Extended Data Fig. 5: Impact of MED14 degradation on Pol II clusters and nascent transcription dynamics.

a, Image quantification related to Fig. 3c. Pie chart: percent of n = 100 large Pol II foci, which overlap MED14-dTAG foci. Mean±s.e.m. with two-sided, unpaired t-test (n = 40 nuclei in DMSO; n = 36 nuclei in dTAG7 condition). b, Control imaging experiment related to Fig. 3c, omitting anti-HA primary antibody to rule out that Pol II foci are an HA channel bleed through artifact. c, Immunofluorescence of large hypo-phosphorylated Pol II foci (8WG16; arrows) in MED14-dTAG KBM7 cells. Maximum intensity projections of 3D images. Scale bars 1 µm. Pie chart: percent of n = 60 large Pol II foci, which overlap MED14-dTAG foci. Dot plots: changes in number of large Pol II foci per cell and integrated nuclear fluorescence intensity. Mean±s.e.m. with unpaired, two-sided t-tests (n = 94 nuclei for DMSO; n = 89 for dTAG7). d, PRO-seq signal of auto-regulatory TFs MYC and MYB, and the expression-matched control gene RAB3GAP1 after 1 h MED14 degradation. Arrows highlight loss of promoter-proximal signal. H3K4me3 and H3K27ac ChIP-seq signal from KBM7 wild-type cells. e, Aggregated PRO-seq coverage over an SE-proximal metagene. TSS, transcription start site; TES, transcription end site. f, Changes in PRO-seq pausing index at n = 7,643 genes after 1 h MED14 degradation. g, Observed vs. expected median Euclidean distance of auto-regulatory TFs from the pause-initiation limit in Fig. 3f. The expected distribution was generated by randomly selecting the same number of genes from bulk. h, Changes in productive initiation rate and pause duration of all 6,791 genes vs. the 24 auto-regulatory TFs. Productive initiation rates selectively decrease for auto-regulatory TFs, while pause duration decreases globally. Box plot elements: medians with interquartile range and 1.5x whiskers.