Non-B-Form DNA Is Enriched at Centromeres.
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ABSTRACT:
SUBMITTER: Kasinathan S
PROVIDER: S-EPMC5889037 | biostudies-literature | 2018 Apr
REPOSITORIES: biostudies-literature
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Project description:Although the pericentromeric regions of chromosomes that are enriched in tandemly repeated satellite DNA represent a significant part of eukaryotic genomes, they remain understudied, which is mainly due to interdisciplinary knowledge gaps. Recent studies suggest their important role in genome regulation, karyotype stability, and evolution. Thus, the idea of satellite DNA as a junk part of the genome has been refuted. The integration of data regarding molecular composition, chromosome behaviour, and the details of the in situ organization of pericentromeric regions is of great interest. The objective of this work was a cytogenetic analysis of the interactions between pericentromeric regions from non-homologous chromosomes in mouse spermatocytes using immuno-FISH. We analysed two events: the associations between centromeric regions of the X chromosome and autosomes and the associations between the centromeric regions of the autosomal bivalents that form chromocenters. We concluded that the X chromosome forms temporary synaptic associations with different autosomes in early meiotic prophase I, which can normally be found until the pachytene-diplotene, without signs of pachytene arrest. These associations are formed between the satellite-DNA-rich centromeric regions of the X chromosome and different autosomes but do not involve the satellite-DNA-poor centromeric region of the Y chromosome. We suggest the hypothetical model of X chromosome competitive replacement from such associations during synaptic correction. We showed that the centromeric region of the X chromosome in association remains free of γH2Ax-dependent chromatin inactivation, while the Y chromosome is completely inactivated. This finding highlights the predominant role of associations between satellite DNA-rich regions of different chromosomes, including the X chromosome. We suppose that X-autosomal transient associations are a manifestation of an additional synaptic disorder checkpoint. These associations are normally corrected before the late diplotene stage. We revealed that the intense spreading conditions that were applied to the spermatocyte I nuclei did not lead to the destruction of stretched chromatin fibers of elongated chromocenters enriched in satellite DNA. The tight associations that we revealed between the pericentromeric regions of different autosomal bivalents and the X chromosome may represent the basis for a mechanism for maintaining the repeats stability in the autosomes and in the X chromosome. The consequences of our findings are discussed.
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Project description:The centromere is the chromosomal locus that ensures fidelity in genome transmission at cell division. Centromere protein A (CENP-A) is a histone H3 variant that specifies centromere location independently of DNA sequence. Conflicting evidence has emerged regarding the histone composition and stoichiometry of CENP-A nucleosomes. Here we show that the predominant form of the CENP-A particle at human centromeres is an octameric nucleosome. CENP-A nucleosomes are very highly phased on ?-satellite 171-base-pair monomers at normal centromeres and also display strong positioning at neocentromeres. At either type of functional centromere, CENP-A nucleosomes exhibit similar DNA-wrapping behavior, as do octameric CENP-A nucleosomes reconstituted with recombinant components, having looser DNA termini than those on conventional nucleosomes containing canonical histone H3. Thus, the fundamental unit of the chromatin that epigenetically specifies centromere location in mammals is an octameric nucleosome with loose termini.
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Project description:Centromeres are essential for cell division and growth in all eukaryotes, and knowledge of their sequence and structure guides the development of artificial chromosomes for functional cellular biology studies. Centromeric proteins are conserved among eukaryotes; however, centromeric DNA sequences are highly variable. We combined forward and reverse genetic approaches with chromatin immunoprecipitation to identify centromeres of the model diatom Phaeodactylum tricornutum We observed 25 unique centromere sequences typically occurring once per chromosome, a finding that helps to resolve nuclear genome organization and indicates monocentric regional centromeres. Diatom centromere sequences contain low-GC content regions but lack repeats or other conserved sequence features. Native and foreign sequences with similar GC content to P. tricornutum centromeres can maintain episomes and recruit the diatom centromeric histone protein CENH3, suggesting nonnative sequences can also function as diatom centromeres. Thus, simple sequence requirements may enable DNA from foreign sources to persist in the nucleus as extrachromosomal episomes, revealing a potential mechanism for organellar and foreign DNA acquisition.
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Project description:Epigenetic mechanisms are thought to specify the identity of centromeres, yet the degree to which DNA sequence contributes to centromere determination remains unresolved. For example, the conserved CENP-B protein binds the 17-bp âCENP-B boxâ motif found in α-satellite arrays at most human centromeres. Although CENP-B is required for artificial centromere function, it is non-essential and some species lack CENP-B boxes entirely. To address this âCENP-B paradox,â we determined the distribution of CENP-B boxes in primates and directly demonstrated the absence of CENP-B at Old World Monkey centromeres. We found extensive interspecific variation in α-satellite arrays including abundance of <10-bp dyad symmetries and of non-B-form DNA structures. We confirmed the presence of non-canonical DNA at human centromeres and neocentromeres and detected similar structures at mouse, chicken and yeast centromeres. To resolve the CENP-B paradox, we propose that CENP-B enhances non-canonical DNA formation, thus providing a general basis for centromere specification.
2018-01-28 | GSE102111 | GEO
Project description:Unzipping of double-stranded nucleic acids by an electric field applied across a wild-type ?-hemolysin (?HL) nanopore provides structural information about different duplex forms. In this work, comparative studies on A-form DNA-RNA duplexes and B-form DNA-DNA duplexes with a single-stranded tail identified significant differences in the blockage current and the unzipping duration between the two helical forms. We observed that the B-form duplex blocks the channel 1.9 ± 0.2 pA more and unzips ?15-fold more slowly than an A-form duplex at 120 mV. We developed a model to describe the dependence of duplex unzipping on structure. We demonstrate that the wider A-form duplex (d = 2.4 nm) is unable to enter the vestibule opening of ?HL on the cis side, leading to unzipping outside of the nanopore with higher residual current and faster unzipping times. In contrast, the smaller B-form duplexes (d = 2.0 nm) enter the vestibule of ?HL, resulting in decreased current blockages and slower unzipping. We investigated the effects of varying the length of the single-stranded overhang, and studied A-form DNA-PNA duplexes to provide additional support for the proposed model. This study identifies key differences between A- and B-form duplex unzipping that will be important in the design of future probe-based methods for detecting DNA or RNA.
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Project description:Centromeres that are essential for faithful segregation of chromosomes consist of unique DNA repeats in many eukaryotes. Although recombination is under-represented around centromeres during meiosis, little is known about recombination between centromere repeats in mitotic cells. Here, we compared spontaneous recombination that occurs between ade6B/ade6X inverted repeats integrated at centromere 1 (cen1) or at a non-centromeric ura4 locus in fission yeast. Remarkably, distinct mechanisms of homologous recombination (HR) were observed in centromere and non-centromere regions. Rad51-dependent HR that requires Rad51, Rad54 and Rad52 was predominant in the centromere, whereas Rad51-independent HR that requires Rad52 also occurred in the arm region. Crossovers between inverted repeats (i.e. inversions) were under-represented in the centromere as compared to the arm region. While heterochromatin was dispensable, Mhf1/CENP-S, Mhf2/CENP-X histone-fold proteins and Fml1/FANCM helicase were required to suppress crossovers. Furthermore, Mhf1 and Fml1 were found to prevent gross chromosomal rearrangements mediated by centromere repeats. These data for the first time uncovered the regulation of mitotic recombination between DNA repeats in centromeres and its physiological role in maintaining genome integrity.
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Project description:Eukaryotic centromeres contain the kinetochore, which connects chromosomes to the spindle allowing segregation. During meiosis, centromeres are suppressed for inter-homolog crossover, as recombination in these regions can cause chromosome missegregation and aneuploidy. Plant centromeres are surrounded by transposon-dense pericentromeric heterochromatin that is epigenetically silenced by histone 3 lysine 9 dimethylation (H3K9me2), and DNA methylation in CG and non-CG sequence contexts. However, the role of these chromatin modifications in control of meiotic recombination in the pericentromeres is not fully understood. Here, we show that disruption of Arabidopsis thaliana H3K9me2 and non-CG DNA methylation pathways, for example, via mutation of the H3K9 methyltransferase genes KYP/SUVH4 SUVH5 SUVH6, or the CHG DNA methyltransferase gene CMT3, increases meiotic recombination in proximity to the centromeres. Using immunocytological detection of MLH1 foci and genotyping by sequencing of recombinant plants, we observe that H3K9me2 and non-CG DNA methylation pathway mutants show increased pericentromeric crossovers. Increased pericentromeric recombination in H3K9me2/non-CG mutants occurs in hybrid and inbred backgrounds and likely involves contributions from both the interfering and noninterfering crossover repair pathways. We also show that meiotic DNA double-strand breaks (DSBs) increase in H3K9me2/non-CG mutants within the pericentromeres, via purification and sequencing of SPO11-1-oligonucleotides. Therefore, H3K9me2 and non-CG DNA methylation exert a repressive effect on both meiotic DSB and crossover formation in plant pericentromeric heterochromatin. Our results may account for selection of enhancer trap Dissociation (Ds) transposons into the CMT3 gene by recombination with proximal transposon launch-pads.
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Project description:Highly conserved repetitive DNA sequence clones, largely consisting of (GGAAT)n repeats, have been isolated from a human recombinant repetitive DNA library by high-stringency hybridization with rodent repetitive DNA. This sequence, the predominant repetitive sequence in human satellites II and III, is similar to the essential core DNA of the Saccharomyces cerevisiae centromere, centromere DNA element (CDE) III. In situ hybridization to human telophase and Drosophila polytene chromosomes shows localization of the (GGAAT)n sequence to centromeric regions. Hyperchromicity studies indicate that the (GGAAT)n sequence exhibits unusual hydrogen bonding properties. The purine-rich strand alone has the same thermal stability as the duplex. Hyperchromicity studies of synthetic DNA variants indicate that all sequences with the composition (AATGN)n exhibit this unusual thermal stability. DNA-mobility-shift assays indicate that specific HeLa-cell nuclear proteins recognize this sequence with a relative affinity greater than 10(5). The extreme evolutionary conservation of this DNA sequence, its centromeric location, its unusual hydrogen bonding properties, its high affinity for specific nuclear proteins, and its similarity to functional centromeres isolated from yeast suggest that this sequence may be a component of the functional human centromere.
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Project description: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.
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Project description:Centromeres are essential for faithful chromosome segregation during mitosis and meiosis. However, the organization of satellite DNA and chromatin at mouse centromeres and pericentromeres is poorly understood due to the challenges of sequencing and assembling repetitive genomic regions. Using recently available PacBio long-read sequencing data from the C57BL/6 strain and chromatin profiling, we found that contrary to the previous reports of their highly homogeneous nature, centromeric and pericentromeric satellites display varied sequences and organization. We find that both centromeric minor satellites and pericentromeric major satellites exhibited sequence variations within and between arrays. While most arrays are continuous, a significant fraction is interspersed with non-satellite sequences, including transposable elements. Additionally, we investigated CENP-A and H3K9me3 chromatin organization at centromeres and pericentromeres using Chromatin immunoprecipitation sequencing (ChIP-seq). We found that the occupancy of CENP-A and H3K9me3 chromatin at centromeric and pericentric regions, respectively, is associated with increased sequence abundance and homogeneity at these regions. Furthermore, the transposable elements at centromeric regions are not part of functional centromeres as they lack CENP-A enrichment. Finally, we found that while H3K9me3 nucleosomes display a well-phased organization on major satellite arrays, CENP-A nucleosomes on minor satellite arrays lack phased organization. Interestingly, the homogeneous class of major satellites phase CENP-A and H3K27me3 nucleosomes as well, indicating that the nucleosome phasing is an inherent property of homogeneous major satellites. Overall, our findings reveal that house mouse centromeres and pericentromeres, which were previously thought to be highly homogenous, display significant diversity in satellite sequence, organization, and chromatin structure.
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