Molecular basis for DNA recognition by the maternal pioneer transcription factor FoxH1

Abstract Nucleosomes are barriers for the binding of most transcription factors, but pioneer factors (PFs) do bind and facilitate subsequent interactions of other proteins during transcription activation. FoxH1 is a maternal PF essential during embryonic development that interacts with specific GK-forkhead targets. How FoxH1 binds DNA targets has remained elusive for decades until now. We have determined high-resolution structures of human, frog and fish proteins bound to four DNAs. We found that the FoxH1 DNA-binding domain is almost twice the size of other FOX proteins, allowing for a highly specific binding to both minor and major grooves. Consistent with its PF activity, we also quantified that the affinity for DNA is even higher for native mononucleosomes than for linear DNA. Our structures illustrate how binding to distinct GK sites allows FoxH1 to avoid cross-regulation by other FOX proteins that also operate during the maternal-zygotic transition and select canonical TT-forkhead sites.