Fig. 4: Tethering RUVBL2 to an active gene promoter enhances Pol II cluster formation.

a Schematic showing the system used for simultaneous imaging of tethered proteins and recruited proteins, as published previously⁶⁰. b Representative images of endogenous RUVBL1 and RUVBL2. Scale bar, 5 μm. c The relative IF signals of the target proteins at the LacO loci under mock (RUVBL1 n = 24, RUVBL2 n = 30) and Dox treatments (RUVBL1 n = 18, RUVBL2 n = 30). Box plots indicate median (middle line), 25th and 75th percentile (box) and 5th and 95th percentile (whiskers), as well as outliers (single black points), differences between groups were analyzed by two-tailed Student’s t tests (****p < 0.0001). d The mean and total intensity of LacR-BFP fusion signals at LacO loci per cell were plotted in bar graphs. The cotransfected cells (n = 30) in different fields from 3 transfection replicates were used for quantification. Differences between groups were analyzed by two-tailed Student’s t tests. “ns” indicates no significant difference. The error bars indicate mean + /− SDs. e Representative images of endogenous Pol II before and after transcriptional activation. Scale bars, 5 μm. f The relative IF signals of Pol II and LacR-BFP fusions at the LacO loci were plotted as boxplot. Cells from a minimum of 10 different fields (BFP n = 11, RUVBL1 n = 28, RUVBL2 n = 29 in mock state, and BFP n = 13, RUVBL1 n = 24, RUVBL2 n = 16 in Dox activation state) were used for quantification. Differences between groups were analyzed with two-tailed Student’s t tests (ns, not significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). g Nuclear Pol II (RPB1) density was quantified from 3 biological replicates. Two-tailed Student’s t tests were performed to determine the significance of differences between groups, and the error bars indicate the mean + /− SD. h IF assay was performed to examine endogenous RUVBL2. Cells (BFP n = 22, CTD n = 19) from a minimum of 10 different fields were used for clock scanning and quantification, and the error bars indicate the mean + /− SDs. The clock scan densities were analyzed with two-tailed Student’s t-tests (****p < 0.0001). Scale bars, 5 μm.