Transgenic analysis of the atrialnatriuretic factor (ANF) promoter: Nkx2-5 and GATA-4 binding sites are required for atrial specific expression of ANF.
The atrial natriuretic factor (ANF) gene is initially expressed throughout the myocardial layer of the heart, but during subsequent development, expression becomes limited to the atrial chambers. Mouse knockout and mammalian cell culture studies have shown that the ANF gene is regulated by combinatorial interactions between Nkx2-5, GATA-4, Tbx5, and SRF; however, the molecular mechanisms leading to chamber-specific expression are currently unknown. We have isolated the Xenopus ANF promoter in order to examine the temporal and spatial regulation of the ANF gene in vivo using transgenic embryos. The mammalian and Xenopus ANF promoters show remarkable sequence similarity, including an Nkx2-5 binding site (NKE), two GATA sites, a T-box binding site (TBE), and two SRF binding sites (SREs). Our transgenic studies show that mutation of either SRE, the TBE or the distal GATA element, strongly reduces expression from the ANF promoter. However, mutations of the NKE, the proximal GATA, or both elements together, result in relatively minor reductions in transgene expression within the myocardium. Surprisingly, mutation of these elements results in ectopic ANF promoter activity in the kidneys, facial muscles, and aortic arch artery-associated muscles, and causes persistent expression in the ventricle and outflow tract of the heart. We propose that the NKE and proximal GATA elements serve as crucial binding sites for assembly of a repressor complex that is required for atrial-specific expression of the ANF gene.
Small
Eric M
EM
Department of Cell Biology and Anatomy, University of Arizona Health Sciences Center, Tucson, AZ 85724, USA.
Krieg
Paul A
PA
D000818:Animals; D030801:Animals, Genetically Modified; D009320:Atrial Natriuretic Factor / Q000235:genetics*; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D050991:GATA4 Transcription Factor; D018507:Gene Expression Regulation, Developmental; D049452:Green Fluorescent Proteins; D006325:Heart Atria / Q000378:metabolism; D000072576:Homeobox Protein Nkx-2.5; D018398:Homeodomain Proteins / Q000378:metabolism*; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics; D051379:Mice; D008955:Models, Cardiovascular; D008969:Molecular Sequence Data; D009154:Mutation; D009206:Myocardium / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000235:genetics; D014157:Transcription Factors / Q000378:metabolism*; D029867:Xenopus Proteins / Q000378:metabolism*; D014982:Xenopus laevis
D004268:DNA-Binding Proteins; D050991:GATA4 Transcription Factor; C494751:GATA4 protein, Xenopus; D000072576:Homeobox Protein Nkx-2.5; D018398:Homeodomain Proteins; D008164:Luminescent Proteins; C092720:NKX2-5 protein, Xenopus; C095552:NKX2-5 protein, human; C484469:Nkx2-5 protein, mouse; D011994:Recombinant Proteins; D014157:Transcription Factors; D029867:Xenopus Proteins; D049452:Green Fluorescent Proteins; D009320:Atrial Natriuretic Factor; D004247:DNA