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.     

Functional rice centromeres are marked by a satellite repeat and a centromere-specific retrotransposon

The centromere of eukaryotic chromosomes is essential for the faithful segregation and inheritance of genetic information. In the majority of eukaryotic species, centromeres are associated with highly repetitive DNA, and as a consequence, the boundary for a functional centromere is difficult to define. In this study, we demonstrate that the centers of rice centromeres are occupied by a 155-bp satellite repeat, CentO, and a centromere-specific retrotransposon, CRR. The CentO satellite is located within the chromosomal regions to which the spindle fibers attach. CentO is quantitatively variable among the 12 rice centromeres, ranging from 65 kb to 2 Mb, and is interrupted irregularly by CRR elements. The break points of 14 rice centromere misdivision events were mapped to the middle of the CentO arrays, suggesting that the CentO satellite is located within the functional domain of rice centromeres. Our results demonstrate that the CentO satellite may be a key DNA element for rice centromere function.     

Molecular and functional dissection of the maize B chromosome centromere

The centromere of the maize (Zea mays) B chromosome contains several megabases of a B-specific repeat (ZmBs), a 156-bp satellite repeat (CentC), and centromere-specific retrotransposons (CRM elements). Here, we demonstrate that only a small fraction of the ZmBs repeats interacts with CENH3, the histone H3 variant specific to centromeres. CentC, which marks the CENH3-associated chromatin in maize A centromeres, is restricted to an approximately 700-kb domain within the larger context of the ZmBs repeats. The breakpoints of five B centromere misdivision derivatives are mapped within this domain. In addition, the fraction of this domain remaining after misdivision correlates well with the quantity of CENH3 on the centromere. Thus, the functional boundaries of the B centromere are mapped to a relatively small CentC- and CRM-rich region that is embedded within multimegabase arrays of the ZmBs repeat. Our results demonstrate that the amount of CENH3 at the B centromere can be varied, but with decreasing amounts, the function of the centromere becomes impaired.     

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 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.     

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.     

Genome‐wide mapping of cytosine methylation revealed dynamic DNA methylation patterns associated with genes and centromeres in rice

We conducted genome‐wide mapping of cytosine methylation using methylcytosine immunoprecipitation combined with Illumina sequencing. The chromosomal distribution pattern of methylated DNA is similar to the heterochromatin distribution pattern on rice chromosomes. The DNA methylation patterns of rice genes are similar to those in Arabidopsis thaliana, including distinct methylation patterns asssociated with gene bodies and promoters. The DNA sequences in the core domains of rice Cen4, Cen5 and Cen8 showed elevated methylation levels compared with sequences in the pericentromeric regions. In addition, elevated methylation levels were associated with the DNA sequences in the CENH3‐binding subdomains, compared with the sequences in the flanking H3 subdomains. In contrast, the centromeric domain of Cen11, which is composed exclusively of centromeric satellite DNA, is hypomethylated compared with the pericentromeric domains. Thus, the DNA sequences associated with functional centromeres can be either hypomethylated or hypermethylated. The methylation patterns of centromeric DNA appear to be correlated with the composition of the associated DNA sequences. We propose that both hypomethylation and hypermethylation of CENH3‐associated DNA sequences can serve as epigenetic marks to distinguish where CENH3 deposition will occur within the surrounding H3 chromatin.     

Composition and structure of the centromeric region of rice chromosome 8

Understanding the organization of eukaryotic centromeres has both fundamental and applied importance because of their roles in chromosome segregation, karyotypic stability, and artificial chromosome-based cloning and expression vectors. Using clone-by-clone sequencing methodology, we obtained the complete genomic sequence of the centromeric region of rice (Oryza sativa) chromosome 8. Analysis of 1.97 Mb of contiguous nucleotide sequence revealed three large clusters of CentO satellite repeats (68.5 kb of 155-bp repeats) and >220 transposable element (TE)-related sequences; together, these account for approximately 60% of this centromeric region. The 155-bp repeats were tandemly arrayed head to tail within the clusters, which had different orientations and were interrupted by TE-related sequences. The individual 155-bp CentO satellite repeats showed frequent transitions and transversions at eight nucleotide positions. The 40 TE elements with highly conserved sequences were mostly gypsy-type retrotransposons. Furthermore, 48 genes, showing high BLAST homology to known proteins or to rice full-length cDNAs, were predicted within the region; some were close to the CentO clusters. We then performed a genome-wide survey of the sequences and organization of CentO and RIRE7 families. Our study provides the complete sequence of a centromeric region from either plants or animals and likely will provide insight into the evolutionary and functional analysis of plant centromeres.     

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.     

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。     

Maize centromeres: organization and functional adaptation in the genetic background of oat

Centromeric DNA sequences in multicellular eukaryotes are often highly repetitive and are not unique to a specific centromere or to centromeres at all. Thus, it is a major challenge to study the fine structure of individual plant centromeres. We used a DNA fiber-fluorescence in situ hybridization approach to study individual maize (Zea mays) centromeres using oat (Avena sativa)-maize chromosome addition lines. The maize centromere-specific satellite repeat CentC in the addition lines allowed us to delineate the size and organization of centromeric DNA of individual maize chromosomes. We demonstrate that the cores of maize centromeres contain mainly CentC arrays and clusters of a centromere-specific retrotransposon, CRM. CentC and CRM sequences are highly intermingled. The amount of CentC/CRM sequence varies from approximately 300 to >2800 kb among different centromeres. The association of CentC and CRM with centromeric histone H3 (CENH3) was visualized by a sequential detection procedure on stretched centromeres. The analysis revealed that CENH3 is always associated with CentC and CRM but that not all CentC or CRM sequences are associated with CENH3. We further demonstrate that in the chromosomal addition lines in which two CenH3 genes were present, one from oat and one from maize, the oat CENH3 was consistently incorporated by the maize centromeres.     

A novel translocation event leads to a recombinant stable chromosome with interrupted centromeric domains in rice

Rice (Oryza sativa L.) centromeres are composed of 155-bp satellite repeats (CentO), centromere-specific retrotransposon (CRR), and a variety of other repeats. Previous studies have shown that CentO and CRR elements are both parts of the functional centromere/kinetochore complex. In this study, a naturally occurring karyotype rearrangement involving a reciprocal translocation between chromosomes 9 and 11 in an indica rice Zhongxian 3037 has been identified. The recombinant centromere in Chr11L?·?9L has two CentO tandem arrays, separated by a long array of 5S rDNAs. Chromatin immunoprecipitation and immunostaining showed that centromere-specific histone H3 (cenH3) variant was bound to the two flanking CentO arrays, but not to the 5S rDNAs residing between the CentO repeats. No obvious difference was detected in H3K4me2 and H3K9ac modification of the 5S rDNAs between the wild type and the mutant. Therefore, the translocation results in a recombinant stable chromosome with interrupted centromeric domains. A lack of cenH3 binding in 5S rDNA sequences residing within the centromeric core suggests that not all centromeric sequences confer centromere identity in rice.     

The transcribed 165-bp CentO satellite is the major functional centromeric element in the wild rice species Oryza punctata

Centromeres are required for faithful segregation of chromosomes in cell division. It is not clear what kind of sequences act as functional centromeres and how centromere sequences are organized in Oryza punctata, a BB genome species. In this study, we found that the CentO centromeric satellites in O. punctata share high homology with the CentO satellites in O. sativa. The O. punctata centromeres are characterized by megabase tandem arrays that are flanked by centromere-specific retrotransposons. Immunostaining with an antibody specific to CENH3 indicates that the 165-bp CentO satellites are the major component for functional centromeres. Moreover, both strands of CentO satellites are highly methylated and transcribed and produce small interfering RNA, which may be important for the maintenance of centromeric heterochromatin and centromere function.     

Centromere inactivation and epigenetic modifications of a plant chromosome with three functional centromeres

A chromosome with two functional centromeres is cytologically unstable and can only be stabilized when one of the two centromeres becomes inactivated via poorly understood mechanisms. Here, we report a transmissible chromosome with multiple centromeres in wheat. This chromosome encompassed one large and two small domains containing the centromeric histone CENH3. The two small centromeres are in a close vicinity and often fused as a single centromere on metaphase chromosomes. This fused centromere contained approximately 30% of the CENH3 compared to the large centromere. An intact tricentric chromosome was transmitted to about 70% of the progenies, which was likely a consequence of the dominating pulling capacity of the large centromere during anaphases of meiosis. The tricentric chromosome showed characteristics typical to dicentric chromosomes, including chromosome breaks and centromere inactivation. Remarkably, inactivation was always associated with the small centromeres, indicating that small centromeres are less likely to survive than large ones in dicentric chromosomes. The inactivation of the small centromeres also coincided with changes of specific histone modifications, including H3K27me2 and H3K27me3, of the pericentromeric chromatin.     

Epigenetic modification of centromeric chromatin: hypomethylation of DNA sequences in the CENH3-associated chromatin in Arabidopsis thaliana and maize

The centromere in eukaryotes is defined by the presence of a special histone H3 variant, CENH3. Centromeric chromatin consists of blocks of CENH3-containing nucleosomes interspersed with blocks of canonical H3-containing nucleosomes. However, it is not known how CENH3 is precisely deposited in the centromeres. It has been suggested that epigenetic modifications of the centromeric chromatin may play a role in centromere identity. The centromeres of Arabidopsis thaliana are composed of megabase-sized arrays of a 178-bp satellite repeat. Here, we report that the 178-bp repeats associated with the CENH3-containing chromatin (CEN chromatin) are hypomethylated compared with the same repeats located in the flanking pericentromeric regions. A similar hypomethylation of DNA in CEN chromatin was also revealed in maize (Zea mays). Hypomethylation of the DNA in CEN chromatin is correlated with a significantly reduced level of H3K9me2 in Arabidopsis. We demonstrate that the 178-bp repeats from CEN chromatin display a distinct distribution pattern of the CG and CNG sites, which may provide a foundation for the differential methylation of these repeats. Our results suggest that DNA methylation plays an important role in epigenetic demarcation of the CEN chromatin.     

Identification of centromeric and telomeric DNA‐binding proteins in rice

Centromeres and telomeres are DNA/protein complexes and essential functional components of eukaryotic chromosomes. Previous studies have shown that rice centromeres and telomeres are occupied by CentO (rice centromere satellite DNA) satellite and G‐rich telomere repeats, respectively. However, the protein components are not fully understood. DNA‐binding proteins associated with centromeric or telomeric DNAs will most likely be important for the understanding of centromere and telomere structure and functions. To capture DNA‐specific binding proteins, affinity pull‐down technique was applied in this study to isolate rice centromeric and telomeric DNA‐binding proteins. Fifty‐five proteins were identified for their binding affinity to rice CentO repeat, and 80 proteins were identified for their binding to telomere repeat. One CentO‐binding protein, Os02g0288200, was demonstrated to bind to CentO specifically by in vitro assay. A conserved domain, DUF573 with unknown functions was identified in this protein, and proven to be responsible for the specific binding to CentO in vitro. Four proteins identified as telomere DNA‐binding proteins in this study were reported by different groups previously. These results demonstrate that DNA affinity pull‐down technique is effective in the isolation of sequence‐specific binding proteins and will be applicable in future studies of centromere and telomere proteins.     

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.