Tissue-Restricted Expression of the Cardiac α-Myosin Heavy Chain Gene Is Controlled by a Downstream Repressor Element Containing a Palindrome of Two Ets-Binding Sites

Abstract

The expression of the α-myosin heavy chain (MHC) gene is restricted primarily to cardiac myocytes. To date, several positive regulatory elements and their binding factors involved in α-MHC gene regulation have been identified; however, the mechanism restricting the expression of this gene to cardiac myocytes has yet to be elucidated. In this study, we have identified by using sequential deletion mutants of the rat cardiac α-MHC gene a 30-bp purine-rich negative regulatory (PNR) element located in the first intronic region that appeared to be essential for the tissue-specific expression of the α-MHC gene. Removal of this element alone elevated (20- to 30-fold) the expression of the α-MHC gene in cardiac myocyte cultures and in heart muscle directly injected with plasmid DNA. Surprisingly, this deletion also allowed a significant expression of the α-MHC gene in HeLa and other nonmuscle cells, where it is normally inactive. The PNR element required upstream sequences of the α-MHC gene for negative gene regulation. By DNase I footprint analysis of the PNR element, a palindrome of two high-affinity Ets-binding sites (CTTCCCTGGAAG) was identified. Furthermore, by analyses of site-specific base-pair mutation, mobility gel shift competition, and UV cross-linking, two different Ets-like proteins from cardiac and HeLa cell nuclear extracts were found to bind to the PNR motif. Moreover, the activity of the PNR-binding factor was found to be increased two- to threefold in adult rat hearts subjected to pressure overload hypertrophy, where the α-MHC gene is usually suppressed. These data demonstrate that the PNR element plays a dual role, both downregulating the expression of the α-MHC gene in cardiac myocytes and silencing the muscle gene activity in nonmuscle cells. Similar palindromic Ets-binding motifs are found conserved in the α-MHC genes from different species and in other cardiac myocyte-restricted genes. These results are the first to reveal a role of the Ets class of proteins in controlling the tissue-specific expression of a cardiac muscle gene.

Eukaryotic cells have developed an elaborate mechanism to ensure that the expression of genes is tightly regulated, thereby allowing only certain genes to be expressed in response to a particular developmental and/or physiologic signal. This selective expression is controlled primarily by activation of gene-specific transcription factors and their interaction with other ubiquitously expressed factors that allows for both the positive and the negative regulation of the target genes. In the last decade, the field of transcription regulation has advanced rapidly, and the initial role played by positively acting factors has been well characterized. However, the importance of the transcription repression process contributed by the negatively acting factors has been recognized only recently (39, 46, 53, 74). Based on several reports, it is becoming apparent that repression at the transcriptional level could restrict cellular gene expression more stringently. Furthermore, a rapid cellular response to changing requirements could be achieved more efficiently by a decrease in activation in conjunction with active repression than by a single process (for a review, see reference 8). In the case of cardiac myocytes, our understanding of the transcriptional-regulation process is still in its infancy. Several transcription factors have recently been characterized and shown to play a role in cardiac muscle cell gene regulation (reviewed in reference 49). However, in contrast to its close counterpart, the skeletal muscle cells, relatively little is known about transcriptional events that define cardiac cell-specific gene expression. As several of the cardiac muscle genes are also expressed in skeletal muscle cells and are regulated developmentally, it is becoming increasingly clear that both divergent and overlapping pathways between cardiac and skeletal muscle cells might be involved in controlling the muscle gene regulation in these two cell types. Recently, several studies have indicated that unique combinatorial regulatory mechanisms are likely to be involved in controlling cardiac-cell-specific gene regulation (22, 25, 37, 38, 65).

The myosin heavy chain (MHC) gene, which encodes a major protein of the contractile apparatus, has served as a model system with which to analyze pathways leading to cardiac-cell-specific transcriptional regulation. Among the several MHC isoforms encoded by this multigene family, only the α- and β-MHC forms are expressed in the cardiac muscle cell (23, 28). In rodents, during development, MHC transcripts are detected as early as at 7.5 to 8 days of gestation and, as development proceeds in late fetal life, α-MHC is expressed in the atria and β-MHC in the developing ventricles (28). Immediately before birth, the α-MHC starts to appear both in the atria and in the ventricles and becomes a predominant isoform during the adulthood of the animal. As the animal ages, the α-MHC mRNA again becomes suppressed, and β-MHC transcripts predominate (28, 49). This antithetic regulation of α- and β-MHC may be, in part, mechanistically controlled in response to changes in the contractile requirements of the cell (6, 23). Indeed, in transient-transfection assays the increase in the levels of cyclic AMP, thyroid hormone, and contractile-cell activity has been shown to upregulate α-MHC gene expression, and the cis regulatory elements that mediate these effects have also been documented (15, 43, 66). Furthermore, several other DNA elements sufficient to direct a significant level of α-MHC gene expression in cardiac myocytes have been identified. These include binding sites of myocyte-specific enhancer factor 2 (MEF-2), transcription enhancer factor 1 (M-CAT), Egr-1, CArG box, GATA box, and E-box binding sequences (18, 25, 35, 38). These elements bind regulatory factors that are expressed not only in cardiac myocytes but also in other cell types. However, a significant expression of the α-MHC gene remains restricted to atrial and ventricular myocytes and to a muscle region of the lung called the pulmonary myocardium (28, 60). A low level of expression of α-MHC transcripts has also been detected by reverse transcriptase-PCR and immunohistochemistry in certain skeletal muscle fibers, where it could be further induced by low-frequency mechanical stimulation of the muscle (38). Thus, these reports suggest that there must be other regulatory mechanisms involved in the control of a high-level expression of this gene in myocardial cells.

Recently, an Ets (E26-transformation specific or E-twenty-six-specific) family of eukaryotic transcription factors that contain a winged helix-turn-helix DNA-binding domain has been identified (reviewed in reference 71). Proteins of this class are found in animals across the phylogenetic spectrum, from lower eukaryotes to humans. Ets proteins recognize a purine-rich DNA motif centered around the core sequence GGA(A/T) and act both as positive and negative regulators of a wide variety of gene promoters (7, 20, 50, 54, 56, 71). Involvement of Ets proteins has been implicated in many cellular functions, including growth control, cell transformation, development, and apoptosis. A significant role played by the Ets family in hematopoiesis and immune cell lineage development has been fairly well established (see reference 71 and references therein). Furthermore, defects in Ets gene expression have been linked to the phenotype of Down’s syndrome, which is also associated with congenital heart malformation (45, 61). Most members of the Ets family are expressed ubiquitously and in a widely differential tissue-restricted expression pattern that includes the heart (27, 32); however, no Ets target gene has thus far been identified in cardiac muscle.

We report here that the cardiac α-MHC gene is a target of Ets transcription factors. A 30-bp downstream region of the α-MHC gene that contains two palindromic Ets protein-binding sites acts as a strong repressor element for the expression of the gene. The two Ets binding sites are found to be mutually dependent on each other for binding of an Ets-like factor from the cardiac nuclear extract. The mutation of these Ets-binding sites resulted not only in a 20- to 30-fold activation of α-MHC gene expression in cardiac myocyte cultures and in in vivo myocardial tissue directly injected with plasmid DNA but also allowed for the expression of this gene in nonmuscle cells, where it is normally inactive. These results suggest that the cardiac-cell-restricted expression of the α-MHC gene may be controlled in part by an Ets-related repressor protein. Expression of factors binding to the Ets-binding site was developmentally regulated and was elevated in hypertrophic myocardium, where α-MHC mRNA levels are known to be suppressed (6, 29, 40). Among different sarcomeric genes expressed in the cardiac myocytes, one or more copies of Ets-binding sites are found conserved. Because the Ets class of proteins is expressed early in the developing heart tube, demonstration that these proteins play a role in cardiac cell-restricted expression of the α-MHC gene raises the possibility that Ets proteins may also be involved in cardiac muscle cell development.

MATERIALS AND METHODS

Plasmid construction.

Deletion mutants were derived from the plasmid MP1.0CAT containing an HindIII fragment of the α-MHC gene from −612 to +420 bp linked immediately upstream to the chloramphenicol acetyltransferase (CAT) reporter gene in the pGCAT-C vector. The plasmid MP0.67CAT was generated by subcloning a PCR fragment comprising the −612-to-+66-bp region of the α-MHC gene. Internal deletion of the purine-rich negative regulatory (PNR) element was performed in the pMP1.0CAT by use of a two-step PCR procedure. In the first step, PCR was performed with two sets of primers. In one set, the forward primer starting at the −612 bp position of the α-MHC gene that included a HindIII cloning site and the reverse primer (IR) flanked the outer boundaries of the PNR element (G CCG GTG GGA GGA GCC CGT GGG ACA GGT CTG GTG CGGT) at the +46-to-+66- and +96-to-+112-bp positions in the α-MHC gene. The second set of primers was comprised of a forward primer that is complementary to IR (ACC GCA CCA GAC CTG TCG GGC TCC TCC CAC CGG C) and the reverse primer starting at the +420-bp position of the gene and including a PstI cloning site. PCR products of the two reactions were gel purified, annealed, and reamplified with a primer starting at −612 bp as a forward and at the +420-bp position as a reverse primer. The PCR product was digested with HindIII and PstI and subcloned into the HindIII and PstI sites of the pGCAT-C vector. Internal deletion was confirmed by the dideoxy sequencing method. The 5′ deletion mutants were generated with an exonuclease III-mung bean nuclease kit from Stratagene, Inc., by the procedure described by the manufacturer. Briefly, the plasmid MHC-CAT was linearized with SalI digestion, and the flanking ends were filled with thio-deoxynucleoside triphosphate and digested with HindIII. DNA was treated with exonuclease at 18 to 19°C for 5 min, and samples were removed at 1-min intervals, digested with 15 U of mung bean nuclease at 30°C for 30 min, and left overnight at 15°C for ligation. The remaining deletion mutants were generated by PCR by using primers from appropriate sites. Point mutations were generated with a Stratagene Quickchange site-directed mutagenesis kit according to the procedure described by the manufacturer. Each of the deletion mutants was confirmed by dideoxy sequence analysis.

Cell culture and transfection.

Primary myocytes were cultured from 18-day-old fetal rat hearts (15). After differential plating was done to eliminate nonmuscle cells, myocytes were plated at a density of 2 × 10⁶ cells/100-mm-diameter culture dish (Falcon; Becton Dickinson Labware) precoated with 0.1% gelatin in Ham’s F-12 medium (Gibco BRL) with 5% calf serum. Cultures generally consisted of more than 90% myocytes, as measured by immunocytofluorescence with anti-myosin antibody. More than 90% of the cells began to contract spontaneously within 24 h of plating. Nonmuscle cells were grown in growth medium containing Dulbecco’s modified Eagle’s medium (DMEM) (Gibco BRL) supplemented with 10% fetal bovine serum in an atmosphere of 5% CO₂. Sol8 muscle cells were grown in DMEM supplemented with 20% fetal bovine serum, and myogenic differentiation of cells was induced by exposure of confluent cultures to differentiation medium containing DMEM plus 10% horse serum. All culture media contained penicillin (5 mg/ml), streptomycin (5 mg/ml), and neomycin (100 mg/ml).

Primary cultures of cardiac myocytes were transfected after 48 h in culture with 15 μg of DNA/plate by using a lipotaxi reagent (Stratagene) according to the protocol given by the manufacturer. All other cell types were transfected by the CaPO₄ precipitation method. All transfections contained a reporter plasmid (α-MHC/CAT or RSV.CAT) plus 2 μg of pCMV.β-gal as a reference plasmid. After 48 h of transfection, cells were harvested (unless indicated otherwise), the cell lysates were prepared, and the CAT and β-galactosidase assays were performed in the same cell extract (2).

In vivo direct DNA injection into heart muscle.

Plasmids to be assayed for in vivo activity were injected directly into the apex of the adult heart as described previously (5). Male Sprague-Dawley rats weighing 400 g were anesthetized by intramuscular injection of ketamine (150 mg/kg) and xylazine (3 mg/kg). For the heart injection, rats were intubated and artificially ventilated with a Harvard model respirator. A left lateral thoracotomy was performed, the heart was exposed, and the pericardium was removed; 100 to 150 μg of plasmid in 30 μl of saline solution was then injected into the apex of the left ventricle with a 27-gauge needle. Typically, the injection contained 120 μg of test plasmid with the CAT reporter gene and 30 μg of pCMV.βgal reference plasmid. The animals were given penicillin G at 30,000 U/100 g of body weight postoperatively and were allowed to recover for 6 days. The rats were then sacrificed by a pentobarbital overdose. The heart was removed, rinsed with saline, and quick-frozen in liquid N₂. After being ground in liquid N₂ with a mortar and pestle, tissue was homogenized in 1 ml of ice-cold lysis buffer containing 0.1 M Tris (pH 7.5) and 0.01 M MgCl₂ with the proteinase inhibitors aprotinin (2 μg/ml) and phenylmethylsulfonyl fluoride (PMSF; 100 μg/ml). Homogenization was performed in a 5-ml glass tissue grinder (Wheaton) by 10 to 15 strokes or until the tissue resistance was minimal. The homogenate was then centrifuged at 10,000 × g for 10 min at 4°C, and the supernatant was used for assay of CAT and β-galactosidase activities.

Preparation of nuclear extract and electrophoretic mobility gel shift assay (EMSA).

Nuclear extracts were prepared from a pool of 75 to 100 neonatal rat hearts, 1 to 2 gm of adult rat heart tissue, or 8 × 10⁸ to 10 × 10⁸ cells according to a procedure described previously (14, 15). HeLa and Y-79 cell nuclear extracts were purchased from Santa Cruz Biotechnology, Inc. For the EMSA, double-stranded oligonucleotides were 5′ end labeled with T4 polynucleotide kinase (Gibco BRL) and [γ-³²P]ATP (2). The analytical binding reaction was carried out in a total volume of 25 μl containing approximately 10,000 cpm (0.1 to 0.5 ng) of the labeled DNA probe, 2 to 5 μg of the nuclear extract (unless indicated otherwise), and 1 μg of poly(dI-dC) (Sigma) as a nonspecific competitor. The binding buffer consisted of 10 mM Tris-HCl (pH 7.4), 100 mM NaCl, 0.1 mM EGTA, 0.5 mM dithiothreitol, 0.3 mM MgCl₂, 8% glycerol, and 0.5 mM PMSF. After incubation at room temperature for 20 min, the reaction mixtures were loaded onto 5% native polyacrylamide gels and electrophoresis was carried out at 150 V in a 0.5× TBE buffer in a cold room. For competition and antibody experiments, unlabeled competitor DNAs or the antibody were preincubated with nuclear extracts at room temperature for 15 to 20 min in the reaction buffers prior to the addition of labeled DNA probe.

UV cross-linking analysis.

EMSA was performed with labeled probes, the binding reaction of the EMSA was scaled up two times, and multiple identical reactions were run on the same polyacrylamide gel. After electrophoresis, glass plates were opened and wet gel (still attached to one plate) was wrapped in Saran Wrap and exposed to 300 nm of UV irradiation for 1 h (UV transilluminator; Fotodyne Industries) at approximately 7 cm above the gel in a cold room. After UV irradiation, the gel was exposed to autoradiography film overnight, and the regions corresponding to specific shifts were excised from the gel and submerged in Laemmli’s protein sample buffer supplemented with 0.2 M NaCl. The cross-linked DNA-protein complex was eluted from the acrylamide by being crushed with a glass stirring rod, incubated at 37°C for 2 h and at 95°C for 2 min, and then spun through a Schleicher & Schuell Centrex spin filter. The filtrate was resolved on a sodium dodecyl sulfate (SDS)–10% polyacrylamide gel; after SDS-polyacrylamide gel electrophoresis (PAGE), the gel was dried and exposed to Kodak XAR film at −80°C.

DNase I footprint analysis.

DNase I footprinting was performed with the Sure-Track footprinting kit of Pharmacia Biotech, Inc., according to the procedure described by the manufacturer. A 130-bp ³²P-end-labeled α-MHC gene fragment containing purine-rich negative regulatory (PNR) element was amplified by PCR with a 5′-end-labeled forward primer, 5′-TAAGAAGGAGTTTAGCGT-3′, and a cold reverse primer, 5′-ATCCAGTAGAACATCCTG-3′. The labeled probe was incubated with nuclear extracts (20 to 40 μg of protein) in a total 50-μl reaction volume containing poly(dI-dC) and binding buffer as in the EMSA. After incubation at room temperature for 30 min, DNase I digestion was performed with freshly diluted DNase I (1 μg/ml; Bethesda Research Laboratories) for 30 s. The reaction was terminated by adding 50 μl of stopping buffer containing 0.2 M NaCl, 0.02 M EDTA, 1% SDS, and 20 mg of carrier tRNA per ml. The mixture was then subjected to phenol-chloroform extraction and analyzed on an 8% sequencing gel. Standard Maxam-Gilbert (G+A) sequencing reactions were run in parallel to identify the protected sequences (52).

Induction of pressure overload hypertrophy.

Cardiac hypertrophy was induced in male Sprague-Dawley rats (300 to 400 g) by coarctation of the ascending aorta as previously described (see reference 6). Surgical procedures were carried out under pentobarbital anesthesia (30 mg/kg, given intramuscularly), and coarctation was performed by placing a silver clip (0.2-mm internal diameter) around the ascending aorta. Sham controls were operated in a similar manner except for the placement of an aortic clip. At 4 weeks after the operation, the animals were sacrificed by an overdose of pentobarbital; their hearts were then harvested and washed in saline, and the atria were removed. The ventricles were weighed and then quick-frozen in liquid nitrogen until use.

RESULTS

Identification of a strong repressor element that controls tissue-restricted expression of the α-MHC gene.

The role of the first intronic sequences in the expression of the rat cardiac α-MHC gene was evaluated by measurement of the activity of the CAT reporter gene after transfection of plasmids into different cell types and after direct injection into the myocardium. As shown in Fig. 1, expression of ∼1 kb of the α-MHC gene fragment that contains the sequence from −612-bp upstream to +420-bp downstream from the transcription initiation site was observed in the primary cultures of cardiac myocytes, in in vivo heart muscle, and in Sol8 muscle cells but not in nonmuscle cells, a finding consistent with many previous reports (5, 25, 37, 38, 66). However, when downstream sequences were deleted up to the +66-bp position, the resulting construct, MP0.67CAT, had 20- to 30-fold-higher activity than the control plasmid (pMP1.0CAT) in each of the three muscle cell systems analyzed, thus indicating the presence of a strong negative regulatory element in the region between bp +420 and +66 of the α-MHC gene. Sol8 myocytes were used in this study because, as in many previous reports, the expression profile of the α-MHC/CAT reporter gene in these cells was identical to that seen in cultured cardiac myocytes (16, 25). In order to examine whether the tissue specificity of the gene was still retained after removal of intronic sequences, we analyzed the expression of plasmid MP0.67CAT in HeLa and other nonmuscle cells. Surprisingly, the construct MP0.67CAT was highly active in all of the nonmuscle cells tested. Since in many previous studies (35, 37, 38, 66), upstream positive regulatory elements located between bp −340 and −39 have been documented to be sufficient to direct cardiac-muscle-restricted expression of the α-MHC gene, finding the expression of plasmid MP0.67CAT in nonmuscle cells was totally unexpected. This result therefore implies that in conjunction with the upstream positive regulatory elements, a repressor sequence located in the region between bp +66 and +420 of the α-MHC gene is also potentially involved in directing the tissue-restricted expression of the gene.

FIG. 1.

Sequences of the first intronic region control tissue-restricted expression of the α-MHC gene. (A) Schematic representation of the α-MHC gene; the shaded boxes show the positions of exons. (B and C) Configuration of plasmids MP1.0CAT and MP0.67CAT. (D) Expression of the CAT reporter gene from both plasmids after transfection in different cell types or after direct injection of DNA into the myocardium. Relative CAT activities were measured by taking plasmids RSV.CAT and CMV.β-gal as positive controls in cultured cells and in the myocardium, respectively. Bars represent mean values of five separate experiments. (E) Representative CAT assays normalized with β-galactosidase activity in the same cell lysate.

In order to localize the exact position of the cis-regulatory element that participates in the repression of the α-MHC gene activity, we constructed a series of 3′ unidirectional deletion mutants from the +420-bp downstream position of the α-MHC gene, and each construct was tested for CAT activity in primary cultures of cardiac myocytes. The results indicated that a 30-bp fragment located in the first intronic region between +66 and +96 bp, which contained a purine-rich sequence motif, was involved in the repression of α-MHC gene activity (Fig. 2). The role of this sequence motif was further examined by creating an internal deletion mutation of a 30-bp sequence between the positions at +66 and +96 bp in the plasmid MP1.0CAT. As shown in Fig. 3, this internal deletion mutation resulted in a marked activation of the CAT activity in cardiac myocytes as well as in HeLa and other nonmuscle cells. We denote this 30-bp region of the α-MHC gene as a purine-rich negative regulatory (PNR) element. Taken together, these results indicate that, in the absence of the PNR element, the upstream sequences within the −612-bp region of the α-MHC gene are also active in the heterologous system.

FIG. 2.

Localization of the negative cis-regulatory element in the first intronic region of the α-MHC gene. (A) Expression of different 3′ deletion mutants of the α-MHC gene in primary cultures of cardiac myocytes. Bars represent the mean value of seven different experiments. (B) Diagram of the α-MHC gene sequences from bp −340 to +117. The regulatory sequences are shown within the labeled box. The positions of the first and second exons are illustrated by black boxes. (C) Sequences of the first exon of the α-MHC gene, which are slightly different from previously published sequences (30).

FIG. 3.

Effect of the PNR element placed in different positions on the α-MHC promoter and on a heterologous promoter-reporter gene. Transient-expression analysis of different CAT reporter constructs was analyzed in different cell types (as indicated). (A) Expression of the CAT reporter gene from constructs with an internal deletion of the PNR element or when the PNR element was cloned at either the 5′ or the 3′ end of the α-MHC gene fragment in the plasmid MP0.67CAT. (B) Effect of the PNR element on the basal promoter activity of the Egr-1/CAT gene. Each bar represents the mean value of five different experiments.

The position dependency of the PNR element in regulating α-MHC gene activity was examined next. A construct was generated in which a 30-bp PNR sequence cassette was cloned at the 5′ end of the α-MHC gene fragment in the plasmid MP0.67CAT, and the expression of the resulting construct, pMPN0.67CAT, was analyzed in both muscle and nonmuscle cells. No noticeable difference in the CAT activity was observed between plasmids MP0.67CAT (which has no PNR element) and MPN0.67CAT (which has a PNR element at the 5′ end of the gene) in cardiac myocytes as well as in Sol8 muscle cells (not shown), indicating that the repressor activity of the PNR element is position dependent (Fig. 3A). However, the presence of the PNR element in the plasmid at the 5′ end of the α-MHC gene was able to suppress the α-MHC/CAT activity in HeLa cells significantly, albeit to a lesser extent than when it was present at the 3′ end of the α-MHC gene fragment. Similar results were obtained when expression of these plasmids was examined in two other nonmuscle cell lines, JEG and NIH 3T3 cells (Fig. 3A). Thus, these data demonstrate that the PNR element acts differently in the two cell types, being position dependent in cardiac and Sol8 myocytes but position independent (which could define it as a silencer [8]) in HeLa and other nonmuscle cells.

To evaluate the mechanism of the transcription repression effect exerted by the PNR element, we first tested whether the PNR sequence could modify the activity of a basal transcription complex. On a minimum heterologous promoter, the Egr-1 minimum promoter-reporter gene, the PNR element was cloned at either end of the Egr-1 gene fragment, and the expression of constructs was tested in cardiac myocytes and HeLa cells. As shown in Fig. 3B, the presence of the PNR sequence did not change the basal expression of the plasmid in either of the cell types tested. A similar result was obtained when the PNR element was cloned to thymidine kinase or simian virus 40 heterologous minimum promoter-reporter genes (data not shown). These results suggest that the repressor protein(s) bound to the PNR sequence does not directly alter the activity of the basal transcription machinery. This observation led us to examine another possibility: whether the PNR sequence could act by interfering with the activity of an upstream positive regulatory element. In order to localize an upstream DNA sequence that might be a target for the PNR-bound repressor protein(s), we constructed progressive 5′ deletion mutants of the α-MHC/CAT reporter gene, with or without the presence of the PNR element, and analyzed for the expression of the reporter gene in the myocardium and in Sol8 myocytes. As shown in Fig. 4, although 5′ progressive deletions up to the −130 bp eliminated several known positive regulatory elements, the presence of the PNR element at the 3′ end of the gene significantly reduced the expression from each of the constructs analyzed. Further deletion up to the position at −74 bp gave rise to the α-MHC gene minimum promoter region. These sequences are sufficient to drive the expression of the reporter plasmid (pMPminCAT) in the myocardium and Sol8 muscle cells significantly above the control (promoterless) plasmid, an observation previously reported by us as well as by others (15, 38). Cloning of the PNR element to the α-MHC gene minimum promoter fragment (−74 to +66 bp) at either end of the gene produced no significant effect (Fig. 4), thus further confirming that the PNR-repressor does not directly affect the activity of the basal promoter sequences. These data indicate that the positive regulatory sequences within the region from −130 to −74 bp of the α-MHC gene are a target for the repressor activity of the PNR element. Interestingly, within this region at least three different regulatory sequences are present: a thyroid response element (from −126 to −111), an Ets (from −100 to −97), and an AP-2-like binding site (from −93 to −85) (see Fig. 2B). Future studies with point mutations in each site will be required to establish the exact location of the regulatory sequence that is being interfered with by the PNR-bound repressor protein.

FIG. 4.

Identification of upstream DNA sequences of the α-MHC gene required for the repressor activity of the PNR element. Transient-expression analysis of different 5′ progressive deletion mutants of the α-MHC gene with or without the PNR element was carried out by direct injection of DNA into the heart muscle. Expression of pCMV.β-gal in the same heart was used as a reference control. Bars represent the means ± standard errors from four separate injections. Open bars, no PNR element; solid bars, PNR element at the 3′ end of the α-MHC gene; shaded bars, PNR element at the 5′ end of the α-MHC gene fragment.

Characterization of nuclear factor(s) binding to the PNR element.

To determine the nuclear factor(s) that binds to the PNR element, we carried out an EMSA with a +66- to +96-bp oligonucleotide as a labeled probe. Since these sequences were active as a repressor element regardless of cell type, nuclear extracts from both muscle and nonmuscle cells were examined for the factor binding to the PNR element. As shown in Fig. 5, a distinct difference in the gel mobility of complexes generated from muscle and nonmuscle cell nuclear extracts was observed. While a relatively faster migrating complex was generated from neonatal and adult rat hearts, as well as from Sol8 muscle cell nuclear extracts, two slow-migrating complexes were formed from the different nonmuscle cell nuclear extracts tested. It should be mentioned that the muscle and nonmuscle cell (JEG) nuclear extracts were prepared by using the same cocktail of proteinase inhibitors (leupeptin, pepstatin, antipain, aprotinin, and PMSF); therefore, a difference in the gel mobilities of the complexes could not be due to proteolytic events occurring during preparation. Among the hearts at different developmental stages, a dual complex was generated from fetal (not shown) and neonatal hearts, whereas a single complex was formed with adult rat heart nuclear extract. With the Sol8 muscle cell nuclear extract, a complex with higher intensity but with a gel mobility identical to that of the adult rat heart complex was observed. Addition of excess cold PNR oligonucleotide successfully competed for each of these complexes, thus documenting their specificity.

FIG. 5.

Different gel mobility PNR complexes formed with muscle and nonmuscle cell nuclear extracts. The end-labeled α-MHC PNR oligonucleotide was incubated with 4 μg of nuclear extract (N.E.) from different sources. DNA-protein complex formation was analyzed on a 5% polyacrylamide gel. Competitor: self, same as probe (50×).

To characterize a factor binding to the PNR oligonucleotide, we carried out a series of EMSAs with different oligonucleotides as competitors or as labeled probes containing different protein binding sites (Fig. 6). Given some base pair similarity with the PNR element, we first selected an oligonucleotide corresponding to the troponin-C gene MEF-3 binding site (19) as a competitor in the gel mobility shift assay. However, no competition occurred with this oligonucleotide for the PNR complex formation (Fig. 6B). Next, we carried out a competition assay using oligonucleotides corresponding to the Ets protein binding sites of polyomavirus and stromelysin genes (27, 72). Polyomavirus Ets-binding site oligonucleotide that has a single Ets binding site could compete only partially, even at a 200- to 500-fold molar excess of the probe, whereas stromelysin Ets-binding site oligonucleotide, which has two Ets binding sites on opposite strands as inverted repeats, successfully inhibited PNR DNA-protein complex formation. Furthermore, when a reverse experiment was carried out in which polyomavirus Ets-binding site oligonucleotide was used as a labeled probe, the formation of a complex with this probe was inhibited completely by a 50-fold molar excess of the PNR oligonucleotide (Fig. 6B). Thus, these results suggest that an Ets class of factor is interacting with the α-MHC PNR motif.

FIG. 6.

Factor(s) binding to polyomavirus Ets-binding site is recognized by the α-MHC gene PNR motif. (A) Sense strand sequence of double-stranded oligonucleotides used in this study. The Ets motif is underlined. Nucleotides in lowercase letters indicate a mutation from the wild-type oligonucleotide. (B) EMSAs were performed with different end-labeled probes and neonatal rat heart nuclear extract (N.E.). The increasing molar excess of unlabeled competitor oligonucleotide is 200× and 500×. Competitors: self, same as probe; p.Ets, polyomavirus Ets-binding site; S.Ets, stromelysin Ets-binding site; MEF-3, troponin-C MEF-3 site; p.Ets-mt., mutation in the polyomavirus Ets site; and S.Ets-mt., mutation in the stromelysin Ets-binding site.

By inspection of the PNR element, three potential Ets-binding sites were recognized: two at the 3′ end of the oligonucleotide (N-1 and N-2) containing a palindrome separated by just two nucleotides, and the third (N-0), a single Ets-binding site toward the 5′ end of the oligonucleotide (see Fig. 8). To identify the precise nucleotides within the PNR element that are involved in DNA-protein interaction, DNase I footprinting analysis was performed with a 130-bp fragment of the α-MHC gene (+35 to +165 bp) that contains the PNR motif and the neonatal rat heart and Sol8 muscle cell nuclear extracts (Fig. 7). Three protected regions (Fig. 7, regions A, B, and C) and one hypersensitive site were detected. Protected regions A and B, spanning the area between +76 and +82 bp, are located within the PNR element, whereas region C is located downstream from the PNR element. Regions A and B contain a palindrome with two Ets-binding sites (N-1 and N-2) with a common sequence motif, GGAAG. In addition, region B also contains a part of the N-0 site of the PNR element. Similar sequence motifs were also identified in the regulatory regions of other sarcomeric genes expressed in cardiac myocytes, thus implying that this motif may play a conserved role in gene transcriptional regulation.

FIG. 8.

Both Ets-binding sites of the palindrome are required for cardiac nuclear factor binding but not for the HeLa cell nuclear factor interaction to the PNR element. (A) Ets-binding sites of the PNR element point mutated in the plasmid MP0.15 CAT (containing the α-MHC gene fragment stretching from 156 bp upstream to 420 bp downstream) and the expression from each construct as determined after transfection into primary cultures of cardiac myocytes and in HeLa cells. (B and C) EMSAs were performed with α-MHC PNR (B) or polyomavirus Ets (C) oligonucleotides used as labeled probes and nuclear extracts (N.E.) from different sources as indicated above each gel. The molar excess of unlabeled competitor oligonucleotide is given above each lane.

FIG. 7.

A palindrome of two Ets-binding sites in the PNR element is protected by DNase I footprinting analysis. A 130-bp ³²P-end-labeled α-MHC gene fragment containing the PNR element was obtained by PCR, incubated with increasing concentrations (10 and 40 μg) of nuclear extracts from neonatal rat heart and Sol8 muscle cells, and then subjected to partial digestion with DNase I (1 μg/ml) as described in Materials and Methods. Lane G+A represents the Maxam-Gilbert sequencing reaction. Lane 0 shows free DNA cleaved with DNase I. The boundaries of the PNR element are on the left, and the sequences of the protected regions A and B are on the right. EBS, Ets-binding site.

To strengthen further the importance of these sequences in gene regulation, we created clustered point mutations within each site. The resulting constructs and the corresponding mutated oligonucleotides were analyzed for CAT activity and for DNA-protein interaction, respectively. As shown in Fig. 8A, three point mutations in either region A (Nmt-1) or B (Nmt-2) resulted in 10- to 12-fold activation of the CAT expression compared to the control plasmid in cardiac myocytes, suggesting that both palindromic Ets-binding sites of the plasmid are equially important in negative regulation of the gene in these cells. However, in HeLa cells, activation of the α-MHC/CAT plasmid could be seen only when both N-1 and N-2 sites were mutated in the same plasmid, indicating that a single Ets-binding site was capable of suppressing the α-MHC gene expression in nonmuscle cells. This observation was also supported by the results obtained from the mobility gel shift competition assay. As shown in Fig. 8B, the PNR complex formed with the neonatal or adult rat heart nuclear extracts was abolished by an excess of oligonucleotide that has two Ets-binding sites but not when either site was mutated (Nmt-1 or Nmt-2). On the other hand, the PNR complex generated by HeLa cell nuclear extract was effectively inhibited by an excess of both Nmt-1 and Nmt-2 oligonucleotides (Fig. 8B). Similarly, a complex generated by the polyomavirus Ets-binding site probe was found to be abolished by the Nmt-1 and Nmt-2 oligonucleotides but not by the Nmt-3 oligonucleotide, which has mutations at both sites of the palindrome (Fig. 8C). These results demonstrate that although a single Ets-binding site (N-1 and N-2) of the palindrome is capable of recognizing a nuclear Ets-like factor from HeLa cells, the cardiac nuclear factor requires both Ets sites of the palindrome for DNA-protein interaction. A similar palindrome composed of two Ets binding sites has also been identified in the enhancer region of the GATA-1 gene, where the spacing between the two Ets-binding sites has been shown to be crucial for the binding of the factor to DNA (20, 56). It is interesting to note that there is also a difference in the gel mobilities of complexes between the HeLa cell and cardiac muscle cell nuclear extracts with the polyomavirus Ets-binding site probe (Fig. 8C), as it was with the α-MHC PNR probe. Thus, collectively these data suggest that different Ets proteins present in HeLa and cardiac muscle cell nuclear extracts are being recognized by the α-MHC PNR element.

To characterize further a factor binding to the PNR sequences, we carried out a gel mobility shift assay in which nuclear extract was preincubated with different antibodies raised against different Ets proteins. As shown in Fig. 9A, preincubation of cardiac nuclear extract with an antibody against ERP protein (27) greatly inhibited PNR complex formation in a concentration-dependent manner but not the troponin-T M-CAT complex, which served as a negative control (Fig. 9B). The HeLa cell nuclear extract-PNR complex was also abolished by the anti-ERP antibody (data not shown). We also tested the ability of the PNR element to bind to the in vitro-synthesized ERP protein, and the results showed that the ERP protein is recognized by these sequences and that this binding is abolished by the ERP antibody, thus confirming the reactivity of the antibody to its protein (Fig. 9C). A slow-migrating complex also occurred with the ERP antibody (Fig. 9A to C), which appeared to be nonspecific since a complex of similar mobility was also formed between the probe and ERP anti-sera (Fig. 9A). In this experiment we also tested antibodies against Ets-1/Ets-2, PEA-3, and Elk-1 proteins (Santa Cruz Biotechnology), and these did not alter the PNR DNA-protein complex generated by the cardiac nuclear extract (Fig. 9D). With the Ets-1/Ets-2 antibody, although the specific PNR complex remained unchanged, a slower-migrating complex became evident at a higher concentration of the antibody (Fig. 9D). However, the Ets-1/Ets-2 antibody itself showed no DNA binding activity (not shown). Because an antibody against Ets-1 protein has been shown previously to induce a stable Ets complex (57), we conclude that the newer (slower) complex is an antibody-induced Ets complex, which does not normally bind to the PNR element. As a positive control, the reactivity of each of the PEA-3 and Elk-1 antibodies to specific Ets protein was tested by either EMSA or Western blot analysis. As shown in Fig. 9E, the PEA-3 antibody used in this experiment has the ability to produce a specific supershift of the Ets complex generated by an oligonucleotide corresponding to PEA-3 recognition sequences. In addition, in the Western blot analysis the Elk-1 antibody was found to interact with a commercially available Elk-1 peptide (Santa Cruz Biotechnology), as well as with a protein present in the cardiac nuclear extract, but not with proteins eluted from the PNR-DNA protein complex (Fig. 9F), thus indicating that although Elk-1 protein is present in the heart nuclear extract it is not a part of the PNR complex. Together, these data demonstrate that a factor immunologically related to an ERP protein is a part of the PNR complex but not of the Ets-1/Ets-2, PEA-3, or Elk-1 proteins.

FIG. 9.

An ERP-related protein is a part of the PNR-protein complex. (A and B) Cardiac nuclear extract was preincubated with the anti-ERP antibody or preimmune serum, and EMSA was performed by using PNR or troponin-T M-CAT oligonucleotide (14) as a labeled probe. (C) EMSA was carried out with 2 μg of ERP protein. (D) EMSA was done with cardiac nuclear extract preincubated with Ets-1/Ets-2, PEA-3, or Elk-1 antibodies (4 μl each). (E) Cardiac nuclear extract was incubated with PEA-3 antibody (3 μl), and EMSA was carried out with an oligonucleotide corresponding to the PEA-3 binding site (5′-GATCTCGAGCAGGAAGTTCGA-3′; Santa Cruz Biotechnology). (F) Western blot analysis. Cardiac nuclear extract (10 μg), Elk-1 protein (1 μg), or proteins eluted from the PNR complex were subjected to SDS-PAGE and transfered to polyvinylidene difluoride membrane. Western blot analysis was performed with 1,000-fold-diluted anti-Elk-1 antibody and 2,000-fold-diluted horseradish peroxidase-labeled anti-rabbit antiserum. Solid arrow, a slow-migrating, nonspecific complex; broken arrow, a specific supershifted band.

In order to find out the molecular weight of the protein binding to the PNR sequence, we performed UV cross-linking analysis. In this experiment, the DNA-protein complex generated with the labeled PNR oligonucleotide was separated from the free probe by the gel mobility shift assay, and the gel was exposed to UV irradiation. Cross-linked DNA-protein complex was eluted from acrylamide and resolved by SDS-PAGE. In the case of nonmuscle cell nuclear extracts, for the two complexes generated in the mobility gel shift assay, the upper band was analyzed in the UV cross-linking analysis. As shown in Fig. 10, bands of different molecular weight were observed with the muscle and nonmuscle cell nuclear extracts. Whereas an identical 50- to 55-kDa band was obtained from nuclear extracts of the neonatal and adult rat hearts as well as the Sol8 muscle cells, a much higher band of ∼85 kDa was detected with HeLa and Y-79 cell nuclear extracts. These results strongly suggest that proteins with different molecular weights present in the muscle and nonmuscle cells bind to the α-MHC gene PNR motif.

FIG. 10.

UV cross-linking analysis: proteins of different molecular sizes from muscle and nonmuscle cell nuclear extracts bind to the PNR motif. DNA-protein complexes formed with the end-labeled oligonucleotide and different nuclear extracts were separated from the free probe by EMSA, and the wet gel was exposed to 300 nm of UV irradiation for 1 h. Cross-linked DNA-protein complex was eluted from the acrylamide gel as described in Materials and Methods and was resolved by SDS–12% PAGE. After being dried, the gel was exposed to Kodak X-ray film for 1 week.

PNR-binding factor(s) is upregulated in the hypertrophied myocardium.

Because the levels of the α-MHC transcripts are known to be downregulated in the pressure overload hypertrophied myocardium, we explored the possibility of whether the activity of the PNR-binding factor could be changed in the heart in response to hypertrophy signals. An acute pressure overload was induced by aortic coarctation, and nuclear extracts were prepared from the ventricles of the hearts harvested 4 weeks after the operation. Only hearts with a weight that was at least 1.3 times higher than that of the average control hearts were used to prepare nuclear extract. Five hearts each from sham-operated and coarcted animals were used for further experiments. Each heart was used separately to prepare nuclear extract; thus, five nuclear extract preparations from controls and five from pressure overload hypertrophied hearts were obtained. A gel mobility shift assay was performed to determine the levels of activity of PNR-binding factor(s) in the two groups. In order to avoid any possible pipetting errors, different concentrations of total proteins from the two groups of hearts were used. An oligonucleotide corresponding to an E-box binding site (BF-2) of the α-MHC gene was used as a negative control, since factors binding to this site were shown previously to be unchanged by pressure overload hypertrophy (37). As seen in Fig. 11, a hypertrophied heart that was 30% larger by LV/BV ratio had a two- to threefold higher activity of the factor binding to the PNR oligonucleotide compared to the control. However, no change was observed in the binding activity of the factor recognized by the BF-2 probe. These experiments were repeated with all five nuclear extract preparations from control and hypertrophied hearts, and similar results were obtained. These data demonstrate that the activity of the factor(s) binding to the PNR motif is upregulated in response to pressure overload hypertrophy. Furthermore, to demonstrate a physiological relevance of the PNR element in hypertrophic myocytes, we analyzed expression of the α-MHC/CAT reporter plasmid in the norepinephrine (NE)-induced hypertrophy of cultured cardiac myocytes (Fig. 11E). Cardiac myocytes were plated at a density 2 × 10⁶ cells/100-mm-diameter dish and transfected with either plasmid MP1.0CAT or MP0.67CAT. Cultures were treated with 4 μM NE for induction of hypertrophy, and parallel untreated plates were used as a control. All cultures received 4 μM of propranolol to block cell β-adrenoceptors. After 72 h of NE treatment cells were examined and only those which showed near doubling in the size of cardiac myocytes were considered to be hypertrophied cells and used for further experiments. As shown in Fig. 11E, in hypertrophic cells the expression of the pMP1.0CAT was found to be repressed by almost 50%, a finding consistent with a previous report (3). However, no change in the expression of the pMP0.67CAT, which is devoid of the PNR element, was observed between hypertrophic and control cells. These results document a role of the PNR element in the downregulation of the α-MHC gene expression during hypertrophy of cardiac myocytes.

FIG. 11.

The activity of PNR-binding factor(s) is upregulated in the hypertrophied heart. An acute pressure overload (PO) was induced by aortic coarctation as described in Materials and Methods, and nuclear extracts obtained from the sham-operated (Sham) and hypertrophied (PO) hearts were used for the EMSAs. (A) Measurement of left ventricular weight (LV) and body weight (BW) of rats subjected to sham operation and aortic coarctation. (B) EMSA was performed with PNR oligonucleotide as a labeled probe and cardiac nuclear extracts from the same rats as in panel A. (C) A probe corresponding to the α-MHC BF-2 site (Fig. 2B) was used as a negative control in the EMSA. (D) Specific DNA-protein complexes obtained from the EMSA in panel B were cut out from the gel, counted for radioactivity, and plotted as a function of the total protein used in the EMSA binding reaction. (E) Primary cultures of cardiac myocytes were transfected with pMP1.0CAT or pMP0.67CAT and treated with 4 μl of NE to induce hypertrophy. After 72 h of NE treatment, the cells were harvested and the CAT activity was measured. Bars represent the mean values of five separate experiments.

DISCUSSION

A significant expression of the rat cardiac α-MHC gene remains restricted to cardiac myocytes. In recent years, several positive regulatory elements and their cognate binding factors, such as TEF-1, GATA-4, MEF-2, SRF, and TR, which are involved in the transcriptional regulation of the α-MHC gene in different pathophysiologic states of the heart have been identified (49). However, none of these factors are restricted to cardiac myocytes, indicating that some other mechanism(s) must be involved that directs cardiac tissue-restricted expression of this gene. In this study we have shown that a 30-bp sequence, PNR, in the first intronic region containing a palindrome with two high-affinity Ets-binding sites acts as a strong negative regulatory element for the expression of the α-MHC gene in cardiac myocytes in vitro and in vivo, as well as in Sol8 muscle cells. More importantly, we have found that deletion of the same 30-bp intronic region (PNR) enables the α-MHC/CAT constructs to be expressed at a significant level in nonmuscle cells, where it is normally inactive, thus documenting an essential role of this repressor element in controlling the tissue-restricted expression of the α-MHC gene in cardiac myocytes.

Although a factor(s) binding to the PNR element was found to be expressed both in muscle and nonmuscle cells, at least four different lines of evidence presented in this study indicate that PNR-binding factors from the two cell types are not identical. (i) In the gel mobility shift assay, an obvious difference in the gel mobilities of complexes formed with myocyte and nonmyocyte nuclear extracts was observed. (ii) In the competition assay, although two inverted repeats of the Ets-binding sites were found to be necessary for the myocyte nuclear factor to bind to the PNR element, the HeLa cell nuclear factor could bind effectively to a single Ets-binding site of the palindrome. (iii) By UV cross-linking analysis, the molecular weight of the protein binding to the PNR element from muscle and nonmuscle cells was apparently different. (iv) In the functional analysis, while a position-dependent effect of the PNR element was observed in cardiac myocytes, in HeLa cells it was capable of repressing gene activity when present either at the upstream or at the downstream position of the α-MHC gene. Thus, these findings indicate that different functions of the PNR element in the two cell types reflect its binding to different factors. However, it should be noted that a single PNR binding factor from each nuclear extract as examined by the UV cross-linking analysis may or may not be a repressor; rather, it may require other interacting factors to constitute a cell-specific repressor complex.

Role of the PNR element in the tissue-specific expression of the α-MHC gene.

The PNR element is located in the middle of the first intron, almost 50 bp from the 5′ (donor) and 498 bp from the 3′ (acceptor) splicing sites (Fig. 2B); therefore, it is unlikely that removal of the PNR element would have affected RNA splicing. This notion was also supported by the fact that merely a three-point substitution mutation in one Ets-binding site of the palindrome was capable of activating α-MHC/CAT expression in cardiac myocytes (Fig. 8), thus revealing a negative regulatory role of the PNR element. There are several examples in which first intronic sequences of muscle genes have been found to contain functionally significant sequence domains that are required for tissue- and differentiation-specific gene expression (10, 59). In our attempts to define nucleotide sequences of the PNR element in other genes, we found that one or more copies of Ets-binding sites are present in the promoter region of almost every cardiac myocyte-expressed gene we analyzed. Furthermore, in the α-MHC genes of different species, such as mouse, rabbit, Syrian hamster, and humans, an identical palindrome of two Ets-binding sites was found to be conserved in the first intronic region of the genes (Table 1), thus documenting a crucial role of these sequences in gene transcriptional regulation.

TABLE 1.

Ets-binding sites in different genes expressed in cardiac myocytes

Source and genea	Sequence	Reference
r, α-MHC	+80 TGTC.TTCCCT.GGAAGT.GGGGCT.CCTCCC	This study
r, α-MHC	−109 CAGA.CAGGAG.GGATGG.GAGGGA.GGGTCC	30
m, α-MHC	+86 TGCC.TTTCCT.GGAAGT.GGGGTT.CAGGCC.GGTC	13
m, α-MHC	−102 AGGA.CAGGAG.GGAAGT.GGGAGG.GAGGGT.CC	13
rb, α-MHC (AS)	+57 TCCC.TGGGAC.GGAAGG.ACCCTC.ACTCGT.CGT	9a
rb, α-MHC	+106 GGGT.TCTGCT.GGAAGC.GCCCTT.CTCCAG.CC	9a
rb, α-MHC	−88 GGGT.CCCTCC.GGAAGG.GCTCCA.AATTTA.GG	9a
h, α-MHC	+149 CCAG.GCCCAG.GGAAGT.TCCCCC.TGACAC.AGGA	62
h, α-MHC	−95 AGGG.TCCTCC.GGAAGG.ACTCCA.AATTTA.GA	62
sh, α-MHC	+86 TGCC.TTCCCT.GGAAGT.GGGGCT.TAGGGC.CGTC	69
sh, α-MHC	−311 GGCC.ATGTGG.GGAAGG.GAGGTG.GCGTGC.TATG	69
r, β-MHC	−147 TTCG.GACAAG.GGAAGG.GGGGGA.GAGTT	65
r, β-MHC (AS)	−174 GGAA.ACAATT.GGAAGT.GGGCGT.CATTGT.TA	65
m, β-MHC	+81 TCCA.GGTTCA.GGAAGT.AATTCC.TCTAGA.ACA	48
m, β-MHC (AS)	−175 GGAA.ACAATT.GGAAGT.GGTCGT.CATTGT.TGT	48
h, β-MHC	−147 GCAC.TGTTTG.GGAAGG.GGGGGA.GCCTCG	26
h, β-MHC (AS)	−177 GGAA.ACAATT.GGAAGT.GGTCGT.CATTGT.TA	26
rb, β-MHC (AS)	−162 GGAA.ACAATT.GGAAGT.GGTCGT.CATTGT.TA	58
rb, β-MHC	+63 TCCC.AGGTTA.GGAAGG.GGCTCC.CCCAGG.AACA	9a
r, MLC-2	−47 TTAA.CCCCAG.GGAAGA.GGTATT.TATTGT.TCCAA	18
r, MLC-2 (AS)	−272 AAGT.AACCCA.GGAAGG.GGAGGG.GGGAGG.AAGA	18
c, MLC-2	−257 CCAG.CAGAGG.GGAAGA.GCACAG.CCTCTG.CCCAC	57
c, C-TnT	−167 CTCC.TGTGGG.GGAAGG.GGGAGC.ACGGAG.GGGG	22
m, C-Tnc	+142 GGTA.CACTAG.GGAAGT.GATGGG.GGACTC.AAAA	44
m, C-Tnc	−645 ACCT.TGCATA.GGAAGT.GTTTCT.TGGCAG.GACTT	44
m, C-TnI	−833 TTCC.TTTCCT.GGAACT.CTCGGT.TGTACT.ATTTT	1
m, C-TnI	−250 TATG.AACATG.GGAAGC.TGAAGT.CTAGAC.T	1
r, ANF	−289 CGAG.CGCCCA.GGAAGA.TAACCA.AGGACT.CTTTT	55
r, ANF	−1139 ATGT.GGGTAT.GGAAGT.CTTCCA.ATAGCC.CATA	55
m, MCK (AS)	−251 CCCT.TCGCCG.GGAACA.TGGAAC.AGTAAT.AC	59
m, MCK	−221 ACTT.AGTTTA.GGAACC.AGTGAG.CAAGTC.AG	59
sh, MHC	−128 AAAA.ATACGT.GGAAGG.GGCCAG.TTCTCA.GCTT	33
sh, MHC	−317 CAGG.GTGGGA.GGAACT.GCAGGA.GTCAAG.GCAG	33
q, slow MHC-3	−395 GCAC.AGTGTG.GGAACT.ATGGGG.CAGAGG.CTT	41
q, slow MHC-3	−324 GGGC.ACAACG.GGAACT.GGGGGG.CAGAGG.G	41
m, C-α-actin (AS)	−386 GTCA.CATGGC.GGAAGA.CTTGGC.GCCCTG.CCCT	34
h, C-α-actin (AS)	−266 AGGG.GGCAGG.GGAAGA.CTAAGT.GACGCC.AGC	34
c, C-α-actin (AS)	−258 GGTC.CCTCGA.GGAAGT.GTAAGC.CAGG	11
m, Sk-α-actin	−339 GTAA.ATCTTG.GGAAGT.ACAGAC.CAGCGG.TCA	21
m, Sk-α-actin	−92 ATAT.GGCTTG.GGAAGG.GCAGCA.ACATTC.TTC	21
r, Sk-α-actin	−90 ATAT.GGCTTG.GGAAGG.GCAACA.ACATTC.CT	21
c, Sk-α-actin (AS)	−46 GCCC.GCGACA.GGAATG.CGACCC.CGG	21
Ets-binding core sequence	GGA (A/T)

^aSources: r, rat; m, mouse; h, human; sh, Syrian hamster; rb, rabbit; q, quail; c, chicken. AS, antisense DNA. Sequences similar to the rat α-MHC gene Ets-binding site are shown boldface and other, adjacent sites are underlined. 

The data presented here are consistent with a previous study in which Cribbs et al. (9) showed that a rabbit α-MHC/CAT reporter gene containing a fragment from −412 to +48 bp of the rabbit α-MHC gene was expressed to a significant level in both myocytes and HeLa cells. As shown in Table 1, the first intronic sequences of the rabbit α-MHC gene possess two perfect inverted repeats of Ets-binding sites located immediately downstream of the +48-bp position of the gene that were lacking in the promoter-reporter constructs previously analyzed by Cribbs et al. (9). Thus, in light of the results presented here, it seemed reasonable to speculate that the lack of tissue specificity of the rabbit α-MHC/CAT gene might have been due to the absence of the first intronic Ets-binding sites of the gene. However, it remains to be proven formally whether, by inclusion of the Ets-binding sites, the tissue specificity of the rabbit α-MHC/CAT construct could be restored. The importance of first intronic sequences in cardiac α-MHC gene regulation has often been ignored in many other earlier studies examining cis-regulatory sequences in different model systems. In one study, Buttrick et al. (5), who used direct injection of DNA into the myocardium, reported that the α-MHC/CAT construct having a fragment of the gene from −612 to +32 bp had an almost 30-fold-higher activity compared to the construct that had gene sequences of −1,696 to +420 bp. The higher activity of the fragment from −612 to +32 bp was interpreted as being due to the presence of a strong negative regulatory element in the region between bp −1696 and −612 of the gene; however, the differences between the downstream sequences of the α-MHC gene in the two constructs were not accounted for. Other studies examining the expression of α-MHC/CAT plasmids containing either a −1696-to-+420- or a −612-to-+420-bp fragment of the α-MHC gene in primary cultures of cardiac myocytes did not show a significant difference in the CAT expression from these two plasmids (16, 66). Although, there is a difference in the model system utilized in the above two studies (i.e., in vivo versus in vitro), our data presented argue that a strong expression of the −612-to-+32-bp construct might have been due to the lack of the PNR element in this region. Furthermore, identification of a strong negative regulatory element in the first intronic region of the gene could also explain, at least partly, why the −138-to-+1071-bp α-MHC/CAT in the mouse transgene was found to be inactive (60), while constructs with shorter intronic sequences, e.g., −161 to +32 and −86 to +32 bp (lacking the PNR element), were found to be significantly active when DNA was injected directly into the myocardium (5, 38).

Tissue-specific expression of several genes, such as neuron-specific type II sodium channel and SCG10 genes (39), the α-chain of the T-cell receptor, and the immunoglobulin K genes (46, 74), have been shown to be controlled by repressor (silencer) elements. In many of these instances, tissue specificity is achieved by a restricted expression of the silencer binding factor in cells where the gene is usually not expressed. However, for the α-MHC gene, based on the criteria discussed above, the PNR element appears to have dual functions: it acts as a negative gene regulator in the homologous cell context (muscle cells), and it acts as a silencer in the heterologous system (nonmuscle cells). Based on studies from different tissues, at least two main modes of transcriptional repression by a negative regulatory element have been described: a passive and an active one (8). In the passive mode of repression, repressor-protein may downregulate the activity of one or more positive activating factors by either competing for their DNA-binding site or interacting with a positive activator, thereby reducing their DNA-binding activity or transcriptional activation ability. On the other hand, an active repressor possesses an intrinsic repressing activity and apparently inhibits the activity of the basal transcription complex directly. In our study, the α-MHC PNR element did not alter the activity of the basal promoter-reporter construct and required upstream regulatory sites for gene repression, thus suggesting that it acts through a passive mode of repression. As measured by EMSA, the amount of the repressor in cardiac myocytes seems to be much higher than that in the nonmuscle cells (Fig. 5). Yet the α-MHC promoter reporter gene is expressed in cardiac myocytes but not in the nonmuscle cells, and even when the PNR element is deleted the gene induction is much greater in cardiac myocytes than in the nonmuscle cells. Hence, it appears that, aside from the issue concerning the difference in the nature of repressors in the two cell types, a deficit of cardiac muscle gene activators in nonmuscle cells also accounts for the tissue-restricted expression of the α-MHC gene.

Deletion analysis of the upstream sequences of the α-MHC gene has revealed that the elements located in the proximal promoter region of the gene located between −130 and −74 bp are required for the negative regulatory activity of the PNR element. Within this region, there are several conserved regulatory elements, including an Ets-binding site present in the α-MHC genes of different species (Fig. 2 and Table 1). Although the mechanism of the PNR element-mediated gene repression remains to be determined, the presence of multiple Ets-binding sites as a palindrome at the downstream position of the α-MHC gene and the involvement of the upstream sites would indicate that it might be a requirement for the physical hindrance and/or conformational rigidity needed for the negative control of gene transcription initiation. Similarly, other studies have shown a requirement of multiple sites for silencing the myosin light chain-2, collagen-II, and vimentin gene expression (10, 12, 53).

An Ets protein binds to the PNR element.

Data obtained from base pair mutation analysis, gel mobility shift competition assay, DNase I footprinting, and the inhibition of DNA-protein complex formation by an antibody against an Ets protein indicate that an Ets-related factor is indeed a part of the α-MHC–PNR complex. To date, at least 20 different Ets family members have been identified; they share homology of a common Ets-DNA-binding domain and bind to the GGA(A/T) DNA motif. Many Ets family members have been shown to bind DNA cooperatively with other transcription factors, such as Ets-1 protein with c-fos and c-jun (70). Other potentially important interactions have been described between Elk-1 and SRF (47), PU.1 and retinoblastoma protein (17), Elf-1 and retinoblastoma protein (68), PU.1 and TF-IID (17), and ERM and androgen receptors (54). In addition, the Ets-1 protein has been shown to interact with a homeodomain protein GHF-1/Pit-1 for pituitary-specific expression of prolactin gene (4). Another Ets factor, PEA-3, has been documented to be activated in conjunction with the MEF-2 factor in response to myogenic stimulation of satellite cells during skeletal muscle regeneration, thus implying a role for Ets protein in myogenesis (63). The Ets proteins have been shown to participate in tissue-restricted gene regulation by utilizing both the gene activation and the repression mechanisms (7, 50, 67).

In this report it appears, based on the ability of the anti-ERP antibody to abolish PNR complex formation, that a factor immunologically related to ERP protein is binding to the PNR element. Transcripts of ERP, also known as Net or Sap-2, have been shown to be expressed in many tissues, including heart, lung, and skeletal muscle (27, 47). ERP/Net has been described as a DNA-binding repressor protein containing a novel inhibitory domain that resembles the helix-loop-helix motif (31). Protein-protein interaction studies have documented that the ERP/Net protein can physically interact with the basic helix-loop-helix protein E-47 (31), which is also an important heterodimeric partner for the MyoD family of proteins in skeletal muscle cells. Furthermore, because ERP/Net belongs to the group of ternary complex factors that includes Elk-1 and Sap-1, which are known to interact with SRF, ERP/Net protein has also been suggested to be an SRF-binding factor (47, 71). Given the importance of E-47 and SRF in muscle gene transcription, the expression pattern of ERP/Net protein in muscle cells and its ability to interact with these partner proteins suggest that ERP/Net protein may also have a role in muscle gene regulation. However, for the following reasons, data obtained in this study do not support the idea that the PNR-binding cardiac nuclear factor might be an ERP/Net protein. (i) ERP/Net has been shown to repress the activity of the basal promoter complex, such as the thymidine-kinase/CAT reporter gene (31), but we have shown, by using three different gene minimum promoter-reporter constructs, that PNR-interacting protein does not alter directly the activity of the basal transcription complex; rather, that it requires upstream gene sequences. (ii) ERP/Net protein has been shown to bind efficiently to a single Ets-binding site (27), whereas the findings presented here indicate that both inverted repeats of the palindrome are essential for the cardiac nuclear factor to interact with the PNR element. (iii) ERP/Net protein is also abundantly expressed in NIH 3T3 fibroblasts (27); however, the complex generated by NIH 3T3 and cardiac muscle cell nuclear extracts showed different gel mobilities. These lines of evidence raise the possibility that the PNR-binding cardiac nuclear factor may be a variant of ERP/Net/Sap-2 protein but is not identical to it. To the best of our knowledge, no Ets protein (except in this study) has been shown thus far to be involved in cardiac-muscle gene regulation. Ets proteins are reported to be expressed in the developing cardiac structures as early as the eighth and ninth days of embryonic development (32), coinciding with the appearance of α-MHC transcripts in the heart. In future studies, identification of the Ets protein responsible for the tissue-restricted expression of the α-MHC gene could provide clues for elucidation of transcriptional events involved in the induction and/or maintenance of the cardiac cell lineage.

Increased DNA-binding activity of PNR-interacting factor in the hypertrophied heart.

A change in the expression of MHC genes during cardiac hypertrophy is of major interest as a model for studying how cardiomyocytes respond to the increased workload and to the changing pattern of cardiac cell growth. During pressure overload cardiac hypertrophy, the expression of α-MHC mRNA has been shown to decrease three- to fourfold in rats, as well as in humans (6, 29). Furthermore, in a recent study, an almost 80% reduction in α-MHC mRNA levels was detected in failing human hearts (40). However, it is not yet clear whether this results from repression of the α-MHC gene expression or from a decrease in mRNA stability. If it is due to repressed α-MHC gene expression, it could be caused by an increased activity of the Ets factor binding to the negative regulatory element. The activity of the Ets class of proteins has been shown to be regulated by various extracellular stimuli both at the transcriptional and posttranscriptional levels. Several members of the Ets class have been documented to be a target for phosphorylation by different signaling pathways; for instance, ERP/Net/Sep-2 and PEA-3 are phosphorylated by extracellularly regulated kinases (ERKs), as well as by Jun N-terminal kinase (JNK); ERM is phosphorylated by ERK kinases and PK-A, and Ets-1 is phosphorylated by ERK kinases, casein kinase, and PK-C (20, 24, 31, 42, 47, 71, 73). Furthermore, an Ets protein, but not Jun, has been shown to be a target of the Ras/raf-1 signaling pathway for pituitary-cell-specific gene expression (7). Because these signaling pathways have also been shown to be activated during mechanical overload of cardiac myocytes (51), it is possible that posttranslational modification of a factor by phosphorylation could contribute to the increased binding activity of the PNR factor. It is interesting to note that the NFAT family of factors has recently been found to be involved in the hypertrophic response of cardiac myocytes (36). The NFAT factors have been shown to be associated with Ets factors for cell-specific gene activation (64, 71), thus further supporting a possibility for a role of the Ets factors in the process of cardiac myocyte growth. Furthermore, some evidence has indicated that the Ets proteins are also involved in regulating the activity of matrix-metalloproteinase genes (54) that control the degradation of the extracellular matrix; thus, it is tempting to consider that an uncoordinated change in the activity of Ets proteins in different cell types of the heart may lead to the development of pathologic hypertrophy.

In summary, this study is the first to identify an Ets protein-mediated negative gene regulation that contributes to the tissue-restricted expression of the cardiac α-MHC gene. Although the precise mechanism of the Ets repression remains to be elucidated, our findings could lead to a further search into diverse mechanisms involved in the regulation of cardiac-muscle-specific gene transcription. Because the function of the Ets family of proteins ranges from their role in cell transformation and cell growth to development and apoptosis, their participation in cardiac-muscle gene regulation reveals that they may also have a major impact on various developmental, physiologic, and disease processes of the heart.