The DNA Binding Diversity of the SIX Homeodomain Transcription Factor Family

Abstract

Transcription factor (TF) DNA‐binding specificity is determined by how their DNA‐binding domain (DBD) interacts with DNA. TFs are identified by the sequence homology shared with described DBDs, which allows them to be classified into families. It is highly widely accepted that similar DBDs recognize similar DNA motifs. However, changes in a TF can lead to changes in its DNA recognition. TFs are critical for development and have been found since the beginning of animals, how their DNA binding changes through time has not been fully studied. TFs members of the sine oculis homeobox (SIX) homeodomain family are found from sponges to humans and are considered atypical members of the homeodomain family. They regulate numerous developmental processes, with phenotypic features spanning from eye development in flies, red color patterning in Heliconius butterfly wings to brain development in humans. How evolutionary related TFs diversify their function has not been fully understood, especially changes to their DNA binding specificity. Using full length SIX proteins from Drosophila melanogaster, Heliconius erato, and Homo sapiens, we performed in vitro Systematic Evolution of Ligands by Exponential Enrichment (SELEX‐seq) to identify the DNA binding specificity. Our preliminary data shows the majority bind to their canonical binding motif (TGATAC), except for six4 members which prefer TGACAC. Interestingly, the way they bind to these motifs differs. Both sine oculis and six4 homologs require a 5’‐GA flanking the core motif. While optix related members prefer a shorter flaking region with less dependence on 5’‐GA. This is interesting since optix is more evolutionarily related to six4 than to sine oculis. In addition, we found that Heliconius erato optix can bind both as a homodimer with a preferred spacing of 2‐bp between binding sites or as a monomer. Our results provide new insights in the binding diversity found in the SIX family of TFs and predict genomic targets of these understudied TF family.