biostudies-literatureLeng FHoward Hughes Medical InstituteNIAID NIH HHSNIH116633https://www.ebi.ac.uk/biostudies/studies/S-EPMC12820563biostudies-literatureUnknown44(12)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 isCell reportsFoxP3 forms a head-to-head dimer in vivo and stabilizes its multimerization on adjacent microsatellites.PMC12820563R01 AI180137R01AI180137Zhang WViennet THur SWang CClark RLeng FWang XArthanari HfalseFoxP3 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 is2025-01-01T00:00:00Z2025 Dec2026-06-06T19:23:35.002Z2026-06-04T03:12:58.931ZS-EPMC128205634131368110.1016/j.celrep.2025.116633