Bispecific Forkhead Transcription Factor FoxN3 Recognizes Two Distinct Motifs with Different DNA Shapes

Transcription factors (TFs) control gene expression by binding DNA recognition sites in genomic regulatory regions. Although most forkhead TFs recognize a canonical forkhead (FKH) motif, RYAAAYA, some forkheads recognize a completely different (FHL) motif, GACGC. Bispecific forkhead proteins recognize both motifs, but the molecular basis for bispecific DNA recognition is not understood. We present co-crystal structures of the FoxN3 DNA binding domain bound to the FKH and FHL sites, respectively. FoxN3 adopts a similar conformation to recognize both motifs, making contacts with different DNA bases using the same amino acids. However, the DNA structure is different in the two complexes. These structures reveal how a single TF binds two unrelated DNA sequences and the importance of DNA shape in the mechanism of bispecific recognition. [Display omitted] •FoxN3 recognizes two different DNA motifs, both in vitro and in cells•Co-crystal structures of FoxN3 with both sites were determined•Differences in DNA shape enable FoxN3 to bind the two sites of different length•Parts of the forkhead domain that do not contact DNA influence binding specificity Transcription factors typically bind DNA sites related by sequence similarity. Some forkhead factors bind two classes of DNA sequences of different length and sequence composition. Rogers et al. show the mechanism of this bispecificity in FoxN3, revealing that DNA conformation differences enable the same protein to bind both sites.