©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Cyclic AMP Response Elements of the Genes for Angiotensin-converting Enzyme and Phosphoenolpyruvate Carboxykinase (GTP) Can Mediate Transcriptional Activation by CREM and CREM (*)

(Received for publication, January 26, 1995; and in revised form, May 10, 1995)

Tauqir Y. Goraya (1) (2),   Sean P. Kessler (1) Paul Stanton (1) Richard W. Hanson (3) Ganes C. Sen (1) (2) (3)(§)

From the (1)Department of Molecular Biology, The Cleveland Clinic Foundation, Research Institute, Cleveland, Ohio 44195 and Departments of (2)Physiology and Biophysics and (3)Biochemistry, Case Western Reserve University Medical School, Cleveland, Ohio 44106

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The potential of the CREM family of proteins to activate transcription of the genes encoding the testis-specific isozyme of angiotensin converting enzyme (ACE(T)) and the gluconeogenic enzyme, phosphoenolpyruvate carboxykinase (GTP) (PEPCK) (EC 4.1.1.32) were investigated. Both CREM and CREMalpha bind efficiently to the putative cyclic AMP response element (CRE) present in the ACE(T) gene (CRET) and to the CRE in the PEPCK gene. In HepG2 cells, the CRE was required for the strong stimulation by CREM of the expression of a chimeric PEPCK (-210 to +73)-chloramphenicol acetyl transferase (CAT) gene. The CRE could be mutated to the CRET sequence without losing the stimulatory effects of CREM. However, a similar chimeric gene driven by the regulatory region of the ACE(T) gene, which contains the CRET site, could only be stimulated by CREM when its imperfect TATA element was mutated to an authentic TATA. Surprisingly, CREMalpha, an alleged inhibitor of CRE-mediated transcription, stimulated the expression of both PEPCK-CAT and ACE(T)-CAT genes in HepG2 cells, a process which required the presence of the CRE and the CRET sites, respectively. In contrast, when the same CRE elements were used to drive the transcription of a chimeric gene containing the thymidine kinase promoter linked to the CAT structural gene, CREMalpha inhibited its expression in HepG2 and JEG3 cells. The expression of the same chimeric gene, however, was stimulated by CREMalpha in F9 embryonal carcinoma cells. These results demonstrated that the nature of the transcriptional effects of CREM isoforms on CRE-mediated transcription depends on the specific gene, the specific cell type and the promoter context of the CRE site.


INTRODUCTION

Angiotensin-converting enzyme (ACE) (^1)is the key enzyme of the renin-angiotensin system which regulates blood pressure(1) . ACE has two isozymic forms. The larger protein, ACE(P), is expressed in vascular endothelial cells, kidney and intestinal epithelial cells, macrophages and brain cells(2, 3, 4, 5) . The smaller isozyme, ACE(T), is expressed exclusively in developing sperm cells(3) . The two isozymes are the products of 5- and 2.5-kilobase mRNAs which are transcribed from the same gene by a tissue-specific choice of two alternative transcription initiation sites and two alternative polyadenylation sites in the gene(6) . Our studies have identified positive and negative regulatory elements present in the 5`-upstream region of the rabbit ACE(P) transcription unit, which are used both in the vascular endothelial and kidney epithelial cells(7) . This report, on the other hand, deals with the regulation of ACE(T) mRNA transcription.

Inspection of the nucleotide sequence 5` to the rabbit ACE(T) transcription initiation site revealed several putative, but no authentic, transcriptional regulatory elements. There is a TATA-like sequence at -27 and a cyclic AMP response element (CRE)-like sequence at -52(8) . Further upstream at -114 and -121, there are sequence motifs similar to one present in the PGK2 gene which is also expressed in sperm cells(9, 10) . The ACE(T) gene contains, in addition, several palindromic and repeat sequences which may contribute to its tissue-specific expression(8) . Experimental analysis of the functional roles of these putative sites has, however, been difficult mainly because of the unavailability of in vitro culture systems of the relevant cell types which can be used for transfection of reporter genes and measurements of their resultant expression. To circumvent these problems, transgenic assays have been used. Mice carrying reporter transgenes driven by putative regulatory regions of the ACE(T) gene have been tested for reporter gene expression in the testis. Such an analysis with the rabbit gene has indicated that a region containing 325 bp 5` to the ACE(T) transcription start site is sufficient for driving sperm-specific expression of the reporter gene. (^2)Similar analysis with the mouse gene has narrowed down the regulatory region to 91 bp upstream of the transcription start site(11) . There are two putative regulatory sites in this region, a CRE-like sequence, CRET, and a TATA-like sequence. We have examined the transcriptional roles, if any, of these elements using a heterologous cell culture system.

Our transfection analyses was performed using the HepG2 cell, a hepatoma cell line which has been widely used for studying transcriptional regulation of cAMP-responsive genes. One such gene encodes the gluconeogenic enzyme, PEPCK. The transcriptional regulation of the PEPCK gene has been extensively studied by us (12, 13, 14) and others(15, 16) . We have shown that a chimeric PEPCK-CAT gene containing a region from -210 to +73 of the PEPCK promoter is responsive to cAMP in hepatoma cells(14) . A CRE present in this region mediates the major portion of the cAMP response(13, 17) . Several trans-acting factors can bind to this element and activate transcription(14, 18, 19) . In the current study, we have analyzed the potential role of CREM and CREMalpha in the regulation of PEPCK gene transcription in HepG2 cells and have compared the results of this analysis with the induction of transcription of the gene for ACE(T).

Since the CRE-like site may be involved in sperm-specific regulation of ACE(T) expression, we were also interested in determining the trans-acting factors which are preferentially expressed in sperm cells and bind to this regulatory element. One such family of proteins, the CREM proteins, has been recently described(20) . This family of proteins originates from the same gene by cell-specific alternative splicing. They all contain DNA-binding domains, which recognize the CRE, and a phosphorylation box which is the target of phosphorylation by cAMP-activated PKA. Some isoforms such as CREM contain, in addition, glutamine-rich domains which are absent from other isoforms such as CREMalpha. CREM is a strong activator of transcription, which is cAMP dependent and mediated by the CRE. On the other hand, other forms lacking the glutamine-rich domain, such as CREMalpha, have been shown to repress CRE-mediated transcription(21) . Thus, the relative levels of CREM and CREMalpha can control the extent of cAMP-mediated gene transcription in cell types which express these proteins. Both activator and repressor isoforms of CREM are found at a low level in immature sperm cells. But during later stages of sperm differentiation, there is a pronounced isoform switch so that a large level of CREM, the activator form, is produced(22, 23) . Since ACE(T) is expressed in maturing sperm, CREM could be a strong candidate for promoting its transcription. This hypothesis was tested in the current study.

Our studies demonstrated that CREM stimulated transcription via both the CRE of the PEPCK gene and the CRET of the ACE(T) gene. Expression of the latter gene in HepG2 cells required, in addition, mutation of the TATA-like sequence to an authentic TATA. Unexpectedly, CREMalpha also stimulated transcription of both genes. Further experiments demonstrated that the ability of CREMalpha to stimulate or repress gene transcription depends on the promoter context of the CRE sequence and the cell type.


EXPERIMENTAL PROCEDURES

Materials

All DNA-modifying enzymes were obtained from either Life Technologies, Inc. or Boehringer Mannheim. Oligonucleotides were obtained from Operon Technologies Inc. [^14C]Chloramphenicol (50.10 mCi/mmol) and [alpha-P]dCTP (3000Ci/mmol) at 10 mCi/ml were purchased from Dupont NEN. Cell culture products were purchased from Life Technologies, Inc. and Cell Culture Laboratories, Cleveland, OH. Sequencing reactions were performed using Sequenase and dideoxy reagents purchased from U. S. Biochemical Corp.

Construction of Plasmids

The PEPCK-CAT expression plasmids have been described previously(12, 24) . Plasmids pSVCREM and pSVCREMalpha were kind gifts of P. Sassone-Corsi. They contain the CREM and CREMalpha cDNAs cloned in the EcoRI site of the pSG5 vector(20, 21) . This vector contains both the SV40 early promoter and the T7 promoter and therefore can be used for both in vivo and in vitro eukaryotic expression(25) . The expression vector for the C subunit of PKA contains the ubiquitously active SRalpha promoter to direct the expression of the catalytic subunit of PKA and was provided by M. Muramatsu(26) .

Site-directed mutagenesis of the PEPCK-CAT and ACET-CAT vectors was performed using the Muta-Gene® phagemid in vitro mutagenesis system from Bio-Rad, strictly following the manufacturer's instructions. All mutations of the PEPCK gene promoter were performed in the single stranded replicative form of the PTZ18R derived PEPCK-CAT expression vectors, as described previously(12, 24) . -210CRE2 mCREmPEPCK-CAT and -210CRE2 mACETPEPCK-CAT constructs were thus obtained by using mutagenizing oligonucleotides, CRE1 mTYGS and CRETOACET, on the -210CRE2 mPEPCK-CAT background, respectively. Mutagenesis of -85ACET gene promoter was performed by subcloning this gene segment in the pBlueScript(KS) vector. TATAm oligonucleotide was used to generate the -85ACETTATAm vector. All subcloning reactions were performed using directional cloning strategies and were confirmed by multiple restriction digestion analysis. All site-directed mutations were confirmed by sequencing using the dideoxy sequencing reaction.

The chimeric TK-CAT gene contains a -109 to +51 segment of the herpes simplex virus thymidine kinase gene (where transcription state site is designated as +1), ligated upstream of the CAT gene. To generate the CRETK-CAT and CRETTK-CAT plasmids, blunted double-stranded 18-bp oligonucleotides containing the CRE and CRET sequences were cloned 5` to the TK promoter in the blunted BamHI site of the TK-CAT vector. Plasmids with single copies of CRE and CRET sequences inserted at the correct position and orientation were selected by sequencing several clones.

In Vitro Transcription and Translation of CREM Transcription Factors

TNT-coupled reticulocyte lysate system from Promega was used to synthesize CREM proteins for electrophoretic mobility-shift assays. Briefly, the reaction consisted of TNT rabbit reiculocyte lysate, 25 µl; TNT reaction buffer, 2 µl; T7 RNA polymerase, 1 µl; 1 mM amino acid mixture minus methionine, 1 µl; 1 mM methionine, 1 µl; RNasin ribonuclease inhibitor (40 units/µl), 1 µl; miniprep plasmid DNA substrate, 2 µl; and diethyl pyrocarbonate-treated H(2)O to a final volume of 50 µl. Incubation was carried out at 30 °C for 90 min. In vitro translated proteins were used either fresh or after storing at -70 °C. It is critical that the substrate plasmid DNA isolated using Qiagen is extracted with phenol:cholroform to remove any traces of RNase A. In order to confirm that CREMalpha and CREM protein of the correct size were produced, the synthesis reaction was also performed by substituting cold methionine with 2 µl of [S]methionine (1000 Ci/mmol, 10 mCi/ml). The radioactive products were visualized by separation on a 4% SDS-polyacrylamide gel electrophoresis and autoradiography (results not shown).

Electrophoretic Mobility Shift Assay

An electrophoretic mobility shift assay was performed using a modification of the method described by Sassone-Corsi et al.(27) . DNA-protein binding was carried out in a final volume of 20 µl. One µl of reticulocyte lysate reaction mixture was incubated with 1 µg of poly(dIbulletdC) in 1 TM buffer at room temperature for 20 min. 5 TM buffer stock solution consists of 250 mM Tris pH 7.9, 62.5 mM MgCl(2), 5 mM EDTA, 5 mM dithiothreitol, and 70% glycerol. A synthetic 18-bp double-stranded oligodeoxynucleotide containing the CRE sequence from the PEPCK gene was labeled with P by filling-in with Klenow using [alpha-P]dCTP (3000 Ci/mmol) at 10 mCi/ml. Approximately 50 pg of the P-labeled probe (40,000 cpm) and any competitor cold DNA were added together to the preincubated protein extracts. After another incubation at room temperature for 10 min, the DNA-protein complexes were separated on a 4% polyacrylamide gel (39:1 acrylamide:bisacrylamide) in 0.25 TBE (1 TBE is 50 mM Tris borate (pH 8.3), 1 mM EDTA). The gels were dried and exposed for autoradiography; quantification of bands was performed using a PhosphorImager.

Double-stranded CRE, CRE2, CREmut, and CRET probes were prepared by annealing synthetic complimentary oligodeoxynucleotides followed by enzymatic filling-in of the shorter strands: ACETCRESEN, GGCCCCTTACGTCAGAGG; ACETCREAS, CCTCTGACGTAAGGGGCC; CRE2S, GGCCGGCCCCTTAGGTCAGAGGCGAGC; CRE2AS, GCCTCTGACCTAAGGGGCC; CREmTYGS, GGCCGGCCCCCTGCGGACAGAGGCGAGC; CREmTYGAS, GCCTCTGTCCGCAGGGGCC; CRE1TOACET, GGCCGGCCCCCTGAGGTCAGAGGCGAGC; ACEtAS, GCCTCTGACCTCAGGGGCC.

Cell Culture

Cell lines derived from American opossum kidney (OPK), JEG-3 cells, mouse F9 embryonal carcinoma cells, and human cervical carcinoma (Hela) were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 2 mML-glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin sulfate (complete medium). F9 cells were cultured on gelatin-coated plates. Human hepatoma (HepG2) cell line was grown in minimum essential medium-alpha supplemented with 10% fetal bovine serum. For maintenance in culture, all cells were grown in 175-mm^2 flasks and split at a ratio of 1:5 upon achievement of confluence. Hela and HepG2 cells were split from one confluent flask into eight 100-mm plates a day before the transfection. This way the cells were at approximately 60% confluence for transfection the next day. F9 cells were trypsinized and replated every other day to prevent their spontaneous differentiation.

DNA Transient Transfection Assays

Plasmid DNA was purified using Qiagen columns (Qiagen, Inc.) and transfected into F9, OPK, Hela, or HepG2 cells using calcium phosphate precipitation method(28) . Transfections were carried out in 100-mm plates using 5 or 10 µg of reporter CAT plasmid and where indicated, 5 µg of Sralpha-PKA expression plasmid and 5 µg of CREM or CREMalpha expression plasmids. Total amount of transfected DNA in each plate was kept constant at 15 or 20 µg by using 5 or 10 µg of pUC 18 plasmid. Cell cultures grown to 60% confluence received DNA-calcium phosphate precipitate in 3 ml of complete medium. Sixteen hours later, cells were shocked with 20% dimethyl sulfoxide in Dulbecco's modified Eagle's medium for 2 min and then incubated in complete medium. Cells were harvested 40 h post-transfection. Extracts were prepared by repeated freeze-thawing, and CAT activity was assayed as described previously (28) . Acetylated and non-acetylated forms of chloramphenicol on the TLC were quantified using a PhosphorImager screen (Molecular Dynamics). Percent acetylation/unit volume of cell extract was normalized to the protein concentrations of cell extracts and expressed as relative CAT activities in arbitrary units. To ensure reproducibility of results, each experiment was performed in duplicate or triplicate and repeated at least three times using different batches of cells and plasmid DNA preparations.


RESULTS

Different Affinities of Different CRE Sequences for Binding to CREM and CREMalpha

In order to evaluate the potential of the CREs present in the genes for ACE(T) and PEPCK, to mediate CREM-driven transcriptional stimulation, we first determined the relative affinities of these sequences for binding two members of the CREM family of proteins: CREM and CREMalpha. These proteins, generated by in vitro transcription/translation of the corresponding cDNAs, were used for electrophoretic mobility shift assays (Fig.1). The radiolabeled probe used for this assay was the CRE present in the PEPCK gene (Table1). CREMalpha produced two prominent shifted complexes (Fig.1, lane 3), which probably represent the probe bound to a monomer or a dimer of CREMalpha. Two slower moving complexes were formed with CREM (lane 4), which is a larger protein than CREMalpha. When competed for binding to the probe, the unlabeled CRE was very effective (lanes 5-8), whereas a cryptic CRE (CRE2) present in the PEPCK gene between -155 and -135, and the CRET sequence present in the ACE(T) gene were less effective. A mutated CRE sequence (CREmut) failed to compete even at the highest concentration tested. The profile for binding of different CRE sequences to CREMalpha was parallel to that for CREM (data not shown). In order to assign quantitative values to the binding efficiencies of the different CREs to CREM and CREMalpha, we determined the molar amounts of each oligonucleotide required to diminish the labeled shifted bands by 50% (Table1). The amount of unlabeled PEPCK CRE required to achieve 50% reduction of the band intensity was assigned the value of 1. To obtain the same level of reduction, 16-fold more CRET was required for binding to CREM and 5-fold more CRET was required for binding to CREMalpha. CRE2 was even less efficient than CRET for binding to either protein, whereas CREmut was at least two orders of magnitude less efficient than the functional CRE from the PEPCK promoter. It is curious to note that CRET has a higher affinity than CRE2, although the latter varies from the functional CRE in the PEPCK promoter in only one position, whereas the former has changes in two nucleotides out of eight. These results suggested that CRE and CRE2 of the PEPCK gene and CRET from the gene for ACET all have the potential to interact with CREM and CREMalpha.


Figure 1: Gel mobility shift assay to determine the different affinities of CREM for the different CRE sequences. A radiolabeled 18-bp oligonucleotide (40,000 cpm, 50 pg) corresponding to the CRE sequence from the PEPCK gene was incubated in the absence or presence of various reticulocyte lysate protein extracts (2 µl) and various molar excesses of cold competitor probes. Lane 1, the probe alone; lane 2, the probe and reticulocyte lysate; lane 3, the probe and in vitro translated CREM alpha; lanes 4 and 9, the probe and in vitro translated CREM; lanes 5-8, the probe, CREM and 20-, 100-, 400, and 2,000-fold molar excess of the unlabeled CRE oligonucleotide; lanes 10-12, the probe, CREM and 400-, 3,000-, and 30,000-fold molar excess of unlabeled CREmut; lanes 13-15, the probe, CREM and 400-, 2,000-, and 20,000-fold molar excess of unlabeled CRET; lanes 16-18, the probe, CREM and 400-, 2,000-, and 20,000-fold molar excess of unlabeled CRE2. Arrows A, B, C, and D indicate the specific DNA-protein complexes.





Transcription Stimulation by CREM via CRE and CRE2 of PEPCK Gene

In the next series of experiments, we tested the ability of the CRE and CRE2 sites of the PEPCK gene to mediate transcriptional activation by CREM, a protein known to activate gene transcription. The transcriptional response of a series of chimeric PEPCK-CAT genes to expression of CREM was determined. These studies were performed using HepG2 cells in which the PEPCK promoter has been previously shown to control the transcription of linked structural genes in a regulated manner. The PEPCK promoter extending from the -210 upstream position to the +73 position relative to the start site (-210PEPCK-CAT) (Fig.2A) drove the expression of the CAT gene at a low level in these cells. Upon cotransfection with the SRalphaPKA plasmid, that encodes the catalytic subunit of PKA under the control of a constitutively active promoter, the level of CAT expression increased by about 2-fold (Fig.2B). Upon cotransfection with pSVCREM the level of CAT expression increased dramatically both in the presence and absence of PKA. As expected, the overall levels of stimulation observed in the presence of PKA were much higher than in its absence.


Figure 2: Transcription stimulation by CREM via the CRE sites of PEPCK gene. A, schematic diagram of the PEPCK-CAT promoter from -210 to +73 positions, where +1 is the transcription start site. Important protein binding domains within this region of the PEPCK gene are outlined above the promoter. B, HepG2 cells were transfected with plasmids containing the PEPCK-CAT with serial 5` deletions in the PEPCK promoter starting at positions indicated below the bars. 5 µg of PEPCK-CAT plasmid were transfected alone (open bar); cotransfected with 5 µg of CREM expression vector (striped bar); cotransfected with 5 µg of an expression plasmid for the C subunit of PKA (hatched bar); cotransfected with SRalpha-PKA and CREM expression plasmids (solid bar). Each bar represents the mean value from a duplicate experiment, and the dot over the bar shows the higher value. Each experiment was performed at least three times using at least two different plasmid DNA preparations and batches of cells. C, transfections were carried out in HepG2 cells using 5 µg of -210PEPCK-CAT plasmids containing specific mutations in the protein binding domains of the promoter. The sites which were block mutated are indicated on the horizontal axis. Open bar, cotransfected with 5 µg of SRalpha-PKA; solid bar, 5 µg of SRalpha-PKA and 5 µg of CREM. Data analysis and presentation are similar to those described for B.



Since the region of the PEPCK promoter between -210 and +73 contains several well characterized cis-acting binding sites (Fig.2A), a series of 5`-deletion mutants were tested to identify the site responsible for the mediation of CREM stimulation. The PEPCK promoter deleted to -174 (-174 to +73) lacks the HNF-1 binding site and was stimulated by the C subunit of PKA and CREM to levels very similar to those seen with the PEPCK promoter deleted to -210 (Fig.2B). The region of the PEPCK promoter between -174 and +73 contains two putative CRE sites (CRE and CRE2) and an NF-1 binding site. Further deletion to -109 upstream position of PEPCK promoter decreased its basal activity to about 15% of that noted with the promoter deleted to -210 but a pronounced stimulation was still observed with the cotransfection of CREM. The PEPCK promoter deleted to -109 contains only the CRE which has previously been shown to bind CREB, Fos/Jun heterodimers and members of C/EBP family of transcription factors. These results suggest that the CRE is sufficient for mediating the effect of CREM on transcription from the PEPCK promoter.

To further elucidate the role of the various cis-elements, mutants of these sites in the PEPCK promoter bretween -210 and +73 were tested. When the CRE2 site was mutated, there was a negligible effect on transcription from the PEPCK promoter in HepG2 cells (Fig.2C). Similarly, mutation of the NF-1 site did not alter either the basal level of transcriuption from the PEPCK promoter or the CREM-induced level of expression. Mutation of the CRE, on the other hand, decreased both levels drastically. Since transcription was stimulated to a slight extent with the CRE mutated, it is possible that at least some effects of CREM on PEPCK gene expression is mediated through the CRE2 site. This observation is consistent with the observed weaker but detectable binding of CRE2 to CREM in gel shift assays (Table1). When both CRE and CRE2 sites were mutated, the response to CREM was completely abrogated.

Transcriptional Stimulation by CREM via CRET

In order to assess whether the putative CRE-like site from the ACE(T) promoter (CRET) supports CREM mediated stimulation in vivo, the CRE site of the PEPCK promoter was converted to the CRET sequence of the ACET gene. This mutation was introduced into the PEPCK promoter in which the CRE2 site had previously been mutated in order to avoid any confusion with the interpretation of results due to the observed weak affinity of CRE2 site for the CREM protein in the EMSA assays. The PEPCK promoter containing the CRET could still be stimulated with CREM, although to levels about 25% of the maximal activity seen with the PEPCK promoter (-210 to +73) containing the authentic CRE (Fig.3). Mutation of the CRE to CREmut, on the other hand, decreased both the basal and CREM-mediated transcription to background values.


Figure 3: CREM can stimulate transcription through CRET. HepG2 cells were transfected with 5 µg of -210PEPCK-CAT vectors containing specific mutations. -210CRE2 mPEPCK-CAT vector contains a disruptive mutation in the CRE2 binding site. In the -210CRETCRE2 mPEPCK-CAT vector, in addition to the CRE2 mutation, the CRE site was mutated to a CRET binding sequence. The -210CREmutCRE2 mPEPCK-CAT vector contains mutations of both the CRE and CRE2 sites. The CAT reporter plasmids were cotransfected along with 5 µg of SRalpha-PKA expression vector alone (open bars); or with 5 µg each of PKA and CREM expression vectors (solid bars). Data analysis and presentation are similar to those described for Fig.2B.



Once we established that CRET can mediate transcriptional stimulation by CREM in the PEPCK gene context, we tested if the same is true in context of the ACE(T) gene. For this purpose, we used a reporter CAT gene driven by 85 bp of the transcriptional regulatory region of the ACE(T) transcription unit (Fig.4A). This region contains the CRET site at -52 position and a putative TATA element at -26 position. The corresponding region from the mouse ACE(T) transcription unit, which has strong sequence homology to the rabbit gene used in our study, has been shown to drive the expression of a reporter gene in the sperm cells of adult transgenic mice(11) . We wanted to test if CREM, which is known to be expressed in the sperm cells, could be the responsible transcription factor. For this purpose, the ACE(T)-CAT gene and the CREM expression vector were cotransfected in HepG2 cells which do not express the gene for this transcription factor. CREM failed to stimulate the expression of this chimeric gene, not only in HepG2 cells (Fig.4B) but also in HeLa and NIH3T3 cells (data not shown). The ACE(T) promoter lacks an authentic TATA sequence but instead contains a TATA-like sequence at -27 position; we determined whether the lack of responsiveness to CREM could be due to the defective TATA element in the ACE(T)-CAT gene. To test this hypothesis, its TCTTATT sequence was mutated to TATAATT. The authentic TATA mutant of the ACE(T)-CAT gene responded to CREM stimulation very efficiently (Fig.4B).


Figure 4: CREM can stimulate the transcription of ACE(T) gene in somatic cells only after mutation of its TATA-like element to an authentic TATA sequence. A, sequence of the upstream region of the ACE(T) transcription unit from position -85 to +30 (transcription start site is designated +1) which was used to construct the ACE(T)-CAT gene. The potential protein-binding sites are underlined. B, transfections were carried out in HepG2 cells using 5 µg of an expression plasmid containing the ACE(T)-CAT gene expression plasmids. ACE(T)TATAm-CAT vector contains a mutation whereby the native TATA-like element of the ACE(T) gene has been mutated to an authentic TATA sequence. Cotransfections were performed with either 5 µg of expression vector containing the SRalpha-PKA gene (open bars) or with 5 µg each of the expressions vectors for the catalytic subunit of PKA and CREM (solid bars). C, same experiment as in B, performed in the ACEp-producing OPK cells. An additional control was 5 µg of CAT reporter plasmid transfected alone (hatched bars). Open and solid bars represent the same conditions as described in B. Data analysis and presentation are similar to those described for Fig.2B.



These results suggest that in somatic cells, such as HepG2, the ACE(T) transcription unit cannot be expressed even if a positive transcription factor such as CREM, which binds to the CRE, is made available. It is possibly because the transcriptional machinery in these cells do not recognize the TATA-like sequence present in this gene. Thus, the tissue-specific expression of the ACE(T) mRNA is influenced by both the CRET element and the TATA-like element which apparently is used in the transcription machinery in sperm cells. In the experiment shown in Fig.4C, we examined the situation in cell types in which the ACE gene is transcriptionally active but only the ACE(P) transcript is synthesized. OPK cells, a kidney cell line, expresses ACE(P) but not ACE(T)(7) . In those cells, ACE(T)-CAT was again not stimulated by CREM unless the TATA-like element was mutated to an authentic TATA (Fig.4C). These results indicate that the tissue-specific expression of ACE(T) is, at least partly, dictated by the defective TATA element not only in cells which do not express the ACE gene at all but also in cells which contain the ACE(P) mRNA, but not the ACE(T) mRNA.

Transcriptional Activation by CREMalpha

CREM and CREMalpha are alternative splicing products from the same gene(21) . CREMalpha is missing an exon present in CREM which is thought to be required for its transcriptional activation property(22) . It has been shown with artificial test genes that CREMalpha cannot stimulate CRE-mediated transcription(20, 21, 22) . Moreover, since the CREM proteins can dimerize (see Fig.1), CREMalpha inhibits the action of CREM by heterodimerization. We thus tested the effects of CREMalpha on ACE(T) TATAm-CAT gene expression in HepG2 cells. To our surprise, CREMalpha did not inhibit the basal level of expression of this gene; instead, it stimulated its expression by more than 2-fold (Fig.5). CREM, however, was a better activator than CREMalpha.


Figure 5: Transcriptional activation of ACE(T) gene by CREMalpha. The effect of cotransfection of CREMalpha expression vector on the transcription of a chimeric ACE(T)TATAm-CAT gene was tested in HepG2 cells. Five µg of ACE(T)TATAm-CAT reporter plasmid was cotransfected with 5 µg of SRalpha-PKA and with 5 µg of either pUC18 plasmid (open bar), CREMalpha expression plasmid (hatched bar), or CREM expression vector (solid bar). Data analysis and presentation are similar to those described for Fig.2B.



Since CREMalpha can bind to the PEPCK CRE sequence better than the CRET sequence of ACE(T) gene (Table1), we tested if CREMalpha could stimulate transcription of the PEPCK-CAT gene. Indeed, there was strong stimulation of CAT expression in response to CREMalpha (Fig.6). Although somewhat weaker, the overall pattern of stimulation was similar to that observed with CREM. The stimulation was enhanced by PKA and mediated mostly by the CRE site of the PEPCK promoter. These results clearly demonstrated that CREMalpha can act as a transcriptional activator in the appropriate cellular context.


Figure 6: Transcriptional activation by CREMalpha. A, HepG2 cells were transfected with the PEPCK-CAT plasmids with serial 5` deletions starting at positions indicated below the bars. 5 µg of PEPCK-CAT plasmid was transfected alone (open bar); cotransfected with 5 µg of CREMalpha expression vector (striped bar); cotransfected with 5 µg of SRalpha-PKA (hatched bar); cotransfected with SRalpha-PKA and CREMalpha expression plasmids (solid bar). B, transfections were carried out in HepG2 cells using 5 µg of -210PEPCK-CAT plasmids containing specific mutations in the protein binding domains of the promoter. The site which was mutated is indicated on the horizontal axis. Open bar, cotransfected with 5 µg of SRalpha-PKA; solid bar, 5 µg of SRalpha-PKA and 5 µg of CREMalpha. Data analysis and presentation are similar to those described for Fig.2B.



Promoter Context-dependent and Cell Line-dependent Effects of CREMalpha

To reconcile our results with the published report of CRE-mediated inhibition of transcription by CREMalpha, we ascertained whether there were major differences in the experimental protocols. Two such differences were apparent. First, HepG2 cells were employed in our experiments, whereas other cell lines were used in previous studies. Second, we used the natural genomic sequences containing the CRE sites for driving the reporter gene, whereas the other study used an artificial reporter gene containing the somatostatin CRE site and the thymidine kinase promoter. To determine if these differences in the test system could account for the observed differences in CREMalpha's effects, we constructed TK-CAT genes driven by either the CRE of the PEPCK promoter or CRET of the ACE(T) gene. The newly constructed test genes were tested in the JEG3 cell line in which the somatostatin CRETK-CAT gene has been shown to be repressed by CREMalpha(20) . As shown in Fig.7A, CREMalpha strongly repressed both the basal and the PKA-stimulated expression of CAT from both CRE1TK-CAT and CRETTK-CAT genes. Similar inhibition by CREMalpha was also observed in HepG2 cells (Fig.7B). These experiments demonstrated that different CRE sequences used in this study respond similarly to CREMalpha. However, whether transcription mediated by these sites would be stimulated or repressed was dependent on the promoter context of the gene. The same two CRE sites were stimulated by CREMalpha in their natural promoter contexts but were repressed by it when placed in front of the TK promoter.


Figure 7: Promoter context-dependent effects of CREMalpha. The effect of CREMalpha on transcription of a chimeric TK-CAT gene was tested in JEG-3 and HepG2 cell lines. The thymidine kinase promoter (-151 to +60) was cloned upstream of the CAT reporter gene to obtain TK-CAT plasmid. CRETK-CAT and CRETTK-CAT vectors contain the CRE sequence from the PEPCK gene and the CRET sequence from the ACE(T) gene subcloned 5` to the TK promoter, respectively. A, JEG-3 cells were transfected with the TK-CAT, CRETK-CAT, and CRETTK-CAT plasmids, as indicated below the bars. Open bar, 5 µg of CAT plasmid was transfected alone; striped bar, cotransfected with 5 µg of CREMalpha expression vector; hatched bar, cotransfected with 5 µg of SRalpha-PKA; solid bar, cotransfected with 5 µg each of SRalpha-PKA and CREMalpha expression plasmids. B, similar experiment using CRETK-CAT and CRETTK-CAT plasmids was performed in HepG2 cells. Symbols for the bars are the same as in A. Data analysis and presentation are similar to those described for Fig.2B.



Possible contributions by the cellular contexts to the observed stimulatory and repressing activities of CREMalpha were further explored by testing the effects of CREM and CREMalpha on the expression of the PEPCK-CAT gene and the CRETK-CAT gene in F9 embryonal carcinoma cells. F9 cells, in contrast to the other cell lines used in this study, do not contain a detectable level of CREB with which CREMalpha is known to be able to form heterodimers(35) . Neither CREM nor CREMalpha could stimulate the expression of the PEPCK-CAT gene in F9 cells, even in the presence of the catalytic subunit of PKA (Fig.8). This was possibly due to the known tissue specificity of the expression of this gene which is controlled by sites other than the CRE. The CRETK-CAT gene was, however, expressed at a low, but detectable, level in the presence of PKA. The level of expression was enhanced by about 10-fold in the presence of CREM and by about 3-fold in the presence of CREMalpha. Thus, expression of the same reporter gene, CRETK-CAT, could either be stimulated or be repressed by CREMalpha in different cell lines suggesting that its interaction with other transcription factors may be a crucial determining factor.


Figure 8: Effects of CREMalpha in F9 cells. Expression of PEPCK-CAT and CRETK-CAT genes was tested in F9 cells. 10 µg of the reporter gene were cotransfected with 5 µg of SRalpha-PKA gene (open bar), with 5 µg of SRalpha-PKA and 5 µg of CREM- (solid bar), or with 5 µg of SRalpha-PKA and 5 µg of CREMalpha (hatched bar). Data analysis and presentation are similar to those described for Fig.2B.




DISCUSSION

The present study provides useful information regarding the possible basis of tissue-specific expression of ACE(T) in the testis. Such a tissue-specific expression has two layers of complexity. In the majority of tissues neither isoform of ACE is expressed at all. It is quite possible that the ACE gene is buried in transcriptionally inactive parts of the chromosome in these tissues. However, there are other tissues such as kidney, vascular endothelial cells, and macrophages in which one isozyme, ACE(P), is exclusively expressed, whereas in sperm cells only the other isozyme, ACE(T), is expressed. The ACE gene cannot be in a transcriptionally inactive environment in these tissues. Our studies indicate that two cis-elements of the ACE(T) transcription unit mediate its tissue-specific expression. The CRET site was shown to be capable of serving as a cAMP-inducible enhancer. The trans-acting factors responsible for mediating this response could be members of the CREM family of transcription factors. Our in vitro transfection expression clearly demonstrated that CRET can bind and respond to CREM, a potent trans-activator of transcription. Previous studies have established that CREM proteins are expressed in testis. Moreover, spermatogenesis is accompanied by a isoform switching of CREM. CREM is abundant in maturing sperm cells, the site of ACE(T) synthesis(22, 23) .

Our studies also indicate another possible tissue-specific cis-element, the TATA-like sequence present in the ACE(T) gene. In the two somatic cell lines used in our study, one, a nonexpressor of ACE, and the other, an expressor of ACE(P), ACE(T) upstream region could only drive transcription after the TATA-like sequence was converted to an authentic TATA element. Thus, in these cells, both the availability of CREM and the presence of authentic TATA were necessary for the expression of ACE(T)-reporter genes. This may explain the lack of expression of ACE(T) in several cell types, other than sperm, in which CREM is highly expressed. Presumably there is a specific transcription factor in ACE(T)-expressing sperm cells which recognizes the TATA-like element present in ACE(T) gene. The existence of such a cell type and sequence-specific TATA-binding factor remains to be documented. Alternatively, it is possible that some other as yet unrecognized cis-element impacts the sperm-specificity. In this scenario, the TATA-like sequence is irrelevant and noncontributing and the ACE(T) gene is driven by a TATA-less promoter like many other known genes(29) . Testing the ability of appropriately constructed reporter genes to be expressed in sperm cells will be necessary for providing definitive answers. Such experiments using transgenic mice are currently in progress.

Our experiments defined the spectrum of interaction between CREM proteins and different CRE sequence. The CREmut sequence which varies from the canonical CRE sequence present in the somatostatin gene, TGACGTCA, in positions 3, 4, and 6, failed to bind to CREM and CREMalpha and did not promote transcription in transfection analysis (Table1). The CRE from the PEPCK gene, which varies only in position 2 from the somatostatin CRE, was highly efficient in both binding CREMs and mediating CREM-driven transcription. CRET, which also varies in only one position, 4, was less efficient than CRE1 by both measures. The cryptic CRE2 from the PEPCK gene, which combines the differences of CRE1 and CRET and varies from the somatostatin CRE at both positions 2 and 4, was even less efficient and it could barely mediate CREM-driven transcription. As expected from their structures, the binding properties of CREM and CREMalpha were parallel although the absolute affinities were different.

The most unexpected observation reported here is the stimulation of CRE-mediated transcription by both CREMalpha and CREM. Furthermore, the observed difference in the stimulatory and the inhibitory phenotypes of CREMalpha, in different cell types or when the same CRE sequence is present in different promoter contexts, indicates a novel mode of regulation of transcription. Similar dichotomy has been previously noted for the chicken ovalbumin upstream promoter-transcription factor which represses the promoter of the ornithine transcarbamylase gene but activates the promoters of several other genes(30) . Consequently, it should be appreciated that conclusions drawn from studying the transcriptional regulation of one gene may not be valid for another gene even if the same cis-element and the same trans-acting factor are involved in regulating the transcription of both genes.

CREMalpha has previously been identified as a repressor of CRE-mediated transcription. The experiments leading to this characterization were generally carried out using artificial promoters containing the CRE of the somatostatin gene and the promoter of the thymidine kinase gene, which is similar to the chimeric promoters we used in the experiment shown in Fig.7. The observed repression of transcription by CREMalpha was rationalized by its structural difference as compared to CREM; CREMalpha does not contain the glutamine-rich domains present in CREM. Such domains can serve as transcriptional activators and they often exist in transcription factor motifs distinct from the DNA-binding and other regulatory domains. Although CREMalpha lacks the glutamine-rich domains, its DNA-binding domains are identical to those of CREM. Moreover, both isoforms share the same phosphorylation box or kinase-inducible domain. Within this domain is serine 117 which is the target of phosphorylation by not only by the C subunit of PKA but also by PKC, calmodulin kinase, p34 and p70(31) . How phosphorylation of this residue promotes the transcriptional activation potential of CREM proteins is not known. However, more is known about the activation mechanism of another CRE-binding transcriptional activator, CREB, upon its PKA-mediated phosphorylation. Such a phosphorylation does not change the affinity of the protein for its cognate DNA element(16, 32) , but changes its affinity for the CREB-binding protein, CBP, which serves as a co-activator of transcription(33) . The kinase-inducible domain (KID) of CREB is also present in CREMalpha and CREM. Thus, it is not unlikely that PKA-activated CREM and CREMalpha also bind CBP and promote transcription as a result of this interaction. Using engineered genes, it has been shown that KID can act as a conditional activator working either through a physically attached activation domain or through a promoter-bound protein in trans(34) . Here, we have uncovered a natural example of these two modes of transactivation using the regulatory regions of the PEPCK and ACE(T) genes. PKA-dependent activation by CREM probably depends on the phosphorylation of KID, resultant binding of CBP or another protein having equivalent properties, and its interaction with the glutamine-rich activation domains present in the CREM itself. In the case of CREMalpha, the first two steps might be the same but the bound CBP, instead of interacting with glutamine-rich domains which are absent for CREMalpha, interacts with neighboring factors bound to other cis-elements present in the gene. This model would postulate that the last crucial protein-protein interaction between CREMalpha-bound CBP and other proteins bound to the transcription unit is different when the PEPCK CRE sequence is present in its natural context versus when it is present in the context of the TK promoter. The exact nature of this difference remains to be identified.

The observed differences in the effects of CREMalpha could also, in principle, be due to a difference in the extent of its hetrodimerization with other CRE-binding factors such as CREB(35) . The observed stimulation of expression of the CRETK-CAT gene by CREMalpha in F9 cells rules out an involvement of CREB in this process, since these cells are devoid of this transcription factor. It appears therefore that CREMalpha, like CREM, can by itself promote transcription through a CRE site present in the right genetic context in the right type of cell.


FOOTNOTES

*
This work was supported in part by National Institutes of Health Grant HL-48258 and by National Institutes of Health Training Grant DK-07678 (to T. Y. G.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by 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; PEPCK, phosphoenolpyruvate carboxykinase (GTP); CRE, cyclic AMP response element; CAT, chloramphenicol acetyl transferase; NF-1, nuclear factor 1; PKA, cyclic AMP-dependent protein kinase; TK, thymidine kinase; TBE, Tris borate EDTA; OPK, opossum kidney; KID, kinase-inducible domain; CBP, CREB-binding protein; bp, base pair(s).

^2
R. Erickson, S. P. Kessler, and G. C. Sen, unpublished results.


ACKNOWLEDGEMENTS

We thank P. Sassone-Corsi for the CREM expression vectors, Richard Goodman for F9 cells, Steve Nizielski for helpful advice, and Dorthy Herzberg for expert manuscript preparation.


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