Fig. 4: Optimizing aromatic dispersion in C/EBPα enhances transactivation.
From: An activity-specificity trade-off encoded in human transcription factors
a, Schematic models of wild-type and mutant C/EBPα proteins (left). The positions of the bZIP DBD (grey box) and aromatic residues (orange dots) are indicated. Omega plots and ΩAro scores (middle). Results of luciferase reporter assays (right). Data are the mean ± s.d. of n = 3 biological replicates with three technical replicates each. b, Representative images of droplet formation of purified C/EBPα IDR–mEGFP fusion proteins at the indicated concentrations in droplet formation buffer. Scale bars, 5 μm. c, Fluorescence intensity of C/EBPα wild type, AroLITE and AroPERFECT IS15 IDR in in vitro droplets before, during and after photobleaching. Data are the mean ± s.d. of n = 15 (wild-type) and 14 (AroPERFECT IS15 and AroPERFECT IS10) droplets from two replicates. d, Fluorescence images of ectopically expressed YFP–RNAPII CTD in live U2OS cells that were cotransfected with the indicated CFP–LacI-C/EBPα IDR fusion constructs. The dashed line represents the nuclear contour. Inserts: magnified views of the regions in the red boxes. Scale bars, 10 μm (main images) and 40 μm (inserts). e, Relative YFP signal intensity in the tether foci. Data are the mean ± s.d. of n = 51 (wild-type YFP, AroPERFECT YFP and wild-type YFP–RNAPII CTD) and 56 (AroPERFECT YFP–RNAPII CTD) nuclei pooled from two independent replicates. f, Results of a C/EBPα IDR tiling experiment using luciferase reporter assays. C/EBPα wild type and AroPERFECT IS15 IDR 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, Results of luciferase reporter assays of the indicated IDR constructs. a,f,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. f,g, Data are the mean ± s.d. of n = 3 biological replicates. a,e,g, P values are from a two-sided unpaired Student’s t-tests.
