Fig. 1. A LacO array can mediate the formation of an LCD hub in live cells, which involves extensive LCD self-interaction and recruits RNA Pol II.

(A) Schematic for a LacO array (n ≈ 50,000 repeats for array 1, n ≈ 15,000 repeats for array 2) in the U20S genome nucleating an LCD hub when EYFP-LCD-LacI is transiently expressed. Alternatively, EYFP-LacI is expressed as a control. NLS, nuclear localization signal. (B) Confocal fluorescence and bright-field images of LacO-containing U20S cells where LacO array 1 (highlighted by circles) is bound by EYFP-labeled LCD-LacI or LacI. LCD-LacI-bound, but not LacI-bound, LacO arrays are visible in bright-field images. (C and D) Copy number of EYFP-labeled (C) or mCherry-labeled (D) TAF15 LCD-LacI (red) or LacI (blue) molecules bound to LacO array 1 (C) or 2 (D) as a function of mean nuclear concentration of the TF. Concentrations were measured by fluorescence intensity comparison. Each dot represents one cell. (E) Averaged FRAP curves at LacO array 1 bound by mCherry-labeled TAF15 LCD-LacI (red) or LacI (blue). Error bars represent SD. a.u., arbitrary units; N, number of cells analyzed. (F) (Top) Schematic of the proteins expressed in the LacO-containing U20S line. (Bottom) Confocal fluorescence images show that Halo-RPB1 (labeled with 200 nM Halo ligand JF500, green) is enriched at LacO array 2 bound by mCherry-EWS LCD-LacI (red). (G) (Left) Averaged Halo-RPB1 images at LacO array 2 bound by mCherry-labeled LacI, EWS LCD-LacI, FUS LCD-LacI, or TAF15 LCD-LacI (N = 55, 69, 81, or 143). (Right) Fluorescence intensity of Halo-RPB1 at the LacO array center in the average images after subtraction of nuclear Halo-RPB1 background (see supplementary methods). ** denotes a statistically significant increase compared with the LacI condition (P < 0.01, two-sample t test). Error bars represent bootstrapped SD (45).