The Super Elongation Complex mediates phase transition of SPT5 during transcriptional pause release (2022)
Figure 7

Figure 7. Disease-causing AFF4 mutations reduces droplet fluidity, but promotes accumulation of elongation factors at the target genes.

A Confocal images showing co-localization of AFF4 with SPT5 in nuclear puncta in AFF4 wild type (WT) or R258W cells.

B Dotplot (left) showing the AFF4 or SPT5 puncta area in AFF4 WT or R258W cells (Fig 7A). Two-tailed, unpaired Student's t test was performed. AFF4 R258W vs. WT, *p = 0.0254. Violin plot (right) showing the Pearson correlation coefficient of co-localization ratio (Fig 7A). Each n > 20. Results are representative of three biological replicates. Two-tailed, unpaired Student's t test was performed. AFF4 R258W vs. WT, *p = 0.0166.

C Droplet experiments showing that CHOPS syndrome patient missense mutations reinforced AFF4-ALF domain droplet formation.

D Dotplot showing the droplet areas in Fig 7C. Fields per condition n = 5. Two-tailed, unpaired Student's t test was performed. T254S, T254A or R258W vs. WT, *p < 0.05, **p < 0.01, ***p < 0.001.

E AFF4-ALF-mCherry condensed fraction as a function of AFF4-ALF-mCherry concentration for experiments in Fig 7C. Error bars represent standard deviations. Fields per condition n = 5. Two-tailed, unpaired Student's t test was performed. T254S, T254A or R258W vs. WT, *p < 0.05, **p < 0.01.

F Western blot analysis for the eGFP-AFF4mut was expressed in a concentration dependent manner. α-Tubulin was used as a loading control.

G Normalized FRAP curves for eGFP-AFF4 (green) and mCherry-ENL (purple/blue) and eGFP-AFF4mut (yellow) in the AFF4/ENL and AFF4mut/ENL heterotypic droplets. The bleaching events occurred at 0 second. Results shown are from three biological replicates.

H Normalized FRAP curves for eGFP-AFF4 + ENL (green) and mCherry-SPT5 (purple/blue) and eGFP-AFF4mut + ENL (yellow) in the AFF4/ENL/SPT5 and AFF4mut/ENL/SPT5 heterotypic droplets. The bleaching events occurred at 0 second. Results shown are from three biological replicates.

I AFF4 WT and R258W HEK-293T cells were heat shocked by incubation at 42°C for indicated times (minutes), and HSP70 mRNA levels were assessed by RT-qPCR and normalized to GAPDH. Error bars represent standard deviations. Results are from two biological replicates. Two-tailed, unpaired Student's t test was performed. For 90 minutes, AFF4 R258W vs. WT, **p = 0.0033. For 120 minutes, AFF4 R258W vs. WT, ***p = 0.0005.

J AFF4 WT and R258W HEK-293T cells were heat shocked by incubation at 42°C for indicated times (minutes), and R258W HEK-293T cells were treated with indicated concentrations of 1,6-hexanediol (1,6-hex) for 30 minutes, and HSP70 mRNA levels were assessed by RT-qPCR and normalized to GAPDH. Error bars represent standard deviations. Results are from two biological replicates. Two-tailed, unpaired Student's t test was performed. For 90 minutes, AFF4 R258W + 1% 1,6-hex vs. AFF4 R258W, **p = 0.0016; AFF4 R258W + 3% 1,6-hex vs. AFF4 R258W, **p = 0.0011. For 120 minutes, AFF4 R258W + 1% 1,6-hex vs. AFF4 R258W, ***p < 0.0001; AFF4 R258W + 3% 1,6-hex vs. AFF4 R258W, ***p < 0.0001.

K-N ChIP-qPCR showing the occupancies of Pol II (K), AFF4 (L), SPT5 (M), and PAF1 (N) at the promoter and gene body of HSP70 in AFF4 WT and R258W HEK-293T cells after heat shock for 2 hours. The HEMO gene serves as a negative control for ChIP-qPCR. Error bars represent standard error of means. Results are plotted with four technical repeats from two biological replicates. Two-tailed, unpaired Student's t test was performed. For Pol II ChIP, HSP70_promoter, ***p < 0.0001; HSP70_gene body, ***p = 0.0002. For AFF4 ChIP, HSP70_promoter, ***p < 0.0001; HSP70_gene body, ***p < 0.0001. For SPT5 ChIP, HSP70_promoter, ***p < 0.0001; HSP70_gene body, **p = 0.0086. For PAF1 ChIP, HSP70_promoter, ***p = 0.0007; HSP70_gene body, **p = 0.0021.

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