CDK12 prevents MYC-induced transcription-replication conflicts [RNA-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 cells were transfected with either non-targeting siRNAs (siLuc) or siRNAs targeting CDK12 (siCDK12). 48 hours post transfection and MycER activation by 300nM OHT cells were collected and processed.

为鉴定Myc过表达状态下维持基因组完整性所必需的基因与通路，我们在同基因细胞系中开展了大规模基于小干扰RNA（siRNA）的筛选实验。我们发现了数个可抑制Myc诱导的DNA损伤应答、且在Myc激活时对细胞存活至关重要的基因。我们鉴定出细胞周期蛋白依赖性激酶12（CDK12）——一种参与转录调控与基因组稳定的周期蛋白依赖性激酶。我们揭示了CDK12在DNA损伤位点近端基因座调控转录中的全新且出乎意料的功能，并解析了其上游调控通路与下游效应分子。机制研究结合复制动力学与DNA双链断裂（DSBs）的全基因组图谱分析，阐明了CDK12如何通过抑制固有转录-复制冲突，从而避免癌细胞中产生具有细胞毒性的DNA损伤。综上，本研究揭示了CDK12的全新功能，以及Myc驱动型癌症的一项新的治疗易损性，该发现可被开发用于治疗用途。
我们将U2OS细胞分为两组，分别转染非靶向小干扰RNA（siLuc）或靶向CDK12的小干扰RNA（siCDK12）。转染48小时后，使用300nM 4-羟基他莫昔芬（OHT）激活MycER，随后收集细胞并进行后续实验处理。