©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Regulation of the Myoblast-specific Expression of the Human -Enolase Gene (*)

(Received for publication, November 18, 1994)

Jane M. Taylor John D. Davies Charlotte A. Peterson (§)

From the Departments of Medicine and Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences and the Geriatric Research, Education, and Clinical Center, McClellan Veterans Hospital, Little Rock, Arkansas 72205

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The muscle-specific beta-enolase gene is expressed in proliferating adult myoblasts as well as in differentiated myotubes. Through deletion-transfection analysis, we identified a 79-base pair enhancer from the beta-enolase gene that leads to high level expression of a reporter gene in myoblasts, but not in fibroblasts. Following myoblast differentiation into myotubes, the activity of the enhancer declined, indicating that beta-enolase gene expression in myotubes is mediated by other regulators, possibly the myogenic helix-loop-helix family of transcription factors. Electrophoretic mobility shift assays indicated that proteins present in myoblast nuclear extracts specifically bind to the 3` half of the 79-base pair enhancer. This region contains an ets DNA-binding motif which is required not only for high level activity in myoblasts, but also for repressing activity in fibroblasts. Furthermore, the beta-enolase myoblast-specific enhancer shows limited similarity to the myoblast-specific enhancer associated with the human desmin gene, suggesting that gene expression in adult myoblasts may be coordinately regulated.


INTRODUCTION

In adult muscle, activation of myoblasts, called satellite cells, is one mechanism for maintaining skeletal muscle mass. Although quiescent in normal muscle, satellite cells proliferate and differentiate in response to muscle damage or degeneration, thereby regenerating muscle fibers. We reported previously that the muscle-specific beta-enolase gene is expressed in proliferating myoblasts of adult, but not fetal human muscle(1) . The appearance of beta-enolase mRNA in myoblasts prior to birth may be indicative of the emergence of adult satellite cells in humans, although they arise somewhat later during development than the population of myoblasts resistant to the phorbol ester 12-O-tetradecanoylphorbol-13-acetate reported previously to represent the emergence of satellite cells(2) . beta-Enolase expressing myoblasts emerge at a comparable stage to the distinct population of adult myoblasts that appear during mid to late fetal development in avian embryos(3, 4) . In cell culture, adult myoblasts differ from fetal myoblasts in responsiveness to 12-O-tetradecanoylphorbol-13-acetate, acetylcholine, and platelet-derived growth factor(5, 6, 7) , and in myosin heavy chain expression following differentiation(8, 9) .

Although fetal and adult myoblasts can be distinguished based on beta-enolase mRNA accumulation, this difference disappears following differentiation so that myotubes derived from both cell types accumulate comparable levels of beta-enolase transcript(1, 10) . Thus, not only do the regulatory mechanisms that control beta-enolase gene expression change with developmental stage, but also with the differentiated state of the cell. It is likely that the helix-loop-helix (HLH) (^1)family of myogenic regulators (MyoD, myogenin, myf-5, and MRF4) are involved in controlling beta-enolase gene expression following differentiation (for review, see (11) and (12) ). Transcripts encoding betaenolase are detectable in the somites (from which skeletal myoblasts are derived) of 8.75-day postcoitum mouse embryos shortly after the first myogenic HLH factors are detected(13) . Moreover, the HLH myogenic regulators have been shown to activate muscle-specific gene expression, including beta-enolase(14) , when expressed in non-muscle cells. In adult muscle fibers in vivo, beta-enolase gene expression is also linked to the contractile and metabolic properties of muscle fibers. beta-Enolase transcripts accumulate to high levels in fast twitch relative to slow twitch muscle(1) . Alteration of the exogenous stimuli which control fiber type such as innervation and hormone levels results in changes in beta-enolase expression(15, 16) .

The myogenic HLH regulators are not likely to control betaenolase gene expression in proliferating myoblasts. Although present in myoblasts, the myogenic HLH proteins appear inactive as transcription factors for muscle-specific genes(17) . Growth factors necessary to maintain myoblasts in a proliferative, undifferentiated state, such as basic fibroblast growth factor and transforming growth factor beta, appear to inhibit the activity of the myogenic HLH proteins(18, 19) . Consistent with this data, beta-enolase mRNA accumulation was unchanged in myoblasts in which the expression of the entire family of HLH myogenic regulators had been abolished(1) . It is likely that the transcription factor(s) that control myoblast-specific gene expression are activated by growth factors, perhaps in a manner analogous to activation of serum response factor that controls expression of many cellular immediate-early genes(20) .

In the present study, we identified a cis-regulatory DNA sequence that controls expression of the beta-enolase gene specifically in adult myoblasts. As this enhancer did not activate transcription following differentiation, distinct regulatory factors control beta-enolase gene expression in proliferating myoblasts and differentiated myotubes. Site-directed mutagenesis indicated that an ets motif within the enhancer is required for transcriptional activation in myoblasts and repression in fibroblasts, suggesting that Ets domain proteins activate or repress transcription of the beta-enolase gene depending on the cellular context.


EXPERIMENTAL PROCEDURES

Construction of Human beta-Enolase/Luciferase Reporter Genes

Human genomic DNA was used as a template for the polymerase chain reaction (PCR) to amplify different regions of DNA flanking the betaenolase gene. The original set of primers was designed based on the sequence of the human beta-enolase gene reported by Peshavaria and Day(21) . Unless otherwise indicated, all primers contained a HindIII restriction site to facilitate cloning of the PCR products. A 979-bp PCR product (-917 to +62) was subcloned into the Bluescript vector (Stratagene), sequenced using the Sequenase kit (U. S. Biochemical Corp.), and was used as a template for further PCR amplifications. PCR products were cloned in both orientations either into luciferase vectors lacking a promoter, pOluc (22) or pGL2-basic (Promega), or into the pGL2-promoter vector (Promega) which encodes the luciferase reporter gene fused downstream of the SV40 minimal promoter. Using the megaprime technique, a double point mutation in the ets motif within the beta-enolase promoter was generated. A 5` megaprimer was generated by Vent polymerase (New England Biolabs) using the mutant single-stranded oligonucleotide shown in Fig. 5, the pGL2 vector sequencing primer 1, and pJC13 (see Fig. 4) as template. The resulting 145-bp DNA fragment was gel purified and used as described above to generate a PCR product (-628 to +62) containing the mutation. This PCR product was directionally cloned following digestion with KpnI and HindIII into the pGL2-basic vector. The wild type and mutant oligonucleotides shown in Fig. 5were also cloned into the pGL2-promoter vector. Equal quantities of single-stranded oligonucleotides were kinased, annealed, and ligated. Gel-purified trimeric oligonucleotide was then ligated into the BglII site to generate a plasmid consisting of three head-to-tail ligated oligonucleotides. All constructs were verified by sequence analysis as described above. Sequences were analyzed using the Genetics Computer Group Program Package(23) . As controls, a 225-bp fragment generated by HindIII digestion of DNA and a 500-bp BamHI fragment containing the enhancer from the rat myosin light chain 1/3 gene (24) were also cloned into pGL2-promoter.


Figure 5: The ets motif within the beta-enolase myoblast-specific enhancer shows limited similarity to a region within the myoblast-specific enhancer from the human desmin gene. The region within the desmin myoblast-specific enhancer important for activity (34) is shown compared with the beta-enolase regulatory region. The consensus ets motif (32) is also shown. The bracket indicates the region protected from DNase digestion by myotube nuclear extracts during footprint analysis of the desmin enhancer (Mb, (34) ). The sequences of the two oligonucleotides (wild type and mutant) used as competitors in EMSA are shown. Bold letters indicate the region that corresponds to -583 to -563 from the beta-enolase gene. Additional bases were added to facilitate cloning. Mutated bases are boxed.




Figure 4: An ets motif within the 79bp myoblast-specific enhancer (-628 to -549) is important for activity. Schematic representation of constructs pJC13-pJC16 in A, and pJC3 and pJC17-pJC19 in B is shown on the left and the relative luciferase activity produced by each following transient transfection into C2C12 myoblasts and 10T1/2 fibroblasts is shown on the right. Hatched boxes represent regions of beta-enolase 5`-flanking DNA, and the thin line represents the SV40 minimal promoter, cloned upstream of the luciferase reporter gene (LUC). The ets motif is represented by the stippled box in B. The double point mutation (GA CT) within the ets motif of pJC18 is indicated. Numbers denote position relative to the start site of transcription of the beta-enolase gene. Relative luciferase values from five separate experiments were averaged.



Transfections and Reporter Gene Assays

C2C12 mouse myoblasts were grown in DMEM with 15% defined/supplemented bovine calf serum (Hyclone) and 5% fetal bovine serum (Hyclone). 10T1/2, NIH, and Swiss 3T3 fibroblasts were grown in DMEM with 10% defined/supplemented bovine serum. For differentiation, confluent myoblasts were exposed to DMEM with 2% horse serum for 3 days.

Subconfluent cells were transfected on either 60- or 100-mm dishes with 6 or 10 µg of luciferase plasmids, respectively, using a modification of the calcium phosphate precipitation method described by Sternberg et al.(25) . The DNA precipitate was allowed to stand on the cells overnight, at which time the cells were washed twice in phosphate-buffered saline and given fresh media. For transient transfections, a vector expressing the lacZ gene under the control of the Rous sarcoma virus long terminal repeat was included as an internal control for transfection efficiency(26) . Approximately 48 h following addition of DNA, total protein content in cell extracts was determined (Bio-Rad Protein Assay) and luciferase assays were performed using the Promega luciferase assay kit and a TD-20e luminometer (Turner Designs), normalizing values to beta-galactosidase activity, quantitated using the Galacto-Light kit (TROPIX, Inc.).

For stable transfections, the pSV2neo vector was included at [1/10] the concentration of the test plasmid. Following overnight incubation with DNA, cells were washed and fresh media was added. After allowing the cells to recover for an additional day, cells were split 1:2 and selected in 400 µg of active G418 (Life Technologies, Inc.)/ml. As colonies began to appear, several hundred were pooled and the cells were split to maintain myoblasts at low density in high serum. Luciferase assays were performed on myoblasts and on myotubes following 3 days of myoblast differentiation as described above.

Electrophoretic Mobility Shift Assays (EMSA)

Nuclear extracts were prepared according to the method of Gossett et al.(27) . DNA fragments were generated by PCR using primers into which unique restriction sites had been incorporated. Following digestion, fragments were made blunt-ended using the Klenow fragment of DNA polymerase I, dATP, dGTP, dTTP, and [alpha-P]dCTP. Approximately 10,000 cpm (approximately 1 ng of DNA) and 4 µg of nuclear protein were incubated in 10 mM HEPES (pH 7.9), 75 mM KCl, 1 mM EDTA, 0.5 mM dithiothreitol, 10% glycerol, 0.15 µg/µl poly(dI-dC)bullet(dI-dC) in a reaction volume of 20-25 µl at room temperature for 30 min or several hours at 4 °C. Reaction products were separated on a nondenaturing 6% acrylamide gel (75:1, acrylamide:bis) containing 0.5 times TBE at 15 °C as described by Carey(28) . For competition experiments, varying concentrations of unlabeled DNA (ranging from 1- to 100-fold molar excess) were added to the reaction mixture prior to addition of extract. The 79-, 58-, and 40-bp competitor DNAs were generated by PCR. Smaller competitor DNAs were synthesized using a 381A DNA synthesizer (Applied Biosystems). The 25-bp DNA containing an Sp1 binding site was purchased from Santa Cruz Biotechnology.


RESULTS

Identification of cis-Regulatory DNA Sequences That Control beta-Enolase Gene Expression in Myoblasts

To identify cis-regulatory DNA sequences responsible for controlling the beta-enolase gene in myoblasts, we sequenced approximately 1 kilobase upstream of the human beta-enolase gene. Using PCR, we constructed plasmids containing serial deletions of 5`-flanking DNA from the beta-enolase gene cloned in both orientations upstream of the firefly luciferase reporter gene (pJC1, pJC3, pJC5, pJC7; Fig. 1). Each construct was tested for activity following transient transfection into C2C12 myoblasts and 10T1/2 fibroblasts. Swiss and NIH 3T3 fibroblasts yielded similar results to 10T1/2 cells (data not shown). In all cases, cells were cotransfected with an RSV/lacZ reporter gene plasmid and luciferase activity within each cell extract was normalized to beta-galactosidase activity to correct for variation due to differences in transfection efficiency. Fig. 1shows that constructs pJC1, pJC3, pJC5, and pJC7 generated low level luciferase activity in 10T1/2 cells. By contrast, the largest construct, pJC1, containing -917 to +62 of beta-enolase DNA cloned in the correct 5` to 3` orientation relative to the luciferase gene, produced approximately 9-fold higher luciferase activity in myoblasts than fibroblasts. All DNAs cloned in the reverse orientation produced no luciferase activity above background (data not shown). pJC3 with -628 to +62 from the beta-enolase gene generated even higher luciferase activity in myoblasts, greater than 20-fold higher than in fibroblasts, suggesting that a negative regulatory element resides between -917 and -628. Further deletion of 5`-flanking DNA reduced luciferase activity significantly, indicating that a myoblast-specific positive regulatory region is present between -628 and -362 relative to the start site of transcription of the beta-enolase gene. The smallest construct tested (-131 to +62) comprises the beta-enolase basal promoter and produced low level myoblast-specific expression.


Figure 1: Identification of beta-enolase 5`-flanking DNA that promotes high level luciferase activity in myoblasts. Schematic representation of constructs (pJC1, pJC3, pJC5, pJC7, pJC10) is shown on the left, and the relative luciferase activity produced by each following transient transfection into C2C12 myoblasts and 10T1/2 fibroblasts is shown on the right. Fragments containing different regions of beta-enolase 5`-flanking DNA (hatched boxes) were generated by PCR and cloned upstream of the luciferase reporter gene (LUC). The thin line in pJC10 represents the SV40 minimal promoter. Numbers denote position relative to the start site of transcription of the beta-enolase gene. Relative luciferase values from five separate experiments were averaged.



To determine if the region of DNA between -628 and -362 will enhance transcription in association with a heterologous promoter, this region was amplified by PCR and cloned into the pGL2 vector containing the luciferase reporter gene downstream of the SV40 minimal promoter (pJC10, Fig. 1). Following transient transfection, pJC10 reproducibly demonstrated significantly higher activity in myoblasts than fibroblasts, suggesting that a myoblast-specific enhancer is present within this 266-bp DNA fragment.

The beta-Enolase Myoblast-specific Enhancer Is Not Active following Differentiation of Myoblasts into Myotubes

The activity of the putative myoblast-specific enhancer identified above was tested following differentiation. For these experiments, two control plasmids were constructed utilizing the pGL2 vector. Into one was cloned a 225-bp nonspecific DNA (pJC11) and the other (pJC12) contained the 500-bp myotube-specific enhancer from the myosin light chain 1/3 gene(24) . Luciferase reporter gene constructs were cotransfected into C2C12 myoblasts with the pSV2neo plasmid, and stable transfectants were selected by resistance to G418. Approximately 200 neomycin-resistant myoblast clones were pooled, and luciferase activity was assayed in extracts derived from subconfluent cultures in high serum or following three days of confluence in low serum, conditions under which fully differentiated myotubes had formed. Pooled clones were analyzed to avoid possible position effects due to the site of integration. Analysis of activity in myoblasts and myotubes following stable transfection demonstrated that the ability of the 266-bp DNA fragment from the beta-enolase gene to enhance transcription specifically in myoblasts was unique to this region of DNA (Fig. 2). Following differentiation, luciferase activity from both pJC3 and pJC10 dropped dramatically, suggesting that the cis-regulatory element present in these constructs is active only in undifferentiated myoblasts. As expected, pJC11 produced similar low level luciferase activity in both myoblasts and myotubes, whereas expression of pJC12 increased following differentiation.


Figure 2: The beta-enolase enhancer located between -628 and -362 is not active following differentiation of myoblasts into myotubes. Schematic representation of constructs (pJC3, pJC310, pJC311, pJC312) is shown on the left and the relative luciferase activity produced by each following stable transfection into C2C12 myoblasts is shown on the right. The thin line in pJC10-pJC12 represents the SV40 minimal promoter. pJC3 and 10 were as described in the legend to Fig. 1. pJC11 contains a 225-bp fragment of DNA, and pJC12 contains the 500-bp myosin light chain 1/3 enhancer. Myoblasts were cotransfected with the luciferase reporter constructs together with pSV2neo and selected with G418. Stable clones were pooled and assayed for luciferase activity while growing in high serum (myoblasts) or following differentiation for 3 days in low serum (myotubes).



Further Delineation of Myoblast-specific Enhancer Elements Controlling beta-Enolase Gene Expression

The sequence of the 266-bp region of DNA between -628 and -362 is shown in Fig. 3. Consensus binding sites for known transcription factors are indicated(29, 30, 31, 32, 33) . To determine the relative importance of the different sites in promoting myoblast-specific gene expression, the 266-bp DNA fragment was subdivided into a 79-bp fragment (-628 to -549, pJC13 and pJC14) and a 187-bp fragment (-549 to -362, pJC15 and pJC16) by PCR and cloned in both orientations into the pGL2 vector (Fig. 4A). Following transient transfection into myoblasts and fibroblasts, the 79-bp region, in either orientation, retained the ability to enhance myoblast-specific expression of the luciferase gene (Fig. 4A). Analysis of luciferase activity in myoblasts or myotubes derived from them following stable transfection of pJC13 and pJC14 demonstrated that the ability of the 79-bp fragment to enhance transcription declined following differentiation (data not shown). pJC15 and pJC16 produced some luciferase activity over background in both myoblasts and fibroblasts (Fig. 4A), likely due to the presence of Sp1 sites within this region (see Fig. 3). Consistent with these results, deletion of the 79-bp DNA fragment from pJC3 (-549 to +62; pJC17, Fig. 4B) reduced luciferase activity in myoblasts by over 20-fold compared with pJC3. Surprisingly, pJC17 was 3-fold more active in 10T1/2 cells than pJC3 (Fig. 4B), suggesting that the 79-bp DNA is involved in repressing the beta-enolase promoter in fibroblasts.


Figure 3: The nucleotide sequence of the 266-bp DNA element that promotes high level myoblast-specific expression of the luciferase reporter gene. Potential binding sites for known transcription factor families are indicated(29, 30, 31, 32, 33) . Numbers indicate position relative to the start site of transcription of the beta-enolase gene. This region was divided at the position indicated by the arrow(-549) into two smaller fragments (79 and 187 bp), and each was tested for activity as described below.



The 79-bp enhancer contains consensus binding sites for GATA and Ets domain transcription factors. In addition, the ets motif within the enhancer showed some homology (10 of 14 bp) with a region within the myoblast-specific enhancer from the human desmin gene shown to be important for activity ((34) ; Fig. 5). We examined the role of the ets motif by mutating 2 base pairs within the GGA core consensus from GA to CT within pJC3 to generate pJC18. Similar to pJC17, this construct was nearly 15-fold less active in myoblasts than pJC3 (Fig. 4B), suggesting that an Ets domain protein may be involved in controlling high level myoblast-specific expression of the beta-enolase gene. However, a construct with three copies of the ets motif upstream of the SV40 minimal promoter (pJC19) was inactive in myoblasts, indicating that this site alone is not sufficient for myoblast-specific activity (Fig. 4B). The ets motif also appeared important for inhibiting activity in fibroblasts as pJC18 containing the mutated ets motif was consistently more active than pJC3 in 10T1/2 cells (Fig. 4B). In fact, pJC17 and 18 were more active in 10T1/2 cells than in C2C12 myoblasts. Thus, the ets motif appears to be involved in controlling tissue-specific expression by enhancing promoter activity in myoblasts and repressing activity in fibroblasts.

Analysis of Protein-DNA Interactions within beta-Enolase cis-Regulatory DNA Sequences

To characterize the proteins that specifically bind to the 79-bp enhancer from the beta-enolase gene, EMSA were performed. The 79-bp DNA fragment (-628 to -549) was radiolabeled, mixed with nuclear extracts, and DNA bound by proteins was resolved from unbound DNA by electrophoresis through native polyacrylamide gels. Two major shifted bands were observed with nuclear extracts from C2C12 myoblasts (Fig. 6, complexes A and B) and from myotubes and 10T1/2 cells (data not shown). The specificity of these protein-DNA interactions was demonstrated by competition analysis with 100-fold molar excess unlabeled competitor DNAs. The unlabeled 79-bp fragment competed for protein binding, resulting in the disappearance of complex A and reduction in complex B (Fig. 6, lane 3). A 25-bp fragment containing an Sp1 binding site failed to compete for protein binding (Fig. 6, lane 4). Overlapping subfragments of the 79-bp DNA varied in their ability to compete for protein binding (Fig. 7). A 40-bp fragment from the 5` end (-628 to -588) was unable to compete (Fig. 6, lanes 6-8), whereas a 58-bp fragment from the 3` end containing the ets motif (-607 to -549; Fig. 7, lanes 9-11) competed efficiently for protein binding. Labeling the 58-bp fragment and using it directly in EMSA confirmed the above results (Fig. 8). The 58-bp fragment produced shifted complexes A and B which were specifically competed with excess unlabeled DNA of the same sequence (Fig. 8, lane 3). Neither the 40-bp DNA fragment (Fig. 8, lane 4) nor a 25-bp DNA fragment containing an Sp1 DNA binding site (Fig. 8, lane 5) competed for protein binding. Furthermore, a double-stranded wild type oligonucleotide including the ets motif (-583 to -563, Fig. 5) abolished complex A (Fig. 8, lane 7), whereas an oligonucleotide containing the double point mutation (GA CT, Fig. 5) had no affect in EMSA (Fig. 8, lane 8). Complex B appeared unaffected by either oligonucleotide competitor, suggesting that in addition to binding to the ets motif, proteins interact with other DNA sequences within the 58-bp DNA fragment.


Figure 6: The 79-bp myoblast-specific enhancer binds to proteins present in myoblast nuclear extracts. EMSA were performed using P-labeled 79-bp DNA (-628 to -362) and nuclear extracts derived from C2C12 myoblasts (lanes 2-4). Two complexes were formed (A and B) which could be specifically competed with unlabeled 79-bp DNA (lane 3) but not with a 25-bp DNA containing an Sp1 binding site (lane 4). A 100-fold molar excess of each competitor was added. Lane 1 shows the migration of labeled fragment in the absence of extract.




Figure 7: Localization of protein binding within the 79-bp DNA. EMSA were performed using P-labeled 79-bp DNA (-628 to -362) and nuclear extracts derived from C2C12 myoblasts (lanes 2-11). Increasing concentrations (1-, 10-, and 100-fold molar excess) of three different unlabeled DNAs were used as competitors: the 79-bp DNA and two overlapping subfragments of the 79-bp DNA, 40-bp (-628 to -588) and 58 bp (-607 to -549) in length. Whereas the 40-bp DNA did not compete (lanes 6-8), the 79-bp fragment (lanes 3-5), and the 58-bp fragment (lanes 9-11) competed efficiently for protein binding in complexes A and B. Lane 1 shows the migration of labeled fragment in the absence of extract.




Figure 8: Protein binding to the myoblast-specific enhancer requires an ets motif. EMSA were performed using P-labeled 58-bp DNA (-607 to -549) and nuclear extracts derived from C2C12 myoblasts (lanes 2-8). Complexes A and B were formed and were specifically competed with unlabeled 58-bp DNA (lane 3) but not with the 40-bp DNA (-628 to -588, lane 4) or a 25-bp DNA containing an Sp1 binding site (lane 5). Complex A was specifically competed with the wild type 25-bp DNA from -583 to -563 (wt, lane 7; also see Fig. 5), whereas a mutated DNA, also shown in Fig. 5, failed to compete for protein binding (lane 8). A 100-fold molar excess of each competitor was added. Lane 1 shows the migration of labeled fragment in the absence of extract.




DISCUSSION

beta-Enolase belongs to a relatively small group of muscle-specific gene products expressed in proliferating myoblasts, as well as differentiated myotubes. Regulatory mechanisms that control gene expression in myoblasts have not been defined, but appear to change with the age of the muscle donor, so that myoblasts from embryonic, fetal, and adult muscle have distinct phenotypes. As a first step to isolating regulators of the adult myoblast phenotype, we identified an enhancer responsible for controlling the adult myoblast-specific expression of the human beta-enolase gene. beta-Enolase mRNA is undetectable in fetal myoblasts which instead accumulate the ubiquitous isoform alpha-enolase(1) . The switch to the muscle-specific isoform occurs prior to birth so that myoblasts derived from postnatal muscle tissues accumulate predominantly beta-enolase. Previous analysis of the DNA sequence flanking the human beta-enolase gene (21, 35) revealed the presence of potential binding sites for several general (Sp1, AP1, and 2) and muscle-specific transcription factors (E box for myogenic HLH factors, CArG box, MEF2 site, and M-CAT site(11, 27, 36, 37) . The latter sites have been shown to be involved in controlling the expression of genes induced during differentiation, such as myosin light chain 1/3 (MLC1/3). The MLC1/3 enhancer contains two E boxes and a MEF2 binding site (38) and promotes low level expression in myoblasts and high level expression in myotubes(24) . Our analysis showed that a myoblast-specific enhancer is present between -628 and -549 upstream of the transcription start site of the beta-enolase gene and that the activity of this enhancer dropped dramatically following removal of serum which induces differentiation of myoblasts into myotubes, in marked contrast to the MLC1/3 enhancer. An ets motif within the enhancer was important for myoblast-specific activity and was bound by proteins present in myoblast nuclear extract. Ets domain proteins comprise a family of transcription factors that bind to purine-rich DNA sequences with a GGA core consensus(39) . They have been shown to be involved in regulating gene expression and controlling growth in a variety of biological systems(32) . The subfamily of Ets domain proteins, including Elk-1 and SAP-1, which bind DNA autonomously and as part of the serum response factor ternary complex, are activated by phosphorylation following growth factor stimulation(20) . Our data suggest that growth factor stimulation may also be required for activation of Ets-dependent beta-enolase gene expression in myoblasts.

In addition to acting as an enhancer in myoblasts, the 79-bp DNA fragment we identified appeared to mediate repression of the beta-enolase gene in fibroblasts. Deletion of the 79-bp enhancer or mutation of the ets motif resulted in elevated transcriptional activity in fibroblasts relative to the parental construct. In fibroblasts, it is possible that an Ets domain protein bound to DNA acts to repress transcription. Net, another member of the Elk/SAP Ets subfamily has negative effects on transcription and Ras expression switches Net activity to positive(40) . It is possible that the same Ets domain protein is acting on the enhancer in myoblasts and fibroblasts, but that activity is modified in response to different signal transduction pathways in the two cell types.

The ets motif alone did not display enhancer function in myoblasts, suggesting that additional cis elements within the enhancer are involved in regulating beta-enolase gene expression. We found that protein complexes formed on the 79-bp DNA outside the ets motif, supporting the idea that multiple DNA-binding proteins must interact for enhancer activity. A common feature of Ets domain proteins is that they interact with other proteins to form multisubunit complexes(20, 32) . Some Ets domain proteins can bind to DNA autonomously, as is the case for Ets1 and the TCRalpha enhancer, but transcriptional activation requires a second non-Ets protein(41) . We have, as yet, been unable to identify additional cis-regulatory elements within the 79-bp DNA (data not shown). Sequences outside the enhancer also appear important for beta-enolase gene expression. Several potential Sp1 sites are located downstream of the enhancer and Sp1 may act alone or cooperatively with the Ets domain protein to produce maximal transcriptional activity. Sp1 sites present within the enhancer of the 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase gene have been proposed to regulate the expression of that gene in myoblasts(42) . Furthermore, as the myoblast-specific enhancer was most effective in association with its endogenous promoter, the beta-enolase initiation complex may play an important role in regulating expression. It is significant that the beta-enolase promoter (-131 to +62) demonstrated considerable myoblast-specific activity. Several studies have suggested that relatively short promoter regions can generate tissue-specific transcription. For example, 133 bp of 5`-flanking DNA from the myogenin gene promotes appropriate temporal and spatial expression of a reporter gene in transgenic animals(43) . Finally, beta-enolase gene expression may also be influenced by a negative regulatory element located upstream of the myoblast-specific enhancer between -917 and -628. The vimentin gene, which is expressed in many cell types, including myoblasts, is down-regulated during myogenesis (44) . The silencer located upstream of the promoter (45, 46, 47) may participate in this process. Further characterization of the putative negative regulatory region associated with the beta-enolase gene is required to determine what, if any, role it plays in regulating its expression.

The regulators that bind to the beta-enolase myoblast-specific enhancer may also control the expression of other muscle-specific gene products. Like beta-enolase, carbonic anhydrase III, the integral membrane protein H36 (alpha7 integrin), the intermediate filament desmin, and MyoD are muscle-specific gene products expressed in proliferating myoblasts (48, 49, 50, 51) . The genes encoding the latter two proteins have been cloned and enhancers active in myoblasts have been identified(52, 53) . No significant homology was found between the beta-enolase and MyoD enhancers. The desmin gene is regulated by separate myotube- and myoblast-specific enhancers that are adjacent to one another between -973 and -693 relative to the transcription start site(34) . The ets motif within the beta-enolase enhancer shares limited homology with the Mb sequence within the desmin myoblast-specific enhancer, shown to be important for activity. The enhancers associated with the human desmin and beta-enolase genes must be examined in greater detail to determine whether they are recognized by similar regulatory factors.


FOOTNOTES

*
This work is supported by Grant AG10523 from the National Institute on Aging and by grants from the Muscular Dystrophy Association, the Arkansas Affiliate of the American Heart Association, and the National Science Foundation Experimental Program to Support Competitive Research (all to C. A. P.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 501-671-2539; Fax: 501-671-2510.

(^1)
The abbreviations used are: HLH, helix-loop-helix; DMEM, Dulbecco's modified Eagle's medium; bp, base pair(s); PCR, polymerase chain reaction; EMSA, electrophoretic mobility shift assay(s).


ACKNOWLEDGEMENTS

We thank Dr. Usha Ponnappon for providing the Sp1 oligonucleotide and David Shin for help in constructing some of the luciferase expression vectors. We also thank Drs. Barry Hurlburt, Patty Wight, and Mark Crew for critical reading of this manuscript.


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