DNA binding analysis of rare variants in homeodomains reveals homeodomain specificity-determining residues

Homeodomains (HDs) are the second largest class of DNA binding domains (DBDs) in eukaryotic sequence-specific transcription factors (TFs) and are the TF structural class with the largest number of disease mutations in the Human Gene Mutation Database (HGMD). Despite numerous structural studies and large-scale analyses of HD DNA binding specificity, HD-DNA recognition is still not fully understood. Here, we analyzed 92 human HD mutants, including disease-associated variants and variants of unknown significance (VUS), for their effects on DNA binding activity. Many of the variants altered DNA binding affinity and/or specificity. Structural analysis identified 14 novel specificity-determining positions, 5 of which do not contact DNA. The same missense substitution at analogous positions within different HDs exhibited different effects on DNA binding activity. Variant effect prediction tools perform moderately well in distinguishing variants with altered DNA binding affinity, but poorly in identifying those with altered binding specificity. Our results highlight the need for biochemical assays of TF coding variants and promote dozens of variants for further investigations into their pathogenicity and the development of clinical diagnostics and precision therapies. 123 homedomains were assayed by protein binding microarray (PBM).