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

## TL;DR

FoxP3 uses two DNA-binding strategies to regulate chromatin structure in Tregs, with head-to-head dimers helping stabilize multimerization on nearby DNA repeats.

## Contribution

Discovery of FoxP3's head-to-head dimerization and its role in stabilizing multimerization on suboptimal DNA repeats.

## Key findings

- FoxP3 binds DNA as both head-to-head dimers and multimers in Tregs.
- Head-to-head dimers seed and stabilize multimerization on adjacent TnG repeats.
- H-H dimerization is unique to FoxP3 orthologs due to its divergent accessory loop.

## Abstract

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.

The transcription factor FoxP3 binds DNA through two modes: multimerization on TnG repeats to stabilize chromatin loops and head-to-head dimerization on inverted forkhead motifs. Pull-down sequencing reveals relaxed motifs enabling head-to-head binding, which seeds FoxP3 multimerization on adjacent microsatellites. This dual strategy expands FoxP3’s sequence repertoire and chromatin-architectural function.