BRD4 Prevents R-Loop Formation and Transcription-Replication Conflicts by Ensuring Efficient Transcription Elongation

Effective spatio-temporal control of transcription and replication during S-phase is paramount to maintaining genomic integrity and cell survival. Dysregulation of these systems can lead to conflicts between the transcription and replication machinery causing DNA damage and cell death. BRD4, a BET bromodomain protein and known transcriptional regulator, interacts with P-TEFb to ensure efficient transcriptional elongation by stimulating phosphorylation of RNA Polymerase II (RNAPII). Here we report that disruption of BET bromodomain protein function causes RNAPII pausing on the chromatin and DNA damage affecting cells in S-phase. We find that this persistent, RNAPII-dependent pausing leads to accumulation of RNA:DNA hybrids (R-loops), which are known to lead to transcription-replication conflicts (TRCs), DNA damage, and cell death. Furthermore, we show that resolution of R-loops abrogates BET bromodomain inhibitor-induced DNA damage, and that BET bromodomain inhibition induces both R-loops and DNA damage at sites of BRD4 occupancy. Finally, we see that the BRD4 C-terminal domain, which interacts with P-TEFb, is required to prevent R-loop formation and DNA damage caused by BET bromodomain inhibition and that oncogenes which promote transcription and replication exacerbate BET bromodomain inhibitor-induced DNA damage. Together, these findings demonstrate that BET bromodomain inhibitors can damage DNA via induction of R-loops and TRCs in highly replicative cells.S期内转录与复制的高效时空调控，对维持基因组完整性与细胞存活至关重要。此类调控系统的失调，可引发转录机器与复制机器之间的冲突，进而导致DNA损伤与细胞死亡。BRD4作为BET溴结构域蛋白（BET bromodomain protein）与已知转录调控因子，可与P-TEFb相互作用，通过刺激RNA聚合酶II（RNA Polymerase II，RNAPII）的磷酸化，保障高效的转录延伸过程。本研究发现，BET溴结构域蛋白功能受损会导致RNAPII在染色质上发生停滞，并引发S期细胞出现DNA损伤。研究表明，这种依赖RNAPII的持续性停滞，会导致RNA:DNA杂交链（RNA:DNA hybrids，R-loops）的积累；而这类结构已知会引发转录-复制冲突（transcription-replication conflicts，TRCs）、DNA损伤与细胞死亡。此外，本研究证实，消解R-loops可消除BET溴结构域抑制剂诱导的DNA损伤；而BET溴结构域抑制会在BRD4结合位点处同时诱导R-loops形成与DNA损伤。最后，本研究发现，BRD4与P-TEFb相互作用的C末端结构域，是预防BET溴结构域抑制引发R-loop形成与DNA损伤的必需结构；而促进转录与复制的癌基因，会加重BET溴结构域抑制剂诱导的DNA损伤。综上，本研究结果表明，BET溴结构域抑制剂可通过在高复制活性细胞中诱导R-loops与TRCs，进而造成DNA损伤。