Interaction between the Skeletal Muscle Type 1 Na+Channel Promoter E-box and an Upstream Repressor Element

We have defined how four elements that regulate expression of the rat skeletal muscle type 1 sodium channel (SkM1) gene cooperate to yield specific expression in differentiated muscle. A basal promoter region containing within it a promoter E-box (−31/−26) is broadly expressed in many cells, including myoblasts and myotubes; mutations within the promoter E-box that disrupt binding of the myogenic basic helix-loop-helix (bHLH) factors reduce expression in all cell types only slightly. Sequential addition of upstream elements to the wild-type promoter confer increasing specificity of expression in differentiated cells, even though all three upstream elements, including a positive element (−85/−57), a repressor E-box (−90/−85), and upstream repressor sequences (−135/−95), bind ubiquitously expressed transcription factors. Mutations in the promoter E-box that disrupt the binding of the bHLH factors counteract the specificity conferred by addition of the upstream elements, with the greatest interaction observed between the upstream repressor sequences and the promoter E-box. Forced expression of myogenin in myoblasts releases repression exerted by the upstream repressor sequences in conjunction with the wild-type, but not mutant, promoter E-box, and also initiates expression of the endogenous SkM1 protein. Our data suggest that particular myogenic bHLH proteins bound at the promoter E-box control expression of SkM1 by releasing repression exerted by upstream repressor sequences in differentiated muscle cells.

Expression of the rat SkM1 1 sodium channel isoform is restricted almost exclusively to skeletal muscle; following a rapid post-natal increase in mRNA and protein levels, SkM1 becomes the predominant voltage-dependent sodium channel expressed in adult skeletal muscle (1,2). The spatial distribution of the channel within a myofiber is also tightly regulated, with the highest density of channel protein found within the folds of the neuromuscular junction, and lower levels throughout the sarcolemma and T-tubular membrane (3)(4)(5). Given the multiple levels at which channel expression is regulated, complex interactions of transcription factors probably govern SkM1 transcription.
We have previously characterized several cis-regulatory elements that control expression of this gene in a primary muscle culture system (6). We found that both positive and negative mechanisms combine to modulate expression, and that two E-boxes play pivotal roles in this process. One E-box, located at Ϫ31/Ϫ26 within the promoter, works with other elements to orchestrate positive regulation of the gene, while a second E-box, located at Ϫ90/Ϫ85 within a larger upstream repressor region, confers muscle-specific expression on the basal promoter that otherwise lacks cell-type specific function.
One of the unresolved issues from our earlier work was the mechanism by which the upstream repressor region achieved muscle-specific function. Transcription factors that bind to this region are present in all cell types examined, and transfer of either the entire repressor or its various sub-components to a heterologous rat brain type II sodium channel (RBII) promoter repressed expression in muscle cells as well as non-muscle cells. We postulated that the native SkM1 promoter influenced the ability of the upstream repressor region to act selectively in non-muscle cells, perhaps through the E-box within the SkM1 promoter.
Several muscle-specific genes, including those for troponin I, desmin, and the acetylcholine receptor (AChR) ␣, ␤, ␥, ␦, and ⑀ subunits, contain E-boxes within their promoter regions that are involved in regulating positive gene expression. These Eboxes function in part through their interaction with myogenic basic helix-loop-helix (bHLH) proteins (7)(8)(9)(10)(11)(12)(13)(14). Although positive regulation through the E-box is common to all these genes, the interplay between the bHLH factors and other transcription factors is more variable, and in some cases has not been completely resolved. The E-box within the promoter of the desmin gene coordinates positive regulation through interaction with a distal enhancer that contains a second E-box and an MEF2 binding site (7), while the E-boxes of the ␤ AChR subunit promoter interact with an M-CAT sequence adjacent to it (10). The AChR ␦ subunit and SkM1 5Ј-flanking sequences have substantial sequence and functional similarities (6,12,13). However, the E-box within the ␦ subunit promoter controls both positive and negative regulation of that gene, while these functions are split between two E-boxes within the SkM1 sequence (6,13).
In this report we demonstrate that the SkM1 promoter E-box influences the ability of the upstream repressor region to function, and that the binding of bHLH factors to the promoter E-box releases repression exerted by this element in the muscle lineage. Furthermore, comparison of repressor function in different muscle cell types and at different developmental stages reveals specificity in the ability of particular myogenic bHLH factors to effect this release of repression.

EXPERIMENTAL PROCEDURES
Generation of Reporter Gene Constructs-The Ϫ174, Ϫ135, Ϫ95, and Ϫ85 promoter E-box mutations were created from previously characterized full-length (Ϫ2800/ϩ249) promoter E-box mutations contained in pCAT-Basic (Promega; 6). Briefly, PCR was performed using either the c/g or tcc/gaa mutant Ϫ2800/ϩ254 as a template. The 5Ј-primer contained 20 base pairs of SkM1 sequence starting at the designated point and a restriction site (either HindIII or PstI) for cloning purposes. The 3Ј-primer was complementary to ϩ56 to ϩ78 of the SkM1 sequence. The PCR products were digested with HindIII and SacI (Ϫ135, Ϫ95, Ϫ85) or PstI and SacI (Ϫ174) and cloned into the same sites of the corresponding wild-type 5Ј-deletion mutant. Mutants generated by PCR were sequenced (Sequenase; U. S. Biochemical Corp.). All other mutations used in this report were created and characterized previously (6).
Cell Culture and Transient Expression Assays-Culture and transfection of primary muscle cells was carried out as reported previously (2,6). The C2C12 cell line was maintained in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) containing 10% fetal bovine serum, 100 units/ml penicillin, and 100 g/ml streptomycin (Life Technologies, Inc.). To initiate and maintain differentiated C2C12 cells, 2% horse serum replaced the fetal bovine serum. LipofectAMINE and Op-tiMEM (Life Technologies, Inc.) were used to transfect C2C12 cells, according to manufacturer directions. To allow comparison between calcium phosphate-transfected primary cultures and LipofectAMINEtransfected C2C12 cells, a constant molar ratio of test DNA (4 -6.4 g) to pCAT-C (0.8 g) was maintained. Normalization and quantitation of CAT assays were carried out as reported previously using the pCAT-Control (Promega) vector expressing the gene for chloramphenicol acetyltransferase driven by the SV-40 promoter and enhancer as a positive control, and the pCAT-Basic vector as a negative control (6).
Cells that were both transfected with reporter gene constructs and infected with the CMV-myogenin internal ribosome entry sequence (IRES) ␤-galactosidase adenovirus were transfected first for 3-4 h, then infected with 2 ϫ 10 10 particles of the adenovirus and maintained overnight (16 -18 h). Control cells were fed medium without adenovirus. Cells were either switched to differentiation medium or maintained in medium containing 10% fetal bovine serum for an additional 28 -30 h prior to harvest. Cells that were not transfected with reporter gene constructs were infected with the CMV-myogenin IRES ␤-galactosidase adenovirus according to the same paradigm and harvested for nuclear extracts or membrane proteins.
Gel-shift Assays-Gel-shift assays and supershift assays were carried out as previously reported (6) with the following modifications and additions. Gel-shift assays for the repressor probes were carried out at 4°C rather than room temperature. The antibodies used to supershift the various bHLH factors were obtained from Santa Cruz (E2A and myf-6), PharMingen (myogenin), and Novocastra (MyoD).
Preparation of Protein Fractions and Western Blotting-Membrane fractions containing sodium channel protein or nuclear extracts containing transcription factors were prepared as reported previously (6,15). Gel electrophoresis and Western blotting were carried out as re-ported using the Western Star kit (Tropix; Ref. 15). The primary antibodies used to detect the bHLH factors were the same as those used in the supershifts. To remove particulate matter and reduced background staining, the myf-6 antibody was treated as follows. The antibody was diluted 1:50 in 10% heat-inactivated horse serum in phosphate-buffered saline and incubated with 60 mg of porcine liver extract (Sigma) for 1 h a 4°C. The extract and particulate matter were removed by centrifugation at 100,000 ϫ g for 2 h. The final antibody solution was diluted 1:250 in 10% horse serum, 0.4% I-block (Tropix), and 0.1% Tween in phosphate-buffered saline. Final dilutions for the MyoD antibody was 1:250, and the myogenin antibody 1:500. The monoclonal antibody (L/D3) used to detect the sodium channel has been extensively characterized and is specific for the SkM1 isoform of sodium channel (16,17).
Generation of Replication-deficient CMV-myogenin IRES ␤-Galactosidase Adenovirus-The dl327 adenovirus and pAd-Link vector used to create the CMV-myogenin IRES ␤-galactosidase adenovirus were obtained from the Vector Core of the University of Pennsylvania (18,19). 293 cells (American Type Cell Culture) were grown in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 100 units/ml penicillin, and 100 g/ml streptomycin.
The polylinker in the pCI expression vector (Promega) was altered to contain restriction sites for the enzymes EcoRI, StyI, NotI, SnaBI, and BclI, and an EcoRI to StyI fragment containing myogenin was inserted. A NotI to BamHI fragment containing an IRES in frame with the ␤-galactosidase gene, obtained from pLIGns (20), was inserted between the NotI and BclI site of CMV-myogenin to yield CMV-myogenin IRES ␤-galactosidase. This construct was digested to completion with BglII, and a partial digest carried out with ClaI to yield a fragment extending from the CMV promoter to the SV40 poly(A) site of the expression vector. This fragment was cloned into the BglII and ClaI sites of pAd-Link and prepared for recombination by linearizing with NheI.
The dl327 adenovirus was grown in 293 cells and prepared for recombination by digestion with ClaI as reported (19). The initial transfection was carried out as reported previously (19), but the standard agar overlay procedure was replaced by plaque-purification using 96well plates. Serial dilutions of the transfected cells were combined with 1 ϫ 10 6 293 cells in a 20-ml total volume and dispensed into 96-well plates using 100 l/well. The plates were maintained for 6 days, then fed with 100 l of medium. After another 5-6 days, plaques were observed by eye. Dilutions resulting in more than 20 plaques/plate were discarded. A total of 30 plaques were screened by Southern blot, and of these, 9 were positive.
Plaque purification was carried out in the same manner, using serial dilutions ranging between 10 Ϫ6 and 10 Ϫ10 . Eleven plaques were screened in a Southern blot, and all were positive. One of these was expanded for large scale production according to published methods (19).

Arrangement and Activity of Cis-regulatory Elements-We
previously characterized several cis-regulatory elements that control expression of the SkM1 gene (6, Fig. 1). For most of the experiments reported here, we focused on four major functional elements between Ϫ174 and ϩ49. Our previous results are summarized as follows. The promoter E-box at Ϫ31/Ϫ26 directs FIG. 1. Cis-regulatory elements that control SkM1 gene expression. The locations of previously characterized cis-regulatory elements are indicated, with elements that modulate positive expression shown in black and those that modulate negative expression shown in white (6). An upstream repressor region or repressor contains both upstream repressor sequences and a repressor E-box. These elements repress SkM1 expression in non-muscle cells in conjunction with the native SkM1 promoter. The Ϫ85/Ϫ57 positive element contains within it two smaller motifs at Ϫ83/Ϫ78 and Ϫ64/Ϫ59 that are required for the binding of the cognate transcription factor; the full element is shown as two connected boxes containing plus signs, indicating the two motifs. The promoter, which by itself is broadly expressed in many cell types, contains within it a promoter E-box that binds the myogenic bHLH factors. In the context of the full-length Ϫ2800/ϩ254 SkM1 regulatory sequence, but not the promoter region itself, mutations in the promoter E-box severely reduce muscle-specific expression, indicating that the promoter E-box works with other elements outside the promoter to control positive modulation of the gene. The 3Ј-positive element located downstream of the transcription initiation sites encompasses part of the 5Ј-untranslated region and part of the first intron. This element confers 10-fold higher levels of reporter gene expression in muscle cells.
positive modulation of the gene through an interaction with elements elsewhere in the SkM1 genomic sequence. Myogenic bHLH proteins play a role in this interaction. The activity of the Ϫ85/Ϫ57 positive element is largely muscle-specific and confers 7-fold higher expression levels on the promoter, although its activity is masked in cultured muscle cells by the repressor E-box immediately upstream at Ϫ90/Ϫ85. DNase footprinting of the transcription factors that bind the upstream repressor region or repressor have shown that this element extends upstream to approximately Ϫ135, and functional studies described below substantiate an independent function for these upstream repressor sequences. A 3Ј-positive element that includes part of the 5Ј-untranslated region and part of the first intron lies between ϩ50/ϩ254; this element increases expression levels 10-fold in muscle cells.
Myogenic bHLH Proteins Bind the Wild-type SkM1 Promoter E-box but Not the Promoter E-box Mutants-The SkM1 promoter E-box binds multiple proteins in both muscle and nonmuscle cells, but the myogenic bHLH proteins MyoD, myogenin, and MRF4 were present only in muscle cells, as indicated by supershift assays with the appropriate antibodies (Fig. 2, A-E). There was heterogeneity in the expression of these myogenic factors in muscle at different stages of development and in different muscle cell types (13). For example, MyoD, but not myogenin, was expressed in C2C12 myoblasts (panel C), while both MyoD and myogenin were expressed in C2C12 myotubes (panel D). Likewise, gel-shifts obtained with nuclear extracts prepared from primary muscle cells after 4 days or 7 days in culture differed in the number of transcriptional complexes formed with the wild-type SkM1 E-box probe (panels A and B). MyoD and myogenin were present at both time points, while MRF4 was detected only on day 7.
We showed previously that two mutants in the promoter E-box either abolished (c/g mutant) or markedly reduced (tcc/ gaa mutant) the ability of this region to orchestrate positive interactions with other elements in the SkM1 gene (6). We tested these mutants directly in gel-shift assays to determine the effect of each mutation on transcription factor binding (Fig.  2). The c/g mutation abolished the binding of most transcription factors, including the myogenic bHLH factors in all cells types (Fig. 2). Functional data published previously (6) and additional data presented below indicate that this mutant is a true null mutation.
The results obtained for the tcc/gaa promoter mutation are more complex. This mutation greatly reduced the binding of the myogenic bHLH proteins, as indicated by the loss of supershifts generated by the antibodies to the factors, although some residual MyoD binding was observed in myoblasts and day 4 primary cultures (Fig. 2). The binding of non-bHLH protein complexes that interact with the wild-type promoter region was not affected by the tcc/gaa mutation. In the experiments that follow, these binding characteristics helped to differentiate between the functional impact of the myogenic bHLH proteins and the action of other transcription factors that bind the promoter E-box in primary muscle cultures. In contrast, the c/g mutation affected the binding of all factors in these nuclear extracts.
The tcc/gaa probe also binds a new factor not observed with the wild-type probe (Fig. 2, A-D). Although this additional complex co-migrated closely with the wild-type band supershifted by the MyoD antibody, this mutant complex did not contain MyoD. However, functional assays reported below indicate that the effects of the tcc/gaa mutation on gene expression reflect loss of function, rather than the gain of function generated by the binding of this new factor.
Although the primary purpose of these experiments was to determine whether or not the myogenic bHLH proteins could bind the promoter E-box mutants, we also noted unexpected complexities in the MyoD supershift patterns. Comparison of the size of the supershift generated by the MyoD antibody in C2C12 myoblast and myotubes extracts, or day 4 and day 7 primary culture extracts, revealed that two alternative supershifted states of MyoD existed, with the higher complex predominating in myoblasts, and the lower in myotubes (Fig. 2F). An antibody to the MyoD dimerization partner, E2A, further underscored the difference between the MyoD complexes in the two developmental states (Fig. 2F). In C2C12 myoblasts and day 4 primary cultures, similar discrete supershifts were observed with both the E2A and MyoD antibodies, while in more mature myotubes, the E2A supershift was similar in location and intensity only to the higher MyoD complex, suggesting that E2A was involved in the higher complex, but not the lower. The E2A antibody did not supershift the complex corresponding to myogenin, again suggesting that myogenin does not dimerize with E2A. Overall, our data suggested that the dimerization partners of the myogenic bHLH proteins vary depending on the state of differentiation, although we did not pursue this point further.
The Transcription Factors That Bind the Ϫ85/Ϫ75 Positive Element, Repressor E-box, and the Upstream Repressor Se- Myogenic bHLH proteins bind the wild-type SkM1 promoter E-box but not the promoter E-box mutants. The wild-type SkM1 promoter E-box region (cagCAGCTGtcc), a c/g mutant promoter E-box region (cagGAGCTGtcc), and a tcc/gaa mutant promoter E-box region (cagCAGCTGgaa) were used in gel-shift assays with nuclear extracts prepared from the following cell types: A, day 4 primary muscle cells; B, day 7 primary muscle cells; C, C2C12 myoblasts; D, C2C12 day 2 myotubes; E, PC12 cells. For all panels, antibodies against the following factors were used for supershift assays: 1, no antibody; 2, MyoD; 3, myogenin; 4, MRF4; 5, E2A. The supershifts are indicated by asterisks. The SkM1 promoter E-box bound multiple proteins in both muscle and non-muscle cells, but the myogenic bHLH proteins were bound only in muscle cells, as indicated by the asterisks. The two mutations in the promoter E-box either abolished (c/g mutant), or markedly reduced (tcc/gaa mutant) the ability of this region to bind myogenic bHLH factors. The c/g mutant also severely reduced the binding of non-bHLH proteins, while the tcc/gaa mutant retained binding for these additional factors. The tcc/gaa mutant also bound an additional factor not observed with the wild-type probe; this new complex co-migrated with the MyoD gel-shift but was not supershifted by the MyoD antibody. In panel F, a direct comparison was made between the supershifts of the MyoD and E2A antibodies in muscle cells at different stages of development. Two alternative supershifted states of MyoD existed, with the higher complex predominating in myoblasts, and the lower in myotubes. An antibody to the MyoD dimerization partner, E2A, demonstrates that E2A was involved only in the higher complex. The E2A antibody did not supershift the complex corresponding to myogenin. The nuclear extracts were prepared from the cell type indicated beneath each set of lanes.
quences Are Expressed in All Cell Types-The Ϫ85/Ϫ57 positive element binds a single complex in all cell types examined, including PC12 and muscle cells (Fig. 3A). This factor was displaced by the wild-type competitor (Fig. 3A, lane 2) but not a mutant competitor that altered two short motifs at Ϫ83/Ϫ78 and Ϫ64/Ϫ59, represented by the connected boxes in Fig. 1 (Fig.  3A, lane 3). Although this factor is present in all cell types, the Ϫ85/Ϫ57 positive element exhibited greater activity in differentiated muscle cells, as published previously and shown below (6).
The upstream repressor region is comprised of two components, a repressor E-box and the upstream repressor sequences. Previously, we identified a broad gel-shift that exhibited an extensive footprint covering sequences between Ϫ135 and Ϫ82 (6). Gel-shift assays carried out at low temperatures revealed an additional factor that associates more uniquely with the repressor E-box. Both the Ϫ135/Ϫ82 probe that included the repressor E-box and the repressor E-box probe alone associated with a sharp gel-shift that runs as the highest complex, while the shorter Ϫ135/Ϫ95 probe that excluded the repressor E-box did not bind this factor. The broad middle gel-shift was found with the probes that include the upstream repressor sequences, while the lowest gel-shift band appeared with all three probes. The longest probe, containing both components of the upstream repressor region, generated the most intense gel-shift, suggesting that these factors stabilize each other on the DNA. The repressor-binding transcription factors were found in all cell types.
The Promoter Determines the Cell Type in Which the SkM1 Repressor Functions-The Ϫ174/ϩ49 SkM1 sequence with the wild-type promoter E-box or the Ϫ174/ϩ49 sequence with either the c/g or tcc/gaa mutant promoter E-box were examined in transient expression assays both in primary muscle cells, which express the SkM1 gene, and PC12 cells, which express the RBII gene (21,22). As reported for other cell types previously (6), the upstream repressor region functioned in the nonmuscle PC12 cell line, but allowed expression in muscle cells. However, mutations in the promoter E-box that interfered with binding of the myogenic bHLH factors caused the repressor to function in muscle cells to the same extent it did in the nonmuscle cell line. These same mutations did not further reduce expression in PC12 cells. These data suggest that the "default" setting of the upstream repressor region is to function except when the promoter E-box binds bHLH factors.
Transfer of the upstream repressor region to the heterologous RBII sodium channel promoter reduced expression of that promoter in muscle cells, while permitting expression in PC12 cells (Fig. 4, bottom panel). The repressor binding-proteins were clearly present in PC12 cells (Fig. 3B), and the repressor was able to function in conjunction with the native SkM1 promoter in these cells (Fig. 4, top panel), indicating that the repressor-binding proteins were functionally active in PC12 cells. Together, these data indicate that the promoter, and specifically the promoter E-box, determines the cell type in which the repressor functions.
Expression of SkM1 Sodium Channel Protein and Myogenic Factors as a Function of Development in C2C12 Cells-We have previously used primary muscle cultures to study the expression of SkM1 mRNA levels and the function of the SkM1 cis-regulatory elements because this system relates most closely to the in vivo setting (1, 2, 6). However, primary muscle cultures express detectable levels of SkM1 mRNA and protein at the earliest times measured in culture, even before myotubes form (2). 2 We therefore examined C2C12 cells as an alternative to primary muscle cultures since C2C12 cells can be main-2 S. D. Kraner, unpublished observation.  For both A and B, the cell types from which the nuclear extracts were prepared is shown beneath the lanes. A, the Ϫ85/Ϫ57 positive element bound a transcription factor expressed in all cell types; this factor was displaced by the wild-type competitor, but not the mutant. 1, no competitor; 2, wild-type competitor (GAAGATTGGC-CCAGTCCTCAGGTTTCACT); 3, mutant competitor (GAGCTAGCGC-CCAGTCCTCAGCCCGGGCT). B, the repressor region bound multiple factors in all cell types. For all cell types, the following probes were used: 1, Ϫ135/Ϫ82 probe containing both upstream repressor sequences and repressor E-box; 2, Ϫ135/Ϫ95 probe containing only upstream repressor sequences; 3, Ϫ93/Ϫ82 probe containing only repressor E-box. The highest complex associated more uniquely with the repressor E-box and was bound to the probes containing the repressor E-box, while the middle complex associated more uniquely with the upstream repressor sequences and was bound to the probes containing these sequences. A lower band associated with both elements, although the intensity of the gel-shift for all factors was greatest on the probe containing both components of the upstream repressor region.
FIG. 4. The promoter determines the cell type in which repression occurs. In the top panel, the Ϫ174/ϩ49 SkM1 sequence with the wild-type promoter E-box or the Ϫ174/ϩ49 sequence with either the c/g or tcc/gaa mutant promoter E-box were inserted into a vector containing the reporter gene for CAT. These constructions were assayed in transient expression assays in primary muscle cells, which express the SkM1 gene, or PC12 cells, which express the RBII gene. Both cell types were transfected with the pCAT-Control (pCAT-C) plasmid as a positive control. In conjunction with its native promoter, the repressor functioned in the non-muscle PC12 cell line, while it allowed expression in muscle cells. Mutations that disrupt the ability of the promoter E-box to bind the myogenic factors caused the repressor to function in primary muscle cells, but did not further reduce gene expression in the negative PC12 cell line. In the lower panel, the Ϫ174/Ϫ50 sequence containing the entire upstream repressor region was transferred onto the heterologous RBII promoter, which also contains an E-box, and analyzed in both cell types. This switch in promoters caused the repressor to function in primary muscle cells rather than PC12 cells. The activity of the RBII promoter without added sequences is also shown in both cell types. tained in culture as myoblasts, yet reliably form myotubes when culture conditions are altered. We first determined the levels of SkM1 sodium channel protein expressed in C2C12 cells at various stages of development (Fig. 5). The SkM1 protein was not detected in myoblasts. Upon differentiation to form myotubes, the level of SkM1 protein gradually increased; highest levels were attained only at days 5 and 7, several days after myotubes had formed. Thus, C2C12 cells differed from primary muscle cultures in that SkM1 protein was not expressed in undifferentiated cells, and the level of expressed protein in myotubes continued to increase with maturation in culture.
We then examined the appearance of the myogenic factors MyoD, myogenin, and MRF4 (Fig. 5). MyoD was present at the highest levels in myoblasts and decreased following differentiation. Myogenin was not present in myoblasts, but appeared within a day following transfer to differentiation medium, prior to the rise in the sodium channel protein level. Myogenin peaked at day 2, and expression decreased at later times. We were unable to detect MRF4 by Western blot in C2C12 cells, although MRF4 could be detected by day 4 in C2C12 myotubes using the more sensitive supershift assay (data not shown). All three myogenic factors were present at higher levels in day 7 primary muscle cultures than day 7 C2C12 myotubes. Since the temporal pattern of channel protein and myogenic factor expression was most clearly defined in C2C12 cells, we carried out further functional studies of the SkM1 cis-regulatory elements in C2C12 myoblasts, day 7 C2C12 myotubes, and day 7 primary muscle cultures.
The Upstream Repressor Sequences Play a Key Role in the Interaction with the SkM1 Promoter E-box-To determine which element(s) interacted with the promoter E-box, we sequentially added upstream elements to the wild-type or promoter mutants. The promoter alone was expressed at high levels in both myoblasts and myotubes (Fig. 6A), consistent with our previous observations that the promoter is broadly expressed in both positive and negative cell types (6). The c/g and tcc/gaa mutations reduced promoter function slightly in primary muscle cells, with less effect in C2C12 myoblasts and myotubes. These data indicate that the promoter E-box does not play an important role in function of the basal promoter and that the promoter E-box by itself does not confer specific expression in differentiated muscle cells.
Addition of the Ϫ85/Ϫ57 element contributed to positive regulation in differentiated myotubes (Fig. 6, A and B; note difference in scale). Although this positive element increased the level of gene expression in both myoblasts and myotubes, consistent with the presence of a ubiquitous transcription factor, the augmentation produced in differentiated muscle cells was 4 -6-fold greater than in myoblasts (Fig. 2B). Both mutations in the promoter E-box significantly reduced this differentiation-specific activity (Fig. 6B), indicating that at least part of the activity was derived through an interaction with the promoter E-box.
Addition of the repressor E-box to the combined promoter and positive element reduced expression in all cells, but to a greater degree in C2C12 myoblasts and myotubes (10-fold) than primary muscle cells (7.5-fold). The level of expression observed in myoblasts approached that reported previously for the negative NIH 3T3 cell line (6), and neither promoter mutation further reduced expression levels in myoblasts. In differentiated cells, the tcc/gaa mutation did not significantly alter repressor activity, while the c/g promoter mutation reduced expression in both C2C12 and primary muscle myotubes to nearly the same level as myoblasts, suggesting that the promoter E-box binding proteins that interact with the repressor E-box are the non-bHLH factors.
The greatest effect of the promoter E-box mutations was observed in constructs that included the upstream repressor sequences (Fig. 6D). Addition of these sequences further reduced expression in both myoblasts and myotubes, but the incremental decrease was much less in myotubes, particularly primary muscle myotubes. Promoter mutations had little effect on the residual expression in myoblasts, but both the c/g and tcc/gaa mutations virtually eliminated expression above background in myotubes, producing a 90% reduction in primary culture myotubes. Our data suggest that the upstream repressor sequences play an important role in the interaction with the promoter E-box, and that mutations in the promoter E-box allow the combined components of the upstream repressor region to function to the same degree in differentiated muscle cells as in non-muscle cells. The combination of all four elements produces the highest degree of developmental specificity of SkM1 expression.
Myogenin Releases Repression of the SkM1 Upstream Repressor Region in C2C12 Myoblasts-To determine if the interaction between the promoter E-box and the upstream repressor sequences was mediated by particular bHLH proteins, we forced expression of myogenin in C2C12 myoblasts using a recombinant adenovirus, and tested the functional impact of this transcription factor on either the wild-type Ϫ174/ϩ49 sequence, or the corresponding c/g or tcc/gaa promoter E-box mutants. The production of myogenin in the infected cells was verified by Western blot (Fig. 7). In the absence of myogenin, myoblasts did not express either the wild-type or mutant Ϫ174/ ϩ49 sequences at levels above background. Introduction of myogenin resulted in expression of the wild-type sequence in both myoblasts and 1 day myotubes at levels 9-fold higher than background (Fig. 7), but the mutations in the promoter E-box interfered with the ability of myogenin to potentiate this increase. The overall level of myogenin-driven expression attained with the Ϫ174/ϩ49 construct in these cells was comparable to that observed in primary muscle cells (Figs. 6D and 7).
Myogenin Is Sufficient to Initiate Expression of the Endogenous Sodium Channel Gene in C2C12 Myoblasts-Although our data indicate that myogenin releases repression exerted by the upstream repressor region in the small segment of SkM1 flanking sequence used in our functional assays, there are additional elements that control expression of the endogenous FIG. 5. The initiation of myogenin expression precedes the onset of SkM1 expression in C2C12 cells. A membrane protein fraction or a nuclear extract protein fraction was isolated from C2C12 myoblasts (MB) or myotubes at the indicated day following application of differentiation medium (D1ϭ day 1, etc.). As positive and negative controls, the same fractions were isolated from day 7 primary muscle cells (D7, PM) or NIH 3T3 cells. After SDS-polyacrylamide gel electrophoresis, membrane proteins were analyzed for the sodium channel using a monoclonal antibody (L/D3) to the SkM1 sodium channel, or nuclear extract proteins were analyzed for the myogenic transcription factors using antibodies against the individual factors. MGN, myogenin.
gene. The ability of myogenin to initiate expression of the endogenous sodium channel gene was therefore assessed by directly measuring sodium channel protein levels in the same experimental paradigm used for the functional assays. In control myoblasts, no SkM1 protein product was detected, while the forced expression of myogenin was sufficient to up-regulate SkM1 protein levels to an extent comparable to control day 1 C2C12 myotubes (Fig. 7, inset).
Later Phases of Sodium Channel Up-regulation Correlate with the Activity of a 3Ј-Positive Element-Although the initiation of SkM1 transcription correlates with the appearance of myogenin in C2C12 myotubes, both MyoD and myogenin levels are relatively low at later times in culture when the highest levels of SkM1 protein are detected, and only low levels of MRF4 are found in these cells, as determined by Western blot (Fig. 5). These observations suggest that additional factors must act to maintain transcription of the SkM1 gene at later times. Since we previously demonstrated the contribution of a 3Ј-positive element to positive regulation of the SkM1 gene (6), we compared the activity of SkM1 constructs with and without the 3Ј-positive element during late myotube development in C2C12 cells (Fig. 8). Although the enhanced expression produced by this positive element in C2C12 cells was less than in primary muscle cultures, the magnitude of the effect did cor-relate with developmental up-regulation of SkM1 protein expression in C2C12 cells. DISCUSSION We have shown previously that multiple cis-regulatory elements control expression of the SkM1 sodium channel gene in primary muscle cells, and two E-boxes located within several larger elements play a dominant role (6). Although we had demonstrated that the promoter E-box interacts with elements outside the promoter to control positive regulation of the gene, we had not determined with what element(s) it interacted or how this positive regulation was achieved. Our earlier experiments also indicated that the native SkM1 promoter controls the cell type in which the upstream repressor region can function, but the mechanism underlying the promoter/repressor interaction was unclear. In this report, we have focused most of our effort on understanding the interactions between four major elements located between Ϫ174 and ϩ49 in order to systematically approach these unresolved issues.
The promoter E-box influences the ability of upstream repressor region to act in differentiated muscle. As we have shown previously and again in this report, the promoter itself is expressed broadly in many cell types, even though the promoter E-box is the one element characterized to date that binds FIG. 6. Mutations in the promoter E-box that disrupt binding of the myogenic factors interfere with the muscle-specific activity of upstream cis-regulatory elements, with the most prominent interaction taking place between the upstream repressor sequences and the promoter E-box. The diagram at the top of each graph schematically depicts the elements that were inserted into the pCAT-Basic vector for the set of assays shown in that graph; arrows indicate the position of the c/g and tcc/gaa mutations in the promoter E-box that disrupt binding of the myogenic bHLH factors. The following abbreviations are used to denote the elements: PEB, promoter E-box; REB,repressor E-box; URS, upstream repressor sequences. The full positive element is denoted by the connected black boxes containing plus signs. Transient expression assays were carried out in the indicated cell types, and CAT activity was normalized relative to the control plasmid (pCAT-C). The designation on the abscissa indicates whether the wild-type, c/g, or tcc/gaa promoter E-box mutation was used. In panel A, the promoter was expressed broadly in all cell types, and the promoter E-box mutations had little effect on expression levels. In panel B, the Ϫ85/Ϫ57 positive element increased expression to the greatest extent in differentiated muscle cells; mutations in the promoter E-box partially counteracted this increase. In panel C, the repressor E-box reduced expression in all cell types, but greater levels of expression remained in differentiated muscle cells, especially primary muscle cells. Although the tcc/gaa mutation did not alter the behavior of this combination of elements, the c/g mutation reduced the expression in differentiated cells to the same level as seen in myoblasts. In panel D, the inclusion of the upstream repressor sequences reduced expression in both myoblasts and myotubes, but the greatest degree of muscle-specific expression was observed with the inclusion of this element, with primary muscle cells exhibiting 10-fold higher expression levels than C2C12 myoblasts. Both the c/g and tcc/gaa mutants reduced level of expression in primary muscle cells to nearly the same level as in myoblasts.
cell-type specific factors, the myogenic bHLH proteins. Mutations in the promoter E-box reduce expression of the basal promoter in all cell types to a relatively small degree, indicating both that the promoter E-box is not important to the function of the basal promoter and that the promoter E-box cannot confer specificity by itself. It is the addition of upstream elements that increase expression in differentiated muscle relative to undifferentiated muscle, even though the upstream elements bind ubiquitously expressed transcription factors. Mutations that disrupt the ability of the promoter E-box to bind either the myogenic bHLH factors or non-bHLH factors interfere with the specificity conferred by these upstream elements, indicating that factors bound at the promoter E-box interact with those bound at upstream elements. The interaction of these factors potentiate higher levels of expression in differentiated muscle relative to myoblasts, especially in primary muscle cells where the amount of the myogenin is highest.
Two different mutations in the promoter E-box were used to help characterize the interaction between the promoter E-box and upstream elements. One mutation (c/g) abolishes the binding of both myogenic bHLH proteins and non-bHLH proteins, while a second (tcc/gaa) severely reduces binding of the bHLH proteins but does not diminish binding of the non-bHLH transcription factors. These mutants distinguish the impact of the bHLH factors from that of the other proteins that bind to the promoter.
The interaction between the promoter E-box and the Ϫ85/ Ϫ57 positive element is a supportive one in that factors bound to the promoter E-box "aid" the action of the transcription factor that binds the positive element. Since both promoter mutations affect this interaction, it appears that the myogenic bHLH proteins are involved.
The upstream repressor region is comprised of two individual components, the repressor E-box and the upstream repressor sequences, that have different relationships with the promoter E-box. For both of these components, factors bound to the promoter E-box inhibit the function of the repressor specifically in differentiated muscle cells, resulting in the retention of positive expression in primary muscle cells, and to a lesser extent in C2C12 myotubes. The factors responsible for the promoter E-box/repressor E-box interaction appear to be the non-bHLH factors, since the c/g mutation, but not the tcc/gaa mutation, affect the interaction. Both promoter E-box mutations allow full repression by the upstream repressor sequences, demonstrating that myogenic bHLH are responsible for this release of repression.
Experiments that we carried out previously, in which various subcomponents of the upstream repressor region were transferred to the heterologous RBII promoter, demonstrated that the repressor E-box was both necessary and sufficient for negative regulation (6). Although the data presented in this paper do not allow us to conclude if the upstream repressor sequences can function without the repressor E-box in the Ϫ174/ϩ49 sequence, it is clear that the upstream repressor sequences play a key and distinct role in interacting with the bHLH proteins.
The promoter determines in what cell type gene expression is allowed. Transfer of the SkM1 repressor to the RBII promoter results in repression in primary muscle cells, while expression is allowed in PC12 cells, which express the RBII gene (21,22). One prediction from these data is that the E-box within the RBII promoter will bind bHLH proteins specific to neuronal cells and that these factors might act in PC12 cells to inhibit the action of the SkM1 repressor much as the myogenic factors do in conjunction with the wild-type SkM1 promoter in myotubes. Indeed, bHLH factors have been found in PC12 cells, and increased levels of specific neuronal bHLH factors have been shown to correlate with increased expression of the RBII gene in this cell type when treated with nerve growth factor, suggesting neuronal bHLH proteins play an important role in the regulation of the RBII gene (21,23). Other neuronal genes FIG. 7. Forced expression of myogenin in C2C12 myoblasts disrupts activity of the upstream repressor and initiates transcription of the endogenous SkM1 gene. The wild-type or mutant Ϫ174/ϩ49 SkM1 flanking sequences were assayed in transient expression assays in either control cells or cells infected with an adenovirus expressing myogenin. Following transfection and infection, cells were either maintained in 10% fetal bovine serum (FBS) or switched to 2% horse serum (HS) for 28 h prior to harvesting. Forced expression of myogenin increased expression of the wild-type Ϫ174/ϩ49 sequence, while the mutations in the promoter E-box blocked this increase either completely (c/g) or partially (tcc/gaa). MGN, myogenin. Inset, the level of myogenin protein in the nuclear extract fraction and the level of SkM1 sodium channel protein in the membrane protein fraction were assayed by Western blotting. Control C2C12 myoblasts did not express either myogenin or sodium channel protein, while treatment with myogenin adenovirus induced expression of both proteins. Treatment with horse serum for 28 h (day 1 myotubes) also induced expression of both proteins, but treatment with myogenin adenovirus further increased the level of both myogenin and sodium channel.
FIG. 8. The late increase in SkM1 protein level in differentiating cultures correlates with the activity of an additional 3positive element. The activity of the entire SkM1 5Ј-flanking sequence (Ϫ2800/ϩ49) either with or without the additional 3Ј-positive element (ϩ50/ϩ254) was assayed as a function of development in C2C12 cells at days 4, 5, and 7, corresponding to the time at which the late increase in sodium channel protein levels occurs. As a positive control, activity was also analyzed in day 7 primary muscle cells. The 3Ј-positive element produced enhanced expression in C2C12 cells most prominently at the latest time in culture, and even higher levels of expression were observed in primary muscle cultures, where this element increased expression 10-fold. expressed in PC12 cells are regulated by interactions between E-boxes and separate regulatory sequences, and this interaction is mediated in part through bHLH proteins (24,25). We anticipate that there may be parallel mechanisms controlling expression of the SkM1 and RBII genes, with regulation by bHLH factors acting as a common theme.
Not all bHLH proteins that bind the promoter E-box are equivalent in their ability to release the repression exerted by the upstream sequences. A complex program initiates development in muscle, with different myogenic factors expressed at different times. C2C12 myoblasts, which do not contain detectable levels of SkM1 protein, express only MyoD partnered with E2A. Upon differentiation, myogenin is rapidly up-regulated. Initiation of SkM1 gene expression takes place at this time. This correlation led us to hypothesize that myogenin plays an important role in the initiation of SkM1 expression.
Direct introduction of myogenin in combination with the entire Ϫ174/ϩ49 sequence into C2C12 myoblasts and day 1 myotubes released repression, leading to expression levels comparable to those observed for this same construct in primary muscle cells. Mutations in the promoter E-box either abolish (c/g) or greatly reduce (tcc/gaa) the release of repression that myogenin can confer, confirming that myogenin binding at the promoter E-box directly affects negative regulation. Myogenin also activates expression of the endogenous SkM1 gene. However, unlike its action on the short regulatory sequences, the effect of myogenin on the endogenous gene is potentiated by culture conditions that induce differentiation, suggesting that myogenin may initiate expression of the endogenous SkM1 gene through multiple mechanisms.
Levels of SkM1 increase at later times in C2C12 differentiation, suggesting later action of another factor, particularly since the levels of the bHLH factors themselves decrease. We previously demonstrated that the 3Ј-positive element plays a major role in tissue-specific SkM1 expression (6), and the activity of this element correlates with the late increase in the endogenous SkM1 protein in C2C12 cells, although it never confers the level of activity in C2C12 myotubes that it does in primary muscle cells. Part of the activity of the 3Ј-positive element is derived through a specific interaction with MRF4, since forced expression of MRF4 in C2C12 cells increases the activity of the 3Ј-positive element to the same level observed in primary muscle cells. 2 The absence of high levels of SkM1 gene expression in the constructs that lack the 3Ј-positive element indicates its important role, particularly for the maintenance of expression in later stages of differentiation.
Although our initial analysis presents the relationship between the promoter E-box-binding proteins and other transcription factors as a one-on-one interaction, this is certainly an oversimplification of an association that is probably far more complex, with changes occurring in the entire transcription initiation complex to switch it from an "inactive" to and "active" state. The myogenic bHLH proteins and perhaps factors that are still unknown may independently contribute to the assembly of the transcription initiation complex. Our data suggest that only specific myogenic bHLH proteins can function in conjunction with the other SkM1 factors, and it may be that the bHLH proteins confer the muscle-specific action to the complex. However, the myogenic bHLH factors cannot act alone. Our future work will be directed toward identifying all of the factors involved and understanding the interplay between them.