Fig. 6: Optimizing aromatic dispersion in NGN2 enhances neural differentiation.
From: An activity-specificity trade-off encoded in human transcription factors
a, Schematic models of wild-type and mutant NGN2 proteins (left). The positions of the bHLH DBD (grey box) and aromatic amino acids (yellow dots) are indicated. Omega plots and ΩAro scores (right). b, Fluorescence intensity of NGN2 wild-type and AroPERFECT IDR in in vitro droplets before, during and after photobleaching. Data are the mean ± s.d. of n = 20 droplets pooled from two independent replicates. c, Schematic model of the NGN2-mediated human iPSC-to-neuron differentiation experiment. ROCKi, Rho-kinase inhibitor. d, Representative fluorescence microscopy images of differentiating human iPSCs expressing the indicated NGN2 proteins. Hoechst dye was used as a nuclear counterstain; mEGFP, NGN2-T2A–mEGFP. Insets: magnified views of the regions in the white boxes. Scale bars, 0.1 mm (main images) and 0.05 mm (insets). e, Number of cells, based on Hoechst nuclear staining, in the NGN2-directed differentiation experiments. f, Neurite density (fraction of tubulin-covered area) in the NGN2-directed differentiation experiments. e,f, Data are the mean ± s.d. of n = 6 images pooled from two independent experiments. P values from a two-sided unpaired Student’s t-test. g, Principal component analysis of the RNA-seq expression profiles of parental ZIP13K2 human iPSCs and human iPSCs expressing the indicated NGN2 transgenes. h, Differential expression analysis of human iPSCs expressing the indicated transgenes. NGN2 target genes are highlighted. P values were determined using the Benjamini–Hochberg method. i, Heatmap representation of ChIP–Seq read densities of cells expressing wild-type, AroLITE and AroPERFECT NGN2 within a 1.5 kb window around all shared NGN2 peaks (top), differentially enriched peaks in AroPERFECT NGN2 (centre) and differentially enriched peaks in wild-type NGN2 (bottom). FE, fold over input. j, NGN2 differential binding at the TMEM97 locus. Genome browser tracks of ChIP–Seq data after 24 and 48 h of NGN2 expression are displayed. The arrowhead highlights a differentially bound peak at 24 h. The coordinates are hg38 genome assembly coordinates. k, Nascent transcription (TT-SLAM-Seq) metagene profiles at approximately 9,000 NGN2 target genes. TSS, transcription start site; TES, transcription end site.
