Browse
Submit Data
Databases
API
Help

Dataset Information

29 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Protocol for High-Throughput Analysis of Sister-Chromatids Contacts.

ABSTRACT: Sister chromatid interactions are a key step to ensure the successful segregation of sister chromatids after replication. Our knowledge about this phenomenon is mostly based on microscopy approaches, which have some constraints such as resolution limit and the impossibility of studying several genomic positions at the same time. Here, we present a protocol for Hi-SC2, a high-throughput sequencing-based method, to monitor sister chromatid contacts after replication at high resolution throughout the genome, which we applied to study cohesion in Vibrio cholerae. For complete details on the use and execution of this protocol, please refer to Espinosa et al. (2020).

SUBMITTER: Espinosa E

PROVIDER: S-EPMC7757560 | biostudies-literature | 2020 Dec

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Protocol for High-Throughput Analysis of Sister-Chromatids Contacts.

Espinosa Elena E Yamaichi Yoshiharu Y Barre François-Xavier FX

STAR protocols 20201209 3

Sister chromatid interactions are a key step to ensure the successful segregation of sister chromatids after replication. Our knowledge about this phenomenon is mostly based on microscopy approaches, which have some constraints such as resolution limit and the impossibility of studying several genomic positions at the same time. Here, we present a protocol for Hi-SC2, a high-throughput sequencing-based method, to monitor sister chromatid contacts after replication at high resolution throughout t ...[more]

PMID: 33377096

Similar Datasets

Persistent mechanical linkage between sister chromatids throughout anaphase.

Project description:In budding yeast, we have found that sister rDNA arrays marked with fluorescent probes can be visualized as two distinguishable strands during metaphase. Upon anaphase, these arm loci are drawn into the spindle, where they adopt a cruciform-like structure and stretch 2.5-fold as they migrate to the poles. Therefore, while sister rDNA arrays appear separated in metaphase, mechanical linkages between sister arm loci persist throughout anaphase in yeast, as shown in grasshopper spermatocytes (Paliulis and Nicklas 2004). These linkages are partially dependent on the protector of cohesin, SGO1. In anaphase, the spatially regulated dissolution of these mechanical linkages serves to prevent premature sister separation and restrain the rate of spindle elongation. Thus, sister separation is temporally controlled and linkages between sister chromatids contribute to the regulation of anaphase spindle elongation.

| S-EPMC2875669 | biostudies-literature

Identification of sister chromatids by DNA template strand sequences.

Project description:It is generally assumed that sister chromatids are genetically and functionally identical and that segregation to daughter cells is a random process. However, functional differences between sister chromatids regulate daughter cell fate in yeast and sister chromatid segregation is not random in Escherichia coli. Differentiated sister chromatids, coupled with non-random segregation, have been proposed to regulate cell fate during the development of multicellular organisms. This hypothesis has not been tested because molecular features to reliably distinguish between sister chromatids are not obvious. Here we show that parental 'Watson' and 'Crick' DNA template strands can be identified in sister chromatids of murine metaphase chromosomes using CO-FISH (chromosome orientation fluorescence in situ hybridization) with unidirectional probes specific for centromeric and telomeric repeats. All chromosomes were found to have a uniform orientation with the 5' end of the short arm on the same strand as T-rich major satellite repeats. The invariable orientation of repetitive DNA was used to differentially label sister chromatids and directly study mitotic segregation patterns in different cell types. Whereas sister chromatids appeared to be randomly distributed between daughter cells in cultured lung fibroblasts and embryonic stem cells, significant non-random sister chromatid segregation was observed in a subset of colon crypt epithelial cells, including cells outside positions reported for colon stem cells. Our results establish that DNA template sequences can be used to distinguish sister chromatids and follow their mitotic segregation in vivo.

| S-EPMC3757939 | biostudies-literature

Cohesin-independent segregation of sister chromatids in budding yeast.

Project description:Cohesin generates cohesion between sister chromatids, which enables chromosomes to form bipolar attachments to the mitotic spindle and segregate. Cohesin also functions in chromosome condensation, transcriptional regulation, and DNA damage repair. Here we analyze the role of acetylation in modulating cohesin functions and how it affects budding yeast viability. Previous studies show that cohesion establishment requires Eco1p-mediated acetylation of the cohesin subunit Smc3p at residue K113. Smc3p acetylation was proposed to promote establishment by merely relieving Wpl1p inhibition because deletion of WPL1 bypasses the lethality of an ECO1 deletion (eco1? wpl1?). We find that little, if any, cohesion is established in eco1? wpl1? cells, indicating that Eco1p performs a function beyond antagonizing Wpl1p. Cohesion also fails to be established when SMC3 acetyl-mimics (K113Q or K112R,K113Q) are the sole functional SMC3s in cells. These results suggest that Smc3p acetylation levels affect establishment. It is remarkable that, despite their severe cohesion defect, eco1? wpl1? and smc3-K112R,K113Q strains are viable because a cohesin-independent mechanism enables bipolar attachment and segregation. This alternative mechanism is insufficient for smc3-K113Q strain viability. Smc3-K113Q is defective for condensation, whereas eco1? wpl1? and smc3-K112R,K113Q strains are competent for condensation. We suggest that Smc3p acetylation and Wpl1p antagonistically regulate cohesin's essential role in condensation.

| S-EPMC3279399 | biostudies-literature

Physical Proximity of Sister Chromatids Promotes Top2-Dependent Intertwining.

Project description:Sister chromatid intertwines (SCIs), or catenanes, are topological links between replicated chromatids that interfere with chromosome segregation. The formation of SCIs is thought to be a consequence of fork swiveling during DNA replication, and their removal is thought to occur because of the intrinsic feature of type II topoisomerases (Top2) to simplify DNA topology. Here, we report that SCIs are also formed independently of DNA replication during G2/M by Top2-dependent concatenation of cohesed chromatids due to their physical proximity. We demonstrate that, in contrast to G2/M, Top2 removes SCIs from cohesed chromatids at the anaphase onset. Importantly, SCI removal in anaphase requires condensin and coincides with the hyperactivation of condensin DNA supercoiling activity. This is consistent with the longstanding proposal that condensin provides a bias in Top2 function toward decatenation. A comprehensive model for the formation and resolution of toxic SCI entanglements on eukaryotic genomes is proposed.

| S-EPMC5065527 | biostudies-literature

Conformation of sister chromatids in the replicated human genome.

Project description:The three-dimensional organization of the genome supports regulated gene expression, recombination, DNA repair, and chromosome segregation during mitosis. Chromosome conformation capture (Hi-C)1,2 analysis has revealed a complex genomic landscape of internal chromosomal structures in vertebrate cells3-7, but the identical sequence of sister chromatids has made it difficult to determine how they topologically interact in replicated chromosomes. Here we describe sister-chromatid-sensitive Hi-C (scsHi-C), which is based on labelling of nascent DNA with 4-thio-thymidine and nucleoside conversion chemistry. Genome-wide conformation maps of human chromosomes reveal that sister-chromatid pairs interact most frequently at the boundaries of topologically associating domains (TADs). Continuous loading of a dynamic cohesin pool separates sister-chromatid pairs inside TADs and is required to focus sister-chromatid contacts at TAD boundaries. We identified a subset of TADs that are overall highly paired and are characterized by facultative heterochromatin and insulated topological domains that form separately within individual sister chromatids. The rich pattern of sister-chromatid topologies and our scsHi-C technology will make it possible to investigate how physical interactions between identical DNA molecules contribute to DNA repair, gene expression, chromosome segregation, and potentially other biological processes.

| S-EPMC7116725 | biostudies-literature

High-throughput sequencing analysis of the relative frequency of Contacts between Sister Copies emerging from a replication fork in Vibrio cholerae

Project description:Paired-end deep-sequencing was used to determine in parallel the status and position of sister-chromatid contact (SC2) reporters and/or site-specific recombinase (SSR) activity reporters inserted at random positions in a library of cells. The SC2 reporters are short recombination cassettes for a topology-independent site-specific recombinase (Cre or Xer). The SSR-activity reporters are long recombination cassettes. The SC2 reporter cassettes are composed of two directly-repeated recombination sites separated by a DNA segment too short to permit their excision by intramolecular recombination. The SSR-activity cassettes are composed of two directly-repeated recombination sites separated by a DNA segment long enough to permit their excision by intramolecular recombination. The assays start with the engineering of a cell line with a conditional expression allele for Cre or Xer and the creation of a library of cells harbouring a cognate SC2 or SSR-activity reporter at different genomic positions. Production of the recombinase was induced for different lengths of time during cell growth and/or at specific stages of the cell cycle. The position of the SC2 reporter harboured by each cell and the recombination status of the recombination cassette it contains are then determined by high-throughput paired-end sequencing.

2020-07-07 | E-MTAB-8946 | biostudies-arrayexpress

Conformation of sister chromatids in the replicated human genome

Project description:The three-dimensional organization of the genome supports regulated gene expression, recombination, DNA repair, and chromosome segregation during mitosis. Chromosome conformation capture (Hi-C) has revealed a complex genomic landscape of internal chromosome structures in vertebrate cells yet how sister chromatids topologically interact in replicated chromosomes has remained elusive due to their identical sequences. Here, we present sister-chromatid-sensitive Hi-C (scsHi-C) based on nascent DNA labeling with 4-thio-thymidine. Genome-wide conformation maps of human chromosomes revealed that sister chromatid pairs interact most frequently at the boundaries of topologically associating domains (TADs). Continuous loading of a dynamic cohesin pool separates sister-chromatid pairs inside TADs and is required to focus sister chromatid contacts at TAD boundaries. We identified a subset of TADs that are overall highly paired, characterized by facultative heterochromatin, as well as insulated topological domains that form separately within individual sister chromatids. The rich pattern of sister chromatid topologies and our scsHi-C technology will make it possible to dissect how physical interactions between identical DNA molecules contribute to DNA repair, gene expression, chromosome segregation, and potentially other biological processes.

2020-06-12 | GSE152373 | GEO

BRCA1 and CtIP suppress long-tract gene conversion between sister chromatids.

Project description:BRCA1 controls early steps of the synthesis-dependent strand annealing (SDSA) pathway of homologous recombination, but has no known role following Rad51-mediated synapsis. Here we show that BRCA1 influences post-synaptic homologous recombination events, controlling the balance between short- (STGC) and long-tract gene conversion (LTGC) between sister chromatids. Brca1 mutant cells reveal a bias towards LTGC that is corrected by expression of wild-type but not cancer-predisposing BRCA1 alleles. The LTGC bias is enhanced by depletion of CtIP but reversed by inhibition of 53BP1, implicating DNA end resection as a contributor to the STGC/LTGC balance. The impact of BRCA1/CtIP loss on the STGC/LTGC balance is abolished when the second (non-invading) end of the break is unable to support termination of STGC by homologous pairing (annealing). This suggests that BRCA1/CtIP-mediated processing of the second end of the break controls the annealing step that normally terminates SDSA, thereby suppressing the error-prone LTGC outcome.

| S-EPMC3838905 | biostudies-literature

H4K20me0 recognition by BRCA1-BARD1 directs homologous recombination to sister chromatids.

Project description:Genotoxic DNA double-strand breaks (DSBs) can be repaired by error-free homologous recombination (HR) or mutagenic non-homologous end-joining1. HR supresses tumorigenesis1, but is restricted to the S and G2 phases of the cell cycle when a sister chromatid is present2. Breast cancer type 1 susceptibility protein (BRCA1) promotes HR by antagonizing the anti-resection factor TP53-binding protein 1(53BP1) (refs. 2-5), but it remains unknown how BRCA1 function is limited to the S and G2 phases. We show that BRCA1 recruitment requires recognition of histone H4 unmethylated at lysine 20 (H4K20me0), linking DSB repair pathway choice directly to sister chromatid availability. We identify the ankyrin repeat domain of BRCA1-associated RING domain protein 1 (BARD1)-the obligate BRCA1 binding partner3-as a reader of H4K20me0 present on new histones in post-replicative chromatin6. BARD1 ankyrin repeat domain mutations disabling H4K20me0 recognition abrogate accumulation of BRCA1 at DSBs, causing aberrant build-up of 53BP1, and allowing anti-resection activity to prevail in S and G2. Consequently, BARD1 recognition of H4K20me0 is required for HR and resistance to poly (ADP-ribose) polymerase inhibitors. Collectively, this reveals that BRCA1-BARD1 monitors the replicative state of the genome to oppose 53BP1 function, routing only DSBs within sister chromatids to HR.

| S-EPMC6420097 | biostudies-literature

Cohesin-mediated DNA loop extrusion resolves sister chromatids in G2 phase

Project description:Genetic information is stored in linear DNA molecules, which are highly folded inside cells. DNA replication along the folded template path yields two sister chromatids that initially occupy the same nuclear region in an intertwined arrangement. Dividing cells must disentangle and condense the sister chromatids into separate bodies such that a microtubule-based spindle can move them to opposite poles. While the spindle-mediated transport of sister chromatids has been studied in detail, the chromosome-intrinsic mechanics pre-segregating sister chromatids have remained elusive. Here, we show that human sister chromatids resolve extensively already during interphase, in a process dependent on the loop-extruding activity of cohesin, but not that of condensins. Increasing cohesin's looping capability increases sister DNA resolution in interphase nuclei to an extent normally seen only during mitosis, despite the presence of abundant arm cohesion. That cohesin can resolve sister chromatids so extensively in the absence of mitosis-specific activities indicates that DNA loop extrusion is a generic mechanism for segregating replicated genomes, shared across different Structural Maintenance of Chromosomes (SMC) protein complexes in all kingdoms of life.

| S-SCDT-10_15252-EMBJ_2023113475 | biostudies-other

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

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