Fig. 6: UTP15 modulates gene transcription by supporting Pol II phase separation.

a Western blot showing HA-mediated UTP15 co-IP in POLR2A-FLAG knock-in cell lines. Representative western blots are shown from two independent experiments with consistent results. b Average read density analysis of Pol II ChIP-seq (NTD, Ser5, Ser2) after IAA induced acute UTP15 degradation in the region from −2 kb upstream of TSS to +2 kb downstream of the TES across UTP15 target genes (n = 685). c Prediction of disorder regions for UTP15 using PONDR. The sequence of UTP15 was submitted to the PONDR server, which predicted regions of disorder with the VL-XT, VL3, VSL2 predictor. d Droplet formation assays of varying concentrations of GFP and GFP-UTP15_IDR in the presence of 16% dextran. Scale bar, 10 μm. e Quantification of nuclear UTP15 signal intensity in control and 1,6-HD treated cells (Ctrl: n = 18; 1,6-HD: n = 28, n represents individual nuclei analyzed). Cells were treated with 3% 1,6-HD for 10 minutes. Box plots show the median (centre line) and 25th-75th percentiles (bounds); whiskers extend to 1.5 × IQR. Statistical significance was assessed using a two-sided Mann-Whitney U test. f Representative immunofluorescence images of UTP15 (green) in UTP15-AID cells following treatment with 3% 1,6-HD for 10 min or control (untreated) conditions. Scale bar, 5 μm. g, h Droplet formation assays of mCherry-CTD (10 μM) with GFP (10 μM) or GFP-UTP15_IDR (10 μM) in the presence of 16% dextran. The incubation time was increased from 30 min to 150 min at room temperature. Quantification and representative pictures are shown in h and g, respectively. In h, y axis shows the sum of fluorescence intensity of mCherry-CTD within droplets in each field (n = 5, n represents individual image analyzed, two-sided Student’s t-test). Scale bar, 10 μm. i Representative wide field and SIM² super-resolution images showing Pol II-NTD (green) and UTP15 (red) in UTP15-AID cells after IAA induced UTP15 degradation. Scale bar, 5 μm. j Quantification of nuclear Pol II clusters per cell using Laplace of Gaussian (LoG) filter method under different durations of IAA treatment (n = 20 per group, n represents individual nuclei analyzed). Box plots show the median (centre line) and 25th-75th percentiles (bounds); whiskers extend to 1.5 × IQR. Statistical significance was assessed using a two-sided Mann-Whitney U test. Source data for Fig. 6a, e, h and j are provided as a Source Data file.