Missense variants in human forkhead transcription factors reveal determinants of forkhead DNA bispecificity

Recognition of specific DNA sequences by transcription factors (TFs) is a key step in transcriptional control of gene expression. While most forkhead (FH) TFs bind either an FKH (RYAAAYA) or an FHL (GACGC) recognition motif, some FHs can bind both motifs. Mechanisms that control whether a FH is monospecific versus bispecific have remained unknown. Screening a library of 12 reference FH proteins, 61 naturally occurring missense variants including clinical variants, and 22 designed mutant FHs for DNA binding activity using universal ("all 10-mer") protein binding microarrays (PBMs) revealed non-DNA-contacting residues that control mono- versus bispecificity. Variation in non-DNA-contacting amino acid residues of TFs is associated with human traits and may play a role in the evolution of TF DNA binding activities and gene regulatory networks. HighlightsO_LIMost forkhead (FH) proteins recognize FKH or FHL motifs, while others are bispecific C_LIO_LIDNA binding activities of 12 reference and 83 variant or designed FHs C_LIO_LIClinical or population FH variants with altered DNA binding affinity or specificity C_LIO_LINon-DNA-contacting amino acid residues synergistically control mono- versus bi- specificity C_LI