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(Circulation Research. 1997;81:558-566.)
© 1997 American Heart Association, Inc.

Articles

Conserved Enhancer Elements in Human and Mouse Renin Genes Have Different Transcriptional Effects in As4.1 Cells

Yan Yan, Craig A. Jones, Curt D. Sigmund, Kenneth W. Gross, , Daniel F. Catanzaro

From the Cardiovascular Center (Y.Y., D.F.C.), Cornell University Medical College, New York, NY; the Department of Physiology (C.D.S.), University of Iowa College of Medicine, Iowa City; and the Department of Molecular Biology (C.A.J., K.W.G.), Roswell Park Cancer Institute, Buffalo, NY.

Correspondence to Daniel F. Catanzaro, PhD, Cardiovascular Center, Cornell University Medical College, 1300 York Ave, Room A863, New York, NY 10021. E-mail dfcatanz{at}mail.med.cornell.edu

	Abstract

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Abstract Despite the strong conservation of proximal 5'-flanking DNA sequences, cell transfection and transgenic animal studies have failed to provide a unifying hypothesis to explain the expression of both mouse and human renin genes. Recently, sequences contained in the mouse Ren-1^C gene 5'-flanking DNA (–2866 to –2625) were shown to contain an enhancer-like element that stimulates Ren-1^C promoter activity in renin-expressing As4.1 cells 80-fold. Earlier studies using transgenic mice had suggested that this same region is required for the cell-specific expression of mouse renin genes. Since existing human renin genomic clones lack sequences homologous to the mouse renin enhancer, we isolated several human P1 and P1 artificial chromosome genomic clones that contain >80 kb spanning the human renin gene. Analysis of these clones by Southern blot hybridization and long-range polymerase chain reaction showed that they contain sequences homologous to the mouse enhancer at 12 kb upstream of the transcription start site. Mouse and human sequences were 59% identical over a 650-bp region that contained the minimal enhancer from the mouse Ren-1^C gene. However, a 1-kb fragment containing the entire human enhancer homology failed to stimulate human renin promoter activity in transiently transfected As4.1 cells. Further deletional analysis showed that a 220-bp region of the human sequence highly conserved in the mouse Ren-1^C gene exhibited up to 47-fold transcriptional stimulation, although this was lower than the maximal effect exhibited by the minimal mouse enhancer (223-fold). Taken together, these observations suggest that sequences surrounding the conserved enhancer core stimulate enhancer activity in the mouse gene but suppress activity in the human gene. The high transcriptional activity of the mouse enhancer may have evolved to support the exceptionally high plasma renin concentrations found in mice. However, the enhancer core and surrounding conserved sequences may play an additional role in directing cell specificity.

Key Words: renin gene expression • enhancer • As4.1 cell

	Introduction

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Because much of our present knowledge about renin gene expression is based on studies carried out in mice and rats, understanding whether common mechanisms direct renin gene expression in different species is crucial to the development of animal models that will be relevant to human hypertension. In strains of laboratory mice, the plasma renin concentration is extraordinarily high compared with that in humans and rats. Several groups have reported mouse plasma renin concentrations exceeding 1000 ng Ang I · mL^-1 · h^-1,¹ whereas the levels are 10 ng Ang I · mL^-1 · h^-1 in rats² and 1 to 5 ng Ang I · mL^-1 · h^-1 in humans.³ Since renin is cleared from the circulation of mice and rats at similar rates,^{4 5} the higher plasma renin concentration found in mice suggests a higher rate of renin synthesis and secretion. However, transgenic mouse and transfection studies have failed so far to provide a unifying hypothesis to explain the differential expression of the renin genes in different species.

Among renin genes from mouse, rat, and human, sequences contained within several hundred base pairs upstream from the transcription start site are highly conserved.⁶ A number of transfection studies have shown that although these conserved renin 5'-flanking DNA sequences can direct expression of a linked reporter gene in cell cultures from various renin-expressing tissues,^{6 7 8 9} their promoter activity is similarly low, suggesting that sequences outside of these conserved regions (proximal to the transcription start site) might be involved in directing renin gene expression at appropriate physiological levels.

Studies of mouse renin gene expression in transgenic animals have produced conflicting results that also suggest the involvement of sequences outside of the conserved proximal 5'-flanking DNAs. Although one study showed that 2.5-kb 5'-flanking DNA was sufficient to direct cell-specific expression of the mouse Ren-2 structural gene,¹⁰ the same 2.5-kb 5'-flanking DNA failed to direct the cell-specific expression of a heterologous reporter (SV40 TAg).¹¹ In another study 4.6 kb 5'-flanking DNA from the mouse Ren-2^d gene was sufficient to direct the juxtaglomerular cell-specific expression of TAg,¹² which displayed correct temporal expression patterns.^{13 14} These apparently conflicting data were interpreted to suggest that sequences contained downstream from the transcription start site may contribute to high-level cell-specific expression, but they may be redundant to sequences further upstream in the 5'-flanking DNA.¹⁵ Recent studies showed that the region of the mouse Ren-1^C gene 5'-flanking DNA between –2866 and –2625 bp upstream from the transcription start site contains an enhancer that stimulates renin gene expression.¹⁶

Studies of hREN gene expression in transgenic animals also indicated that sequences outside the genomic regions that were tested might be required for fully appropriate cell- and tissue-specific expression. In two studies that examined human renin gene expression in transgenic mice^{17 18} renin genomic fragments containing coding, intervening, 0.9- or 3-kb 5'-flanking DNA, and 0.4- or 1.2-kb 3'-flanking DNA were found to be sufficient for expression in juxtaglomerular cells. However, renin mRNA was also detected at extrarenal sites, including some where renin mRNA had not previously been detected. Similarly, in a transgenic rat model¹⁹ using the same genomic sequences as in one of the mouse models described above,¹⁷ hREN was detected in juxtaglomerular cells and a number of extrarenal sites.

In the present study, we tested the hypothesis that the hREN gene contains an enhancer sequence similar to the mouse by isolating a number of P1 and PAC human renin genomic clones that contain extended flanking sequences. At 12 kb upstream from the transcription start site, we identified a region with 77% identity to the 243-bp mouse Ren-1^C gene minimal enhancer sequences. Despite this conservation of sequence, dramatic differences were observed in the ability of mouse and human sequences to stimulate renin promoter activity in transiently transfected As4.1 cells.²⁰ The much stronger enhancer activity of the mouse sequences may reflect the different transcription rates required to maintain the higher plasma renin concentrations in mice. However, enhancer sequences from both species may be involved in determining cell-specific expression.

	Materials and Methods

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Construction and Screening of Human P1 and PAC Genomic Libraries
Screening of P1 and PAC human genomic libraries was contracted to Genome System Inc by PCR using the following primers spanning the proximal 5'-flanking DNA region of the human renin gene: GGT ACC CTT CAC CCA CCT AGC TCT G (–147/–123) and GAT CCA CTG AGG TTC TGT GGC TCC C (–7/+18); in exon 3, TGA CAC TGG TTC GTC CAA TG; and in exon 4, ATA GCG GAG GGT GAG TTC TG. PAC libraries were constructed as described by Ioannou et al²¹ using the pCYPAC-2 vector and human genomic library RPCI-1 (prepared in the laboratory of Dr Pieter de Jong, Department of Human Genetics, Roswell Park Cancer Institute). The library was screened by hybridization with a mouse renin cDNA probe.²²

Southern Blot Analysis
P1 DNA was prepared using an alkaline lysis procedure^{23 24} from the IPTG-induced culture. P1 plasmid DNA was digested with various restriction enzymes, and the fragments were separated by either constant or pulse-field gel electrophoresis.²⁵ DNA was transferred to colony/plaque screen membranes (No. NEF-978, DuPont NEN) as specified by the vendor. Blots were hybridized to probes from human renin 5'-flanking DNA (–891/–148), hREN cDNA,²⁶ and mouse Ren-1^C enhancer (–3.1/–2.6 kb) according to the protocol provided by the vendor. Probes were prepared by randomly primed incorporation of [-³²P]dCTP. The membranes were washed with excess 2x SSC twice for 10 minutes at room temperature and then washed twice with 2x SSC and 1% SDS at 65°C for 30 minutes. The membranes then were covered with plastic wrap and exposed to Kodak X-OMAT AR film at room temperature or –70°C.

Subcloning and Sequences Analysis
P1 plasmid DNA was digested with Pst I overnight, extracted with phenol/chloroform, precipitated with ethanol, and ligated into a pBluescript vector. Colonies were screened by filter hybridization with the mouse Ren-1^C enhancer probe (–3.1/–2.6-kb fragment) labeled by random priming by following the same protocol as used for Southern blot analysis. Double-stranded DNA from positive clones was sequenced directly using universal M13 forward and reverse primers and primers 1 and 5, which are shown in Fig 3A.

View larger version (19K): [in this window] [in a new window]   Figure 3. Localization of the human renin enhancer (hENH) by long-range PCR. A, The strategy and primers used for the long-range PCR. Numbers above the arrows correspond to the primers listed below. Numbers below the arrows indicate the positions of the primers in the enhancer and promoter sequences. The direction of the arrows indicate the 5' to 3' orientation of each primer. B, Long-range PCR results. Numbers on the left indicate the 1-kb DNA size marker. Primers used were as follows: 1, AAG GAG CAG GGA GAG AAC AAG TTG G; 2, CGC GGT ACC TGA CCC CAT CTC TGA CAG TCC TCC; 3, ACC CAC CCA GGT TCA AGC CC; 4, TAG GAC GTG GCT GTG GAT AG; 5, CGC GGT ACC TCT AGC CAC CTT ACA TTC CAC AG; 6, CAG CTG AGT GCT CAA CAC ATA GAA C; 7, CAG AGC TAG GTG GGT GAA GGG TAC C; and 8, GAT CCA CTG AGG TTC TGT GGC TCC C.

Long-Range PCR
Long-range PCR was carried out using the P1 plasmid DNA as the template and by following the protocol in the XL PCR kit (No. N808-0182, Perkin-Elmer). The positions of the primers used are shown in Fig 3A. A typical long-range PCR reaction contained 1 µL of P1 plasmid DNA (10⁷ copies), 200 µmol/L each dNTP, 1x XL buffer, 1.1 mmol/L Mg(OAc)₂, 40 pmol of each primer, and 4 U rTth DNA polymerase in a total volume of 100 µL. AmpliWax PCR Gem 100 (No. N808-0100, Perkin-Elmer) was used for automated hot starts. The program for the long-range PCR was as follows: 94°C for 3 minutes, 1 cycle; 94°C for 1 minute, 62°C for 10 minutes, 16 cycles; 94°C for 1 minute, 62°C for 10 minutes (increment, 15 seconds per cycle), 12 cycles; and 72°C for 10 minutes, 1 cycle. The PCR products were stored at 4°C until they were analyzed on a 0.6% agarose gel.

Constructions and Transfections
Constructions were prepared that contained various mouse and human enhancer fragments in either the –148 hREN.luc construct,⁶ in which the downstream BamHI site was modified (GGATCCGAATTC). The 1-kb Pst I HEL was cloned into the BamHI site of –148 hREN.luc using a Pst I–BamHI adapter. The 1.5-kb (–4.1 to –2.6 kb) MEL was isolated as a BamHI fragment and inserted into the Kpn I site using the Kpn I–BamHI adapter. The MES fragment was inserted into the Sma I site after treatment of HindIII-digested DNA with Klenow. The shorter enhancer fragments, HEM and MEM, were obtained by PCR, subcloned into a pCRII vector (Invitrogen), and then excised as BamHI fragments that were inserted at the BamHI site upstream from human renin promoter in –148 hREN.luc. The minimal 220-bp HEMM and the corresponding MEMM were obtained by PCR with BamHI restriction sites produced at both ends and inserted at the BamHI site of –148 hREN.luc. The human and mouse renin sequences contained in these clones were confirmed by direct sequencing.

As4.1 cells were grown in DMEM containing 10% FBS. Cells were plated in Opti-MEM (GIBCO) containing 2% FBS 72 hours before transfection, and media were changed 24 hours before transfection. For each construction, 2x10⁷ cells were transfected by electroporation with 50 µg plasmid DNA.¹⁶ Each transfection also contained 2.5 µg pCMV–ß-gal²⁷ to control for transfection efficiency. Media were changed 12 to 18 hours after transfection. Cells were harvested and assayed for luciferase and ß-gal activities as described previously.^{6 28} Statistical analysis of the data was carried out using Statview 4.5 (Abacus Concepts).

RNA Preparation and Ribonuclease Protection Assay
To isolate total RNA, cells were washed twice with PBS and then scraped from 10-cm dishes into 1 mL PBS. Cells were pelleted by centrifugation and resuspended in 200 µL lysis buffer containing 150 mmol/L NaCl and 50 mmol/L Tris-Cl (pH 8.0), to which 14 µL of 10% NP-40 was added. After vortexing, the mixture was centrifuged for 1 minute in a microcentrifuge. To the supernatant were added 20 µL 10% SDS and 3 µL proteinase K (25 mg/mL); incubation was at 37°C for 30 minutes. After phenol/chloroform extraction and ethanol precipitation, the pellet was resuspended in 100 µL diethyl pyrocarbonate–treated dH₂O, to which was added 100 µL 100 mmol/L Tris-Cl (pH 7.5), 20 mmol/L MgCl₂, and 0.4 U RNase-free DNaseI (Promega). The mixture was incubated at 37°C for 30 minutes, then 100 µL buffer containing 30 mmol/L EDTA and 0.6% SDS plus 1 µL proteinase K (25 mg/mL) was added, and the incubation was continued for an additional 30 minutes. The mixture was then extracted with phenol/chloroform and RNA-precipitated with ethanol.

Ribonuclease protection assays were performed using an RPA II kit (Ambion) and following the procedure recommended by the manufacturer. To generate human renin riboprobes, a 172-bp internal fragment Acc I–EcoRI from phR1100²⁶ was cloned into pBluescript, linearized with EcoRI, and transcribed with T3 polymerase to generate a 202-bp transcript. To generate renin/luciferase riboprobes, the sequences from -148 hREN.luc (–148/+210) were amplified by PCR and cloned into pBluescript. The resulting plasmid was linearized with Spe I and transcribed with T7 polymerase to produce a 472-nt riboprobe containing 66 nucleotides from vector sequences at the 3' end.

	Results

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Identification and Characterization of hREN P1 Clone
The human renin genomic clones currently available contain <3-kb 5'-flanking DNA sequences.^{29 30 31} Southern blot analysis showed that the existing human renin genomic clones lack sequences homologous to the mouse enhancer (data not shown). To extend the flanking sequences, we screened several human P1 and PAC genomic libraries that resulted in the identification of five independent clones containing the human renin genomic sequences.

Southern blot analysis of restriction digests of the single P1 clone showed that it contains all coding and intervening sequences plus at least 13 kb of 5'-flanking DNA and 3 kb of 3'-flanking DNA (Fig 1A). Except for the Kpn I digest, the human renin cDNA probe hybridized the same large fragments as did the human renin 5'-flanking DNA probe and the mouse enhancer probe. The hybridizing fragments included a 97-kb Not I fragment, a 64-kb Sal I fragment, and a 45-kb Not I–Sal I fragment. The human renin cDNA probe hybridized an 11-kb Kpn I fragment, whereas the mouse enhancer probe and human renin 5'-flanking DNA probe hybridized a longer Kpn I fragment (at least 13 kb). These two Kpn I fragments are contiguous; the enhancer probe detects a fragment ending at –148, and the cDNA probe detects a fragment beginning at –148. Taken together, these data showed that the P1 clone contains an 80-kb insert that extends the hREN 5'-flanking DNA sequences to at least 13 kb. This extended 5'-flanking DNA contains sequences homologous to the mouse enhancer element.

View larger version (20K): [in this window] [in a new window]   Figure 1. Identification of sequences with identity to the mRen-1C enhancer in hREN 5'-flanking DNA. A, The P1 DNA containing the hREN genomic sequences was digested with the restriction enzymes listed, applied to the pulse-field gel electrophoresis (gel shown at the left), and then probed as indicated. Numbers at the left indicate the high molecular weight range DNA size marker. N indicates Not I; S, Sal I; and K, Kpn I. EtBr indicates ethidium bromide. B, Restriction maps of a P1 clone and several PAC clones containing the hREN gene are shown. The location of known Kpn I sites in the hREN gene are also shown. The horizontal arrow indicates the orientation of the hREN coding sequences 5'3'. The vertical arrow indicates the position of the human enhancer (ENH) homology determined in Fig 3.

Fig 1B shows the restriction maps of this P1 clone (from the Cornell University Medical College group) and three PAC clones (from the Roswell Park Cancer Institute group). Another PAC clone (from the University of Iowa group) contained the enhancer homology but was not entirely mapped. Although these four hREN genomic clones obtained from two different human genomic libraries contained different lengths of flanking DNA sequences, the common restriction fragments contained in these clones indicate that they do not contain gross chromosomal deletions, insertions, or rearrangements.

Subcloning and Sequence Analysis of the Human Enhancer–Like Fragment
To obtain smaller DNA fragments for sequence analysis and functional studies, the P1 plasmid DNA was subjected to digestion by a number of frequently cutting restriction enzymes and probed with the mouse enhancer probe (results not shown). We found that a 1-kb Pst I fragment contains the mouse enhancer–like element, which was subcloned and fully sequenced (Fig 2A). An optimal alignment between mouse and human enhancer sequences is shown in Fig 2B. The overall homology between the aligned mouse and human renin enhancer sequences is 59%, although the additional bases present in the human sequence toward the center of the aligned regions reduce the overall identity. At the 3' end of the alignment, a fragment of 220 bp showed 77% identity to the corresponding mouse enhancer region.

View larger version (64K): [in this window] [in a new window]   Figure 2. Sequence analysis of the human renin enhancer fragment. A, DNA sequence of a 1-kb Pst I fragment containing the human renin enhancer. B, The sequence shown in panel A was aligned with the sequence of a 1477-bp BamHI fragment (–4.1 to –2.6 kb of the mouse Ren-1C gene 5'-flanking DNA) containing the mouse enhancer. Numbers on the left indicate the positions in the Pst I fragment (human [H]) and BamHI fragment (mouse [M]) at which the aligned residues occur. The end points of some of the constructs shown in Fig 4 are indicated by vertical arrows.

Localization of the hREN Enhancer–Like Fragment by Long-Range PCR
Long-range PCR was carried out to localize and determine the orientation of the mouse enhancer–like fragment in the P1 hREN genomic clone (Fig 3A). All the PCRs gave consistent results (Fig 3B): oligonucleotide pairs oriented in the same direction did not yield PCR products (eg, pair 4+6 and pair 4+8); PCRs using only one primer (eg, primer 5 or 2 alone) did not yield the products; and the sizes of the PCR products were consistent with their spacing in the human sequences (eg, pair 2+8 yielded a product that was 900 bp longer than pair 5+8, and pair 2+8 yielded a product of 12 kb, which was 3 kb longer than pair 2+6). These results are consistent with the fact that primers 2 and 5 and primers 6 and 8 are 900 bp and 3 kb apart, respectively. Analysis of the fragment sizes generated (eg, 12 kb PCR product from the oligo pair 2+8) suggested that the cloned Pst I fragment lies 12 kb upstream from the hREN transcription start site. A number of fainter bands were also produced by some of the oligonucleotide pairs. These may be the result of mispriming or might arise from similar sites present elsewhere in the vicinity of the hREN gene. The nature or origins of these bands were not further investigated.

Long-range PCR results also showed that the Pst I hREN genomic fragment, when in its native configuration, is located in the hREN genomic clones with the same 5' to 3' orientation as the mouse enhancer relative to the transcription start site. The location of the hREN enhancer homology was confirmed by restriction mapping and Southern blot analysis of the PAC clones illustrated in Fig 1B and by its absence from the PAC clone No. 150H15 DNA that contains only 10-kb 5'-flanking DNA sequences (data not shown).

The Human Enhancer–Like Sequence Can Stimulate Human Renin Promoter Activity in As4.1 Cells
To determine the transcriptional role of the human enhancer–like sequence, constructions were made in which the human sequences were inserted upstream of the hREN 5'-flanking DNA (–148 to +18) in the luciferase expression vector –148 hREN.luc. Our earlier studies showed that the mouse enhancer could stimulate expression of –148 hREN.luc in renin-expressing As4.1 cells, although the effect appeared to be orientation dependent.³² However, preliminary testing of the 1020-bp Pst I fragment containing the human enhancer homology (Fig 2A) showed that this fragment had no apparent enhancer activity.

The orientation dependence of the mouse enhancer sequence and the lack of activity of the Pst I fragment containing the human homologue suggested that these fragments might also contain inhibitory sequences. Thus, if stimulatory and inhibitory sequences could be separated, enhancer activity in the human enhancer homology might be unmasked. To test this hypothesis, we prepared a series of constructs that contained truncated fragments of either human or mouse sequences. The sequences used in these constructions are indicated in Fig 2 and shown schematically in the left panel of Fig 4. Among the human sequences tested were the 1-kb Pst I fragment (HEL, 1 to 1020 [coordinates from Fig 2A]) and truncated fragments (HEM, 450 to 733; HEMM, 450 to 666). The HEM fragment was truncated at the 5' end to the homologous position in a fragment of the mouse enhancer that retained full activity.¹⁶ However, at its 3' end, HEM retained 68 bp not conserved between human and mouse genes (see Fig 2B). The HEMM fragment truncated the 3' end of the HEM sequences to eliminate these unconserved sequences. Constructs were also made that contained similar truncated sequences from the mouse Ren-1^C gene (MEM and MEMM) as well as sequences extending MEM in the 5' direction, MES and MEL (Fig 4).

View larger version (30K): [in this window] [in a new window]   Figure 4. Enhancer activity of human and mouse genomic sequences. Left, Mouse and human genomic sequences inserted upstream from the human renin 5'-flanking DNA in the –148 hREN.luc construct.6 Arrowheads show the orientation of each construct (indicated as + [forward] or – [reverse]). Shading indicates sequences conserved between mouse and human renin genes. Right, Activities of the transfected constructs expressed as a percentage of RSV (%RSV [±SEM]) and as the fold induction over the –148 hREN.luc (fold-148). The activity of RSV.luc was 326 151±112 048 light units and ranged from 45 815 to 1 644 328 light units in 16 separate experiments. The activity of a cotransfected CMV–ß-gal construct varied <30% between transfections in each experiment. pZ and –148 hREN denote the promoterless vector pZluc33 and –148 hREN.luc, respectively. Fold inductions were analyzed by a Wilcoxon signed rank test. *P<.05; **P<.005.

The various constructs were transfected into renin-expressing As4.1 cells. Cells were harvested 48 hours after transfection, and cell extracts were assayed for luciferase and ß-gal activity. Luciferase activities were first normalized for transfection efficiency (ß-gal activity) and were then normalized to the activity of pRSV.luc³³ (%RSV in Fig 4) or –148 hREN.luc⁶ (fold–148 in Fig 4) in each transfection. However, ANOVA by an F test revealed unequal variances in the fold inductions, especially between constructs containing the more active and the less active enhancer sequences. This was mostly due to increases in the fold inductions that occurred as the transfections were carried out over a 2-month period. Analysis of the data using a nonparametric Kruskal-Wallis test showed that the there were significant differences in the fold inductions between constructs (P<.0001). To determine the significance of the stimulation for each construct, pairwise comparisons were made by a Wilcoxon signed rank test. Fold stimulations by all sequences except HEL were greater than one (the normalized value for –148 hREN.luc) (Fig 4). Truncation of mouse enhancer sequences at the 5' end had no effect on the relative orientation dependence. In each case, the MEL, MES, and MEM fragments were more active in the forward (+) orientation than in the reverse (-) orientation. However, the MEM construct exhibited a 2-fold-greater stimulation than did either the MEL or MES constructs (P<.05), which did not differ significantly in fold stimulation from one another.

Truncation of the human (HEL) sequence at both 5' and 3' ends to yield the fragment HEM resulted in a relatively small stimulation (3- to 4-fold) when placed in either orientation. However, this effect was minuscule compared with the 200-fold stimulation afforded by the MEM(+) fragment it most closely resembled.

Deletion of the 3'-nonconserved regions from the HEM and MEM sequences resulted in an increase in activity of the human sequences but a decrease in activity of the mouse sequences, resulting in a similar level of activity in the forward orientation. However, in the reverse orientation, the activity of the HEMM sequence was much lower than that of its MEMM counterpart. Notably, the activity of the MEM fragment in the reverse orientation was similar to the activity of the MEMM fragment in either orientation. This would suggest that the effect mediated by the 23 bp deleted from the 3' end of MEM in the MEMM sequence is orientation dependent. The finding that deletion of 68 bp from the 3' end of the HEM sequence resulted in an increase in activity suggests either that this sequence has inhibitory effects or that in the context of the test system used, the activity of the human enhancer homology is sensitive to distance, orientation, or both. In the absence of the hREN promoter, both mouse and human enhancer sequences failed to elicit luciferase expression, indicating that expression was not due to a cryptic promoter within the enhancer sequences (not shown).

Ribonuclease protection assays (Fig 5) showed that transcripts from luciferase constructs containing human renin promoter sequences (–148/+18) were initiated at the native start site +1. Both mouse and human enhancer sequences increased the amount of correctly initiated transcripts in proportion to the luciferase activities shown in Fig 4. The protected fragment from RSV.luc was 18 bp shorter, consistent with the length of the renin sequence transcribed from hREN.luc constructs. In each case, the amount of total RNA from the cells transfected with various constructs are very similar, as shown by the relatively similar strength of the bands resulting from the expression of a cotransfected human renin expression vector phR1100²⁶ (not shown).

View larger version (50K): [in this window] [in a new window]   Figure 5. Transcripts stimulated by mouse and human enhancer sequences are correctly initiated in the human promoter. Ribonuclease protection assays were carried out as described in "Materials and Methods" using RNA from cells transfected with selected constructs shown in Fig 4. P indicates the full-length probe; C contains tRNA as a negative control; pZ and –148 denote the promoterless vector pZluc33 and –148 hREN.luc, respectively; +1 indicates correctly initiated transcripts; +18 shows the luciferase sequence protected from the RSV.luc transcript; and + and -, forward and reverse constructs, respectively.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
In the present study, we isolated several human P1 and PAC genomic clones containing all the coding and intervening sequences of the hREN gene plus various lengths of 5'- and 3'-flanking DNA. The flanking DNAs present in these clones greatly extend the sequences surrounding the human renin gene that had previously been cloned in lambda vectors.^{29 30 31} Within the extended 5'-flanking DNA sequences, we identified a region of 650 bp lying at 12 kb upstream from the transcription start site that had very high homology with a region of the mouse Ren-1^C enhancer. However, a 1-kb hREN genomic fragment containing this 650-bp conserved region failed to stimulate expression from the hREN promoter in As4.1 cells. Within this 1-kb human genomic fragment, a 220-bp fragment was 77% identical to the minimal mouse Ren-1^C enhancer, with the exception of 23 bp that were not conserved at the 3' end of the human sequence. Although this 220-bp conserved region from both mouse and human genes resulted in similar stimulation of hREN promoter activity when placed in the forward orientation, this stimulation was <20% of the maximum provided by the intact mouse enhancer and, with the human sequence, was greatly reduced in the reverse orientation. Taken together, these observations suggest that although mouse and human renin genes have conserved some common transcriptional control elements, changes within and around the human enhancer homology suppress its activity as a classical enhancer, at least in the context of transiently transfected As4.1 cells. The highly active mouse enhancer could provide the transcriptional drive to sustain elevated plasma renin levels in mice.

The major determinant of activity from the mouse sequences was the 23 bp 3' to the conserved region that were absent from the human sequence. Notably, the positive effect of this 3' sequence was orientation dependent, suggesting either that this region binds one or more factors that may function in an orientation-dependent manner³⁴ or that deletion of this sequence results in deleterious changes in the relative alignment or spacing of enhancer and promoter elements.^{35 36} Screening of this 23-bp sequence against a library of transcription factor binding sites³⁷ revealed the presence of a consensus binding site for GATA-2, a factor that has been reported to direct gene expression in vascular tissues.³⁸ In a previous study,¹⁶ the mouse enhancer (MEM fragment) was shown to have an orientation-independent effect on the mouse renin promoter. Because the human renin promoter used in the present study was 50 bp longer than the mouse renin promoter tested previously, it is also possible that the directional effects we observed depend on interactions between the enhancer and factor(s) binding in this 50-bp region. Differences in orientation dependence between the 220-bp conserved sequences in mouse and human genes may be due to qualitative and quantitative differences in their ability to bind trans-acting factors.

The identification of the enhancer homology in the same distal position in several human renin genomic clones isolated from different P1 and PAC libraries suggests that this is its natural location in the human gene and not an artifact of cloning. Southern blot analysis of human genomic DNA using the human enhancer homology as a probe indicated that it is present as a single copy contained in the same restriction fragment as the human renin 5'-flanking DNA (not shown). Screening of the GenBank with the human enhancer homology identified the mouse enhancer but no other sequences with significant extended homologies. Taken together with the conservation of some transcriptional activity, conservation of this unique human homologue to the mouse enhancer at a position considerably further upstream than in the mouse renin gene suggests that it may play an important functional role in directing expression of the human renin gene.

Transgenic studies have shown expression of human renin genes at appropriate sites,^{17 18 19 39} even though the genomic fragments tested lacked the enhancer sequences. However, in many of these lines, expression was also detected in brain, heart, lung, pancreas, spleen, thymus, and intestine. Some of these extrarenal sites may be bona fide sites of hREN gene expression, whereas others may represent ectopic expression due to the lack of appropriate control elements or to the site of transgene integration. For example, after identification of hREN mRNA in the transgenic intestine, renin mRNA was also shown in normal intestines of both humans and mice.⁴⁰ On the other hand, hREN mRNA was identified in adipose tissue of four separate transgenic lines¹⁸ but has never been reported in normal human or rodent adipose. Whether appropriate or ectopic, extrarenal hREN expression at common sites in different independent transgenic lines suggests that tissue specificity is determined by the transgene sequences rather than their sites of integration. Since regions of the mouse gene spanning the enhancer appear to limit the expression of an heterologous reporter gene at extrarenal sites,^{10 11 12 41} we propose that one function of the enhancer, or the associated negatively acting elements we identified in the present study, might be to suppress renin gene expression at inappropriate sites. Such a repressor function has been described for the interaction of MyoD with the IgH enhancer.⁴²

Enhancer sequences in combination with other elements may form so-called LCRs that confer alterations in chromatin structure permitting position-independent gene expression.^{43 44} In the absence of transcriptional activation, truncated immunoglobulin µ enhancer sequences together with flanking nuclear MARs have been shown to induce alterations in chromatin structure,⁴⁵ suggesting that LCR activity and transcriptional activation are separable. Although the enhancer is sufficient to activate immunoglobulin µ gene transcription in cell-transfection studies, for enhancer-mediated transcription of the immunoglobulin µ gene to occur in transgenic mice in vivo requires the presence of the MAR.⁴⁶ In this respect, it is noteworthy that the human genomic sequence reported herein contains a highly AT-rich region at the 3'-end (bases 931 to 990, 70% A/T, Fig 2A) that may function as an MAR.⁴⁷

In summary, the present study shows that although common sequence elements with similar stimulatory activities are conserved between mouse and human renin genes, changes within and around these common sequences result in different transcriptional effects. In the context of transiently transfected As4.1 cells, the activity of the mouse enhancer and the inactivity of the human enhancer homology may reflect the different levels of transcriptional activity required to maintain physiological levels of renin gene expression in vivo. However, both the mouse enhancer and its human counterpart may be important in directing fully appropriate cell-specific renin gene expression. We are carrying out studies in transgenic animals to determine the role of the human renin enhancer homology in vivo.

	Selected Abbreviations and Acronyms

 

ß-gal = ß-galactosidase Ang I = angiotensin I CMV = cytomegalovirus h (as prefix) = human HEL = human renin enhancer fragment (long) HEM = human enhancer fragment (minimum) HEMM = human renin enhancer fragment (minimum truncated) LCR = locus control region m (as prefix) = mouse MAR = matrix attachment region MEL = mouse renin enhancer fragment (long) MEM = mouse enhancer fragment (minimum) MEMM = mouse enhancer fragment (minimum truncated) MES = mouse enhancer fragment (short) PAC = P1 artificial chromosome RSV = Rous sarcoma virus SV = simian virus

	Acknowledgments

 
This study was supported by a Grant-in-Aid from the American Heart Association, New York City Affiliate, Inc; the Hypertension SCOR HL-18323 (Dr Catanzaro); National Institutes of Health grants DK-45982 (Dr Catanzaro), HL-48459 (Dr Gross), and HL-48058 (Dr Sigmund); and the generous support of the Michael Wolk Heart Foundation (Dr Catanzaro). Drs Catanzaro and Sigmund are Established Investigators of the American Heart Association. We thank Dr Timothy Reudelhuber for helpful discussions and Julie A. Lang for technical assistance. We also thank Dr Pieter de Jong for access to and Dr Eirik Frengen for screening of the human genomic PAC library (Department of Energy grant DE-FG02-94ER61883).

Received February 10, 1997; accepted June 25, 1997.

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