DSBCapture: in situ capture and direct sequencing of dsDNA breaks
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE78172 | GEO | 2016/07/18
SECONDARY ACCESSION(S): PRJNA312824
REPOSITORIES: GEO
Similar Datasets
Project description:Double-strand DNA breaks (DSBs) continuously arise and are a source of mutations and chromosomal rearrangements. Here, we present DSBCapture, a sequencing-based method that captures DSBs in situ and directly maps these at single nucleotide resolution enabling the study of DSB origin. DSBCapture shows substantially increased sensitivity and data yield compared to other methods. Employing DSBCapture, we uncovered a striking relationship between DSBs and elevated transcription within nucleosome-depleted chromatin. 6 library samples, 75 base pairs (50 bp for the EcoRV library) custom protocol (DSBCapture or BLESS) sequenced as paired-end reads on Illumina NextSeq 500 (MiSeq for EcoRV library): 1 replicate for the EcoRV library, 1 replicate for the library coming from the U2OS AID-DlvA cell line with AsiSI restriction enzyme, 2 replicates for the BREAk-seq NHEK libraries and 2 replicates for the BLESS NHEK libraries. 4 RNA-Seq library samples from HEK Gibco cells, single-end sequencing on the Illumina NextSeq 500, 75 base pairs.
2016-07-18 | E-GEOD-78172 | biostudies-arrayexpress
Project description:Background: Of the many types of DNA damage, DNA double-strand breaks (DSB) are probably the most deleterious. Mounting evidence points to an intricate relationship between DSBs and transcription. A cell system in which the impact on transcription can be investigated at precisely mapped genomic DSBs is essential to study this relationship. Methods: Here in a human cell line, we map genome-wide and at high-resolution the DSBs induced by a restriction enzyme and we characterize their impact on gene expression by four independent approaches by monitoring steady-state RNA levels, rates of RNA synthesis, transcription initiation and RNA polymerase II elongation. Results: We consistently observe transcriptional repression in proximity to DSBs. Downregulation of transcription depends on ATM kinase activity and it is sensitive to the distance from the DSB. Conclusions: Our study couples for the first time high-resolution mapping of DSBs with multilayered transcriptomics to dissect the events shaping gene expression after DSB induction at multiple endogenous sites.
2017-06-01 | GSE97589 | GEO
Project description:Chromatin acts as a key regulator of DNA related processes such as DNA damage repair. While ChIP-chip is a powerful technique to provide high-resolution maps of protein-genome interactions, its use to study DNA Double Strand Break (DSB) repair has been hindered by the limitations of the available damage induction methods. We have developed a human cell line that permits induction of multiple DSBs randomly distributed and unambiguously positioned within the genome. Using this system, we have generated the first genome-wide mapping of gammaH2AX around DSBs. We found that all DSBs trigger large gammaH2AX domains, which extend from the DSB in a bidirectional, discontinuous and not necessarily symmetrical manner. Strikingly we uncovered that, within domains, gammaH2AX distribution is highly influenced by gene transcription since parallel mapping of RNA Polymerase II and strand specific expression revealed that ?H2AX does not propagate on active genes. In addition, we demonstrate that transcription is accurately maintained within gammaH2AX domains, indicating that mechanisms may exist to protect genes transcription from gammaH2AX spreading and from the chromatin rearrangements induced by DSBs.
2010-04-20 | E-MEXP-1769 | biostudies-arrayexpress
Project description:Efficient repair of DNA double-strand breaks (DSBs) requires a coordinated DNA Damage Response (DDR), which includes phosphorylation of histone H2Ax, forming γH2Ax. This histone modification spreads beyond the DSB into neighboring chromatin, generating a DDR platform that protects against end disassociation and degradation, minimizing chromosomal rearrangements. However, mechanisms that determine the breadth and intensity of γH2Ax domains remain unclear. Here, we show that chromosomal contacts of a DSB site are the primary determinants for γH2Ax landscapes. DSBs that disrupt a topological border permit extension of γH2Ax domains into both adjacent compartments. In contrast, DSBs near a border produce highly asymmetric DDR platforms, with γH2Ax nearly absent from one broken end. Collectively, our findings lend insights into a basic DNA repair mechanism and how the precise location of a DSB may influence genome integrity.
2020-05-13 | GSE150384 | GEO
Project description:DNA double-strand breaks (DSBs) initiate meiotic recombination. Past DSB-mapping studies have used rad50S or sae2? mutants, which are defective in break processing, to accumulate DSBs, and report large (= 50 kb) “DSB-hot” regions that are separated by “DSB-cold” domains of similar size. Substantial recombination occurs in some DSB-cold regions, suggesting that DSB patterns are not normal in rad50S or sae2? mutants. We therefore developed novel methods that detect DSBs using ssDNA enrichment and microarray hybridization, and that use background-based normalization to allow cross-comparison between array datasets, to map genome-wide the DSBs that accumulate in processing-capable, repair-defective dmc1î and dmc1î rad51î mutants. DSBs were observed at known hotspots, but also in most previously-identified “DSB-cold” regions, including near centromeres and telomeres. While about 40% of the genome is DSB-cold in rad50S mutants, analysis of meiotic ssDNA from dmc1? shows that most of these regions have significant DSB activity. Thus, DSBs are distributed much more uniformly than was previously believed. Southern-blot assays of DSBs in selected regions in dmc1?, rad50S and wild-type cells confirm these findings. Comparisons of DSB signals in dmc1, dmc1 rad51, and dmc1 spo11 mutant strains identify Dmc1 as the primary strand transfer activity genome-wide, and Spo11-induced lesions as initiating all meiotic recombination. Keywords: DSB mapping, ChIP-chip, single strand DNA , BND cellulose We use two different strategies to map the genome-wide distribution of meiotic DSBs in the yeast Saccharomyces cerevisiae. The first is a chromatin immunoprecipitation (ChIP) based approach that targets the Spo11p protein, which remains covalently attached to DSB ends in the rad50S mutant background. The second approach involves BND cellulose enrichment of the single strand DNA (ssDNA) recombination intermediate formed by end-resection at DSB sites following Spo11p removal. We use dmc1 and dmc1 rad51 mutants that accumulates meiotic single strand DNA intermediates
2007-12-30 | E-GEOD-8981 | biostudies-arrayexpress
Project description:Translocations, that occur when two DNA Double Strand breaks (DSBs) are abnormally rejoined, represent highly deleterious genome rearrangements favoring cancer apparition and progression. However, the mechanisms that drive their formation are yet poorly deciphered. One prerequisite for translocation is the juxtaposition of two distant DSBs, an event that would be favored if DSB cluster, i.e. are brought together in close spatial proximity within the nucleus. Whether DSB cluster in higher eukaryotes has been subjected to a strong controversy over the past decade, due to conflicting results obtained using microscopy based methods1-9. Here we used for the first time a high throughput chromosome conformation capture assay (Capture Hi-C10) to investigate DSB clustering. We unambiguously found that DSBs do cluster in human nuclei but only when induced in transcriptionally active genes. Clustering of damaged genes mainly occur during the G1 cell cycle phase and coincide with delayed repair. Moreover DSB clustering depends on the MRN complex, as well as the Formin 2 (FMN2) nuclear actin organizer and the LINC (LInker of Nuclear and Cytoplasmic skeleton) complex, suggesting that active mechanisms promote DSB clustering. This work reveals that when damaged, active genes exhibit a very peculiar behavior compared to the rest of the genome, being mostly left unrepaired and clustered in G1 while being repaired by homologous recombination in post-replicative cells.
2017-04-19 | E-MTAB-4846 | biostudies-arrayexpress
Project description:DNA Double-Strand Breaks (DSBs) are highly detrimental since they can lead to mutations and chromosomes rearrangements (amplification, deletion, translocation and chromosome loss). Here, we set to assess the tridimensional genome organization around DSBs and its role in DSB repair foci formation. We performed Hi-C experiments before and after DSB induction and upon ctrl or SCC1 depletion, 4C-seq experiments before and after DSB induction and upon cohesin (SCC1) depletion or ATM inhibition. We also performed ChIP-seq of pATM(S1981), CTCF, P-SMC3(S1083), MDC1 and a calibrated ChIP-seq of SCC1 with or without damage. ChIP-chip of SMC3 (S1083) and SMC1 (S966) were used to show the recruitment of these marks on DNA repair foci. ChIP-chip of gammaH2AX was realized upon SCC1 depletion and ChIP-seq of gammaH2AX was realized upon SCC1 or WAPL to show the role of the cohesin complex in gammaH2AX foci formation. ChIP-seq of gammaH2AX was realized upon ATM or ATR inhibition to show that ATM is the major kinase that phosphorylates H2AX at clean breaks in human cells.
2021-01-29 | E-MTAB-8851 | biostudies-arrayexpress
Project description:The nonrandom distribution of meiotic recombination shapes patterns of inheritance and genome evolution, but chromosomal features governing this distribution are poorly understood. Formation of the DNA double-strand breaks (DSBs) that initiate recombination results in accumulation of Spo11 protein covalently bound to small DNA fragments. We show here that sequencing these fragments provides a genome-wide DSB map of unprecedented resolution and sensitivity. We use this map to explore the influence of large-scale chromosome structures, chromatin, transcription factors, and local sequence composition on DSB distributions. Our analysis supports the view that the recombination terrain is molded by combinatorial and hierarchical interaction of factors that work on widely different size scales. Mechanistic aspects of DSB formation and early processing steps are also uncovered. This map illuminates the occurrence of DSBs in repetitive DNA elements, repair of which can lead to chromosomal rearrangements. We discuss implications for evolutionary dynamics of recombination hotspots. We deep sequenced 4 samples of Spo11 oligos on Roche 454 platform. Three samples are technical replicates of Spo11 oligo products prepared from one meiotic culture and the fourth sample was prepared from an independent culture.
2011-03-07 | E-GEOD-26449 | biostudies-arrayexpress
Project description:DNA double-strand breaks (DSBs) initiate meiotic recombination. Past DSB-mapping studies have used rad50S or sae2? mutants, which are defective in break processing, to accumulate DSBs, and report large (= 50 kb) “DSB-hot” regions that are separated by “DSB-cold” domains of similar size. Substantial recombination occurs in some DSB-cold regions, suggesting that DSB patterns are not normal in rad50S or sae2? mutants. We therefore developed novel methods that detect DSBs using ssDNA enrichment and microarray hybridization, and that use background-based normalization to allow cross-comparison between array datasets, to map genome-wide the DSBs that accumulate in processing-capable, repair-defective dmc1î and dmc1î rad51î mutants. DSBs were observed at known hotspots, but also in most previously-identified “DSB-cold” regions, including near centromeres and telomeres. While about 40% of the genome is DSB-cold in rad50S mutants, analysis of meiotic ssDNA from dmc1? shows that most of these regions have significant DSB activity. Thus, DSBs are distributed much more uniformly than was previously believed. Southern-blot assays of DSBs in selected regions in dmc1?, rad50S and wild-type cells confirm these findings. Comparisons of DSB signals in dmc1, dmc1 rad51, and dmc1 spo11 mutant strains identify Dmc1 as the primary strand transfer activity genome-wide, and Spo11-induced lesions as initiating all meiotic recombination. Keywords: DSB mapping, ChIP-chip, single strand DNA , BND cellulose
2007-12-31 | GSE8981 | GEO
Project description:Pre-B cell receptor (pre-BCR) signals initiate immunoglobulin light (Igl) chain gene assembly leading to RAG-mediated DNA double-strand breaks (DSBs). These signals also promote cell cycle entry, which could cause aberrant DSB repair and genome instability in pre-B cells. Here we show that RAG DSBs inhibit pre-BCR signals through the ATM- and NF-κB2-dependent induction of SPIC, a hematopoietic-specific transcriptional repressor. SPIC inhibits expression of the SYK tyrosine kinase and BLNK adaptor to prevent the pre-BCR from inducing additional Igl chain gene rearrangements and driving pre-B cells with RAG DSBs into cycle. We propose that pre-B cells toggle between pre-BCR signals and this RAG DSB-dependent checkpoint to maintain genome stability while iteratively assembling Igl chain genes. Three independent IL-7 cultures for each genotype (Rag1-/-:μIgH:Bcl2, Art-/-:μIgH:Bcl2 and Art-/-:Nfkb2-/-:μIgH:Bcl2) were withdrawn from IL-7 for 2 days. RNA was isolated using RNeasy (Qiagen). Gene expression profiling was performed using Illumina MouseRef-8 expression microarrays.
2016-01-13 | E-GEOD-67854 | biostudies-arrayexpress