Cooperativity between head-to-head dimerization and head-to-tail multimerization of FoxP3 transcription factor

FoxP3, an essential transcription factor for regulatory T cells (Tregs), forms higher-order multimers on TnG microsatellites in the genome. Although a previous study has shown that FoxP3 can form head-to-head (H-H) dimers on inverted repeats of forkhead motifs (IR-FKHM), in vivo evidence for this configuration was elusive. An unbiased FoxP3 pull-down sequencing approach identified a wide range of sequences supporting H-H dimerization, enabling systematic genomic analysis. This work revealed that FoxP3 binds DNA in Tregs as both H-H dimers and higher-order multimers, often incorporating one half-site of the H-H dimer into TnG repeats. A robust H-H interaction can seed multimerization on adjacent TnG repeats, enhancing assembly even on suboptimal sequences. Unlike other FoxP transcription factors, H-H dimerization is unique to FoxP3. Chimeric construct analyses identified a loop adjacent to the forkhead domain as the determinant for this distinctive function, underscoring a unique synergism between two binding modes. Overall design: In vitro pulldown assay using a synthetic dsDNA oligonucleotide library to identify FoxP3-binding motifs. MBP-tagged FoxP3 and FoxP1 were used to pull down DNA sequences with potential binding affinity. The input library was included as a control. Pulldown-enriched DNA was PCR-amplified and sequenced using paired-end 150 bp reads on an Illumina NovaSeq 6000 platform. Follow-up in vitro pulldown experiment using the same synthetic dsDNA library, focused on comparative binding between FoxP3 and FoxP1. Samples were sequenced using single-end 130-cycle reads on an Illumina NextSeq 2000 platform.