Abstract
Homeodomain factor Nkx2-5 is a central component of the transcription factor network that guides cardiac development; in humans, mutations in NKX2.5 lead to congenital heart disease (CHD). We have genetically defined a novel conserved tyrosine-rich domain (YRD) within Nkx2-5 that has co-evolved with its homeodomain. Mutation of the YRD did not affect DNA binding and only slightly diminished transcriptional activity of Nkx2-5 in a context-specific manner in vitro. However, the YRD was absolutely essential for the function of Nkx2-5 in cardiogenesis during ES cell differentiation and in the developing embryo. Furthermore, heterozygous mutation of all nine tyrosines to alanine created an allele with a strong dominant-negative-like activity in vivo: ES cell<-->embryo chimaeras bearing the heterozygous mutation died before term with cardiac malformations similar to the more severe anomalies seen in NKX2.5 mutant families. These studies suggest a functional interdependence between the NK2 class homeodomain and YRD in cardiac development and evolution, and establish a new model for analysis of Nkx2-5 function in CHD.
MeSH terms
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Amino Acid Sequence
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Animals
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Animals, Newborn
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Blotting, Western
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Cell Line
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Cells, Cultured
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Cephalopoda
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Conserved Sequence
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Electrophoretic Mobility Shift Assay
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Embryo, Mammalian
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Embryo, Nonmammalian
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Gene Expression Regulation, Developmental
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Gene Targeting
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Genes, Reporter
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Glutathione Transferase / metabolism
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Green Fluorescent Proteins / metabolism
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Heterozygote
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Homeobox Protein Nkx-2.5
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Homeodomain Proteins / chemistry
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Homeodomain Proteins / genetics*
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Homeodomain Proteins / metabolism*
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In Situ Hybridization
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Luciferases / metabolism
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Mice
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Molecular Sequence Data
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Mutation
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Myocardium / cytology
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Myocardium / metabolism*
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Myocardium / pathology
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Myocytes, Cardiac / cytology
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Myocytes, Cardiac / metabolism
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Phylogeny
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Protein Structure, Tertiary
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Recombinant Fusion Proteins / metabolism
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Reverse Transcriptase Polymerase Chain Reaction
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Sequence Homology, Amino Acid
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Transcription Factors / chemistry
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Transcription Factors / genetics*
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Transcription Factors / metabolism*
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Transcription, Genetic*
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Transcriptional Activation
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Tyrosine / chemistry*
Substances
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Homeobox Protein Nkx-2.5
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Homeodomain Proteins
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Nkx2-5 protein, mouse
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Recombinant Fusion Proteins
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Transcription Factors
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Green Fluorescent Proteins
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Tyrosine
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Luciferases
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Glutathione Transferase