Centromeric chromatin exhibits a histone modification pattern that is distinct from both euchromatin and heterochromatin

Post-translational histone modifications regulate epigenetic switching between different chromatin states. Distinct histone modifications, such as acetylation, methylation and phosphorylation, define different functional chromatin domains, and often do so in a combinatorial fashion. The centromere is a unique chromosomal locus that mediates multiple segregation functions, including kinetochore formation, spindle-mediated movements, sister cohesion and a mitotic checkpoint. Centromeric (CEN) chromatin is embedded in heterochromatin and contains blocks of histone H3 nucleosomes interspersed with blocks of CENP-A nucleosomes, the histone H3 variant that provides a structural and functional foundation for the kinetochore. Here, we demonstrate that the spectrum of histone modifications present in human and Drosophila melanogaster CEN chromatin is distinct from that of both euchromatin and flanking heterochromatin. We speculate that this distinct modification pattern contributes to the unique domain organization and three-dimensional structure of centromeric regions, and/or to the epigenetic information that determines centromere identity.     

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.     

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。     

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.     

Nucleosome depletion alters the chromatin structure of Saccharomyces cerevisiae centromeres.

Saccharomyces cerevisiae centromeric DNA is packaged into a highly nuclease-resistant chromatin core of approximately 200 base pairs of DNA. The structure of the centromere in chromosome III is somewhat larger than a 160-base-pair nucleosomal core and encompasses the conserved centromere DNA elements (CDE I, II, and III). Extensive mutational analysis has revealed the sequence requirements for centromere function. Mutations affecting the segregation properties of centromeres also exhibit altered chromatin structures in vivo. Thus the structure, as delineated by nuclease digestion, correlated with functional centromeres. We have determined the contribution of histone proteins to this unique structural organization. Nucleosome depletion by repression of either histone H2B or H4 rendered the cell incapable of chromosome segregation. Histone repression resulted in increased nuclease sensitivity of centromere DNA, with up to 40% of CEN3 DNA molecules becoming accessible to nucleolytic attack. Nucleosome depletion also resulted in an alteration in the distribution of nuclease cutting sites in the DNA surrounding CEN3. These data provide the first indication that authentic nucleosomal subunits flank the centromere and suggest that nucleosomes may be the central core of the centromere itself.     

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.     

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.     

Epigenetics regulate centromere formation and kinetochore function

The eukaryote centromere was initially defined cytologically as the primary constriction on vertebrate chromosomes and functionally as a chromosomal feature with a relatively low recombination frequency. Structurally, the centromere is the foundation for sister chromatid cohesion and kinetochore formation. Together these provide the basis for interaction between chromosomes and the mitotic spindle, allowing the efficient segregation of sister chromatids during cell division. Although centromeric (CEN) DNA is highly variable between species, in all cases the functional centromere forms in a chromatin domain defined by the substitution of histone H3 with the centromere specific H3 variant centromere protein A (CENP-A), also known as CENH3. Kinetochore formation and function are dependent on a variety of regional epigenetic modifications that appear to result in a loop chromatin conformation providing exterior CENH3 domains for kinetochore construction, and interior heterochromatin domains essential for sister chromatid cohesion. In addition pericentric heterochromatin provides a structural element required for spindle assembly checkpoint function. Advances in our understanding of CENH3 biology have resulted in a model where kinetochore location is specified by the epigenetic mark left after dilution of CENH3 to daughter DNA strands during S phase. This results in a self-renewing and self-reinforcing epigenetic state favorable to reliably mark centromere location, as well as to provide the optimal chromatin configuration for kinetochore formation and function.     

Strong epigenetic similarity between maize centromeric and pericentromeric regions at the level of small RNAs, DNA methylation and H3 chromatin modifications

Both kinetochore function and sister chromatid cohesion can depend upon pericentromere chromatin structure, and factors associated with heterochromatin have been proposed to have general, conserved roles in distinguishing centromeres and pericentromeres and in conferring pericentromere-intrinsic functions. We applied genome-wide sequencing approaches to quantify RNA expression, DNA methylation and histone modification distributions in maize (Zea mays), focusing on two maize chromosomes with nearly fully sequenced centromeres and pericentromeres. Aside from the presence of the Histone H3 variant common to all centromeres, Centromeric Histone H3 (CENH3), we found no RNA expression or chromatin modifications that clearly differentiate pericentromeres from either centromeres or from chromosome arms, nor did we identify an epigenetic signature that accurately predicts CENH3 location. RNA expression and chromatin modification frequencies were broadly associated with distance from centromeres, gradually peaking or dipping toward arms. When interpreted in the context of experimental data from other systems, our results suggest that centromeres may confer essential functions (such as cohesion retention) to flanking sequence regardless of the local heterochromatin profile.     

Propagation of centromeric chromatin requires exit from mitosis

Centromeres direct chromosomal inheritance by nucleating assembly of the kinetochore, a large multiprotein complex required for microtubule attachment during mitosis. Centromere identity in humans is epigenetically determined, with no DNA sequence either necessary or sufficient. A prime candidate for the epigenetic mark is assembly into centromeric chromatin of centromere protein A (CENP-A), a histone H3 variant found only at functional centromeres. A new covalent fluorescent pulse-chase labeling approach using SNAP tagging has now been developed and is used to demonstrate that CENP-A bound to a mature centromere is quantitatively and equally partitioned to sister centromeres generated during S phase, thereby remaining stably associated through multiple cell divisions. Loading of nascent CENP-A on the megabase domains of replicated centromere DNA is shown to require passage through mitosis but not microtubule attachment. Very surprisingly, assembly and stabilization of new CENP-A-containing nucleosomes is restricted exclusively to the subsequent G1 phase, demonstrating direct coupling between progression through mitosis and assembly/maturation of the next generation of centromeres.     

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.     

Formation of a Functional Maize Centromere after Loss of Centromeric Sequences and Gain of Ectopic Sequences

The maize (Zea mays) B centromere is composed of B centromere–specific repeats (ZmBs), centromere-specific satellite repeats (CentC), and centromeric retrotransposons of maize (CRM). Here we describe a newly formed B centromere in maize, which has lost CentC sequences and has dramatically reduced CRM and ZmBs sequences, but still retains the molecular features of functional centromeres, such as CENH3, H2A phosphorylation at Thr-133, H3 phosphorylation at Ser-10, and Thr-3 immunostaining signals. This new centromere is stable and can be transmitted to offspring through meiosis. Anti-CENH3 chromatin immunoprecipitation sequencing revealed that a 723-kb region from the short arm of chromosome 9 (9S) was involved in the formation of the new centromere. The 723-kb region, which is gene poor and enriched for transposons, contains two abundant DNA motifs. Genes in the new centromere region are still transcribed. The original 723-kb region showed a higher DNA methylation level compared with native centromeres but was not significantly changed when it was involved in new centromere formation. Our results indicate that functional centromeres may be formed without the known centromere-specific sequences, yet the maintenance of a high DNA methylation level seems to be crucial for the proper function of a new centromere.     

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.     

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.     

Stable Patterns of CENH3 Occupancy Through Maize Lineages Containing Genetically Similar Centromeres

While the approximate chromosomal position of centromeres has been identified in many species, little is known about the dynamics and diversity of centromere positions within species. Multiple lines of evidence indicate that DNA sequence has little or no impact in specifying centromeres in maize and in most multicellular organisms. Given that epigenetically defined boundaries are expected to be dynamic, we hypothesized that centromere positions would change rapidly over time, which would result in a diversity of centromere positions in isolated populations. To test this hypothesis, we used CENP-A/cenH3 (CENH3 in maize) chromatin immunoprecipitation to define centromeres in breeding pedigrees that included the B73 inbred as a common parent. While we found a diversity of CENH3 profiles for centromeres with divergent sequences that were not inherited from B73, the CENH3 profiles from centromeres that were inherited from B73 were indistinguishable from each other. We propose that specific genetic elements in centromeric regions favor or inhibit CENH3 accumulation, leading to reproducible patterns of CENH3 occupancy. These data also indicate that dramatic shifts in centromere position normally originate from accumulated or large-scale genetic changes rather than from epigenetic positional drift.     

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.