Transcription of metazoan genes by RNA Polymerase II (Pol II) controlled promoters is a highly dynamic process whereby upstream signalling cascades ultimately converge onto gene promoters to relay cell state information. This process requires the coordinated activity of several megadalton-scale protein complexes which exert regulatory control at multiple levels spanning chromatin accessibility through to Pol II termination. Co-transcriptional processes (e.g. splicing and export) impart additional layers of control over gene expression through physical and functional coupling to the transcriptional machinery. Here, the mechanism(s) by which diverse input signals from upstream regulatory factors and co-transcriptional processes are interpreted and integrated by downstream transcription factors and their associated co-activators to govern transcriptional output remains poorly understood.
To investigate these transactivation related processes in greater detail, we constructed a real-time flow cytometry-based reporter of c-MYC, an archetypal proto-oncogene regulated at multiple stages of transcription. Using this system, we performed systematic CRISPR/Cas9 screens to identify chromatin factors that impact endogenous c-MYC expression following small molecule inhibition of different stages of transcription. We find that the loss of key members the SF3b complex, an intrinsic component of the U2 spliceosome, desensitises cells to the inhibition of factors acting upstream of the Pol II pause release checkpoint including CDK7, p300/CBP and the BET bromodomain proteins. By integrating nascent transcriptomics, long-read sequencing and ChIP-sequencing, we demonstrate that c-MYC is transcriptionally re-activated and fully spliced upon acute ablation of these SF3b factors despite ongoing and potent transcriptional inhibition. Further analysis reveals a suite of over 600 genes that follow similar reactivation kinetics suggesting a common mechanism for transcriptional control mediated by the SF3b complex of the U2 spliceosome. Together, these data reveal new insights into a novel regulatory checkpoint that coordinates regulatory inputs from the splicing, transcriptional co-activation and basal transcriptional machinery to regulate gene expression.