DNA binding specificity of cardiac transcription factor complex GATA4 and NKX2‐5

Abstract

Transcription factors (TFs) are sequence‐specific DNA binding proteins responsible for cellular differentiation and development. Eukaryotic TFs are notorious for not working alone and they often bind DNA as multiprotein complexes to regulate gene expression, raising the possibility that complex formation might change their individual DNA binding specificities. Transcription factors GATA4 and NKX2‐5 are central components of the gene regulatory network that controls heart development. However, the DNA‐binding specificity of the cooperative complex between GATA4:NKX2‐5 remain undetermined. We want to identify the intrinsic DNA binding preferences and the emergent properties that result from this complex. In this study, we expressed full‐length GST‐Tagged GATA4 and NKX2‐5 using wheat germ cell‐free system. We validated DNA‐binding activity of GATA4 and NKX2‐5 through Electrophoretic Mobility Shift Assays (EMSA). We successfully determined the in vitro DNA‐binding specificity of GST‐GATA4, GST‐NKX2‐5 and GST‐GATA4:GST‐NKX2‐5 complex using Cognate Site Identification by High‐Throughput Systematic Evolution of Ligands by Exponential Enrichment (SELEX‐seq). DNA‐specificity models for GATA4, NKX2‐5 and GATA4:NKX2‐5 complex were generated using various computational tools. Specificity models will be used to make genome‐wide binding predictions. Findings of this study will help understand the spatial and temporal gene regulation rules involved in normal heart development.Support or Funding InformationThis project is funded by National Science Foundation Bridge to the Doctorate Award HRD‐1400868 and National Institutes of Health SC1GM127231.