biostudies-arrayexpressVincent ROCHERHomo sapienshttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-12712Repair 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.biostudies-arrayexpressLibrary Construction - For ChIP: Sequencing libraries were prepared from IP of PER2, BMAL1, SUN1 and SUN2 samples using the NEBNext® Ultra™ II Library Prep Kit (NEB)for Illumina.Nucleic Acid Extraction - Hi-C experiments were performed in DIvA cells using the Arima Hi-C kit (Arima Genomics) according to the manufacturer’s instructions. 1 million cells were used by condition. Briefly, cells were cross-linked with 2% formaldehyde for 10 min at RT, lysed and chromatin was digested with two different restriction enzymes included in the kit. Ends were filled-in in the presence of biotinylated nucleotides, followed by subsequent ligation. Ligated DNA was sonicated using the Covaris S220 to an average fragment size of 350 bp with the following parameters (Peak incident power: 140; Duty factor: 10%; Cycles per burst: 200; Treatment time: 70s). DNA was then subjected to a double-size selection to retain DNA fragments between 200 and 600 bp using Ampure XP beads (Beckman Coulter). Biotin-ligated DNA was precipitated with streptavidin-coupled magnetic beads (included in the kit).Sample Treatment - For AsiSI-dependent DSB induction, cells were treated with 300 nM OHT (Sigma; H7904) for 4h. siRNA transfection were performed using the 4D Nucleofector X (kit SE cell line) according to the manufacturer's instructions and subsequent treatment(s) were performed 48 h later. siRNA: CTRL: siGENOME Non-targeting Control #2 Dharmacon (D-001206-13), siPER2 Dharmacon (M-012977-01)Sequencing - ChIP-seq samples were sequenced using Illumina NextSeq 500 (single-end, 85-130bp reads) at EMBL Genomics core facilities (Heidelberg, Germany).Nucleic Acid Extraction - For ChIP: Cell lysis was performed by incubation in cell lysis buffer (5 mM Pipes pH 8, 85 mM KCl, 0.5% IGEPAL® CA-630) followed by Dounce homogenization. Nuclei were harvested by centrifugation and incubated in nuclear lysis buffer: (50 mM Tris pH 8.1, 10 mM EDTA, 1% SDS) and sonicated 10 times for 10 s at a power setting of 5 and 50% duty cycle (Branson Sonifier 250). Samples were diluted 10 times in ChIP dilution buffer (16.7 mM Tris pH 8.1, 167 mM NaCl, 1.2 mM EDTA, 1.1% Triton X‐100, 0.01% SDS) and precleared using a mixture of protein A-agarose (Pierce) and protein G-sepharose (Sigma) previously blocked with BSA (Sigma). Precleared samples (200 μg) were incubated overnight at 4°C with indicated antibodies. Immune complexes were recovered by incubation with blocked protein A/protein G beads for 2 h at 4°C on a rotating wheel. Beads were washed once in dialysis buffer (50 mM Tris pH 8, 2 mM EDTA, 0.2% Sarkosyl), five times in wash buffer (100 mM Tris pH 8.8, 500 mM LiCl, 1% IGEPAL® CA-630, 1% NaDoc) and twice with TE (10mM Tris-HCl, pH 8, 1mM EDTA). Beads were resuspended in TE supplemented with 50µg/ml DNase-free RNase A (Abcam) and incubated for 30 minutes at 37°C. SDS (final concentration 0.5%) was added and crosslink was reversed by overnight incubation at 70°C. Following a 2 hours treatment with 200 µg/ml proteinase K (Roche) at 45°C, DNA was purified by phenol/chloroform extraction and recovered by ethanol precipitation in presence of 5 µg glycogen (Invitrogen). DNA pellet was resuspended in water. Prior to next-generation sequencing library preparation, samples from multiples ChIP experiments were pooled and sonicated for 5 cycles (30 seconds on, 30 seconds off, high setting) on a Bioruptor (Diagenode) then concentrated with a vacuum concentrator (Eppendorf). Antibodies: PER2 Proteintech 20359-1-AP (5µg), BMAL1 Abcam ab3350 (2µg), SUN1 Sigma HPA008346 (5µg), SUN2 Abcam ab124916 (5µg).Sample Collection - For Hi-C: 10x106 cells were harvested by trypsin and resuspend in PBS. Formaldehyde was added at a final concentration of 2% and crosslinking was allowed to proceed for 10 min at room temperature. Reaction was stopped by adding Stop solution from Arima Hi-C kit (Arima Genomics) and incubation for 5 minutes at room temperature then incubate for 15 min on ice. Cells were resuspended in PBS and 1x106 cells per aliquot were freeze in liquid nitrogen.Sample Collection - For ChIP: Formaldehyde was added to the culture medium at a final concentration of 1% and crosslinking was allowed to proceed for 15 min at room temperature. Reaction was stopped by adding glycine (0.125M final concentration) and incubation for 5 minutes at room temperature. Cells were washed twice with PBS and harvested by scraping.Growth Protocol - DIvA (AsiSI-ER-U20S) cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with antibiotics, 10% FCS (Invitrogen) with 1 µg/mL puromycin (DIvA cells) at 37°C under a humidified atmosphere with 5% CO2.Sequencing - The final Hi-C libraries were subjected to 100 bp paired-end sequencing on a NextSeq2000 P2 platform at the EMBL Genomics core facility (Heidelberg, Germany).Sequencing - ChIP-seq samples were sequenced using Illumina NextSeq 2000 (single-end, 100bp reads) at EMBL Genomics core facilities (Heidelberg, Germany).Library Construction - Hi-C library was prepared on beads using the NEBNext® Ultra™ II DNA Library Prep Kit for Illumina and NEBNext® Multiplex Oligos for Illumina (New England Biolabs) following instructions from the Arima Hi-C kit.OrganizationMINSEQE ScoreAssays and DataProcessed DataMAGE-TAB FilesSequence Alignment - ChIP-seq protocol. FastQ files was aligned using bwa aln and bwa samse (bwa0.7.12-r1039) on their reference genome (hg19) with default settings. SAM files was then converted into BAM with samtools view v1.9 (filtering low quality reads <25), sorted with samtools sort(default settings) and duplicates was removed with samtools rmdups(default settings).Sequence Alignment - Firstly, restriction enzyme position was extracted using a python script(https://github.com/aidenlab/juicer/blob/master/misc/generate_site_positions.py)for DpnII on hg19. Juicer pipeline (juicer.sh) was then used on Hi-Cfastq files on hg19 genome with DpnII as retrsiction enzyme (-s) and positions specified by the -y argument.Data Transformation - BIGWIG coverage file was then computed using deeptools bamCoverage v3.4.3 with a custom binsize (-bs 50) and with the normalization CPM (--normalizeUsing CPM)and the option exactScaling (--exactScaling).Data Transformation - In order to reduce biases arising from GC content, fragment size, mappability, or other unspecified sources, the contact matrices were balanced using the Knight-Ruiz matrix balancing algorithm. Using this approach the Hi-C maps are normalised so that interactions for each bin sum up to one, and therefore the values can be interpreted as contact probabilities.MetabolomicsUnknownTranscriptomicsGenomicsProteomicsNextSeq 500NextSeq 2000Hi-CHomo sapiensVincent ROCHERThomas CLOUAIREBenjamin LE BOZECSarah COLLINSfalseMammalian PERIOD complex regulates repair of DSB in active chromatin through anchoring to the nuclear envelopeRepair 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.2025-11-12T00:00:00Z2026-05-26T13:06:18.697Z2023-02-22T18:55:54.6ZE-MTAB-12712ERP183003EFO_0007693EFO_0002944EFO_0004170EFO_0003789EFO_0004917EFO_0005518EFO_0003816EFO_0004184EFO_0003969