(Hypertension. 1999;33:303-311.)
© 1999 American Heart Association, Inc.
Scientific Contribution |
Transcriptional Regulation of the Rat Renin Gene by Regulatory Elements in Intron I
From the Max-Delbrück-Center for Molecular Medicine (MDC), Berlin-Buch, Germany (T.V., M.B., D.G.); Institute for Clinical Pharmacology, University Clinic Benjamin Franklin, Free University Berlin, Berlin, Germany (D.G.); and Transgenics in Berlin-Buch GmbH, Berlin, Germany (M.B.). T. Voigtländer is now at the Institute of Neurology, University of Vienna, Austria.
Correspondence to Dr Michael Bader, Max-Delbrück-Center for Molecular Medicine (MDC), Robert-Rössle-Str. 10, D-13122 Berlin-Buch, Germany. E-mail mbader{at}mdc-berlin.de
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Abstract |
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Abstract—Renin catalyzes the rate-limiting step in the enzymatic cascade leading to the vasoactive peptide angiotensin II. Therefore, the activity of the renin-angiotensin system in a tissue is regulated significantly at the level of transcription of the renin gene. Besides transcription factor binding sites in the promoter region, the renin genes of human and rat contain regulatory elements also in intron I. Inclusion of intron I in reporter gene constructs with the renin promoter leads to a marked down-regulation of gene expression in nonrenin expressing 293 human embryonic kidney cells but has hardly any effect in renin-expressing L8 rat skeletal myoblasts. In combination with the cytomegalovirus immediate early gene promoter, the silencing occurs in both cell lines but is less pronounced in L8 cells. By partially deleting intron I in these constructs, we describe 5 negative (I-NRE) and 2 positive (I-PRE) regulatory elements responsible for these effects. Using gel-retardation and methylation-interference assays with 293-nuclear extracts, we detected a pseudo-palindromic protein-binding sequence between position +159 and +171 relative to the transcriptional start site. Binding of transcription factors to this sequence may be important for the tissue-specific silencing of the renin gene outside the juxtaglomerular cells of the kidney.
Key Words: angiotensin • renin • transcription • silencer • intron • gene
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Introduction |
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The renin-angiotensin system plays a key role in the control of blood pressure and electrolyte homeostasis in mammals. In rats and humans, renin represents the rate-limiting factor in the catalytic generation of the effector peptide angiotensin (ANG) II. Therefore, the understanding of its transcriptional regulation under basal as well as stimulatory conditions is of particular scientific interest and has been a major goal for the past 10 years.
Renin-gene expression studies are confronted with several difficulties. The major problem is the lack of suitable renin-producing cell lines. Using a transgenic approach, Sigmund et al1 have generated As4.1 cells that resemble juxtaglomerular cells and secrete renin in the culture medium. However, these cells have not yet been utilized broadly for gene-regulation studies. Instead, renin-secreting cell lines from extrarenal renin production sites as well as nonrenin secreting cell lines have often been used in the past to study transcriptional regulation of the renin gene. So far the interest has focused primarily on the 5' region and several different cis-acting regulatory elements like RP-2, RU-1,2 PRE, NRE,3 cAMP response element (CRE)3 and Pit-1 sites4 have been described in mouse and human renin genes. However, because of the use of different cell lines and renin genes of different species, the results concerning the location of a functional CRE are contradictory, whereas some functional elements have been demonstrated only in 1 defined cell type (for review see reference 5).
When the human and the rat renin gene were compared, a high degree of interspecies homology was discovered not only in the promoter and the coding region, but also in at least the first 120 bp of intron I.6 The degree of homology within the intronic sequences even exceeded that of the promoter and of exon I sequences. In a previous study,7 we have extended this analysis to the mouse Ren-1c sequence, confirming the former results. A high degree of homology can be an indicator for a specific function of a sequence even if it is located in an intron. In recent years, an increasing number of intronic sequences has been identified exerting a stimulatory8 9 or inhibitory10 11 function in terms of gene regulation.
Further evidence for regulatory elements in the transcribed region of renin genes comes from transgenic animals. Several studies12 in transgenic mice and rats have demonstrated that correct tissue specificity of the Ren-2 gene expression is directed by DNA sequences residing between 2.5 kb 5'-flanking and 3 kb 3'-flanking regions. Likewise, 4.6 kb of the 5'-flanking sequence of the Ren-2 gene conferred tissue specificity to a SV40 large T-antigen reporter gene in transgenic mice.13 In contrast, 2.5 kb 5'-flanking region of Ren-2 failed to direct tissue specificity to the same reporter gene,14 leading to the hypothesis that functional important regulatory elements may reside downstream of the transcription start site.
Taken together, these results suggest that intron I of the renin gene may be an interesting sequence to search for regulatory elements. In a previous report,7 we have presented some preliminary results concerning the effect of rat renin intron I on gene regulation, demonstrating for the first time the existence of a potent silencer within the intron I. In the meantime, the presence of a silencer has been demonstrated by other groups in the human renin gene.15 16 We now report a more detailed study dissecting several regulatory regions in intron I and identifying by gel-retardation experiments and methylation-interference assays a DNA sequence that is bound by nuclear proteins.
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Methods |
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Library Screening
A Sprague-Dawley (SD) genomic library (Stratagene) was screened by the use of standard methods17 and an EcoRI/XbaI fragment of the rat renin gene comprising 1.2 kb 5'- flanking region, exon I, and 316 bp intron I as a radiolabeled probe. Four clones (r1, r4.1, r4.2, and r8) could be isolated and mapped by restriction analysis (Figure 1
). A 9.7 kb SacI fragment
from clone r8 containing 3.9 kb 5'-flanking region, exon I, and
the entire intron I was then subcloned to generate the basic renin gene
vector.
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Site-Directed Mutagenesis of the 2 Translation Start
Codons
PCR-directed site-specific mutagenesis of the 2 translation
start codons ATG I and ATG II (at position +33 bp and +48 bp with
respect to the transcription start site18 was performed by
the use of a 4 primer system with a pair of overlapping internal
primers. The total length of the sequence remained unchanged. The
primers were designed as follows: 5' primer rR12 [5' TCT AGA GAC ACG
CAT AAT CAG TGC 3'] and 3' primer rR13 [5' CAG AGA GGC GGC
CGC CTC CCG CCG ATC TGG TCC 3'] for mutagenesis
of start codons ATG I/II and 5' primer rR14 [5' CGG GAG GCG
GCC GCC TCT CTG GG 3'] and 3' primer rR15 [5' CCT GCA AAA
CTC TAG AGG GGC CG 3'] for mutagenesis of start codon ATG II (bold
letters indicate mutated residues). In addition to the mutagenesis of
ATG II, a new NotI restriction site was introduced in the
sequence. PCR products were subcloned in a T-vector system
(Promega), sequenced, combined at the NotI restriction site,
and integrated as a 1.8 kb XbaI fragment in the basic renin
gene vector, replacing the former genomic part with functional ATG
start codons.
Reporter Gene Constructs
Two reporter genes, ß-galactosidase from E. coli
(lacZ) and firefly luciferase, were used in parallel. The
basic lacZ vectors contained a 121 bp nuclear translocation
signal (NTS) fused in frame to the lacZ gene and either a
850 bp intron and polyadenylation signal (pA) cassette from SV40 or a
splice acceptor site (SAS) from the c-myc gene and a pA
cassette of SV40 in a pGEM4 plasmid backbone (Promega). The basic
luciferase vector, p
Lux, was generated as
described.19 For constructs containing introns,
pLux was modified by adding the SAS of the c-myc gene
upstream of the luciferase gene.
Reporter gene constructs with parts of the renin gene or the cytomegalovirus immediate early gene (CMV) promoter were cloned by standard methods. Restriction enzymes or Bal31 nuclease17 was applied for the shortening of the intron-I fragments.
Cell Culture and Transfection Experiments
Two cell lines, 293 (human embryonic kidney
cells20 ) and L8 (rat skeletal muscle
myoblasts21 ) were used in the present study. Both were
cultured under optimal conditions and transfected with 5 µg of the
respective plasmids by the CaPO4 coprecipitation
method.19 As an internal control, 2 µg of
pUHC13-122 (for lacZ plasmids) or pCH110
(Pharmacia, for luciferase plasmids) containing the lacZ
gene under control of the SV40 early gene promoter were added to the
precipitation mixture. Both reporter gene activities were measured in
each cell extract 40 hours after transfection as
described.19 The lacZ activity measured
was normalized to the luciferase activity of the same extract if the
promoter to test was linked to lacZ and vice versa. The
values were statistically analyzed by use of ANOVA.
Preparation of Nuclear Extracts and Gel-Retardation
Experiments
293 and L8 cells were grown to 75% confluence, harvested after
a short trypsin treatment, and pelleted at low speed. Nuclear extracts
were prepared as described by Dignam et al,23 except that
HEPES was replaced by Tris-HCl, pH 7.9. Protein content was quantified
according to Bradford24 by the use of the BioRad protein
assay system. Salt content was determined by conductivity measurement.
Gel-retardation experiments were performed as follows: In 20 µL total
reaction volume shift buffer (20 mmol/L HEPES, pH 8.4, 60
mmol/L KCl, 4% Ficoll) was mixed with 2 mmol/L DTT, 2 µg bovine
serum albumin, 2 µg poly(dI-dC) heteropolymer, 2.5 to 7 µg
nuclear extract, and 15 000 to 25 000 cpm end-labeled probe. After 30
minutes incubation at 30°C, samples were loaded to a 4% to 6%
(39:1) polyacrylamide gel in 0.25x TBE (25 mmol/L
Tris-HCl, 25 mmol/L boric acid, 0.05 mmol/L EDTA) and
electrophoresed at 15 V/cm. Gels were dried and exposed to Kodak XAR-5
X-ray film.
DE 52 Purification and Heparin-Sepharose
Chromatography of Nuclear Extracts
Purification and chromatographic separation was
carried out at 4°C essentially as described by Scheidereit et
al.25 A DEAE cellulose column (DE 52, Whatman, 1 mL packed
column volume [cv] per 4 to 10 mg protein load) was equilibrated in
buffer C (20 mmol/L Tris-HCl, pH 7.9, 25% glycerol, 0.42 M NaCl,
1.5 mmol/L MgCl2, 0.2 mmol/L EDTA,
0.5 mmol/L DTT, and 0.5 mmol/L PMSF). Crude nuclear extracts
were adjusted to 0.42 M NaCl, passed over the column, eluted with 1
additional cv buffer BC 500 (20 mmol/L Tris-HCl, pH 7.9, 20%
glycerol, 0.5 M KCl, 0.2 mmol/L EDTA, 0.5 mmol/L DTT, and
0.5 mmol/L PMSF) and dialysed against 60x volume buffer BC 100
(buffer BC with 0.1 M KCl). The purified material was loaded onto a
heparin-sepharose column (1 mL cv per 3.5 mg protein load, equilibrated
with 6 cv BC 100) at 2 cv per hour, washed with 2 cv BC 100 at 2 cv per
hour and eluted with a linear gradient (5 cv) from 0.1 to 0.5 M KCl,
again at 2 cv per hour. In a final step, the column was washed with 0.3
cv of BC 1000 (buffer BC with 1 M KCl) to elute remaining proteins. The
gradient was collected in 32 fractions and protein and salt content was
analyzed as described above.
Methylation-Interference Assay
For the methylation-interference assay, probes were labeled by a
fill-in reaction of restriction sites by the use of exo-Klenow
polymerase (Stratagene) and [32P]dGTP or
[32P]dATP. Partial methylation of the probes
was subsequently performed essentially as described,17 by
the use of 2x107 cpm probe and 1 µg
poly(dI-dC) as carrier for the methylation reaction at 25°C and with
1.5 M sodium acetate/1 M 2-mercaptoethanol for reaction
termination. After 2 ethanol precipitations and rinsing, the partially
methylated DNA was used for gel-retardation assays as described above
except that the gels were not dried before X-ray film exposure. All
visible retarded complexes as well as the unbound DNA fraction were cut
out from the gel, purified, and cleaved in 100 µL 10% piperidine at
90°C for 30 minutes. After precipitation and rinsing, the DNA was
loaded onto an 8% polyacrylamide-sequencing gel and visualized
by autoradiography.
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Results |
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Cloning of the Rat Renin Gene
Previously,18 1.2 kb of the rat renin promoter region had been cloned. For the present study, a longer 5'-flanking region was considered to be necessary to include as many regulatory elements of the promoter region as possible. By screening of a rat genomic library (Stratagene), 4 clones (r1, r4.1, r4.2, and r8) could be isolated, comprising 16.5 kb 5'-flanking region, all exons and introns and 5.8 kb 3'-flanking region (Figure 1
). A comparison between
these clones revealed a BglII restriction fragment length
polymorphism probably due to different amounts of the tandem
repeats present at this site in intron I of the rat renin
gene.18 26
Transcriptional Effects of Intron I
Conversion of the coding exon I of the rat renin gene into a
noncoding one by PCR-directed site-specific mutagenesis of the 2
translation start sites at position +33 and +48, respectively, served
as a starting point for the subsequent construction of 30 different
reporter gene constructs, 16 containing the 3.9 kb renin 5'-flanking
region and 14 containing the 760 bp CMV promoter element. For each
promoter fragment, experiments with 2 reporter genes (lacZ
and luciferase) were performed yielding essentially identical
results.
All constructs were transfected into 2 different cell types, the human
embryonic kidney cell line 293 not expressing renin2 and
the rat skeletal myoblast cell line L8 containing renin transcripts
detectable by RT-PCR (not shown). Schematic representations of
all constructs used in the transfection experiments and their
transcriptional activity are shown in Figure 2 (renin promoter) and Figure 3 (CMV promoter).
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In 293 cells, addition of parts of the rat renin intron I to reporter
gene constructs driven by the homologous renin 5'-flanking region
resulted in a down-regulation of reporter gene activity (Figure 2A). Integration of the first 65 bp of intron I (rRI1) led to
a significant 2-fold decrease of lacZ or luciferase activity
from 100±6.7% to 51.5±2.5%. Elongation of intron I to 117 bp
(rRI3) and further to 275 bp (rRI5) resulted in additional
reductions of reporter gene activities to 17.2±1.9% and 9.0±2.8%,
respectively. Although longer sequences of intron I (rRI7,9,10,11)
showed only insignificant additional changes in reporter gene activity,
inclusion of almost the entire intron I, except for the last 40 bp at
the 3' end, (rRI1) led to a further significant reduction of the
transcription level to 2.7±0.9%. This is exactly in the range of
negative control constructs bearing the renin sequences in antisense
orientation (as1 and asI1).
Transfection of the same constructs into L8 cells revealed a completely
opposite pattern of reporter gene activity (Figure 2B).
Integration of the first 65 bp of intron I (rRI1) increased
lacZ or luciferase activity 3-fold from 100±2.0% to
296±12%. Elongation of intron I to 117 bp (rRI3) decreased
transcription activity by a factor of 2.3 to 131.5±6.5%. Variations
between 117 and 410 bp length of intron I did not display any
significant changes. Further elongation of intron I to 538 bp (rRI9)
and 750 bp (rRI11), however, resulted first in a significant
decrease in reporter gene activity to 79.6±2.0%, followed by an
increase to 178.5±3.5%. Inclusion of the full-length intron (rRI1)
significantly decreased reporter gene activity to 77.9±4.2%, compared
with the intron-less construct (rR1).
Transfection experiments with renin intron constructs controlled by the
heterologous CMV promoter in 293 cells displayed a similar but even
stronger inhibitory influence of intron I on
transcriptional activity as seen in the corresponding renin constructs
(Figure 3A). Integration of the first 65 bp of intron I (CI1)
decreased the lacZ or luciferase activity 5-fold to
16.9±1.5% (instead of 2-fold in the case of construct rRI1).
Elongating intron I to 103 bp caused a reduction of reporter gene
activity to 6.3±0.4%. However, further elongation elicited no
additional significant effect except for the addition of the
full-length intron I lowering the transcription level to
1.1±0.3%.
The transcriptional activities of the CMV promoter/renin intron
constructs transfected into L8 cells were basically comparable to the
ones in 293 cells, but contrasted to the results obtained with the
corresponding renin promoter/renin intron constructs in L8 cells
(Figure 3B). Integration of the first 65 bp of intron I (CI1)
decreased reporter gene activity by a factor of 3.2 to 30.8±5.2%, an
activity level that was also found in intron deletion constructs with a
length of intron I between 103 and 432 bp. Further elongation of intron
I to 538 bp (CI9), 750 bp (CI11), and 5.3 kb (CI1) resulted again
in the wavy pattern of reporter gene activity seen with renin
promoter/renin intron constructs with activity levels of 9.4±1.6%,
28.5±2.8%, and 9.5±2.1%, respectively. Thus, the addition of the
full-length renin intron reduced transcriptional activity 10-fold for
the CMV promoter but only by 22% for the renin promoter in L8
cells.
DNA-Protein Interactions in Intron I
The sequence causing the most obvious changes in reporter gene
activity, ie, the coding part of exon I (79 bp) and the first part of
intron I (65 bp) was further analyzed for possible DNA-protein
interactions. Nuclear extracts of both cell lines were fractionated on
heparin-sepharose columns and the fractions were analyzed by
gel-retardation assays. As probes, different end-labeled fragments were
used containing either the exon I-intron I sequence mentioned above
(+45 to +189 bp) or a part of it.
The elution profile of the heparin-sepharose column using 293 nuclear
extracts is depicted in Figure 4A, a
typical gel-retardation experiment with gradient-separated extracts and
a fragment covering the first 65 bp of intron I (+124 to +189 bp) as
probe is shown in Figure 4B. The protein-DNA complexes separated
by the shift experiment were named B1 to B18 with regard to their
degree of retardation and their affiliation to low salt (B1-B8) or high
salt (B9-B18) portions of the gradient. Although most complexes were
common to both 293 and L8 nuclear extracts, B4, represented
by faint (Figure 4B) to moderate (data not shown) signals, was
exclusively restricted to 293 extracts. On the other hand, a complex
named B15', presenting a weak and variable signal, could only
be observed in L8 extracts (data not shown).
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) and the
Bradford method (
), respectively (A), and for DNA-binding activity
by a gel-retardation assay (B) with 1 µL of each fraction and the
first 65 nucleotides of intron I endlabelled with
32P. Triangles mark the complexes used for the
methylation-interference assay (see Figure 5
To determine the sequence specificity of the different binding
activities methylation-interference assays were performed. The first 65
bp of intron I were labeled at the 5'-end and randomly methylated at an
average of 1 guanine (G) or (to a lesser extent) adenine (A) residue
per probe molecule. After incubation with the heparin-sepharose
fractions 14, 20, 24, and 30 derived from 293 nuclear extract,
protein-DNA complexes (B1-B18) were separated by
polyacrylamide-gel electrophoresis, purified from the gel,
cleaved at methylated residues and subsequently analyzed on a
denaturing sequencing gel as outlined in Methods (Figure 5). The methylated probe cleaved only by
piperidine without prior incubation with nuclear extracts and
separation by electrophoresis as well as the free probe served as
controls. Both displayed an identical G/A sequence pattern indicating
that the reaction procedures have no effect per se on the methylated
probe. Analyzing the sequence pattern of all complexes only B4 showed
significant differences in the signal intensity of G and A residues
(Figure 5). The signal intensities of the residues at positions
+162 (G), +163 (A), +166 (G), and +167 (G) decreased markedly whereas
that of +168 (A) increased significantly indicating that methylation at
these nucleotide positions interfered with
sequence-specific protein binding. Analysis of the surrounding
sequence profile of the putative binding region revealed a
dodecamer sequence (positions +159 to +171) with 8 out of
12 nucleotide positions arranged in a palindrome-like
configuration. Neither B12 (Figure 5) nor any other protein-DNA
complex in the gel-retardation experiments exhibited sequence-specific
binding in the methylation-interference assay (data not shown).
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Discussion |
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In the present study, nonrenin-expressing 293 cells and renin-expressing L8 cells were used in parallel to investigate transcriptional regulation of reporter-gene constructs. The 293 cells are well established regarding transcription analysis of renin gene constructs.2 27 The paradox that their endogenous renin gene is silent while most transfected renin-gene constructs can be transcribed with high efficiency may be explained by the expression of the adenoviral E1A protein,20 which is capable of activating many eukaryotic promoters when transfected as episomal DNA constructs, whereas the activation of endogenous genes is variant between different genes.28 L8 cells were used for the first time in expression studies of the renin gene. However, a related rat skeletal muscle myoblast cell line, L6, has already been shown to express renin.29
Transcription analysis of the different constructs revealed a
complex pattern of 7 potential cis-regulating elements within the first
850 bp of intron I, 5 negative regulatory elements I-NRE 1 to 5 and 2
positive regulatory elements I-PRE 1 and 2. Their approximate positions
within intron I and their functional relevance considering cell type
and promoter region are outlined in Figure 6. In summarizing our data, we propose
the following hypothetical model for the transcriptional control of the
rat renin gene by intron I:
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Two regulatory elements (I-PRE 1 and I-NRE 1) are located within the
first 65 bp of intron I demonstrating antagonistic
regulatory effects. Whereas I-NRE 1 acts promoter-independently, I-PRE
1 represents as a renin-promoter dependent enhancer in both
cell types with barely any effect in constructs controlled by the CMV
promoter. I-NRE 2, located in the segment between 66 and 103 bp of
intron I, represents a promoter-independent negative regulatory
element in both cell types. I-NRE 3, located between
nucleotides 138 and 275 bp of intron I, acts as a
renin-promoter and 293-cell specific negative regulatory element.
Almost promoter independent but L8-cell specific are the regulatory
effects of I-NRE 4 and I-PRE 2. I-NRE 4, located in the DNA segment
between 432 and 538 bp of intron I, reduces the transcription activity
of the renin and the CMV-promoter, whereas no effect is seen in 293
cells. I-PRE 2 however, located more downstream between
nucleotides 618 and 750 of intron I, antagonizes the
silencing effect of I-NRE 4 in L8 cells irrespective of the origin of
the controlling promoter region. Again, there is no obvious regulatory
effect in 293 cells. I-NRE 5 was localized to the segment between 750
and 848 bp of intron I as reporter gene activities of 12 deletion
mutants (Figure 1) exhibited the same transcriptional activities
as full-length intron constructs (data not shown). I-NRE 5 influences
transcription regulation of either promoter in either cell line. Taken
together, I-NRE 2, 4, and 5 may be responsible for the low expression
of the endogenous renin gene in L8 cells and, together with
I-NRE 1 and 3, also for the inactivity of this gene in 293 cells.
Splicing artifacts as explanation for the differential reporter gene expression in both cell lines are unlikely as all intron-bearing constructs were cloned in a modular manner guaranteeing an identical nucleotide sequence around the SAS. Moreover, RT-PCR analysis of RNA isolated from cells transfected with the different constructs revealed amplification products of the expected size for a correct splicing process (data not shown). Furthermore, the differential interactions of some of the intron-I elements with both promoters can hardly be explained on a post-transcriptional level.
Regulatory elements in intronic sequences of mammalian genes are a common phenomenon.8 9 10 11 After our initial report of a transcriptional silencer in the rat renin gene,7 Lang et al15 described a strong orientation- and position-independent repressor in intron A of the human renin gene active in Calu-6 cells, a renin-producing human lung carcinoma cell line. In contrast to our study, the authors tested the transcriptional influence of intron A only in combination with the homologous human renin promoter and could only demonstrate a regulatory effect on a promoter fragment of at least 0.9 kb in length. More recently, Germain et al16 published a similar study confirming the existence of this silencer active in Calu-6 cells. In addition, they demonstrated a comparable silencing effect of human renin intron A on the heterologous SV40 promoter in these cells. In contrast to our results, the authors found a stimulatory effect of the first intron transfecting these constructs into the nonrenin expressing cell lines CHO and HeLa. The discrepancy regarding nonrenin producing cell lines may be explained by the use of different cell lines and renin genes of different origin. Nevertheless, it should be emphasized that it is difficult to understand why nonrenin expressing cell lines should express the transfected constructs.
Analysis of protein binding to the DNA region containing I-PRE 1 and I-NRE 1 by gel-retardation experiments and methylation-interference analyses revealed a number of complexes, 1 of them demonstrating sequence-specificity. The responsible cis-acting DNA sequence consisted of 12 nucleotides (AACTGGCCTCTT, +160 to +171 bp) of which 8 (underlined) form a palindromic structure, which is typical for binding sites of dimeric transcription factors.
The protein-binding DNA sequence in the rat renin intron I contains (on the opposite strand) the pentamer GAGGC (+169 to +165 bp). This sequence has been shown to bind T antigens of several oncogenic viruses, like SV40 and polyomavirus.30 31 By binding to this pentamer the T antigens regulate viral replication and transcription. However, the sequence has not yet been shown to be involved in the action of nonviral transcription factors and therefore its functional significance in the rat renin gene is elusive.
The sequence CCTCT (+166 to +170 bp) is located on the other strand, overlapping with the pentamer. This represents the central and most conserved part of several transcriptional silencers found in a variety of genes including the exon 1 silencer of the rat osteocalcin gene.32 In the human homolog of the same gene, exon 1 carries another silencer element (TGGCCCT),33 which resembles the sequence TGGCCTC (+163 to +169 bp) located in the center of the palindromic protein-binding region of renin intron I. It has to be clarified whether the same transcription factors bind the exon 1 elements of the osteocalcin gene or another described silencer and the DNA-element found in the 5'-end of rat renin intron I.
In conclusion, transcriptional analysis of the rat renin gene in 2 cell lines differentially expressing the endogenous renin gene revealed a complex pattern of regulation by transcription factors binding to intron I of this gene. The physiological role of such a complex machinery for transcriptional regulation in intron I of the renin gene needs further investigation. As potential function we suggest that it offers the opportunity to easily regulate and precisely modulate cell-type specific expression of the renin gene, thereby facilitating the differential transcriptional regulation of renin in different tissues for instance in response to environmental stimuli.
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Acknowledgments |
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We thank C. Scheidereit for help in the analysis of the protein-DNA interactions and for critically reading the manuscript, and we acknowledge the excellent technical assistance of A. Ripperger. T. Voigtländer was a recipient of a Max-Delbrück-Center fellowship.
Received September 15, 1998; first decision October 12, 1998; accepted October 23, 1998.
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References |
|---|
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
1. Sigmund CD, Okuyama K, Ingelfinger J, Jones CA, Mullins JJ, Kane C, Kim U, Wu C, Kenny L, Rustum Y, Dzau VJ, Gross KW. Isolation and characterization of renin-expression cell lines from transgenic mice containing a renin-promoter viral oncogene fusion construct. J Biol Chem. 1990;265:19916–19922.
2.
Tamura K, Tanimoto K, Murakami K, Fukamizu A. A
combination of upstream and proximal elements is required for efficient
expression of the mouse renin promoter in cultured cells. Nucl
Acids Res. 1993;20:3617–3623.
3.
Burt DW, Nakamura N, Kelley P, Dzau VJ. Identification
of negative and positive regulatory elements in the human renin gene.
J Biol Chem. 1989;264:7357–7362.
4.
Sun J, Oddoux C, Lazarus A, Gilbert MT, Catanzaro DF.
Promoter activity of human renin 5'-flanking DNA sequences is
activated by the pituitary-specific transcription factor Pit-1.
J Biol Chem. 1993;268:1505–1508.
5. Morris BJ. Molecular biology of renin. In: Lindpaintner K, Ganten D, eds. Molecular Reviews in Cardiovascular Medicine. London: Chapman & Hall; 1996: 12–32.
6. Smith DL, Morris BJ. Transient expression analyses of DNA extending 2.4 kb upstream of the human renin gene. Mol Cell Endocrinol. 1991;80:139–146.[Medline] [Order article via Infotrieve]
7. Voigtländer T, Ripperger A, Ganten D, Bader M. Transcriptional silencer in intron I of the rat renin gene. Adv Exp Med Biol. 1995;377:285–292.[Medline] [Order article via Infotrieve]
8.
Handen JS, Rosenberg HF. Intronic enhancer activity of
the eosinophil-derived neurotoxin (RNS2) and eosinophil cationic
protein (RNS3) genes is mediated by an NFAT-1 consensus binding
sequence. J Biol Chem. 1997;272:1665–1669.
9. Jenuwein T, Forrester WC, Fernandez Herrero LA, Laible G, Dull M, Grosschedl R. Extension of chromatin accessibility by nuclear matrix attachment regions. Nature. 1997;385:269–272.[Medline] [Order article via Infotrieve]
10. Umezawa A, Yamamoto H, Rhodes K, Klemsz MJ, Maki RA, Oshima RG. Methylation of an ETS site in the intron enhancer of the keratin 18 gene participates in tissue-specific repression. Mol Cell Biol. 1997;17:4885–4894.[Abstract]
11. Sawada S, Scarborough JD, Killeen N, Littman DR. A lineage-specific transcriptional silencer regulates CD4 gene expression during T lymphocyte development. Cell. 1994;77:917–929.[Medline] [Order article via Infotrieve]
12. Bader M, Ganten D. Transgenic rats: tools to study the function of the renin-angiotensin system. Clin Exp Pharmacol Physiol. 1996;23(suppl 3):S81–S87.
13. Sigmund CD, Jones CA, Fabian JR, Mullins JJ, Gross KW. Tissue and cell specific expression of a renin promoter-reporter gene construct in transgenic mice. Biochem Biophys Res Commun. 1990;170:344–350.[Medline] [Order article via Infotrieve]
14. Sola C, Tronik D, Dreyfus M, Babinet C, Rougeon F. Renin-promoter SV40 large T-antigen transgenes induce tumors irrespective of normal cellular expression of renin genes. Oncogene Res. 1989;5:149–153.[Medline] [Order article via Infotrieve]
15.
Lang JA, Ying L-H, Morris BJ, Sigmund CD.
Transcriptional and posttranscriptional mechanisms regulate human renin
gene expression in Calu-6 cells. Am J Physiol. 1996;271:F94–F100.
16. Germain S, Philippe J, Fuchs S, Lengronne A, Corvol P, Pinet F. Regulation of human renin secretion and gene transcription in Calu-6 cells. FEBS Lett. 1997;407:177–183.[Medline] [Order article via Infotrieve]
17. Sambrook J, Fritsch EF, Maniatis T. Molecular cloning: A Laboratory Manual. Cold Spring Harbour, NY: Cold Spring Harbour Laboratory; 1989.
18. Fukamizu A, Nishi K, Cho T, Saitoh M, Nakayama K, Ohkubo H, Nakanishi S, Murakami K. Structure of the rat renin gene. J Mol Biol. 1988;201:443–450.[Medline] [Order article via Infotrieve]
19.
Pesquero JB, Lindsey CJ, Zeh K, Paiva ACM, Ganten D,
Bader M. Molecular structure and expression of the rat bradykinin B2
receptor gene: evidence for alternative splicing. J Biol
Chem. 1994;269:26920–26925.
20.
Graham FL, Smiley J, Russell WC, Nairn R.
Characteristics of a human cell line transformed by DNA from human
adenovirus type 5. J Gen Virol. 1977;36:59–74.
21. Richler C, Yaffe D. The in vitro cultivation and differentiation capacities of myogenic cell lines. Dev Biol. 1970;23:1–22.[Medline] [Order article via Infotrieve]
22.
Deuschle U, Pepperkok R, Wang F, Giordano TJ,
McAllister WT, Ansorge W, Bujard H. Regulated expression of foreign
genes in mammalian cells under the control of coliphage T3 RNA
polymerase and lac repressor. Proc Natl Acad Sci U S A. 1989;86:5400–5404.
23.
Dignam JD, Lebowitz RM, Roeder RG. Accurate
transcription initiation by RNA polymerase II in a soluble extract from
isolated mammalian nuclei. Nucl Acids Res. 1983;11:1475–1489.
24. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976;72:248–254.[Medline] [Order article via Infotrieve]
25. Scheidereit C, Heguy A, Roeder RG. Identification and characterization of a human lymphoid-specific octamer-binding protein (OTF-2) that activates transcription of an immunoglobulin promotor in vitro. Cell. 1987;51:783–793.[Medline] [Order article via Infotrieve]
26. Rapp JP, Dene H, Deng AY. Seven renin alleles in rats and their effects on blood pressure. J Hypertens. 1994;12:349–355.[Medline] [Order article via Infotrieve]
27.
Sun J, Oddoux C, Gilbert MT, Yan Y, Lazarus A, Campbell
WG, Catanzaro DF. Pituitary specific transcription factor (Pit-1)
binding site in the human renin gene 5'-flanking DNA stimulates
promoter activity in placental cell primary cultures and pituitary
lactosomatotropic cell lines. Circ Res. 1994;75:624–629.
28. Shenk T, Flint J. Transcriptional and transforming activities of the adenovirus E1A proteins. Adv Cancer Res. 1991;57:47–85.[Medline] [Order article via Infotrieve]
29.
Dostal DE, Rothblum KN, Chernin MI, Cooper GR, Baker
KM. Intracardiac detection of angiotensinogen and renin: a
localized renin-angiotensin system in neonatal rat heart.
Am J Physiol. 1992;263:C838–C850.
30. Tjian R. The binding site on SV40 DNA for a T antigen-related protein. Cell. 1978;13:165–179.[Medline] [Order article via Infotrieve]
31.
Pomerantz BJ, Hassell JA. Polyomavirus and simian virus
40 large T antigens bind to common DNA sequences. J
Virol. 1984;49:925–937.
32. Frenkel B, Mijnes J, Aronow MA, Zambetti G, Banerjee C, Stein JL, Lian JB, Stein GS. Position and orientation-selective silencer in protein-coding sequences of the rat osteocalcin gene. Biochemistry. 1993;32:13636–13643.[Medline] [Order article via Infotrieve]
33.
Li YP, Chen W, Stashenko P. Characterization of a
silencer element in the first exon of the human osteocalcin gene.
Nucl Acids Res. 1995;23:5064–5072.
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