Abstract 1962 Determination of the DNA-binding specificity of the extant SIX transcription factor family

Gene expression is defined as an intricate process that interprets the information encoded in our DNA. The regulation of gene expression is crucial to allow cell differentiation and organism development. Gene regulation is primarily controlled by transcription factors (TFs), sequence-specific proteins that bind to DNA in locations called cis-regulatory elements (CREs) to enhance or suppress the expression of genes. Our research focuses on a family of evolutionary conserved animal TFs known as the sine oculis homeobox (SIX) family. Initial investigations identified SIX TFs in the fruit fly Drosophila melanogaster as playing a crucial role in the development of the eye. In humans, at least six TFs from the SIX family play a critical role in the development of several organs and tissues, including the retina, brain, kidney, and muscle. Initial findings identified the sequence GTAANNTGATAC as the DNA-binding site for members of the SIX TF family. This investigation aims to express the sole SIX TFs family members found in the marine sponge Amphimedon queenslandica and the three SIX TFs found in the cnidarian Nematostela vectensis, the most distant ancestors where these TFs have been identified. We achieved the expression of these proteins using a eukaryotic cell-free system, and the DNA-binding activity of proteins was evaluated using an Electrophoretic Shift Mobility Assay (EMSA). Determination of specific DNA-binding sites for these TFs will be assessed through the Systematic Evolution of Ligands by Exponential Enrichment followed by DNA sequencing (SELEX-seq). This study will help us understand the protein-DNA interactions of SIX TFs and how they have changed through evolutionary time. This research was supported by NIH Grant # SC1GM127231, RISE #5R25GM061151-22, and Fideicomiso para Ciencia, Tecnología e Investigación de Puerto Rico