Extended Data Fig. 5: Optimizing aromatic dispersion enhances the activity of multiple TF IDRs.
From: An activity-specificity trade-off encoded in human transcription factors
a. AlphaFold models of OCT4, PDX1 and FOXA3. b. (left) Schematic models of OCT4 (top), PDX1 (middle) and FOXA3 (bottom) wild type and mutant sequences. (right) Results of luciferase reporter assays. Note that shown AroPERFECT IDRs have stronger transactivation capacity as their respective wild type sequences. c. Western blot of GAL4-DBD and GAL4-DBD-OCT4-IDR- (top), GAL4-DBD-PDX1-IDR- (middle) and GAL4-DBD-FOXA3-IDR- (bottom) fusion proteins in HEK293T cells 24 hours after transfection using a GAL4-DBD specific antibody. HSP90: loading control. Wild type and AroPERFECT mutants are expressed at comparable levels. d. Results of a OCT4 C-IDR tiling experiment by using luciferase reporter assays. Sequences were tiled into fragments of 40 amino acids with 20 amino acid overlaps. The activities of the full-length IDRs are indicated with dashed horizontal lines. e. (left) Schematic model of EGR1 IDR wild type and mutant sequences. Aromatic amino acids are highlighted as orange dots. (right) Results of luciferase reporter assays. f. Results of a EGR1 IDR tiling experiment by using luciferase reporter assays. Sequences were tiled into fragments of 40 amino acids with 20 amino acid overlaps. The activities of the full-length IDRs are indicated with dashed horizontal lines. g. (left) Schematic model of HOXB1 IDR wild type and AroPERFECT sequences. Aromatic amino acids are highlighted as orange dots. (middle) Omega plots and ΩAro scores of the IDRs. (right) Results of luciferase reporter assays. In b., e., g. 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. N = 3 for OCT4, N = 2 for FOXA3 and N = 2 for PDX1 from independent replicates. P-values are from two-sided unpaired t-tests. *: P < 0.05, ***: P < 10−3. DBD: DNA-binding domain; IDR: intrinsically disordered region; AD: activation domain.
