Fig 2: 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.

Fig 3: Contribution of hydrophobic cores of AFF4-THD to dimerization.a, b Analytical gel filtration revealed that the hydrophobic cores are critical to the dimerization of AFF1/4. Left: gel filtration chromatography, the horizontal axis is elution volume, and the vertical axis is UV absorption. The molecular weight standards (kD) are denoted on top of the curves. Right: SDS-PAGE results of peak fractions of corresponding gel filtration chromatography, and the elution volume in gel filtration is denoted below. a The curve of wild-type AFF4-THD, AFF4F1014A, AFF4Y1096A, and AFF4F1014A+Y1096A are colored in cyan, orange, blue, and magenta, respectively. b Wild-type AFF1-THD, AFF1F1063A, AFF1Y1145A, and AFF1F1063A+Y1145A+V1146A+V1152A are colored in cyan, orange, blue, and magenta, respectively. c, d Sedimentation velocity analytical ultracentrifugation results. Curve of wild type of AFF1/4 are shown in cyan, while the mutations of AFF1/4 are shown in magenta. The calculated molecular weight is denoted on top of these peaks. Horizontal axis: sedimentation coefficient. Vertical axis: continuous sedimentation coefficient distribution.

Fig 4: 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.