Abstract 2441 Determining the DNA-binding sites for the SIX family of transcription factors in the Drosophila melanogaster free of chromatin landscape environment

Transcription factors (TF) are DNA-binding proteins that orchestrate DNA transcription, either promoting or repressing gene expression. Therefore, TFs are essential to maintain control of the amount of RNA generated, acting as an early mode of gene regulation. Sine oculis homeobox (SIX) TFs found in both vertebrae and invertebrates were initially associated with retina development, nonetheless these TFs have been found to be involved in the early development of mesodermal, skeletal, and connective tissue in embryogenesis. Although these TFs are essential for gene regulation, there is no data on DNA-binding sites across the complete Drosophila melanogaster genome without the potential effects of chromatin. The objective of this study is to identify the DNA-binding preferences of the SIX family members in the Drosophila melanogaster genome free of the chromatin landscape environment. We focused on performing DNA-affinity purification followed by sequencing (DAP-seq). Firstly, we created a Drosophila melanogaster genomic library with Illumina adapters. Additionally, the genomic library was subjected to several rounds of polymerase chain reaction (PCR) to remove methylated DNA. In parallel, we expressed sine oculis, optix, and Six4, the SIX family TFs from Drosophila melanogaster, using a cell-free system. These expressed TFs were shown to be functionally active through Electrophoretic Mobility Shift Assay (EMSA). Following the completion of the genomic library and TFs expression, DAP-seq will be performed by equilibrating the TFs and genomic library to form TF-DNA complexes. The mixture will be subjected to immunoprecipitation to isolate the protein-DNA complexes. Once isolated, the DNA from the complexes will be separated and amplified using PCR and will be sequenced using next-generation sequencing. The resulting sequencing will be analyzed through peak calling to obtain the TFs DNA-binding sites across the genome. Evaluation of these binding sites will provide regions of regulations important to biological function in Drosophila melanogaster.