒是染色体重要结构域,介导动粒装配。不同物种间着丝粒重复序列快速趋异进化,着丝粒功能保守,确保有丝分裂和减数分裂过程中染色体正确分离和准确传递。伴随染色质免疫共沉淀技术(Chromatin immunoprecipitation, ChIP)、ChIP 与高密度芯片相结合技术(ChIP-chip)、ChIP 与高通量测序相结合技术(ChIP-seq)的应用,植物着丝粒研究获得里程碑式进展:某些模式植物着丝粒DNA 序列、蛋白质结构、功能获得大量新认识;着丝粒基本蛋白质组蛋白H3 被用来界定着丝粒大小和边界;某些非着丝粒区域被激活为新着丝粒,在世代传递中保持稳定性。本文对植物着丝粒结构、功能、进化研究进行了综述,并探讨了植物着丝粒研究存在的问题。     

Pericentric heterochromatin: dynamic organization during early development in mammals

Abstract Constitutive heterochromatin in mammals is essentially found at centromeres, which are key chromosomal elements that ensure proper chromosome segregation. These regions are considered to be epigenetically defined, given that it is not sequence composition but chromatin organization that defines centromere function. How such an epigenetically defined domain, like the centromere, can be established during development and maintained during somatic cell life are fundamental questions. This review discusses the most recent insights into centromeric heterochromatin organization and replication. We further highlight the plasticity of this domain by describing the large-scale re-organization that occurs during development.     

Structure, divergence, and distribution of the CRR centromeric retrotransposon family in rice

The centromeric retrotransposon (CR) family in the grass species is one of few Ty3-gypsy groups of retroelements that preferentially transpose into highly specialized chromosomal domains. It has been demonstrated in both rice and maize that CRR (CR of rice) and CRM (CR of maize) elements are intermingled with centromeric satellite DNA and are highly concentrated within cytologically defined centromeres. We collected all of the CRR elements from rice chromosomes 1, 4, 8, and 10 that have been sequenced to high quality. Phylogenetic analysis revealed that the CRR elements are structurally diverged into four subfamilies, including two autonomous subfamilies (CRR1 and CRR2) and two nonautonomous subfamilies (noaCRR1 and noaCRR2). The CRR1/CRR2 elements contain all characteristic protein domains required for retrotransposition. In contrast, the noaCRR elements have different structures, containing only a gag or gag-pro domain or no open reading frames. The CRR and noaCRR elements share substantial sequence similarity in regions required for DNA replication and for recognition by integrase during retrotransposition. These data, coupled with the presence of young noaCRR elements in the rice genome and similar chromosomal distribution patterns between noaCRR1 and CRR1/CRR2 elements, suggest that the noaCRR elements were likely mobilized through the retrotransposition machinery from the autonomous CRR elements. Mechanisms of the targeting specificity of the CRR elements, as well as their role in centromere function, are discussed.     

The smooth and stable operation of centromeres

The centromere functions as a unique chromosomal attachment site for microtubules. Appropriate microtubule attachment is fundamental for organized chromosome behavior during mitosis and meiosis. Hence, centromeres must function both smoothly and stably. However, centromeric DNA sequences are poorly conserved between species despite common functions and similar centromeric protein composition, which leads us to the question: how are centromeres established and maintained? In this review, we summarize the recent progress in deciphering the mechanisms of centromere function. Specifically, we focus our attention on mechanisms closely-related to centromeric DNA and chromatin. By gathering such information, we hope to reveal a new dimension to the true nature of centromeres.     

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.