Octameric nucleosomes are present within the inner kinetochore of human centromeres
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA531000 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR8856351_subreads.fastq.gz | Fastqsanger.gz | |||
| SRR8856352_subreads.fastq.gz | Fastqsanger.gz | |||
| SRR8856353_subreads.fastq.gz | Fastqsanger.gz | |||
| SRR8856354_subreads.fastq.gz | Fastqsanger.gz |
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Project description:The centromere is a vital locus on each chromosome which seeds the kinetochore, allowing for a physical connection between the chromosome and the mitotic spindle. At the heart of the centromere is the centromere-specific histone H3 variant CENP-A/CENH3. Throughout the cell cycle the constitutive centromere associated network is bound to CENP-A chromatin, but how this protein network modifies CENP-A nucleosome dynamics in vivo is unknown. Here, using a combination of biophysical and biochemical analyses we provide evidence for the existence of two populations of structurally distinct CENP-A nucleosomes that co-exist at human centromeres. These two populations display unique sedimentation patterns in a glycerol gradient, and CENP-A nucleosomes that are physically associated with the inner kinetochore appear stabilized in an octameric conformation, with restricted access to the nucleosomal DNA by DNase I. In contrast, the bulk population of CENP-A nucleosomes have diminished heights and weakened DNA interactions. These data suggest that, in vivo, a reserve pool of immature CENP-A nucleosomes exist which wrap DNA loosely, whereas an active inner kinetochore complex associates with stabilized CENP-A nucleosomes. Our data have implications for a function for CENP-A that may be independent of its role in mitotis.
2020-04-02 | GSE129351 | GEO
Project description:We use high-resolution chemical cleavage mapping and both native and cross-linked chromatin immunoprecipitation with paired-end sequencing to elucidate the profile of nuceleosomes containing the centromere-specific variant of H3 (cenH3), known as CENP-A or Cnp1 in fission yeast. We find that in the central domain of fission yeast centromeres H3 nucleosomes are nearly absent and CENP-A nucleosomes are more widely spaced that nucleosomes elsewere. CENP-A (Cnp1), CENP-C (Cnp3), CENP-T (Cnp20) and CENP-I (Mis6) are highly enriched at every position in the central domain except at tRNA genes, with weak enrichment in the flanking heterochromatin where these proteins show no evidence of the positioning that has been seen in point centromeres and in the satellite-rich centromeres of plants and animals. Our findings suggest that classical regional centromeres are distinguished from other centromere classes by the absence of cenH3 nucleosome positioning.
2015-08-17 | GSE64294 | GEO
Project description:Kinetochores are macromolecular protein complexes that ensure accurate chromosome segregation by linking chromosomes to spindle microtubules and integrating safeguard mechanisms. In yeast, the inner kinetochore, also known as Constitutive Centromere Associated Network (CCAN), is specifically established at point centromeres and has been implicated in contributing to Aurora-BIpl1 function. In an attempt to gain a more detailed picture of the budding yeast kinetochore architecture, crosslink-guided in vitro reconstitution revealed novel direct interactions of the inner kinetochore assembled on Cse4CENP-A nucleosomes. The Ame1/Okp1CENP-U/Q heterodimer selectively bound Cse4CENP-A nucleosomes through the Cse4 N-terminus, providing an explanation for the essential role of the COMA complex in budding yeast. Moreover, the Sli15/Ipl1 core chromosomal passenger complex was found to directly interact with COMA in vitro, suggesting a hitherto unknown role of the COMA complex in establishing biorientation. In line with this finding, in vivo artificial tethering of Sli15 to inner kinetochore proteins rescued synthetically lethal subunit deletion phenotypes in a Sli15 centromere targeting deficient mutant. This study reveals characteristics of the inner kinetochore architecture assembled at point centromeres and its implications on chromosomal passenger complex function.
2019-04-12 | PXD011236 | Pride
Project description:To faithfully segregate chromosomes during vertebrate mitosis, kinetochore-microtubule interactions must be restricted to a single site on each chromosome. Prior work on pair-wise kinetochore protein interactions has been unable to identify the mechanisms that prevent kinetochore formation in regions with a low density of CENP-A nucleosomes. To investigate the impact of higher-order assembly on kinetochore formation, we generated defined oligomers of the inner kinetochore protein CENP-T using two distinct, genetically engineered systems in human cells. Although individual CENP-T molecules interact poorly with other kinetochore proteins, oligomers that mimic the centromeric density of CENP-T trigger the robust formation of functional, cytoplasmic kinetochore-like particles. Both in cells and in vitro, each molecule of oligomerized CENP-T recruits substantially higher levels of outer kinetochore components than monomeric CENP-T molecules. Thus, the density-dependence of CENP-T restricts outer kinetochore recruitment to centromeres, where densely packed CENP-A recruits a high local concentration of CENP-T.
2023-11-09 | PXD042174 | Pride
Project description:Plant and animal centromeres comprise megabases of highly repeated satellite sequences, yet centromere function can be specified epigenetically on single-copy DNA by the presence of nucleosomes containing a centromere-specific variant of histone H3 (cenH3). We determined the positions of cenH3 nucleosomes in rice (Oryza sativa), which has centromeres composed of both the 155-bp CentO repeat and single-copy non-CentO sequences. We find that cenH3 nucleosomes protect 90-100 bp of DNA from micrococcal nuclease digestion, sufficient for only a single wrap of DNA around the cenH3 nucleosome core. cenH3 nucleosomes are translationally phased with 155-bp periodicity on CentO repeats, but not on non-CentO sequences. CentO repeats have a ~10-bp periodicity in WW dinucleotides and in micrococcal nuclease cleavage, providing evidence for rotational phasing of cenH3 nucleosomes on CentO, and suggesting that satellites evolve for translational and rotational stabilization of centromeric nucleosomes. Examination of measured size of rice centromere nucleosome
2013-11-06 | E-GEOD-50755 | biostudies-arrayexpress
Project description:Kinetochores are macromolecular protein complexes that ensure accurate chromosome segregation by linking chromosomes to spindle microtubules and integrating safeguard mechanisms. In yeast, the inner kinetochore, also known as Constitutive Centromere Associated Network (CCAN), is specifically established at point centromeres and has been implicated in contributing to Aurora-BIpl1 function. In an attempt to gain a more detailed picture of the budding yeast kinetochore architecture, crosslink-guided in vitro reconstitution revealed novel direct interactions of the inner kinetochore assembled on Cse4CENP-A nucleosomes. The Ame1/Okp1CENP-U/Q heterodimer selectively bound Cse4CENP-A nucleosomes through the Cse4 N-terminus, providing an explanation for the essential role of the COMA complex in budding yeast. Moreover, the Sli15/Ipl1 core chromosomal passenger complex was found to directly interact with COMA in vitro, suggesting a hitherto unknown role of the COMA complex in establishing biorientation. In line with this finding, in vivo artificial tethering of Sli15 to inner kinetochore proteins rescued synthetically lethal subunit deletion phenotypes in a Sli15 centromere targeting deficient mutant. This study reveals characteristics of the inner kinetochore architecture assembled at point centromeres and its implications on chromosomal passenger complex function.
2019-04-12 | PXD011235 | Pride
Project description:We find that CENP-T acts as a bridge between two well-positioned CENP-A nucleosomes that are present on young alpha-satellite dimers that dominate functional human centromeres. CENP-T is centered over the CENP-B box, where it interacts with the CENP-B/CENP-C complex. Upon cross-linking, the entire CENP-A/CENP-C/CENP-T-containing complex is recovered as a nuclease-protected particle over an alpha-satellite dimer that comprises the fundamental unit of kinetochore chromatin. Our work reveals that CENP-A/CENP-C and CENP-T branches of kinetochore assembly are physically integrated.
2016-05-24 | GSE69839 | GEO
Project description:A key element for defining the centromere identity is the incorporation of a specific histone H3, CENP-A, known as Cnp1p in S. pombe. Previous studies have suggested that functional S. pombe centromeres lack nucleosome arrays and may involve chromatin remodeling as a key step of kinetochore assembly. We used tiling microarrays to show that nucleosomes are in fact positioned in regular intervals in the core of centromere 2, providing the first high resolution map of regional centromere chromatin. Nucleosome locations are not disrupted by mutations in kinetochore proteins cnp1, mis18, mis12, nuf2, mal2, overexpression of Cnp1p, or deletion of ams2. Bioinformatic analysis of the centromere sequence indicates certain enriched motifs in linker regions between nucleosomes and reveals a sequence-bias in nucleosome positioning. We conclude that centromeric nucleosome positions are stable and may be derived from the underlying DNA sequence. In addition, sequence analysis of nucleosome-free regions identifies novel binding sites for the GATA-like protein Ams2p, which participates in CENP-A incorporation. Keywords: Nucleosome Mapping Study Entire cnt regions and histone-related genes were tiled at 1-5 bp spacing using 60-mer probes.
2008-05-30 | E-GEOD-10742 | biostudies-arrayexpress
Project description:Plant and animal centromeres comprise megabases of highly repeated satellite sequences, yet centromere function can be specified epigenetically on single-copy DNA by the presence of nucleosomes containing a centromere-specific variant of histone H3 (cenH3). We determined the positions of cenH3 nucleosomes in rice (Oryza sativa), which has centromeres composed of both the 155-bp CentO repeat and single-copy non-CentO sequences. We find that cenH3 nucleosomes protect 90-100 bp of DNA from micrococcal nuclease digestion, sufficient for only a single wrap of DNA around the cenH3 nucleosome core. cenH3 nucleosomes are translationally phased with 155-bp periodicity on CentO repeats, but not on non-CentO sequences. CentO repeats have a ~10-bp periodicity in WW dinucleotides and in micrococcal nuclease cleavage, providing evidence for rotational phasing of cenH3 nucleosomes on CentO, and suggesting that satellites evolve for translational and rotational stabilization of centromeric nucleosomes.
2013-11-06 | GSE50755 | GEO
Project description:Centromeres are the regions of eukaryotic chromosomes where kinetochores are assembled and direct the correct segregation of chromosomes. Active centromeres are defined by presence of nucleosomes containing CENP-A, a histone H3 variant, which alone is sufficient to direct kinetochore assembly. Once assembled at a location CENP-A chromatin and the kinetochore is maintained at that location though a positive feedback loop where kinetochore proteins recruited by CENP-A promote deposition of new CENP-A following replication. Although CENP-A chromatin itself is a heritable entity, it is normally associated with specific sequences such as human alpha satellite arrays. Such analyses suggest that properties of centromeric DNA itself may favour assembly of CENP-A rather than H3 nucleosomes. To investigate the innate properties of centromeric DNA we have examined histone dynamics on this DNA assembled in CENP-A chromatin at endogenous centromeres and when assembled only in H3 chromatin at an ectopic location. We demonstrate that H3 occupancy on centromeric DNA is innately low while H3 turnover is high. Moreover, even at an ectopic location centromeric DNA programs H3 deposition in S phase and its eviction during G2 when CENP-A is otherwise deposited. G2 accumulation of RNAPII on centromeric DNA during G2 is consistent with transcription-coupled destabilisation of H3 nucleosomes to favour CENP-A deposition.
2018-11-29 | GSE106492 | GEO