Fig. 5: Optimizing aromatic dispersion in C/EBPα enhances macrophage reprogramming, and leads to stronger and more promiscuous genomic binding.

a, Schematic models of wild-type and mutant C/EBPα proteins. The transactivation data are identical to the data displayed in Fig. 4a. P values are from two-sided unpaired Student’s t-tests. b, Schematic model of C/EBPα-mediated transdifferentiation of B cells to macrophages. c, FACS quantification of GFP⁺ RCH-rtTA cells encoding C/EBPα overexpression cassettes. The proportions of CD19⁻ Mac1⁺ cells were measured 48, 96 and 168 h after transgene induction. Data are the mean ± s.d. of n = 5 (wild type and AroPERFECT IS15) and 3 (AroLITE and AroPERFECT IS10) independent experiments. d, Graph-based clustering (uniform manifold approximation and projection, UMAP) of the scRNA-seq data of C/EBPα-mediated transdifferentiation. Clusters were annotated based on marker genes. Overlayed is the partition-based graph abstraction (PAGA) showing the cell trajectory based on dynamic modelling of RNA velocity. Inset: pseudotime plot. e, Proportion of mEGFP⁺ cells in the macrophage clusters (colour-coded as in d). f, Heatmap representation of ChIP–Seq read densities of wild-type and AroPERFECT IS15 C/EBPα within a 1.5-kb window around all shared C/EBPα peaks and differentially enriched peaks in AroPERFECT IS15 C/EBPα. ‘Peaks unique to IS15 and reported before’ denotes binding sites differentially enriched in IS15 binding that overlap C/EBPα peaks reported in previous literature. FE, fold enrichment. g, Enrichment scores of bZIP TF motifs and adjusted (adj.) P values of enrichment at the three indicated peak sets. P values were determined using the Benjamini–Hochberg method. h,j, AroPERFECT IS15 C/EBPα shows enhanced binding at the FAM98A (h) and GBP5 (j) loci. Displayed are genome browser tracks of ChIP–Seq data of C/EBPα 24 and 48 h after C/EBPα induction. The coordinates are hg38 genome assembly coordinates. i,k, UMAPs coloured on FAM98A (i) and GBP5 (k) expression. The numbers denote the mean ± s.d. expression in the whole samples. l, Luciferase assays using the indicated reporter plasmids cotransfected with expression vectors encoding either wild-type or AroPERFECT IS15 C/EBPα. Luciferase values were normalized to an internal Renilla control and the values are displayed as percentages of the activity measured using the ‘basic’ vector. Data are the mean ± s.d. of four biological replicates. P values are from two-sided unpaired Student’s t-tests.