Project description:sBLISS (in-suspension Break Labeling In Situ and Sequencing) is a versatile and widely applicable method for identification of endogenous and induced DNA double-strand breaks (DSBs), in any cell type that can be brought into suspension. After in situ labeling, DSB ends are linearly amplified followed by next-generation sequencing and DSB landscape analysis. Here, we present a step-by-step experimental protocol for sBLISS, followed by a basic computational analysis. The main advantages of sBLISS are (i) the suspension setup, which circumvents the need to work with delicate coverslips as in the original BLISS, (ii) the possibility for adaptation to a high-throughput robotics or single-cell workflow, and (iii) its flexibility and applicability to virtually every cell type, including patient-derived cells, organoids, and isolated nuclei. The wet-lab protocol can be completed in 1.5 weeks, and it is suitable for researchers with intermediate expertise in molecular biology and genomics. For the computational analyses, basic-to-intermediate bioinformatics expertise is required.
Project description:The number and position of breaks using BLISS-seq was measured in MCF-7 cells upon irradiation with 10 Gy and with or without the use of the antioxidant (N-acetylcysteine, NAC, 10mM).
Project description:Transcriptional activation leads to transient accumulation of DNA Double Strand Breaks (DSBs), which might promote formation of chromosomal translocations. We report here the genomic distribution of DSBs in non-transformed mammary cells grown under unperturbed conditions, using genome-wide Breaks Labeling In Situ and Sequencing (BLISS). We found thousands of high-confidence DSBs, which were enriched at the promoters of a subset of moderately- to highly-transcribed genes (fragile promoters) and co-localized with Topoisomerase II beta. Analyses of DSB-predictive features identified gene length and paused RNA Polymerase II, but not transcription, as critical factors (84% prediction accuracy). Analyses of DSB signaling and repair factors showed high levels of XRCC4, but not of gammaH2AX and NBS1, and no RAD51. Finally, the observed DSBs were predictive of a significant fraction of the recurrent translocations found in human breast cancers. These data suggest that, in normal cells, basal transcription from a specific class of promoters entails accumulation of TOP2B, leading to unresolved DSBs and increased probability of chromosomal translocations.
Project description:Transcriptional activation leads to transient accumulation of DNA Double Strand Breaks (DSBs), which might promote formation of chromosomal translocations. We report here the genomic distribution of DSBs in non-transformed mammary cells grown under unperturbed conditions, using genome-wide Breaks Labeling In Situ and Sequencing (BLISS). We found thousands of high-confidence DSBs, which were enriched at the promoters of a subset of moderately- to highly-transcribed genes (fragile promoters) and co-localized with Topoisomerase II beta. Analyses of DSB-predictive features identified gene length and paused RNA Polymerase II, but not transcription, as critical factors (84% prediction accuracy). Analyses of DSB signaling and repair factors showed high levels of XRCC4, but not of gammaH2AX and NBS1, and no RAD51. Finally, the observed DSBs were predictive of a significant fraction of the recurrent translocations found in human breast cancers. These data suggest that, in normal cells, basal transcription from a specific class of promoters entails accumulation of TOP2B, leading to unresolved DSBs and increased probability of chromosomal translocations.
Project description:Doxorubicin is a widely used chemotherapeutic drug that intercalates between DNA base-pairs and posions Topoisomerase II, although the mechanistic basis for cell killing remains speculative. Here we show that both anthracyclines and Topoisomerase II poison cause enhanced DNA double-strand breaks around CpG island promoters of active genes genome-wide. We propose that torsion-based enhancement of nucleosome turnover exposes promoter DNA, ultimately causing DNA breaks around promoters that contributes to cell killing. We have analyzed mouse squamous cell carcinoma cells treated with doxorubicin, aclarubicin and etoposide. The direct in situ Breaks Labeling, Enrichment on Streptavidin (BLESS, PMID 23503052) method was used for mapping DNA double-strand breaks genome-wide.
Project description:The depletion of HMGB1 reduces the deposition of histones on naked DNA (Celona et al, 2011). in primary MEFs and yeast. We verified that the downmodulation of HMGB1 decreases histone level also in a model of murine mesothelioma. HMGB1 was KD in Murine Mesothelioma AB22 cells by the expression of a shRNA. Nucleosome positioning and occupancy were tested by ATAC-seq. Since a reduced amount of nucleosomes increase the cell sensitivity to gamma irradiatio, we tested the number and position of breaks using BLISS-seq.
Project description:The depletion of HMGB1 reduces the deposition of histones on naked DNA (Celona et al, 2011). in primary MEFs and yeast. We verified that the downmodulation of HMGB1 decreases histone level also in a model of murine mesothelioma. HMGB1 was KD in Murine Mesothelioma AB22 cells by the expression of a shRNA. Nucleosome positioning and occupancy were tested by ATAC-seq. Since a reduced amount of nucleosomes increase the cell sensitivity to gamma irradiatio, we tested the number and position of breaks using BLISS-seq.
