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.     

Distinct dynamics of HISTONE3 variants between the two fertilization products in plants

Sexual reproduction involves epigenetic reprogramming comprising DNA methylation and histone modifications. In addition, dynamics of HISTONE3 (H3) variant H3.3 upon fertilization are conserved in animals, suggesting an essential role. In contrast to H3, H3.3 marks actively transcribed regions of the genome and can be deposited in a replication-independent manner. Although H3 variants are conserved in plants, their dynamics during fertilization have remained unexplored. We overcame technical limitations to live imaging of the fertilization process in Arabidopsis thaliana and studied dynamics of the male-gamete-specific H3.3 and the centromeric Histone Three Related 12 (HTR12). The double-fertilization process in plants produces the zygote and the embryo-nourishing endosperm. We show that the zygote is characterized by replication-independent removal of paternal H3.3 and homogeneous incorporation of parental chromatin complements. In the endosperm, the paternal H3.3 is passively diluted by replication while the paternal chromatin remains segregated apart from the maternal chromatin (gonomery). Hence epigenetic regulations distinguish the two products of fertilization in plants. H3.3-replication-independent dynamics and gonomery also mark the first zygotic divisions in animal species. We thus propose the convergent selection of parental epigenetic imbalance involving H3 variants in sexually reproducing organisms.     

Loading of Arabidopsis centromeric histone CENH3 occurs mainly during G2 and requires the presence of the histone fold domain

The centromeric histone H3 (CENH3) substitutes histone H3 within the nucleosomes of active centromeres in all eukaryotes. CENH3 deposition at centromeres is needed to assemble the kinetochore, a complex of conserved proteins responsible for correct chromosome segregation during nuclear division. Histones of regular nucleosomes are loaded during replication in S phase, while CENH3 deposition deviates from this pattern in yeast, human, and Drosophila melanogaster cells. Little is known about when and how CENH3 targets centromeric loci. Therefore, we determined the location and quantity of recombinant enhanced yellow fluorescent protein (EYFP)-CENH3 in mitotic root and endopolyploid leaf nuclei of transgenic Arabidopsis thaliana cells. Our data indicate significant loading of A. thaliana CENH3 during G2 (before splitting into sister kinetochores) rather than during the S or M phase of the cell cycle. The histone fold domain of the C-terminal part of CENH3 is sufficient to target A. thaliana centromeres. A. thaliana EYFP-CENH3 can recognize and target three different centromeric repeats of Arabidopsis lyrata but not field bean (Vicia faba) centromeres.     

Adaptive evolution of the histone fold domain in centromeric histones

Centromeric DNA, being highly repetitive, has been refractory to molecular analysis. However, centromeric structural proteins are encoded by single-copy genes, and these can be analyzed by using standard phylogenetic tools. The centromere-specific histone, CenH3, replaces histone H3 in centromeric nucleosomes, and is required for the proper distribution of chromosomes during cell division. Whereas histone H3s are nearly identical between species, CenH3s are divergent, with an N-terminal tail that is highly variable in length and sequence. Both the N-terminal tail and histone fold domain (HFD) are subject to adaptive evolution in Drosophila. Similarly, comparisons between Arabidopsis thaliana and Arabidopsis arenosa detected adaptive evolution, but only in the N-terminal tail. We have extended our evolutionary analyses of CenH3s to other members of the Brassicaceae, which allowed the detection of positive selection in both the N-terminal tail and in the HFD. We find that adaptively evolving sites in the HFD can potentially interact with DNA, including sites in the loop 1 region of the HFD that are required for centromeric targeting in Drosophila. Other adaptively evolving sites in the HFD can be localized on the structure of the nucleosome core particle, revealing an extended surface in addition to loop 1 in which conformational changes might alter histone-DNA contacts or water bridges. The identification of adaptively evolving sites provides a structural basis for the interaction between centromeric DNA and the protein that is thought to underlie the evolution of centromeres and the accumulation of pericentric heterochromatin.     

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。     

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.     

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.     

Ecotype-specific and chromosome-specific expansion of variant centromeric satellites in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Despite the conserved roles and conserved protein machineries of centromeres, their nucleotide sequences can be highly diverse even among related species. The diversity reflects rapid evolution, but the underlying mechanism is largely unknown. One approach to monitor rapid evolution is examination of intra-specific variation. Here we report variant centromeric satellites of Arabidopsis thaliana found through survey of 103 natural accessions (ecotypes). Among them, a cluster of variant centromeric satellites was detected in one ecotype, Cape Verde Islands (Cvi). Recombinant inbred mapping revealed that the variant satellites are distributed in centromeric region of the chromosome 5 (CEN5) of this ecotype. This apparently recent variant accumulation is associated with large deletion of a pericentromeric region and the expansion of satellite region. The variant satellite was bound to HTR12 (centromeric variant histone H3), although expansion of the satellite was not associated with comparable increase in the HTR12 binding. The results suggest that variant satellites with centromere function can rapidly accumulate in one centromere, supporting the model that the satellite repeats in the array are homogenized by occasional unequal crossing-over, which has a potential to generate an expansion of local sequence variants within a centromere cluster. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

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.     

Meiosis-specific loading of the centromere-specific histone CENH3 in Arabidopsis thaliana

Centromere behavior is specialized in meiosis I, so that sister chromatids of homologous chromosomes are pulled toward the same side of the spindle (through kinetochore mono-orientation) and chromosome number is reduced. Factors required for mono-orientation have been identified in yeast. However, comparatively little is known about how meiotic centromere behavior is specialized in animals and plants that typically have large tandem repeat centromeres. Kinetochores are nucleated by the centromere-specific histone CENH3. Unlike conventional histone H3s, CENH3 is rapidly evolving, particularly in its N-terminal tail domain. Here we describe chimeric variants of CENH3 with alterations in the N-terminal tail that are specifically defective in meiosis. Arabidopsis thaliana cenh3 mutants expressing a GFP-tagged chimeric protein containing the H3 N-terminal tail and the CENH3 C-terminus (termed GFP-tailswap) are sterile because of random meiotic chromosome segregation. These defects result from the specific depletion of GFP-tailswap protein from meiotic kinetochores, which contrasts with its normal localization in mitotic cells. Loss of the GFP-tailswap CENH3 variant in meiosis affects recruitment of the essential kinetochore protein MIS12. Our findings suggest that CENH3 loading dynamics might be regulated differently in mitosis and meiosis. As further support for our hypothesis, we show that GFP-tailswap protein is recruited back to centromeres in a subset of pollen grains in GFP-tailswap once they resume haploid mitosis. Meiotic recruitment of the GFP-tailswap CENH3 variant is not restored by removal of the meiosis-specific cohesin subunit REC8. Our results reveal the existence of a specialized loading pathway for CENH3 during meiosis that is likely to involve the hypervariable N-terminal tail. Meiosis-specific CENH3 dynamics may play a role in modulating meiotic centromere behavior.     

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 retroelements and satellites interact with maize kinetochore protein CENH3

Maize centromeres are composed of CentC tandem repeat arrays, centromeric retrotransposons (CRs), and a variety of other repeats. One particularly well-conserved CR element, CRM, occurs primarily as complete and uninterrupted elements and is interspersed thoroughly with CentC at the light microscopic level. To determine if these major centromeric DNAs are part of the functional centromere/kinetochore complex, we generated antiserum to maize centromeric histone H3 (CENH3). CENH3, a highly conserved protein that replaces histone H3 in centromeres, is thought to recruit many of the proteins required for chromosome movement. CENH3 is present throughout the cell cycle and colocalizes with the kinetochore protein CENPC in meiotic cells. Chromatin immunoprecipitation demonstrates that CentC and CRM interact specifically with CENH3, whereas knob repeats and Tekay retroelements do not. Approximately 38 and 33% of CentC and CRM are precipitated in the chromatin immunoprecipitation assay, consistent with data showing that much, but not all, of CENH3 colocalizes with CentC.     

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.     

Isolation of centromeric-tandem repetitive DNA sequences by chromatin affinity purification using a HaloTag7-fused centromere-specific histone H3 in tobacco

The centromere is a multi-functional complex comprising centromeric DNA and a number of proteins. To isolate unidentified centromeric DNA sequences, centromere-specific histone H3 variants (CENH3) and chromatin immunoprecipitation (ChIP) have been utilized in some plant species. However, anti-CENH3 antibody for ChIP must be raised in each species because of its species specificity. Production of the antibodies is time-consuming and costly, and it is not easy to produce ChIP-grade antibodies. In this study, we applied a HaloTag7-based chromatin affinity purification system to isolate centromeric DNA sequences in tobacco. This system required no specific antibody, and made it possible to apply a highly stringent wash to remove contaminated DNA. As a result, we succeeded in isolating five tandem repetitive DNA sequences in addition to the centromeric retrotransposons that were previously identified by ChIP. Three of the tandem repeats were centromere-specific sequences located on different chromosomes. These results confirm the validity of the HaloTag7-based chromatin affinity purification system as an alternative method to ChIP for isolating unknown centromeric DNA sequences. The discovery of more than two chromosome-specific centromeric DNA sequences indicates the mosaic structure of tobacco centromeres.     

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.