(A, J, S) Graphs plotting intrinsic disorder for HOXA13, RUNX2 and TBP. The IDRs cloned for subsequent experiments are highlighted with a purple bar.
(B, K, T) Representative images of HEK-293T nuclei expressing the indicated TF IDR-mCherry-CRY2 fusion proteins. Cells were stimulated with 488nm laser every 20s for 3 minutes.
(C, L, U) Quantification of the fraction of the nuclear area occupied by droplets of the indicated TF IDR-mCherry-CRY2 fusion proteins in HEK-293T nuclei over time. Data displayed as mean+/−SEM.
(D, M, V) Fluorescence intensity of droplets of the indicated TF IDR-mCherry-CRY2 fusion proteins before, during and after photobleaching. For the HOXA13 +7A IDR and the RUNX2 +10A IDR the spontaneously formed droplets were bleached, for all other fusion proteins the light-induced droplets were bleached. Data displayed as mean+/−SD.
(E, N) Representative images of droplet formation by purified TF IDR-mCherry fusion proteins in droplet formation buffer.
(F, O) Phase diagram of TF IDR-mCherry fusion proteins. Every dot represents a detected droplet. The inset depicts the projected average size of the droplets as mean+/− SD (middle circle: mean, inner and outer circle: SD). n.d.: not detected
(G, P) Representative images of droplet formation by purified MED1-IDR-GFP and TF IDR-mCherry fusion proteins in droplet formation buffer with 10% PEG-8000.
(H, Q) Quantification of GFP and mCherry fluorescence intensity in TF IDR-mCherry containing droplets in the indicated MED1 IDR-GFP mixing experiments. Each dot represents one droplet, and the size of the dot is proportional to the size of the droplet.
(I, R) (left): GAL4 activation assay schematic. The luciferase reporter plasmid, and the expression vector for the GAL4 DBD-TF IDR fusion proteins were transfected into HEK-293T cells. (right): Luciferase reporter activity of the indicated TF IDRs fused to GAL4-DBD. p <10−3 for both wt/mutant comparisons (Student’s t-test).
See also Figure S6.