INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

ADULT MOUSE HINDLIMB skeletal muscles express four major myosin heavy chain (MyHC) isoforms (fast IIb, IIx/d, IIa, and slow type I) whose differential expression pattern has contributed to the broad classification scheme that distinguishes four primary fiber types termed fast type IIb, IIx/d, IIa, and slow type I (or ). Each MyHC isoform is thought to serve a specific physiological role; therefore, variation in the proportion and spatial arrangement of each fiber type contributes to the biochemical and functional specialization of each muscle. The notion that each MyHC serves a physiological role is supported by the classic findings that actin-activated myosin ATPase and unloaded shortening velocity (V_max) are highly correlated to the amount and type of native isomyosin or MyHC comprising a given muscle or muscle fiber (2). For example, type I fibers primarily populate slow-twitch muscles, rely on oxidative metabolism, and express the MyHC, which is highly efficient in energy utilization while maintaining tension. Thus slow-twitch muscles are primarily used in chronic activities such as posture maintenance and for sustained locomotor activity. On the other hand, fast type IIb and IIx/d fibers are used for high-force burst activities, primarily populate fast-twitch muscles, rely on glycolytic energy production, and are less efficient in energy utilization while maintaining tension (4, 36).

Once established, the adult skeletal muscle phenotype is not static but instead retains the ability to adjust to variations in load bearing and contractile usage patterns, resulting in profound adaptations in morphology, phenotype, and contractile properties (1, 4). The removal of body loading in the microgravity environment of space flight results in decreased bone density, a marked degree of muscle atrophy, and an altered protein phenotype that correlates with a slow-to-fast change in contractile and metabolic properties for both rodents and humans alike (1, 10, 11, 14). Likewise, qualitatively comparable results have been obtained from animal studies by using a rodent ground-based model of simulated microgravity imposed by hindlimb unloading [non-weight bearing (NWB)] (1, 10, 11, 14). In addition to altered muscle mass, strength, and endurance, alterations in the pattern of motor nerve activity have been reported (3, 34). Thus it is not surprising that chronically innervated postural muscles such as the slow-twitch soleus are most susceptible to the effects of a microgravity or simulated microgravity environment. In agreement with the aforementioned findings, our studies on the NWB mouse soleus muscle have documented a loss in mass, a histochemical slow-to-fast fiber type shift, and a decrease in endogenous MyHC mRNA expression (29).

Transcriptional regulation is a fundamental mechanism by which adult-stage skeletal muscle phenotype and its adaptation are controlled. To identify regulatory elements that control MyHC gene transcription during NWB activity, we have performed a transgenic deletion and mutational analysis of the MyHC promoter. An expression analysis of transgenes comprised of either 5,600 or 600 base pairs (bp) of wild-type MyHC promoter revealed that expression had significantly decreased in response to NWB; however, this response was found to be substantially blunted when the MyHC proximal promoter control region (300/170; Fig. 1A) distal muscle-CAT (dMCAT), C-rich, and proximal MCAT (pMCAT) elements were simultaneously mutated (29). Further analysis of transgenes comprising either 350 or 293 bp of wild-type MyHC promoter revealed that transgene 350wt, which contains a negative element at its 5'-terminal end, did not express under basal or malleable (NWB or functional overload) conditions, whereas transgene 293wt was unexpectedly upregulated in response to NWB activity (30). The latter observation was of particular importance because it revealed that the 600/294 region of the MyHC promoter contained a NWB responsive element(s), a regulatory role possibly served by the negative (350/294) element. Our subsequent biochemical analysis of this sequence led to the elucidation of the first putative NWB element, termed dNRE-S (332/311), that bound two distinct proteins highly enriched in NWB soleus (NWB-S) nuclear extract (Fig. 1A; Ref. 30).

View larger version (20K): [in this window] [in a new window]   Fig. 1.   A: schematic representation of -myosin heavy chain (MyHC) proximal promoter. Depicted is the non-weight-bearing (NWB)-responsive region (600-294) containing the dNRE-S site, which may act as a NWB element. Also shown is the downstream control region (300/170) comprised of the distal muscle-CAT (dMCAT), A/T-rich, C-rich, proximal MCAT (pMCAT), and E-box/nuclear factor of activated T cells (NFAT) elements, which are highly conserved in sequence and location across species. B: schematic illustration of human MyHC transgenes. Wild-type transgene comprises 293 base pairs (bp) of the human MyHC proximal promoter plus 120 bp of 5'-untranslated region (+1 to +120) linked to the 5'-end of bacterial chloramphenicol acetyltransferase (CAT) gene-simian virus 40 poly(A) cassette (transgene 293wt). The small box to the left of the CAT gene represents exon 1. Regulatory elements comprising the MyHC proximal control region are shown from the 5'- to 3'-end and are termed dMCAT, A/T-rich, pMCAT, and E-box/NFAT. Mutant transgene 293Mm is identical to transgene 293wt, except that it harbors a mutated dMCAT element that was generated by bp substitution at nucleotide sites previously shown to be critical for protein-DNA interaction by footprint and scanning mutagenesis analysis (44). Bp substitutions (mutation) are depicted by lowercase letters and are presented 5' to 3'.

Our current questions regarding MyHC NWB regulation arise from two intriguing observations obtained from our above-cited transgenic analysis. First, the loss of normal NWB regulation of wild-type transgene 293wt occurred when the upstream NWB-responsive region (600/294, containing dNRE-S) was eliminated. Thus it is conceivable that deletion of this NWB-responsive region may have disrupted critical interactions between this element and those elements within the MyHC control region. In support of this notion, numerous studies using transgenic mice have shown that distinct regulatory modules (enhancers) and/or individual cis-acting elements are required to accurately duplicate diverse aspects of endogenous muscle-specific gene expression (9, 13, 16, 20, 25, 26, 32). Second, the partial loss of NWB regulation of the 5,600-bp MyHC promoter occurred when the MyHC control region (300/170) dMCAT, C-rich, and pMCAT elements were simultaneously mutated, indicating that these elements, or some combination of these elements, may provide key regulatory contributions necessary for complete NWB responsiveness. Because previous work has shown the involvement of MCAT elements in mediating both muscle-specific and inducible (muscle loading and hormonal stimuli) gene expression, they represent likely candidate elements for a regulatory role during NWB (6, 23, 24, 28, 44). Furthermore, an extensive electrophoretic mobility shift assay (EMSA) analysis to determine whether MCAT elements might be involved in NWB regulation has not been investigated. Therefore, one aim of this study was to investigate by EMSA analysis the potential contribution of the distal and proximal MCAT elements to the regulation of MyHC expression in response to NWB activity. Another goal was to determine the mechanistic basis underlying the unexpected upregulation of transgene 293wt after NWB. Thus we performed a transgenic mutagenesis analysis to test the hypothesis that the strong positive dMCAT element drives upregulation of transgene 293wt in response to NWB in the absence of the upstream NWB-responsive region (Fig. 1A).

Our transgenic analyses demonstrate conclusively that the dMCAT element underlies the atypical NWB response of transgene 293wt and, importantly, reveal that previously unidentified sequence(s) located downstream from the dMCAT element participate in NWB regulation. In addition, our EMSA analyses suggest that the dMCAT element contributes, at least in part, to NWB regulation of the MyHC gene by incurring decreased binding of transcriptional activator proteins, thus allowing negative regulation to proceed in the presence of other intact NWB-responsive elements. As regards the proximal MCAT element, our EMSA results indicate that the rat pMCAT element, but not the human pMCAT element, is likely involved in NWB regulation. Our EMSA analyses also reveal that the human and rat MyHC pMCAT elements display very different nuclear protein binding properties that are likely due to differences in nucleotide composition, because these differences were consistently observed whether using mouse or rat nuclear extract or in vitro synthesized human TEF-1 proteins. These data provide the first evidence that combinatorial interactions are required to confer complete NWB regulation of the human MyHC gene and that specific combinatorial interactions may differ with species.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Transgenes and site-directed mutagenesis. Wild-type transgene 293wt consists of 293 bp of human MyHC promoter sequence and 120 bp of 5'-untranslated region (UTR; includes exon 1), fused to the 5'-end of the bacterial chloramphenicol acetyltransferase (CAT) gene (Fig. 1B). The distal MCAT element within the human 293 promoter was mutated within the plasmid p293CAT using the QuikChange site-directed mutagenesis kit (Stratagene) according to the manufacturer's instructions. Detailed procedures for PCR-mediated incorporation of mutant distal MCAT sequence have been described elsewhere (44). Briefly, complementary oligonucleotide primers harboring mutations (bold lowercase) within the distal MCAT were designed as follows: 5'-GCATAGTTAAGCCAGCCAGCTGcGtctTagGAGGCCTGGCCTGGG-3. Bp substitutions were incorporated at nucleotides determined by our previous diethylpyrocarbonate interference footprinting to be crucial DNA protein contact sites (45). Unintentional transcription factor recognition sites were not created by these mutations as assessed by cross-referencing the mutated primers against the Eukaryotic Transcription Factor database (TFD) available on the Wisconsin Package [version 10.0; Genetics Computer Group (GCG), Madison, WI]. Successful incorporation of the mutation was verified via automated sequencing of both strands (Applied Biosystems, model 377). Wild-type (293wt) and mutant (293Mm) transgenes were isolated and purified as described previously (44).

Transgenic mice. Transgenic mice were generated by microinjection of purified transgene DNA into pronuclei of zygotes as described previously (40). Transgenic founder mice were identified by Southern blot analysis, and copy number was then estimated (47). Transgene-positive offspring were identified by PCR amplification by using primers specific for the CAT gene (47). All lines were maintained in a heterozygous state by backcrossing to the nontransgenic FVB/N parental mouse strain.

Animal care and NWB procedure. The Animal Care Committee for the University of Missouri-Columbia approved the NWB procedure used in this study, and the NWB mice were housed in an Association for the Assessment and Accreditation of Laboratory Animal Care International-accredited animal facility. All animals were provided with food and water ad libitum and were housed at room temperature (24°C) with a 12:12-h light-dark cycle in standard rodent cages (control animals) or in cages designed for head-down tilt hindlimb suspension (NWB) as described in detail previously (29). Adult female wild-type 293 (293wt) transgenic mice from line 2 are used in this study to show 1) control vs. NWB body and muscle weight and 2) basal and NWB expression levels (Tables 1 and 2). Basal and NWB expression levels for transgenic mice lines 99, 96, and 5 have been reported previously (30) and are used here to facilitate comparisons against basal and NWB expression data collected for transgenic mice carrying mutant transgene 293Mm (Table 2). Adult female 293Mm transgenic mice from lines 4, 6, 7, and 9 (22 g) were assigned to one of two groups: 1) a NWB group that used 2 wk of hindlimb suspension to impose NWB conditions (NWB; n = 8), and 2) a group that served as cage ambulatory controls for the NWB group (control; n = 8). After a 2-wk experimental period, both cage control and NWB mice were anesthetized and weighed, and their control soleus (CS) and NWB-soleus (NWB-S) muscles were collected for further study (Tables 1 and 2). At the same time, we collected gastrocnemius muscle (123 mg) from 40 adult (22g) control mice to be used for the isolation of nuclear extract. The gastrocnemius muscle was used for the isolation of nuclear extract because of the small size of the CS (7-8 mg) muscle. All control and NWB muscles were trimmed clean of fat and connective tissue, weighed, and stored at 80°C until assayed for CAT-specific activity (CS and NWB-S) or used for the isolation of nuclear extract (control gastrocnemius). All mice designated for terminal sample collection were anesthetized by using 2.5% avertin at a dosage of 0.017 ml/g of body wt. Mice were euthanized by cervical dislocation while under anesthesia.

View this table: [in this window] [in a new window]   Table 2.   Response of mutant transgene 293Mm to NWB.

CAT assays. CAT assays were performed as previously described (41). Muscle extracts were prepared from transgenic tissues by using a glass tissue homogenizer to disrupt tissues in 250 mM Tris · HCL (pH 7.8) and 5 mM EDTA. All muscle extracts were prepared from frozen tissue, and each n value represents the number of pooled soleus muscles from one mouse. The protein concentration of the extracts was determined by the method of Bradford (5). Muscle extracts were heated at 65°C for 10 min, followed by centrifugation at 10,000 g for 20 min. Because different transgenic lines exhibit inherently different transgene expression levels, it was necessary to use different amounts of tissue extract and variable incubation times so that the CAT enzyme activities could be determined within a linear range (30% conversion) as described previously (30, 39, 41, 48). The percent conversion of [¹⁴C]chloramphenicol to the acetylated form was quantified by using a PhosphorImager (Storm860) with ImageQuant version 5.1 software. Direct comparisons between and within transgenic lines representing both control and experimental groups (CS and NWB-S) were facilitated by presenting the data as specific CAT activity (picomoles per microgram of protein per minute) (Table 2).

Preparation of nuclear protein extract from adult skeletal muscle. Nuclear extracts (NE) were isolated from adult rat CS and NWB-S muscle and from mouse control gastrocnemius muscle as previously described (30). Three independent batches of CS, NWB-S, and control gastrocnemius nuclear extract were isolated and used in EMSA analysis and yielded the same results. Protein concentration was determined according to Bradford (5).

EMSA. All oligonucleotide probes used in this study are listed in Table 3. EMSAs were carried out as previously described (30, 43, 44). The double-stranded human and rat proximal MCAT oligonucleotide probes were labeled by fill-in reaction using Klenow fragment pf Escherichia coli DNA polymerase I (Stratagene, La Jolla, Ca) and [-³²P]dCTP (3,000Ci/mmol). The human distal MCAT oligonucleotide probe was end-labeled by T4 polynucleotide kinase (New England Biolabs, Beverly, Ma) and [-³²P]dATP (6,000Ci/mmol). All probes were purified by polyacrylamide gel electrophoresis before use in EMSAs. Binding reactions were performed by using either rat CS or NWB-S nuclear extract (dMCAT probe = 4.0 µg, pMCAT probe = 5.0 µg) or mouse control gastrocnemius nuclear extract (pMCAT probe = 5.5 µg) and 20,000 cpm of labeled probe for 20 min at room temperature in a 25-µl total volume. Where indicated, binding reactions contained 1 µl (rat pMCAT element) or 5 µl (human pMCAT probe) of in vitro-translated human NTEF-1 protein in place of muscle nuclear extract. 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 µl of either preimmune serum (PI) or the corresponding antibody for 30 min at room temperature, followed by the addition of the ³²P-labeled DNA probe. After electrophoresis, the gels were dried, and DNA protein complexes were visualized by autoradiography at 80°C.

In vitro transcription/translation. In vitro-translated protein was produced by using 1 µg of TEF-1 expression plasmid in the T7 transcription/translation (TNT) rabbit reticulocyte-coupled TnT system according to the instructions provided by the manufacturer (Promega). The expression plasmid corresponded to NTEF-1 [pXJ40-TEF-1, open reading frame (ORF) of human TEF-1] (21, 51). Parallel TNT reactions were performed in the presence of [³⁵S]methionine (Perkin Elmer). Efficient translation and expected molecular weights of the protein products were verified by resolving the radiolabeled reaction products on a sodium dodecyl sulfate-12% polyacrylamide gel (SDS-PAGE). Equivalent reactions of lysate not programmed with plasmid DNA were used as negative controls (unprogrammed lysate, UL).

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); poly(ADP-ribose) polymerase (PARP), rabbit polyclonal antibody raised against full-length human PARP (Boehringer Mannheim); and Max, rabbit polyclonal antibody raised against full-length mouse Max (Upstate Biotechnology). All antibodies listed hereafter were purchased from Santa Cruz Biotechnology 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) rat myogenin; upstream stimulatory factor-1 (USF-1), rabbit polyclonal antibody raised against carboxy-terminal amino acids 291-310 of human USF-1; E2A.E12, rabbit polyclonal raised against a peptide in the carboxy terminus of human E47 and corresponding to amino acids 422-439 of human E12; and HEB, rabbit polyclonal antibody raised against a peptide in the carboxy-terminal domain of human HEB (HTF 4).

Statistical analysis. All statistical analyses were performed by using SPSS Graduate Pack 10.0 program (SPSS, Chicago, IL). A Levene's test for equality of variances was performed, followed by a two-tailed independent-samples t-test used to assess differences between group means. Where the Levene's test was rejected (significance  0.05), the separate variance t-test for means was used, where equal variances were not assumed. All data are reported as means ± SE. The lowest significance level accepted was P < 0.05.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The formation of a highly specific and enriched low-mobility complex at the MyHC dMCAT decreases after 2 wk of NWB. To determine whether the binding properties of the MyHC dMCAT element differed between control and NWB conditions, we performed a direct and competition EMSA analysis using either CS or NWB nuclear extract. Binding reactions containing the ³²P-labeled wild-type dMCAT probe (Table 3) and either CS or NWB-S nuclear extract showed the formation of multiple binding complexes termed low-mobility complex (LMC), intermediate-mobility complex (IMC), and high-mobility complex (HMC) (Fig. 2). The formation of a LMC when using NWB-S nuclear extract was substantially reduced compared with the LMC formed when using CS nuclear extract, whereas those comprising the HMC increased (Fig. 2, lane 1 vs. 6). The addition of 100-fold molar excess cold wild-type MyHC dMCAT probe to binding reactions containing either CS or NWB-S nuclear extract completely abolished complex formation, indicating that these complexes are specific (Fig. 2, lanes 2 and 7). Interestingly, the addition of 100-fold molar excess cold cTnT MCAT1 probe [an element previously shown to form LMC, IMC, and HMC binding complexes (28)] to binding reactions containing either CS or NWB-S nuclear extract prevented IMC and HMC formation and partially competed away the LMC formation (Fig. 2, lane 3 vs. 8). The addition of 100-fold molar excess cold muscle creatine kinase (MCK) transcriptional regulatory factor x (Trex) element [an element that has relative sequence similarity to consensus MCAT site but that is shown not to bind TEF-1 (12)] to binding reactions containing either CS or NWB-S nuclear extract did not effectively compete for complex formation (Fig. 2, lane 4 vs. 9). Because the immediate 5'-flanking region of the MyHC dMCAT element contains a consensus E-box element, we examined whether E-box binding proteins were components of the LMC. The addition of 100-fold molar excess high-affinity MCK E-box as competitor to binding reactions containing either CS or NWB-S nuclear extract did not alter complex formation (Fig. 2, lane 5 vs. 10). These data show that specific binding complex formation at the dMCAT element differed when using CS vs. NWB-S nuclear extract, and this difference was characterized by a striking reduction in LMC formation only when using NWB extract. Also, known muscle regulatory factors (MyoD, myogenin) previously shown to bind the MCK E-box are likely not components of the specific binding complex formed at the MyHC dMCAT element.

View larger version (74K): [in this window] [in a new window]   Fig. 2.   Competition electrophoretic mobility shift assay (EMSA) assessment of sequence-specific DNA-protein binding at the MyHC distal MCAT element. 32P-labeled MyHC distal MCAT (dMCAT) oligonucleotide probe was incubated with 4.0 µg of either control soleus (CS; lanes 1-5) or NWB-soleus (NWB-S; lanes 6-10) nuclear extract. To characterize the sequence specificity of binding at the dMCAT, the following cold (nonradioactive) competitors were added to the reaction mixture at a 100-fold molar excess before the addition of the probe: dMCAT (lanes 2 and 7), cardiac troponin T (cTnT) MCAT (lanes 3 and 8), muscle creatine kinase (MCK) Trex (lanes 4 and 9), and the MCK high-affinity right E-box (lanes 5 and 10). Lane 11 contains no extract (free). Low-, intermediate-, and high-mobility complexes are designated LMC, IMC, and HMC, respectively. Free probe represents excess unreacted radiolabeled probe. A decrease in LMC formation and a concomitant increase in HMC formation was consistently observed with NWB-S nuclear extract (lane 6) relative to CS (lane 1). These experiments were repeated using 3 independent batches of nuclear extracts, which yielded the same results.

dMCAT element core nucleotides are required for DNA-protein binding complex formation. To determine whether different nucleotides within the 21-bp MyHC dMCAT element interact with CS vs. NWB-S nuclear protein, we performed competition EMSA analysis using scanning mutagenesis. In these experiments, we introduced nucleotide substitutions 2 bp at a time, starting within the immediate 5'-flanking region (E-box) and extending throughout the core MCAT element and its immediate 3'-flanking region (Fig. 3, A and B). The resulting unlabeled oligonucleotides were then added in 100-fold molar excess to binding reactions containing either CS or NWB-S nuclear extract or the ³²P-labeled wild-type dMCAT probe (Fig. 3A). As shown previously, the addition of excess wild-type MyHC dMCAT probe to binding reactions containing either CS or NWB-S nuclear extract completely abolished the formation of all binding complexes (Fig. 3A, lanes 2 and 12). Similarly, all binding complexes were effectively competed away by the addition of either dMCAT mut-1, mut-2 (mutations within E-box), or mut-7 (3'-flanking region) to binding reactions containing either CS (Fig. 3A, lane 1 vs. 3, 4, and 9) or NWB-S (Fig. 3A, lane 11 vs. 13, 14, and 19) nuclear extract. Importantly, mutant MCAT probes carrying nucleotide substitutions within the core MCAT element (mut-3, 4, 5, and 8) did not act as effective competitors of complex formation when added to binding reactions containing CS (Fig. 3A, lane 1 vs. 5-7 and 10) or NWB-S (Fig. 3A, lane 11 vs. 15, 16, 18, and 20) nuclear extract. Interestingly, the addition of dMCAT mut-6 probe to binding reactions containing CS (Fig. 3A, lane 1 vs. 8) or NWB-S (Fig. 3A, lane 11 vs. 18) nuclear extract partially competed for LMC formation, but not IMC or HMC. These data show that nucleotides comprising the core MyHC dMCAT element are critical for the formation of all binding complexes when using either CS or NWB-S nuclear extract (Fig. 3C).

View larger version (54K): [in this window] [in a new window]   Fig. 3.   Competition EMSA determination of specific nucleotides involved in DNA-protein interactions at the MyHC dMCAT element. A: 32P-labeled MyHC dMCAT oligonucleotide was incubated with 4.0 µg of either CS (lanes 1-10) or NWB-S (lanes 11-20) nuclear extract. Where indicated, 100-fold molar excess of either competitor (nonradiolabeled), dMCAT oligonucleotide (lanes 2 and 12) or various dMCAT mutant oligonucleotides (B) (mut 1-8, lanes 3-10 and 13-20), were added to the reaction before the addition of the probe. B: summary of competition EMSA with dMCAT mutant oligonucleotides. Wild-type dMCATwt represents dMCAT sequence devoid of mutations. The core MCAT sequence is indicated in gray stipple. Mutated bases are in lowercase and bold type, and bp that are unchanged from wt sequence are represented as dots. +++, Competition as effective as dMCATwt sequence; ++, partially effective competition; , no detectable competition. C: the dMCAT oligonucleotide is shown with MCAT core sequence in gray, and E-box is in bold face. The 2-bp numbering (1-7) above the oligonucleotide sequence corresponds to that particular mutant oligonucleotide shown in B. The heavy underline under LMC delineates nucleotides whose mutation renders the dMCAT an ineffective competitor, especially of the LMC, and suggests that this region is the putative binding site for the LMC. These results were found for each of 3 independent batches of isolated nuclear extract.

NTEF-1, PARP, and Max comprise the LMC formed at the MyHC distal MCAT element. Although we have shown previously that NTEF-1, PARP, and Max comprise the LMC formed at the dMCAT element when using CS nuclear extract, it is possible that different nuclear proteins form the LMC under NWB conditions (44). Thus we performed antibody EMSAs using polyclonal antibodies that recognize NTEF-1, PARP, or Max to determine whether these proteins comprised the LMC formed at the MyHC dMCAT element when using NWB-S nuclear extract or whether other proteins were components of this complex. The specific binding complexes formed at the ³²P-labeled human MyHC dMCAT element when reacted with NWB-S nuclear extract were not altered by preincubation with PI (Fig. 4, lane 2 vs. 3). The addition of polyclonal NTEF-1 antibody to binding reactions containing NWB-S nuclear extract supershifted only the top band of the IMC doublet and the prominent HMC band, whereas the LMC was partially abolished (Fig. 4, lane 2 vs. 4). Interestingly, the addition of either polyclonal PARP or Max antibody to binding reactions using NWB-S extract essentially immunodepleted the LMC, which resulted in an embellishment of the top band of the IMC, which entirely comprises NTEF-1 protein (Fig. 4, lane 2 vs. 5 and 6). When all combinations of the three polyclonal antibodies were added to binding reactions using NWB-S nuclear extract, the LMC was completely immunodepleted (Fig. 4, lane 2 vs. 7). The preincubation of binding reactions containing either CS or NWB-S nuclear extract with antibodies recognizing other E-box-binding bHLH (basic helix-loop-helix) and bHLH-Zip proteins (MyoD, myogenin, E2A, HEB, and USF) did not alter MyHC dMCAT element binding complex formation or mobility (unpublished observation). These data support several noteworthy conclusions: 1) proteins antigenically related to NTEF-1, PARP, and Max comprise the LMC formed at the human MyHC dMCAT element; 2) protein antigenically related to NTEF-1 likely constitutes the prominent HMC band and the top band of the IMC doublet; and 3) decreased binding of NTEF-1, PARP, and Max at the dMCAT element under NWB conditions may, in part, contribute to NWB decreases in MyHC gene expression.

View larger version (69K): [in this window] [in a new window]   Fig. 4.   Antibody EMSA analysis of NWB-S nuclear extract binding complexes at the MyHC dMCAT element. 32P-labeled MyHC dMCAT oligonucleotide probe was incubated with 4.0 µg of either CS (lane 1) or NWB-S (lanes 2-7) nuclear extract. EMSA analysis was performed by preincubation of NWB-S nuclear extract with 2 µl of either preimmune serum (PI; lane 3), NTEF-1 (lane 4), poly(ADP-ribose) polymerase (PARP; lane 5), or Max (lane 6) antibody for 30 min at room temperature before the addition of the probe. All 3 antibodies (NTEF-1, PARP, and Max) were included in the reaction mixture as shown in lane 7. The addition of an antibody to the reaction mixture is denoted (+) above the corresponding lane. Transcription enhancer factor (TEF)-1 SS marks the supershifted TEF-1 components of the IMC and HMC. Free probe represents excess unreacted radiolabeled probe. This assay was performed by using 3 different batches of nuclear extract, which yielded the same results.

Morphological changes after NWB. Body and soleus muscle weight data reported for transgenic mice harboring mutant transgene 293Mm represent pooled weights from transgenic lines 4, 6, 7, and 9 (Table 1). Compared with control values, 2 wk of NWB activity imposed by hindlimb suspension resulted in a significant decrease in the body wt of 293Mm (15.8%) and 293wt (19.3%) transgenic mice (Table 1). Similarly, significant decreases were measured in both absolute (293Mm = 27.1%; 293wt = 31.9%) and normalized (293Mm = 17.1%; 293wt = 16%) NWB-S muscle weight compared with control values (Table 1). These data are consistent with our previous findings showing that a 2-wk NWB treatment produces statistically significant decreases in both mouse body and muscle mass (29, 30).

Mutation of distal MCAT element restores NWB regulation and alters basal slow muscle expression of wild-type transgene 293wt. We examined the hypothesis that the dMCAT element is responsible for the unexpected upregulation of transgene 293wt in the absence of the upstream NWB-region by studying multiple independent lines of two distinct classes of transgenic mice (Fig. 1B). The first class of transgenic mouse carried a wild-type transgene comprised of 293 bp of human MyHC promoter fused to the CAT reporter gene (293wt). The second class carried the same transgene, except that the highly conserved dMCAT element was mutated (293Mm). Importantly, if this mutation restores NWB regulation, we will have elucidated the mechanistic basis underlying the atypical response of transgene 293wt after NWB and revealed that previously unidentified sequence(s) located downstream of the dMCAT element participate in NWB regulation.

Dissimilar binding of rat nuclear extract between human and rat MyHC pMCAT elements suggests a species difference in regulatory roles. The pMCAT element (210/203) resides downstream from the dMCAT element (290/284) and has been shown to play a regulatory role in the downregulation of injected rat MyHC reporter plasmids in the soleus muscle of spinal cord isolated rats (19). Because spinal cord isolation and NWB are associated with decreased soleus muscle MyHC gene expression, we have used an EMSA analysis to evaluate whether the human pMCAT element (210/203) serves a regulatory role in response to NWB. Incubation of ³²P-labeled human pMCAT element with rat CS nuclear extract resulted in the formation of three binding complexes (termed C1, C2, C3) that did not change in intensity when rat NWB-S nuclear extract was used (Fig. 5A, lanes 1 and 6). The addition of 100-fold molar excess cold wild-type human pMCAT probe to binding reactions containing rat CS nuclear extract competed away complex C2 only, whereas competition with 100-fold molar excess of mutant human pMCAT (pMCATm) element did not compete away complex C2 but did compete away complexes C1 and C3, indicating that only complex C2 represents specific binding (Fig. 5A, lanes 2 and 3). When binding reactions containing rat CS or NWB-S nuclear extract were preincubated with PI, complex formation at the human pMCAT element was not altered (Fig. 5A, lanes 1 vs. 4 and 6 vs. 7), whereas addition of polyclonal NTEF-1 antibody led to a supershift (SS) of only complex C2 (Fig. 5A, lanes 1 vs. 5 and 6 vs. 8). These data indicate that a nuclear protein antigenically related to NTEF-1 forms the low-intensity C2 specific binding complex at the human pMCAT element. However, because the intensity of this complex did not differ between control and NWB conditions, it is unlikely that the human pMCAT element plays a regulatory role under NWB conditions.

View larger version (67K): [in this window] [in a new window]   Fig. 5.   Competition and antibody EMSA analysis of human and rat pMCAT elements using CS or NWB-S extract. A: 32P-labeled human pMCAT probe was incubated with 5.0 µg of either CS (lanes 1-5) or NWB-S (lanes 6-8) nuclear extract. EMSA analysis of sequence specific binding at the human pMCAT element was determined by the addition of 100-fold molar excess nonradioactive (cold) wild-type or mutant human pMCAT element before the addition of the 32P-labeled human pMCAT probe. Note that only complex C2 is competed away by the addition of wild-type pMCAT element (lane 1 vs. 2), whereas the addition of mutant pMCATm probe does not compete for complex C2 (lane 1 vs. 3). For antibody supershift, EMSA rat CS or NWB-S nuclear extract was preincubated with 2 µl of PI (lanes 4 and 7) or polyclonal NTEF-1 antibody (lanes 5 and 8) for 30 min at room temperature before the addition of the probe. B: 32P-labeled rat pMCAT probe was incubated with 5.0 µg of either CS (lanes 9-13) or NWB-S (lanes 14-16) nuclear extract. Specificity of binding at the rat pMCAT element was determined by the addition of 100-fold molar excess nonradioactive (cold) wild-type or mutant rat pMCAT element before the addition of the 32P-labeled rat pMCAT probe. Note that only complex C2 is competed away by the addition of wild-type pMCAT element (lane 1 vs. 2), whereas the addition of mutant pMCATm probe does not compete for complex C2 (lane 1 vs. 3). For antibody supershift, EMSA rat CS or NWB-S nuclear extract was preincubated with 2 µl of PI (lanes 12 and 15) or polyclonal NTEF-1 antibody (lanes 13 and 16) for 30 min at room temperature before the addition of the probe. The anti-NTEF-1 antibody produced a supershift of binding complex C2 (human pMCAT, lanes 5 and 8; rat pMCAT, lanes 13 and 16). This assay was performed by using 3 different batches of nuclear extract which yielded the same results. C: alignment of MCAT core elements is delineated in bold type. Nucleotide differences between the human vs. rat pMCAT element are underlined on the human sequence. The overlapping rat pMCAT element is boxed.

Use of mouse nuclear extracts or in vitro-synthesized human TEF-1 proteins confirms a species difference in binding properties of TEF-1 at the human vs. rat MyHC pMCAT elements. To clarify whether the dissimilar binding of nuclear TEF-1 at the human vs. rat pMCAT element was due to differences in nucleotide composition and not species difference in nuclear TEF-1 protein, we performed an EMSA analysis using either mouse nuclear extract or in vitro synthesized human TEF-1 protein. EMSA analysis of binding reactions containing mouse control gastrocnemius nuclear extract and a ³²P-labeled human pMCAT element revealed three binding complexes (C1, C2, C3; Fig. 6) that were of very low intensity like those obtained when using rat nuclear extract. When 100-fold molar excess cold wild-type pMCAT element was added to the binding reaction, only complex C2 was abolished (Fig. 6, lanes 1 vs. 2). The addition of PI to the binding reaction did not alter the complex formation (Fig. 6, lane 3), whereas addition of polyclonal NTEF-1 antibody resulted in a SS of complex C2 only (Fig. 6, lane 4). Similarly, three binding complexes formed at the ³²P-labeled rat pMCAT element when mouse control gastrocnemius nuclear extract was used, and only complex C2 was competed away with the addition of 100-fold molar excess unlabeled wild-type pMCAT probe (Fig. 6, lane 5 vs. 6). In contrast, formation of complex C2 at the rat pMCAT element was highly enriched compared with the formation of complex C2 when using the human pMCAT element (Fig. 6, lane 1 vs. 5). Preincubation of binding reactions with PI did not alter complex formation at the rat pMCAT element (Fig. 6, lane 7), whereas addition of polyclonal NTEF-1 antibody resulted in a SS of complex C2 (Fig. 6, lane 8).

View larger version (67K): [in this window] [in a new window]   Fig. 6.   Analyses of mouse nuclear extract binding at the human and rat pMCAT elements. All binding reactions contained 5.5 µg of control mouse gastrocnemius (CG) nuclear extract and either 32P-labeled human pMCAT (lanes 1-4) or rat pMCAT (lanes 4-8) element. Specific binding was determined by the addition of 100-fold molar excess cold wild-type human (lane 2) or rat (lane 6) pMCAT before the addition of the 32P-labeled human or rat pMCAT probe. Note that formation of specific complex C2 is highly enriched when reacting the rat pMCAT element with control mouse gastrocnemius nuclear extract, whereas complex C2 is barely detectable when using the human pMCAT element. Antibody supershift EMSA was performed by preincubation of mouse control gastrocnemius nuclear extract with 2 µl of PI (lanes 3 and 7) or polyclonal NTEF-1 antibody (lanes 4 and 8) for 30 min at room temperature before the addition of the probe. Free probe (lane 9) represents the rat pMCAT probe resolved in the absence of mouse nuclear extract.

View larger version (80K): [in this window] [in a new window]   Fig. 7.   EMSA analyses of human and rat pMCAT element binding of recombinant NTEF-1. Inset reveals the correct size of [35S]methionine-labeled human NTEF-1. Rabbit reticulocyte lysate system was programmed with 1 µg of circular NTEF-1 in the presence of [35S]methionine. The transcription/translation product was resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and exposed to film. Molecular weight markers (in kDa) are shown at left. The lane marked UL (unprogrammed lysate) represents parallel reaction not programmed with TEF-1 expression plasmid. EMSA of 32P-labeled human pMCAT and rat pMCAT element reacted with in vitro-translated human NTEF-1 TnT product. Binding assays contained 1 µl of UL (lanes 1 and 6) or 5 µl of NTEF-1 TnT protein for the human pMCAT probe (lanes 2-5) or 1 µl of NTEF-1 TnT protein for the rat pMCAT probe (lanes 7-10). Sequence specific binding was determined by the addition of 100-fold molar excess cold wild-type human (lane 3) or rat (lane 8) pMCAT before the addition of the 32P-labeled human or rat pMCAT probe. Note the enriched binding of NTEF-1 at the rat pMCAT element (lane 7) vs. the human pMCAT element (lane 2). Antibody supershift EMSA was performed by preincubation of in vitro synthesized human NTEF-1 antibody(lanes 5 and 10) for 30 min at room temperature before the addition of the probe. Lane 11 represents free labeled human pMCAT probe, whereas lanes 12 and 13 show that NTEF-1 antibody reacted with either the human or rat pMCAT element, respectively, does not form a complex.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The genetic regulatory programs that control the adaptation of adult-stage skeletal muscle phenotype in response to decreased weight bearing are complex and remain incompletely understood. To gain insight into this process, our current study utilizes a transgenic mutagenesis and EMSA analysis to investigate whether two distinct MyHC proximal promoter MCAT elements (dMCAT and pMCAT) serve regulatory roles during NWB. Our transgenic study provides the first in vivo evidence that: 1) the dMCAT element is responsible for the unexpected upregulation of human transgene 293wt in response to NWB, and 2) regulatory sequence(s) located downstream from the dMCAT element are sufficient to direct NWB responsiveness. Our EMSA experiments are the first to reveal the existence of a species (human vs. rat) difference in MyHC pMCAT element binding of NTEF-1 under both control and NWB conditions, suggesting a possible species divergence in functional roles served by this element.

Transgenic mutagenesis analysis reveals that the dMCAT element is required to achieve high-level basal expression of transgene 293wt. MCAT regulatory elements are frequently found in the control region of numerous muscle genes and have been shown to mediate muscle-specific and inducible gene transcription. In our previous transgenic mutagenesis study, we found that mutation of the dMCAT element did not abolish induction of transgene 293Mm expression in the functionally overloaded adult plantaris muscle; however, basal expression levels of this transgene were significantly reduced (44). Likewise, our current transgenic analysis shows that mutation of the dMCAT element significantly decreases transgene 293Mm expression in the adult CS muscle, indicating that in the context of a 293-bp MyHC promoter, this element acts as a strong positive cis-acting sequence (Table 2).

Transgenic mutagenesis analysis reveals that the dMCAT element is responsible for the increase in transgene 293wt expression after NWB. Our observation that the dMCAT element acts as a strong positive cis-acting sequence offers insight into how the dMCAT element mediates the uncharacteristic upregulation of transgene 293wt in response to NWB. In this regard, it is plausible that in the absence of the upstream NWB-responsive region (600/294; Fig. 1A), interactions required for NWB downregulation would be disrupted, thereby allowing the dMCAT element (290/284) to exert a dominant positive effect on transgene 293wt expression in response to NWB. The transgenic results presented here support this notion by showing NWB decreased expression of a transgene containing a mutated dMCAT element (293Mm). In fact, decreases in transgene 293Mm expression were similar to those previously reported by us for larger (5,600 or 600 bp) MyHC promoter/transgenes (Table 2; Ref. 29), decisively demonstrating in vivo that the dMCAT element is responsible for the unexpected upregulation of wild-type transgene 293wt in response to NWB.

EMSA analysis suggests a role for the dMCAT element during NWB regulation of the MyHC gene. In this study, we have used EMSA analysis to investigate how a strong positive cis-acting regulatory element might function during a physiological process that normally results in negative regulation. One conceivable mechanism by which a strong positive cis-acting element can contribute to decreased expression levels of a given gene is by incurring changes in the type and/or amount of transcription factor binding. As concerns change in the type of transcription factor binding, our current antibody EMSA experiments show that the proteins interacting at the dMCAT element do not differ between control and NWB conditions. Specifically, antibody EMSA analysis determined that proteins antigenically related to PARP, NTEF-1, and Max comprised the LMC that formed at the MyHC dMCAT element when using either control or NWB nuclear extract (Fig. 4). This analysis also showed that other factors, such as the E-box binding proteins MyoD, myogenin, E2A, HEB, and USF, were not components of the binding complexes formed at the dMCAT element (data not shown). Furthermore, we showed by EMSA scanning mutagenesis that no difference existed in protein-DNA interactions at the dMCAT element because the same nucleotides comprising only the core MCAT element were involved in both control and NWB nuclear protein binding (Fig. 3).

Previously unidentified NWB element(s) reside downstream of the dMCAT element. In addition to determining that the dMCAT element directs the uncharacteristic upregulation of transgene 293wt, our transgenic analysis showing downregulation of transgene 293Mm expression after NWB provides the first in vivo evidence that previously unidentified NWB responsive element(s) reside downstream from the dMCAT element (Fig. 1A). Candidate elements that may fulfill this role that are located downstream from the dMCAT element (290/284) are the A/T-rich (269/258), C-rich (242/231), pMCAT (210/203, examined herein), and E-box/NFAT (179/171) elements. Our previous transgenic analysis revealed that the independent mutation of the A/T-rich element in the context of a 293-bp MyHC promoter/transgene resulted in the complete loss of expression in all 21 independent lines examined (CAT = 15 lines, luciferase = 6 lines) (44, 46). Similarly, E-box/NFAT mutation in 11 independent lines resulted in 4 lines that did not express and 7 lines whose basal expression levels were very low-rendering results, with NWB being unreliable (unpublished observation). Interestingly, a molecular model that accounts for slow fiber-specific gene expression and involves both A/T-rich and NFAT elements has been recently proposed. This model suggests that sustained elevations of intracellular calcium coactivate the calcineurin and calmodulin-dependent protein kinase signaling pathways and the subsequent transcriptional activation of slow fiber genes by various members of the myocyte enhancer factor-2 (MEF-2) and/or nuclear factor of activated T-cell (NFAT) transcription factor families (31, 33, 49, 50). Because NWB has been shown to result in a decrease in total muscle electrical activity and a slow-to-fast fiber-type transition (3, 34), it is conceivable that a decrease in nuclear protein binding at the A/T-rich and/or NFAT elements may contribute to reduced MyHC expression under NWB conditions. In fact, we have previously reported reduced levels of NFAT protein binding at the MyHC NFAT element (46). Although not all agree with the proposed model of slow fiber-specific expression (7), it will be instructive to reevaluate the involvement of the A/T-rich and E-box/NFAT elements in regulating MyHC expression during the NWB induced slow-to-fast fiber-type by using larger MyHC promoter/transgenes.

Divergence in pMCAT element sequences underlies species difference in NTEF-1 binding. The pMCAT element (210/203) is located downstream from the dMCAT element and represents a viable element that could confer NWB regulation. Previous EMSA analysis has revealed robust binding at this element when reacted with nuclear extracts isolated from a variety of differentiated myogenic cell lines (37, 38). In addition, mutation of this element within a 600-bp MyHC promoter representing several species (rat, human, rabbit, mouse) resulted in a significant reduction and/or abolished MyHC reporter gene expression when assayed in the in vitro context (37, 38). The pMCAT element has also been shown to confer inducible expression of the rat MyHC gene in response to ₁-adrenergic treatment of primary rat neonatal cardiomyocytes (23, 24). Mechanistically, the latter was shown by immunological techniques to involve the formation of a single binding complex (termed C2) that was comprised of TEF-1 protein. More recently, it was reported that the pMCAT element plays a role in the downregulation of rat MyHC plasmids injected into the soleus muscle of spinal cord isolated rats (19).