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DNA‐Binding Properties of TBX5 Mutants Associated with Congenital Heart Defects
Author(s) -
Cesar Brenda I.,
Carrasquillo Emmanuel I.,
Zazaboi Jasmin,
Rodriguez Jose A.
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.s1.05530
Subject(s) - mutant , mutagenesis , gene , transcription factor , dna , electrophoretic mobility shift assay , dna binding domain , primer (cosmetics) , genetics , biology , context (archaeology) , mutation , dna sequencing , computational biology , chemistry , paleontology , organic chemistry
Congenital Heart Diseases (CHD) affects over 150,000 persons every year. CHDs are characterized by abnormal structural heart formation during embryonic development. Previous studies have identified several TBX5 mutations in CHD patients. TBX5 is a transcription factor (TF) that has been identified as an important regulator during heart development. TFs are sequence‐specific DNA binding proteins that regulate gene expression and control cellular state. We selected 9 non‐synonymous TBX5 mutants discovered in CHD patients. The consequences on the interactions between these mutated cardiac TFs and DNA have not formerly been investigated. Evaluation of DNA‐recognition properties of TBX5 is fundamental for understanding its possible roles in controlling gene regulatory networks and could potentially help us know more about the role of TFs in the context of CHD. Our lab plans to determine the sequences our TFs prefer and how this specificity changes when it mutates. In order to test this, we have cloned TBX5 and its T‐Box domain that contain a His‐ and Strep‐Tag affinity tags that we will use for purifying after we overexpress it in competent E. coli . cells. We have also generated all 9 sequence‐verified TBX5 mutants using site‐directed mutagenesis. In site‐directed mutagenesis, mutations are introduced by the use of a PCR primer pair carrying the intended mismatch. We have also optimized experimental conditions for the overexpression and affinity‐purification of the TBX5 T‐Box domain. The DNA‐binding activity of TBX5 DNA binding domain T‐box has been confirmed through electrophoretic mobility shift assays (EMSA) using the TBX5 cognate site. This assay allows us to visualize DNA‐protein affinity through electrophoretic gel shifts. The mutation I54T shows more affinity towards the TBX5 cognate site and the mutation G80R shows no affinity towards it. Identifying the effects of non‐synonymous mutations on the DNA‐binding properties of TBX5 will contribute to our understanding of TBX5 role during heart development in congenital heart defects. Support or Funding Information This research was funded by SC1GM127231