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Dev Biol
2003 Sep 01;2611:116-31. doi: 10.1016/s0012-1606(03)00306-3.
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Transgenic analysis of the atrialnatriuretic factor (ANF) promoter: Nkx2-5 and GATA-4 binding sites are required for atrial specific expression of ANF.
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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.
Fig. 2. The 625-bp wild-type Xenopus ANF promoter fragment recapitulates the endogenous ANF expression pattern. (A) In situ hybridization analysis of
stage 45 nontransgenic heart showing ANF expression throughout the atrium, ventricle, and outflow tract. (B) Stage 49 nontransgenic Xenopus heart showing
restriction of ANF transcripts to the atrial myocardium. (C) Stage 44 transgenic embryo showing GFP reporter expression driven by the wild-type 625-bp
ANF promoter. This is a ventrolateral view of a living embryo under combined UV and visible light. GFP fluorescence can be observed in the eye, driven
from the control -crystallin promoter and throughout the heart. (D) Fluorescent image of embryo in (C), highlighting GFP reporter expression. (E) Heart
of living stage 44 transgenic embryo, with GFP expression driven from the wild-type 625-bp ANF promoter. Fluorescence is visible throughout the atrium,
ventricle, and outflow tract. (F) Fluorescent image of dissected stage 48 transgenic heart, showing that GFP reporter expression is now limited to the atria. (G) In situ hybridization analysis of dissected stage 45 transgenic heart, showing distribution of GFP transcripts throughout the heart. (H) In situ hybridization analysis of GFP transcripts in dissected stage 49 transgenic heart. Note restriction of transgene expression to the atria, confirming the expression pattern observed using fluorescence. Abbreviations: o, outflow tract; a, atria; v, ventricle.
Fig. 3. Mutational analysis of the Xenopus ANF promoter in transgenic embryos. The different ANF promoter constructions are illustrated at the left of the
figure. Numbers on the top line indicate the distance of the regulatory element upstream of the transcription start site. The number of independently generated
transgenic embryos is indicated in the first column and the relative activity of the mutant constructions (expressed to the nearest 5%) is presented in the second column. This number is calculated as the proportion of mutant transgene embryos showing visible reporter expression relative to the number of wild type transgene embryos showing reporter expression (see Materials and methods).
Fig. 4. Mutation of the NKE within the ANF promoter results in ectopic transgene expression outside of the heart. All transgenic embryos shown are mutant for the NKE, but identical results are observed for the GATAp mutant promoter. (A) Fluorescent image of a living stage 32 transgenic embryo. Ectopic
expression of reporter GFP is visible in the developing facial muscles (indicated by an arrow head). Expression in the brain, from the control crystallin
promoter, as indicated by an arrow. Apparent fluorescence in the flank of the embryo is background due to autofluorescence of yolk granules. Cardiac
expression of the transgene has not commenced at this stage. (B) In situ hybridization detection of GFP reporter gene expression in stage 32 embryo also shows transcripts in developing facial muscles (arrowhead). (C) Combined brightfield and UV image of stage 45 transgenic embryo. Ventrolateral view shows ectopic reporter expression in facial muscles and muscles associated with the third and fourth aortic arch artery. (D) Fluorescent image of the tadpole in (C) showing ectopic GFP expression in the interbranchialis I and II (ib1 and ib2) adjacent to the third and forth aortic arch arteries, the subarcualis obliquus (so) muscle, and the orbitohyoideus (or) muscle. Expression is also observed in the kidneys (ki). Transgene expression is strong in the heart (arrow). (E) Ventral
view of the head of a different transgenic embryo shows additional facial muscles expressing GFP. The orbitohyoideus (or) associated with the musculus
quadratohyangularis (qha) and the musculus interhyoideus (ih) are indicated. Control -crystallin promoter-driven GFP is observed in the eye. (F)
Dorsal-view of stage 45 transgenic embryo showing ectopic GFP expression in the kidneys (ki).
Fig. 5. Mutations of the NKE or proximal GATA (GATAp) element within the ANF promoter result in persistent transgene expression throughout the ventricle and
outflow tract. (A) In situ hybridization analysis of dissected nontransgenic stage 49 heart, showing restriction of endogenous ANF transcripts to the atrium. Note
patchy expression of endogenous gene. (B) Fluorescent image of GFP expression in dissected stage 49 heart transgenic for the wild-type ANF promoter. Reporter expression is restricted to atria and also shows patchy expression. (C) Fluorescent image of dissected stage 52 transgenic heart. Mutation of the NKE within the ANF promoter results in persistent expression of the GFP reporter throughout the ventricle, and outflow tract. This ectopic expression persists for at least 3 weeks after expression of endogeneous ANF, or the wild-type ANF transgene, has restricted to the atria. (D) Fluorescent image of GATAp mutant transgene expression in the heart of a living stage 50 embryo. Expression is visible throughout the ventricle and outflow tract, exactly as observed with the NKE mutant transgene.
Fig. 6. A model of ANF transcriptional regulation by Nkx2-5, GATA-4, and SRF. (A) In the early myocardium and in the mature atria activation of ANF
transcription is proposed to be regulated by a complex of Nkx2-5, SRF and cofactor myocardin, and GATA-4. (B) Inhibition of ANF transcription in the
ventricle may occur due to posttranslational modification of Nkx2-5, which results in exposure of an inhibitory domain within the protein. This modification
must also abolish Nkx2-5–SRF interactions since mutation of the Nkx2-5 binding site within the ANF promoter results in loss of inhibition in the ventricle. (C) A second inhibitory model relies on a hypothetical repressor protein that becomes expressed (or activated) in the ventricle after stage 47. This repressor protein would bind to the NKE and displace Nkx2-5 from the ANF promoter. (D) A third possible inhibitory mechanism results from the binding of a ventricle-specific repressor protein directly to Nkx2-5. This interaction between Nkx2-5 and the candidate repressor must abolish the ability of Nkx2-5 to interact with SRF, and therefore, to be tethered to the promoter in the absence of a functional NKE
