Chorionic enhancer is dispensable for regulated expression of the human renin gene

Abstract

We tested the hypothesis that a transcriptional chorionic enhancer (CE), previously identified to increase human renin expression in choriodecidual cells is required to mediate tissue-specific, cell-specific, and regulated expression of human renin in transgenic mice. Recombineering was used to delete the CE upstream of the renin gene alone or in combination with the kidney enhancer (KE) in a large artificial chromosome construct containing the entire human renin gene and extensive flanking sequences. Deletion of the CE had no qualitative or quantitative effect on the tissue-specific expression of human renin, nor on the cellular localization of human renin in the kidney or placenta. Combined deletion of both the CE and KE caused a decrease in the level of renal renin expression consistent with the established role of the KE. We also considered the possibility that the CE is a downstream enhancer of the KiSS1 gene, which lies directly upstream of renin and is also expressed in the placenta. Deletion of the CE alone, or the CE and KE together, had no effect on the level of KiSS1 expression in the placenta. These data provide convincing evidence that the CE is silent in vivo, at least in the mouse. The absence of a phenotype caused by deletion of the CE is consistent with the observation that the sequence is not evolutionarily conserved.

Figure 1. Generation of the PAC160 Transgenes

Schematic representation of the PAC160 and mutant transgenes showing the hREN gene (blue), the kidney enhancer (KE, red) and its replacement by a lox511 site (red crosshatched arrow), the chorionic enhancer (CE, green) and its replacement by a lox2272 site (green crosshatched arrow), and neighboring genes (closed arrows). The direction of transcription is indicated by the direction of the arrows. The two terminal genes PEPP3 and Sox13 gene are truncated by the end of PAC160 as indicated by open boxes. The GOLT1A and KiSS1 genes lie upstream of hREN, whereas ETNK2 lies directly downstream. The LoxP site present in the PAC parent vector is indicated by the solid yellow arrowhead.

Figure 2. Southern Blot of PAC160ΔKE Transgenic Mice

A. Schematic representation of the region upstream of the hREN gene showing the location of the four probes (a-d) used in the Southern blots shown in B and C. B and C. Southern blots of genomic DNA isolated from mice carrying wildtype PAC160 (WT), lines carrying PAC160ΔCE, and lines carrying PAC160ΔCEKE transgenes. The numbers (8, 12, 16, 17,18) indicate the line numbers. The probes are indicated to the left of each blot. The DNA was digested with BamHI (B) and MscI (C). The blots in B initially probed with probe b was striped and re-probed with probe c.

Figure 3. Expression of the PAC160 Transgenes

A. Representative multiplex RNase Protection Assays of hREN, GOLT1A, ETNK2 and 28S expression from total tissue RNA (50 μg) from male or female WT, PAC160ΔCE lines 14 and 16, PAC160ΔCEKE lines 8, 12 and 17 is shown. The location of the protected fragments is indicated. Tissues labels are: brain (B), heart (H), kidney (K), liver (L), lung (Lg), skeletal muscle (S), submandibular gland (Sg), testis (T) and uterus (U). Like PAC160CE and PAC160CEKE, hREN is expressed in the uterus of PAC160-WT mice (data not shown).

Figure 4. Influence of CE and CEKE Deletion on hREN Expression

A. A representative RPA was performed with total kidney RNA from independent kidney samples from a representative line of wild type PAC160, PAC160ΔCEKE, and PAC160ΔCE mice. The position of the hREN, ETNK2, and Cyclophilin protected products is indicated. B. RPAs were quantified using the phosphorimager software and ratio of hREN/ETNK2 is shown. *, P<0.001 vs wildtype by ANOVA. The N is indicated within each bar. The RPAs in A were taken from a single large gel.

Figure 5. Induction of Renal hREN and mREN mRNA in Response to Captopril

Total kidney mRNA was isolated from the indicated lines and analyzed by RNase protection assay (RPA) using probes specific for either mREN or hREN and normalized for expression of β-actin.

Figure 6. Immunostaining of Human Renin in Kidney

Representative immunofluorescent images of human renin staining in the kidney of non-transgenic (NT) and PAC160ΔCEKE17, PAC160ΔCE14 and PAC160ΔCE16. The arrows indicate human renin stained cells.

Figure 7. Co-localization of hREN and mREN in Kidney

Representative immunofluorescent images of mREN (red) and hREN (green) staining in the kidney of non-transgenic (NT), PAC160 wildtype (WT), PAC160ΔCE16, and PAC160ΔCEKE17.

Figure 8. Placental Expression of hREN and KiSS1

Representative RNase Protection Assays of hREN (A) and KiSS1 (B) expression from total placental RNA (18.5 gd) from the indicated lines of WT, PAC160ΔCE, PAC160ΔCEKE mice are shown. The location of the protected fragments are indicated. The individual samples are derived from a mix of transgenic (expressing) and non-transgenic (non-expressing, i.e. lane 3) placentas.

Figure 9. In Situ Hybridization of Human Renin in Kidney and Placenta

Representative in situ hybridization images of human renin mRNA in the kidney (A,B) and placenta (C-F) of PAC160 wildtype (WT), PAC160ΔCEKE, and PAC160ΔCE. AS, antisense probe; S, sense probe. The image in panel B is a higher magnification from the same slide as panel A.

Figure 10. In Situ Hybridization of Human Renin and PL2 in Placenta

Representative dual in situ hybridization images of human renin mRNA (blue) and PL2 mRNA (brown) in the placenta of PAC160 wildtype (WT), PAC160ΔCEKE, PAC160ΔCE, and nontransgenic (NT) mice. Only PL2 expression is seen in the non-transgenic placenta. Open arrowhead denote PL2 expressing cells; Closed arrowhead denotes hREN expressing cell; regular arrows denotes cells co-expressing hREN and PL2.