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.

由RNA聚合酶II（RNA Polymerase II，Pol II）催化的基因表达调控，需要大量辅助因子以确保细胞在环境胁迫下的生长、分化与存活。本研究利用植物生长素诱导降解系统（auxin-inducible degradation，AID），研究溴结构域及额外末端结构域家族（bromodomain and extra terminal domain，BET）与超级延伸复合物（Super Elongation Complex，SEC）的转录活性，发现包含CDK9的BRD4复合物可促进多数基因的Pol II从启动子近端暂停状态释放，而包含CDK9的SEC则是热休克应答中激活转录所必需的。

本研究同时采用靶向降解嵌合体（Proteolysis-targeting chimera，PROTAC）dBET6与AID系统，结果显示dBET6处理会产生两种主要效应：一是因BRD4缺失导致的Pol II暂停水平升高，二是因BRD2缺失引发的增强子活性降低。

在热休克应答过程中，尽管SEC的AFF4亚基经生长素介导的降解后，其功能缺陷程度较AFF1降解更为严重，但同时降解AFF1与AFF4会导致热休克应答的抑制效果更强，这与使用SEC抑制剂KL-1处理的实验结果一致，提示SEC家族成员间可能存在功能冗余。

本研究凸显了正交性急性降解/抑制策略在区分Pol II延伸因子旁系同源物的独特功能与冗余功能方面的应用价值。

整体实验设计：通过ChIP-seq检测人类细胞中RNA Pol II的动态变化，这些细胞分别通过生长素诱导降解标签（auxin-inducible degron）快速降解AFF1、AFF4或BET溴结构域蛋白。本研究使用PROTAC dBET6降解所有BET家族蛋白，并使用SEC干扰剂KL-1靶向包含P-TEFb复合物的AFF1与AFF4。此外，还对BRD4降解标签细胞与AFF1/AFF4双降解标签细胞开展了添加/不添加生长素的RNA-seq实验。