Abstract
AimsTranscription factor GATA4 is a dosage sensitive regulator of heart development and alterations in its level or activity lead to congenital heart disease (CHD). GATA4 has also been implicated in cardiac regeneration and repair. GATA4 action involves combinatorial interaction with other cofactors such as NKX2-5, another critical cardiac regulator whose mutations also cause CHD. Despite its critical importance to the heart and its evolutionary conservation across species, the structural basis of the GATA4-NKX2-5 interaction remains incompletely understood.Methods and ResultsA homology model was constructed and used to identify surface amino acids important for the interaction of GATA4 and NKX2-5. These residues were subjected to site-directed mutagenesis, and the mutant proteins were characterized for their ability to bind DNA and to physically and functionally interact with NKX2-5. The studies identify 5 highly conserved amino acids in the second zinc finger (N272, R283, Q274, K299) and its C-terminal extension (R319) that are critical for physical and functional interaction with the third alpha helix of NKX2-5 homeodomain. Integration of the experimental data with computational modeling suggests that the structural arrangement of the zinc finger-homeodomain resembles the architecture of the conserved DNA binding domain of nuclear receptors.ConclusionsThe results provide novel insight into the structural basis for protein-protein interactions between two important classes of transcription factors. The model proposed will help to elucidate the molecular basis for disease causing mutations in GATA4 and NKX2-5 and may be relevant to other members of the GATA and NK classes of transcription factors.
Highlights
A homology model was constructed and used to identify surface amino acids important for the interaction of GATA4 and NKX2-5. These residues were subjected to site-directed mutagenesis, and the mutant proteins were characterized for their ability to bind DNA and to physically and functionally interact with NKX2-5
These residues were subjected to site-directed mutagenesis and the mutant proteins were characterized for their ability to bind DNA and to physically and functionally interact with NKX2-5 and with another GATA4 cofactor, Krüppellike zinc finger KLF13 [32]
The results identify specific residues within the GATA4 second zinc finger domain and C-terminal extension that are differentially required for NKX2-5 interaction
Summary
Such networks are commonly regulated through combinatorial interactions of transcription factors. GATA family transcriptional regulators and their co-factors control cell fate decisions in multiple tissues from worms to mammals [1,2,3]. In the developing murine heart, GATA4 is one of the earliest-expressed transcription factors [4] and is indispensable for normal cardiac development [5, 6]. GATA4 is expressed in the adult heart, acting as a key transcriptional regulator of numerous cardiac genes including those encoding atrial natriuretic peptide (ANP), B-type natriuretic peptide (BNP), α-myosin heavy chain (α-MHC), β-MHC, and many others [13, 14]. GATA4 acts as a critical regulator of hormone response and mechanical stress as well as cardiomyocyte survival and myocardial remodeling [15,16,17,18,19,20,21,22]
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