Abstract
The 36 Paired-like homeodomain transcription factors in humans are required for the development of many cell types, tissues, and organs as missense variants in 24 Paired-like genes have been associated with numerous diseases and developmental disorders. How these factors identify distinct genomic targets using highly similar DNA binding domains is not fully understood. Here, we focus on determining how the human Paired-like homeodomain factors gain DNA binding specificity by cooperatively binding palindromic sites spaced three base pairs apart (P3 site). Through structural, biochemical, and bioinformatic approaches, we define 11 rules that describe homeodomain residues that are critical, permissive, and inhibitory to cooperativity on the P3 site. Applying these rules, we successfully alter the cooperative behavior of Paired-like factors, identify residues that prevent the related Antennapedia class of homeodomains from binding cooperatively, and predict that thirty-eight disease-associated missense variants across ten Paired-like proteins alter cooperativity. Using quantitative DNA binding assays, we confirm eleven of twelve of these disease-associated variants impact cooperativity but not DNA binding affinity. These findings reveal the importance of cooperativity in defining DNA binding specificity and highlight how missense variants associated with as many as fifteen different diseases can selectively disrupt cooperative DNA binding.