Fig. 3. PHF3 drives liquid-liquid phase separation of phosphorylated Pol II, colocalizes in Pol II clusters in cells and associates with Pol II genome-wide.

a Representative images of in vitro LLPS assays with 5 µM unphosphorylated or phosphorylated mEGFP-CTD, 5 µM mCherry-SPOC, 3 µM Alexa594-PHF3, 3 µM phosphorylated Alexa488-Pol II, 1.5 µM phosphorylated Alexa647-Pol II + 1.5 µM Alexa488-PHF3. Scale bar = 5 µm. The experiments were repeated three times and the representative images are shown. b Quantification of condensate area (µm²). N = 556 (CTD); 64 (phCTD); 89 (phCTD+SPOC); 582 (phPol II); 480 (PHF3); 580 (PHF3+phCTD); 588 (PHF3+phPol II). Data are presented as median with interquartile range. Mann-Whitney test was used to determine p-values (Supplementary Data 7). c Representative Airyscan high resolution images of PHF3-GFP (IF staining with rabbit anti-GFP + Alexa Fluor 488, green) and Pol II pS2 or pS5 (Alexa Fluor 594, red). Co-localization analysis of clusters that overlap in both channels (white). Scale bar = 5 µm or 200 nm for zoomed regions. d Quantification of the fraction of Pol II pS2 (N = 28) and Pol II pS5 (N = 23) co-localizing with PHF3. Box and whiskers plot with error bars representing 10 and 90 percentiles are shown. Each experiment was repeated three times with comparable results. Statistics are indicated in detail in Supplementary Data 7. e ChIP-seq analysis shows that PHF3 travels with Pol II across the length of genes. Relative enrichment of PHF3, Pol II pS2, pS5, and pS7 on TSS-gene body region (TSS viewpoint; left panel) and gene body-pA region (pA viewpoint; right panel) for genes that showed Pol II occupancy with the F-12 antibody (minimal gene body RPKM of 5 in F-12 ChIP-seq). f Scatter plots showing PRO-seq nascent transcription levels at gene body relative to TSS in WT cells. Blue dots indicate PHF3-bound genes at transcription start sites (TSS, left), gene body (Body, middle) or polyadenylation sites (pA, right).