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Am J Physiol Renal Physiol 295: F642-F653, 2008. First published July 16, 2008; doi:10.1152/ajprenal.00384.2007
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Dysregulated human renin expression in transgenic mice carrying truncated genomic constructs: evidence supporting the presence of insulators at the renin locus

Xiyou Zhou,1,* Eric T. Weatherford,2,* Xuebo Liu,3 Ella Born,3 Henry L. Keen,3 and Curt D. Sigmund1,2,3

1Molecular and Cellular Biology Graduate Program, Departments of 2Molecular Physiology and Biophysics and of 3Internal Medicine, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, Iowa

Submitted 13 August 2007 ; accepted in final form 9 July 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
We previously generated transgenic mice carrying a large P1 artificial chromosome (PAC160) encompassing a 160-kb segment containing the human renin gene, two upstream genes, and one downstream gene. We also previously generated mutant PAC160 constructs lacking the distal enhancer and concluded it is required to maintain baseline expression of human renin, but is not required for tissue-specific, cell-specific, and regulated expression of renin in vivo. We now report two additional transgenic lines carrying random truncations of PAC160 upstream of the renin gene. Southern and PCR mapping studies indicate that the truncation break points in the two lines are located ~10.4 and 2.5 kb upstream of the renin gene causing a deletion of all DNA upstream of the break. We tested the hypothesis that large-scale deletion of DNA upstream of the human renin gene including the enhancer would cause dysregulation of human renin expression. Phenotypically, these truncations cause a severe dysregulation of human renin expression, but remarkably, a preservation of the normal tissue-specific expression of the human ethanolamine kinase 2 (ETNK2) gene which lies immediately downstream of renin. Several functional binding sites for CTCF, a mammalian insulator protein, were identified in and around the renin and ETNK2 loci by gel shift and chromatin immunoprecipitation. We conclude that there are sequences in and around the renin and ETNK2 loci which act as boundaries between neighboring genes which insulate them from each other. The study illustrates the value of taking a much wider genomic perspective when studying mechanisms regulating gene expression.

P1 artificial chromosome; gene expression; chromatin; transcription

RENIN IS THE FIRST and rate-limiting enzyme in renin-angiotensin system (RAS) cascade. The end products of the RAS, including ANG II and ANG-(1–7), control blood pressure by exerting opposing effects on vasoconstriction, natriuresis, and sympathoexcitation. The angiotensin peptides are also required during development as evidenced by lethality in knockout mice lacking the renin, angiotensinogen, and both AT1 receptor genes (12, 14, 38). Given its importance both developmentally and in adults, it is not surprising that expression of renin is under tight transcriptional and posttranscriptional control (1, 26, 36).

In vitro, expression of the renin gene is controlled by both proximal and distal regulatory elements. The proximal elements include the binding sites for developmentally regulated transcription factors including HoxD10, Ets-1, and CBF1, an effector of the notch signaling pathway (25, 28). Regulation of renin expression by the interaction between PPAR{gamma} and the proximal promoter has also been recently reported (41), although its importance in vivo is yet to be established (42). The distal elements include the binding sites for members of the ligand-activated family of steroid hormone receptors such as retinoic acid receptor (RAR{alpha}), as well as binding sites for the E-box proteins USF-1/2 and the cAMP response element binding protein (CREB) (17, 24, 30, 31). The binding sites for these proteins are closely clustered together and constitute the distal or kidney enhancer (KE). This enhancer strongly stimulates transcription in kidney-derived renin-expressing As4.1 cells (29). Although the KE is conserved upstream of the mouse, rat, and human renin genes, its activity differs substantially across species (11, 43). Mutation of the CRE, E-box, or the steroid hormone response element (HRE) significantly attenuates enhancer-mediated transcriptional activity (24, 31). However, the enhancer also appears to bind transcription factors involved in negative regulation (17, 30) and may be required to mediate the negative regulatory response to cytokines (4, 18, 27).

Despite an extensive evaluation of the KE in vitro, only recently has its function been examined in vivo. For example, a marked decrease in renal renin under baseline and low-salt diet conditions was reported in REKO mice engineered to lack the endogenous mouse renin KE and additional sequences (2, 20). As a complementary approach, we used homologous recombination to precisely delete only the 241-bp KE located ~11 kb upstream of the human renin gene in the context of a large P1 artificial chromosome (PAC160) (22). PAC160 contains 160 kb of DNA from human chromosome-1 consisting of the human renin gene, two upstream genes (GOLT1A and KISS1), and one downstream (ETNK2) gene (23). PAC160{Delta}KE is identical to PAC160 except that the KE was replaced with a single loxP511 site, a heterospecific loxP variant used in gene targeting when a loxP site already exists (32, 46). We observed a significant decrease in human renin mRNA in the kidney at baseline in PAC160{Delta}KE transgenic mice. However, despite the striking decrease in baseline human renin expression, its tissue specificity, juxtaglomerular (JG) cell specificity, and physiologically regulated expression were preserved. Similar results were obtained in two independent lines of transgenic mice carrying either a single or several copies of the transgene. Our results suggest that elements other than the KE may be necessary to regulate expression of the renin gene.

Interestingly, in addition to the two lines of mice which carry complete copies of the PAC160{Delta}KE transgene, we identified two additional lines which carry forms of the transgene which were truncated upstream of the human renin gene. Both lines lack the upstream GOLT1A and KISS1 genes but retain the human renin and downstream ETNK2 genes. Analysis of these lines which exhibit dysregulated renin expression but preserved ETNK2 expression suggests that sequences surrounding the renin and ETNK2 loci which bind the insulator protein CTCF may insulate them from neighboring genes (7). These boundaries may act to protect the unique tissue-specific expression profile of closely linked genes. These data serve to illustrate how large genomic constructs carrying multiple genes can be employed to provide a wider genomic perspective than possible when examining the expression of single genes on their own.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Generation of KE-deficient PAC160 transgenic mice. Construction of the KE deletion mutant of PAC160 and of transgenic mice containing PAC160 and PAC160{Delta}KE was previously reported (32, 46). All mice were fed with standard mouse chow (LM-485; Teklad Premier Laboratory Diets) and water ad libitum. Captopril (CAP) was dissolved in drinking water (0.5 mg/ml) and administered to mice for 10 days. Care and use of mice met the standard set forth by the National Institutes of Health and all procedures were approved by the University Animal Care and Use Committee at the University of Iowa.

Southern blot, PCR, and gene expression assays. Three probes were generated for use in Southern blot analysis. Probe a is a hREN cDNA derived from pRHR1100-FM (gift of T. L. Reudelhuber, Clinical Research Institute of Montreal). Probe b is derived from a 550-bp DNA sequence located immediately upstream of the chorionic enhancer reported to be active in choriodecidual cells (9). It was cloned from purified PAC160 DNA using the primers listed in Supplemental Table S. 1 (the online version of this article contains supplemental data). Probe c is similarly derived from a 503-bp DNA sequence immediately upstream of the KE using the primers listed in Supplemental Table S. 1. Southern blots were performed as previously reported (46). PCR was performed using the primer pairs listed in Supplemental Table S. 1 to identify the location of the truncation breakpoint in lines PAC160{Delta}KE3 and PAC160{Delta}KE6. One hundred to two hundred nanograms of genomic template DNA isolated from spleen were used with high-fidelity Taq polymerase in each reaction (Invitrogen).

Tissues were homogenized in Tri-Reagent (Molecular Research Center, Cincinnati, OH) and the RNA was isolated and subjected to RNase protection as previously described (46). Superscript III one-step RT-PCR with Platinum Taq (Invitrogen) was used according to the manufacturer's instructions using the primers listed in Supplemental Table S. 1.

Immunohistochemistry. Immunohistochemistry on frozen kidney sections was performed as described previously (46).

In vitro transcription translation and EMSA. Recombinant CTCF protein corresponding to the zinc finger domain (hCTCFZnF11; ~45 kDa) was obtained using similar methods to those previously described (3, 7, 15). Briefly, human cDNA encoding the full-length 11 zinc finger domain of CTCF was amplified from Calu-6 cDNA and cloned into pQE-30 (Qiagen) (33). The cloned fragment was PCR amplified to generate a template for in vitro translation using Platinum Taq DNA polymerase high-fidelity (Invitrogen) using the primers listed in Supplemental Table S. 1. The presence of a single specific band was confirmed on a 1% agarose gel. Five microliters of the PCR reaction were used as a template for in vitro transcription using reticulocyte lysate TNT T7 Quick for PCR kit (Promega). Parallel [35S]methionine labeling reactions were carried out to confirm production of a specific protein product of the appropriate size. Labeled [35S]protein was resolved on 10% SDS-PAGE gels.

EMSAs were performed as previously described (3, 7, 15). Probes were constructed by annealing two single-strand oligos with 5'-GATC overhangs and the resultant double-strand probe was labeled with [{alpha}-32P]dATP. Probe sequences for the 5' hypersensitive site 5 of the β-globin locus (HS5), human renin intron (probe 1), and the intergenic sites between ethanolamine kinase 2 (ETNK2) and SRY-box 13 (SOX13; probes 2 and 3) were obtained from the UCSC genome browser. The HS5 probe has been previously described (7) and potential CTCF binding sites were identified by loading a track accessible at http://licr-renlab.ucsd.edu/download.html as referenced in Ref. 15. Binding reactions were carried out in 1x PBS solution with 5 mM MgCl2, 0.1 mM ZnSO4, 1 mM DTT, 0.1% Nonidet P-40, 10% glycerol, and 50 ng/µl of poly (dI-dC) plus a 44-mer double-strand nonspecific competitor and the indicated probes were listed in Supplemental Table S. 1. Each reaction contained 2 µl of programmed extract and 20–40 fmol of labeled probe. Competition reactions contained 100-fold molar excess of cold probes. Each reaction was incubated at room temperature for 30 min followed by separation of complexes on 5% polyacrylamide gels in 0.5x TBE.

Identification of the site of PAC160{Delta}KE6 insertion. Genomic DNA from a {Delta}KE6 mouse and a synthetic double-strand adapter were digested in separate reactions with BamHI, SalI, NruI, and EcoRI. The sequence of the top strand of the double-strand adaptor is provided in Supplemental Table S. 1. Fragments were purified using Qiagen's Qiaquick PCR purification kit and genomic DNA fragments were subsequently digested with PacI. Separate ligation reactions were carried out with digested genomic DNA and the complementary adapter. Aliquots of the ligation reactions were then used for PCR with transgene and adapter-specific primers. PCR products were separated on a gel and an enriched fragment from the SalI reaction was purified, subjected to another round of PCR with nested primers, and gel purified again. The purified PCR product was sequenced and results were submitted for BLAT against the mouse genome using the UCSC genome browser (February 2006 Assembly). Based on the BLAT result, two forward primers (Supplemental Table S. 1) were designed specific to the gene hypothesized to contain the transgene insertion. PCR reactions were performed to confirm the site of transgene insertion using those primers in combination with a transgene-specific reverse primer. One {Delta}KE6-specific band was then sequenced and analyzed by BLAT to confirm the site of transgene insertion. Primers used to detect Zbtb20-renin fusion transcript are listed in Supplemental Table S. 1. The RNase protection probe used to identify Zbtb20-renin fusion transcript was derived from amplification of {Delta}KE6 RNA with the primer set indicated in Supplemental Table S. 1 and cloned into pCR4-TOPO2 (Invitrogen).

Chromatin immunoprecipitation. Mouse liver tissue for ChIP assays was harvested from ({Delta}KE6) transgenic and nontransgenic mice. Tissues were minced on ice and suspended in PBS containing 1% (vol/vol) formaldehyde at room temperature for 15 min. Reactions were stopped by the addition of glycine (0.125 M, 5 min, room temperature), homogenized on ice, and then rinsed with ice-cold PBS three times. The final washed pellet was resuspended in lysis buffer with protease inhibitors (EZ-CHIP kit, Millipore) and sonicated on ice under the following conditions (Amplitude 50%, time 15 s, cooling 15 s, 10–15 times using a Sonic Dismembrator model 500, Fisher Scientific). The size of the sonicated chromatin was verified as between 400 and 700 bp by electrophoresis. The chromatin immunoprecipitation was performed following the instructions provided by the manufacturer using CTCF antibody (Upstate Biotechnology, 07–729), rabbit normal IgG as negative control, and 2% input as a positive control. The primers used for PCR are shown in Supplemental Table S. 1. The mouse H19 gene was used as a positive control for CTCF binding (21).


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Identification of truncated PAC160 transgenes. We generated four lines of transgenic mice carrying the human PAC160 transgene (termed PAC160{Delta}KE) where the KE was removed and replaced with a loxP511 site (Fig. 1A) (46). Analysis of two lines carrying completely intact PAC160{Delta}KE transgenes (lines 2 and 4) was previously reported (46). Here, we report on two additional lines (lines 3 and 6) carrying extensive truncations in the PAC160{Delta}KE transgene.


Figure 1
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  		Fig. 1. Generation of the PAC160{Delta}KE transgenes. A: schematic representation of the PAC160{Delta}KE transgenes showing the exons of the hREN gene (vertical black lines), the kidney enhancer replaced by a loxP511 site (cross-hatched arrow), the chorionic enhancer (CE; checkerboard square), and neighboring genes (gray arrows) in PAC 160. The direction of transcription is indicated by the direction of the arrows. The 2 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 arrowhead. B: schematic map of the resultant insertions of PAC160{Delta}KE in the genomes of lines {Delta}KE3 and {Delta}KE6. The deletion in {Delta}KE6 included all DNA in PAC160{Delta}KE upstream of the KE including the GOLT1A and KISS1 genes, and extending to a point ~10.4 kb upstream of hREN (0.6 kb downstream of the KE). The deletion in {Delta}KE3 extended further to a point ~2.5 kb upstream of hREN.

 
Transgenic founders were initially identified by PCR using primers present within the human renin gene and then Southern blotting was performed to determine whether each 160-kb transgene remained intact after insertion into the genome. Consistent with the initial PCR, Southern blots using a human renin cDNA probe correctly identified three HindIII bands in genomic DNA from all four lines confirming the presence of the human renin gene (Fig. 2, probe a). Interestingly, the hybridization signal was lost in line {Delta}KE3 when we used a probe located ~6.3 kb upstream of the human renin gene (Fig. 2, probe b). The hybridization signal was also lost in lines {Delta}KE3 and {Delta}KE6 when a probe located 11.2 kb upstream was employed (Fig. 2, probe c). There was no signal in either line when a cDNA derived from the upstream GOLT1A gene was used as a probe (data not shown). The absence of GOLT1A DNA is consistent with the loss of its expression in all tissues examined from these transgenic mice. These data suggested that truncation of the PAC160{Delta}KE transgene occurred in lines 3 and 6.


Figure 2
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  		Fig. 2. Southern blot of PAC160{Delta}KE transgenic mice. A: schematic representation of the region upstream of the hREN gene showing the location of the 3 probes (a, b, c) used in the Southern blots shown in B. B: Southern blots of genomic DNA isolated from mice carrying wild-type PAC160 (WT), lines carrying intact copies of PAC160{Delta}KE ({Delta}2 and {Delta}4), and lines carrying the truncated PAC160{Delta}KE transgenes ({Delta}3 and {Delta}6). The probes are indicated on the left of each blot. The DNA was digested with HindIII (top), BamHI (middle), and MscI (bottom). M, male; F, female; h, human renin; m, mouse renin.

 
To determine the breakpoint of the truncation upstream of human renin in lines {Delta}KE3 and {Delta}KE6, we developed primer sets distributed between the KE and the transcription start site and performed PCR on genomic DNA (Fig. 3A, Supplemental Table S. 1). No specific PCR products were detected in genomic DNA from nontransgenic mice, and all the expected products were identified in genomic DNA from transgenic mice carrying an intact wild-type PAC160 transgene (Fig. 3B). The presence or absence of a PCR product allowed us to determine the approximate location of the truncation in {Delta}KE6 (Fig. 3B) and {Delta}KE3 (Fig. 3C). The truncation in line {Delta}KE3 was identified ~2,500 bp upstream of the transcription start site, whereas in line {Delta}KE6 the truncation was identified 10,440 bp upstream of the transcription start and ~0.6 kb downstream of where the KE was replaced with a loxP511 site (Fig. 1B).


Figure 3
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  		Fig. 3. Identification of truncation break points in PAC160{Delta}KE3 and PAC160{Delta}KE6. A: schematic representation of the 5' flanking region of the hREN gene and the location of primer sets P2-P6 and PA-PE. The approximate location of the truncations is indicated by an X. B: PCR analysis of genomic DNA from nontransgenic (N), PAC160{Delta}KE6 ({Delta}6), and mice carrying an untruncated PAC160 transgene (W) using the indicated primer sets. C: PCR analysis of genomic DNA from nontransgenic (N), PAC160{Delta}KE3 ({Delta}3), and mice carrying an untruncated PAC160 transgene (W) using the indicated primer sets. The gels are representatives of at least 3 PCR reactions with different mice for each PCR test.

 
Expression of human renin in transgenic mice carrying truncated PAC160. We next examined the tissue-specific expression of these human transgenes. We previously reported that human renin in wild-type PAC160 and full-length PAC160{Delta}KE mice is expressed almost exclusively in the kidney with much lower levels of renin mRNA in lung and brain (32, 46, 47). Human renin mRNA in line {Delta}KE3 was predominantly observed in the lung and very low levels of renin mRNA were identified in kidney (Fig. 4A). We previously reported that in the lung, expression of renin mRNA is initiated from an alternative promoter located in intron A, which generates a new first exon (termed exon 1c) (34). Given our previous results and the truncation of DNA close to the renin promoter, we predicted that transcription would be derived entirely from the exon 1c promoter. Consequently, we performed RT-PCR using primer sets designed to distinguish exon 1a from exon 1c renin mRNA. These assays were performed with each primer set alone (Fig. 4B) or together (Fig. 4C) in the PCR reaction. Our data clearly indicate that exon 1c was the exclusive form of renin mRNA found in the lung. These data confirm our previous hypothesis that renin expression in the lung is derived from a alternative promoter located entirely in the first intron of the human renin gene (34).


Figure 4
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  		Fig. 4. Expression of the PAC160{Delta}KE3 transgene. A: representative multiplex RNase protection assay of hREN, GOLT1A, ETNK2, and 28S expression from total tissue RNA (50 µg) from a male PAC160{Delta}KE3 mouse. The location of the protected fragments is indicated. The RPA is representative of 2 mice. Tissues labels are brain (B), heart (H), kidney (K), liver (L), lung (Lg), skeletal muscle (S), submandibular gland (Sg), and testis (T). B–C: RT-PCR of RNA derived from kidney and lung of PAC160{Delta}KE3 ({Delta}KE3) and transgenic mice carrying an untruncated PAC160 transgene (WT). The presence and absence of reverse transcriptase are indicated by + or –. RT-PCR was performed with primer sets designed to amplify either exon-1a or exon-1c renin mRNA (B) or competitively (C). The position of those products is indicated. Identical results were obtained using a different exon-1c-specific primer set (data not shown). Size markers in B are 100-bp ladder. 1a, Exon-1a (kidney specific); 1c, exon-1c (lung specific).

 
We next examined the tissue-specific expression of human renin in {Delta}KE6 mice. Contrary to our expectation, human renin was ubiquitously expressed in all tissues (Fig. 5). This observation was confirmed by both RNase protection (Fig. 5A) and RT-PCR (Fig. 5C). For comparison purposes, human renin mRNA was evident in the kidney, but not liver in mice carrying the full-length (untruncated {Delta}KE4 transgene), but in both the kidney and liver of {Delta}KE6 mice (Fig. 5B). Whereas expression of human renin in the kidney was not responsive to CAP (0.08 ± 0.008 vs. 0.09 ± 0.004, 1.1-fold, P = 0.32), there was an appropriate induction of endogenous mouse renin mRNA (0.03 ± 0.003 vs. 0.18 ± 0.01, 6-fold, P < 0.001) in the kidney of these mice, and of human renin mRNA (0.38 ± 0.04 vs. 0.75 ± 0.01, 2-fold, P < 0.001) in the kidney of mice carrying an intact PAC160 transgene in response to CAP (Fig. 6). At the cellular level, a few foci of JG cells containing low levels of human renin protein were observed by immunostaining in the kidney of {Delta}KE6 mice under baseline conditions (Fig. 7). There was no human renin protein detected in the kidney of nontransgenic mice, and a normal pattern of JG cell staining of human renin in the kidney from mice expressing a full-length PAC160 transgene. In response to CAP, the intensity of renin immunostaining and the extent of staining along the afferent arteriole were increased in mice containing wild-type PAC160. However, consistent with the RNase protection assays data, there was no induction in kidney from {Delta}KE6 mice.


Figure 5
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  		Fig. 5. Expression of the PAC160{Delta}KE6 transgene. A: representative multiplex RNase protection assays of hREN, GOLT1A, ETNK2, and 28S expression from total tissue RNA (50 µg) from male (left) and female (right) PAC160{Delta}KE6 mice. The location of the protected fragments is indicated. The RPA is representative of 6 different mice. B: representative multiplex RNase protection assays of hREN, GOLT1A, ETNK2, and 28S expression from total kidney and liver RNA comparing the untruncated full-length {Delta}KE4 (4) line with the truncated {Delta}KE6 (6) line. C: RT-PCR analysis of human renin expression in captopril (CAP)-treated PAC160{Delta}KE6 mice. U, uterus; NT, nontransgenic. WT1 and WT2 are 2 different lines of untruncated PAC160 mice.

 

Figure 6
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  		Fig. 6. Induction of hREN and mREN mRNA in response to CAP in PAC160{Delta}KE6. Total kidney RNA was isolated from vehicle- or CAP-treated PAC160{Delta}KE6 transgenic ({Delta}KE6) mice and transgenic mice carrying an untruncated PAC160 transgene (WT). RPA was performed with probes designed to specifically detect hREN and mREN using β-actin as loading control. A representative portion of the RPA gels are shown. A and B: PAC160{Delta}KE6. C: wild-type.

 

Figure 7
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  		Fig. 7. Immunostaining of human renin in kidney. Representative confocal immunofluorescent images of frozen kidney sections from nontransgenic (NT), transgenic mice carrying an untruncated PAC160 transgene (WT), and PAC160{Delta}KE6 ({Delta}KE6) under vehicle (VEH) or after CAP. These are representative sections from at least 2–3 mice per treatment. Photomicrographs were taken with x10 lens. Arrows point to juxtaglomerular (JG) apparatuses positively stained for renin.

 
Identification of site of insertion of PAC160{Delta}KE6. The ubiquitous expression and loss of CAP responsiveness despite the maintenance of a substantial 5' flanking region suggested to us that the {Delta}KE6 transgene may have come under the influence of regulatory elements at the site of transgene insertion (i.e., a position effect). To test this hypothesis, we cloned and identified the site of transgene insertion using a method analogous to the 5' RACE protocol used to clone the 5' end of mRNAs (Fig. 8A). Sequence analysis of the resulting PCR product revealed 1) that the deletion breakpoint occurred precisely 10,440 bp upstream of the renin transcription start site, 2) that the transgene inserted into a relatively gene sparse region of mouse chromosome 16 near the Zbtb20 locus (NM_019778 [GenBank] ), and 3) the insertion occurred between exons 1 and 2 of the Zbtb20 gene, 115,204 bp downstream of its annotated first exon. The direction of human renin and ETNK2 transcription was in the same orientation as Zbtb20. The site of insertion was confirmed by using two different primer sets consisting of an upstream primer present in Zbtb20 DNA adjacent to the insertion and a downstream primer in the renin locus. Specific PCR products of 1,677 and 1,047 bp were only observed in PAC160{Delta}KE6 genomic DNA, but not in genomic DNA from nontransgenic mice or mice carrying a full-length (untruncated) PAC160 transgene (Fig. 8B).


Figure 8
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  		Fig. 8. Identification of the insertion site in PAC160{Delta}KE6. A: schematic of the method used to identify the {Delta}KE6 insertion site as described in MATERIALS AND METHODS. R, restriction site present in DNA upstream of the insertion and also present in the adaptor; Pac1, an anchor restriction site in the renin locus near the breakpoint. The bold line represents known renin locus DNA, whereas the dotted line represents unknown DNA upstream of the insertion. The cross-hatched box represents the adaptor and the arrows indicate the PCR primers. B: PCR was performed on genomic DNA isolated from nontransgenic (N), transgenic mice carrying the untruncated full-length PAC160 transgene (W), and PAC160{Delta}KE6 (6) using primer sets designed to amplify either a 1,677- or 1,047-bp segment flanking the insertion. The upstream primer was in Zbtb20 genomic DNA and the downstream primer was in PAC160{Delta}KE6 DNA close to the break point.

 
It is noteworthy that like the ubiquitous expression of renin in {Delta}KE6 mice, Zbtb20 also appears to be ubiquitously expressed. This is based on an analysis of 173 gene expression profiling data sets deposited in the publicly accessible Gene Expression Omnibus Database (GEO, http://www.ncbi.nlm.nih.gov/projects/geo/) using entire tissue RNA as the query probe. Positive calls for Zbtb20 expression were noted in nearly 100% of the data sets where RNA from brain (n = 24), kidney (n = 21), heart (n = 33), liver (n = 33), lung (n = 36), muscle (n = 5), or testes (n = 21) was queried. On the contrary, a positive call for renin was found in 100% of data sets using kidney RNA as the query, in ~20% of the data sets querying brain or heart, and in less than 3% of the sets querying liver or lung. Based on this, we determined whether the ubiquitous expression of renin was due to a fusion of the Zbtb20 and renin transcripts or was due to influences of Zbtb20 regulatory sequences on the renin promoter. PCR primers were designed in Zbtb20 exon 1 and human renin exon 5. Two RT-PCR products, one major and one minor, were identified in all tissues from {Delta}KE6 but not from nontransgenic mice or tissues from mice carrying untruncated wild-type PAC160 transgenes (Fig. 9A). Based on this result, we designed an RNase protection assay to determine the relative abundance of renin transcripts initiated at the renin or Zbtb20 promoters (Fig. 9B). Although this varied among tissues, ~50% of the renin transcripts in tissues from {Delta}KE6 mice initiated at the Zbtb20 promoter while 50% did not contain Zbtb20 sequences and most likely initiated at the renin promoter. These data suggest that ubiquitous expression in this line was due to both Zbtb20 promoter activity and influences of Zbtb20 regulatory elements on the renin promoter.


Figure 9
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  		Fig. 9. Identification of fusion transcripts between Zbtb20 and human renin in PAC160{Delta}KE6. A: RT-PCR was performed using primers present in Zbtb20 exon 1 and human renin exon 5. Specific products were only identified in tissues from PAC160{Delta}KE6 mice. Sequence analysis of the major form reveals the transcript is a fusion between Zbtb20 exon 1 splicing directly to human renin exon 2. B: RNase protection assay using a probe derived from a fusion transcript identified by RT-PCR between Zbtb20 exon 1 and human renin exon 2. The 246 nucleotide protected product is indicative of a fusion transcript between Zbtb20 and human renin, whereas the 197 protected product does not hybridize to Zbtb20 sequences and therefore consists of only renin mRNA. WT2, PAC160 containing the kidney enhancer; {Delta}KE2, full-length (untruncated) PAC160{Delta}KE2; {Delta}KE6, truncated PAC160{Delta}KE6.

 
Expression of ETNK2 in transgenic mice carrying truncated PAC160. Perhaps one of the most interesting observations was that despite the loss of renin expression in the kidney of {Delta}KE3 mice and ubiquitous expression of human renin in {Delta}KE6 mice, that the normal pattern of tissue-specific expression of the immediate downstream gene ETNK2 was preserved. ETNK2 expression was observed in kidney, liver, and testes of both {Delta}KE3 (Fig. 4A) and {Delta}KE6 (Fig. 5A) mice exactly as we described for wild-type PAC160 and full-length PAC160{Delta}KE (46). Therefore, the correct pattern of human ETNK2 expression was preserved despite dysregulation of human renin expression. As expected, given the location of the truncation, there was no expression of GOLT1A mRNA. These data suggest the possibility that there may be insulators surrounding the renin and/or ETNK2 locus.

Identification of CTCF binding sites in the hREN and ETNK2 loci. Although mammalian insulator sequences remain poorly defined, the CTCF protein (also known as CCCTC binding factor) has been reported to act as an insulator (7). Kim et al. (15) examined the location of CTCF binding sites genome wide using a combination of chromosome immunoprecipitation followed by genome tiling microarrays. They identified six CTCF binding sites in and around the renin locus (arrows in Fig. 10A). We generated double-strand oligonucleotides to three conserved CTCF binding sites located in the first intron of the renin gene (site 1) and downstream of ETNK2 (sites 2 and 3) and performed EMSA using the 11-zinc-finger motif from CTCF generated by coupled in vitro transcription and translation (Fig. 10B). We used the β-globin hypertensive site 5 (HS5) which has been reported to bind CTCF and act as an insulator (7) as a control. A clear gel shift was obtained with HS5 using programmed reticulocyte extract but not with unprogrammed extract (+ vs. – in Fig. 10C). Excess cold oligonucleotide (S) effectively competed for binding, whereas a probe with mutations in the CTCF binding site was ineffective as a competitor. Similarly, CTCF was able to bind to the sites within the renin intron and downstream of ETNK2. There was no binding when mutant oligonucleotides were used as probes.


Figure 10
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  		Fig. 10. CTCF binds to sites in the renin and ETNK2 genes. A: schematic representation of the genes encoded on PAC160 showing the location of CTCF binding sites previously reported in a whole genome scan (15). B: coupled in vitro transcription-translation of [S35]methionine labeled CTCF zinc-finger DNA binding domain. The reticulocyte extract was either programmed with CTCF template (+) or without (–) template. C: EMSA of unlabeled CTCF zinc-finger binding domains with the indicated labeled double-strand oligonucleotides. –, EMSA with unprogrammed reticulocyte extract; +, EMSA with CTCF-programmed reticulocyte extract; S, 100-fold excess cold competitor identical in sequence to the probe; M, 100-fold excess of mutant competitor. The last 2 lanes utilize labeled double-strand mutant oligonucleotides as probes.

 
To determine whether the CTCF binding sites identified above are physiologically relevant in vivo, we performed chromatin immunoprecipitation (ChIP) on chromatin isolated from the liver of {Delta}KE6 transgenic and nontransgenic control. Liver was chosen as it is a relative homogenous tissue and an endogenous site of ETNK2 expression. Recall that tissue-specific expression of ETNK2 was preserved in {Delta}KE6 mice. Primers were chosen to amplify DNA in the H19 gene, a control gene known to bind CTCF (21), and the CTCF binding sites in intron A of hREN (site 1 in Fig. 11A) and downstream of ETNK2 (site 2 in Fig. 11A). A PCR product was detected on the input DNA (labeled I in Fig. 11C) for transgenic and nontransgenic for endogenous H19, but only in transgenic DNA for site 1 and site 2. Similarly, a positive ChIP signal using CTCF antisera (labeled {alpha}C) was evident for endogenous H19 whether derived from transgenic or nontransgenic chromatin. A ChIP signal was only detected for site 1 and site 2 when chromatin was purified from transgenic mice. There was no ChIP signal when nonimmune serum (labeled –) was tested on transgenic chromatin. Interestingly, an alignment of the composite CTCF binding site detected by Kim et al. (15) revealed a potential CTCF in the intergenic region between hREN and ETNK2 (Fig. 11B). Although double-strand oligonucleotides containing this sequence did not result in a gel shift using in vitro transcribed and translated zinc-finger motif from CTCF (data not shown), a positive albeit weaker ChIP signal was detected on this sequence in liver chromatin from transgenic but not nontransgenic mice (labeled site RE). It required 40 cycles of PCR to detect the ChIP signal from site RE, whereas it took only 30 cycles for the other sites. Because of the apparent difference in efficiency, we repeated this study using real-time quantitative PCR comparing the efficiency of amplification of the CTCF ChIP and IgG ChIP with input ({Delta}CT). Figure 11D shows that the {Delta}CT with IgG was on average 10.6 ± 0.5, whereas the {Delta}CT with CTCF antisera varied depending on the primer set. Consistent with above, the ChIP signal for site RE was weaker than sites 1 or 2. Nevertheless, the CTCF ChIP signal for all primer sets was much stronger than for a negative control site in the CD3 gene which does not bind CTCF. The enrichment in the CTCF ChIP signal based on the {Delta}{Delta}CT was 315- and 445-fold for sites 1 and 2, respectively, and 21-fold for site RE (Fig. 11E). These data clearly identify CTCF binding sites in the renin gene, in the intergenic region between renin and ETNK2, and intergenic region between ETNK2 and SOX13.


Figure 11
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  		Fig. 11. CTCF binds to chromatin in the renin and ETNK2 genes. A: schematic representation of the genes encoded on PAC160{Delta}KE6 showing the location of CTCF binding sites previously reported in a whole genome scan (15). B: clustal W alignment of CTCF binding site core motifs flanked by 10 bp 5' and 3'. Shading was done using a server located at http://www.ch.embnet.org/software/BOX_form.html running Boxshade 3.21. Black represents a base identical to the consensus sequence and gray indicates a similar base. The mH19 sequence is as described in Ref. 13; all other sequences were obtained from the UCSC Genome Browser-Human May 2004 (hg17) Assembly. C: ChIP performed on the indicated sites using 2% input (I), CTCF antisera ({alpha}C), or nonimmune IgG (–) on liver chromatin from KE6 transgenic (Tg+) and nontransgenic control (Tg–). D–E: ChIP was performed as in C but quantified with real-time QPCR. D: {Delta}CT comparing the individual ChIP signals from CTCF (solid) and IgG (gray) vs. input. *P < 0.001 vs. all other ChIP signals. E: {Delta}{Delta}CT comparing the CTCF ChIP from the indicated primer sets with the negative control CD3. The fold-enrichment in the signal is indicated above each bar.

 

   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
We tested the in vivo function of the distal KE in the 5' flanking region of the human renin gene using homologous recombination (recombineering) of the PAC160 construct in bacteria coupled with the generation of transgenic mice employing the altered constructs. From an examination of two transgenic lines carrying full-length PAC160{Delta}KE transgenes, we recently reported that the KE is dispensable for cell-specific, tissue-specific and physiologically regulated expression, but is required to maintain baseline expression of renin (46). This conclusion is consistent with that reported using the "REKO" mouse, a complementary model consisting of a targeted deletion of the KE and surrounding sequences upstream of the endogenous mouse renin gene (2, 20). In addition to the two transgenic lines carrying intact PAC160{Delta}KE transgenes, we unexpectedly obtained two transgenic lines carrying extensive truncations of the construct. The truncations in these two lines were unidirectional, that is, DNA upstream of human renin was deleted, whereas the DNA downstream remained intact. Although it is likely that independent deletions in other transgenic founders were also generated, they probably went undetected because the initial screen and selection for transgenic founders were based on PCR using primers within human renin. Consequently, any deletion which included human renin would not be identified.

Expression of human renin in transgenic mice that carry truncated constructs. Instead of discarding these two transgenic founders, we reasoned that the spontaneous deletion mutants may provide an unanticipated tool for identifying, in an unbiased way, important regulatory sequences controlling human renin expression. We therefore examined them for expression of human renin and, as an internal control, the immediate downstream gene ETNK2. Truncations of genomic sequence upstream of the human renin gene resulted in two vastly different renin expressing phenotypes. In {Delta}KE3, where the truncation extended to within 2.5 kb of the renin promoter, human renin expression was primarily detected in the lung. Moreover, the form of renin mRNA observed in the lung was exclusively the exon 1c renin mRNA previously identified by us using 5' RACE (34). Exon 1c is an alternative first exon made up by the terminal 78 bp of the first intron which in the mRNA is fused directly to exon 2 without the need for splicing. These data confirm the identification of this alternative mRNA and suggest that despite an extensive truncation upstream of renin, the lung-specific promoter was left intact. It is interesting to note that this phenotype of lung-specific renin expression is quite similar to the expression of human renin in transgenic mice containing short genomic transgenes consisting of either 896 or 149 bp of the renin promoter (35). Multiple lines of 896-hREN or 149-hREN transgenic mice exhibited much higher renin expression in lung than kidney, whereas in wild-type PAC160, expression in the lung was much lower than in kidney (46). The physiological significance of renin expression in the lung, and the lung-specific expression of this isoform predicted to be translated into a protein lacking the signal peptide, remains unknown. That lung is thought to be a bonafide site of renin expression is evidenced by its expression in a pulmonary carcinoma cell line (16) and in tumors of pulmonary origin (8, 37, 40).

Unlike the lung-specific expression of renin observed in {Delta}KE3, renin expression in the {Delta}KE6 line was completely ubiquitous. Moreover, there was a loss of responsiveness of the transgene to CAP. It is interesting that the more "severe" phenotype was observed in the line which retained a larger amount of 5' flanking DNA. We determined the site of insertion to be between exons 1 and 2 within the Zbtb20 locus with transcription of renin and ETNK2 to be in the same direction as Zbtb20. The Zbtb20 gene apparently encodes a zinc finger transcription factor which is ubiquitously expressed (10, 45). Altogether, these data suggest the possibilities that 1) there was a loss of critical regulatory elements located somewhere upstream of the truncation breakpoint which normally prevents renin expression in nonrenin expressing tissues, 2) that the renin transcript detected in our assays was a fusion transcript initiated at the Zbtb20 promoter and consisting of Zbtb20 1st exon fused to renin exon 2, or 3) that the renin gene was now being strongly influenced by Zbtb20 regulatory elements located near the site of transgene insertion. Our RT-PCR and RNase protection assay results support the latter two hypotheses and suggest that the ubiquitous expression of human renin mRNA in the {Delta}KE6 mice was due to both transcription initiation at the Zbtb20 promoter and at the renin promoter. We hypothesize that transcripts initiated at the renin promoter fell under the regulatory influences of elements which normally control Zbtb20.

Evidence for the presence of insulators in and around the renin locus. Why would expression of renin become influenced by regulatory elements controlling another gene? Locus control regions (LCRs) are thought to function through an ability to manipulate the structure of chromatin (6). Large genomic clones (such as those on BACs or PACs as utilized in this study) are more likely to contain LCR sequences. Canonical to this is the β-globin LCR which consists of a series of DNase I-hypersensitive sites which confer high level, copy number-proportional and position-independent expression in transgenic mice (39). We previously demonstrated that expression of human renin and ETNK2 in the untruncated and unmutated PAC160 construct is proportional to copy number (32, 46). It is significant that genomic DNA containing the hypersensitive sites in the β-globin LCR also have the property of acting as insulators when placed between an enhancer and promoter in transfected erythroid cells (5). Insulators are defined as cis-acting elements which can "shield" genes from the effects of neighboring silencers and enhancers. In this fashion, one of the most interesting findings from the current study was a preservation of the normal tissue-specific expression pattern of ETNK2 in the kidney, liver, and testes despite dysregulation of human renin expression. This was true under conditions where renin expression was observed only in lung ({Delta}KE3) or when renin expression was ubiquitous ({Delta}KE6). This suggests the possibility that sequences within the renin and ETNK2 locus may function as an insulator.

Interestingly, the mammalian insulator protein CTCF has been reported to bind to several of the hypersensitive sites flanking the β-globin gene in chicken, mouse, and human, and mutation of its binding site eliminates its ability to act as an insulator (7). A genome wide scan for CTCF binding sites has been reported (15). Their data reveal potentially conserved CTCF binding sites in the renin gene, downstream of ETNK2 and upstream of GOLT1A. Our data show that the three sites tested all have the capacity to bind CTCF in vitro. Chromatin immunoprecipitation reveals that these sites actually bind CTCF at least in liver where ETNK2 is expressed. We did not test whether they also bind to chromatin in renin expressing cells in kidney because JG cells represent less than 0.1% of the cells in the kidney. Interestingly, a sequence with homology to the core CTCF binding site was identified in the intergenic region between renin and ETNK2. Although this site did not bind the zinc-finger motif of CTCF in vitro, it was identified as a weak CTCF binding site in vivo based on ChIP.

Conceptually, these results are interesting because they suggest that the CTCF binding sites may act as chromatin boundaries providing insulation between genes. If this is true, then the loss of the CTCF binding sites upstream of the renin locus in {Delta}KE3 and {Delta}KE6 may have left the renin gene unprotected from the influences of regulatory elements at or near their site of insertion, whereas the preservation of the CTCF binding sites upstream and downstream of ETNK2 may have protected its expression even in the face of dysregulated renin expression. That the CTCF binding site in the first intron of the human renin gene was preserved suggests that it is insufficient on its own to act as an insulator for renin. Instead, in conjunction with the CTCF binding site in the renin-ETNK2 intergenic region, it may act to insulate ETNK2 from transcriptional signals from genes lying upstream including renin. Interestingly, many of the CTCF sites identified in the human genome are at remote locations from transcription start sites, suggesting the interactions can be long range, whereas others are found within genes (15). CTCF binding sites found in the intergenic region between two genes may either control both genes such as the two MHC class II genes HLA-DRB1 and HLA-DQA1 (19) or differentially regulate the genes they separate such as Igf2-H19 (44). Interestingly, Igf2 and H19 share common enhancer elements located downstream of H19. As such, the insulator separates the Igf2 promoter from the shared enhancer and may control access to it.

Perspectives and significance. Typically, investigators examining mechanisms regulating gene expression make fusions between the promoter of that gene and some easily detectable reporter gene such as luciferase. Similar fusion constructs are often used to generate transgenic mice; and this type of analysis is often used to confirm the identity of cis-acting sites previously identified through "promoter bashing" studies in cell lines. However, the interpretation of these studies is complicated because expression of the transgene can vary widely among lines due to influences of both copy number and insertion site. Our approach using PAC (or BAC) clones containing renin and closely linked neighboring genes is attractive because the transgenes better emulate the structure of the native gene found in the genome, and the neighboring genes provide a critical internal control lacking in so many experiments. We feel that the use of large transgenes provides a broader genomic perspective that helps develop a more complete picture of gene regulation. Indeed, it is now becoming clear that in addition to considering cis-acting elements which bind transcription factors, investigators must also consider "longer range" effects on gene expression implicit in the structure and chemical modification of chromatin.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Institutes of Health (NIH) Grants HL-48058, HL-61446, and HL-55006. We acknowledge D. Davis for support with experiments involving captopril treatment of mice. Transgenic mice were generated at the University of Iowa Transgenic Animal Facility directed by C. D. Sigmund, Ph.D., and supported in part by grants from NIH and from the Roy J. and Lucille A. Carver College of Medicine. We gratefully acknowledge the generous research support of the Roy J. Carver Trust.


   ACKNOWLEDGMENTS
 
We thank N. Sinclair, P. Yarolem, and J. Schwarting for technical expertise in generating transgenic mice. We also thank the University of Iowa Central Microscopy Facility.


   FOOTNOTES
 

Address for reprint requests and other correspondence: C. D. Sigmund, Depts. of Internal Medicine and Physiology and Biophysics, 3181B Medical Education and Biomedical Research Facility, Roy J. and Lucille A. Carver College of Medicine, Univ. of Iowa, Iowa City, IA 52242 (e-mail: curt-sigmund{at}uiowa.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

* X. Zhou and E. T. Weatherford contributed equally to the data presented in this paper (co-first authors). Back


   REFERENCES
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 MATERIALS AND METHODS
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 DISCUSSION
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