Fig. 3: Evidence for the direct activation and modulation of target transcription by TF clustering.

a Assay design. Note that U2OS-7TetO cells were used for the following experiments. b–c DNA fluorescence in situ hybridization (DNA FISH) assay for analyzing gene locus-specific binding of TF clusters. Three-color images of cells with arrows indicating co-localization events between TF clusters and Cy5-labeled DNA probes targeting the reporter gene loci (b, left), and the corresponding fluorescence intensity line profiles (b, right). Dot plot showing the number of co-localization events per cell for two culture conditions, i.e., with or without 0.3 μg/mL doxycycline and both with 100 nM rapamycin (c, n = 16 cells for each condition and p value from two-sided t test). d–e Time-lapse analysis of spatiotemporal interactions between TF clusters and nascent transcription sites of reporter genes. Snapshots (d) and the intensity trajectories of arrow-indicated reporter locus (e). f–g Evidence supporting the direct transcriptional activation by TF clusters. Two-color images showing the spatial co-localization between TF clusters and nascent transcriptional sites and corresponding fluorescence intensity line profiles (f). The fraction of TF clusters co-localizing with nascent transcriptional signals were calculated for two culture conditions (with or without 0.3 μg/mL doxycycline, and both with 100 nM rapamycin) (g). Fraction of co-localization was calculated by bootstrap (resampled 1000 times with replacement), and more than 2500 TF clusters were used for each calculation. n = 615 (with dox) and 897 (without dox) cells. h, i Evidence supporting the direct modulation of transcriptional dynamics by TF clustering propensity. Cells were cultured at indicated rapamycin concentrations, and doxycycline (0.3 μg/mL) was added. Both the fraction of ON time for each nascent site (h) and the number of nascent sites per cell (i) vary with rapamycin concentration. Dot numbers are 236, 378, 362, and 348 from left to right in h. Cell numbers are 36, 46, 40, and 37 from left to right in i. p values were calculated by one-way ANOVA and TukeyHSD. For boxplots, horizontal line indicates median and box ranges from first to third quartile, with whiskers extending up to 1.5 × interquartile range. Source data are provided.