Human Molecular Genetics Advance Access originally published online on May 25, 2005
Human Molecular Genetics 2005 14(14):1965-1975; doi:10.1093/hmg/ddi202
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Published by Oxford University Press 2005
Functional dissection of sequence-specific NKX2-5 DNA binding domain mutations associated with human heart septation defects using a yeast-based system
1Chromosome Stability Section, Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, NIH, PO Box 12233, Research Triangle Park, NC 27709, USA and 2Department of Drug Research and Medical Biotechnology, Fraunhofer Institute of Toxicology and Experimental Medicine, Nikolai-Fuchs-Strasse 1, D-30625 Hannover, Germany
* To whom correspondence should be addressed. Email: resnick{at}niehs.nih.gov and Juergen.Borlak{at}item.fraunhofer.de
Received March 18, 2005; Revised May 10, 2005; Accepted May 20, 2005
Human heart development requires an orderly coordination of transcriptional programs, with the homeodomain protein NKX2-5 being one of the key transcription factors required for the differentiation of mesodermal progenitor cells. Indeed, lack of Nkx2-5 in mice arrests heart development prior to looping, resulting in embryonic lethality. There are 28 germline NKX2-5 mutations identified in humans that are associated with congenital heart disease, and we recently reported multiple somatic mutations in patients with complex cardiac malformations. To address the functional consequences of single and multiple mutations of NKX2-5, we developed a functional assay in the budding yeast Saccharomyces cerevisiae, which could determine transactivation capacity and specificity of expressed NKX2-5 alleles towards targeted response element (RE) sequences. We focused on mutants of the third helix, which provides DNA binding specificity, and characterized mutations that were highly associated with either ventricular (VSD) or atrioventricular (AVSD) septal defects. Individual mutants exhibited partial to complete loss of function and differences in transactivation capacity between the various REs. The mutants also exhibited gene dosage rather than dominant effects on transcription. Surprisingly, all AVSD patients (22/23) had a single K183E mutation in the DNA binding domain, which resulted in transcriptional inactivation. None of the VSD patients had this mutation; yet 14/29 had at least one mutation in the third helix leading to either inactivation or reduction of NKX2-5 transactivation. Therefore, mutations of somatic origin in the binding domains of NKX2-5 were associated specifically with AVSD or VSD and resulted in loss of protein function.
Present address: Laboratory of Experimental Oncology B, Department of Translational Oncology, National Cancer Research Institute, IST, Largo R. Benzi X, 16132 Genoa, Italy.
These two authors contributed equally as Senior Authors.
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