Mapping breakome of multiple cancer cell-lines
Cancer is characterized by uncontrolled proliferation accompanied by the hypertranscription of oncogenes, leading to transcription stress, a key source of DNA double-strand breaks (DSBs) that jeopardize genomic stability. Yet, transcription stress is still underexplored. In this study, we utilized maps of DSBs identified through in-suspension break labeling in situ and sequencing (sBLISS), along with transcription stress markers, revealing that transcription stress regions coincide with the super-enhancer regulatory landscape. Notably, ?H2AX mapping indicates its enrichment at transcription stress sites while not all DSB-enriched genes show equal ?H2AX marking, but those with DSBs tied to transcription stress are distinctly marked. Intriguingly, genes with high-DSBs marked by ?H2AX exhibited significantly higher DSB turnover and repair than those with ?H2AX-low genes, manifesting vulnerability to mutagenesis. These findings underscore super-enhancer activity as a determinant of the transcription stress landscape in cancer, posing a threat to the genomic stability of oncogenes in cancer. Overall design: MCF7 (HTB-22) and T-47D (HTB-133) cells were grown in RPMI supplemented with 10% (vol/vol) fetal bovine serum FBS (GIBCO), glutamine, and penicillin/streptomycin. HEK-293 (CRL-1573), MDA-MB-468 (HTB-132), MDA-MB-231 (HTB-26), and HeLa cells (CCL-2) were grown in DMEM supplemented with 10% (vol/vol) FBS, glutamine, and penicillin/streptomycin. A-4098 cells (HTB-44) were grown in. EMEM supplemented with 10% FBS, glutamine, and penicillin/streptomycin. SH-SY5Y (CRL-2266) were grown in DMEM/F12 supplemented with 10% FBS, glutamine, and penicillin/streptomycin. HMLE cells were grown in Promocell mammary epithelial cell basal media (C-21010) with added supplements (c-93110) whereas MCF10A were grown on DMEM/F12 supplemented with 5% Horse serum, 20 ng/ml EGF, 0.5 mg/ml Hydrocortisone, 100 ng/ml Cholera toxin, 10 mg/ml Insulin and Pen/Strep. MCF-7 cells were synchronized in the G1 cell cycle stage by culturing in serum-free media for 72 hours followed by replacing the media with 5% FBS media for 5 hours. Cells were grown at 37°C under a humidified atmosphere with 5% CO2. Cells were routinely authenticated by STR profiling, and tested for mycoplasma, and cell aliquots from early passages were used. Cells were treated with DMSO (Sigma-Aldrich), dBET6 (Sigma-Aldrich, SML2638), or CDK9i (NVP-2) (MCE, Cat# Y-12214A) 100 nM for 4 hours. Cells were then treated with DMSO or ATMi (20 uM, KU-55933, Sigma) for 5 hours. Transient transfection of siXRCC4 (Dharmacon; SMARTpool siRNA, Catalog ID: L-004494-00-0005) and siSc (Dharmacon; D-00181010) was carried out using Lipofectamine (ThermoFisher). On the day of transfection, cells were seeded to achieve a confluency of 60-70%. The transfection solution was prepared by mixing 1.5 ml of serum and antibiotic-free RPMI with 30 µL of Lipofectamine and incubating for 5 minutes before adding a mixture of 1.5 ml RPMI and 0.6 nmoles of siRNA, followed by a 20-minute incubation at room temperature. This mixture was then added to cells in a 10 cm plate cultured in antibiotic and serum-free media. The media was replaced with fully supplemented media after 5-6 hours, and the cells were incubated in the incubator for 48 hours.