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Protein‐DNA Interactomes of NKX2‐5 Mutants Identified in Congenital Heart Defects
Author(s) -
Carrasquillo-Dones Emmanuel Antonio,
César Brenda,
Rodriguez-Martinez Jose Arcadio
Publication year - 2020
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2020.34.s1.06732
Subject(s) - homeobox , transcription factor , biology , genetics , mutant , dna , gene , mutagenesis , mutation , tbx1 , dna sequencing , electrophoretic mobility shift assay , computational biology , gene expression , promoter
Congenital Heart Defects (CHDs) are one of the most common birth defects, 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. NKX2‐5 is cardiac transcription factors (cardiac TFs) that play a critical role in developmental stages and physiological process of the heart. Transcription factors regulate gene expression via sequence‐specific interactions with DNA. Genetic analyses of CHD patients have identified various non‐synonymous mutations within the DNA‐binding domain (DBD) of NKX2‐5. We hypothesize that non‐synonymous mutations in NKX2‐5’s DBD will alter their DNA‐binding properties ultimately affecting transcriptional pathways necessary for proper heart development. To address this hypothesis, we want to study how the cardiac TFs DNA‐protein interactomes are affected by non‐synonymous mutations found in CHD patients. We have successfully cloned, over‐expressed and purified the homeodomain (HD) DBD of NKX2‐5. Functional analysis protocols using Electrophoretic Mobility Shift Assay (EMSA) confirms protein binding to its DNA binding sites. Additionally, we generated 7 non‐synonymous NKX2‐5 CHD mutants (A148E, E154G, R161P, L171R, T178M, Q181H, R189P) via site directed mutagenesis. Comprehensive protein‐DNA interactomes of NKX2‐5 and its mutants will be determined by Systematic Evolution of Ligands and Exponential Enrichment (SELEX‐seq). Bioinformatics tools will be used predict changes in DNA‐binding preferences and putative gene targets between wild‐type and mutant TFs. Support or Funding Information This research was supported: NIH grant award SC1GM127231 Research Initiative for Scientific Enhancement (RISE) Grant #5R25GM061151‐18