ARID1A recruits non-homologous end joining factors to DNA breaks induced by G4 ligands
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PROVIDER: PRJNA1255162 | ENA |
REPOSITORIES: ENA
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Project description:ARID1A, a subunit of the BAF chromatin remodeler, is frequently mutated in cancer. Predicting how ARID1A loss impacts cancer therapy response is challenging because it influences many cellular pathways. G quadruplex (G4) binding ligands, such as pyridostatin, show anticancer effects, but the genetic determinants influencing the response to G4 ligands are not fully understood. Here, we show that ARID1A-deficient cells are selectively sensitive to pyridostatin compared to isogenic controls. This was apparent in ovarian and colorectal cancer cell line models, and in vivo studies suggest that G4 ligands hold promise for ARID1A-deficient cancers. While ARID1A modulates pyridostatin-induced transcriptional responses, we show that toxicity in ARID1A-deficient cells arises from the defective repair of topoisomerase-induced breaks. Notably, these cells fail to efficiently accumulate non-homologous end joining proteins on chromatin following pyridostatin exposure. These data uncover a role for ARID1A in the cellular response to G4 ligands, linking remodeling to G4 ligand-induced responses.
2025-08-13 | GSE295547 | GEO
Project description:ARID1A is a subunit of the BAF chromatin remodelling complex that is frequently mutated in cancer. However, it is challenging to predict how ARID1A loss might impact on responses to cancer therapies because it participates in many different cellular pathways. G quadruplex (G4) binding ligands, such as pyridostatin, have shown anticancer effects, but the pathways and genetic determinants involved in the response to G4 ligands are still not fully understood. Here, we show that ARID1A deficient cells are selectively sensitive to G4 binding ligands when compared with isogenic controls. Sensitivity to G4 ligands was apparent in both ovarian clear cell and colorectal cancer cell line models, and in vivo studies suggest that G4 ligands might hold promise for treating ARID1A deficient cancers. While we find that ARID1A loss impacts on the transcriptional response to pyridostatin, we find that the influence of ARID1A on pyridostatin-mediated toxicity is driven by the DNA repair response to topoisomerase activity. Specifically, we find that ARID1A deficient cells are unable to efficiently accumulate nonhomologous end joining proteins onto chromatin following exposure to pyridostatin, providing mechanistic insights into their impaired survival. These data uncover a role for ARID1A in the cellular response to G4 ligands, and link chromatin remodelling to G4 ligand-induced DNA damage responses and genome instability.
2025-08-15 | PXD063556 | Pride
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:The reliability of the BG4-IP-seq protocol, along with the straightforward access to rice genomic DNA, makes this system ideal for comparing the pull-down efficiency of the antibody BG4 versus the small-molecular baits BioTASQ and BioCyTASQ. These two TASQs (for template-assembled synthetic G-quartets) are biotinylated biomimetic ligands that were successfully employed for fishing G4-RNAs out from human cell lysates, in a protocol referred to as G4RP-seq, which belong to the toolbox of seq-based techniques used to interrogate G4-RNA prevalence, comprising rG4-seq, DMS-seq, Keth-seq and SHALiPE-seq. We reasoned that the ability of TASQs to isolate G4-DNA deserves both to be investigated and to be compared to that of BG4. We thus report here on these investigations. which provides the very first comparison of the efficiency of the immuno- versus chemo- precipitation of G4s, under strictly identical experimental conditions.
2023-08-10 | GSE200171 | GEO
Project description:G-quadruplexes (G4s) and R-loops are non-canonical DNA structures that can play regulatory functions of basic nuclear processes and can trigger DNA damage and genome instability. We here show that specific G4 ligands can stabilize G4s and simultaneously increase R-loop levels in human cancer cells likely by spreading DNA:RNA heteroduplexes to adjacent regions containing G4 structures. DNA cleavage and DNA damage response induced by G4 ligands were rescued by overexpression of exogenous RNaseH1 in cancer cells independently of BRCA2 status. The data thus show that R-loops are involved in the induction of DNA damage by chemical G4 stabilization. In addition, G4 ligands trigger the formation of micronuclei, particularly in BRCA2-silenced cancer cells, in an R-loop dependent manner. Our results uncover the mechanism of genome instability caused by G4 ligands and can open to the development of unexpected anticancer strategies using G4-targeted agents.
2018-12-27 | GSE115957 | GEO
Project description:During B cell development, RAG endonuclease cleaves immunoglobulin heavy chain (IgH) V, D, and J gene segments and orchestrates their fusion as deletional events that assemble a V(D)J exon in the same transcriptional orientation as adjacent Cμ constant region exons. In mice, six additional sets of constant region exons (CHs) lie 100-200kb downstream in the same transcriptional orientation as V(D)J and Cμ exons. Long repetitive switch (S) regions precede Cμ and downstream CHs. In mature B cells, class switch recombination (CSR) generates different antibody classes by replacing Cμ with a downstream CH. Activation Induced Cytidine Deaminase (AID) initiates CSR by promoting deamination lesions within Sμ and a downstream acceptor S region; these lesions are converted into DNA double strand breaks (DSBs) by general DNA repair factors. Productive CSR must occur in a deletional orientation by joining the upstream end of an Sμ DSB to the downstream end of an acceptor S region DSB. However, the relative frequency of deletional to inversional CSR junctions had not been measured. Thus, whether orientation-specific joining is a programmed mechanistic feature of CSR as it is for V(D)J recombination and, if so, how this is achieved was unknown. To address this question, we adapted high throughput genome wide translocation sequencing (HTGTS) into a highly sensitive DSB end-joining assay and applied it to endogenous AID-initiated S region DSBs. We find that CSR indeed is programmed to occur in a productive deletional orientation and does so via an unprecedented mechanism that involves in cis IgH organizational features in combination with frequent S region DSBs initiated by AID. We further implicate ATM-dependent DSB response (DSBR) factors in enforcing this mechanism and provide a solution to the enigma of why CSR is so reliant on the 53BP1 DSBR factor. We performed high-throughput genome-wide translocation sequencing (HTGTS) with different B cell genotypes that induces either I-SceI or AID-initiated DSBs as bait to study their joining pattern to AID-initiated S region breaks Please note that the 'ΔSγ1_2xI/ΔSµ_2xI-3' raw data files were analyzed twice with different reference assemblies to address specific points, and associated with both 'ΔSγ1_2xI_ΔSµ_2xI-3'.txt and Sγ1_2xI_ΔSµ_2xI-3'_trans-Sm.txt processed data files. As for the reference genome mm9_129_IgHC, it is a custom built generated based on mm9 (Bl6 line based mouse genome) and the partial genome sequence of another mouse line 129sv. It is not currently available in any public database however can be easily obtained either from the authors or self-built using information provided in the associated manuscript.
2015-08-26 | E-GEOD-71005 | biostudies-arrayexpress
Project description:Geobacillus sp. G4 strain:G4 Genome sequencing
| PRJNA1124973 | ENA
Project description:Although both homologous recombination (HR) and nonhomologous end joining can repair DNA double-strand breaks (DSBs), the mechanisms by which one of these pathways is chosen over the other remain unclear. Here we show that transcriptionally active chromatin is preferentially repaired by HR. Using chromatin immunoprecipitation-sequencing (ChIP-seq) to analyze repair of multiple DSBs induced throughout the human genome, we identify an HR-prone subset of DSBs that recruit the HR protein RAD51, undergo resection and rely on RAD51 for efficient repair. These DSBs are located in actively transcribed genes and are targeted to HR repair via the transcription elongation-associated mark trimethylated histone H3 K36. Concordantly, depletion of SETD2, the main H3 K36 trimethyltransferase, severely impedes HR at such DSBs. Our study thereby demonstrates a primary role in DSB repair of the chromatin context in which a break occurs.
| S-EPMC4300393 | biostudies-literature
Project description:Chromosomal rearrangements are essential events in the pathogenesis of both malignant and nonmalignant disorders, yet the factors affecting their formation are incompletely understood. We developed a zinc finger nuclease translocation reporter (ZITR) and screened for factors that modulate rearrangements in human cells. We identified UBC9 and RAD50 as suppressors and 53BP1, DDB1, and PARP3 as promoters of chromosomal rearrangements across human cell types. We focused on poly(ADP)ribose polymerase 3 (PARP3) as it is dispensable for murine viability and has druggable catalytic activity. We found that PARP3 regulated G quadruplex (G4) DNA in response to DNA damage, which suppressed repair by nonhomologous end-joining and homologous recombination. Chemical stabilization of G4 DNA in PARP3-/- cells led to widespread DNA double-strand breaks and synthetic lethality. We propose a model in which PARP3 suppresses G4 DNA and facilitates DNA repair by multiple pathways.
2017-03-01 | GSE94588 | GEO
Project description:The DNA-PK complex is essential for non-homologous end-joining (NHEJ) to repair DNA double-strand breaks (DSB) in a template-independent way. The association of Ku70/80 with the DSB ends facilitates the formation of the DNA-PK holoenzyme. Many questions remain regarding the dynamics that DNA-PK is attached and stabilized at broken DNA ends. Here we revealed that PRRX1, a homeodomain containing protein, mediates chromatin localization and subsequent activation of the DNA-PK. PRRX1 oligomerizes to enable its simultaneous associations with both double-strand DNA and the SAP domain of Ku70, thereby enhancing Ku anchoring at DSBs to stabilize DNA-PK for efficient NHEJ repair. Low expression and pathogenic mutations of PRRX1 are associated with genomic instability and defective NHEJ repair. The peptide interfering PRRX1 oligomerization compromises NHEJ and hampers cell survival on irradiation. These findings deepen our understanding of how the NHEJ machinery is activated and have implications for optimizing cancer therapies.
2025-03-26 | GSE265857 | GEO