Project description:To determine the distribution of centromere units in the genome of holocentric Chionographis japonica, we performed CENH3-ChIPseq using the customized species-specific CENH3 antibody. We mixed the chromatins of C. japonica and Secale cereal (inbred line Lo7) to dilute the highly abundant centromeric Chio satellite repeats (16%) in the C. japonica genome before immunoprecipitation. In addition, to determine the large-scale genome organization, we performed ChIPseq by targeting the evolutionarily conserved eu- and heterochromatin-specific histone marks H3K4me2 and H33K9me2
Project description:Kinetochores are essential macromolecular complexes anchoring chromosomes to the mitotic spindle, ensuring faithful cell division. Despite their critical role, the structural organization of kinetochores over large centromeric regions and across diverse species remains poorly understood. We present the inner kinetochore (CCAN) structure of the silkmoth Bombyx mori, an insect without the canonical centromeric CENP-A gene and with chromosome-wide centromeric activity (holocentric). Our analyses reveal a ring-shaped complex with structural parallels to the known human and yeast kinetochores. Notably, B. mori CCAN incorporates four previously uncharacterized proteins, Centromeric Subunits 1-4, which have unexpected evolutionary relationships to the outer kinetochore Dam1/DASH complex. We demonstrate that CCAN binds DNA as a distinctive head-to-head dimer, folding the DNA into a loop and generating an alternative point-centromere-like architecture poised for chromosome segregation. Our work establishes this self-contained CCAN dimer as a key structural unit that forms the basis of a holocentric organization and suggests that large-scale centromere architectures can emerge from the modular arrangement of such discrete kinetochore units.
Project description:Centromeric repetitive DNA sequences are highly variable during evolution, which are the hub for genome stability in almost all the eukaryotic organisms. However, how centromeric repeat sequences diverge rapidly among closely related species and populations, and how polyploidy contributed to the diversity of centromere among co-evolved subgenomes are largely unknown. Here, we applied the Brachypodium system to investigate the track of centromere evolution within this taxa, and their adaptation to alloploidization process. Subgenome divergent centromeric satellite repeat were discovered in tetraploid B. hybridum, and this divergent was originated form their two diploid progenitors. Furthermore, differential sequences influence the association sites with CENH3 nucleosomes on the monomer satellite repeats, and positioning of CENH3 nucleosomes on the satellite DNA are stable in each subgenome after alloploidization. Only minor intrasubgenomic variations were observed on these satellite repeats from diploid to tetraploid in B. hybridum, and no evident intersubgenomic transfer of centromeric satellite repeats after alloploidization. Pan-genome analysis reveals that the general principle of centromere dynamic within the populations in Brachypodium genomes with different polyploidy level. Our results provide an unprecedented information regarding the genomic and functional diversity of centromeric repeat DNA during evolution.
Project description:We report the sequences bound to CENP-A in the dog genome (Canis familiaris) for high-throughput characterization of centromeric sequences. We compare these ChIPSeq reads (72 bp, single read) against a reference centromeric satellite DNA domain database for the dog genome, resulting in the annotation of sequence variation and estimated abundance of seven satellite families together with adjacent, non-satellite sequences. To study global patterns of sequence diversity and characterizing the subset of sequences correlated with centromere function, these sequences were evaluated relative to a comprehensive centromere sequence domain k-mer library. From this analysis, we identify functional sequence features from two satellite families (CarSat1 and CarSat2) that are defined by distinct arrays subtypes. Sequences bound to CENP-A in MDCK (dog) cell line
Project description:We report the sequences bound to CENP-A in the dog genome (Canis familiaris) for high-throughput characterization of centromeric sequences. We compare these ChIPSeq reads (72 bp, single read) against a reference centromeric satellite DNA domain database for the dog genome, resulting in the annotation of sequence variation and estimated abundance of seven satellite families together with adjacent, non-satellite sequences. To study global patterns of sequence diversity and characterizing the subset of sequences correlated with centromere function, these sequences were evaluated relative to a comprehensive centromere sequence domain k-mer library. From this analysis, we identify functional sequence features from two satellite families (CarSat1 and CarSat2) that are defined by distinct arrays subtypes.
Project description:Specification and propagation of the centromeres of eukaryotic chromosomes is determined by epigenetic mechanisms. Unfortunately, the epigenetic characteristics of centromeric DNA and chromatin are difficult to define because the centromeres are composed of highly repetitive DNA sequences in most eukaryotic species. Several rice centromeres have been fully sequenced, making rice an excellent model for centromere research. We conducted genome-wide mapping of cytosine methylation using methylcytosine immunoprecipitation combined with Illumina sequencing. The DNA sequences in the core domains of rice Cen4, Cen5, and Cen8 showed elevated methylation levels compared to the sequences in the pericentromeric regions. In addition, elevated methylation levels were associated with the DNA sequences in the CENH3-binding subdomains compared to 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 to 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. mCIP-seq of one sample of rice seedling
Project description:CUT&RUN experiment was carried out on bovine semeimbranosous muscle satellite cells to identify the locus of the accumulation of the centromeric protein A (CENP-A) on the cattle Y-chromosome. This was carried out to support the annotation of the DNA signal comprising tandem array of a centromeric satellite signal which characterizes the centromere on the Y-chromosome.
Project description:The centromere is a defining feature of eukaryotic chromosomes and is essential for the segregation of chromosomes during cell division. Centromeres are universally marked by the histone variant cenH3 and are restricted to specialized chromatin that most commonly localized to a single position along the chromosome. However, the DNA on which centromeric nucleosomes assemble is not conserved and varies greatly in size and composition. It ranges from genetically defined point centromeres that assemble a single cenH3-containing nucleosome to epigenetically defined regional centromeres embedded in megabases of tandemly repeated DNA to holocentromeres that extend along the length of the entire chromosomes. The organization of regional and holocentric centromeres has so far been elusive, as the precise locations of cenH3-containing sequences could not be determined. Our results show that the point centromere is the basic unit of holocentromeres and provide a basis for understanding how centromeric chromatin is maintained. We use high-resolution mapping of cenH3-associated DNA to show that Caenorhabditids elegans holocentromeres are organized as dispersed but discretely localized point centromeres.
Project description:We report that DNA2 predominantly bound to the centromeric α-satellite regions. The centromeric regions contained 58% of the DNA2-associated DNA, representing a 33.5-fold enrichment over genomic input. To define the under-replicated DNA regions in DNA2-null cells, we conducted whole-genome DNA sequencing of the under-replicated BrdU negative DNA. After normalization to genomic input DNA from the same cells, 12.9% of the peaks from the under-replicated DNA aligned with the centromeric DNA regions, representing an 8.5-fold enrichment. In addition, among the peaks that overlapped between the DNA2 pull-down and under-replicated regions, two thirds fell into the centromeric regions, representing a 48-fold enrichment.