INTRODUCTION

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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 - and -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, -MHC is expressed in the atria and -MHC in the developing ventricles (28). Immediately before birth, the -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 -MHC mRNA again becomes suppressed, and -MHC transcripts predominate (28, 49). This antithetic regulation of - and -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 -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 -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 -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 -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 -MHC gene is a target of Ets transcription factors. A 30-bp downstream region of the -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 -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 -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 -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 -MHC gene raises the possibility that Ets proteins may also be involved in cardiac muscle cell development.

	MATERIALS AND METHODS

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Plasmid construction. Deletion mutants were derived from the plasmid MP1.0CAT containing an HindIII fragment of the -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 -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 -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 -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).

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.gal 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 -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 [-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 -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

Top Abstract Introduction Materials & Methods Results Discussion References

Identification of a strong repressor element that controls tissue-restricted expression of the -MHC gene. The role of the first intronic sequences in the expression of the rat cardiac -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 -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 -MHC gene. Sol8 myocytes were used in this study because, as in many previous reports, the expression profile of the -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 -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 -MHC gene is also potentially involved in directing the tissue-restricted expression of the gene.

View larger version (21K): [in this window] [in a new window]   FIG. 1.   Sequences of the first intronic region control tissue-restricted expression of the -MHC gene. (A) Schematic representation of the -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.-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 -galactosidase activity in the same cell lysate.

View larger version (42K): [in this window] [in a new window]   FIG. 2.   Localization of the negative cis-regulatory element in the first intronic region of the -MHC gene. (A) Expression of different 3' deletion mutants of the -MHC gene in primary cultures of cardiac myocytes. Bars represent the mean value of seven different experiments. (B) Diagram of the -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 -MHC gene, which are slightly different from previously published sequences (30).

View larger version (24K): [in this window] [in a new window]   FIG. 3.   Effect of the PNR element placed in different positions on the -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 -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.

View larger version (16K): [in this window] [in a new window]   FIG. 4.   Identification of upstream DNA sequences of the -MHC gene required for the repressor activity of the PNR element. Transient-expression analysis of different 5' progressive deletion mutants of the -MHC gene with or without the PNR element was carried out by direct injection of DNA into the heart muscle. Expression of pCMV.-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 -MHC gene; shaded bars, PNR element at the 5' end of the -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.

View larger version (58K): [in this window] [in a new window]   FIG. 5.   Different gel mobility PNR complexes formed with muscle and nonmuscle cell nuclear extracts. The end-labeled -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×).

View larger version (42K): [in this window] [in a new window]   FIG. 6.   Factor(s) binding to polyomavirus Ets-binding site is recognized by the -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.

View larger version (61K): [in this window] [in a new window]   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 -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.

View larger version (48K): [in this window] [in a new window]   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 -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 -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.

View larger version (35K): [in this window] [in a new window]   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.

View larger version (45K): [in this window] [in a new window]   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 -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 -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 -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 -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 -MHC gene expression during hypertrophy of cardiac myocytes.

View larger version (37K): [in this window] [in a new window]   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 -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

Top Abstract Introduction Materials & Methods Results Discussion References

A significant expression of the rat cardiac -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 -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 -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 -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 -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 -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 -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 -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 -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.

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 -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).

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 -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 -MHC mRNA levels was detected in failing human hearts (40). However, it is not yet clear whether this results from repression of the -MHC gene expression or from a decrease in mRNA stability. If it is due to repressed -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.