Three-dimensional proximity mapping of the DRGFP DNA double-strand break site in U2OS cells
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE58563 | GEO | 2014/08/22
SECONDARY ACCESSION(S): PRJNA252903
REPOSITORIES: GEO
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Project description:We mapped the DNA sequences located in physical proximity to an I-SceI endonuclease-dependent DNA double-strand break (DSB) site to be able to study the impact of DSBs on the break-associated chromatin micro-environment. The I-SceI DSB site is part of a DRGFP transgene that serves as a reporter for homology-directed DSB repair and was stably integrated into U2OS human osteosarcoma cells (Pierce et al., Genes Dev, 1999). DRGFP-U2OS cells were modified to carry a stable Doxycycline (Dox)-inducible I-SceI transgene (TRE-I-SceI) as well as the Tet-ON vector from Contech. Circular chromosome conformation capture (4C) was used to identify DSB-proximal DNA. 4C amplified DNA was subjected to Illumina Hi-Seq 200 100 bp paired end sequencing and reads were mapped to the human genome. The resulting genome-wide map of I-SceI/DRGFP-proximal DNA allows for the investigation of changes in DSB-surrounding chromatin features following I-SceI-mediated DSB induction. 4C library of I-SceI (DRGFP)-proximal DNA contacts in DRGFP-U2OS cells was generated using Illumina Hi-Seq 2000 sequencing, data sets are from two independent experiments.
2014-08-22 | E-GEOD-58563 | biostudies-arrayexpress
Project description:Antibody class switch recombination (CSR) in B lymphocytes joins two DNA double-strand breaks (DSBs) lying 100 to 200 kilobases (kb) apart within switch (S) regions in the immunoglobulin heavy chain locus (IgH). CSR-activated B lymphocytes generate multiple S-region DSBs in the donor Sm and in a downstream acceptor S region, with a DSB in Sm being joined to a DSB in the acceptor S region at sufficient frequency to drive CSR in a large fraction of activated B cells. Such frequent joining of widely separated CSR DSBs could be promoted by IgH-specific or B cell-specific processes or by general aspects of chromosome architecture and DSB repair. Previously, we found that B cells with two yeast I-SceI endonuclease targets in place of Sg1 undergo I-SceI-dependent class switching from IgM to IgG1 at 5-10% of normal levels. Now, we report that B cells in which Sg1 is replaced with a 28 I-SceI target array, designed to increase I-SceI DSB frequency, undergo I-SceI-dependent class switching at almost normal levels. High-throughput genome-wide translocation sequencing revealed that I-SceI-generated DSBs introduced in cis at Sm and Sg1 sites are joined together in T cells at levels similar to those of B cells. Such high joining levels also occurred between I-SceI-generated DSBs within c-myc and I-SceI- or CRISPR/Cas9-generated DSBs 100 kb downstream within Pvt1 in B cells or fibroblasts, respectively. We suggest that CSR exploits a general propensity of intra-chromosomal DSBs separated by several hundred kb to be frequently joined together and discuss relevance of this finding for recurrent interstitial deletions in cancer. Comparison of frequency of long-range joining between I-SceI-induced DSBs at IgH and c-myc loci in different cell types by HTGTS
2014-01-03 | E-GEOD-53755 | biostudies-arrayexpress
Project description:Antibody class switch recombination (CSR) in B lymphocytes joins two DNA double-strand breaks (DSBs) lying 100 to 200 kilobases (kb) apart within switch (S) regions in the immunoglobulin heavy chain locus (IgH). CSR-activated B lymphocytes generate multiple S-region DSBs in the donor Sm and in a downstream acceptor S region, with a DSB in Sm being joined to a DSB in the acceptor S region at sufficient frequency to drive CSR in a large fraction of activated B cells. Such frequent joining of widely separated CSR DSBs could be promoted by IgH-specific or B cell-specific processes or by general aspects of chromosome architecture and DSB repair. Previously, we found that B cells with two yeast I-SceI endonuclease targets in place of Sg1 undergo I-SceI-dependent class switching from IgM to IgG1 at 5-10% of normal levels. Now, we report that B cells in which Sg1 is replaced with a 28 I-SceI target array, designed to increase I-SceI DSB frequency, undergo I-SceI-dependent class switching at almost normal levels. High-throughput genome-wide translocation sequencing revealed that I-SceI-generated DSBs introduced in cis at Sm and Sg1 sites are joined together in T cells at levels similar to those of B cells. Such high joining levels also occurred between I-SceI-generated DSBs within c-myc and I-SceI- or CRISPR/Cas9-generated DSBs 100 kb downstream within Pvt1 in B cells or fibroblasts, respectively. We suggest that CSR exploits a general propensity of intra-chromosomal DSBs separated by several hundred kb to be frequently joined together and discuss relevance of this finding for recurrent interstitial deletions in cancer.
2014-01-03 | GSE53755 | GEO
Project description:Genome modification in mouse embryonic stem cells (mESCs) has provided the versatile platform to analyzing gene functions in mammalian cells. Bloom helicase-deficient mESCs showed elevated rate of loss of heterozygosity (LOH) through mitotic recombination. By utilizing this characteristic of Blm deficiency, we established an efficient method to introduce homozygous mutations into mESCs, which can be used for phenotype-driven recessive screen. It is generally thought that mitotic recombination is initiated by double-strand break. DNA repair machinery, however, possesses mechanisms that suppress deleterious crossovers between homologous chromosomes. Bloom helicase plays a central role to suppress such crossover; hence its deficiency causes elevated frequency of mitotic recombination. To directly answer if DSB can initiate mitotic recombination, we established a reporter cell line, in which the recognition site of I-SceI endonuclease was introduced in the Aprt gene in Blm-deficient mESCs. We used two types of I-SceI expression vectors; one expresses a mammalian codon-optimized I-SceI (ISceIo) and the other expresses I-SceIo fused with N-terminal 110-amino acids of Geminin (Gemi). Geminin-fused I-SceI can be stabilized only in lateS/G2 phase. After I-SceI transfection and subsequent selection, we isolated Aprt-deficient mutants. Half of the mutants showed LOH in the whole telomeric region from I-SceI site, but the other half showed local LOH near the I-SceI site. Further analyses suggested that large deletion was introduced and caused LOH, which was not seen in spontaneous LOH. We designed CGH probes in 300-bp resolution in 8Mb telomeric region of chr 8 (The Aprt gene locates 7Mb from the telomere) to map the deleted regions in these mutants.
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Project description:Rif1 is involved in telomere homeostasis, DNA replication timing, and DNA double-strand break (DSB) repair pathway choice from yeast to human. The molecular mechanisms that enable Rif1 to fulfill its diverse roles remain to be determined. Here, we demonstrate that Rif1 is S-acylated within its conserved N-terminal domain at cysteine residues C466 and C473 by the DHHC family palmitoyl acyltransferase Pfa4. Rif1 S-acylation facilitates the accumulation of Rif1 at DSBs, the attenuation of DNA end-resection, and DSB repair by non-homologous end-joining (NHEJ). These findings identify S-acylation as a posttranslational modification regulating DNA repair. S-acylated Rif1 mounts a localized DNA-damage response proximal to the inner nuclear membrane, revealing a mechanism of compartmentalized DSB repair pathway choice by sequestration of a fatty acylated repair factor at the inner nuclear membrane.
2019-04-15 | PXD012137 | Pride
Project description:During Class Switch Recombination (CSR) B cells replace the Igh Cμ/δ exons with another downstream constant region exon (CH), altering the antibody isotype. CSR occurs through the introduction of AID-mediated double strand break (DSBs) in switch regions and subsequent ligation of broken ends. Here we developed an assay to investigate the dynamics of DSB introduction in individual cells. We demonstrate that the upstream switch region Sμ is always targeted first during recombination and that the mechanism underlying this control relies on 53BP1. Surprisingly, regulation of break order occurs through residual binding of 53BP1 to chromatin before the introduction of damage and independent of its established role in DNA repair. Using chromosome conformation capture we show that 53BP1 mediates changes in chromatin architecture that affect break order. Finally, our results explain how changes in Igh architecture in the absence of 53BP1 could promote inversional rearrangements that compromise CSR. High Resolution 4C was performed in resting and activated B cells using a bait on the Eμ enhancer
2016-06-16 | E-GEOD-81180 | biostudies-arrayexpress
Project description:Recent observations show that the single-cell response of p53 to ionizing radiation (IR) is “digital” in that it is the number of oscillations rather than the amplitude of p53 that shows dependence on the radiation dose. We present a model of this phenomenon. In our model, double-strand break (DSB) sites induced by IR interact with a limiting pool of DNA repair proteins, forming DSB–protein complexes at DNA damage foci. The persisting complexes are sensed by ataxia telangiectasia mutated (ATM), a protein kinase that activates p53 once it is phosphorylated by DNA damage. The ATM-sensing module switches on or off the downstream p53 oscillator, consisting of a feedback loop formed by p53 and its negative regulator, Mdm2. In agreement with experiments, our simulations show that by assuming stochasticity in the initial number of DSBs and the DNA repair process, p53 and Mdm2 exhibit a coordinated oscillatory dynamics upon IR stimulation in single cells, with a stochastic number of oscillations whose mean increases with IR dose. The damped oscillations previously observed in cell populations can be explained as the aggregate behavior of single cell
2020-05-13 | MODEL2005130001 | BioModels
Project description:Investigating the induction of the SOS response after the introduction of a single dsDNA break per genome by expressing the SceI endonuclease. The endonuclease is under the control of a xylose-inducible promoter.
2008-11-21 | GSE13545 | GEO
Project description:In bacteria, double-strand break (DSB) repair via homologous recombination is thought to be initiated through the bi-directional degradation and resection of DNA ends by a helicase-nuclease complex such as AddAB. The activity of AddAB has been well-studied in vitro, with translocation speeds between 400-2000 bp/s on linear DNA suggesting that a large section of DNA around a break site is processed for repair. However, the translocation rate and activity of AddAB in vivo is not known, and how AddAB is regulated to prevent excessive DNA degradation around a break site is unclear. To examine the functions and mechanistic regulation of AddAB inside bacterial cells, we developed a next-generation sequencing-based approach to assay DNA processing after a site-specific DSB was introduced on the chromosome of Caulobacter crescentus. Using this assay we determined the in vivo rates of DSB processing by AddAB and found that putative chi sites attenuate processing in a RecA-dependent manner. This RecA-mediated regulation of AddAB prevents the excessive loss of DNA around a break site, limiting the effects of DSB processing on transcription. In sum, our results, taken together with prior studies, support a mechanism for regulating AddAB that couples two key events of DSB repair-the attenuation of DNA-end processing and the initiation of homology search by RecA-thereby helping to ensure that genomic integrity is maintained during DSB repair.
2017-05-16 | GSE86913 | GEO
Project description:Meiotic recombination is initiated by developmentally programmed DNA double-strand breaks (DSBs). In S. cerevisiae, the vast majority of DSBs occur in the nucleosome-depleted regions at gene promoters, where transcription factors (TFs) bind. It has been proposed that TF binding can stimulate DSB formation nearby by modulating local chromatin structure. However, a prior study in TF bas1 mutant suggested that the role of TF binding in determining break formation is complex. Here, we examined fine-scale DSB distributions in TF mutant (bas1Δ and ino4Δ) strains. In bas1Δ mutants, 239 out of the 2468 hotspots showed reduced DSB activity, whereas 87 hotspots showed increased DSB activity. Similarly, in ino4Δ mutant, 415 out of the 2468 hotspots showed reduced DSB activity, whereas 322 hotspots showed increased DSB activity. We also mapped Bas1 and Ino4 binding sites in meiosis and found that only a small portion of the affected hotspots contained TF binding sites. This indicates that TF can influence DSB distribution both directly and indirectly. Surprisingly, these DSB changes in TF mutants did not correlate with change in chromatin structure and histone H3K4me3 modification, suggesting that the role of TF on DSB distribution cannot be simply explained by affecting local chromatin status. Nine samples total: two wild type, four bas1 mutant and three ino4 mutant (each an independent culture)
2015-08-21 | E-GEOD-67910 | biostudies-arrayexpress