Protein‐DNA Interactomes of NKX2‐5 and TBX5 Mutants Identified in Congenital Heart Defects

Abstract

Congenital Heart Defects (CHDs) are the most common birth defect from children to adults in the United States. CHDs are characterized by malformations in the heart’s chambers, walls, and great vessels, sometimes leading to embryonic death. Cardiac Transcription Factors (cardiac TFs) such as NKX2‐5 and TBX5 play a critical role in developmental stages and physiological processes of the heart by regulating gene expression through DNA‐protein interactions. Genetic analyses have identified various non‐synonymous mutations within the homeodomain (HD) DNA‐binding domain (DBD) of NKX2‐5 and the (Tbox) DBD of TBX5 in associated with CHD. We hypothesize that non‐synonymous mutations in cardiac TFs DBD will alter their DNA‐binding properties ultimately affecting transcriptional pathways necessary for proper heart development. To address this hypothesis, we studied how the cardiac TFs DNA‐protein interactomes are affected by non‐synonymous mutations found in CHD patients. We expressed and purified the DBD of NKX2‐5 (HD), TBX5 (Tbox), also NKX2‐5 and TBX5 non‐synonymous mutations (A148E, E154G, R161P, T178M, Q181H, R190C, Y191C), and (I54T, M74V, G80R, I101F, R113K, R237Q, R237W) respectively. Functional analysis using Electrophoretic Mobility Shift Assay (EMSA) confirms HD and Tbox binding to cognate DNA binding sites. DNA‐binding activity as assessed by EMSA, shows retention of DNA activity in the case of NKX2‐5 and TBX5 mutants A148E, R161P, Y191C and I54T, R113K respectively. Mutants T178M, Q181H, R190C, M74V, G80R, I101F, R237Q, R237W showed complete loss in DNA binding activity towards their specific cognate sites. We measured the comprehensive protein‐DNA interactomes of NKX2‐5 and TBX5 non‐synonymous mutants using High‐Throughput Systematic Evolution of Ligands and Exponential Enrichment (SELEX‐seq) to identify changes in DNA targets between wild‐type and mutant TFs. Mutants A148E, R161P and Y191C bind to similar binding sites as NKX2‐5 (HD). Interestingly, mutant Y191C showed an altered DNA binding site significantly different from NKX2‐5 (HD). Mutants I54T, R113K bind to similar binding sites as TBX5 (Tbox). Mutants E154G, T178M, Q181H, R190C, M74V, G80R, I101F, R237Q and R237W showed no significant enrichment for specific DNA binding sites. Integrative analysis with available NKX2‐5 and TBX5 genomic binding sites data (ChIP‐seq), human and mouse cardiac enhancers and promoters catalogued in the literature will facilitate the identification of putative genes mutant cardiac TFs are regulating and how different are from their wild‐type TFs.