Fig. 1: A synthetic gene regulatory system with chemically controlled TF clustering propensity.

a, b Schematic of the system design. Our design is built upon a previously reported rapamycin-inducible clustering mechanism. The synthetic TF contains DNA binding domain (e.g., rTetR) and trans-activation domain (e.g., VP48), fused with FRB, EGFP, and a homo-oligomeric tag HOTag6 (a). This synthetic TF is co-translated with a “clustering mediator” composed of FKBP, EGFP, and HOTag3. The cognate reporter of the TF contains iRFP and 24x PP7 stem loops. The system also contains a plasmid encoding PCP-3xmCherry for visualizing nascent transcription (not shown). TF clustering is mediated by rapamycin-mediated binding between FRB and FKBP (b). c–f Rapamycin modulates TF clustering propensity. Representative confocal images (maximum projection of z slices) of monoclonal U2OS cells containing the preceding system were treated with rapamycin at indicated concentrations (c). See “Methods” for details on imaging. Scale bars indicate 10 μm. The fraction of cells containing detectable TF clusters (d), the averaged detected cluster number per cell (e), and the averaged cluster diameter (f) are quantified. The rapamycin concentrations are 0.05, 0.1, 0.3, 0.5, 0.7, 1, 3, 5, 7, 10, 30, 50, 70, 100, 300, 500, 700 and 1000 nM (left to right), and the cell numbers are between 70 and 117 (see source data for the exact cell number). The shaded regions indicate 95% CI by bootstrap. See also Supplementary Fig. 1b, c. Source data are provided.