Abstract 1545 Synergistic Properties of TBX5 and GATA4 on Cardiac Gene Regulatory Activity

Transcription factors (TFs) are sequence-specific DNA-binding proteins that control biological processes, such as cellular differentiation and development, by regulating gene expression. In eukaryotes, gene expression is a complex process that is highly regulated and occurs in a tissue-specific manner dictated by TF interactions. One way that TFs drive gene expression is by recognizing and binding to cis-regulatory elements (CREs), such as enhancers and promoters. Cardiac TFs TBX5 and GATA4 are master regulators of cardiac gene expression and heart development and have been shown to work cooperatively as a heterodimeric protein complex to regulate cardiac gene expression. However, the exact manner by which these TFs interact with each other and bind to their respective CREs is still unknown. In this work, we tested the heterodimeric protein capacities of TBX5 and GATA4 to understand how they interact together to bind to its respective DNA sequence and promote or inhibit cardiac gene expression. Firstly, we expressed TBX5 and GATA4 proteins using a cell-free wheat germ system and verified their expression via Western Blot. Secondly, their DNA-binding activity was tested by Electrophoretic Mobility Shift Assays (EMSA). Lastly, using in vitro DNA-binding specificity data CREs were validated through EMSA. We selected the calmodulin binding transcription activator 1 (Camta1) gene, which is a possible target gene regulated by TBX5 and GATA4. We tested TBX5 and GATA4's heterodimeric protein binding properties to Camta1 DNA sequence via EMSA. Our results show both TBX5 and GATA4 as a monomer bind to Camta1. In addition, the heterodimeric binding of TBX5 and GATA4 to Camta1 DNA shows increased binding, which illustrates the synergic properties of TBX5 and GATA4. This approach evaluated the regulatory capacities of cardiac TFs that work cooperatively to regulate gene expression. Our findings will help us understand the intricacies and molecular mechanisms that TFs play in eukaryotic gene regulation. U-RISE Rio Piedras Program (R25GM061151), the National Science Foundation GRFP Award (1744619), and National Institute of Health (SC1GM127231).