DNA‐Binding Specificity of Cardiac Transcription Factor Complex Formed by NKX2‐5 and TBX5

Abstract

Transcription factors (TFs) are sequence-specific DNA-binding proteins responsible for cellular differentiation and development. Eukaryotic TFs are known for binding DNA as multimeric complexes to regulate gene expression. Transcription factors NKX2-5 and TBX5 are fundamental elements of the gene regulatory networks that control mammalian heart development. More importantly, NKX2-5 and TBX5 can bind to DNA as a cooperative complex. Our aim was to identify the intrinsic DNA-binding preferences and the motif spacing and orientation pattern of the NKX2-5:TBX5 heterodimer complex. In this study, we expressed full-length GST-tagged NKX2-5 and TBX5 using a wheat germ cell-free system. We successfully determined the in vitro DNA-binding specificity of the NKX2-5:TBX5 complex using Systematic Evolution of Ligands by Exponential Enrichment (SELEX-seq). Our preliminary results shows NKX2-5:TBX5 binding in a head-to-tail orientation with 0bp and 3bps spacing between core binding sites: GGTGT for TBX5 and CACTT for NKX2-5. The spacing preference is consistent with ChIP-exo data from cardiomyocytes and cardiac precursors. Our results highlight the utility of using in vitro techniques to determined DNA-binding specificity to understand genomic binding. The findings of our study will help understand the spatial and temporal gene regulation rules involved in normal heart development.