Journal of Receptors and Signal Transduction
ISSN: 1079-9893 (Print) 1532-4281 (Online) Journal homepage: http://www.tandfonline.com/loi/irst20
A mouse renin distal enhancer is essential for blood pressure homeostasis in BAC-rescued reninnull mutant mice Keiji Tanimoto, Sumiyo Kanafusa, Aki Ushiki, Hitomi Matsuzaki, Junji Ishida, Fumihiro Sugiyama & Akiyoshi Fukamizu To cite this article: Keiji Tanimoto, Sumiyo Kanafusa, Aki Ushiki, Hitomi Matsuzaki, Junji Ishida, Fumihiro Sugiyama & Akiyoshi Fukamizu (2014) A mouse renin distal enhancer is essential for blood pressure homeostasis in BAC-rescued renin-null mutant mice, Journal of Receptors and Signal Transduction, 34:5, 401-409 To link to this article: http://dx.doi.org/10.3109/10799893.2014.908917
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Date: 12 September 2015, At: 16:30
http://informahealthcare.com/rst ISSN: 1079-9893 (print), 1532-4281 (electronic) J Recept Signal Transduct Res, 2014; 34(5): 401–409 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/10799893.2014.908917
RESEARCH ARTICLE
A mouse renin distal enhancer is essential for blood pressure homeostasis in BAC-rescued renin-null mutant mice Downloaded by [Karolinska Institutet, University Library] at 16:30 12 September 2015
Keiji Tanimoto1,2, Sumiyo Kanafusa3, Aki Ushiki3, Hitomi Matsuzaki1,2, Junji Ishida1,2, Fumihiro Sugiyama4, and Akiyoshi Fukamizu1,2 1
Faculty of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki, Japan, 2Life Science Center of Tsukuba Advanced Research Alliance (TARA), University of Tsukuba, Tsukuba, Ibaraki, Japan, 3Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki, Japan, and 4Laboratory Animal Resource Center, University of Tsukuba, Tsukuba, Ibaraki, Japan
Abstract
Keywords
Renin is predominantly expressed in juxtaglomerular cells in the kidney and regulates blood pressure homeostasis. To examine possible in vivo functions of a mouse distal enhancer (mdE), we generated transgenic mice (TgM) carrying either wild-type or mdE-deficient renin BACs (bacterial artificial chromosome), integrated at the identical chromosomal site. In the kidneys of the TgM, the mdE contributed 80% to basal renin promoter activity. To test for possible physiological roles for the mdE, renin BAC transgenes were used to rescue the hypotensive renin-null mice. Interestingly, renal renin expression in the TgBAC:renin-null compound mice was indistinguishable between the wild-type and mutant BAC carriers. Surprisingly, however, the plasma renin activity and angiotensin I concentration in the mdE compound mutant mice were significantly lower than the same parameters in the control mice, and the mutants were consistently hypotensive, demonstrating that blood pressure homeostasis is regulated through transcriptional cis elements controlling renin activity.
Bacterial artificial chromosome, blood pressure, renin, transcriptional regulation, transgenic mice
Introduction The renin-angiotensin system (RAS) plays a pivotal role in the regulation of blood pressure and electrolyte homeostasis. Renin, synthesized primarily in the juxtaglomerular (JG) cells of the kidney, cleaves its unique substrate, angiotensinogen, a plasma protein synthesized predominantly in the liver, to produce the decapeptide angiotensin I (AI). AI is further cleaved by angiotensin-converting enzyme (ACE) to produce an octapeptide, angiotensin II (AII), which increases blood pressure through vasoconstriction and aldosterone secretion by binding to angiotensin receptors. In this cascade, renin catalyzes the first, rate-limiting reaction and thus plays a central role in regulation of the overall activity of RAS. A number of physiological stimuli, including renal sympathetic activity, sodium balance, and pharmacological blockade of AII synthesis or receptor function modulate renin secretion and transcription in the kidney (1). Although the levels expressed elsewhere are much lower than in the kidney, renin is also expressed in extra-renal tissues, such as the adrenal gland, testes, ovaries, heart, and submandibular gland (SMG).
Address for correspondence: Keiji Tanimoto, Faculty of Life and Environmental Sciences, University of Tsukuba, Tennoudai 1-1-1, Tsukuba, Ibaraki 305-8577, Japan. Tel/Fax: (+81) 29-853-6070. E-mail: [email protected]
History Received 27 January 2014 Revised 23 March 2014 Accepted 24 March 2014 Published online 15 April 2014
In order to understand pathogenesis of hypertension associated with the RAS activity, it is crucial to understand transcriptional mechanisms of renin, which is characterized by its cell-type specificity and strong inducibility by cAMP (2,3). A number of candidate cis regulatory sequences have been identified in the renin gene promoters by cell transfection assays (4–8) and some of them have also been evaluated in vivo. Although usual DNA constructs (less than 20 kb) have been widely used to investigate the regulation of gene activity in transgenic mice (TgM), they often carry less regulatory information and are more susceptible to position-of-integration site effects, which can most often generate significant line-to-line heterogeneity in the level of transgene (Tg) expression. Gene knock-outs also have a potential drawback, i.e. mutating a cis-regulatory element that is essential for efficient gene expression may lead to compensatory activation of other cis elements for the gene, which may mask transcriptional phenotypes primarily caused by mutations. To avoid such situations, in our previous work we employed bacterial artificial chromosomes (BAC) to generate TgM to evaluate putative cis regulatory element activities in vivo (9,10). As anticipated, identical transgene constructs, when integrated at distinct chromosomal loci, exhibited similar (but never identical) levels of transgene expression. To fully circumvent possible position-of-integration site effects, we further employed a transgene coplacement strategy, in which
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activities of wild-type and mutant sequences can be directly compared at the identical chromosomal integration position (9,11,12). Importantly, by using a set of wild-type and mutant transgenes for rescuing knock-out mice of the gene, the physiological significance of cis-regulatory sequences can also be examined. Using this BAC transgenic coplacement methodology, we have previously evaluated two of the putative renin regulatory sequences, CNRE (overlapping the cAMP-responsive element [CRE] and the negative regulatory element [NRE]) and RP-2 (renin proximal promoter element-2), both of which are potentially responsive to cAMP. The results clearly demonstrated that the CNRE sequence was dispensable for basal and cell-type specific renin gene transcription, as well as for its responsiveness to several physiological stimuli, and for blood pressure homeostasis. On the other hand, RP-2 sequence was indispensable for basal level transcription in the kidney, as well as for blood pressure homeostasis (10). In the present study, we focused on another potential cAMP-responsive, cis-regulatory element, the mdE (mouse distal enhancer; (13)), and examined its functional significance, again in BAC TgM. The mdE, defined by 240 bp DNA sequence, was identified by transient transfection of 50 deletion constructs of the mouse Ren-1c gene into As4.1 cells. This tissue-specific enhancer is located 2.6 kb upstream (2866 to 2625 bp) of the transcriptional start site of the mouse renin gene and its homologous sequence is present about 12 kb upstream of the human renin start site of transcription (14,15). The mdE bears clustered binding sites for multiple transcription factors, including E-box proteins Figure 1. Experimental design. (A) The RPCI23-240p23 BAC (192 kb insert) carries all the nine exons of the Ren-1c gene. The FRT (shaded oval), as well as SfiI restriction enzyme sites have been introduced into 30 -untranslated region of the gene (10). BstBI restriction enzyme fragment (155 kb) with approximately 66 kb and 80 kb of 50 - and 30 -flanking sequences, respectively, was used for generating TgM. (B) Structural analysis of the transgenic mdE region. In vivo Cre-loxP recombination shown in the left was confirmed by Southern blot analysis of tail DNA from parental, wt, and mut TgM lines, in which the DNA was digested with EcoRI (E), separated on agarose gels, transferred to a nylon membrane, and hybridized to the probe (shaded rectangles in the left panel; from nt 2635 to 2214 relative to transcription start site). The sizes of the expected bands are indicated (in kb) in the right panel and those expected from the endogenous locus are in parentheses. (C) Wild-type (wt, top) and mutant (mut, bottom) murine mdE sequences. Several transcription factor-binding sites are located within the deleted portion (broken line) (4).
J Recept Signal Transduct Res, 2014; 34(5): 401–409
USF1/USF2 (16), CREB/CREM (16), NF-Y (15), RAR/RXR (17), Ear2 (18), Coup-TFII (19), etc. (20,21) (wt in Figure 1C), and activates renin gene transcription by 50-fold on transfection into As4.1 cells (13). Our transgenic approach disclosed that the mdE contributed 80% to basal renin promoter activity in the kidney, while it was dispensable for responsiveness to various stimuli, such as high-salt loading, dehydration and captopril (a potent ACE inhibitor) administration. To our surprise, this approach revealed that renin mRNA levels in the kidneys of Renin-null mutant mice that had been rescued by wild-type or mutant (missing mdE) BAC transgenes were indistinguishable, even though the plasma renin activity (PRA), AI concentration and blood pressure were all significantly lower in the compound mice rescued by the mutant BAC. The results clearly demonstrate that blood pressure homeostasis is transcriptionally regulated through the mdE.
Methods BAC mutagenesis A mouse Ren-1c BAC clone (RPCI23-240p23, GenBank accession no. AC068906, Figure 1A) was obtained as described elsewhere (10). Transcriptional start site of the Ren-1c gene (22) corresponded to a nucleotide (nt) position 89 005 of this clone, and all the nt positions hereafter were expressed as relative to this site (+1). BAC DNA modifications were performed in the EL250 E. coli strain (gift from Dr. N. A. Jenkins) by employing prophage-recombination system (23).
BAC-rescued renin-null mutant mice
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The SfiI recognition as well as FRT sequences were introduced into 30 -untranslated region of the gene (Figure 1A, (10)). Floxed kanamycin resistance gene (Kanr) cassette (EcoRI-SacII) was generated as described elsewhere (9). Wild-type mdE fragment (Figure 1B and C) was PCRamplified with following primer set and 240p23 BAC DNA as template, and digested with AccI and EcoRI: 50 -ACGTGTAGACATAACTTCGTATAGGATACTTTATACGA AGTTATACCAGGAGATGACCTTGGCCT-30 (AccI) and 50 GGAATTCGCTATCACAACCAGCCACTCA-30 (EcoRI). Mutant mdE fragment (i.e. blank sequences with loxP site, Figure 1B and C) was generated by annealing the following phosphorylated oligonucleotides, which creates SacII and SphI compatible ends: 50 -GGATAACTTCGTATAGTACACAT TATACGAAGTTATGCATG-30 and 50 -CATAACTTCGTATAA TGTGTACTATACGAAGTTATCCGC-30 . Restriction enzyme sites and loxP sequences are underlined and italicized, respectively, in each oligonucleotide. 50 -Regulatory sequences of the mouse Ren-1c gene (ApaIMscI, from 3228 to 2214) were cloned into ApaI/MscI sites of pBluescriptII KAS (+) (10). The wild-type, Kanr, and mutant DNA fragments were linked in this order (from 50 to 30 ) and used for substituting the corresponding portion (AccISphI, from 2873 to 2635) of the above plasmid to derive pTmRn/DmdE (Supplementary Figure 1A). An ApaI-BsmI (from 3228 to 2504) fragment from the pTmRn/DmdE was used for BAC targeting.
Institutions under the jurisdiction of the Ministry of Education, Culture, Sports, Science and Technology of Japan.
Transgenic mice
RT-PCR analysis
The DNA insert was released by BstBI digestion of modified BAC DNA (Figure 1A), as described elsewhere (10). Purified DNA was microinjected into the pronuclei of fertilized eggs from ICR mice (Ren-2 colony; Charles Liver, Yokohama, Japan). Transgene carriers were screened by PCR and Southern blot analyses. Long- and short-range structural analyses of transgene were done essentially as described elsewhere (10). In addition to the one we introduced into 30 UTR of the Ren-1c gene (Figure 1A), there are five SfiI sites within 155 kb Ren-1c Tg DNA fragment (‘‘transgenic locus’’ in Supplementary Figure 1B). Corresponding region from the Ren-2 locus carries five SfiI sites (‘‘endogenous locus’’ in Supplementary Figure 1B) and restriction fragment lengths are different from those in the Tg locus, which enables discrimination of DNA fragments from the two loci. High molecular weight thymic DNA of TgM sub-lines (W: wild-type and M: mutant in Supplementary Figure 1C) was digested with SfiI and subjected to PFGE followed by Southern blot hybridization with five probes (II-VI) spanning the locus (Supplementary Figure 1B). Most of the detected bands had expected sizes, while some resulted from partial digests, indicating that transgenes are fundamentally intact. Animal experiments were performed in a humane manner and approved by the Institutional Animal Experiment Committee of the University of Tsukuba. Experiments were conducted in accordance with the Regulation of Animal Experiments of the University of Tsukuba and the Fundamental Guidelines for Proper Conduct of Animal Experiments and Related Activities in Academic Research
Total RNA was converted to cDNA using random hexamers and reverse transcriptase (ReverTraAce, TOYOBO, Osaka, Japan). The cDNA was analyzed by semi-quantitative hot RT-PCR (94 C for 5 min; 25 [renin] or 18 [gapdh] cycles of 94 C for 30 s, 60 C for 30 s, 72 C for 1 min; and 72 C for 5 min). Products were separated on an 8% polyacrylamide gel and analyzed by phosphorimager. For the basal level expression analysis, the Tg Ren-1c signal was normalized to that of endogenous Ren-2&1 d signal. Primer sequences, common to all three genes, are as follows: 50 -CAGTACGGACTACGTGCTACA-30 (+8736 +8756) and 50 -AGCTTGGCTTGGCCTAGGGT-30 (+9483 +9502). Since primers were designed to span 30 FRT sequences (Figure 1A), PCR products from endogenous- and trans-genes were separable on the gel, and sizes of which are 223 and 356 bp for endogenous and transgenes, respectively (Figure 2A and C). For induction experiments (Figures 2D, 3A and B, and Supplementary Figure 2A and B), endogenous and transgenic renin, as well as gapdh gene expressions were separately analyzed by the following specific primer sets: endogenous renin, 50 -GCCCTCTGCCACCCAGTAA-30 (+9465+9483) and 50 -CAAAGCCAGACAAAATGGCCC-30 (+9514 + 9534), 70 bp amplicon; transgenic renin, 50 -TAACTTCTC CATGGTAGCCTC-30 (this primer was set in the 30 -FRT region) and 50 -CAAAGCCAGACAAAATGGCCC-30 (+9514 + 9534), 104 bp; gapdh, 50 -TCACTGGCATGGCC TTCC-30 and 50 -CAGGCGGCACGTCAGATC-30 , 65 bp (nt 723787, MUSGAPDH, GenBank accession no. M32599). Signals of transgenic or endogenous renin product were normalized to gapdh signals.
Generation of Renin-KO::Ren-1c BAC Tg compound mouse The Ren-1c-null allele was originally generated in the C57BL/ 6 strain (24). To facilitate genotyping, we first back-crossed the Ren-1c-null mouse to the ICRRen2 mouse in several generations. The resultant congenic mouse, heterozygous for Ren-1c-null and Ren-2 alleles, was bred with ICRRen2 mouse carrying the Ren-1c-BAC transgene. The F1 progeny, which were heterozygous for Ren-1c-null and Ren-2 alleles and hemizygous for the BAC, were back-crossed with the mouse heterozygous for Ren-1c-null and Ren-2 alleles. F2 progeny were screened to derive mouse homozygous for the Ren-1c-null alleles, either with (Vwt and Vmut) or without (IV) the Ren-1c BAC locus ((10) and data not shown). Northern blot analysis Total RNA was isolated from tissues of 8-week-old animals using ISOGEN (Nippon Gene) and analyzed as described elsewhere (10). An 820 bp DNA fragment (KpnI-NcoI), corresponding to the exon 3 to 9 of the Ren-1c gene was [a-32P]-labeled and used as a probe. Mouse gapdh gene expression analyzed with a 453 bp DNA fragment from the mouse cDNA (nt 5651,017; MUSGAPDH, GenBank accession no. M32599) was used as an internal control.
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Radio immuno assay (measurement of PRA and AI) Blood samples were withdrawn from anesthetized mice (8-week-old) and collected into ice-cold tubes containing EDTA, which were immediately centrifuged to isolate plasma. PRA was estimated by measuring the rate of AI formation. The concentration of AI was determined by radioimmunoassay (25). Blood pressure
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Systolic blood pressure was measured by a programmable sphygmomanometer (BP-98A; Softron, Japan) using the tailcuff method as described previously (26). Statistical analysis for the comparison of blood pressure was performed using two-tailed Student’s t test. In situ hybridization
Figure 2. Renin gene expression in the TgM. (A) Basal renin gene expression. Kidneys were isolated from 8-week-old BAC TgM (4 males [M] and 4 females [F] of each genotype, wild-type [wt] and mutant [mut]), and the RNA was analyzed by semi-quantitative hot RT-PCR in the linear amplification range. Each value represents the ratio of Tg Ren1c (Tg) expression to that of endogenous Ren-2&1d (endo.), which served as an internal control. Relative Tg/endo. values for each individual are shown after normalization to the average value of the wt group, which was arbitrarily set at 100. Each sample was analyzed at least 3 times, and the means ± SD are shown. (B) The means ± SD of each group in A were calculated. P values were determined by 2-tailed Student’s t test. (C) Renin gene expression in the wt- and mut-mdE TgM, treated with high-sodium diet (HS) or dehydration (DH). Two groups of male TgM (8-week-old) were fed high-sodium (8%, HS) or normalsodium (0.6%; C for control) diets for 5 days. In another group, access to drinking water was restricted for 1 day (DH). RNA was isolated from the kidney and analyzed by semi-quantitative hot RT-PCR (upper) using 2 sets of primer pairs, one co-amplifying Tg Ren-1c (Tg) and endogenous Ren-2&1d (endo.) genes and another specific for the gapdh gene. Relative amount of renin mRNA after normalization to that of gapdh was determined by 3 independent RT-PCR for 3 individuals in each group (bottom). Expression value of untreated control animals in each group was arbitrarily set at 100. (D) Renin gene expression in the wt- and mut-mdE TgM, treated with or without captopril. Animals (8-week-old) were divided into two groups, and one of them was treated for 7 days with captopril dissolved in drinking water (0.5 mg/ml). Kidneys were isolated, and RNA was analyzed as described above. Expression value of untreated wt animals was arbitrarily set at 100. Lines in D indicate that lanes were run on the same gel but were noncontiguous. *p50.05; **p50.01.
The mouse kidney was dissected after perfusion, fixed with Tissue Fixative (Genostaff, Tokyo), embedded in paraffin (by their proprietary procedures), and sectioned at 6 mm. For ISH, tissue sections were de-waxed with xylene, and rehydrate through an ethanol series and PBS. The sections were fixed 4% paraformaldehyde in PBS for 15 min and then washed with PBS. The sections were treated with 10 mg/ml ProteinaseK in PBS for 30 min at 37 C, washed with PBS, refixed with 4% paraformaldehyde in PBS, again washed with PBS, and placed in 0.2 M HCl for 10 min. After washing with PBS, the sections were acetylated by incubation in 0.1 M triethanolamine-HCl, pH8.0, 0.25% acetic anhydride for 10 min. After washing with PBS, the sections were dehydrated through a series of ethanol. Hybridization was performed with probes at concentrations of 100 ng/ml in the Probe Diluent (Genostaff) at 60 C for 16 h. After hybridization, the sections were washed in 5 HybriWash (Genostaff), equal to 5 SSC, at 60 C for 20 min and then in 50% formamide, 2 HybriWash at 60 C for 20 min, followed by RNase treatment in 50 mg/ml RNaseA in 10 mM Tris-HCl, pH8.0, 1 M NaCl and 1 mM EDTA for 30 min at 37 C. Then the sections were washed twice with 2 HybriWash at 60 C for 20 min, twice with 0.2 HybriWash at 60 C for 20 min, and once with TBST (0.1% Tween20 in TBS). After treatment with 0.5% blocking reagent (Roche, Basel, Switzerland) in TBST for 30 min, the sections were incubated with anti-DIG AP conjugate (Roche) diluted 1:1000 with TBST for 2 h. The sections were washed twice with TBST and then incubated in 100 mM NaCl, 50 mM MgCl2, 0.1% Tween20, 100 mM Tris-HCl, pH9.5. Coloring reactions were performed with BM purple AP substrate (Roche) overnight and then washed with PBS. The sections were counterstained with Kernechtrot stain solution (Mutoh, Tokyo, Japan), dehydrated, and then mounted with Malinol (Mutoh).
Results Generation of Tg BAC lines in which wild-type or mutant Ren-1c loci are present at the identical chromosomal site Inbred mouse strains can be classified into two groups by which renin allele they carry; one (ex. C57BL/6; called Ren-1 strains, hereafter) carrying a single renin gene, Ren-1c, and
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the another (ex. DBA/2; Ren-2 strains) carrying two tandemly-arrayed genes, Ren-2&1d (Supplementary Figure 1B). We used a mouse Ren-1c BAC (RPCI23-240p23, Figure 1A) to generate TgM. To distinguish transgenic from endogenous sequences in structural and expression analyses, we marked the 30 -untranslated region of the Ren-1c gene within the BAC by inserting 133 bp FRT and SfiI restriction enzyme sites via homologous recombination in E. coli (Figure 1A; (10)). For employing the coplacement strategy (10), the promoter region was also modified to carry a kanamycin resistance gene (Kanr, selectable in E. coli), as well as the wild-type (wt) and mutant (mut) mdE sequences, both flanked by a set of loxP sequence variants, loxP2272 and loxP5171 (‘‘parental’’ in Figure 1B and Supplementary Figure 1A) (27). A 240 bp sequence was deleted to generate the mdE mutant BAC (mut, Figure 1C). Purified BAC DNA (155 kb BstBI fragment, Figure 1A and Supplementary Figur 1A) was injected into fertilized mouse eggs collected from the Ren-2 mouse strain, ICR, to facilitate structural analyses of the transgene. Tail DNA from offspring was analyzed by PCR and Southern blotting to screen for a line carrying a single copy of the parental BAC (data not shown). The degree of integrity of the BAC was determined by long-range structural analysis of the transgene (Supplementary Figure 1B and C). The parental TgM line was crossed with mice ubiquitously expressing Cre recombinase to generate pups carrying both transgene structures (Supplementary Figure 1A). Genotypes of the sibling TgM lines were determined by a restriction fragment length polymorphism as shown in Figure 1B. After Cre-loxP recombination, a 2.26 kb EcoRI fragment in the parental locus changes to 0.95 or 3.35 kb fragments in the wild-type or the mutant loci, respectively, and both diagnostic fragments can be detected with an appropriate probe (Figure 1B). Analysis of tail DNA from parental and sibling TgM by Southern blotting revealed that recombination took place as expected. Therefore, any two resulting sister lines contained either wild-type or mutant transgenes that had integrated at the identical genomic position. It should be noted that the systolic blood pressure (SBP) did not differ significantly between the wild-type and mutant Tg animals (data not shown). Basal level transgene expression in the kidney Total RNA was prepared from kidneys of wild-type and mutant TgM lines and expression of transgenic Ren-1c, as well as endogenous Ren-2&1d genes was analyzed by semiquantitative RT-PCR (Figure 2A and B). Because the PCR primer set was designed to span the FRT sequences in the 30 UTR of the transgene (Figure 1A), it simultaneously amplifies the products of all the genes, but the transgenic and endogenous gene products are separable by gel electrophoresis (10). To rigorously control the data, signals generated by the Tg (single copy) were normalized to those from the endogenous renin genes (endo, Ren-2&1d, four copies). The expression level of the Tg in the mutant mdE TgM was 80% lower than that in the wild-type mdE TgM (Figure 2A and B), demonstrating that the mdE significantly
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contributes to basal level Ren-1c gene transcription in the kidney. Additionally, total RNA from extra-renal tissues that also produce renin, including SMG, adrenal, heart, testis, ovary, liver, brain, and lung, were also analyzed for renin gene expression by semi-quantitative RT-PCR. Although accurate quantitative comparison was difficult because of the quite low expression levels from these tissues, extra-renal Ren-1c gene expression in the mutant and wild-type mdE TgM was roughly comparable (Supplementary Figure 2A). Renal Tg expression in response to physiological stimuli We next asked whether the renin promoter, in the absence of mdE, was still responsive to physiological stimuli known to modulate renin gene expression. Semi-quantitative RT-PCR analysis of RNA from the kidney revealed that a high-salt diet suppressed, and dehydration induced, endogenous renin (Ren2&1d) transcription by 50% and 30–60%, respectively (‘‘endo.’’ in Figure 2C). Renin gene transcription from the mutant mdE, as well as the wild-type transgenic loci responded normally to both stimuli (‘‘Tg’’ in Figure 2C), demonstrating that the mdE is dispensable for the response to these physiological stimuli. Next, to test whether renin gene expression in these TgM was normally regulated by the ACE inhibitor captopril that enhances both per cell renin production and recruitment of renin-producing cells, renin mRNA levels in the kidney of 8-week-old mice were examined. RT-PCR analysis revealed that the level of endogenous renin mRNA accumulation (in both wt and mut Tg carriers) increased 2- to 3-fold upon captopril treatment (‘‘endo.’’ in Figure 2D). In the mutant mdE TgM, the basal Tg renin mRNA level, when normalized to gapdh, was less than half of that in the wild-type mdE TgM (cap ; Figure 2D). This ‘‘fold-difference’’ was maintained after the mice were treated with captopril (cap +; Figure 2D), demonstrating that mdE is also dispensable for the induction of renin gene expression by captopril. Generation of Ren-1c-null::Ren-1c-BAC-Tg compound mouse To further examine the physiological significance of the mdE, we introduced one copy of the BAC transgene (wild-type or mutant) onto the Ren-1c-null genetic background (24) by breeding (Vwt and Vmut, respectively, in Figure 3A, bottom: these rescued animals therefore carry no endogenous renin genes). Congenic Ren-1c +/+ (VI), +/ (VII), and / (IV) strains were also generated and used as controls (Figure 3A, bottom). In the crosses, genotypes were confirmed by allelespecific Southern blot analysis (data not shown; (10)). Expression of the Ren-1c BAC transgenes in the absence of the endogenous renin gene We first analyzed the renin gene expression in the kidneys of the compound mutant mice by Northern blotting (Figure 3A, top). While endogenous Ren-1c mRNA in the Ren-1c-null mouse (IV) was not detectable, transcript in mice carrying either one (VII) or two (VI) copies of the endogenous Ren-1c gene was abundant and not distinguishable from each other (24). In rescued animals carrying a single wild-type BAC
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Figure 3. BAC-Tg-mediated rescue of the Ren-1c-null mouse. (A) Northern blot analysis of renin gene expression in the kidney of compound mice. Single copy of Ren-1c BACTg carrying either wild-type (wt) or mutant (mut) mdE sequences was introduced into the Ren-1c–null (-/-) genetic background by breeding, as described previously (10). Total RNA from the kidney of 8-week-old animals was analyzed by mouse Ren-1c or mouse gapdh cDNA probes. endo., endogenous Ren1c allele genotype. M, male; F, female. (B) RNA was isolated from the kidney of wt and mut mdE compound mice (8-week-old), and Tg Ren-1c (Tg) and gapdh genes expression was analyzed by semi-quantitative hot RT-PCR as described in the legend to Figure 2C. Bar graph shows means ± SD; n ¼ 7 per group. Expression values in the wt male (M) animals were arbitrarily set at 100. (C-E) Systolic blood pressure (C, SBP, 6week-old), plasma renin activity (D, PRA, 8-week-old), and angiotensin I (AI) contents (E, 8-week-old) of the compound mice. Data are means ± SD; numbers are shown below each bar. p Values were determined by 2-tailed Student’s t test. ND, not detected; N.S., not significant. Lines in B indicate that lanes were run on the same gel but were noncontiguous.
transgene (Vwt in Figure 3A), abundant Ren-1c gene expression was observed, at a level that was similar to that in animals heterozygous for the Ren-1c allele (VII). As described above, transgenic renin gene expression in the mutant mdE allele was reduced by 80% when compared with that in the wild-type mdE TgM in the presence of the endogenous renin alleles (Figure 2A and B). However, in the absence of endogenous renin, no apparent difference in renin gene expression was observed between the BAC-Tg rescued animals carrying the wild-type or mutant mdE sequences (Vwt and Vmut, respectively, in Figure 3A). To more precisely quantify the mutant and wild-type BAC expression levels in the Renin null mice, RNA samples from 7 males and 7 females of each genotype were analyzed by semiquantitative RT-PCR (Figure 3B), and the expression was virtually equivalent from the wild-type (wt) and mutant (mut) loci. This observation can be explained by the feedback response to the reduced blood pressure phenotype (26). As shown in Figure 2D, the level of transgenic Ren-1c gene expression in the mutant mdE locus, in response to captoprilinduced hypotension, was well above that seen in the wildtype mdE TgM with no captopril treatment. Thus, in the mutant mdE-rescued animal, transgenic renin gene expression was likely to be upregulated to a level that would be comparable to that in the wild-type mdE-rescued animal in order to maintain normal blood pressure. We also analyzed renin gene expression in extra-renal tissues (SMG, adrenal, testis, and ovary) of the wild-type and mutant mdE rescued animals (Supplementary Figure 2B). Moderate, yet significant differences (p ¼ 0.045) were observed only in the ovary. Blood pressure, PRA, and AI contents in the compound animals The SBP of the Ren-1c-null animals rescued by the wild-type Ren-1c BAC (Vwt in Figure 3C) was comparable to that of
the heterozygous Ren-1c-null (VII) mutant, as well as the wild-type (VI, data not shown) animals. The SBP of the homozygous Ren-1c-null mice (IV) was 20–25 mmHg lower than that of the heterozygous Ren-1c-null mice (VII and (24)). All these results were very consistent with the RNA expression levels in the kidney (Figure 3A). Quite surprisingly, however, SBP of the animals rescued by the mutant mdE BAC (Vmut in Figure 3C) was significantly lower than that of the wild-type mdE-rescued animals (Vwt), and comparable to that of the Ren-1c-null mice (IV), which result is apparently inconsistent with the RNA expression results (Figure 3A). We next measured PRA (Figure 3D) and the serum concentration of angiotensin I (AI, Figure 3E) by radioimmunoassays in the compound mutant mice. In accordance with the renin gene expression level in the kidney (Figure 3A), these parameters were not significantly different between the heterozygous Ren-1c-null animals (VII in Figure 3D and E) and the homozygous Ren-1c-null animals rescued by the wild-type Ren-1c BAC (Vwt). Consistent with the SBP, yet in conflict with the renal renin gene expression level, both PRA and AI concentration in the homozygous Ren-1c-null mice rescued by the mutant Ren-1c BAC (Vmut) were considerably lower than those in the animals rescued by the wild-type Ren-1c BAC (Vwt). These results demonstrated that while the mdE was indispensable for maintenance of blood pressure homeostasis, it was dispensable for renin mRNA accumulation in the kidney. In situ hybridization analysis It is generally established that renin, synthesized predominantly in JG cells of the kidney, is responsible for blood pressure homeostasis. In JG cells, inactive protein is partly processed into active renin, stored in secretory granules, and released into circulation under appropriate stimuli.
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Figure 4. In situ hybridization analysis of the Ren-1c gene expression in the kidney of Ren-1c-null animals (8-week-old) rescued either by the wild-type Tg (wt, ID1455 and 1481) or by the mutant Tg (mut, 1440 and 1441). Kidney sections were hybridized with the mouse Ren-1c antisense (left panels) and sense (right) probes.
As a possible basis for the discrepancy between renal renin gene expression and renin enzyme activity observed in the Ren-1c-null mouse rescued by the mutant Ren-1c BAC, we reasoned that a mdE-null Ren-1c gene might be ectopically expressed in non-JG cells of the kidney and may not be transformed into an active form. To test this possibility, we conducted in situ hybridization analysis of the kidney of the Ren-1c-null animals, rescued either by wild-type or mutant BAC transgenes. As a control, no renin mRNA signal was detected in the Ren-1c-null mouse (Figure 4, top). Transgenic Ren-1c expression was strictly restricted to JG cells of the afferent glomerular arteriole in the Ren-1c-null animals rescued by the wild-type BAC (Figure 4, middle). Essentially the same cell-type specificity of renin gene
expression was observed in the mutant BAC-rescued animals (Figure 4, bottom). Thus, cell-type specificity of the renin gene expression in the kidney was not compromised by mdE deletion from the BAC.
Discussion In vivo assessment of transcriptional phenotypes of mutations introduced into gene regulatory sequences is generally executed by comparing the expression of transgenic to endogenous reference genes. When mouse genome is used as a host, however, a position-of-integration-site effect often hampers precise quantitative comparison of transcriptional activities of different constructs. The mdE has been
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shown to harbor powerful enhancer activity in transfected As4.1 cells (13,28) and is conserved across species. To examine its in vivo function, some groups have employed a mouse knock-out strategy (28,29) and others have examined the expression of PAC (30) or BAC (31) transgenes. Adams et al. deleted a 727 bp DNA region, consisting of a core mdE (241 bp) sequence and its upstream (349 bp) and downstream (137 bp) flanking sequences, from the endogenous Ren-1c locus in mouse ES cells (REKO)(28,29). They reported, using in situ hybridization, that renin mRNA accumulation in JG cells of the kidney decreased severely in the REKO mouse. However, when analyzed by real-time qPCR (29), the mRNA level in the whole kidney of the mutant mouse was only slightly lower than that of the control animals (72% of the control) and a statistically significant difference was not observed. The latter result is consistent with the qRT-PCR (Figure 3A), as well as in situ hybridization results (Figure 4) presented here, in which no significant difference in renin mRNA levels was observed between wild-type and mdE-deleted BAC-rescued animals. To our surprise, we found severely compromised PRA in the mutant BAC-rescued animal (70% lower than the control; Figure 3D), while it was only 16% lower in the REKO animal (29). Phenotypic differences between the two reports may stem from the amount of the sequence that was deleted (240 bp versus 727 bp) or from the experimental strategies employed, i.e. transgenic rescue of the renin-null mice versus endogenous sequence knock-out in mice. However, because the animals exhibited a low blood pressure phenotype in both cases (28), it can be mutually derived that renin mRNA accumulation in the kidney was not sufficient to maintain normal blood pressure homeostasis. Several explanations could account for this unexpected observation. First, renin gene expression in extra-renal tissues may be under the control of the mdE and their adequate transcription may be also essential for maintaining normal blood pressure. This scenario might explain the only modest reduction of PRA in the REKO animals (84% of the control), as renin gene expression in some of the extra-renal tissues was diminished in those mutants (28,29). However, because far less renin is produced in extra-renal tissues than in the kidney, it is uncertain whether diminished renin production, if any, in these tissues (Supplementary Figure 2B) could lead to the observed decrease in PRA (31% of the control) in the mutant mdE-rescued animals described here (Figure 3D). Second, Markus et al. (29) found that activation of the REKO promoter by ‘‘low salt + enalapril (ACE inhibitor)’’ treatment was diminished by 54%, when compared with the wild-type control. Accordingly, the renin stores in the kidney of ‘‘treated’’ REKO mouse were significantly depleted (5% of the wild-type control), and the induced level of PRA also dropped by 52%, which they proposed as a possible reason for the observed 9.9 mmHg lower mean arterial blood pressure (MAP) in the REKO mice. In the current report, however, we observed similar responses to ‘‘captopril (ACE inhibitor)’’ in the wild-type and mutant mdE transgenic alleles, when analyzed in the presence of endogenous, intact renin loci (Figure 2D). In our case, therefore, the mutant renin BAC in the kidney of compound mutant mice might respond to hypotensive (or reduced AII) stimulus and suffused kidney
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cells with renin mRNA in the steady state, while its level was not sufficient to be released as active protein into circulation. By using a human renin gene TgM carried in a P1 artificial chromosome (PAC, 160 kb), Zhou et al. demonstrated that the human distal enhancer (hdE, 241 bp) was responsible for 3-to-10-fold potentiation of renal renin gene expression (30), which is in accordance with our finding examining the mouse renin gene. Therefore, despite over 100-fold difference in renal renin gene expression in the mouse and humans (i.e. the former is higher), as well as their differences in DNA sequence, mouse and human distal enhancers have similar transcriptional roles. On the other hand, it is not known how much the hdE contributes to blood pressure homeostasis, because their assessment was done in the presence of intact, endogenous mouse renin alleles. Fuchs et al. identified a nucleotide variant at position 5312 of transcription initiation site of the human renin gene (32). Although located outside of the core hdE region (5777 to 5552), this variant (REN 5312C/T), significantly affected an activity of the neighboring hdE in reporter assays in cultured human chorionic cells (32). In addition, it has been demonstrated that this polymorphism was associated with increased susceptibility to hypertension in the human population (33,34). Therefore, the REN-5312C/T variant might control blood pressure phenotype by modulating the hdE activity in human. In summary, by employing the transgene coplacement strategy, we compared the transcriptional activities of wildtype and mutant renin genes carried in a BAC at the same mouse genomic integration sites. When analyzed in the presence of the endogenous renin genes, the mdE contributed 80% to the basal transcriptional activity, while it was dispensable for both JG cell-specific and captopril-regulated renin gene expression. When analyzed in the absence of the endogenous renin genes, the mdE was essential for maintenance of PRA, AI content, and blood pressure homeostasis, while it was dispensable for renin mRNA accumulation in the kidney. The results are qualitatively consistent with those obtained from endogenous mdE-null mouse (28,29), yet the underlying molecular mechanism for these unexpected observations still remains to be determined. We have now completed in vivo assessment of mouse renin regulatory elements, CNRE, RP-2 and mdE, in the context of transcriptional regulation of the gene ((10) and this work). We therefore summarized their putative roles, determined by transgene coplacement strategy in Supplementary Table 1.
Acknowledgements We thank Dr Doug Engel (University of Michigan) and Eszter To´th for critical reading of the manuscript, and Akiko Sugiura, Naoto Masui and Tomoko Saitoh for help with experiments.
Declaration of interest This work was supported by grants from the Astellas Foundation for Research on Metabolic Disorders (K.T.), the Japan Heart Foundation (K.T.), the Uehara Memorial Foundation (K.T.), the Tokyo Biochemical Research Foundation (K.T.) and the Takeda Science Foundation (K.T.). The authors report no declaration of interest.
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ISSN: 1079-9893 (Print) 1532-4281 (Online) Journal homepage: http://www.tandfonline.com/loi/irst20
A mouse renin distal enhancer is essential for blood pressure homeostasis in BAC-rescued reninnull mutant mice Keiji Tanimoto, Sumiyo Kanafusa, Aki Ushiki, Hitomi Matsuzaki, Junji Ishida, Fumihiro Sugiyama & Akiyoshi Fukamizu To cite this article: Keiji Tanimoto, Sumiyo Kanafusa, Aki Ushiki, Hitomi Matsuzaki, Junji Ishida, Fumihiro Sugiyama & Akiyoshi Fukamizu (2014) A mouse renin distal enhancer is essential for blood pressure homeostasis in BAC-rescued renin-null mutant mice, Journal of Receptors and Signal Transduction, 34:5, 401-409 To link to this article: http://dx.doi.org/10.3109/10799893.2014.908917
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Date: 12 September 2015, At: 16:30
http://informahealthcare.com/rst ISSN: 1079-9893 (print), 1532-4281 (electronic) J Recept Signal Transduct Res, 2014; 34(5): 401–409 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/10799893.2014.908917
RESEARCH ARTICLE
A mouse renin distal enhancer is essential for blood pressure homeostasis in BAC-rescued renin-null mutant mice Downloaded by [Karolinska Institutet, University Library] at 16:30 12 September 2015
Keiji Tanimoto1,2, Sumiyo Kanafusa3, Aki Ushiki3, Hitomi Matsuzaki1,2, Junji Ishida1,2, Fumihiro Sugiyama4, and Akiyoshi Fukamizu1,2 1
Faculty of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki, Japan, 2Life Science Center of Tsukuba Advanced Research Alliance (TARA), University of Tsukuba, Tsukuba, Ibaraki, Japan, 3Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki, Japan, and 4Laboratory Animal Resource Center, University of Tsukuba, Tsukuba, Ibaraki, Japan
Abstract
Keywords
Renin is predominantly expressed in juxtaglomerular cells in the kidney and regulates blood pressure homeostasis. To examine possible in vivo functions of a mouse distal enhancer (mdE), we generated transgenic mice (TgM) carrying either wild-type or mdE-deficient renin BACs (bacterial artificial chromosome), integrated at the identical chromosomal site. In the kidneys of the TgM, the mdE contributed 80% to basal renin promoter activity. To test for possible physiological roles for the mdE, renin BAC transgenes were used to rescue the hypotensive renin-null mice. Interestingly, renal renin expression in the TgBAC:renin-null compound mice was indistinguishable between the wild-type and mutant BAC carriers. Surprisingly, however, the plasma renin activity and angiotensin I concentration in the mdE compound mutant mice were significantly lower than the same parameters in the control mice, and the mutants were consistently hypotensive, demonstrating that blood pressure homeostasis is regulated through transcriptional cis elements controlling renin activity.
Bacterial artificial chromosome, blood pressure, renin, transcriptional regulation, transgenic mice
Introduction The renin-angiotensin system (RAS) plays a pivotal role in the regulation of blood pressure and electrolyte homeostasis. Renin, synthesized primarily in the juxtaglomerular (JG) cells of the kidney, cleaves its unique substrate, angiotensinogen, a plasma protein synthesized predominantly in the liver, to produce the decapeptide angiotensin I (AI). AI is further cleaved by angiotensin-converting enzyme (ACE) to produce an octapeptide, angiotensin II (AII), which increases blood pressure through vasoconstriction and aldosterone secretion by binding to angiotensin receptors. In this cascade, renin catalyzes the first, rate-limiting reaction and thus plays a central role in regulation of the overall activity of RAS. A number of physiological stimuli, including renal sympathetic activity, sodium balance, and pharmacological blockade of AII synthesis or receptor function modulate renin secretion and transcription in the kidney (1). Although the levels expressed elsewhere are much lower than in the kidney, renin is also expressed in extra-renal tissues, such as the adrenal gland, testes, ovaries, heart, and submandibular gland (SMG).
Address for correspondence: Keiji Tanimoto, Faculty of Life and Environmental Sciences, University of Tsukuba, Tennoudai 1-1-1, Tsukuba, Ibaraki 305-8577, Japan. Tel/Fax: (+81) 29-853-6070. E-mail: [email protected]
History Received 27 January 2014 Revised 23 March 2014 Accepted 24 March 2014 Published online 15 April 2014
In order to understand pathogenesis of hypertension associated with the RAS activity, it is crucial to understand transcriptional mechanisms of renin, which is characterized by its cell-type specificity and strong inducibility by cAMP (2,3). A number of candidate cis regulatory sequences have been identified in the renin gene promoters by cell transfection assays (4–8) and some of them have also been evaluated in vivo. Although usual DNA constructs (less than 20 kb) have been widely used to investigate the regulation of gene activity in transgenic mice (TgM), they often carry less regulatory information and are more susceptible to position-of-integration site effects, which can most often generate significant line-to-line heterogeneity in the level of transgene (Tg) expression. Gene knock-outs also have a potential drawback, i.e. mutating a cis-regulatory element that is essential for efficient gene expression may lead to compensatory activation of other cis elements for the gene, which may mask transcriptional phenotypes primarily caused by mutations. To avoid such situations, in our previous work we employed bacterial artificial chromosomes (BAC) to generate TgM to evaluate putative cis regulatory element activities in vivo (9,10). As anticipated, identical transgene constructs, when integrated at distinct chromosomal loci, exhibited similar (but never identical) levels of transgene expression. To fully circumvent possible position-of-integration site effects, we further employed a transgene coplacement strategy, in which
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activities of wild-type and mutant sequences can be directly compared at the identical chromosomal integration position (9,11,12). Importantly, by using a set of wild-type and mutant transgenes for rescuing knock-out mice of the gene, the physiological significance of cis-regulatory sequences can also be examined. Using this BAC transgenic coplacement methodology, we have previously evaluated two of the putative renin regulatory sequences, CNRE (overlapping the cAMP-responsive element [CRE] and the negative regulatory element [NRE]) and RP-2 (renin proximal promoter element-2), both of which are potentially responsive to cAMP. The results clearly demonstrated that the CNRE sequence was dispensable for basal and cell-type specific renin gene transcription, as well as for its responsiveness to several physiological stimuli, and for blood pressure homeostasis. On the other hand, RP-2 sequence was indispensable for basal level transcription in the kidney, as well as for blood pressure homeostasis (10). In the present study, we focused on another potential cAMP-responsive, cis-regulatory element, the mdE (mouse distal enhancer; (13)), and examined its functional significance, again in BAC TgM. The mdE, defined by 240 bp DNA sequence, was identified by transient transfection of 50 deletion constructs of the mouse Ren-1c gene into As4.1 cells. This tissue-specific enhancer is located 2.6 kb upstream (2866 to 2625 bp) of the transcriptional start site of the mouse renin gene and its homologous sequence is present about 12 kb upstream of the human renin start site of transcription (14,15). The mdE bears clustered binding sites for multiple transcription factors, including E-box proteins Figure 1. Experimental design. (A) The RPCI23-240p23 BAC (192 kb insert) carries all the nine exons of the Ren-1c gene. The FRT (shaded oval), as well as SfiI restriction enzyme sites have been introduced into 30 -untranslated region of the gene (10). BstBI restriction enzyme fragment (155 kb) with approximately 66 kb and 80 kb of 50 - and 30 -flanking sequences, respectively, was used for generating TgM. (B) Structural analysis of the transgenic mdE region. In vivo Cre-loxP recombination shown in the left was confirmed by Southern blot analysis of tail DNA from parental, wt, and mut TgM lines, in which the DNA was digested with EcoRI (E), separated on agarose gels, transferred to a nylon membrane, and hybridized to the probe (shaded rectangles in the left panel; from nt 2635 to 2214 relative to transcription start site). The sizes of the expected bands are indicated (in kb) in the right panel and those expected from the endogenous locus are in parentheses. (C) Wild-type (wt, top) and mutant (mut, bottom) murine mdE sequences. Several transcription factor-binding sites are located within the deleted portion (broken line) (4).
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USF1/USF2 (16), CREB/CREM (16), NF-Y (15), RAR/RXR (17), Ear2 (18), Coup-TFII (19), etc. (20,21) (wt in Figure 1C), and activates renin gene transcription by 50-fold on transfection into As4.1 cells (13). Our transgenic approach disclosed that the mdE contributed 80% to basal renin promoter activity in the kidney, while it was dispensable for responsiveness to various stimuli, such as high-salt loading, dehydration and captopril (a potent ACE inhibitor) administration. To our surprise, this approach revealed that renin mRNA levels in the kidneys of Renin-null mutant mice that had been rescued by wild-type or mutant (missing mdE) BAC transgenes were indistinguishable, even though the plasma renin activity (PRA), AI concentration and blood pressure were all significantly lower in the compound mice rescued by the mutant BAC. The results clearly demonstrate that blood pressure homeostasis is transcriptionally regulated through the mdE.
Methods BAC mutagenesis A mouse Ren-1c BAC clone (RPCI23-240p23, GenBank accession no. AC068906, Figure 1A) was obtained as described elsewhere (10). Transcriptional start site of the Ren-1c gene (22) corresponded to a nucleotide (nt) position 89 005 of this clone, and all the nt positions hereafter were expressed as relative to this site (+1). BAC DNA modifications were performed in the EL250 E. coli strain (gift from Dr. N. A. Jenkins) by employing prophage-recombination system (23).
BAC-rescued renin-null mutant mice
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The SfiI recognition as well as FRT sequences were introduced into 30 -untranslated region of the gene (Figure 1A, (10)). Floxed kanamycin resistance gene (Kanr) cassette (EcoRI-SacII) was generated as described elsewhere (9). Wild-type mdE fragment (Figure 1B and C) was PCRamplified with following primer set and 240p23 BAC DNA as template, and digested with AccI and EcoRI: 50 -ACGTGTAGACATAACTTCGTATAGGATACTTTATACGA AGTTATACCAGGAGATGACCTTGGCCT-30 (AccI) and 50 GGAATTCGCTATCACAACCAGCCACTCA-30 (EcoRI). Mutant mdE fragment (i.e. blank sequences with loxP site, Figure 1B and C) was generated by annealing the following phosphorylated oligonucleotides, which creates SacII and SphI compatible ends: 50 -GGATAACTTCGTATAGTACACAT TATACGAAGTTATGCATG-30 and 50 -CATAACTTCGTATAA TGTGTACTATACGAAGTTATCCGC-30 . Restriction enzyme sites and loxP sequences are underlined and italicized, respectively, in each oligonucleotide. 50 -Regulatory sequences of the mouse Ren-1c gene (ApaIMscI, from 3228 to 2214) were cloned into ApaI/MscI sites of pBluescriptII KAS (+) (10). The wild-type, Kanr, and mutant DNA fragments were linked in this order (from 50 to 30 ) and used for substituting the corresponding portion (AccISphI, from 2873 to 2635) of the above plasmid to derive pTmRn/DmdE (Supplementary Figure 1A). An ApaI-BsmI (from 3228 to 2504) fragment from the pTmRn/DmdE was used for BAC targeting.
Institutions under the jurisdiction of the Ministry of Education, Culture, Sports, Science and Technology of Japan.
Transgenic mice
RT-PCR analysis
The DNA insert was released by BstBI digestion of modified BAC DNA (Figure 1A), as described elsewhere (10). Purified DNA was microinjected into the pronuclei of fertilized eggs from ICR mice (Ren-2 colony; Charles Liver, Yokohama, Japan). Transgene carriers were screened by PCR and Southern blot analyses. Long- and short-range structural analyses of transgene were done essentially as described elsewhere (10). In addition to the one we introduced into 30 UTR of the Ren-1c gene (Figure 1A), there are five SfiI sites within 155 kb Ren-1c Tg DNA fragment (‘‘transgenic locus’’ in Supplementary Figure 1B). Corresponding region from the Ren-2 locus carries five SfiI sites (‘‘endogenous locus’’ in Supplementary Figure 1B) and restriction fragment lengths are different from those in the Tg locus, which enables discrimination of DNA fragments from the two loci. High molecular weight thymic DNA of TgM sub-lines (W: wild-type and M: mutant in Supplementary Figure 1C) was digested with SfiI and subjected to PFGE followed by Southern blot hybridization with five probes (II-VI) spanning the locus (Supplementary Figure 1B). Most of the detected bands had expected sizes, while some resulted from partial digests, indicating that transgenes are fundamentally intact. Animal experiments were performed in a humane manner and approved by the Institutional Animal Experiment Committee of the University of Tsukuba. Experiments were conducted in accordance with the Regulation of Animal Experiments of the University of Tsukuba and the Fundamental Guidelines for Proper Conduct of Animal Experiments and Related Activities in Academic Research
Total RNA was converted to cDNA using random hexamers and reverse transcriptase (ReverTraAce, TOYOBO, Osaka, Japan). The cDNA was analyzed by semi-quantitative hot RT-PCR (94 C for 5 min; 25 [renin] or 18 [gapdh] cycles of 94 C for 30 s, 60 C for 30 s, 72 C for 1 min; and 72 C for 5 min). Products were separated on an 8% polyacrylamide gel and analyzed by phosphorimager. For the basal level expression analysis, the Tg Ren-1c signal was normalized to that of endogenous Ren-2&1 d signal. Primer sequences, common to all three genes, are as follows: 50 -CAGTACGGACTACGTGCTACA-30 (+8736 +8756) and 50 -AGCTTGGCTTGGCCTAGGGT-30 (+9483 +9502). Since primers were designed to span 30 FRT sequences (Figure 1A), PCR products from endogenous- and trans-genes were separable on the gel, and sizes of which are 223 and 356 bp for endogenous and transgenes, respectively (Figure 2A and C). For induction experiments (Figures 2D, 3A and B, and Supplementary Figure 2A and B), endogenous and transgenic renin, as well as gapdh gene expressions were separately analyzed by the following specific primer sets: endogenous renin, 50 -GCCCTCTGCCACCCAGTAA-30 (+9465+9483) and 50 -CAAAGCCAGACAAAATGGCCC-30 (+9514 + 9534), 70 bp amplicon; transgenic renin, 50 -TAACTTCTC CATGGTAGCCTC-30 (this primer was set in the 30 -FRT region) and 50 -CAAAGCCAGACAAAATGGCCC-30 (+9514 + 9534), 104 bp; gapdh, 50 -TCACTGGCATGGCC TTCC-30 and 50 -CAGGCGGCACGTCAGATC-30 , 65 bp (nt 723787, MUSGAPDH, GenBank accession no. M32599). Signals of transgenic or endogenous renin product were normalized to gapdh signals.
Generation of Renin-KO::Ren-1c BAC Tg compound mouse The Ren-1c-null allele was originally generated in the C57BL/ 6 strain (24). To facilitate genotyping, we first back-crossed the Ren-1c-null mouse to the ICRRen2 mouse in several generations. The resultant congenic mouse, heterozygous for Ren-1c-null and Ren-2 alleles, was bred with ICRRen2 mouse carrying the Ren-1c-BAC transgene. The F1 progeny, which were heterozygous for Ren-1c-null and Ren-2 alleles and hemizygous for the BAC, were back-crossed with the mouse heterozygous for Ren-1c-null and Ren-2 alleles. F2 progeny were screened to derive mouse homozygous for the Ren-1c-null alleles, either with (Vwt and Vmut) or without (IV) the Ren-1c BAC locus ((10) and data not shown). Northern blot analysis Total RNA was isolated from tissues of 8-week-old animals using ISOGEN (Nippon Gene) and analyzed as described elsewhere (10). An 820 bp DNA fragment (KpnI-NcoI), corresponding to the exon 3 to 9 of the Ren-1c gene was [a-32P]-labeled and used as a probe. Mouse gapdh gene expression analyzed with a 453 bp DNA fragment from the mouse cDNA (nt 5651,017; MUSGAPDH, GenBank accession no. M32599) was used as an internal control.
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Radio immuno assay (measurement of PRA and AI) Blood samples were withdrawn from anesthetized mice (8-week-old) and collected into ice-cold tubes containing EDTA, which were immediately centrifuged to isolate plasma. PRA was estimated by measuring the rate of AI formation. The concentration of AI was determined by radioimmunoassay (25). Blood pressure
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Systolic blood pressure was measured by a programmable sphygmomanometer (BP-98A; Softron, Japan) using the tailcuff method as described previously (26). Statistical analysis for the comparison of blood pressure was performed using two-tailed Student’s t test. In situ hybridization
Figure 2. Renin gene expression in the TgM. (A) Basal renin gene expression. Kidneys were isolated from 8-week-old BAC TgM (4 males [M] and 4 females [F] of each genotype, wild-type [wt] and mutant [mut]), and the RNA was analyzed by semi-quantitative hot RT-PCR in the linear amplification range. Each value represents the ratio of Tg Ren1c (Tg) expression to that of endogenous Ren-2&1d (endo.), which served as an internal control. Relative Tg/endo. values for each individual are shown after normalization to the average value of the wt group, which was arbitrarily set at 100. Each sample was analyzed at least 3 times, and the means ± SD are shown. (B) The means ± SD of each group in A were calculated. P values were determined by 2-tailed Student’s t test. (C) Renin gene expression in the wt- and mut-mdE TgM, treated with high-sodium diet (HS) or dehydration (DH). Two groups of male TgM (8-week-old) were fed high-sodium (8%, HS) or normalsodium (0.6%; C for control) diets for 5 days. In another group, access to drinking water was restricted for 1 day (DH). RNA was isolated from the kidney and analyzed by semi-quantitative hot RT-PCR (upper) using 2 sets of primer pairs, one co-amplifying Tg Ren-1c (Tg) and endogenous Ren-2&1d (endo.) genes and another specific for the gapdh gene. Relative amount of renin mRNA after normalization to that of gapdh was determined by 3 independent RT-PCR for 3 individuals in each group (bottom). Expression value of untreated control animals in each group was arbitrarily set at 100. (D) Renin gene expression in the wt- and mut-mdE TgM, treated with or without captopril. Animals (8-week-old) were divided into two groups, and one of them was treated for 7 days with captopril dissolved in drinking water (0.5 mg/ml). Kidneys were isolated, and RNA was analyzed as described above. Expression value of untreated wt animals was arbitrarily set at 100. Lines in D indicate that lanes were run on the same gel but were noncontiguous. *p50.05; **p50.01.
The mouse kidney was dissected after perfusion, fixed with Tissue Fixative (Genostaff, Tokyo), embedded in paraffin (by their proprietary procedures), and sectioned at 6 mm. For ISH, tissue sections were de-waxed with xylene, and rehydrate through an ethanol series and PBS. The sections were fixed 4% paraformaldehyde in PBS for 15 min and then washed with PBS. The sections were treated with 10 mg/ml ProteinaseK in PBS for 30 min at 37 C, washed with PBS, refixed with 4% paraformaldehyde in PBS, again washed with PBS, and placed in 0.2 M HCl for 10 min. After washing with PBS, the sections were acetylated by incubation in 0.1 M triethanolamine-HCl, pH8.0, 0.25% acetic anhydride for 10 min. After washing with PBS, the sections were dehydrated through a series of ethanol. Hybridization was performed with probes at concentrations of 100 ng/ml in the Probe Diluent (Genostaff) at 60 C for 16 h. After hybridization, the sections were washed in 5 HybriWash (Genostaff), equal to 5 SSC, at 60 C for 20 min and then in 50% formamide, 2 HybriWash at 60 C for 20 min, followed by RNase treatment in 50 mg/ml RNaseA in 10 mM Tris-HCl, pH8.0, 1 M NaCl and 1 mM EDTA for 30 min at 37 C. Then the sections were washed twice with 2 HybriWash at 60 C for 20 min, twice with 0.2 HybriWash at 60 C for 20 min, and once with TBST (0.1% Tween20 in TBS). After treatment with 0.5% blocking reagent (Roche, Basel, Switzerland) in TBST for 30 min, the sections were incubated with anti-DIG AP conjugate (Roche) diluted 1:1000 with TBST for 2 h. The sections were washed twice with TBST and then incubated in 100 mM NaCl, 50 mM MgCl2, 0.1% Tween20, 100 mM Tris-HCl, pH9.5. Coloring reactions were performed with BM purple AP substrate (Roche) overnight and then washed with PBS. The sections were counterstained with Kernechtrot stain solution (Mutoh, Tokyo, Japan), dehydrated, and then mounted with Malinol (Mutoh).
Results Generation of Tg BAC lines in which wild-type or mutant Ren-1c loci are present at the identical chromosomal site Inbred mouse strains can be classified into two groups by which renin allele they carry; one (ex. C57BL/6; called Ren-1 strains, hereafter) carrying a single renin gene, Ren-1c, and
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the another (ex. DBA/2; Ren-2 strains) carrying two tandemly-arrayed genes, Ren-2&1d (Supplementary Figure 1B). We used a mouse Ren-1c BAC (RPCI23-240p23, Figure 1A) to generate TgM. To distinguish transgenic from endogenous sequences in structural and expression analyses, we marked the 30 -untranslated region of the Ren-1c gene within the BAC by inserting 133 bp FRT and SfiI restriction enzyme sites via homologous recombination in E. coli (Figure 1A; (10)). For employing the coplacement strategy (10), the promoter region was also modified to carry a kanamycin resistance gene (Kanr, selectable in E. coli), as well as the wild-type (wt) and mutant (mut) mdE sequences, both flanked by a set of loxP sequence variants, loxP2272 and loxP5171 (‘‘parental’’ in Figure 1B and Supplementary Figure 1A) (27). A 240 bp sequence was deleted to generate the mdE mutant BAC (mut, Figure 1C). Purified BAC DNA (155 kb BstBI fragment, Figure 1A and Supplementary Figur 1A) was injected into fertilized mouse eggs collected from the Ren-2 mouse strain, ICR, to facilitate structural analyses of the transgene. Tail DNA from offspring was analyzed by PCR and Southern blotting to screen for a line carrying a single copy of the parental BAC (data not shown). The degree of integrity of the BAC was determined by long-range structural analysis of the transgene (Supplementary Figure 1B and C). The parental TgM line was crossed with mice ubiquitously expressing Cre recombinase to generate pups carrying both transgene structures (Supplementary Figure 1A). Genotypes of the sibling TgM lines were determined by a restriction fragment length polymorphism as shown in Figure 1B. After Cre-loxP recombination, a 2.26 kb EcoRI fragment in the parental locus changes to 0.95 or 3.35 kb fragments in the wild-type or the mutant loci, respectively, and both diagnostic fragments can be detected with an appropriate probe (Figure 1B). Analysis of tail DNA from parental and sibling TgM by Southern blotting revealed that recombination took place as expected. Therefore, any two resulting sister lines contained either wild-type or mutant transgenes that had integrated at the identical genomic position. It should be noted that the systolic blood pressure (SBP) did not differ significantly between the wild-type and mutant Tg animals (data not shown). Basal level transgene expression in the kidney Total RNA was prepared from kidneys of wild-type and mutant TgM lines and expression of transgenic Ren-1c, as well as endogenous Ren-2&1d genes was analyzed by semiquantitative RT-PCR (Figure 2A and B). Because the PCR primer set was designed to span the FRT sequences in the 30 UTR of the transgene (Figure 1A), it simultaneously amplifies the products of all the genes, but the transgenic and endogenous gene products are separable by gel electrophoresis (10). To rigorously control the data, signals generated by the Tg (single copy) were normalized to those from the endogenous renin genes (endo, Ren-2&1d, four copies). The expression level of the Tg in the mutant mdE TgM was 80% lower than that in the wild-type mdE TgM (Figure 2A and B), demonstrating that the mdE significantly
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contributes to basal level Ren-1c gene transcription in the kidney. Additionally, total RNA from extra-renal tissues that also produce renin, including SMG, adrenal, heart, testis, ovary, liver, brain, and lung, were also analyzed for renin gene expression by semi-quantitative RT-PCR. Although accurate quantitative comparison was difficult because of the quite low expression levels from these tissues, extra-renal Ren-1c gene expression in the mutant and wild-type mdE TgM was roughly comparable (Supplementary Figure 2A). Renal Tg expression in response to physiological stimuli We next asked whether the renin promoter, in the absence of mdE, was still responsive to physiological stimuli known to modulate renin gene expression. Semi-quantitative RT-PCR analysis of RNA from the kidney revealed that a high-salt diet suppressed, and dehydration induced, endogenous renin (Ren2&1d) transcription by 50% and 30–60%, respectively (‘‘endo.’’ in Figure 2C). Renin gene transcription from the mutant mdE, as well as the wild-type transgenic loci responded normally to both stimuli (‘‘Tg’’ in Figure 2C), demonstrating that the mdE is dispensable for the response to these physiological stimuli. Next, to test whether renin gene expression in these TgM was normally regulated by the ACE inhibitor captopril that enhances both per cell renin production and recruitment of renin-producing cells, renin mRNA levels in the kidney of 8-week-old mice were examined. RT-PCR analysis revealed that the level of endogenous renin mRNA accumulation (in both wt and mut Tg carriers) increased 2- to 3-fold upon captopril treatment (‘‘endo.’’ in Figure 2D). In the mutant mdE TgM, the basal Tg renin mRNA level, when normalized to gapdh, was less than half of that in the wild-type mdE TgM (cap ; Figure 2D). This ‘‘fold-difference’’ was maintained after the mice were treated with captopril (cap +; Figure 2D), demonstrating that mdE is also dispensable for the induction of renin gene expression by captopril. Generation of Ren-1c-null::Ren-1c-BAC-Tg compound mouse To further examine the physiological significance of the mdE, we introduced one copy of the BAC transgene (wild-type or mutant) onto the Ren-1c-null genetic background (24) by breeding (Vwt and Vmut, respectively, in Figure 3A, bottom: these rescued animals therefore carry no endogenous renin genes). Congenic Ren-1c +/+ (VI), +/ (VII), and / (IV) strains were also generated and used as controls (Figure 3A, bottom). In the crosses, genotypes were confirmed by allelespecific Southern blot analysis (data not shown; (10)). Expression of the Ren-1c BAC transgenes in the absence of the endogenous renin gene We first analyzed the renin gene expression in the kidneys of the compound mutant mice by Northern blotting (Figure 3A, top). While endogenous Ren-1c mRNA in the Ren-1c-null mouse (IV) was not detectable, transcript in mice carrying either one (VII) or two (VI) copies of the endogenous Ren-1c gene was abundant and not distinguishable from each other (24). In rescued animals carrying a single wild-type BAC
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Figure 3. BAC-Tg-mediated rescue of the Ren-1c-null mouse. (A) Northern blot analysis of renin gene expression in the kidney of compound mice. Single copy of Ren-1c BACTg carrying either wild-type (wt) or mutant (mut) mdE sequences was introduced into the Ren-1c–null (-/-) genetic background by breeding, as described previously (10). Total RNA from the kidney of 8-week-old animals was analyzed by mouse Ren-1c or mouse gapdh cDNA probes. endo., endogenous Ren1c allele genotype. M, male; F, female. (B) RNA was isolated from the kidney of wt and mut mdE compound mice (8-week-old), and Tg Ren-1c (Tg) and gapdh genes expression was analyzed by semi-quantitative hot RT-PCR as described in the legend to Figure 2C. Bar graph shows means ± SD; n ¼ 7 per group. Expression values in the wt male (M) animals were arbitrarily set at 100. (C-E) Systolic blood pressure (C, SBP, 6week-old), plasma renin activity (D, PRA, 8-week-old), and angiotensin I (AI) contents (E, 8-week-old) of the compound mice. Data are means ± SD; numbers are shown below each bar. p Values were determined by 2-tailed Student’s t test. ND, not detected; N.S., not significant. Lines in B indicate that lanes were run on the same gel but were noncontiguous.
transgene (Vwt in Figure 3A), abundant Ren-1c gene expression was observed, at a level that was similar to that in animals heterozygous for the Ren-1c allele (VII). As described above, transgenic renin gene expression in the mutant mdE allele was reduced by 80% when compared with that in the wild-type mdE TgM in the presence of the endogenous renin alleles (Figure 2A and B). However, in the absence of endogenous renin, no apparent difference in renin gene expression was observed between the BAC-Tg rescued animals carrying the wild-type or mutant mdE sequences (Vwt and Vmut, respectively, in Figure 3A). To more precisely quantify the mutant and wild-type BAC expression levels in the Renin null mice, RNA samples from 7 males and 7 females of each genotype were analyzed by semiquantitative RT-PCR (Figure 3B), and the expression was virtually equivalent from the wild-type (wt) and mutant (mut) loci. This observation can be explained by the feedback response to the reduced blood pressure phenotype (26). As shown in Figure 2D, the level of transgenic Ren-1c gene expression in the mutant mdE locus, in response to captoprilinduced hypotension, was well above that seen in the wildtype mdE TgM with no captopril treatment. Thus, in the mutant mdE-rescued animal, transgenic renin gene expression was likely to be upregulated to a level that would be comparable to that in the wild-type mdE-rescued animal in order to maintain normal blood pressure. We also analyzed renin gene expression in extra-renal tissues (SMG, adrenal, testis, and ovary) of the wild-type and mutant mdE rescued animals (Supplementary Figure 2B). Moderate, yet significant differences (p ¼ 0.045) were observed only in the ovary. Blood pressure, PRA, and AI contents in the compound animals The SBP of the Ren-1c-null animals rescued by the wild-type Ren-1c BAC (Vwt in Figure 3C) was comparable to that of
the heterozygous Ren-1c-null (VII) mutant, as well as the wild-type (VI, data not shown) animals. The SBP of the homozygous Ren-1c-null mice (IV) was 20–25 mmHg lower than that of the heterozygous Ren-1c-null mice (VII and (24)). All these results were very consistent with the RNA expression levels in the kidney (Figure 3A). Quite surprisingly, however, SBP of the animals rescued by the mutant mdE BAC (Vmut in Figure 3C) was significantly lower than that of the wild-type mdE-rescued animals (Vwt), and comparable to that of the Ren-1c-null mice (IV), which result is apparently inconsistent with the RNA expression results (Figure 3A). We next measured PRA (Figure 3D) and the serum concentration of angiotensin I (AI, Figure 3E) by radioimmunoassays in the compound mutant mice. In accordance with the renin gene expression level in the kidney (Figure 3A), these parameters were not significantly different between the heterozygous Ren-1c-null animals (VII in Figure 3D and E) and the homozygous Ren-1c-null animals rescued by the wild-type Ren-1c BAC (Vwt). Consistent with the SBP, yet in conflict with the renal renin gene expression level, both PRA and AI concentration in the homozygous Ren-1c-null mice rescued by the mutant Ren-1c BAC (Vmut) were considerably lower than those in the animals rescued by the wild-type Ren-1c BAC (Vwt). These results demonstrated that while the mdE was indispensable for maintenance of blood pressure homeostasis, it was dispensable for renin mRNA accumulation in the kidney. In situ hybridization analysis It is generally established that renin, synthesized predominantly in JG cells of the kidney, is responsible for blood pressure homeostasis. In JG cells, inactive protein is partly processed into active renin, stored in secretory granules, and released into circulation under appropriate stimuli.
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Figure 4. In situ hybridization analysis of the Ren-1c gene expression in the kidney of Ren-1c-null animals (8-week-old) rescued either by the wild-type Tg (wt, ID1455 and 1481) or by the mutant Tg (mut, 1440 and 1441). Kidney sections were hybridized with the mouse Ren-1c antisense (left panels) and sense (right) probes.
As a possible basis for the discrepancy between renal renin gene expression and renin enzyme activity observed in the Ren-1c-null mouse rescued by the mutant Ren-1c BAC, we reasoned that a mdE-null Ren-1c gene might be ectopically expressed in non-JG cells of the kidney and may not be transformed into an active form. To test this possibility, we conducted in situ hybridization analysis of the kidney of the Ren-1c-null animals, rescued either by wild-type or mutant BAC transgenes. As a control, no renin mRNA signal was detected in the Ren-1c-null mouse (Figure 4, top). Transgenic Ren-1c expression was strictly restricted to JG cells of the afferent glomerular arteriole in the Ren-1c-null animals rescued by the wild-type BAC (Figure 4, middle). Essentially the same cell-type specificity of renin gene
expression was observed in the mutant BAC-rescued animals (Figure 4, bottom). Thus, cell-type specificity of the renin gene expression in the kidney was not compromised by mdE deletion from the BAC.
Discussion In vivo assessment of transcriptional phenotypes of mutations introduced into gene regulatory sequences is generally executed by comparing the expression of transgenic to endogenous reference genes. When mouse genome is used as a host, however, a position-of-integration-site effect often hampers precise quantitative comparison of transcriptional activities of different constructs. The mdE has been
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shown to harbor powerful enhancer activity in transfected As4.1 cells (13,28) and is conserved across species. To examine its in vivo function, some groups have employed a mouse knock-out strategy (28,29) and others have examined the expression of PAC (30) or BAC (31) transgenes. Adams et al. deleted a 727 bp DNA region, consisting of a core mdE (241 bp) sequence and its upstream (349 bp) and downstream (137 bp) flanking sequences, from the endogenous Ren-1c locus in mouse ES cells (REKO)(28,29). They reported, using in situ hybridization, that renin mRNA accumulation in JG cells of the kidney decreased severely in the REKO mouse. However, when analyzed by real-time qPCR (29), the mRNA level in the whole kidney of the mutant mouse was only slightly lower than that of the control animals (72% of the control) and a statistically significant difference was not observed. The latter result is consistent with the qRT-PCR (Figure 3A), as well as in situ hybridization results (Figure 4) presented here, in which no significant difference in renin mRNA levels was observed between wild-type and mdE-deleted BAC-rescued animals. To our surprise, we found severely compromised PRA in the mutant BAC-rescued animal (70% lower than the control; Figure 3D), while it was only 16% lower in the REKO animal (29). Phenotypic differences between the two reports may stem from the amount of the sequence that was deleted (240 bp versus 727 bp) or from the experimental strategies employed, i.e. transgenic rescue of the renin-null mice versus endogenous sequence knock-out in mice. However, because the animals exhibited a low blood pressure phenotype in both cases (28), it can be mutually derived that renin mRNA accumulation in the kidney was not sufficient to maintain normal blood pressure homeostasis. Several explanations could account for this unexpected observation. First, renin gene expression in extra-renal tissues may be under the control of the mdE and their adequate transcription may be also essential for maintaining normal blood pressure. This scenario might explain the only modest reduction of PRA in the REKO animals (84% of the control), as renin gene expression in some of the extra-renal tissues was diminished in those mutants (28,29). However, because far less renin is produced in extra-renal tissues than in the kidney, it is uncertain whether diminished renin production, if any, in these tissues (Supplementary Figure 2B) could lead to the observed decrease in PRA (31% of the control) in the mutant mdE-rescued animals described here (Figure 3D). Second, Markus et al. (29) found that activation of the REKO promoter by ‘‘low salt + enalapril (ACE inhibitor)’’ treatment was diminished by 54%, when compared with the wild-type control. Accordingly, the renin stores in the kidney of ‘‘treated’’ REKO mouse were significantly depleted (5% of the wild-type control), and the induced level of PRA also dropped by 52%, which they proposed as a possible reason for the observed 9.9 mmHg lower mean arterial blood pressure (MAP) in the REKO mice. In the current report, however, we observed similar responses to ‘‘captopril (ACE inhibitor)’’ in the wild-type and mutant mdE transgenic alleles, when analyzed in the presence of endogenous, intact renin loci (Figure 2D). In our case, therefore, the mutant renin BAC in the kidney of compound mutant mice might respond to hypotensive (or reduced AII) stimulus and suffused kidney
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cells with renin mRNA in the steady state, while its level was not sufficient to be released as active protein into circulation. By using a human renin gene TgM carried in a P1 artificial chromosome (PAC, 160 kb), Zhou et al. demonstrated that the human distal enhancer (hdE, 241 bp) was responsible for 3-to-10-fold potentiation of renal renin gene expression (30), which is in accordance with our finding examining the mouse renin gene. Therefore, despite over 100-fold difference in renal renin gene expression in the mouse and humans (i.e. the former is higher), as well as their differences in DNA sequence, mouse and human distal enhancers have similar transcriptional roles. On the other hand, it is not known how much the hdE contributes to blood pressure homeostasis, because their assessment was done in the presence of intact, endogenous mouse renin alleles. Fuchs et al. identified a nucleotide variant at position 5312 of transcription initiation site of the human renin gene (32). Although located outside of the core hdE region (5777 to 5552), this variant (REN 5312C/T), significantly affected an activity of the neighboring hdE in reporter assays in cultured human chorionic cells (32). In addition, it has been demonstrated that this polymorphism was associated with increased susceptibility to hypertension in the human population (33,34). Therefore, the REN-5312C/T variant might control blood pressure phenotype by modulating the hdE activity in human. In summary, by employing the transgene coplacement strategy, we compared the transcriptional activities of wildtype and mutant renin genes carried in a BAC at the same mouse genomic integration sites. When analyzed in the presence of the endogenous renin genes, the mdE contributed 80% to the basal transcriptional activity, while it was dispensable for both JG cell-specific and captopril-regulated renin gene expression. When analyzed in the absence of the endogenous renin genes, the mdE was essential for maintenance of PRA, AI content, and blood pressure homeostasis, while it was dispensable for renin mRNA accumulation in the kidney. The results are qualitatively consistent with those obtained from endogenous mdE-null mouse (28,29), yet the underlying molecular mechanism for these unexpected observations still remains to be determined. We have now completed in vivo assessment of mouse renin regulatory elements, CNRE, RP-2 and mdE, in the context of transcriptional regulation of the gene ((10) and this work). We therefore summarized their putative roles, determined by transgene coplacement strategy in Supplementary Table 1.
Acknowledgements We thank Dr Doug Engel (University of Michigan) and Eszter To´th for critical reading of the manuscript, and Akiko Sugiura, Naoto Masui and Tomoko Saitoh for help with experiments.
Declaration of interest This work was supported by grants from the Astellas Foundation for Research on Metabolic Disorders (K.T.), the Japan Heart Foundation (K.T.), the Uehara Memorial Foundation (K.T.), the Tokyo Biochemical Research Foundation (K.T.) and the Takeda Science Foundation (K.T.). The authors report no declaration of interest.
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