Project description:C8orf33-proficient and deficient DIvA cells were treated with 4-hydroxy tamoxifen (4OHT) to induce DNA double strand breaks (DSB) at several loci within the human genome. following 4OHT treatment cells were subject to ChIP-seq analysis for KAT8 acetyltransferase to map its enrichment at DSB sites in C8orf33 proficient deficient cells.
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.
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.
Project description:DNA Double Strand Breaks (DSBs) repair is essential to safeguard genome integrity but little is known about the contribution of chromosome folding into these processes. Here we unveiled basic principles of chromosome dynamics occurring post-DSB both locally and at a genome wide scale in mammalian cells. We report that topologically associating domains (TAD) that experience a DSB undergo acute ATM-dependent but DNAPK- independent changes. Within these damaged TADs, DSB-induced loop extrusion ensures local transcriptional regulation in response to DSBs. Damaged TADs further coalesce in an ATM-dependent manner, forming a new “D” compartment, where upregulated genes of the DNA damage response (DDR) physically localize suggesting a function of DSB clustering in activating the DNA damage Response. However, these alterations of chromosome folding induced by DSB also come at the expense of an increased translocations rate. Our work highlights the critical impact of chromosome conformation in the maintenance of genome integrity.
Project description:RNA:DNA hybrids accumulate at the vicinity of DNA double-strand breaks (DSBs) and were shown to regulate homologous recombination repair. The mechanism responsible for the formation of these non-canonical RNA:DNA structures remains unclear although they were proposed to arise consequently to RNA Polymerase II or III loading followed by DSB-induced de novo transcription at the break site. Here, we found no evidence of RNA polymerases recruitment at DSBs. Rather, strand-specific R-loop mapping revealed that RNA:DNA hybrids are mainly generated at DSBs occurring in transcribing loci, from the hybridization of pre-existing RNA to the 3’ overhang left by DNA end resection. We further identified the H3K4me3 reader Spindlin 1 and the transcriptional regulator PAF1 as promoting RNA:DNA hybrid accumulation at DSBs, through their role in mediating transcriptional repression in cis to DSBs. Altogether, we provide evidence that RNA:DNA hybrids accumulate at DSBs occurring in transcribing loci as a result of DSB-induced transcriptional shut-down.
Project description:The aim was to compare the levels of histone H3 dimethylation at lysine 9 (H3K9me2) in wild-type and dbl2 knock-out Schizosaccharomyces pombe cells. Fission yeast cultures were grown in the complex YES medium to exponential phase and chromatin immunoprecipitation was carried out using anti-H3 and anti-H3K9me2 antibodies. Two independent biological replicates were performed. The resulting IP and input DNA samples were sequenced using Illumina sequencing (35 nt PE). H3K9me2 occupancy in each sample was normalized to the corresponding total H3 occupancy, and normalized H3K9me2 levels were compared between WT and dbl2 knock-out.
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.
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). ChIP-chip of P-SMC3 (S1083) and P-SMC1 (S966) were used to show the recruitment of these marks on DNA repair foci. ChIP-chip of gammaH2AX upon CRISPR induction at different positions within a single TAD was also performed.
Project description:Repair of DNA Double Strand Breaks produced in transcriptionally active chromatin occurs through a mechanism, Transcription-Coupled DSB repair (TC-DSBR), that is yet poorly characterized. Here, using a screening approach scoring multiple outputs in human cells, we identified the PER complex, a key module ensuring circadian oscillations, as a novel TC-DSBR player, being enriched at DSB occurring in transcribed loci, as compared to DSB induced in un-transcribed loci. We further found that PER2 contributes to target TC-DSBs at the nuclear envelope (NE) and to foster Rad51- mediated repair. PER2 deficiency triggers decreased DSB anchoring to NE, resulting in an increase of DSB clustering, checkpoint activation and translocation frequency. In agreement, we found that the circadian clock also regulates DSB anchoring to the NE, checkpoint activation, and HR usage. Our study provides a direct link between the circadian clock and the response to DNA Damage, opening new therapeutic strategies for chemotherapies based on topoisomerase poisons that induce DSBs in active loci.
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.