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Abstract
Abstract—We have used comparative sequence analysis to evaluate a putative silencer element that has been proposed to be involved in the differential tissue-expression of the murine renin genes: Ren-1 and Ren-2. In the mouse, these genes share a similar pattern of tissue-specific renin expression. One significant difference is seen in the submandibular gland (SMG) where renin expression from the Ren-2 locus is 100-fold greater than the expression from the Ren-1 locus. One model proposes that this differential expression arises from the interplay among a negative regulatory element and a cAMP responsive element, their respective binding factors, and the disruption of the negative regulatory element by an insertion (M2) that is found in Ren-2 but not in Ren-1. The abrogation of the negative regulatory element’s function as a result of the M2 insertion was proposed to be specifically responsible for the higher level of Ren-2 expression in the SMG as compared with Ren-1. We have assessed this hypothesis by looking at an allelic variant in the closely related mouse species M. hortulanus. This species shares the same high level of Ren-2 expression in the SMG as seen in other Ren-2 positive mouse strains. However, the Ren-2 M. hortulanus allele does not appear to contain the disruptive M2 element according to restriction-enzyme mapping. Our sequence analysis confirms that the M. hortulanus Ren-2 allele contains the same sequence elements present in the DBA/2 Ren-2 allele except for the M2 element. Moreover, the proposed negative regulatory element is intact at the sequence level in Ren-2 M. hortulanus allele. This analysis suggests that any involvement of the negative regulatory element in differential Ren-1 and Ren-2 expression in the SMG is not as straightforward as previously hypothesized.
Renin is an important regulatory enzyme involved in blood pressure maintenance and electrolyte homeostasis. It catalyzes the first reaction in the production of angiotensin II, a potent vasoactive hormone that is directly involved in both the circulating and tissue-specific renin-angiotensin systems. Many inbred strains of mice, such as DBA/2, which are derived from M. domesticus and M. musculus, possess an additional copy of the renin gene. This second locus arose as a tandem duplication to Ren-1.1 2 Precisely the same duplication event is observed in the closely related subspecies M. hortulanus and M. spretus. 3 In these mouse species, the 2 renin genes, Ren-1 and Ren-2, share overlapping but non-identical patterns of tissue-specific expression.
The primary site of synthesis for circulating renin in all mouse strains is the kidney. Multiple alleles from both Ren-1 (Ren-1c = Renin-1 BALB/C allele, Ren-1d = Renin-1 DBA/2J allele, Ren-1h = Renin-1 M. hortulanus allele) and Ren-2 (Ren-2d = Renin-2 DBA/2J allele, Ren-2h = Renin-2 M. hortulanus allele) are expressed at approximately equal levels in the kidneys from 1- or 2-gene strains of mice.3 4 In addition, a limited spectrum of extrarenal sites of tissue-renin production, including the adrenal gland, testes, ovaries, coagulating gland, liver, and submandibular gland (SMG), has also been identified.5 The most dramatic difference in Ren-1 and Ren-2 expression has been noted in the SMG. In 2-gene strains of mice (DBA2/J and M. hortulanus), Ren-2 expression is >100-fold higher than expression of Ren-1 transcripts. Overall, the Ren-2d and Ren-2h alleles show a similar expression pattern as judged by the examination of a number of tissues, including SMG, kidney, testes, coagulating gland, and liver. Ren-1d and Ren-1h also share a similar pattern of renin expression, differing only in the increased expression of Ren-1h in the liver relative to Ren-1d. 5
Dzau and his colleagues6 7 8 9 have proposed that the similarities seen for Ren-2 and Ren-1 expression in the kidney, versus the differences seen in the SMG of DBA/2J, result from the complex interplay among a negative regulatory element (NRE), a cAMP responsive element (CRE), and their associated trans-acting factors. Their model of renin expression proposes that the CRE-binding factor (CREB) and the NRE-binding factor (NREB) compete for an overlapping sequence element found in the 5′ flanking sequence of both Ren-1d and Ren-2d. They further propose that CREB possesses a greater affinity for the site than does NREB. Therefore, when both factors are present in a cell, CREB will drive renin expression. However, when CREB is functionally absent, NREB binding will further suppress expression of renin. Hence in the kidney, where both binding factors are present, there is equivalent expression of Ren-1d and Ren-2d. In the SMG, their model proposes that CREB is sequestered by an inhibitory protein, which permits NREB to bind to its target sequence without competition and thus curtail renin expression. Therefore, the sequestration of CREB and the presence of an intact NRE/NREB protein factor complex lead to suppression of Ren-1d expression. In this model, Ren-2 escapes from suppression due to the presence of a disruptive insertional element, M2, which integrated adjacent to the NRE upstream of Ren-2d. This insertion would therefore ablate the NRE’s suppressive action on Ren-2d expression in the SMG.6 7 8 9
However, Gross and colleagues3 10 have noted that the corresponding Ren-2h allele appears to lack the proposed disruptive M2 insertion found in Ren-2d. Nevertheless, as noted above, the M. hortulanus allele still produces equivalently high levels of Ren-2 mRNA in the SMG (Figure 1⇓). These observations are thus in apparent conflict with the model invoked to explain the locus-specific differences of renin expression in the SMG and kidney.9 The assertion that Ren-2h lacks the M2 insertion is based on a previous comparative restriction-enzyme mapping of Ren-2h and Ren-2d (Figure 1⇓). These same studies revealed that the B2 and M3 elements, corresponding to a mouse type-2 repetitive element and a transposon respectively, are conserved between the 2 species.3 Thus at this level of sequence resolution, Ren-2d and Ren-2h share a similar structure in their 5′ flanking sequences, except for the absence of the M2 insertion. The NREB/CREB model of renin expression stems from data derived by experimental manipulation of artificial constructs in vitro and in vivo. However, both the in vitro and in vivo experiments used constructs which included upstream renin sequences and a non-renin-based thymidine kinase promoter. In addition, the initial in vitro experiments used cell lines that do not ordinarily produce renin.6 7 8 9 We have elected to evaluate in greater detail the naturally available construct of the M. hortulanus allele that exhibits a paradoxical structure/expression pattern in respect to the proposed model. To rule out specific sequence disruption and inactivation of the M. hortulanus NRE/CRE site, we have determined the sequence upstream from Ren-2h, including the corresponding M2 insertion site, and directly compared the sequence with its Ren-2d counterpart.
Schematic representation of the insertions in the 5′ and 3′ flanking regions of the renin genes. The positions of the M2, M3, and B2 insertions are displayed. The M3 element is present in all of the renin genes. The B2 element is located in both Ren-2 alleles, whereas the M2 element is located only in the Ren-2d allele. The approximate positions of the NRE and the BamHI (B) restriction-enzyme sites are displayed.3 A summary of the relative mRNA expression level of each renin gene in the expressing cells of the kidney and SMG is displayed to the right of each schematic representation. (Derived from Abel et al3 , Figure 5; and Fabian et al10 , Figures 1 and 5.) Approximately 100-fold more cells express renin in the SMG. *Ren-1d is expressed at extremely low levels in the SMG.5
Methods
Mice
M. hortulanus (Pancevo) mice were maintained as an outbred stock derived from animals trapped by R. Sage (Museum of Vertebrate Zoology, Berkeley, California).
Genomic Clones
Clones were isolated from a M. hortulanus genomic library prepared using the λ vector EMBL-3B and a 10–25 kb fraction of Sau3A partially digested DNA from a single adult male after CO2 inhalation and cervical dislocation.3 Screening of libraries was done according to Benton and Davis.11 DNA probes containing renin exon sequences were nick translated to a specific activity of ≈1.7×106 Bq/kg and included in the hybridization mix at a concentration of 1.7×107 Bq/L.12 Screening of ≈2.5×106 recombinant phage yielded a total of 14 clones encompassing both renin loci plus 10–20 kb of sequences flanking each locus. The M. hortulanus sequence shown in Figure 2⇓ was derived from the clone λHRenII-9, which includes 8 kb of 5′ flanking sequences and exons 1–5 of Ren-2h.
Intact NRE sequences of the Ren-1c, Ren-1d, Ren-2d, and Ren-2h genes. Multiple sequence alignment of the renin genes were created using the Align X program of the Vector NTI suite. The arrows indicate the 5′ and 3′ extent of the B2, M2, and M3 elements. The carets indicate the 5′ and 3′ extent of the “functional CRE” site8 that is disrupted by the M2 insertion in Ren-2d. The B2 element is found in the alleles of Ren-2 gene and is not seen in the alleles of the Ren-1 gene. The M2 element is found only in Ren-2d, whereas the M3 element is found in both Ren-1 and Ren-2 alleles. Ren-1c and Ren-1d share 2 short sequence elements that are not present in either Ren-2d or Ren-2h. The short sequence elements that are specific to the Ren-1 alleles are highlighted in bold, whereas the elements that are specific to Ren-2 alleles are underlined. The NRE is identical in Ren-1c, Ren-1d, Ren-2h, and Ren-2d. However, the expression of Ren-2 mRNA is not suppressed in the M. hortulanus or the DBA2/J SMG. The positions of intact restriction-enzyme sites are indicated by an underline: BamHI, Pvu II, and XbaI. Sequence mismatches are indicated by lowercase letters.
Subcloning and Sequencing
A 2.5 kb KpnI-HindIII (−1429 to +1126) restriction fragment from λHRenII-9 was subcloned into the plasmid vector pGEM-4Z (Promega). This clone, designated pGEMR2H-5, was used to generate 4 subclones in pGEM-4Z vectors which spanned the region between −903 and +832. A XbaI-BamHI restriction digest of pGEMR2H-5 yielded the inserts for the following clones: pGEMR2H-4, −903 to −557 (XbaI-XbaI), pGEMR2H-2, −562 to −112 (XbaI-BamHI), pGEMR2H-1, −117 to +442 (BamHI-XbaI), and pGEMR2H-3, +437 to +832 (XbaI-BamHI). pGEMR2H-1 through pGEMR2H-5 were sequenced at the Roswell Park Cancer Institute Biopolymer facility using the M13 forward and reverse primers in a Applied Biosystems Model 373A DNA sequencer. Two oligonucleotides derived from pGEMR2H-4 and pGEMR2H-3 were created to complete the sequence of pGEMR2H-5: 5′-CAGAGCAGAGTGGTGGC-3′ (−842 to −858) and 5′-GAACGTTAAGCCTGCAA-3′ (+835 to +851). A clone (pCATNOT R2-4.6) from the cosmid cosDBA-1 was used to determine the sequences from the DBA2/J Ren-2 allele.13 Sequences from each clone were deposited into GenBank with the following accession numbers: Ren-2h (AF237861) and Ren-2d (AF237860).
Results
Sequence Analysis of Murine Renin Alleles
Extensive comparative restriction-enzyme analysis of the M. hortulanus and M. domesticus renin duplication has previously suggested that the B2 element is found upstream of both Ren-2h and Ren-2d but is absent from the corresponding Ren-1 locus. Ren-2h also appears to retain the M3 element common to both the Ren-1 and Ren-2 loci.1 2 3 Significantly, the M2 element could not be detected in the 5′ flanking sequences of Ren-2h. A clone designated λHRenII-9 contained the pertinent sequences from the Ren-2h locus. The restriction-enzyme maps generated from the λHRenII-9 and its subclone, pGEMR2H-5, are consistent with the established genomic restriction map of Ren-2h, further suggesting that the M2 element is absent and that the M3 and B2 elements are present as expected.3 In addition, a BamHI site located at −117 bp in Ren-2d (specific to the Ren-2 locus) can also be found in Ren-2h as expected from restriction mapping but is absent in the corresponding Ren-1 alleles (Figures 1⇑ and 2⇑). These data strongly argue that the clone derived from λHRenII-9 is from the Ren-2 locus and not from the Ren-1 locus.
Sequencing of the Ren-2 alleles confirmed the presence of the M3 and B2 elements in both Ren-2h and Ren-2d, and the specific absence of the M2 element in Ren-2h as previously suggested by restriction-enzyme analysis. Two alleles from each of the 2 murine renin loci were compared throughout the sequenced region of the Ren-2h allele (Figure 2⇑). Sequences for Ren-1c (L78789) and Ren-1d (M32352) were retrieved from GenBank,14 15 whereas the sequence for Ren-2h was derived from pGEMR2H-5, and the sequence for Ren-2d was derived from pCATNOT R2-4.6. As noted above, the M3 element is present in both Ren-1 and Ren-2 loci, whereas the B2 element is found in the alleles of Ren-2 and not in the alleles of Ren-1. The comparison revealed the expected significant homology among all of the renin sequences (results not shown). However, in the region of the M3 insertion, the B2 element is only present in Ren-2h and Ren-2d. Additionally, both Ren-1c and Ren-1d share 2 short sequence elements that are not present in either Ren-2d or Ren-2h (Figure 2⇑). Inspection of sequence 5′ to the M2 insertion site indicates that Ren-2h most closely resembles Ren-2d.
Figure 2⇑ shows the sequences surrounding the NRE/CRE element and M2 insertion (or lack of) for each of the 4 renin sequences examined. The alignment of the renin genes clearly shows the absence of the M2 element in the 5′ flanking sequences of Ren-2h. The M2 insertion site is immediately adjacent to the NRE/CRE site and is located near position −600 in both Ren-1 alleles and near position −800 in both Ren-2 alleles. The B2 insertional element is responsible for the offset in position of the M2 insertion site in both Ren-2 alleles relative to the site in the Ren-1 alleles. Ren-2d is the only renin gene that retains the M2 element upstream of position −800.
Our reexamination of the renin genes revealed that Ren-1c, Ren-1d, Ren-2d, and Ren-2h are identical in the region of the NRE consensus sequence and correspond precisely to the reported NRE consensus sequence (Figure 2⇑). Therefore all of the studied Ren-1 and Ren-2 alleles retain a paradoxically functional NRE that is not suppressing expression of Ren-2 mRNA in the SMG. In addition, the “functional CRE” site as described in Horiuchi et al 8 is disrupted only in the Ren-2d gene by the M2 element (Figure 2⇑).
Discussion
By direct sequencing, we have confirmed the previous suggestions from Southern blot analysis that indicated that the M2 insertion was absent from the Ren-2h allele.3 Ren-2h closely resembles Ren-2d upstream and downstream of the M2 insertion site. In Ren-2h, where the M2 insertional element is absent, the B2 element is present when it is compared with both Ren-1 alleles, and there are no alterations that would mimic the presence of M2 in Ren-2h. In fact, sequence analysis unexpectedly (given the previously published report7 ) shows that both Ren-2d and Ren-2h contain an intact copy of the presumed NRE in the 5′ flanking region, which is identical to the homologous NREs found in both Ren-1c and Ren-1d. The CRE site directly overlaps the NRE site; however, it does not conform to the consensus CRE sequence.16 Our analysis also reveals that the M2 insertion splits the proposed “functional CRE” site in half only in the Ren-2d gene and not in any of the other renin genes evaluated here.
In mice carrying the Ren-2d allele, the high level of Ren-2d mRNA in the SMG has been attributed to ablated NRE function due to the presence of the M2 insertion. Plasmid constructs harboring Ren-2 sequences, from which the M2 element has been specifically removed, have been shown to exhibit suppressed reporter expression when introduced into SMG of DBA2/J mice by direct gene transfer.9 Therefore, based on the NRE model of renin expression, low levels of Ren-2h mRNA would be expected in the SMG if the NRE were functional in M. hortulanus. However, both the Ren-2d and the Ren-2h alleles produce the same high level of renin transcripts in vivo in conflict with the proposed model.3 10 Because the M. hortulanus allele retains an intact NRE and a high level of Ren-2 expression in the SMG, it seems unlikely that the model proposed for NRE function plays a straightforward role in regulating renin expression in its natural setting.
Acknowledgments
We thank Colleen Kane for her help in producing this work. We would also like to thank Al Cairo for his work in sequencing the M. hortulanus allele. The Biopolymer Facility is supported, in part, by a Cancer Center Core Grant, CA 16056. This work was supported by NIH Grant HL48459 and Cancer Center Grant CA16056. J. Pablo Abonia was supported by a Summer Research Fellowship from the State University of New York at Buffalo.
- Received April 2, 2000.
- Revision received April 13, 2000.
- Accepted July 6, 2000.
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