Browse
Submit Data
Databases
API
Help

Dataset Information

25 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

R-loops at centromeric chromatin contribute to defects in kinetochore integrity and chromosomal instability in budding yeast.

ABSTRACT: R-loops, the byproduct of DNA-RNA hybridization and the displaced single-stranded DNA (ssDNA), have been identified in bacteria, yeasts, and other eukaryotic organisms. The persistent presence of R-loops contributes to defects in DNA replication and repair, gene expression, and genomic integrity. R-loops have not been detected at centromeric (CEN) chromatin in wild-type budding yeast. Here we used an hpr1∆ strain that accumulates R-loops to investigate the consequences of R-loops at CEN chromatin and chromosome segregation. We show that Hpr1 interacts with the CEN-histone H3 variant, Cse4, and prevents the accumulation of R-loops at CEN chromatin for chromosomal stability. DNA-RNA immunoprecipitation (DRIP) analysis showed an accumulation of R-loops at CEN chromatin that was reduced by overexpression of RNH1 in hpr1∆ strains. Increased levels of ssDNA, reduced levels of Cse4 and its assembly factor Scm3, and mislocalization of histone H3 at CEN chromatin were observed in hpr1∆ strains. We determined that accumulation of R-loops at CEN chromatin contributes to defects in kinetochore biorientation and chromosomal instability (CIN) and these phenotypes are suppressed by RNH1 overexpression in hpr1∆ strains. In summary, our studies provide mechanistic insights into how accumulation of R-loops at CEN contributes to defects in kinetochore integrity and CIN.

SUBMITTER: Mishra PK

PROVIDER: S-EPMC8098821 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Centromeric R loops contribute to defects in kinetochore assembly and chromosomal instability

Project description:We mapped ssDNA formation in WT and hpr1delta cells at permissive (30C) and non-permissive (37C) temperature. We showed that hpr1 cells accumulate ssDNA show elevated levels of ssDNA at centromeric regions. Additionally, we show replication fork-associated ssDNA at origins of replication is more confined to the origins in hpr1 cells than WT cells, suggesting a defect in replication fork progression.

2020-12-31 | GSE151849 | GEO

Centromeric R loops contribute to defects in kinetochore assembly and chromosomal instability

Project description:Centromeric R loops contribute to defects in kinetochore assembly and chromosomal instability

| PRJNA637395 | ENA

Epigenetic rewriting at centromeric DNA repeats leads to increased chromatin accessibility and chromosomal instability.

Project description:BackgroundCentromeric regions of human chromosomes contain large numbers of tandemly repeated α-satellite sequences. These sequences are covered with constitutive heterochromatin which is enriched in trimethylation of histone H3 on lysine 9 (H3K9me3). Although well studied using artificial chromosomes and global perturbations, the contribution of this epigenetic mark to chromatin structure and genome stability remains poorly known in a more natural context.ResultsUsing transcriptional activator-like effectors (TALEs) fused to a histone lysine demethylase (KDM4B), we were able to reduce the level of H3K9me3 on the α-satellites repeats of human chromosome 7. We show that the removal of H3K9me3 affects chromatin structure by increasing the accessibility of DNA repeats to the TALE protein. Tethering TALE-demethylase to centromeric repeats impairs the recruitment of HP1α and proteins of Chromosomal Passenger Complex (CPC) on this specific centromere without affecting CENP-A loading. Finally, the epigenetic re-writing by the TALE-KDM4B affects specifically the stability of chromosome 7 upon mitosis, highlighting the importance of H3K9me3 in centromere integrity and chromosome stability, mediated by the recruitment of HP1α and the CPC.ConclusionOur cellular model allows to demonstrate the direct role of pericentromeric H3K9me3 epigenetic mark on centromere integrity and function in a natural context and opens interesting possibilities for further studies regarding the role of the H3K9me3 mark.

| S-EPMC8317386 | biostudies-literature

Bridgin connects the outer kinetochore to centromeric chromatin.

Project description:The microtubule-binding outer kinetochore is coupled to centromeric chromatin through CENP-CMif2, CENP-TCnn1, and CENP-UAme1 linker pathways originating from the constitutive centromere associated network (CCAN) of the inner kinetochore. Here, we demonstrate the recurrent loss of most CCAN components, including certain kinetochore linkers during the evolution of the fungal phylum of Basidiomycota. By kinetochore interactome analyses in a model basidiomycete and human pathogen Cryptococcus neoformans, a forkhead-associated domain containing protein "bridgin" was identified as a kinetochore component along with other predicted kinetochore proteins. In vivo and in vitro functional analyses of bridgin reveal its ability to connect the outer kinetochore with centromeric chromatin to ensure accurate chromosome segregation. Unlike established CCAN-based linkers, bridgin is recruited at the outer kinetochore establishing its role as a distinct family of kinetochore proteins. Presence of bridgin homologs in non-fungal lineages suggests an ancient divergent strategy exists to bridge the outer kinetochore with centromeric chromatin.

| S-EPMC7794384 | biostudies-literature

Deviant kinetochore microtubule dynamics underlie chromosomal instability.

Project description:The persistent malattachment of microtubules to chromosomes at kinetochores is a major mechanism of chromosomal instability (CIN) [1, 2]. In normal diploid cells, malattachments arise spontaneously and are efficiently corrected to preserve genomic stability [3]. However, it is unknown whether cancer cells with CIN possess the ability to efficiently correct attachment errors. Here we show that kinetochore microtubule attachments in cancer cells with CIN are inherently more stable than those in normal diploid RPE-1 cells. The observed differences in attachment stability account for the persistence of malattachments into anaphase, where they cause chromosome missegregation. Furthermore, increasing the stability of kinetochore microtubule attachments in normal diploid RPE-1 cells, either by depleting the tumor suppressor protein APC or the kinesin-13 protein MCAK, is sufficient to promote chromosome segregation defects to levels comparable to those in cancer cells with CIN. Collectively, these data identify that cancer cells have a diminished capacity to correct erroneous kinetochore microtubule attachments and account for the widespread occurrence of CIN in tumors [4].

| S-EPMC2787757 | biostudies-literature

Tripartite organization of centromeric chromatin in budding yeast.

Project description:The centromere is the genetic locus that organizes the proteinaceous kinetochore and is responsible for attachment of the chromosome to the spindle at mitosis and meiosis. In most eukaryotes, the centromere consists of highly repetitive DNA sequences that are occupied by nucleosomes containing the CenH3 histone variant, whereas in budding yeast, a ?120-bp centromere DNA element (CDE) that is sufficient for centromere function is occupied by a single right-handed histone variant CenH3 (Cse4) nucleosome. However, these in vivo observations are inconsistent with in vitro evidence for left-handed octameric CenH3 nucleosomes. To help resolve these inconsistencies, we characterized yeast centromeric chromatin at single base-pair resolution. Intact particles containing both Cse4 and H2A are precisely protected from micrococcal nuclease over the entire CDE of all 16 yeast centromeres in both solubilized chromatin and the insoluble kinetochore. Small DNA-binding proteins protect CDEI and CDEIII and delimit the centromeric nucleosome to the ?80-bp CDEII, only enough for a single DNA wrap. As expected for a tripartite organization of centromeric chromatin, loss of Cbf1 protein, which binds to CDEI, both reduces the size of the centromere-protected region and shifts its location toward CDEIII. Surprisingly, Cse4 overproduction caused genome-wide misincorporation of nonfunctional CenH3-containing nucleosomes that protect ?135 base pairs and are preferentially enriched at sites of high nucleosome turnover. Our detection of two forms of CenH3 nucleosomes in the yeast genome, a singly wrapped particle at the functional centromere and octamer-sized particles on chromosome arms, reconcile seemingly conflicting in vivo and in vitro observations.

| S-EPMC3252899 | biostudies-literature

ChromoShake: a chromosome dynamics simulator reveals that chromatin loops stiffen centromeric chromatin.

Project description:ChromoShake is a three-dimensional simulator designed to find the thermodynamically favored states for given chromosome geometries. The simulator has been applied to a geometric model based on experimentally determined positions and fluctuations of DNA and the distribution of cohesin and condensin in the budding yeast centromere. Simulations of chromatin in differing initial configurations reveal novel principles for understanding the structure and function of a eukaryotic centromere. The entropic position of DNA loops mirrors their experimental position, consistent with their radial displacement from the spindle axis. The barrel-like distribution of cohesin complexes surrounding the central spindle in metaphase is a consequence of the size of the DNA loops within the pericentromere to which cohesin is bound. Linkage between DNA loops of different centromeres is requisite to recapitulate experimentally determined correlations in DNA motion. The consequences of radial loops and cohesin and condensin binding are to stiffen the DNA along the spindle axis, imparting an active function to the centromere in mitosis.

| S-EPMC4694754 | biostudies-literature

Structural integrity of centromeric chromatin and faithful chromosome segregation requires Pat1.

Project description:The kinetochore (centromeric DNA and associated protein complex) is essential for faithful chromosome segregation and maintenance of genome stability. Here we report that an evolutionarily conserved protein Pat1 is a structural component of Saccharomyces cerevisiae kinetochore and associates with centromeres in a NDC10-dependent manner. Consistent with a role for Pat1 in kinetochore structure and function, a deletion of PAT1 results in delay in sister chromatid separation, errors in chromosome segregation, and defects in structural integrity of centromeric chromatin. Pat1 is involved in topological regulation of minichromosomes as altered patterns of DNA supercoiling were observed in pat1Δ cells. Studies with pat1 alleles uncovered an evolutionarily conserved region within the central domain of Pat1 that is required for its association with centromeres, sister chromatid separation, and faithful chromosome segregation. Taken together, our data have uncovered a novel role for Pat1 in maintaining the structural integrity of centromeric chromatin to facilitate faithful chromosome segregation and proper kinetochore function.

| S-EPMC3781966 | biostudies-literature

The budding yeast silencing protein Sir1 is a functional component of centromeric chromatin.

Project description:In fission yeast and multicellular organisms, centromere-proximal regions of chromosomes are heterochromatic, containing proteins that silence gene expression. In contrast, the relationship between heterochromatin proteins and kinetochore function in the budding yeast Saccharomyces cerevisiae remains largely unexplored. Here we report that the yeast heterochromatin protein Sir1 is a component of centromeric chromatin and contributes to mitotic chromosome stability. Sir1 recruitment to centromeres occurred through a novel mechanism independent of its interaction with the origin recognition complex (ORC). Sir1 function at centromeres was distinct from its role in forming heterochromatin, because the Sir2-4 proteins were not associated with centromeric regions. Sir1 bound to Cac1, a subunit of chromatin assembly factor I (CAF-I), and helped to retain Cac1 at centromeric loci. These studies reveal that although budding yeast and mammalian cells use fundamentally different mechanisms of forming heterochromatin, they both use silencing proteins to attract the histone deposition factor CAF-I to centromeric chromatin.

| S-EPMC218072 | biostudies-literature

Human condensin function is essential for centromeric chromatin assembly and proper sister kinetochore orientation.

Project description:Condensins I and II in vertebrates are essential ATP-dependent complexes necessary for chromosome condensation in mitosis. Condensins depletion is known to perturb structure and function of centromeres, however the mechanism of this functional link remains elusive. Depletion of condensin activity is now shown to result in a significant loss of loading of CENP-A, the histone H3 variant found at active centromeres and the proposed epigenetic mark of centromere identity. Absence of condensins and/or CENP-A insufficiency produced a specific kinetochore defect, such that a functional mitotic checkpoint cannot prevent chromosome missegregation resulting from improper attachment of sister kinetochores to spindle microtubules. Spindle microtubule-dependent deformation of both inner kinetochores and the HEC1/Ndc80 microtubule-capturing module, then results in kinetochore separation from the Aurora B pool and ensuing reduced kinase activity at centromeres. Moreover, recovery from mitosis-inhibition by monastrol revealed a high incidence of merotelic attachment that was nearly identical with condensin depletion, Aurora B inactivation, or both, indicating that the Aurora B dysfunction is the key defect leading to chromosome missegregation in condensin-depleted cells. Thus, beyond a requirement for global chromosome condensation, condensins play a pivotal role in centromere assembly, proper spatial positioning of microtubule-capturing modules and positioning complexes of the inner centromere versus kinetochore plates.

| S-EPMC2730017 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data