BACH2 restrains TCR-driven AP-1 binding in Treg cells. (A) De novo consensus motif (top motif) and selected known motifs from the JASPAR database (bottom motifs) enriched within BACH2 binding sites in iTreg cells (binomial distribution FDR < 5%). Average motif positions relative to BACH2 peak centers, the significance of their enrichment within BACH2 binding sites, and the number BACH2 binding sites containing the specified motifs (of 9,133 total sites) are shown. E values, multiple hypergeometric test using randomly shuffled input sequences. (B) Genome-wide colocalization analysis of BACH2 and JunD binding sites (binomial distribution FDR < 5%) in iTreg cells. The number of overlapping and nonoverlapping sites are shown by the Venn diagram. (C) Colocalization histogram showing the distribution of distances between BACH2 peak summits and nearest JunD peak. (D) Representative alignments of ChIP-Seq measurements showing binding of BACH2, JunD, and p300 at the indicated gene loci. Selected putative regulatory elements at which BACH2 and JunD bind are indicated with arrows. (E) Enrichment of JunD at the indicated putative regulatory elements in naive FACS-sorted CD4⁺ T cells from Bach2^+/+Foxp3^{EGFP-Cre-ERT2} and Bach2^fl/flFoxp3^{EGFP-Cre-ERT2} animals cultured under iTreg conditions in the presence of 4-hydroxytamoxifen. Input-normalized JunD enrichment was assessed by ChIP-qPCR after brief (2 h) stimulation with anti-CD3 relative to negative control locus Ankrd52. Data representative of two pooled experiments with two culture replicates per genotype. (F) Analysis of known changes in mRNA expression following stimulation of WT or Bach2-deficient naive CD4⁺ T cells cultured under iTreg conditions and harvested at the indicated time points (Sidwell et al., 2020). ns, not significant; * P < 0.05; ** P < 0.01; ***, P < 0.001; ****, P < 0.0001; Wilcoxon–Mann–Whitney test (E) and unpaired two-tailed Student’s t test (F). Numbers in gates show percentages. Bars and error show mean and SEM.