Ladder-like multimerization of FoxP3 enables microsatellite recognition and DNA bridging

FoxP3 is a transcription factor (TF) essential for development of regulatory T cells (Tregs), a branch of T cells that suppress excessive inflammation and autoimmunity1-5. Molecular mechanisms of FoxP3, however, remain elusive. We here show that FoxP3 utilizes the forkhead domain--a DNA binding domain (DBD) that is commonly thought to function as a monomer or dimer--to form a higher-order multimer upon binding to TnG repeat microsatellites. A cryo-electron microscopy structure of FoxP3 in complex with T3G repeats reveals a ladder-like architecture, where two double-stranded DNA molecules form the two "side rails" bridged by five pairs of FoxP3 molecules forming "rungs". Each FoxP3 subunit occupies TGTTTGT within the repeats in the manner indistinguishable from that of FoxP3 bound to the forkhead consensus motif (FKHM; TGTTTAC). Mutations in the "intra-rung" interface impair TnG repeat recognition, DNA bridging and cellular functions of FoxP3, all without affecting FKHM binding. FoxP3 can tolerate variable "inter-rung" spacings, explaining its broad specificity for TnG repeat-like sequences in vivo and in vitro. Both FoxP3 orthologs and paralogs show similar TnG repeat recognition and DNA bridging. These findings thus reveal a new mode of DNA recognition that involves TF homo-multimerization and DNA bridging, and further implicates microsatellites in transcriptional regulation and diseases.