Extended Data Fig. 4: Characterization of periodic TF IDR mutants.
From: An activity-specificity trade-off encoded in human transcription factors
a. Representative images of fluorescence recovery after photobleaching (FRAP) experiments with HOXD4 IDR–mEGFP droplets. b. Western blot of GAL4-DBD and GAL4-DBD-HOXD4-IDR-fusion proteins in HEK293T cells 24 hours after transfection using a GAL4-DBD specific antibody. HSP90: loading control. Except for AroLITE A, GAL4-DBD-HOXD4-IDR fusion proteins are expressed at comparable levels. c. Schematic models of HOXD4 wild type and mutant IDRs. Omega plots of the HOXD4 IDRs and ΩAro scores are shown next to the schematic models. d. Results of luciferase reporter assays. The YPWM motif does not contribute to the transactivation potential of the HOXD4 IDR. e. The activity of HOXD4 IDRs (left) and C/EBPα IDRs (right) scales with the number of small inert residues adjacent to aromatic residues in the IDR constructs. f. (left) Schematic models of wild type and AroPERFECT HOXC4 IDRs. (middle) Omega plots and ΩAro scores of the IDRs. IDR: intrinsically disordered region (right). Results of luciferase reporter assays. g. Western blot of GAL4-DBD and GAL4-DBD-HOXC4-IDR fusion proteins in HEK293T cells 24 hours after transfection using a GAL4-DBD specific antibody. HSP90: loading control. h. Representative images of droplet formation of purified HOXC4 IDR–mEGFP proteins. Scale bar: 5 μm. For the wild type IDR, the exact same images are displayed in Fig. 1g. i. The relative amount of condensed protein per concentration quantified in the droplet formation assays. Data are displayed as mean ± SD. N = 10 images per condition pooled from two independent replicates. The curve was generated as a nonlinear regression to a sigmoidal curve function. j. Representative images FRAP experiments with HOXC4 IDR–mEGFP droplets. k. Fluorescence intensity of HOXC4 wild type IDR and HOXC4 AroPERFECT IDR in vitro droplets before, during and after photobleaching. Data displayed as mean ± SD. N = 20 images from two replicates. In d., f. luciferase values were normalized against an internal Renilla control, and the values are displayed as percentages normalized to the activity measured using an empty vector. Data are displayed as mean ± SD from three biological replicates. P values are from two-sided unpaired t-tests.
