BRCA1 mutational complementation induces synthetic viability
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE147468 | GEO | 2020/03/24
REPOSITORIES: GEO
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Project description:BRCA1 promotes the DNA end resection and RAD51 loading steps of homologous recombination (HR). Whether these functions can be uncoupled, and whether mutant proteins retaining partial activity can complement one another, is unclear and could affect the severity of BRCA1-associated Fanconi anemia (FA). Here we generated a Brca1CC mouse with a coiled-coil (CC) domain deletion. Brca1CC/CC mice are born at low frequencies, and post-natal mice have FA-like abnormalities, including bone marrow failure. Intercrossing with Brca1Δ11, which is homozygous lethal, generated Brca1CC/Δ11 mice at Mendelian frequencies that were indistinguishable from Brca1+/+ mice. Brca1CC and Brca1Δ11 proteins were individually responsible for counteracting 53BP1-RIF1-Shieldin activity and promoting RAD51 loading, respectively. Thus, Brca1CC and Brca1Δ11 alleles represent separation-of-function mutations that combine to provide a level of HR sufficient for normal development and hematopoiesis. Because BRCA1 activities can be genetically separated, compound heterozygosity for functional complementary mutations may protect individuals from FA.
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Project description:BRCA1 mutational complementation induces synthetic viability
| PRJNA614940 | ENA
Project description:We report DNA resection length after induced double stranded breaks in specific genomic loci with the expression of the AsisI restriction enzyme
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Project description:To test whether MiDAC-promoted H2AK5/K9 deacetylation is implicated in DSB response, we took advantage of an improved DSB-induced via AsiSI (AiDIvA) system, in which the endonuclease restriction enzyme AsiSI is fused to the estrogen receptor (ER) hormone-binding domain and its nuclear expression in HeLa cells is controlled by doxycycline and 4-hydroxy tamoxifen (4-OHT). The precise distribution of gH2AX, H2AK5ac, H2AK9ac, FLAG-DNTTIP1, FLAG-MIDEAS, and LIG4 at AsiSI-induced DNA double-strand breaks was determined by chromatin immunoprecipitation coupled high throughput sequencing (ChIP-seq). ChIP signals from ER-AsiSI-expressing AiDIvA cells (DSB group) were normalized to ER-expressing control cells (Ctrl group).
2024-02-16 | GSE235845 | GEO
Project description:Control and CDYL1-depleted U2OS-DIvA cells were treated with 4-hydroxy tamoxifen (4OHT) to induce multiple DNA double-strand breaks (DSBs) at defined loci in the genome via AsiSI restriction enzyme. This system was used to map the changes in lysine crotonylation (Kcr) and ENL at DSB sites in control and CDYL1-deficient cells.
2022-01-22 | E-MTAB-10550 | biostudies-arrayexpress
Project description:We have investigated whether components of Polycomb repressive complex 1 (PRC1) are recruited to double-strand breaks (DSBs) generated by inducible expression of the AsiSI restriction enzyme in normal human fibroblasts. Using chromatin immunoprecipitation and deep sequencing (ChIP-seq), which detects PRC1 proteins at common sites throughout the genome, we did not find evidence for recruitment of PRC1 components to AsiSI-induced DSBs. In contrast, the S2056 phosphorylated form of DNA-PKcs and other DNA repair proteins were detected at a subset of AsiSI sites that are predominantly at the 5’ ends of transcriptionally active genes. Our data question the idea that PcG protein recruitment provides a link between DSB repairs and transcriptional repression. Single chromatin preparations from treated and untreated cells (+/- OHT), immunoprecipitated with two antibodies (pDNA-PKcs and MEL18) were sequenced as technical replicates, along with the corresponding input DNAs
2014-07-06 | E-GEOD-55605 | biostudies-arrayexpress
Project description:We have investigated whether components of Polycomb repressive complex 1 (PRC1) are recruited to double-strand breaks (DSBs) generated by inducible expression of the AsiSI restriction enzyme in normal human fibroblasts. Using chromatin immunoprecipitation and deep sequencing (ChIP-seq), which detects PRC1 proteins at common sites throughout the genome, we did not find evidence for recruitment of PRC1 components to AsiSI-induced DSBs. In contrast, the S2056 phosphorylated form of DNA-PKcs and other DNA repair proteins were detected at a subset of AsiSI sites that are predominantly at the 5’ ends of transcriptionally active genes. Our data question the idea that PcG protein recruitment provides a link between DSB repairs and transcriptional repression.
2014-07-06 | GSE55605 | GEO
Project description:Determining the role of DDX17 in the formation of DNA:RNA-hybrids around active DNA double-strand breaks (DSBs) using DRIP-seq in the damaged induced via AsiSI (DIvA) cell system that induced DSBs at known genomic loci in response to hydroxytamoxifen (OHT) treatment via and AsiSI enzyme fused to an oestrogen receptor. Sequencing was done using either control or DDX17 siRNA, and mock or 4 hours 300nM OHT treatment. Paired-end 150 cycles was completed on an Illumina NextSeq 500 and library prep was completed using the NEB NEBNext Ultra II library prep kit.
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Project description:Cells have developed effective mechanisms, namely homologous recombination (HR) and non-homologous end-joining (NHEJ), to repair DNA double-strand breaks (DSBs), which are considered to be the most deleterious type of damage that can challenge genome integrity. While these pathways coexist to repair DSBs, the mechanisms by which one of these pathways is chosen to repair a particular DSB remain unclear. Here, we show that the chromatin context in which a break occurs participates in this choice and that transcriptionnaly active chromatin channels repair to HR. By using a human cell line expressing a restriction enzyme fused to the ligand binding domain of the oestrogen receptor (AsiSI-ER)2,3, together with a genome wide chromatin immunoprecipitation-sequencing (ChIP-seq) approach, we establish that distinct DSBs induced across the genome are not necessarily repaired by the same pathway. Indeed, 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 repair at such DSBs can be switched to RAD51-independent repair pathway upon transcriptional inhibition. Moreover, we show that HR is targeted to transcribed loci thanks to the elongation-associated H3K36me3 histone mark. Indeed depletion of HYPB, the main H3K36 tri methyltransferase severally impedes the use of HR at those DSBs. Our study, thereby demonstrates a clear role for chromatin in DSB repair pathway choice in human cells.
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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.
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