EXPERIMENT_TITLE: Chromatin compartmentalization regulates the response to DNA damage
EXPERIMENT_DESIGN_LIBRARY_DESCRIPTOR_LIBRARY_CONSTRUCTION_PROTOC: DIvA (AsiSI-ER-U20S) cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with antibiotics, 10% FCS (InVitrogenInvitrogen) with 1 µg/mL puromycin (DIvA cells) at 37°C under a humidified atmosphere with 5% CO2. For AsiSI-dependent DSB induction, cells were treated with 300 nM OHT (Sigma; H7904) for 4h or 24h. For ATM inhibition, cells were pretreated for 1 h with 20μM KU-55933 (Sigma, SML1109) and during subsequent 4OHT treatment. For DNA-PK inhibition, cells were pretreated for 1 h with 2μM NU-7441 (Selleckchem, S2638) and during subsequent 4OHT. 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 sequences: CTRL: CAUGUCAUGUGUCACAUCU; or SCC1: GGUGAAAAUGGCAUUACGG:   or SMC1 (siSMC1): UAGGCUUCCUGGAGGUCACAUUUAA; or 53BP1 (si53BP1): GAACGAGGAGACGGUAAUA; or SUN2 (siSUN2): CGAGCCTATTCAGACGTTTCA; or ARP2 (siARP2): GGCACCGGGUUUGUGAAGU or SETX (siSETX): GAGAGAAUUAUUGCGUACU. For cell synchronization, cells were incubated for 18 h with 2 mM thymidine (Sigma, T1895), then released during 11 h, followed by a second thymidine treatment for 18 hr. S, G2 and G1 cells were then respectively treated with OHT at, 0, 6 or 11 h following thymidine release and harvested 4 h later.  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).  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.
STUDY_DESCRIPTOR_STUDY_ABSTRACT: 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.
SAMPLE_TITLE: HiC_siCTRL_pOHT
SAMPLE_SAMPLE_ATTRIBUTES_SAMPLE_ATTRIBUTE: [{"TAG": "ENA-FIRST-PUBLIC", "VALUE": "2023-11-16T12:38:08Z"}, {"TAG": "ENA-LAST-UPDATE", "VALUE": "2023-11-16T12:38:08Z"}, {"TAG": "External Id", "VALUE": "SAMEA114625832"}, {"TAG": "INSDC center name", "VALUE": "MCD, UMR5077, CBI"}, {"TAG": "INSDC first public", "VALUE": "2023-11-16T12:38:08Z"}, {"TAG": "INSDC last update", "VALUE": "2023-11-16T12:38:08Z"}, {"TAG": "INSDC status", "VALUE": "public"}, {"TAG": "Submitter Id", "VALUE": "E-MTAB-10865_3:HiC_siCTRL_pOHT"}, {"TAG": "broker name", "VALUE": "ArrayExpress"}, {"TAG": "cell_line", "VALUE": "U2OS"}, {"TAG": "cell_type", "VALUE": "epithelial cell"}, {"TAG": "collection date", "VALUE": "not collected"}, {"TAG": "common name", "VALUE": "human"}, {"TAG": "disease", "VALUE": "osteosarcoma"}, {"TAG": "genotype", "VALUE": "AsiSI-ER"}, {"TAG": "geographic location (country and/or sea)", "VALUE": "not collected"}, {"TAG": "isolate", "VALUE": "not applicable"}, {"TAG": "organism part", "VALUE": "bone"}, {"TAG": "protocol", "VALUE": "Hi-C"}, {"TAG": "sample name", "VALUE": "E-MTAB-10865_3:HiC_siCTRL_pOHT"}, {"TAG": "scientific_name", "VALUE": "Homo sapiens"}]
Pool_Member_@attributes_sample_title: HiC_siCTRL_pOHT
RUN_SET_RUN_Pool_Member_@attributes_sample_title: HiC_siCTRL_pOHT
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