Abstract 1652 Investigating the DNA-binding specificity of cardiac transcription factors complexes

Transcription factors (TFs) are sequence-specific DNA-binding proteins responsible for cellular differentiation and development. Eukaryotic Transcription factors are known for binding DNA as multimeric complexes to regulate gene expression cooperatively. Transcription factors GATA4, NKX2-5, and TBX5, are fundamental elements of the gene regulatory network that controls heart development. More importantly, GATA4, NKX2-5, and TBX5 synergize to activate target genes. However, little is known about the impact of cooperative DNA-binding on specificity and its role in transcriptional regulation. Our objective is to identify the intrinsic DNA-binding preferences of the heterodimers complexes formed by NKX2-5:TBX5, NKX2-5:GATA4, and GATA4:TBX5, including binding site orientation and spacing patterns. In this work, I determined the in vitro DNA-binding specificity of the NKX2-5:TBX5 complex using Systematic Evolution of Ligands by Exponential Enrichment (SELEX-seq). Our SELEX-seq results show that this heterodimer binds DNA with specific binding site orientation and spacing. Specifically, NKX2-5:TBX5 prefers a spacing preference of -1bp, 3bp, and 1bp between binding sites. Identified binding sites were validated by Electrophoretic Mobility Shift Assays (EMSA). Published data from ChIP-exo confirmed that these binding sites occur in bound genomic regions. Candidate Cis-Regulatory Elements (CREs) will be evaluated using a dual-luciferase reporter assay to determine whether these complexes can activate gene expression in a synergistic manner. These findings will help understand the spatial and temporal gene regulation during heart development. National Science Foundation GRFP Award 1744619, National Institutes of Health SC1GM127231 and RISE 5R25GM061151-22.