BACH2 represses genes associated with aTreg differentiation. (A) Distribution of genome-wide BACH2 binding sites identified by ChIP-Seq in iTreg cells relative to annotated features of known genes. Peaks called based on enrichment of ChIP-Seq reads over input reads (binomial distribution FDR < 5%). (B) Histogram showing differences in gene expression as measured by bulk RNA-Seq of all expressed transcripts within EGFP⁺ Treg cells from tamoxifen-treated animals of the indicated genotypes. Transcripts are categorized into those that do not have, or have one or more, BACH2 binding sites within 50 kb of their TSS (binomial distribution FDR < 5% and fivefold enrichment over input). Significant difference in the distribution of BACH2-bound and nonbound genes was assessed using Wilcoxon rank sum test with continuity correction. (C) Heatmap showing expression of differentially expressed genes in Treg cells from animals of indicated genotypes (fold-change > 1.5, FDR < 0.2; Wald test with Benjamini–Hochberg correction). Frequency of BACH2 binding sites within 50 kb of TSS of identified genes indicated to the right of the expression heatmap. Fragments per kilobase of transcript per million mapped reads normalized to row maxima. Replicates and genes are hierarchically clustered on the x and y axes, respectively. Peaks called based on enrichment over input (binomial distribution FDR < 5% and 20-fold enrichment over input). (D) Representative alignments of ChIP-Seq measurements showing binding of BACH2 and p300 at the indicated gene loci, and RNA-Seq measurements from EGFP⁺ Treg cells of tamoxifen-treated Bach2^+/+Foxp3^{EGFP-Cre-ERT2} (WT) and Bach2^fl/flFoxp3^{EGFP-Cre-ERT2} (CKO) animals; RNA-Seq from three independent biological replicates per genotype are shown.