Prediction of cooperative homeodomain DNA binding sites from high-throughput-SELEX data

• Published in: Nucleic acids research 2023-07, Vol.51 (12), p.6055-6072
• Main Authors: Cain, Brittany, Webb, Jordan, Yuan, Zhenyu, Cheung, David, Lim, Hee-Woong, Kovall, Rhett A, Weirauch, Matthew T, Gebelein, Brian
• Format: Article
• Language: English
• Subjects: Animals; Binding Sites; DNA - chemistry; Gene regulation, Chromatin and Epigenetics; High-Throughput Nucleotide Sequencing; Homeodomain Proteins - metabolism
• ISSN: 0305-1048; 1362-4962
• DOI: 10.1093/nar/gkad318

Abstract Homeodomain proteins constitute one of the largest families of metazoan transcription factors. Genetic studies have demonstrated that homeodomain proteins regulate many developmental processes. Yet, biochemical data reveal that most bind highly similar DNA sequences. Defining how homeodomain proteins achieve DNA binding specificity has therefore been a long-standing goal. Here, we developed a novel computational approach to predict cooperative dimeric binding of homeodomain proteins using High-Throughput (HT) SELEX data. Importantly, we found that 15 of 88 homeodomain factors form cooperative homodimer complexes on DNA sites with precise spacing requirements. Approximately one third of the paired-like homeodomain proteins cooperatively bind palindromic sequences spaced 3 bp apart, whereas other homeodomain proteins cooperatively bind sites with distinct orientation and spacing requirements. Combining structural models of a paired-like factor with our cooperativity predictions identified key amino acid differences that help differentiate between cooperative and non-cooperative factors. Finally, we confirmed predicted cooperative dimer sites in vivo using available genomic data for a subset of factors. These findings demonstrate how HT-SELEX data can be computationally mined to predict cooperativity. In addition, the binding site spacing requirements of select homeodomain proteins provide a mechanism by which seemingly similar AT-rich DNA sequences can preferentially recruit specific homeodomain factors. Graphical Abstract Graphical Abstract Cooperativity Predictor utilizes HT-SELEX data to predict if a homeodomain (HD) transcription factor has the capability to bind to DNA cooperatively and generates cooperative dimer site DNA motifs for each prediction. The pipeline was validated through quantitative EMSAs and analysis of available genomic binding data.