Multiprotein complex formation at the beta myosin heavy chain distal muscle CAT element correlates with slow muscle expression but not mechanical overload responsiveness.

To examine the role of the beta-myosin heavy chain (betaMyHC) distal muscle CAT (MCAT) element in muscle fiber type-specific expression and mechanical overload (MOV) responsiveness, we conducted transgenic and in vitro experiments. In adult transgenic mice, mutation of the distal MCAT element led to significant reductions in chloramphenicol acetyltransferase (CAT) specific activity measured in control soleus and plantaris muscles when compared with wild type transgene beta293WT but did not abolish MOV-induced CAT specific activity. Electrophoretic mobility shift assay revealed the formation of a specific low migrating nuclear protein complex (LMC) at the betaMyHC MCAT element that was highly enriched only when using either MOV plantaris or control soleus nuclear extract. Scanning mutagenesis of the betaMyHC distal MCAT element revealed that only the nucleotides comprising the core MCAT element were essential for LMC formation. The proteins within the LMC when using either MOV plantaris or control soleus nuclear extracts were antigenically related to nominal transcription enhancer factor 1 (NTEF-1), poly(ADP-ribose) polymerase (PARP), and Max. Only in vitro translated TEF-1 protein bound to the distal MCAT element, suggesting that this multiprotein complex is tethered to the DNA via TEF-1. Protein-protein interaction assays revealed interactions between nominal TEF-1, PARP, and Max. Our studies show that for transgene beta293 the distal MCAT element is not required for MOV responsiveness but suggest that a multiprotein complex likely comprised of nominal TEF-1, PARP, and Max forms at this element to contribute to basal slow fiber expression.

pression is detected in the heart, and subsequently its expression becomes primarily restricted to adult stage type I skeletal muscle fibers (1)(2)(3)(4). Once myofibers have differentiated and acquired a distinct adult phenotype, the molecular properties of the fiber are not static but instead are remarkably malleable in response to a broad spectrum of physiological signals (5). A well documented example of this phenotypic plasticity is the induction of ␤MyHC expression in fast type II fibers in response to increased neuromuscular activity imposed by mechanical overload (MOV) (5,6). The regulated control of ␤MyHC expression has been shown to involve the summation of inputs from multiple cis-acting modules located within both the ␤MyHC proximal promoter and more distal regulatory regions (3, 6 -11). However, the precise contribution of each discrete element comprising these regions has not been systematically analyzed in the in vivo context. In addition, the identity of the cellspecific and/or ubiquitous transcription factors that direct ␤MyHC slow fiber restricted expression or MOV responsiveness in adult stage skeletal muscle have not been determined as yet.
To investigate the mechanistic basis controlling both slow fiber restricted expression and MOV responsiveness of the ␤MyHC gene, we have conducted an extensive in vivo deletion analysis of the human ␤MyHC promoter in the context of the transgenic mouse (4, 6, 8 -11). These studies defined a minimal promoter region comprised of 293 bp of the ␤MyHC promoter (transgene ␤293WT) that closely mimics the expression pattern of the endogenous ␤MyHC gene at all developmental stages, and in response to MOV (4,9). Most striking was the finding that the deletion of an 89-bp region (Ϫ293 to Ϫ205) resulted in the loss of transgene ␤293WT expression and MOV responsiveness, thus indicating that this 89-bp region contains regulatory elements important for both basal slow fiber expression and MOV responsiveness of the ␤MyHC gene (9). Further analysis of this 89-bp region led to the identification of a putative MOV element (␤A/T-rich element: Ϫ269 to Ϫ258) that formed an enriched multiprotein binding complex only when MOV-plantaris nuclear extract was used (8). In addition to this A/T-rich element, the 89-bp region also contains a muscle CAT (MCAT) site. Whether this regulatory element plays a functional role in conferring ␤MyHC basal slow fiber expression and/or MOV responsiveness has not been investigated as yet.
MCAT elements are commonly located in the control region of many striated (skeletal and cardiac) muscle-specific genes (Ref. 13 and references within). A detailed molecular analysis of the cTnT promoter has elucidated the mechanistic basis by which two closely positioned MCAT elements participate in directing embryonic skeletal muscle-specific gene transcription of the cTnT gene (20,21). These two MCAT elements (MCAT1 and MCAT2) bind several protein complexes that display high, intermediate, and low migrating mobility in electrophoretic mobility shift assays (EMSA). All binding complexes contain TEF-1 protein; however, the low mobility complex (LMC) formed at the MCAT1 element contains an additional protein identified as poly(ADP-ribose) polymerase (PARP) (20 -22). Mutation of the 5Ј-flanking nucleotides of either the MCAT1 or MCAT2 elements revealed their integral requirement for achieving both skeletal muscle-specific expression of cTnT reporter genes, and the formation of a LMC at these MCAT elements when using embryonic chicken skeletal muscle nuclear extract (20). Likewise, Gupta et al. (23) have reported that the positive regulation of ␣MyHC gene expression in cardiomyocytes requires an E-box/MCAT composite element that involves the cobinding of NTEF-1 and the basic helix-loop-helix leucine zipper transcription factor Max. Additionally, Ojamaa et al. (24) have demonstrated that the ␣MyHC E-box/MCAT element can serve as a contractile/mechanical responsive element in cardiomyocytes and under conditions of increased contractile activity binds the basic helix-loop-helix leucine zipper protein, upstream stimulatory factor (USF).
The ␤MyHC gene is regulated by mechanical stimuli and contains within its proximal promoter two closely spaced MCAT elements; a distal MCAT (Ϫ290/Ϫ284) and a proximal MCAT (Ϫ210/Ϫ203). These two distinct MCAT elements are highly conserved and located within a region required for both slow fiber expression and MOV responsiveness of a 293-bp human ␤MyHC transgene (␤293) in adult skeletal muscle (4). Our previous EMSA analysis revealed that only the distal MCAT element formed a series of protein complexes displaying low, intermediate, and high migrating mobility similar to those formed at the cTnT MCAT element, and in addition, the low mobility complex was enriched only when using MOV-P nuclear extract (25). On the basis of these data obvious questions arise. First, does the distal MCAT element (Ϫ293/Ϫ284) bind both TEF-1 and PARP? Second, does this element contribute to both basal slow fiber expression and MOV responsiveness of the minimal 293-bp human ␤MyHC transgene?
In the current study we have generated multiple independent transgenic mouse lines harboring either a wild type 293-bp human ␤MyHC transgene (␤293WT) or a 293-bp MCAT element mutant transgene (␤293Mm). These mice were used in studies aimed at determining whether the distal MCAT ele-ment is required for: 1) basal slow fiber expression, 2) musclespecific expression, and 3) MOV responsiveness. We also conducted a detailed molecular analysis to determine what nuclear protein(s) comprise the low mobility binding complex formed at the distal MCAT element. Our findings show that the ␤MyHC distal MCAT element contributes to basal slow fiber expression of transgene ␤293 but is not required for MOV-responsive expression of transgene ␤293 in adult transgenic mice. The multiprotein complex formed at the ␤MyHC distal MCAT element is comprised of NTEF-1, PARP, and Max. DNA binding studies indicate that PARP and Max are bound to the distal MCAT element via TEF-1, whereas protein-protein interaction assays revealed that these three proteins physically interact in vitro. Collectively, these data support the notion that the assembly of TEF-1, PARP, and Max at the ␤MyHC distal MCAT element (LMC) contributes to basal slow fiber expression of transgene ␤293 in adult stage skeletal muscle.

EXPERIMENTAL PROCEDURES
Transgenes and Site-directed Mutagenesis-The wild type ␤MyHC 293-bp transgene (␤293) used in this study has been described previously (4,9). Briefly, ␤MyHC transgenes consist of 293 bp of human ␤MyHC 5Ј-promoter sequence and 120 bp of 5Ј-untranslated region (includes exon 1), fused to the 5Ј-end of the bacterial CAT gene (see Fig.  1). The transgenes were released free of pSVOCAT vector sequence by NdeI/BamHI restriction digest, and the digest products were fractionated by agarose gel electrophoresis. The transgene insert DNA was electroeluted from the gel, concentrated by ethanol precipitation, and dialyzed against microinjection buffer (10 mM Tris-HCl, pH 7.4, 0.15 mM EDTA) for 48 h at 4°C. As a final step in transgene construct purification, the dialyzed DNA was passed through a 0.2-m filter.
The distal MCAT site in the human ␤293 promoter was mutated within the plasmid p␤293CAT using the QuikChange TM site-directed mutagenesis kit (Stratagene) according to the manufacturer's instructions. Complementary oligonucleotide primers harboring mutations within the MCAT site were designed to meet the length and melting temperature requirements specified by the manufacturer (mutated bases are underlined): 5Ј-GCATAGTTAAGCCAGCCAAGCGCGTCT-TAGGAGGCCTGGCCTGGG-3Ј. Base pair substitutions were incorporated at nucleotides determined by our previous diethylpyrocarbonate interference footprinting to be crucial DNA-protein contact sites (25). Unintentional transcription factor recognition sites were not created by these mutations as assessed by cross-referencing the mutated primers against the eukaryotic transcription factor data base available on the Wisconsin Package, version 10.0, Genetics Computer Group (Madison, WI). The PCR-mediated incorporation of mutant sequence was performed using 5 ng of double-stranded p␤293CAT DNA template utilizing the manufacturer's recommended conditions. The PCR product was transformed into Epicurian coli XL1-Blue supercompetant cells (Stratagene), and the resulting plasmid DNA was isolated using anion exchange columns (Qiagen EndoFree TM ). Successful incorporation of the mutation was verified via automated sequencing of both strands (Applied Biosystems model 377). The distal MCAT mutant transgene (␤293Mm) was isolated and purified as described above.
Transgenic Mice-Transgenic mice were generated by microinjection of purified transgene DNA into pronuclei of single cell fertilized embryos as described previously (26). Transgenic founder mice were identified by Southern blot analysis. Subsequent transgene-positive offspring were identified by PCR amplification of genomic DNA using primers specific for the CAT gene. In the present study, multiple independent transgenic lines representing each transgene (wild type ␤293WT and mutant ␤293Mm) were analyzed. All lines were maintained in a heterozygous state by continual outbreeding to nontransgenic FVB/n mice.
The in vivo presence of the MCAT mutant sequence was verified by PCR amplification of ␤293WT and ␤293Mm mouse genomic DNA. PCR conditions were as described above, and the reactions performed were with both a sense strand primer harboring the mutated MCAT sequence on the 3Ј terminus (5Ј-GCATAGTTAAGCCAGCCAAGCGCGT-CTTAG-3Ј) and an antisense strand primer specific to sequences within the CAT gene (5Ј-GGATATATCAACGGTGGTAT-3Ј). Under these conditions, only genomic DNA harboring the transgene ␤293Mm sequence was amplified resulting in a 450-bp PCR product (see Fig. 1D).
Transgene copy number was estimated as described previously (26). Briefly, 10 g of transgenic mouse genomic DNA was digested with EcoRI, followed by fractionation through a 1% agarose gel and transfer onto a nylon membrane. The membrane was hybridized with a 32 Plabeled CAT cDNA probe used to identify each transgene. The intensity of each signal was quantified with the use of a PhosphorImager (Storm860 with IMAGEQUANT version 5.1 software, Molecular Dynamics) and was compared with that of a previously reported singlecopy transgenic line (human ␤1285, line 41) (10). The membrane was stripped and rehybridized with a 32 P-labeled c-myc (single copy gene) cDNA probe (350 bp of exon 2) to verify that each lane contained equal amounts of DNA.
Animal Care and MOV Surgical Procedure-The ablation surgery used in this study was performed as described previously (27). All procedures were conducted using adult female transgenic mice (22-26 g) and were approved by the Animal Care Committee for the University of Missouri-Columbia. MOV was imposed on the hindlimb fast twitch plantaris muscle by surgical removal of the gastrocnemius and soleus muscles. All mice demonstrated normal mobility shortly after recovering from anesthesia. The post-surgical experimental period lasted 8 weeks after which time the overloaded plantaris muscle (MOV-P) was removed. Sham operated mice served as controls for the plantaris (CP) and soleus (CS) muscles.
CAT Assays-CAT assays were performed as described previously (9,27) and the percent conversion of [ 14 C]chloramphenicol to the acetylated form was quantified using a PhosphorImager (Storm860) with IMAGEQUANT version 5.1 software.
Preparation of Nuclear Protein Extract from Adult Skeletal Muscle-Nuclear extracts were isolated from adult rat CP, MOV-P, and CS muscle as described previously (8). Protein concentration was determined according to Bradford (28).
Electrophoretic Mobility Shift Assay-All oligonucleotide probes used in this study are listed in Table I. EMSAs were carried out as described previously (8). Binding reactions were performed using either 3.5 g of CP, MOV-P, or CS nuclear extract and 20,000 cpm of labeled probe for 20 min at room temperature in a 25-l total volume. Where indicated, either in vitro transcribed/translated (TnT) NTEF-1, RTEF-1, embryonic TEF-1, divergent TEF-1, PARP, or Max protein were used in place of muscle nuclear extract (see figure legends for specific quantities). The binding reactions were resolved on a 5% nondenaturing polyacrylamide gel at 220 volts for 2.5 h at 4°C. Supershift EMSAs were performed by first preincubating skeletal muscle nuclear extract with 2 l of the corresponding antibody for 30 min at room temperature followed by the addition of the 32 P-labeled DNA probe. Following electrophoresis, the gels were dried, and DNA-protein complexes were visualized by autoradiography at Ϫ80°C.
Antibodies-The antibodies used in this study were as follows: NTEF-1, mouse polyclonal antibody raised against amino acids 86 -199 of human TEF-1 (BD Transduction Laboratories); PARP, rabbit polyclonal antibody raised against full-length human PARP (Roche Molecular Biochemicals); and Max, rabbit polyclonal antibody raised against full-length mouse Max (Upstate Biotechnology). All antibodies listed hereafter were purchased from Santa Cruz Biotechnology, Inc. and include: MyoD, rabbit polyclonal antibody raised against full-length, (amino acids 1-318) mouse MyoD; Myogenin, rabbit polyclonal antibody raised against full-length (amino acids 1-225) of rat myogenin; USF-1, rabbit polyclonal antibody raised against carboxyl terminus amino acids 291-310 of human USF-1; E2A.E12, rabbit polyclonal raised a peptide in the carboxyl terminus of human E47 and corresponds to amino acids 422-439 of human E12; and HEB, rabbit polyclonal antibody raised against a peptide in the carboxyl-terminal domain of human HEB (HTF 4).
Production and Purification of GST Fusion Proteins-The GST-TEF-1 expression plasmid has been described elsewhere (23). Briefly, full-length rat TEF-1 cDNA was subcloned into the XbaI and XhoI sites of the bacterial expression vector pGEX-KG. Full-length Max cDNA was amplified from pVZ1-p21-Max (23) in PCR reactions using the following primers: sense strand, 5Ј-CCGCTCCCTGGGCGGATCCAAA-TGAGCGATACC-3Ј and antisense strand, 5Ј-TGGCCTGCCCCGCTC-GAGTTAGCTGGCCTC-3Ј. The amplified cDNA was subcloned into the BamHI and XhoI sites of pGEX-5X-1 (Amersham Pharmacia Biotech). The GST fusion proteins were expressed in Escherichia coli and purified according to the manufacturer's (Amersham Pharmacia Biotech) instructions with modifications. Briefly, bacteria (DH5␣) containing the pGEX-KG-TEF-1 and PGEX-5X-1-Max plasmids were grown for 15 h at 37°C in LB-Amp medium. Subsequently, the cells were diluted 1:100 in fresh medium, grown for 4 h (A 600 ϳ0.7), and then induced to express the fusion protein at 37°C for 4 h, using 0.4 mM isopropyl-␤-D-thiogalactopyranoside. Following induction, the bacteria were pelleted by centrifugation at 5,000 ϫ g for 10 min at 4°C, and resuspended in 1ϫ phosphate-buffered saline supplemented with protease inhibitors. The cells were lysed by sonication for two 40-s pulses (while on ice). The protein was solubilized (30 min, 4°C) by the addition of Triton X-100 to a final concentration of 1% in the sonicated lysate and then centrifuged at 20,000 ϫ g for 10 min at 4°C to remove insoluble material. Fusion proteins were immobilized by the addition of glutathione-Sepharose 4B beads (Amersham Pharmacia Biotech) to the supernatant, and the binding reaction was allowed to proceed at 4°C for 30 min. The beads were pelleted at 500 ϫ g for 5 min at 4°C and washed five times with 1ϫ phosphate-buffered saline. The proteins were quantified by SDS-PAGE using known concentrations of bovine serum albumin as a standard and stained with Coomassie Blue.
In Vitro Protein-Protein Interactions-[ 35 S]Methionine labeled TnT proteins corresponding to NTEF-1, PARP, and Max were incubated with 2 g of immobilized GST, GST-TEF-1, or GST-Max with continuous rocking for 3 h at 4°C in a modified 1ϫ protein interaction buffer described by Gupta et al. (23); 20 mM HEPES, pH 7.5, 75 mM KCl, 1 mM EDTA, 2 mM MgCl 2 , 2 mM dithiothreitol, and 1% Triton X-100. Unprogrammed lysate that was also transcribed and translated in the presence of [ 35 S]methionine was used as the negative control. After incubation the glutathione-Sepharose beads were pelleted by centrifugation at 500 ϫ g and washed five times with ice-cold 1ϫ protein interaction buffer. The fusion proteins were eluted off the beads by heating at 95°C for 3 min in the presence of 1ϫ SDS sample buffer and analyzed on a 4 -20% gradient SDS-PAGE gel. The gel was dried and exposed to film for 15 h at room temperature.

Mutation of the Distal MCAT Element Decreased Slow Fiber
Expression of Transgene ␤293Mm-We initiated our investigation into the in vivo functional role served by the ␤MyHC distal MCAT element in adult stage skeletal muscle by generating multiple independent lines of two classes of transgenic mice (Fig. 1, A-C). The first class of transgenic mouse carries a wild type transgene comprised of 293 bp of human ␤MyHC promoter fused to the CAT reporter gene (termed transgene ␤293WT), whereas the second class carries the same transgene except that the highly conserved distal MCAT element has been mutated (termed transgene ␤293Mm) (Fig. 1, A-C). Nucleotide mutations were introduced into the core distal MCAT element by base pair substitution and reflected sites of strong DNAprotein interaction as assessed by our previous diethylpyrocarbonate interference footprint analysis (25).
To assess whether the distal MCAT element was solely responsible for directing both muscle-specific and high levels of slow fiber expression, we measured the CAT specific activity (pmol/g of protein/min) in muscle and nonmuscle tissues obtained from transgenic mice carrying mutant transgene ␤293Mm. The CAT specific activity measured in the CS and CP muscles of mice representing each of the four independent ␤293Mm lines was significantly decreased as compared with CAT specific activities measured in the CS and CP muscles obtained from mice representing each of the three independent ␤293WT lines (Fig. 2, A and B, and Table II). Our analysis of the ␤293Mm transgenic lines did not reveal measurable levels of CAT specific activity in any nonmuscle tissue examined even under conditions where excess protein and extended incubation times were used (50 g protein/overnight incubation) (data not shown). These data indicate that the distal MCAT element is not required to direct muscle specific expression of transgene ␤293 in the chromosomal context. Because this result was observed for all four independent transgenic lines each carrying different transgene copy numbers, it is unlikely that chromosomal position or transgene copy number accounts for these results.

Mutation of the Distal MCAT Element Does Not Eliminate
MOV Responsiveness of Transgene ␤293Mm-Our previous transgenic analysis has revealed that the ␤MyHC distal MCAT element (Ϫ290/Ϫ284) is located within an 89-bp region (Ϫ293/ Ϫ205) that is required for MOV responsiveness of transgene ␤293WT (4). Therefore, to determine whether the distal MCAT element functions as an ␤MyHC MOV-responsive element, CAT specific activity was measured in sham operated CP and MOV-P muscles of transgenic mice carrying either transgene ␤293WT or ␤293Mm following an 8-week period of MOV. For mice representing each of the three independent lines carrying transgene ␤293WT, expression assays revealed that the CAT specific activity measured in MOV-P muscle extract was 2.9 -7.5-fold higher than that measured in sham operated CP muscle extract (Table II). Interestingly, following the 8-week MOV period, the CAT specific activity measured in the MOV-P muscle obtained from mice representing each of the four independent ␤293Mm transgenic lines was also 1.5-7.1-fold higher than that measured in corresponding CP muscles (Table II).
Collectively, our transgenic analysis demonstrates that the ␤MyHC distal MCAT element is required for basal slow muscle fiber expression levels of transgene ␤293 in both soleus and plantaris muscles. However, this element is not required for MOV responsiveness because its mutation did not alter the relative fold induction of transgene ␤293Mm in the MOV-P muscle.
Multiple Nuclear Protein Binding Complexes Form at the ␤MyHC Distal MCAT Element-To determine whether the nuclear protein binding properties of the distal MCAT element differed between muscles comprised of different proportions of histochemically classified slow type I fibers, we performed an EMSA analysis using nuclear extracts isolated from rat CS (78 -90% type I fibers), CP (4 -8% type I fibers), and MOV-P (30% type I fibers) muscles. EMSA analysis of binding reactions containing the 21-bp double-stranded 32 P-labeled wild type distal MCAT probe (Table I) and 3.5 g of either CP, MOV-P, or CS nuclear extract revealed the formation of multiple binding complexes (Fig. 3). These binding complexes are referred to as LMC, intermediate mobility complex (IMC), and high mobility complex (HMC). The LMC appeared as a single band that varied in quantity concordant to the proportion of slow type I fibers populating the muscles from which the nuclear extract was isolated, that is, CS Ͼ MOV-P Ͼ CP. The IMC consisted of two distinct bands whose intensity appeared to vary inversely to the amount of LMC that was formed. The high mobility complex consisted of multiple bands; however, only one band was easily distinguishable in our experiments regardless of the amount of nuclear extract used or the length of time the gel was exposed to film.
The LMC Formed at the ␤MyHC and cTnT Distal MCAT Elements Are Similar-Interestingly, Larkin et al. (20) have recently reported a similar binding pattern (LMC, IMC, HMC) as assessed by EMSA analysis using the cardiac troponin T (cTnT) MCAT elements (MCAT1 and MCAT2) and chicken embryonic skeletal muscle nuclear extract. Therefore, to address the possibility that the same proteins bind to the ␤MyHC distal MCAT and cTnT MCAT elements, we performed competition EMSA using as competitor the cTnT and ␣MyHC MCAT elements (Fig. 4). The addition of 100-fold molar excess cold wild type distal ␤MyHC MCAT probe to binding reactions containing either CP (Fig. 4, lane 1 versus lane 2), MOV-P (lane 6 versus lane 7), or CS (lane 11 versus 12) nuclear extract completely abolished complex formation, indicating that these complexes are specific. Interestingly, the addition of 100-fold molar excess cold cTnT MCAT1 probe to binding reactions containing either CP (Fig. 4, lane 1 versus lane 3 As an additional competitor, we used the muscle creatine kinase transcriptional regulatory factor x element that has previously been shown not to bind TEF-1 when using TEF-1-containing MM14 nuclear extract, despite its relative sequence homology to MCAT consensus elements (32). The addition of 100-fold molar excess cold transcriptional regulatory factor x element to binding reactions containing either CP   Because adjacent E-box elements have been shown to be important for nuclear protein binding at the ␣MyHC and cTnT MCAT elements, we used the high affinity MCK E-box as competitor to determine whether the E-box in the immediate 5Ј-flanking region of the ␤MyHC distal MCAT element is required for complex formation. Addition of 100-fold molar excess cold MCK E-box element to binding reactions containing either CP (Fig. 4, lane 1 versus lane 5), MOV-P (lane 6 versus lane 10), or CS (lane 11 versus lane 15) nuclear extract slightly abolished formation of the LMC only. Collectively, our direct and competition EMSA data provide evidence that: 1) nuclear protein binding at the ␤MyHC distal MCAT element is specific, 2) distinct patterns of nuclear protein binding complexes were formed when using either CP, MOV-P, or CS nuclear extracts, and 3) the nuclear proteins forming the LMC preferentially bind the ␤MyHC distal MCAT element versus the cTnT and ␣MyHC MCAT elements. This difference is likely due to sequence specific differences between these three MCAT elements (Table I).
Nucleotides Comprising the ␤MyHC Distal Core MCAT Element Are Essential for Skeletal Muscle Nuclear DNA-Protein Binding Complex Formation-To identify nucleotides within the 21-bp ␤MyHC distal MCAT oligonucleotide probe that interact with MOV-P and CS nuclear protein to form the LMC, IMC, and HMC, we performed competition EMSA using scanning mutagenesis. In these experiments, we introduced nucle-otide substitutions two base pairs at a time starting within the immediate 5Ј-flanking region (E-box) and extending throughout the core MCAT element and its immediate 3Ј-flanking region (Fig. 5A). The resulting unlabeled oligonucleotides were then added in 100-fold molar excess to binding reactions containing either MOV-P or CS nuclear extract and the 32 P-labeled wild type distal MCAT probe (Fig. 5B). As shown previously, the addition of excess wild type ␤MyHC distal MCAT probe to binding reactions containing either MOV-P (Fig. 5B,  lane 1 versus lane 2) or CS (lane 11 versus lane 12) nuclear extract completely abolished the formation of all binding complexes. Similarly, all binding complexes were effectively competed away by the addition of either distal MCAT mut-1, mut-2 (contain mutations within E-box), or mut-7 (3Ј-flanking region) to binding reactions containing either MOV-P (Fig. 5B, lane 1  versus lanes 3, 4, and 9) or CS ( lane 11 versus lanes 13, 14, and  19) nuclear extract. Importantly, mutant MCAT probes carrying nucleotide substitutions within the core MCAT element (mut-3, mut-4, mut-5, and mut-8) did not act as effective competitors of complex formation when added to binding reactions containing MOV-P (Fig. 5B, lane 1 18) nuclear extract completely abolished LMC formation but not IMC or HMC formation. These data confirm our previous findings using diethylpyrocarbonate interference footprinting (25) by showing that nucleotides comprising the core ␤MyHC distal MCAT element are critical for the formation of all binding complexes when using either MOV-P or CS nuclear extract (Fig. 5C).
NTEF-1, PARP, and Max Comprise the LMC Formed at the ␤MyHC Distal MCAT Element-Previous work has shown that TEF-1 and Max interact at the ␣MyHC E-box/MCAT composite element, and that TEF-1 and PARP associate at the cTnT MCAT1 elements (22,23). In addition, Max has been identified as a component of the cTnT MCAT binding complex formed when using neonatal cardiomyocyte nuclear extracts (23). Thus, to assess whether these nuclear proteins were components of the LMC, IMC, and HMC formed at the ␤MyHC distal MCAT element, we performed antibody EMSAs using polyclonal antibodies that recognize each of the aforementioned proteins. The specific binding complexes formed at the 32 Plabeled human ␤MyHC distal MCAT element when reacted with CP (Fig. 6, lane 1  In addition, the enriched LMC observed when using MOV-P nuclear extract appeared to be nearly immunodepleted, whereas the highly enriched LMC formed when using CS nuclear extract was partially abolished. Interestingly, the addition of either polyclonal PARP or Max antibody to binding reactions using MOV-P nuclear extract essentially immunodepleted the LMC, which resulted in an embellishment of the top band of the IMC, which is comprised entirely of NTEF-1 protein (Fig. 6,  lane 4 versus lanes 7 and 8). Identical results were obtained when CS nuclear extract was used in binding reactions with the exception that the highly enriched LMC was again only partially abolished (Fig. 6, lane 13 versus lanes 16 and 17). When all combinations of the three polyclonal antibodies were added to binding reactions using MOV-P (Fig. 6, lane 4 6 -10), or CS (lanes 11-15) nuclear extract was incubated in the presence of the 32 P-labeled distal MCAT oligonucleotide. For analysis of sequence specificity of binding at this MCAT element, the following cold (nonradioactive) competitors were added to the reaction mixture at a 100-fold molar excess prior to the addition of the probe: distal MCAT (lanes 2, 7, and 12), cTnT MCAT (lanes 3, 8, and 13), MCK transcriptional regulatory factor x (lanes 4, 9, and 14), and the MCK high affinity right E-box (lanes 5, 10, and 15).

FIG. 5. Competition EMSA determination of specific nucleotides involved in DNA-protein interactions at the distal MCAT element.
A, summary of MCAT mutant oligonucleotides used in competition EMSA shown in B. wt, wild type or nonmutated distal MCAT sequence. The core MCAT sequence is indicated with shading. Mutated bases are lowercase and in boldface type. ϩϩϩ, competition was as effective as distal MCAT wild type sequence; ϩϩ, partially effective competition; Ϫ, no detectable competition. B, 32 P-labeled distal MCAT oligonucleotide was incubated in the presence of either MOV-P (lanes 1-10) or CS (lanes 11-20). Where indicated, 100-fold molar excess of either distal MCAT oligonucleotide (lanes 2 and 12) or various distal MCAT mutant oligonucleotides (harboring base pair mutations shown in A) were added to the reaction prior to the addition of the probe. C, distal MCAT oligonucleotide with MCAT core sequence is shaded, and E-box sequence is in boldface type. Two base pair numbering above the oligo sequence represents specific nucleotide mutation. The black box delineates nucleotides whose mutation renders the distal MCAT an ineffective competitor (especially of the LMC), suggesting this region as a putative binding site for the LMC. Diethylpyrocarbonate interference footprint (25) is shown here to illustrate the DNA-protein binding profile. Open circles denote partial interference; closed circles depict complete interference. Sequence numbering begins at the 5Ј-end of the sense strand.
MCAT element binding complex formation, or mobility (data not shown). These data support three notable conclusions: 1) the LMC formed at the human ␤MyHC distal MCAT element is comprised of at least three proteins antigenically related to NTEF-1, PARP and Max, 2) the prominent HMC band and the top band of the IMC doublet are comprised of protein antigenically related to NTEF-1 or possibly TEF-1 isoprotein(s) recognized by the NTEF-1 polyclonal antibody used in these experiments, and 3) the bottom band of the IMC doublet was not supershifted/immunodepleted by the addition of any antibodies used herein and thus may be comprised by a TEF-1 isoform not recognized by the polyclonal NTEF-1 antibody or an as yet unidentified nuclear protein(s).
TEF-1, but Not PARP or Max, Specifically Binds to the ␤MyHC Distal MCAT Element in Vitro -Because our antibody supershift assays indicate that proteins antigenically related to Max, NTEF-1, and PARP comprise the LMC formed at the ␤MyHC distal MCAT element, it was important for us to determine the ability of these three factors to independently bind to the 32 P-labeled human ␤MyHC distal MCAT element. EMSA analysis of binding reactions containing the 32 P-labeled human ␤MyHC distal MCAT element and rabbit reticulocyte lysategenerated in vitro translated TEF-1 isoproteins (Fig. 7A, inset shows expected 54-kDa TEF-1 proteins) revealed the formation of specific binding complexes that were different from the nonspecific complex formed when unprogrammed lysate was used (Fig. 7A, lane 1 versus lanes 2-5). In contrast, a binding complex was not formed when either [ 35 S]methionine-labeled in vitro translated PARP (Fig. 7B, inset shows expected 120-kDa PARP protein) or Max (Fig. 7C, inset shows expected 22-kDa Max protein) were reacted with the ␤MyHC distal MCAT element (Fig. 7B, lanes 1-4, Max, and data not shown). The inability of PARP or Max to form a binding complex with the ␤MyHC distal MCAT element remained, even when using 10 -100 ng of either purified recombinant baculovirus expressed PARP (data not shown) or Max (Fig. 7C, lanes 1-3). Further, we were unable to reconstitute the formation of a ␤MyHC distal MCAT LMC in our in vitro binding assays despite the simultaneous use of TEF-1, PARP, and Max, in binding reactions (data not shown). We were, however, able to detect DNAprotein binding when adding either 5 or 10 ng of purified Max to binding reaction containing the CM-1 oligonucleotide (con-tains (33) previously shown Myc/Max E-box binding site CACGTG (23, 34)) (Fig. 7C, lanes 4 -6), indicating that the purified Max protein used in our LMC reconstitution experiments possessed DNA binding capabilities.
NTEF-1, PARP, and Max Stably Interact-Our DNA-protein binding assays revealed that of the three proteins tested, only NTEF-1 binds the ␤MyHC distal MCAT element; therefore, the protein-protein interactions within our LMC remained to be identified. Importantly, previous work has shown a stable protein-protein interaction between RTEF-1 and PARP (22) and between NTEF-1 and Max (23), and, therefore, it was logical to test for protein-protein interactions between NTEF1, PARP, and Max. To this end, we performed in vitro pairwise proteinprotein interaction assays wherein [ 35 S]methionine-labeled in vitro translated Max, NTEF-1, or PARP protein were individually reacted with either bacterially expressed GST-NTEF-1 or GST-Max fusion proteins that were bound to glutathione-Sepharose beads (Fig. 8). Following incubation, the pelleted glutathione-Sepharose beads/fusion protein complex was extensively washed, and this complex was analyzed for bound [ 35 S]methionine-labeled in vitro translated protein by using SDS-PAGE (Fig. 8). Importantly, [ 35 S]methionine-labeled in vitro translated PARP protein was found to bind both GST-Max (Fig. 8A, lane 3) and GST-NTEF-1 (Fig. 8C, lane 3) but not the 26-kDa GST protein (Fig. 8, A and C, lane 2), revealing that this interaction was specific between each respective fusion protein and in vitro translated PARP protein. Similarly, [ 35 S]methionine-labeled in vitro translated NTEF-1 and Max were found to bind specifically to GST-Max (Fig. 8B, lane 2  versus lane 3) and GST-NTEF-1 (Fig. 8D, lane 2 versus lane 3), respectively. In all analyses described above, the [ 35 S]methionine-labeled in vitro translated protein, bound by the GST fusion protein, migrated to a position in the SDS-polyacrylamide gel that aligned with the [ 35 S]methionine-labeled in vitro translated protein that was not reacted with GST fusion protein (Fig. 8, A-D, lanes 1 versus lanes 3). When unprogrammed rabbit reticulocyte lysate was reacted with either GST, GST-NTEF-1, or GST-Max, a [ 35 S]methionine-labeled in vitro translated protein product was not observed, further demonstrating specificity of the interactions between the fusion protein and the in vitro translated product (Fig. 8, A-D, lanes  4 -6). Collectively, these data indicate that the assembly of the ␤MyHC distal MCAT multiprotein LMC involves DNA binding of TEF-1, and that TEF-1, PARP, and Max are capable of protein-protein interactions between each other. However, it remains to be determined conclusively whether the LMC involves a ternary complex comprised of these proteins or alternatively two subsets of paired protein complexes (see "Discussion"). DISCUSSION MCAT elements are known to participate in directing muscle-specific expression. Our transgenic findings herein show that the control region distal MCAT element (Ϫ290/Ϫ284) contributes significantly to basal slow muscle expression of chromosomally integrated transgene ␤293 but is not absolutely required for MOV responsiveness. Furthermore, our in vivo protein-protein interaction studies elucidate the possibility that in adult skeletal muscle NTEF-1, PARP, and Max may physically interact to form a ternary binding complex at the ␤MyHC distal MCAT element.
Transgenic Mouse Analyses of the in Vivo Function of the Human ␤MyHC Distal MCAT Element Reveals That It Contributes to Basal Slow Fiber Expression but Is Not Absolutely Required for MOV Responsiveness-In adult mice, basal expression levels of the endogenous ␤MyHC gene is high in the slow twitch soleus muscle, whereas it is not expressed to any appreciable degree in the fast twitch plantaris muscle, an expression pattern mimicked by wild type transgene ␤293WT (4,9). Our analysis of mutant transgene ␤293Mm basal expression revealed that mutation of the distal MCAT element significantly decreased but did not abolish expression in the slow twitch soleus muscle (Fig. 2, A and B, and Table II). In fact, even though the magnitude of mutant transgene ␤293Mm expression was decreased, its expression pattern (soleus Ͼ plantaris) still mirrored that of wild type transgene ␤293WT. These results showing low, yet persistent expression of mutant transgene ␤293Mm strongly suggest that other cis-acting elements within the 293-bp human ␤MyHC basal promoter must be acting in concert to preserve wild type levels of slow muscle expression. Candidate sites within the ␤MyHC proximal control region (Ϫ293/Ϫ170) that may act in combination with the distal MCAT element to direct high levels of slow muscle expression are an A/T-rich (Ϫ269/Ϫ258), CCAC/Sp1 (C-rich, Ϫ242/Ϫ231), and NFAT (Ϫ179/Ϫ171). In this regard, our results from EMSA and transgenic mouse analysis indicate that the ␤MyHC NFAT and ␤A/T-rich elements contribute to slow muscle expression because their independent mutation in the context of transgene ␤293 (␤293Nm, ␤293A/Tm) significantly decreased slow muscle expression. 2 Our data provide evidence that the combined inputs from the distal MCAT, ␤A/T-rich, and ␤NFAT elements underlie transgene ␤293WT slow muscle gene expression in adult transgenic mice. Indirect support for this notion comes from recent findings implicating NFAT and MEF2 proteins as downstream mediators of calcium-activated intracellular signaling pathways that have been proposed to regulate the slow gene program (Ref. 39 and references within). Additional work is underway to clarify whether these three elements collaborate to direct slow muscle specific expression.
Because MCAT elements have been shown to function as inducible promoter elements in response to mechanical stimuli (13)y and the distal MCAT element resides within the 89-bp region required for MOV responsiveness of transgene ␤293WT, we investigated its role in MOV-inducible expression. Our analysis revealed that the range of MOV-induced CAT specific activity measured in the MOV-P muscle of mice harboring mutant transgene ␤293Mm was almost identical to those carrying wild type transgene ␤293WT, indicating that the distal MCAT element is not absolutely required for MOV responsiveness of transgene ␤293 (Table II). Although MOV induction could still be achieved when the distal MCAT element was mutated (␤293Mm), this element still appears to be necessary to achieve high levels of transgene ␤293 induction in response to MOV (Table II), implicating the requirement for additional element(s). In support of this notion, we have recently identified by EMSA analysis a putative MOV element (A/T-rich element (Ϫ269/Ϫ258)) that demonstrates enriched binding of two unidentified nuclear proteins (44 and 48 kDa) only under MOV conditions (8). Thus the ␤A/T-rich and distal MCAT elements may participate collaboratively to confer MOV responsiveness to transgene ␤293, a study currently under investigation.
In addition to our mutant MCAT transgenic findings, our EMSA analysis provides supportive evidence that the distal MCAT element contributes to slow fiber expression in the control soleus and MOV-P muscle. In direct and competition EMSA experiments we observed the formation of a specific LMC that varied in intensity according to the proportion of slow type I fibers within the adult stage rat skeletal muscles from which nuclear extract was isolated (Fig. 3). This EMSA binding pattern (CP Ͻ MOV-P Ͻ CS) is relevant to the in vivo expression pattern of our transgenes because the mouse and rat display a qualitatively similar pattern of slow type I fibers within these muscles.
Mutant Transgene ␤293Mm Does Not Display a Loss of Muscle Specific Expression-Previous studies by Rindt et al. (7) reported that mutation of the distal MCAT element within a 600-bp mouse ␤MyHC promoter (␤0.6MCAT) led to the loss of muscle specific expression (7). Regardless of the aforementioned findings, we did not detect measurable levels of transgene ␤293Mm in any nonmuscle tissue examined (data not shown). Several explanations may account for these different observations. First, the loss of mouse ␤MyHC transgene ␤0.6MCAT muscle specific expression was examined in only one transgenic line; therefore, this result may reflect chromosomal integration site effects as opposed to the loss of promoter regulation. Second, the levels of human transgene ␤293Mm studied herein are on average lower than those of mouse transgene ␤0.6MCAT, which may have resulted in our missing possible aberrant (nonmuscle) expression. Last, our mutation was restricted to a 5-nucleotide substitution within the core MCAT element, whereas the mutation within mouse transgene ␤0.6MCAT involved substitution of 19 nucleotides that spanned the core MCAT as well as both 5Ј and 3Ј regions (7). Based on the findings of Larkin et al. (20) concerning the requirement of the MCAT 5Ј-flanking nucleotides to achieve cTnT muscle specific gene expression, it is reasonable to suggest that mutation of the immediate 5Ј-flanking region may have altered muscle specific expression.
The ␤MyHC Distal MCAT LMC Is a Multiprotein Complex and Is Likely Comprised of TEF-1, PARP, and Max-Because of the relevance of previous findings showing physical interactions between NTEF-1 and Max (23) as well as RTEF-1 and PARP (22), it was important that we determine whether these same proteins comprise the LMC that forms at the ␤MyHC distal MCAT element when using adult stage MOV-P or soleus nuclear extract. Our EMSA and protein-protein interaction assays provide multiple lines of evidence that TEF-1, PARP, and Max most likely comprise a ternary protein complex that binds the ␤MyHC distal MCAT element to form the LMC. First, EMSA analysis revealed the formation of a LMC with a mobility that resembled the LMC formed at the cTnT element (Fig. 3). Second, in competition EMSAs, oligonucleotides containing either the ␣MyHC E-box/MCAT site or the cTnT MCAT1 element abolished the formation of most but importantly not all of the LMC formed at the ␤MyHC distal MCAT element. This is most apparent when using CS nuclear extract (Fig. 4, lane 13). Alternatively, these findings might also suggest that the proteins comprising the LMC at these three different MCAT elements may be the same but reflect differences in binding affinity. Third, the use of polyclonal anti-NTEF-1, PARP and Max, antibodies revealed that the LMC contained proteins antigenically related to TEF-1, PARP, and Max (Fig. 6). Fourth, our in vitro protein-protein interaction assays show that NTEF-1, PARP, and Max all physically interact with each other (Fig. 7). Alternatively, when considering the third and fourth issues above, it remains possible that two different populations of LMC exists in the form of TEF-1/PARP and TEF-1/Max. Although this is possible, it seems unlikely because there would be a difference of approximately 100-kDa between these two distinct LMCs. For example, a PARP (120 -140 kDa)/TEF-1 (54 kDa) protein complex would have a mass of 174 -194 kDa, whereas a TEF-1/Max (22 kDa) protein complex would have a mass of 76 kDa. This 100 -120 kDa difference in mass would likely be resolved in our EMSA assays. Last, in DNA binding assays, our experiments reveal that of the three proteins tested, only in vitro synthesized TEF-1 isoproteins bind to the ␤MyHC distal MCAT element (Fig. 8A), indicating that the putative ternary protein complex is tethered to this element via TEF-1 DNA-protein binding.
Despite the fact that some of the nuclear proteins that interact at the ␤MyHC, cTnT, and ␣MyHC MCAT elements are the same, our experiments show that the DNA binding properties of the ␤MyHC distal MCAT element differ, possibly explaining why Max and PARP did not bind this element. This important distinction is supported by differences in footprint patterns obtained for the ␤MyHC, cTnT, and ␣MyHC MCAT elements. Our footprint analysis (25) in conjunction with our current scanning mutagenesis results provide evidence that strong DNA-protein interactions occurred throughout the core MCAT element with weaker interactions in the flanking regions (Fig.  5). In contrast, similar analysis of the cTnT MCAT1 element revealed strong DNA-protein interactions occurring at the 5Јflanking nucleotides, the proposed site (5Ј-TGTTG-3Ј) of PARP binding that is conspicuously absent from the ␤MyHC distal MCAT oligonucleotide (Refs. 20 and 22 and Table I). Likewise, analysis of the ␣MyHC E-box/MCAT hybrid element revealed strong DNA-protein interactions spanning the 5Ј-flanking nucleotides containing the high affinity Max target binding site (CACGTG) that is also absent from the ␤MyHC distal MCAT oligonucleotide (Ref. 40 and Table I). The lack of Max DNA binding to our MCAT element was not completely unexpected because Max was identified as a neonatal cardiomyocyte nuclear protein component of a binding complex that formed at a cTnT core MCAT oligonucleotide devoid of an E-box element (23). Consequently, this same oligonucleotide was shown to be incapable of binding purified Max protein, showing that Max can participate in transcriptional regulation without binding DNA.
Although not an absolute certainty, we suggest that the conditions of our in vitro binding assays do not adequately emulate the in vivo biological conditions reflected in our adult stage skeletal muscle nuclear extract, thereby precluding independent binding by both Max and PARP, as well as the reconstitution of a LMC. For example, it is possible that something in the rabbit reticulocyte lysate may have inhibited DNA binding by both PARP and Max. Alternatively, these proteins may require a post-translational modification that occurs only in the context of intact adult stage skeletal muscle. Nevertheless, our data provide evidence that the ␤MyHC distal MCAT element LMC is primarily bound by TEF-1 protein and that PARP and Max may weakly interact with flanking nucleotide.
In summary, our results provide convincing evidence that formation of the ␤MyHC distal MCAT LMC when using either MOV-P or soleus nuclear extract is comprised of TEF-1, PARP, and Max and that this interaction correlates with slow muscle expression but is not required for MOV responsiveness of wild type transgene ␤293WT. Mechanistically, our findings suggest that TEF-1 binds to the core MCAT nucleotides where it serves to activate transgene ␤293WT transcription. Because Max does not contain a transactivation domain, its presence in the LMC may serve to mediate favorable interactions between the transcription initiation complex, as well as transcription factors bound at adjacent cis-acting elements via protein-protein interactions. PARP, on the other hand, has been shown to participate in diverse processes that include DNA repair, chromatin remodeling, and gene transcription (41). Thus, it is reasonable to speculate that nuclear PARP is recruited to the ␤MyHC distal MCAT element to activate ␤MyHC gene expression in slow muscle by altering local chromatin structure, or by modulating transcription factor activity via poly(ADP)-ribosylation. Support for the later notion comes from recent evidence showing the involvement of calcium in the activation of nuclear PARP in neuronal cells (42) and calcium-activated intracellular signaling pathways in regulating, in part, the slow skeletal muscle gene program (39). In our studies, calcium-activated mechanisms should be carefully considered because slow type I skeletal muscle fibers have been reported to have 2-6-fold higher basal levels of intracellular calcium than fast type II fibers (see Ref. 39 and references within), and MOV is associated with an increased proportion of slow type I fibers (6). Thus, it is plausible that the higher basal levels of intracellular calcium within the soleus muscle and within the induced slow type I fibers populating the plantaris following MOV resulted in elevated levels of activated nuclear PARP and thus enhanced LMC formation (Figs. 3 and 4). The physiological importance of the formation of the ␤MyHC distal MCAT LMC is underscored by our transgenic mouse analysis wherein mutation of the distal MCAT element led to a significant decrease in slow muscle expression of chromosomally located transgene ␤293Mm. The investigation into the involvement of calciumactivated pathways in the MOV induced increased proportion of slow type I fibers in adult skeletal muscle is currently underway.