CDK12 prevents MYC-induced transcription-replication conflicts [EU-seq]

In order to identify genes and pathways necessary to preserve genome integrity upon Myc over-expression, we conducted a large siRNA-based screen in isogenic lines. We discovered several genes that suppressed the Myc-induced DNA-damage response and that were essential for the cell survival upon Myc activation. We idntified CDK12, a cyclin dependent kinase involved in transcriptional control and genome stability. We uncovered a novel and unexpected role of CDK12 in controlling transcription at loci proximal to DNA damaged sites and dissected its upstream regulatory pathways and downstream effectors. Mechanistic studies and genome-wide mapping of replication dynamics and DSBs revealed how CDK12 is essential to suppress intrinsic transcription-replication conflicts, thus avoiding cytotoxic DNA damage in cancer cells. Overall, this study uncovers a novel role for CDK12 and a new liability of Myc-driven cancers, which could be exploited for therapeutic purposes. U2OS MycER cells (U20S cells expressing the MycER chimera) were infected with virus expressing non-targeting shRNAs (shNT) or shRNA targeting CDK12 (shCDK12). ShRNA expression was activated with 1 µg/mL doxycycline, while MycER was activated with 300 nM OHT for 40h. Cells were then treated with 100 ng/ml nocodazole (Sigma, Cat. No. SML-1665) for 8h to induce mitotic arrest. G2/M arrested cells were isolated by shake-off, washed with PBS and released in warm medium for 12 hours. 0.5 mM EU (5-ethynyl-uridine, Jena Biosciences, Cat. No. CLK-N002-10) was added to the cells 2h before cells collection.