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Am J Physiol Cell Physiol 291: C300-C307, 2006. First published March 22, 2006; doi:10.1152/ajpcell.00345.2005
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GROWTH, DIFFERENTIATION, AND APOPTOSIS

An E box in the exon 1 promoter regulates insulin-like growth factor-I expression in differentiating muscle cells

A. S. McLellan,1 T. Kealey,1,2 and K. Langlands1,2

1University of Cambridge, Department of Clinical Biochemistry, Addenbrooke’s Hospital, Cambridge; and 2Clore Laboratory, University of Buckingham, Buckingham, United Kingdom

Submitted 12 July 2005 ; accepted in final form 17 February 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Insulin-like growth factor (IGF)-I expression is subject to complex temporal and spatial regulation. Endocrine synthesis occurs in the liver, where transcription is initiated from promoters located in either exon 1 (P1) or in exon 2 (P2), whereas local transcription is mainly initiated from P1. IGF-I is expressed in a range of tissues and, in particular, is an important regulator of skeletal muscle mass, although the mechanisms of tissue-specific regulation remain to be fully characterized. Gene regulation in skeletal muscle is associated with the E box DNA element (5'-CANNTG-3') recognized by myogenic regulatory factors (MRFs), such as MyoD1. Transcription element profiling identified a hypothetical myogenic E box (sequence 5'-CAGCTG-3') within P1, immediately upstream of the major muscle transcriptional start site, and we sought to test its activity in differentiating C2C12 myoblasts. We found P1-driven IGF-I mRNA expression to be associated with myogenic differentiation and, moreover, that a single base-pair mutation in the E box specifically reduced expression in myofibers. A synthetic enhancer construct containing a triplet repeat of the E box was active in muscle cells and strongly induced in myofibers. The capacity of a double-stranded IGF-I E box probe (but not one bearing a single-base pair alteration) to bind C2C12 nuclear lysates increased with myogenesis, and a transactivation assay demonstrated that the E box was recognized by E protein-MRF heterodimers. Mechanisms of tissue-specific gene activation are of increasing biological interest, and we have identified a cis-element able to direct muscle-specific IGF-I gene expression.

muscle development; gene expression regulation; transcription factor; myogenic regulatory factor

INSULIN-LIKE GROWTH FACTOR I (IGF-I or somatomedin C) is a small, basic, single-chain polypeptide of 70 amino acids with relative molecular mass of 7.5 kDa with both autocrine and paracrine actions in numerous cell types (5, 17). IGF-I is implicated in a variety of physiological processes, including, notably, the regulation of both proliferation and differentiation, and its expression is subject to exquisite control at both the transcriptional and translational levels (37). Endocrine IGF-I is synthesized in the liver, with its message being transcribed from promoters located in either exon 1 (P1) or exon 2 (P2) (36). IGF-I synthesis also occurs in peripheral tissues where its transcription is generally initiated from the exon 1 promoter, in which case exon 2 is spliced from the pre-mRNA (15, 42). While exons 3 and 4 encode the mature peptide, the alternative usage of exons 5 or 6 (which encode the E peptide) is associated with the synthesis of the propeptides IGF-Ib or IGF-Ia, respectively (34). The functionally inactive propeptides are cleaved at a unique pentabasic prohormone cleavage motif in the NH2-terminus of each E peptide to yield the functional, secreted forms of IGF-I (7).

Deletional analysis has identified a number of sequences upstream of exon 1 involved in tissue-specific IGF-I expression; indeed, this 5'-untranslated region (UTR) of the IGF-I gene maintains the highest degree of conservation through evolutionary time (25). Wang et al. (42) identified two minimal promoters within a 495-bp region responsible for maximal basal exon 1 activity in a range of cell lines. This region contains four potential transcriptional start sites (TSSs) and a number of transcription factor-binding sites, including those recognizing hepatocyte nuclear factor 1 (Hnf1), CCAAT/enhancer-binding proteins (C/EBP), and GATA elements (26, 27, 41). In particular, core promoter elements responsible for gene expression in a variety of cell types appear to cluster around TSS3 (42). Interestingly, a potential MyoD1-like E box sequence lies ~76 nt downstream of the first TSS (TSS1, numbered according to the human sequence) and ~62 nt upstream of TSS3, which is also the major TSS used in skeletal muscle (11, 18, 36). Furthermore, this element and its flanking nucleotides are highly conserved through evolutionary time; thus we hypothesized that this element may have a role in the regulation of IGF-I expression in skeletal muscle.

IGF-I is a key regulator of skeletal muscle mass, and IGF-I knockout mice display skeletal muscle hypoplasia, whereas ectopic IGF-I expression is associated with hypertrophy (2, 37). A transcriptional variant of IGF-I known as mechanogrowth factor (MGF) is transiently expressed in skeletal muscle immediately after mechanical damage and is thought to be involved in satellite cell activation (12). Myogenic differentiation or muscle loading is also accompanied by an increase in systemic IGF-I mRNA (IGF-Ia), although the cis-elements regulating this process remain to be elucidated (8, 24). It has been previously shown that ectopic MyoD1 increased activity from exon 1 promoter constructs in vitro, although the elements mediating this effect are also unknown (24).

The differentiation of myoblasts into contractile myofibers is specified by a sequential pattern of basic helix-loop-helix (bHLH) transcription factor activation (for a review, see Ref. 29). The myogenic regulatory factors (MRFs; MyoD1, Myf5, myogenin, and Myf6) form heterodimers with the widely expressed E proteins (E12, E47, ITF2, and HEB), and these complexes bind to distinct E box DNA elements (with the consensus sequence 5'-CANNTG-3') located in the promoters of muscle-associated genes to provide temporal and spatial control of gene expression (23). A number of Web-based computational tools can assist in the identification of potential regulatory elements in noncoding regions of DNA, notably, MatInspector (32), TRANSFAC (19), and the transcription element search system (TESS; http://agave.humgen.upenn.edu/tess/index.html). Informed by previously characterized cis-elements, such applications use precompiled weight matrixes to assess the likelihood that any DNA sequence is a potential target for DNA binding proteins. Scanning any promoter sequence inevitably returns multiple hits, and thus it is incumbent upon the investigator to refine the search strategy to minimize this background noise and so reduce the number of targets for subsequent analysis. Rather than using a preexisting weight matrix populated by a broad range of myogenic E box elements, we derived a bespoke matrix from functional skeletal muscle elements sharing a specific core, thereby improving both the specificity and relevance of our search. Moreover, as genetic drift is strongly selected against in regulatory noncoding regions over evolutionary time, conservation provides an additional clue to function. We sought to combine these approaches to improve our discrimination of any uncharacterised E box located in the IGF-I exon 1 promoter.

IGF-I expression in peripheral tissues is specified by a great many factors and so provides an excellent model for the study of gene activation. The characterization of cis-elements in the IGF-I promoter will, therefore, contribute to our understanding of IGF-I biology in particular and gene regulation in general. We tested the ability of differentiating mouse muscle cell line (C2C12) preparations to activate a range of reporter constructs and bind E box DNA elements to determine if a hypothetical E box in the 5'-UTR of IGF-I exon 1 was involved in the temporal and spatial regulation of IGF-I expression.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
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 REFERENCES
 
All reagents were obtained from Sigma-Aldrich (Poole, UK) unless otherwise indicated. Wherever possible, HUGO standard gene nomenclature is used (www.gene.ucl.ac.uk/nomenclature).

Tissue culture. C2C12 mouse myoblasts, OVCAR-3 human ovarian carcinoma, and HeLaB human cervical adenocarcinoma cancer cell lines were obtained from the American Type Culture Collection. Cells were maintained as standard in either RPMI (OVCAR-3) or DMEM (C2C12 and HelaB cells) supplemented with 10% FBS. C2C12 differentiation was induced by substituting 2% horse serum for 10% bovine serum, and cultures were maintained for 2 days (enriching for myotubes) or 4 days (myofibers).

Real-time RT-PCR. RNA was extracted from C2C12 myoblasts, myotubes, and myofibers in triplicate using Qiagen RNAeasy columns (Qiagen; Crawley, UK). Adult female CD1 mouse livers and skeletal muscle (hindlimb) were homogenized in 1 ml TRI-reagent, and total RNA was isolated as a standard. First-strand cDNA was synthesised using 1 µg total RNA as a template in a 20-µl reaction using random hexamer priming and Moloney murine leukemia virus reverse transcriptase (Invitrogen; Paisley, UK). PCR primers for IGF-I promoters located in either exon 1 (P1) or in exon 2 (P2) were each paired with primers for IGF-I exon 4 (Table 1) to measure IGF-I P1- and P2-derived expression, respectively. IGF-I expression data were normalized to the expression of the housekeeping gene hypoxanthine-guanine phosphorybosyl transferase-1 (HPRT1; Table 1). Real-time PCR was performed using an ABI PRISM 7900 sequence detection system (Applied Biosystems; Warrington, UK) and a QuantiTect SYBR Green PCR Kit (Qiagen) in a total volume of 12 µl in triplicate wells, with each containing 1 µl cDNA and 600 nM of each primer. A no-template control was also prepared in triplicate for each primer set. Furthermore, a duplicate 12-step 1:2 serial dilution series was prepared for each primer set, starting with a mixture of 1 µl liver and 1 µl skeletal muscle cDNA per 12-µl reaction. This allowed the ABI SDS software to prepare a standard curve from which cycle threshold values were transformed into arbitrary quantity data. The following PCR protocol was used: a denaturation program (95°C for 15 min) with 40 cycles of an amplification and quantification program (95°C for 15 s, 55°C for 30 s, and 72°C for 45 s with a single fluorescence measurement taken during the extension stage) and a melting curve program (60–95°C with a heating rate of 1.0°C/30 s and continuous fluorescence measurement). Thereafter, PCR product quality was assessed by generating a melting curve, which was also used to verify the absence of PCR artifacts (primer-dimers) or nonspecific PCR products. Results are described as mean IGF-I expression relative to mean HPRT1 expression.


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  		Table 1. Oligonucleotide sequences

 
Promoter analysis. Human, mouse, rat, cow, goat, sheep, salmon, and zebrafish exon 1 sequences were aligned using CLUSTALW, and a consensus sequence was created from the resulting multiple alignment using the European Molecular Biology Open Software Suite program CONS (33, 38). A bespoke weight matrix was constructed from 11 functional skeletal muscle-specific E box elements sharing the 5'-CAGCTG-3' core, along with four flanking nucleotides each side (Table 2). TESS software readily accepts the use of user-defined weight matrixes, and this package was used to scan the consensus promoter sequence for high scoring matches (scores > 10).


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  		Table 2. Construction of a myogenic E box weight matrix

 
Plasmids. The IGF-I630/Luc construct, containing nucleotides –1630 to +322 of the human IGF-I exon 1 sequence cloned upstream of a luciferase (Luc) reporter gene, was a kind gift from Peter Rotwein (Oregon Health Sciences University, Portland, OR) (18). The IGF-I promoter was subcloned as a PstI fragment into the pGL3-Basic reporter vector (Promega; Southampton, UK), which lacks promoter and enhancer elements, to create pGL1630. The pGL3-basic polylinker was previously modified by the insertion of a cassette containing BglII, NdeI, EcoRV, PstI, and HindIII sites into the BglII/HindIII-digested vector, creating pGL3+. The wild-type (WT) 5'-CAGCTG-3' E box sequence in the pGL1630 construct was altered to 5'-AAGCTG-3' with QuickChange site-directed mutagenesis (Stratagene; Cambridge, UK) according to the manufacturer’s recommendations to generate pGL1630E–. This substitution removed a PvuII site, facilitating ready identification of mutants, although the integrity of the altered sequence was also verified by sequencing. The synthetic E box enhancer vector containing three copies of the putative IGF-I E box (pGLEbox) was created by inserting a double-stranded oligonucleotide containing the multimerized E box sequence (5'-CAGCTG-3' core flanked by 10 nucleotides) into a pGL3-promoter vector (Promega). The muscle creatine kinase (CKM) enhancer region [which contains three E boxes as well as myocyte-specific enhancer factor (MEF)-2 binding sites (13)] was PCR amplified from rat genomic DNA and cloned as a Sal1/BamH1 fragment into the pGL3-promoter vector. All other plasmids (full-length MyoD1, Myf6, and E47 cDNAs in a pRcCMV background) were as previously described (20). Oligonucleotides (MWG Biotech; Munich, Germany) are detailed in Table 1.

Transient transfections and reporter assays. Cells were cotransfected with 1 µg of each relevant plasmid and 1 µg of a beta-galactosidase reporter (BD Clontech; Oxford, UK) using FuGeneII reagent (Roche; Lewes, UK) at a 3:1 ratio of FuGeneII-DNA in six-well tissue culture plates. Unless a time course experiment was being performed, cells were harvested 48 h posttransfection. Luc activity in cell lysates was determined using a Promega Luciferase Assay Kit as recommended. Transfection efficiencies were normalized relative to beta-galactosidase activity, and all experiments were performed in duplicate.

EMSAs. Nuclear proteins were prepared using the method of Schreiber et al. (35), and shifts were performed using the Pearce LightShift Chemiluminescent EMSA kit (Pearce). Binding was performed in 1x EMSA buffer [25 mM HEPES (pH 7.9), 40 mM KCl, 3 mM MgCl2, 0.1 mM EDTA, 1 mM DTT, and 10% glycerol] containing 50 ng/µl polydIdC, 4 µg nuclear extract, and 1 fmol/µl double-stranded biotinylated probe (Table 1; each strand was synthesized with a single 5'-biotin moiety). After the samples were incubated for 20 min at room temperature, 5x loading buffer (Pearce) was added, and reactions were resolved by 6% polyacrylamide gel electrophoresis (Novex, Invitrogen) before a semidry transfer to Hybond N+ membranes (Amersham Pharmacia; Little Chalfont, UK). Shifts were visualized with LightShift reagents, according to the manufacturer’s recommendations, and subsequent exposure to X-ray film (BioMax, Amersham Pharmacia). Densitometric analysis of images was performed using AlphaEase imaging software (Alpha Innotech; San Leandro, CA). Quantitative binding values were extracted from triplicate independent experiments.

Statistical analysis. Unless otherwise stated in the text, Luc levels obtained from promoter constructs and promoterless parental vectors in the ectopic transfection assays were compared using paired t-tests. For real-time RT-PCR and quantitative EMSA analysis, values obtained from either myotubes or myofibers were compared with those obtained from myoblasts. All graphs represent mean values ± SE, and statistical analysis was performed using Excel (Microsoft).


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
IGF-I expression increases with myogenesis. Real-time RT-PCR analysis indicated a 4.7-fold increase in P1 transcripts with myogenic differentiation (P = 0.0008; Fig. 1) and, moreover, that P1-derived transcripts were the most abundant species in adult mouse skeletal muscle. Whereas higher levels of P1 transcripts were evident in liver RNA compared with muscle, the predominant RNA species in the former tissue were transcribed from the P2 promoter. Low levels of P2 transcripts were detected in C2C12 RNAs, both before and after differentiation, and in skeletal muscle samples.


Figure 1
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  		Fig. 1. IGF-I expression in C2C12 cells and primary mouse tissues. Real-time RT-PCR was performed using either exon 1 transcript-specific (P1) or exon 2 (P2) transcript-specific primers in conjunction with an exon 4 reverse primer (P4). A 4.7-fold increase in P1 activation was detected in both myotubes and myofibers relative to myoblasts. Significant increases in gene expression are indicated as follows: *P < 0.05 and **P < 0.001.

 
Analysis of a candidate E box in the IGF-I exon 1 promoter. Running a promoter scan under TESS using standard TRANSFAC matrixes identified 12 potential E boxes with the consensus 5'-CANNTG-3' in a 2.5-kb region of the human exon 1 promoter. A subsequent TESS search using the TRANSFAC myogenic E box weight matrixes identified four high-scoring elements (each with a likelihood score > 10). However, before any experimental evaluation was performed, we sought to increase the likelihood that any elements identified were functional. Multiple alignments of exon 1 promoter sequences from diverse species identified a region of ~2-kb that is highly conserved through evolutionary time. A subsequent TESS scan of a consensus promoter derived from this multiple alignment identified two highly conserved 5'-CAGCTG-3' elements (at positions –837 and +76 relative to the first TSS of the human IGF-I gene). A final TESS search using our bespoke myogenic matrix (Table 2) identified one high-scoring element at position +76 as significant (generating a likelihood score 14.7 of a maximum of 18). The sequence of this element, as well as its conservation in a range of other species, is shown in Table 3. For clarity, the results of the initial searches are not shown.


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  		Table 3. Phylogenetic relationships of the IGF-I E box and flanking sequences

 
IGF-I P1 is active in OVCAR-3, HeLaB, and C2C12 myoblasts. The 1952-bp exon 1 fragment significantly increased Luc activity in OVCAR-3 (12-fold, P = 0.014), C2C12 (7-fold, P = 0.05), and HeLaB cells (9-fold, P = 0.007) over background (Fig. 2). A single-base pair mutation in the putative E box region had no significant effect on Luc expression in any cell line relative to the WT promoter; however, the synthetic enhancer construct containing three tandem repeats of this E box was highly induced in C2C12 myoblasts (11-fold, P = 0.003). Little induction of the multimerized E box enhancer construct over background was detected in OVCAR-3 cells, although an increase in activity was also detected in HeLaB cells (6.5-fold, P = 0.033).


Figure 2
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  		Fig. 2. IGF-I promoter activity in cell lines. Activation of IGF-I promoter constructs and parental vector controls (pGL3+ for the pGL1630 vectors and pGL-promoter for the pGLEbox vector) in OVCAR-3, C2C12, and HeLaB cells is shown. The significance of luciferase induction relative to parental vector is indicated as follows: *P < 0.05, **P < 0.02, ***P < 0.01, and ****P < 0.005.

 
IGF-I promoter activity during C2C12 differentiation. Luc expression induced from the exon 1 promoter fragment was detected in proliferating myoblasts and maintained during myogenesis (Fig. 3). However, the destruction of the putative IGF-I E box in the exon 1 promoter resulted in a significant decrease in Luc activity in myofibers (2.2-fold, P = 0.015). The increase in ectopic Luc expression stimulated by the multimerized IGF-I E box enhancer element during C2C12 differentiation was more dramatic (6-fold, P = 0.007), although the magnitude of the change was not as great as CKM enhancer-driven expression (134-fold, P = 0.03) due to a higher level of E box enhancer vector transcription in myoblasts.


Figure 3
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  		Fig. 3. IGF-I promoter activity during myogenic differentiation. Luciferase activation resulting from ectopic IGF-I exon 1 and muscle creatine kinase (CKM) enhancer construct activity in C2C12 myoblasts and myofibers is shown. The significance of increased luciferase levels relative to parental vector is indicated as follows: *P < 0.05, **P < 0.02, ***P < 0.01, and ****P < 0.001. Results from C2C12 cells maintained in nondifferentiating medium (DMEM supplemented with 10% bovine serum) for 4 days after reaching confluence (postconfluent) are also shown.

 
EMSAs. The ability of C2C12 nuclear lysates prepared at various differential stages to retard the migration of biotinylated probes derived from the CKM right E box, the WT IGF-I E box, or the mutated IGF-I E box bearing a single-base pair alteration was evaluated using a nonradioactive EMSA method (Fig. 4). As expected, an increase in CKM E box binding characterized myogenic differentiation, whereas only low levels of binding to the mutated IGF-I probe were seen. Densitometric analysis of WT IGF-I probe EMSAs indicated a 1.5-fold increase in binding of C2C12 myofiber nuclear proteins relative to myoblasts (P = 0.045).


Figure 4
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  		Fig. 4. Binding of C2C12 nuclear lysates to E box probes. EMSAs were performed using biotinylated CKM-R E box (CKM), IGF-I E box [wild type (WT)] and mutated (MUT) IGF-I E box probes incubated with C2C12 nuclear lysates prepared from cells at preconfluence (myoblast) and at 2 and 4 days of differentiation (myotube and myofiber, respectively). A typical experiment is shown.

 
bHLH protein activation of IGF-I promoters. Reporter gene activation by any ectopic bHLH factor in isolation led to only small increases in Luc activation over background (relative to parental promoter vectors) with the exception of MyoD1 homodimers, which were able to significantly activate the CKM (5-fold, P = 0.006), IGF-I exon 1 (8-fold, P = 0.017), and multimerized E box constructs (1.6-fold, P = 0.015) (Fig. 5). Myf6 was also able to activate the CKM promoter over background in the absence of an ectopic binding partner (2.2-fold, P = 0.017). However, higher levels of transactivation were obtained by the expression of heterodimers with E47-MyoD1 complexes inducing the CKM enhancer (4-fold, P = 0.002), multimerized IGF-I E box (2.6-fold, P = 0.011), and WT promoter 1 construct (21-fold, P = 0.0004). E47-Myf6 complexes also activated the exon 1 promoter (6-fold, P = 0.007) and CKM enhancer (4-fold, P = 0.02), although the effect of this complex on the synthetic IGF-I E box enhancer was not found to be significant. The single-base pair change in the exon 1 promoter E box element consistently resulted in a significant decrease in Luc activity relative to the WT sequence in response to MyoD1 homodimer and heterodimer and Myf6 heterodimer action (2-fold, P = 0.04; 3-fold, P = 0.03; and 1.5-fold, P = 0.007, respectively).


Figure 5
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  		Fig. 5. Transactivation of IGF-I and CKM promoter vectors by basic helix-loop-helix (bHLH) factors. HeLaB cells were cotransfected with various promoter constructs and full-length myogenic regulatory factors (MyoD1 or Myf6) or E protein (E47) expression vectors in either homo- or heterodimeric combinations. The significance of increased luciferase expression relative to the appropriate parental vector controls is indicated as follows: *P < 0.02, **P < 0.01, ***P < 0.002, and ****P < 0.0005. {dagger}Significant reduction in reporter activity resulting from a single-base pair mutation in the IGF-I exon 1 promoter (P < 0.05).

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
An increasing number of cis-regulatory elements are known to initiate both local and endocrine IGF-I expression, particularly those associated with the well-conserved exon 1 5'-UTR (18, 26, 39, 41, 42). However, a clear understanding of the mechanisms specifying compartmental IGF-I expression is yet to be achieved. Particularly, IGF-I is an important regulator of skeletal muscle physiology, and its study is important toward the treatment of muscle wasting or atrophy associated with age, with wider implications for the elucidation of tissue-specific gene regulatory networks.

The exon 1 5'-UTR is the most highly conserved region of the IGF-I gene, suggesting that the most fundamental regulatory elements map to this area (25). While promoter-scanning programs provide valuable tools in the identification of those regions capable of specifying gene expression, it is imperative that a clear understanding of the underlying biology informs any search strategy. The use of such a rational approach facilitated our characterization of a potential myogenic E box element immediately upstream of the major exon 1 TSS. The conservation of this E box and its flanking nucleotides from zebrafish to humans increased our confidence that this element was functional and so merited functional investigation. Real-time PCR analysis showed that the induction of IGF-I mRNA species characterized C2C12 differentiation and, moreover, that this transcription was initiated from P1, the majority peripheral promoter. An ~1.9-kb fragment of the human IGF-I exon 1 5'-UTR highly active in the neuroblastoma cell line SK-N-MC, which express mainly IGF-I exon 1 transcripts, was also shown to be active (albeit to a lesser extent) in OVCAR-3 cells, which express mainly IGF-I exon 2 transcripts (15). We used a related 1952-bp fragment (which includes additional 3'-exon 1 DNA), which was also shown to be highly active in SK-N-MC cells (18). However, we found reporter activity to be low when we introduced this construct into OVCAR-3 cells. Assuming this to be a limitation of the vector backbone, we subcloned the entire fragment into a pGL3-basic Luc reporter vector. This produced detectable Luc activity in OVCAR-3 cells with minimal activition in HeLaB cells, which express little IGF-I (data not shown), whereas proliferating C2C12 cells activated the reporter to an intermediate level. The E box knockout had little influence in any of these cell lines; however, the synthetic E box enhancer construct strongly induced Luc expression in C2C12 cells. This suggests that the E box is not active in myoblasts but has the potential to be activated by myogenic factors.

It appears contradictory that the upregulation in IGF-I message was not accompanied by P1 activation during myogenic differentiation. Indeed, McCall et al. (24) reported that a series of P1 fragments were also unresponsive during C2C12 differentiation. This is consistent with the 1952-kb IGF-I fragment spanning a minimal basal promoter region, and its activation in myoblasts is, at least in part, as a consequence of the lack of distal repressor elements and not due to activation of the E box per se (further confirmed by the minimal effect of destroying this element). Differentiation is, however, accompanied by myogenic activation of the E box because its mutation did significantly reduce P1 activity in myofibers but not in any other cell line studied. More compelling evidence for E box-mediated expression was provided by the muscle-specific activation of the multimerized IGF-I E box construct, which generated Luc levels comparable with those obtained by the CKM enhancer. Strong E box-driven activation of skeletal muscle-associated genes is usually associated with occupancy of one or more E box elements as well as the cooperative binding of proximal accessory factors such MEF2, serum response factor, or Sp1 (for a review, see Ref. 43). Intuitively, one would predict that searching for clusters of closely mapping promoter elements would increase predictive power. However, such clustering analysis was shown to identify <50% of known skeletal muscle promoters; thus such a strategy is of only limited value in the evaluation of previously uncharacterised cis-elements (43). Our observations are consistent with a relatively weak (in its natural context) yet highly specific regulatory sequence that would ensure the fine control of IGF-I protein levels in skeletal muscle.

C2C12 myoblast nuclear extracts were able to bind synthetic olignucleotides derived from both CKM and, to a lesser extent, IGF-I E box (WT) sequences. Because myoblasts express high levels of ID proteins, which sequester free E proteins, these shifted bands may indicate MyoD1 homodimer binding (16). Consistent with the differentiation-associated downregulation in ID expression, increased binding was detected in myofiber protein preparations, presumably as a result of E protein-MRF heterodimer formation. However, it is difficult to predict the precise composition of these DNA-binding complexes as any of the four E proteins can form heterodimers with the four MRFs (20). Furthermore, DNA binding does not necessarily indicate efficient transactivation, so we used an ectopic expression assay to assess the influence of E proteins and MRFs, either in isolation or as pairs. MyoD1 expression is associated with commitment to myogenic differentiation, and Myf6 is a later-acting protein whose expression is maintained in terminally differentiated muscle fibers, whereas E47 is more widely expressed (23, 29). These experiments were performed in a HeLaB background because these cells have a relatively low ID and E protein background [Refs. 4 and 14, and data not shown], but we cannot exclude the possibility that apparent MyoD1 homodimer transactivation was facilitated by endogenous E protein dimerization.

Ectopic MyoD1 was able to transactivate each promoter above background levels, with the greatest induction seen in the activity of IGF-I P1, followed by the CKM enhancer, with a small but significant increase in the activity of the pGLEbox construct. As expected, dimerization with E47 greatly facilitated activation, with both native P1 and multimerized E box construct activity increasing in particular. Myf6 was able to transactivate only weakly as a homodimer, but E47 dimerization facilitated a dramatic rise in CKM and P1 activation. The predominant MRF after 4 days of C2C12 differentiation is myogenin; however, we were unable to generate a myogenin vector with comparable expression levels to those of MyoD1 and Myf6 (data not shown). Myf6 expression is sustained in mature myofibers [indeed, it is the only MRF detected at this stage (29)], and thus it has the potential to maintain low levels of IGF-I expression in terminally differentiated tissue.

There has been a postgenomic perspective shift from gene function to gene regulation with the increasing availability of distal regulatory elements that may map many kilobases from the transcriptional start site of a gene. Myogenic regulation of IGF-I activity is complex, but we have identified a single element conferring muscle-specific gene expression, although the full constellation of promoter-binding factors remains to be elucidated. The study of IGF-I expression provides an excellent model for tissue-specific gene activation as its transcription is regulated by multiple elements located in multiple promoters active in multiple cell types. Furthermore, phylogenetic analysis facilitates the identification of those sequences subject to the strongest selection pressure and so central to physiological control. A greater understanding of those cis-acting IGF-I elements active in the myogenic program will expand the promoter map and so enrich our understanding of regulatory networks. Moreover, the growth in gene regulatory network analysis must be accompanied by progressively sophisticated methods to confidently identify cis-elements if predictive developmental maps are to be realized (3). It is hoped that the rational approach described herein to the identification of a myogenic control element will complement such analysis.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
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This work was funded by a grant from Unilever Research.


   ACKNOWLEDGMENTS
 
We are grateful to Dr. P. Rotwein for the kind gift of the IGF-I630/Luc construct and to Dr. Tom Moore (University of Cork) for providing real-time RT-PCR facilities and support.

Present address of A. S. McLellan: Dept. of Biochemistry, Univ. College Cork, Cork, Ireland.


   FOOTNOTES
 

Address for reprint requests and other correspondence: K. Langlands, Clore Laboratory, Univ. of Buckingham, Hunter St., Buckingham, MK18 1EG, UK (e-mail: kenneth.langlands{at}buckingham.ac.uk)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
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C. M. Alfieri, H. J. Evans-Anderson, and K. E. Yutzey
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