Acute perturbation strategies in interrogating RNA Polymerase II elongation factor function in gene expression. 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. Overall design: 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.