Fig. 2: Increasing aromatic dispersion in TF IDRs enhances transactivation.
From: An activity-specificity trade-off encoded in human transcription factors
a, Schematic models of HOXD4 IDRs (left). Aromatic residues (orange dots) and alanine mutations (white dots) are highlighted. Additionally introduced tyrosines are also shown as red dots. Omega plots of the HOXD4 IDRs and ΩAro scores (middle). Results of luciferase reporter assays (right). Data are from three biological replicates. b, Representative images of droplet formation of purified HOXD4 IDR–mEGFP fusion proteins at the indicated concentrations in droplet formation buffer. Scale bars, 5 μm. c, Relative amount of condensed protein per concentration quantified in the droplet formation assays. Data are the mean ± s.d. of n = 15 images from three replicates. The curves were generated as nonlinear regressions to a sigmoidal curve function. d, Fluorescence intensity of wild-type and AroPERFECT HOXD4 in vitro droplets before, during and after photobleaching. Data are the mean ± s.d. of n = 20 images from two replicate imaging experiments. e, Results of a HOXD4 IDR tiling experiment 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. A predicted activation domain (AD) in the HOXD4 wild-type IDR is highlighted (light blue bar). Luciferase activity is reported as the fold change relative to cells transfected with empty vector. f, Results of luciferase reporter assays of the indicated HOXD4 IDR constructs. The position of the 40-mer tile containing the AD in e is illustrated. Data are from three biological replicates. g, Schematic models of synthetic sequences (left); tyrosine residues are highlighted (orange dots). Results of luciferase reporter assays (right). Data are from two (bottom) or three (top) biological replicates. a,e–g, Luciferase values were normalized to an internal Renilla control and the values are displayed as percentages normalized to the activity measured using an empty vector. Data are the mean ± s.d. *P < 0.05, **P < 0.01 and ***P < 1 × 10−3; two-sided unpaired Student’s t-test.
