Acute perturbation strategies in interrogating RNA Polymerase II elongation factor function in gene expression

The regulation of gene expression catalyzed by RNA Polymerase II (Pol II) requires a host of accessory factors to ensure cell growth, differentiation, and survival under environmental stress. Here, using the auxin-inducible degradation (AID) system to study transcriptional activities of the bromodomain and extra terminal domain (BET) and Super Elongation Complex (SEC) families, we found that the CDK9-containing BRD4 complex is required for the release of Pol II from promoter-proximal pausing for most genes, while the CDK9-containing SEC is required for activated transcription in the heat shock response. By using both the Proteolysis-targeting chimera (PROTAC) dBET6 and the AID system, we found that dBET6 treatment results in two major effects: the increased pausing due to BRD4 loss, and reduced enhancer activity attributable to BRD2 loss. In the heat shock response, while auxin-mediated depletion of the AFF4 subunit of SEC has a more severe defect than AFF1 depletion, simultaneous depletion of AFF1 and AFF4 leads to a stronger attenuation of the heat shock response, similar to treatment with the SEC inhibitor KL-1, suggesting a possible redundancy among SEC family members. This study highlights the usefulness of orthogonal acute depletion/inhibition strategies to identify distinct and redundant biological functions among Pol II elongation factor paralogs. Examination of RNA Pol II dynamics by ChIP-seq in human cells subjected to rapid depletion of AFF1, AFF4 or BET bromodomain proteins using an auxin-inducible degron. The PROTAC dBET6 was used to degrade all BET proteins and the SEC disruptor KL-1 was used to target AFF1 and AFF4 containing P-TEFb complexes. RNA-seq +/- auxin was also performed for BRD4 degron cells and for AFF1/AFF4 double degron cells.