DNA-binding specificity of cardiac transcription factor complexes

Abstract

Transcription factors (TFs) are sequence-specific DNA-binding proteins responsible for cellular differentiation and development. In eukaryotes, TFs often function as multimeric complexes to regulate gene expression. NKX2-5, GATA4, and TBX5 are key TFs in the gene regulatory network that controls heart development. More importantly, NKX2-5, GATA4, and TBX5 are known to synergize in activating cardiac target genes. However, the impact of cooperative DNA-binding on specificity and transcriptional regulation remains poorly understood. This thesis aimed to identify the intrinsic DNA-binding preferences of the complexes formed by NKX2-5:TBX5, GATA4:TBX5, and NKX2-5:GATA4. Using Systematic Evolution of Ligands by Exponential Enrichment followed by sequencing (SELEX-seq), we characterized the in vitro DNA-binding specificity of these complexes, including binding site orientation and spacing patterns. To the best of our knowledge, this represents the first comprehensive SELEX-seq analysis of these three cardiac TF complexes. Our results show that these heterodimers bind DNA with specific binding site orientation and spacing. Specifically, NKX2-5:TBX5 prefers a spacing preference of -1 bp, 1 bp, 3 bp, and 4 bp between binding sites. GATA4:TBX5 binds specifically to sequences with spacing of -1 bp, 2 bp, and 3 bp between binding sites. NKX2-5:GATA4 prefers spacing from 1 bp to 5 bp between binding sites. We used the MOODS algorithm to predict candidate cis-regulatory elements (cCREs) matching the position weight matrices (PWMs) of the heterodimer complex, identifying 6,552 candidate cCREs genome-wide. Thirty prioritized cCREs were validated via Electrophoretic Mobility Shift Assay (EMSA) and functionally tested in dual-luciferase reporter assays. Our results show that these TF complexes can bind as a heterodimer complex and increase or decrease gene expression depending on motif spacing. NKX2-5:TBX5 primarily represses at -1 bp and 1 bp spacing between binding sites. GATA4:TBX5 exhibits context-dependent activity, and NKX2-5:GATA4 drives activation as spacing increases between binding sites. Together, these findings demonstrate that DNA-binding grammar, specifically, the orientation and spacing of TF binding sites, plays a critical role in heterodimer binding and transcriptional regulation. This work offers new insights into the combinatorial logic of gene regulation during heart development and its implications for the molecular basis of congenital heart diseases.