Extended Data Fig. 7: RBPJ binds to the FOXP3 promoter and modulates histone acetylation.
From: Genome-wide CRISPR screen in human T cells reveals regulators of FOXP3
a, Comparison of FOXP3hi versus FOXP3lo gRNA enrichment scores within specific domains, related to the saturation mutagenesis experiment in Fig. 4a. Regions surrounding the amino acid positions 235 and 362 have been removed from the BTD and CTD domains, respectively. BTD, β-trefoil DNA-binding domain; AA, amino acid. P-values by a two-tailed Wilcoxon signed-rank test. b, RBPJ-overexpressing human CD4+ iTreg cells (n = 3 independent donors) were assessed for FOXP3 expression and quantified for %FOXP3+ (left) and FOXP3 MFI (bottom). c, RBPJ overexpression has minimal effects on FOXP3 in nTregs, as assessed by flow cytometry (n = 4 independent donors). d, Electromobility shift assays (EMSA) showing binding of RBPJ to the FOXP3 promoter. Lysate was prepared from RBPJ-overexpressing iTreg cells (Methods). Data is representative of two independent experiments. e, Analysis of %FOXP3+ cells (left) and FOXP3 MFI (right) after iTreg polarization in HDAC3-deficient human CD4+ T cells (n = 4 donors). f,g, Summary of H3K9ac ChIP-seq signal enrichment at FOXP3. All data are plotted as ± s.e.m. Repeated measures one-way ANOVA with Dunnett’s multiple comparison test for c; two-tailed paired Student’s t-test in b. Values plotted in a represent CRISPRO gRNA enrichment scores calculated using data from three independent biological replicates.
