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

Abstract

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 extraterminal 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: 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 the 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.

Keywords: BRD2; BRD4; PROTAC; auxin-inducible degradation; enhancer activity; heat shock; super elongation complex; transcription elongation.

Figure 1.

BET bromodomain protein BRD4 is required for global basal transcription by RNA polymerase II. (A) Design of BET protein degrons. CRISPR-mediated introduction of the mAID tag into BET proteins BRD2, BRD3, and BRD4. Cotransfection of donor plasmids bearing neomycin and hygromycin are used to ensure the selection of homozygous knock-in clones. (B) Western blot analysis showing the degradation of each BET protein in each of the degron cell lines at 2-h and 24-h auxin treatment showing the specific knockdown of the tagged protein, while treatment with dBET6 (250 nM) for 2 h and 24 h leads to degradation of BRD2, BRD3, and BRD4. (C) Heat map of mAID and H3K27ac occupancy around the TSS of 6244 Pol II transcribed genes ranked by the H3K27ac signal. Note that BRD3 and BRD4 have much lower occupancy on chromatin than BRD2, consistent with what is seen with the mAID antibody in Western analysis (see Supplemental Fig. S1B). (D) Peak annotations of mAID ChIP-seq for the BET proteins at different locations in the genome. Venn diagram showing the overlap of the three BET proteins, with most of the peaks for the less abundant BRD3 and BRD4 overlapping with the peaks called for the much more abundant BRD2. (E) ChIP-seq analysis of Pol II occupancy ±auxin or dBET6-mediated BET protein degradation (heat maps ranked by Pol II occupancy in parental cells). ECDF plots (bottom panels) show that BRD4 degradation, either through dBET6 treatment or auxin-inducible degradation, leads to a widespread increase in promoter-proximal pausing of Pol II, while BRD2 depletion leads to the release of Pol II into gene bodies for a subset of genes.

Figure 2.

BRD2 depletion results in loss of Pol II at BET-bound enhancers. (A) Venn diagram showing the overlap between each BET protein and the UCSC annotated hg19 coding gene regions. Note that BRD4 has the lowest percentage of inter-genic peaks, while BRD2 has the highest percentage of inter-genic peaks (Fig. 1D). (B) Clustering of H3K27ac, H3K4me1, and H3K4me3 ChIP-seq at the 8421 inter-genic peaks called for one or more of the BRDs using the mAID antibody. (C) Heat map of mAID ChIP-seq ± auxin for BET proteins and sorted by H3K27ac occupancy at the enhancer regions defined by clusters 2 and 3. (D) Genome browser views of Pol II ChIP-seq at two different enhancer regions upon auxin treatment in BET degron cells and dBET6 treatment in parental DLD-1 cells for 2 h. H3K27ac ChIP-seq is shown for comparison. (E) Heat map of Pol II ChIP-seq at enhancer regions before and after auxin treatment in BET degron cells or dBET6 treatment in parental DLD-1 cells for 2 h. (F) Box plot showing the log₂RPKM of Pol II ChIP-seq corresponding to the conditions in E. Auxin treatment in BRD2-AID cells and dBET6 treatment in parental cells exhibit significant reductions in Pol II occupancy at enhancer regions. (****) P < 2.210 × ⁻¹⁶, (ns) not significant. Mann–Whitney U-test.

Figure 3.

BET proteins are not required for rapid transcriptional induction by the heat shock response. (A) Schematic showing that DLD-1 cells were treated with or without auxin for 2 h followed by heat shock for 1 h at 42°C before processing cells for ChIP-seq of Pol II. (B) Genome browser overlays of Pol II occupancy at representative non-heat shock (YTHDF2) and heat shock-induced (BAG3) genes in the 42°C condition in parental DLD-1 cells before (dark color) and after (light color) heat shock. (C) Genome browser overlays of Pol II occupancy at the representative heat shock genes in BET degron cells ±auxin for 2 h, followed by non-heat shock (dark color) and heat shock for 1 h (light color). (D) ECDF plots of the log₂ pause release ratio (PRR) of the 227 HS-induced genes in BET degron cells ±auxin for 2 h, followed by heat shock for 1 h. A decreased heat shock response due to BET protein depletion would be expected to show a leftward shift of the ECDF curves after auxin treatment. (E) Western blots showing normal HSP70 induction after 6-h heat shock in the absence of BRD4. Cells were treated with H₂O or auxin for 2 h at 37°C before 42°C heat shock for 6 h. (F) Quantification of HSP70 protein levels ±auxin for three independent replicates as in E. Western signal intensity of HSP70 was first converted to fold change of actin within groups and then normalized to the condition of H₂O treatment without heat shock. Circled dots represent the median. While each replicate showed a different level of HSP70 induction, the overall level of induction for each replicate was the same for H₂O and auxin treatments. Replicate 1 is shown in E.

Figure 4.

The AFF4 form of the super elongation complex is preferentially required for rapid transcriptional induction upon heat shock. (A) Design of auxin degron tagging of the super elongation complex (SEC) scaffolding proteins, AFF1 and AFF4. AFF1-AID and AFF4-AID single degron cells were generated independently using the double selection strategy shown for the BET degron cells. (B) Western blotting with the indicated antibodies shows the degradation of nuclear AFF1 or AFF4 in the corresponding degron cells within 2 and 24 h of auxin treatment. (C) Genome browser overlay of Pol II occupancy at the representative heat shock-induced genes before (dark color) or after 1h heat shock (light colors) in AFF1 or AFF4 degron cells ±auxin for 2 h. (D) ECDF plots of the log₂ fold change of PRR for the 227 HS-induced genes after 1 h heat shock. AFF4 depletion by auxin treatment leads to reduced heat shock induction as indicated by the leftward shift in the PRR, while AFF1 depletion by auxin treatment does not affect heat shock induction. (E) Metagene analysis of the log₂ fold change (FC) of Pol II occupancy of 42°C over 37°C for the 227 HS-induced genes ±auxin for 2 h in AFF1-AID and AFF4-AID cells or ±6-h KL-1 (20 µM) treatment in AFF4-AID cells.

Figure 5.

The SEC is essential for rapid transcriptional induction by heat shock. (A) Design of AFF1-AFF4 double degron DLD-1 cells. Neomycin was used for selection of AFF1-AID homozygous knock-in clones, which were subsequently used for a homozygous knock-in of the mAID tag for AFF4 using hygromycin selection. (B) Western blot analysis demonstrating the degradation of both AFF1 and AFF4 after auxin treatment for 2 h, compared with the parental DLD-1 cells. (C) Metagene analysis showing the log₂FC of Pol II occupancy of 42°C over 37°C for the 227 HS-induced genes before and after simultaneous depletion of AFF1 and AFF4. Decreased induction of Pol II occupancy in the gene bodies during heat shock is observed. (D) Genome browser overlay of Pol II occupancy at representative heat shock genes before (dark color) and after (light color) heat shock for 1 h, ±auxin depletion of AFF1 and AFF4 for 2 h, or KL-1 treatment (20 µM) for 6 h. (E) Heat map showing the log₂ fold change of Pol II coverage around the TSS for the 227 HS-induced genes for auxin treatment of the AFF1–AFF4 double degron cells and KL-1 treatment of AFF4 degron cells. (F) Venn diagram showing the overlap of the 227 HS-induced genes whose induction is impaired by auxin depletion of AFF1 and AFF4 or by KL-1 treatment or both treatments. (G) Western blotting with the indicated antibodies showing attenuated HSP70 induction after 6-h heat shock after dual depletion of AFF1 and AFF4. Cells were treated with H2O or auxin for 2 h at 37°C before 42°C heat shock for 6 h. (H) Quantification of HSP70 protein levels ±auxin for three independent replicates as in G. Western signal intensity of HSP70 was first converted to fold change of actin within groups and then normalized to the condition of H2O treatment without heat shock. Circled dots represent the median. Replicate 2 is shown in G.