From the Department of Molecular Biology, The Lerner
Research Institute, The Cleveland Clinic Foundation, Cleveland, Ohio
44195, the § Department of Pharmacology, University of
Pennsylvania Medical Center, Philadelphia, Pennsylvania 19104, and the
¶ Departments of Pediatrics and Molecular and Cellular Biology,
University of Arizona, College of Medicine, Tucson, Arizona 85724
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ABSTRACT |
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The angiotensin-converting enzyme (ACE) gene produces two mRNA species from tissue-specific promoters. The transcription start site of the mRNA for the smaller testicular isozyme (ACET) is located within an intron of the larger transcription unit that encodes the pulmonary isozyme (ACEP).We have previously demonstrated that a 298-base pair DNA fragment, 5' to the rabbit ACET mRNA transcription initiation site, can activate the testicular expression of a transgenic reporter gene. In the current study, using the same transgenic reporter system, we identified a putative cyclic AMP response element present within this DNA fragment to be absolutely essential for transcriptional activation. Moreover, we observed that ACET mRNA was not expressed in the testes of mice homozygous for a null mutation in the transcription factor CREM. However, in the same mice, ACEP mRNA was abundantly expressed in the lung. Our observations indicate that ACET mRNA expression in the testes is regulated by the putative cyclic AMP response element present 5' to the transcription start site and the corresponding transcription factor CREM.
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INTRODUCTION |
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Angiotensin-converting enzyme (ACE)1 is a carboxyl-terminal dipeptidyl exopeptidase that converts angiotensin I to angiotensin II, a potent vasopressive hormone (1). The two isoforms of ACE, ACEP and ACET, have identical enzymatic activity and are encoded by distinct mRNAs transcribed from the same gene through tissue-specific choice of transcription initiation and polyadenylation sites (2-4). The genomic organization is such that the ACET transcription unit is nested within the ACEP transcription unit (5-7). In addition, the first exon of ACET mRNA is unique to the ACET transcription unit. The ACET promoter and first ACET exon reside in the 12th ACEP intron, which is spliced out of the primary ACEP transcript (2, 8). Rabbit ACEP is a glycoprotein of 140 kDa and is produced by vascular endothelial cells, intestinal brush-border cells, renal proximal tubular cells, monocytes, and Leydig cells (9-14). The ACET isoform has a molecular mass of 100 kDa and is produced exclusively in adult testis by developing sperm cells, specifically late pachytene spermatocytes (13, 15).
We have previously demonstrated that the 5' proximal 298 bp of DNA
upstream of the ACET transcription initiation site are sufficient to provide correct tissue-specific expression of the rabbit
ACET message (8). Within this region lies a cyclic AMP response element-like site (CRET) at 52 and a TATA-like binding site
at
27, which are homologous to the murine ACET CRE-like and TATA-like sites (8). It was previously reported that the ACET TATA sequence binds TATA-binding protein from
non-testicular nuclear extracts. In addition, mutation of the
ACET promoter TATA-like element to a consensus TATA
sequence did not alter the testes-specific gene expression in
transgenic mice (16). These data suggest that the ACET
TATA-like site is not responsible for tissue-specific expression of
ACET mRNA, and the focus should be directed at the role
of CRET in ACET gene transcription.
Cyclic AMP response in differentiating sperm cells is mediated by the
CREM gene family. All members contain a CRE binding domain and a
kinase-inducible domain that is phosphorylated by cAMP-activated cyclic
AMP-dependent protein kinase. CREM isoforms differ in their
ability to stimulate or repress transcription due to the presence
(CREM) or absence (CREM
) of glutamine-rich domains (17). In
addition, CREM isoform transcriptional effects are gene, cell-type, and
promoter specific (18). Though immature sperm cells contain both
isoforms at low levels, differentiated sperm cells contain markedly
increased levels of CREM
protein (19, 20). The physiological
importance of CREM has recently been demonstrated through murine
gene-targeting methodologies (21). Male mice devoid of all CREM
isoforms are sterile due to dramatic reduction in post-meiotic
sperm-specific gene expression and failed spermatogenesis.
Transcription of protamine 1, protamine 2, TP1, TP2, calspermin,
Krox-20, Kox-24, proacrosin, MCS, and RT7 genes was absent in CREM
/
mice (21, 22). There is no other phenotypic alteration of the
physiology of these male mice nor in homozygous CREM mutant females
that retain fertility (21). The ACET CRET binds both
CREM
and CREM
proteins and directs proper cAMP stimulation of a
heterologous promoter in vitro (18).
In the current study, we investigated the role of the CRET site in
directing ACET mRNA transcription by mutating this site in a transgenic reporter gene that is expressed in sperm cells. In
addition, we assessed the role of the CREM family of transcription factors, in ACE gene expression, by measuring the levels of the two
mRNAs in different tissues of CREM /
mice. Our results showed that both the cis-element CRET and the transacting factor CREM are
necessary for sperm-specific expression of ACET
mRNA.
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EXPERIMENTAL PROCEDURES |
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Materials--
DNA-modifying enzymes were purchased from Life
Technologies, Inc. or Boehringer Mannheim. Oligonucleotides were
purchased from Operon Technologies, Inc. All radioisotopes were
purchased from NEN Life Science Products. Total RNA was isolated and
prepared from tissues (brain, kidney, lung, and testes) of wild type
(+/+) or homozygous (/
) CREM mutant adult mice as described
previously (21). The p-TRI-cyclophilin-mouse antisense control template was purchased from Ambion Inc. RNAzol B was purchased from Tel-Test, Inc.
Plasmid Construction--
The 298 ACET gene
promoter was mutated utilizing the Muta-Gene phagemid in
vitro mutagenesis system (Bio-Rad) and the oligonucleotide rACETCREMN (CCTGCAGTGTGTCCGCATAGAGCAG). Mutagenesis was
confirmed by sequencing. The mutated ACET promoter was
cloned into PSVOCAT to yield pACET-CRENull (23). The ACE
probe used for ribonuclease protection assay (RPA) was cloned by
reverse transcriptase-polymerase chain reaction utilizing 1 µg of
total mouse testes RNA, the mouse ACE
2A (TCTGAAGCTTCTTTATGATCC), and
the mouse ACE-1S (ATGGGCCAAGGTTGGGCTA) oligonucleotides. The polymerase
chain reaction product was subcloned into pBlueScript KS to yield
plasmid PM045.
Transgenic Mice-- The pACET-CRENull plasmid was digested with XbaI and BamHI to release the 2.03-kilobase DNA fragment illustrated in Fig. 1. The DNA fragment was injected into C57BL/6JXSJL/J F2 zygotes by standard techniques (8). Positive transgenics and their progeny were identified by Southern blot hybridization.
Southern Blot Hybridization-- Mouse genomic DNA (15 µg) was digested with HindIII and ScaI or BanI. The samples were electrophoresed through a 0.8% agarose gel in TBE and transferred to HybondTM-N+ (Amersham Pharmacia Biotech) in 0.4 N NaOH. The membrane was prehybridized in 20 ml of C buffer (0.5 M NaPO4, pH 7.2, 7% SDS, 1% bovine serum albumin, 1 mM EDTA, 250 µg/ml denatured salmon sperm DNA) for 2 h at 65 °C. The 551-bp CAT gene probe was released from PSVOCAT by HindIII and NcoI digestion and radiolabeled as described in the random-primed DNA labeling kit (Boehringer Mannheim). The denatured probe was added to 20 ml of C buffer. The membrane was hybridized for 16 h at 65 °C and then washed as follows: solution 1 (40 mM NaPO4, pH 7.2, 5% SDS, 0.5% bovine serum albumin, 1 mM EDTA) for 30 min at 25 °C and 30 min at 55 °C, and solution 2 (40 mM NaPO4, pH 7.2, 1% SDS, 1 mM EDTA) for 30 min at 57 °C.
For copy number determination, the BanI-digested CAT gene-probed membrane was exposed to a PhosphorImager screen, stripped, and rehybridized with the 450-bp ACE probe. The washing conditions were as follows: solution 1 for 30 min at 25 °C and 56 °C for 30 min, and solution 2 for 30 min at 58 °C. The membrane was re-exposed to the PhosphorImager screen. Each CAT mRNA signal was normalized to the ACE-specific mRNA signal using ImageQuant software.Chloramphenicol Acetyltransferase Assay-- Tissues were analyzed according to previously described methods (8, 23). Fifty micrograms of protein was assayed for 2 h at 37 °C. The samples were spotted on a TLC plate and resolved in a 95:5 (v/v) chloroform to methanol solvent system. The radiolabeled acetylated chloramphenicol was visualized by autoradiography.
Ribonuclease Protection Assay--
Total mRNA was isolated
from CREM +/+ and CREM /
mouse tissues using the RNAzol B system
according to the manufacturer's instructions. The cyclophilin probe
was transcribed from the p-TRI-cyclophilin-mouse antisense control
template (Ambion Inc., Austin, TX) to generate a 165-bp riboprobe. The
470-bp ACE probe was transcribed from the plasmid PM045 described
above. The assay was performed as described (24). Antisense RNA was
transcribed in vitro using T7 RNA polymerase (Boehringer
Mannheim), ATP, CTP, GTP, and [
-32P]UTP (800 Ci/mmol)
(NEN Life Science Products). Riboprobe containing 1.0 × 105 Cerenkov counts was hybridized overnight at 45 °C
with 5.0 µg of total RNA from CREM +/+ and
/
mouse brain, kidney,
lung, and testes in 30 µl of hybridization buffer (40 mM
PIPES, pH 6.4, 400 mM NaCl, 1 mM EDTA, pH 8.0, 80% formamide). Following hybridization, the probe was digested with
40 µg/ml RNaseA (Boehringer Mannheim) and 2 µg/ml RNase T1
(Boehringer Mannheim) in 400 µl of ribonuclease digestion buffer (10 mM Tris-Cl, pH 7.5, 5 mM EDTA, 300 mM NaCl) for 1 h at 30 °C. Samples were then
incubated with 100 µg of proteinase K and 0.5% SDS for 20 min at
37 °C. The products were extracted, loaded onto a 6%
polyacrylamide, 8 M urea gel, and visualized by
autoradiography. The mRNA levels were determined by PhosphorImager analysis using ImageQuant software.
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RESULTS |
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Mutation of the CRET Site--
Previous reports have implicated
the ACET CRE-like site (CRET) as being a putative positive
regulator of ACET expression (18, 25). The 298-bp rabbit
ACET promoter, which gave correct tissue-specific reporter
gene expression in transgenic mice, contains a CRE-like site (CRET)
(8). To study the role of this CRET site, we utilized site-directed
mutagenesis to alter the normal ACET CRET (TGAGGTCA) to a
null-CRE site (TGCGGACA) (Fig.
1A). We have previously
demonstrated that this mutation abolishes the binding of CREM isoforms
and
to the altered CRE site in gel mobility shift assays (18). Four independent ACET-CRENull mouse lines were established
by injecting the DNA fragment illustrated (Fig. 1A) into
C57BL/6JXSJL/J F2 zygotes. Positive transgenics and their
progeny were confirmed by Southern blot (Fig. 1B). Genomic
DNA from mouse 5090 (line A), 5091 (line C), 5108 (line D), and 5066 (line E) was digested with
HindIII and ScaI, which releases a 701-bp CAT
gene fragment (Fig. 1B). The probe was the 551-bp
HindIII
NcoI CAT gene fragment (Fig.
1A). To verify that the ACET-CRENull promoter
and CAT gene integrated intact, the genomic DNA was digested with
BanI (Fig. 1A) and reprobed with the
HindIII
NcoI CAT gene probe (Fig. 1C). The transgene copy number of each line was determined
as described under "Experimental Procedures." Both CAT and ACE
probes had the same specific activity. The signals from the CAT
hybridization were normalized to the ACE-specific signal obtained in
the mouse ACE hybridization. The approximate copy number for each line
was as follows: line A, 35-40; line C, 30-35;
line D, 5-10; and line E, 320-330 (Fig.
1C).
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Chloramphenicol Acetyltransferase Assay on Transgenic Mice
Tissue--
The testicular isoform of ACE is expressed exclusively in
sperm (13, 15). Previously, we reported that the 298 ACET
promoter drives CAT gene expression confined to the testes of the mouse containing the pACET-CAT2 transgene (8). When the
ACET-CRENull mice were assayed for transgene expression, no
CAT activity was observed in the testes of any of the four lines (Fig.
2). Our positive controls were testes
extract from the pACET-CAT 2, line F (mouse
2010) (8), and purified CAT enzyme. To determine if the CRENull
mutation causes ubiquitous CAT expression, we assayed CAT activity in
the lung, kidney, brain (Fig. 2) spleen, and liver (data not shown).
All tissues tested were negative for CAT activity.
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Ribonuclease Protection Assay on CREM Knock-out Mice--
Having
established that mutation of the CRET site abolishes rabbit
ACET promoter function in transgenic mice testes, we
focused on the trans-acting factors that bind to the CRET site (18). To
address this point, we studied the endogenous ACE expression in a mouse
devoid of all CREM isoforms. The method used to knock out the CREM gene
disrupts the coding region of all CREM family members (21). Total RNA
was isolated from age-matched CREM +/+ and CREM /
mouse brain,
kidney, lung, and testes. The mouse ACET mRNA level in
each of these tissues was determined by RPA utilizing a mouse ACE probe
that distinguishes between mouse ACEP mRNA and mouse
ACET mRNA levels (Fig.
3). This 470-bp mouse ACE antisense RNA
probe was produced by transcribing mouse cDNA containing ACET-specific exon 12T and ACET and
ACEP shared exons 13 and 14. The protected ACET
message was 450 bp due to the protection of the 12T exon present in
normal ACET mRNA. The protected ACEP
message was 260 bp due to the absence of the 12T exon in the normal
ACEP mRNA (Fig. 3A). This assay clearly
demonstrated that the ACET message was absent in CREM
/
testes (Fig. 3C). To confirm the integrity of the testicular
total RNA preparation, a mouse cyclophilin RNA probe was included in
the RPA. No decrease in cyclophilin mRNA level was observed in CREM
/
testes. The levels of ACEP mRNA was not reduced
in CREM
/
brain, kidney, and lung (Fig. 3, B and
C). However, we did observe a reduction in the
ACEP message in CREM
/
testes (Fig. 3C).
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DISCUSSION |
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In this study, we investigated the mechanism of testis-specific expression of the ACET mRNA. In adult males, this mRNA is exclusively expressed in maturing sperm cells, although the ACEP mRNA, which arises from the same gene, is expressed in many other tissues (9-15). Using tissue-cultured cells, the rabbit ACEP transcription unit has been extensively analyzed, and the presence of several positive regulatory sites and a strong silencer element has been noted (26-28). For analyzing the transcriptional promoter of the ACET transcription unit, we had to resort to a transgenic assay system because the relevant sperm cells cannot be cultivated in vitro. Using a CAT reporter gene, we previously demonstrated that a 298-bp fragment upstream of the rabbit ACET mRNA transcription start site is capable of driving testis-specific and developmentally regulated expression of CAT (8). Among several putative regulatory sites present within this region is the CRET element, which resembles a consensus cyclic AMP response element (29). Using in vitro assays, we demonstrated that this site is capable of binding to the CREM transcription factors and initiating their transcriptional stimulatory effects (18). In the current study, we investigated whether the above observations are also true in vivo in the ACET-expressing adult testes. For this purpose, we used a combination of transgenic and gene knock-out mice.
Results from the CRENull transgenic mice clearly showed that the CRET
site is necessary for in vivo functioning of the
ACET promoter. Five independent CRENull transgenic lines
(line B and data not shown) failed to express the reporter
gene in adult testes. Although earlier studies have suggested that the
CRET site is functional in vitro (16, 18, 25), the current
study has demonstrated its absolute requirement in the context of the
ACET promoter functioning in vivo. Because the
CREM gene family of cAMP-activating transcription factors has been
shown to be important for sperm gene expression, we examined the ACE
mRNA expression profile in CREM /
mice (21, 22). Like several
other cAMP-dependent testicular mRNAs, ACET
mRNA expression was totally absent in CREM
/
cells. These
results strongly suggest that CREM is the relevant physiological
transcription factor that binds to the CRET site of the ACE gene and
activates transcription of the ACET mRNA.
Although the results presented here provide an understanding of the mechanism of activation of the ACET promoter, it is still not clear why this transcription unit is not activated in other tissues. Why is the CRET site not recognized by any of the multiple cAMP-activated transcription factors (e.g. CREM, CREB, c/EBP, etc.) and the ACET mRNA transcribed? CREB is more abundant in lung and kidney than CREM and is capable of binding to the ACET CRE site to activate transcription (30). Alternatively, does active transcription of the ACEP mRNA preclude the use of the ACET promoter in some way?
Our results revealed another apparent anomaly. ACEP
mRNA is normally expressed in vascular endothelial cells and Leydig
cells of the testes (9, 13). However, ACEP mRNA was
poorly transcribed in the testes, but not the lung, of the CREM /
mouse. It was not due to a global deficiency in transcription because
many mRNAs, including the cyclophilin mRNA that was used as an
internal control in our experiment, were transcribed normally in the
same tissue. In addition, CREM
/
mutant mice possess normally
developed Sertoli and Leydig cells (22). Does that mean that
ACET and ACEP mRNA transcription in the
testes are somehow coupled? This may be the case, albeit indirectly, if
the decreased expression of ACEP mRNA in the CREM
/
mice reflects the dependence of its expression on paracrine factors in
the testis. Cultures of Sertoli cells, with and without germ cells,
have suggested that spermatocytes and early spermatids may have
important regulatory influences on Sertoli cells (31). In turn, there
is abundant evidence that Sertoli cell factors modulate Leydig cell
steroidogenesis. These influences have been thought to be both positive
and negative, depending on the study (32). This suggests that although
mature sperm-deficient CREM
/
mice have Leydig cells that appear
normal (22), perhaps their ability to produce ACEP mRNA
is impaired. Therefore, ACEP mRNA levels in the testes
would be more dependent on sperm cell development as a whole rather
than directly on ACET expression itself. It is also
possible that CREM proteins are directly needed for ACEP
mRNA transcription as well. However, that requirement must be
obviated in the lung. No clear-cut explanation emerges from the
available information, and further investigations will be required for
resolving these issues.
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ACKNOWLEDGEMENTS |
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We gratefully acknowledge Diana Davis and Sean Pocock for assistance with the CRENull transgenic mouse lines and Paul Stanton for site-directed mutagenesis techniques. We thank the members of the GCS laboratory for helpful discussions.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grant HL-48258 and by a fellowship from the American Heart Association, Northeast Ohio Affiliate. The University of Arizona transgenic facility is partially supported by Cancer Center Support Grant P30 CA23074 and Southwest Environmental Health Sciences Center Grant P30 ES06694.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.
To whom correspondence should be addressed: Dept. of Molecular
Biology, The Cleveland Clinic Foundation, 9500 Euclid Ave., NC20,
Cleveland, OH 44195. Tel.: 216-444-0636; Fax: 216-444-0512.
1 The abbreviations used are: ACE, angiotensin-converting enzyme; ACET, testicular angiotensin-converting enzyme; ACEP, pulmonary angiotensin-converting enzyme; CRE, cyclic AMP response element; CAT, chloramphenicol acetyltransferase; CREM, cyclic AMP response element modulator; RPA, ribonuclease protection assay; bp, base pair(s); PIPES, 1,4-piperazinediethanesulfonic acid.
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REFERENCES |
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