FoxP3 recognizes microsatellites and bridges DNA through multimerization

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, with each pair forming a "rung". 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.