FoxP3 forms a head-to-head dimer in vivo and stabilizes its multimerization on adjacent microsatellites.

FoxP3, the master regulator of Tregs, employs two DNA-binding modes to recognize diverse DNA sequences. It multimerizes on long TnG repeats (n = 2-5) to bridge DNA segments and stabilize chromatin loops, and it forms head-to-head (H-H) dimers on inverted repeat forkhead motifs (IR-FKHM) without bridging DNA. Although genomic data confirm its multimeric role, in vivo evidence for H-H dimerization has been elusive. Here, unbiased pull-down sequencing uncovers a range of relaxed motifs that drive H-H dimerization, enabling systematic genome-wide analysis. We demonstrate that FoxP3 binds genomic DNA as both H-H dimers and multimers in Tregs, with H-H binding often seeding and stabilizing multimerization on adjacent TnG repeats-especially on shorter, suboptimal repeats. While multimerization is conserved across FoxP family members, H-H dimerization is unique to FoxP3 orthologs, conferred by its divergent accessory loop. This dual-mode strategy broadens FoxP3's sequence repertoire and enhances its architectural function in chromatin looping.