Protein‐DNA Interactomes of NKX2‐5 and TBX5 Mutants Associated with Congenital Heart Defects

Abstract

Congenital Heart Defects (CHDs) is the most common neonatal disease in the U.S. with over 100,000 new reported cases each year. CHDs are characterized by malformations in the heart's chambers, walls and great vessels, sometimes leading to embryonic death. Cardiac Transcription Factors (cardiac TFs) NKX2-5 and TBX5 play a critical role in developmental and physiological processes of the heart by regulating gene expression through DNA-protein interactions. Genetic analyses have identified various non-synonymous mutations within the DNA-binding domain (DBD) of NKX2-5 (Homeodomain, HD) and TBX5 (T-box) in association with CHDs. We hypothesize that non-synonymous mutations in cardiac TFs DNA-binding domain (DBD) will alter their DNA-binding properties ultimately affecting transcriptional pathways necessary for proper heart development. To address this hypothesis, we study how the cardiac TFs DNA-protein interactomes are affected by non-synonymous mutations found in CHD patients. We have cloned, over-expressed and purified functional DBDs of wild type (wt) NKX2-5 (HD) and TBX5 (T-box). In addition, we generated 8 non-synonymous mutations (A148E, E154G, R161P, T178M, Q181H, R189P, R190C, Y191C) of NKX2-5 HD and 6 non-synonymous mutations (I54T, M74V, G80R, I101F, R113K, R237Q) of TBX5 T-box identified in CHD. Functional analysis of NKX2-5 HD mutants A148E, R161P, T178M, Q181H, R189P, Y191C showed retention of DNA binding. Similarly, TBX5 T-box mutants I54T, M74V, G80R, I101F, R113K, R237Q displayed DNA binding activity. This loss in TF DNA binding activity and recognition suggests a link of non-synonymous mutations deleterious effects in TF functions, which can lead to CHD. To comprehensively measure the protein-DNA interactomes of NKX2-5 HD and TBX5 T-box non-synonymous mutants, we performed High-Throughput Systematic Evolution of Ligands and Exponential Enrichment sequencing (SELEX-seq) and bioinformatics tools to predict changes in gene targets between wt and mutant TFs. Interestingly, SELEX-seq on TBX5 T-box wt and its mutant I54T reveals that both TF variants bind to similar AGGTGTCG and GGAGGTGT motifs. Further bioinformatic analysis will determine underlining differences in TF DNA binding activity that impact cardiac TFs’ crucial functions, ultimately revealing molecular mechanisms in CHD.