A tyrosine-rich domain within homeodomain transcription factor Nkx2-5 is an essential element in the early cardiac transcriptional regulatory machinery.
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.
Elliott
David A
DA
Victor Chang Cardiac Research Institute, Darlinghurst, Sydney 2010, Australia.
Solloway
Mark J
MJ
Wise
Natalie
N
Biben
Christine
C
Costa
Mauro W
MW
Furtado
Milena B
MB
Lange
Martin
M
Dunwoodie
Sally
S
Harvey
Richard P
RP
D000595:Amino Acid Sequence; D000818:Animals; D000831:Animals, Newborn; D015153:Blotting, Western; D002460:Cell Line; D002478:Cells, Cultured; D049830:Cephalopoda; D017124:Conserved Sequence; D024202:Electrophoretic Mobility Shift Assay; D004622:Embryo, Mammalian; D004625:Embryo, Nonmammalian; D018507:Gene Expression Regulation, Developmental; D018390:Gene Targeting; D017930:Genes, Reporter; D005982:Glutathione Transferase / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006579:Heterozygote; D000072576:Homeobox Protein Nkx-2.5; D018398:Homeodomain Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D017403:In Situ Hybridization; D008156:Luciferases / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D009206:Myocardium / Q000166:cytology / Q000378:metabolism* / Q000473:pathology; D032383:Myocytes, Cardiac / Q000166:cytology / Q000378:metabolism; D010802:Phylogeny; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014443:Tyrosine / Q000737:chemistry*
D000072576:Homeobox Protein Nkx-2.5; D018398:Homeodomain Proteins; C484469:Nkx2-5 protein, mouse; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D049452:Green Fluorescent Proteins; D014443:Tyrosine; D008156:Luciferases; D005982:Glutathione Transferase