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The muscle creatine kinase (MCK) gene is transcriptionally induced when skeletal muscle myoblasts differentiate into myocytes. The gene contains two muscle-specific enhancer elements, one located 1,100 nucleotides (nt) 5' of the transcriptional start site and one located in the first intron. We have used gel mobility shift assays to characterize the trans-acting factors that interact with a region of the MCK gene containing the 5' enhancer. MM14 and C2C12 myocyte nuclear extracts contain a sequence-specific DNA-binding factor which recognizes a site within a 110-nt fragment of the MCK enhancer region shown to be sufficient for enhancer function. Preparative mobility shift gels were combined with DNase I footprinting to determine the site of binding within the 110-nt fragment. Site-directed mutagenesis within the footprinted region produced a 110-nt fragment which did not bind the myocyte factor in vitro. The mutant fragment had about 25-fold-less activity as a transcriptional enhancer in myocytes than did the wild-type fragment. Complementary oligomers containing 21 base pairs spanning the region protected from DNase degradation were also specifically bound by MM14 and C2C12 myocyte nuclear factors. The oligomer-binding activity was not found in nuclear extracts from the corresponding myoblasts, in nuclear extracts from a variety of nonmuscle cell types (including differentiation-defective MM14-DD1 cells and IOTI/2 mesodermal stem cells), or in cytoplasmic extracts. Both the 5' and intron 1 enhancer-containing fragments competed for factors that bind the oligomer probe, while total mouse genomic DNA and several DNA fragments containing viral and cellular enhancers did not. Interestingly, a 5' MCK proximal promoter fragment that also contains muscle-specific positive regulatory elements did not compete for factor binding to the oligomer. We have designated the factor which interacts with the two MCK enhancers myocyte-specific enhancer-binding nuclear factor 1 (MEF 1). A consensus for binding sites in muscle-specific regulatory regions is proposed.Muscle creatine kinase (MCK) gene expression is developmentally controlled and restricted to certain tissues. High levels of MCK protein are found in vertebrate cardiac and skeletal myocytes but not in the myoblast precursors of these cells. The regulation of muscle-specific genes. such as MCK, can be conveniently studied in several permanent cell lines, including the mouse skeletal muscle satellite cell line MM14 (35). MM14 myoblasts do not express the musclespecific genes associated with terminal differentiation when fibroblast growth factor (FGF) is present in the medium. but they rapidly commit to the myocyte phenotype and express these genes upon FGF withdrawal (9). submitted). (is-Acting regulatory elements of other cloned muscle-specific genes. such as oa-skeletal actin (4. 44. 59). a-cardiac actin (39. 41. 42). troponin I (33). myosin light chain 1/3 (5. 13). myosin light chain 2 (1). acetylcholine receptor cx subunit (30. 60a), and myosin heavy chain (7).have al...

Muscle creatine kinase (MCK) is induced to high levels during skeletal muscle differentiation. We have examined the upstream regulatory elements of the mouse MCK gene which specify its activation during myogenesis in culture. Fusion genes containing up to 3,300 nucleotides (nt) of MCK 5' flanking DNA in various positions and orientations relative to the bacterial chloramphenicol acetyltransferase (CAT) structural gene were transfected into cultured cells. Transient expression of CAT was compared between proliferating and differentiated MM14 mouse myoblasts and with nonmyogenic mouse L cells. The major effector of high-level expression was found to have the properties of a transcriptional enhancer. This element, located between 1,050 and 1,256 nt upstream of the transcription start site, was also found to have a major influence on the tissue and differentiation specificity of MCK expression; it activated either the MCK promoter or heterologous promoters only in differentiated muscle cells. Comparisons of viral and cellular enhancer sequences with the MCK enhancer revealed some similarities to essential regions of the simian virus 40 enhancer as well as to a region of the immunoglobulin heavy-chain enhancer, which has been implicated in tissue-specific protein binding. Even in the absence of the enhancer, low-level expression from a 776-nt MCK promoter retained differentiation specificity. In addition to positive regulatory elements, our data provide some evidence for negative regulatory elements with activity in myoblasts. These may contribute to the cell type and differentiation specificity of MCK expression.The differentiation of skeletal muscle in culture provides a model system for the study of developmental regulatory processes at the cellular level. The earliest phase of muscle cell differentiation, the transition from pluripotential mesodermal stem cells to myoblasts, can be studied using the 1OT1/2 cell line (33,35,67), and the appearance of distinct mnyoblast types during embryogenesis can be monitored and analyzed using primary and subclonal populations of limb bud cells (58,59). In later phases of myogenesis, the extracellular signals and ensuing intracellular events which control the transition from proliferating myoblasts to differentiated myocytes can be studied in various primary cultures and permanent myogenic cell lines (lla, llb, 21, 44, 46, 47). Since a major transition in the pattern of protein synthesis occurs during myogenic terminal differentiation (6), this system can be used to investigate the mechanisms of coordinate gene expression through comparisons of the regulation of different muscle-specific genes (8,13). Eventually, the understanding of a broad range of cellular processes in various myogenic cell culture systems should allow the formulation of a coherent model of muscle development.Several muscle-specific genes are now known to be activated transcriptionally during myogenesis (21,28,32,33,39,44,47), and transcriptional regulation has been inferred for others from the accumulation...

We have used transient transfections in MM14 skeletal muscle cells, newborn rat primary ventricular myocardiocytes, and nonmuscle cells to characterize regulatory elements of the mouse muscle creatine kinase (MCK) gene. Deletion analysis of MCK 5'-flanking sequence reveals a striated muscle-specific, positive regulatory region between-1256 and-1020. A 206-bp fragment from this region acts as a skeletal muscle enhancer and confers orientation-dependent activity in myocardiocytes. A 110-bp enhancer subfragment confers high-level expression in skeletal myocytes but is inactive in myocardiocytes, indicating that skeletal and cardiac muscle MCK regulatory sites are distinguishable. To further delineate muscle regulatory sequences, we tested six sites within the MCK enhancer for their functional importance. Mutations at five sites decrease expression in skeletal muscle, cardiac muscle, and nonmuscle cells. Mutations at two of these sites, Left E box and MEF2, cause similar decreases in all three cell types. Mutations at three sites have larger effects in muscle than nonmuscle cells; an AlT-rich site mutation has a pronounced effect in both striated muscle types, mutations at the MEF1 (Right E-box) site are relatively specific to expression in skeletal muscle, and mutations at the CArG site are relatively specific to expression in cardiac muscle. Changes at the AP2 site tend to increase expression in muscle cells but decrease it in nonmuscle cells. In contrast to reports involving cotransfection of 1OT1/2 cells with plasmids expressing the myogenic determination factor MyoD, we show that the skeletal myocyte activity of multimerized MEF1 sites is 30-fold lower than that of the 206-bp enhancer. Thus, MyoD binding sites alone are not sufficient for high-level expression in skeletal myocytes containing endogenous levels of MyoD and other myogenic determination factors. The mammalian muscle creatine kinase (MCK) gene is expressed primarily in skeletal and cardiac muscle. The mouse MCK gene contains an enhancer located approximately 1,100 bp 5' of the transcription start site. A 206-bp enhancer-containing fragment of the mouse gene and analogous regions of other mammalian MCK genes confer muscle-specific expression in cultured cells and transgenic mice (30, 36-38, 76, 81, 92). In vitro and in vivo footprinting have identified a variety of binding sites within the rat and mouse MCK enhancers (10, 28, 30,31, 57). MEF1, a skeletal myocyte-specific nuclear factor, was identified by gel shift assays and DNase footprinting using the MCK enhancer (10). The MEF1 or Right site contains the E-box sequence, CAnnTG (16, 23), characteristic of the recognition sites of the helix-loop-helix (HLH) family of DNA-binding proteins (58). The MEF1 complex contains the HLH myogenic determination factor, MyoD (41). MyoD, as well as the skeletal muscle determination factors myogenin, Myf5, and MRF4/herculin/Myf6, can bind the MEF1 site in vitro with various affinities, either alone or in combination with other HLH partners (2, 6, 9, 11, 12, 43, 59...

The muscle-specific form of creatine kinase (MCK) is induced in differentiating myoblast cultures, and a dramatic increase in mRNA levels precedes and parallels the increase in MCK protein. To study this induction, the complete MCK gene was cloned and characterized. The transcription unit was shown to span 11 kilobases and to contain seven introns. The splice junctions were identified and shown to conform to the appropriate consensus sequences. Close homology with branchpoint consensuses was found upstream of the 3' splice sites in six of seven cases. Transcriptional regulation of the gene in differentiating myoblast cultures was demonstrated by nuclear run-on experiments; increases in transcription accounted for a major part of the increased mRNA levels. Regulated expression of a transfected MCK gene containing the entire transcription unit with 3.3 kilobases of 5'-flanking sequence was also demonstrated during differentiation of the MM14 mouse myoblast cell line. The MCK 5'-flanking region was sufficient to confer transcriptional regulation to a heterologous structural gene, since chloramphenicol acetyl transferase activity was induced during differentiation of cultures transfected with an MCK-chloramphenicol acetyl transferase fusion construct. Examination of the DNA sequence immediately upstream of the transcription start site revealed a 17-nucleotide element which occurred three times. Comparisons with other muscle-specific genes which are also transcriptionally regulated during myogenesis revealed upstream homologies in the ca-actin and myosin heavy chain genes, but not in the myosin light-chain genes, with the regions containing these repeats. We suggest that coordinate control of a subset of muscle genes may occur via recognition of these common sequences.

Regulatory regions of the mouse muscle creatine kinase (MCK) gene, previously discovered by analysis in cultured muscle cells, were analyzed in transgenic mice. The 206-bp MCK enhancer at nt ؊1256 was required for high-level expression of MCK-chloramphenicol acetyltransferase fusion genes in skeletal and cardiac muscle; however, unlike its behavior in cell culture, inclusion of the 1-kb region of DNA between the enhancer and the basal promoter produced a 100-fold increase in skeletal muscle activity. Analysis of enhancer control elements also indicated major differences between their properties in transgenic muscles and in cultured muscle cells. Transgenes in which the enhancer right E box or CArG element were mutated exhibited expression levels that were indistinguishable from the wild-type transgene. Mutation of three conserved E boxes in the MCK 1,256-bp 5 region also had no effect on transgene expression in thigh skeletal muscle expression. All of these mutations significantly reduced activity in cultured skeletal myocytes. However, the enhancer AT-rich element at nt ؊1195 was critical for expression in transgenic skeletal muscle. Mutation of this site reduced skeletal muscle expression to the same level as transgenes lacking the 206-bp enhancer, although mutation of the AT-rich site did not affect cardiac muscle expression. These results demonstrate clear differences between the activity of MCK regulatory regions in cultured muscle cells and in whole adult transgenic muscle. This suggests that there are alternative mechanisms of regulating the MCK gene in skeletal and cardiac muscle under different physiological states.The muscle creatine kinase (MCK) gene is transcriptionally activated during striated muscle differentiation and is expressed at high levels in adult heart and skeletal muscles (11,39). Previous cell culture analyses of MCK gene regulation have implicated both a 5Ј muscle-specific enhancer (bp Ϫ1256 to Ϫ1050) and the adjacent 1-kb region of DNA (bp Ϫ1049 to ϩ7) as playing important roles in expression of the MCK gene in skeletal and cardiac muscle (24,29,63). The MCK enhancer contains a number of conserved DNA motifs, which are also found in the regulatory regions of many other muscle-specific genes (reviewed in references 12, 22, and 49). These motifs bind trans-acting factors in vitro and are critical control elements in cultured muscle cells (2,3,8,9,24). The MCK enhancer sites include E boxes, which contain the core consensus binding sequence CANNTG for the myogenic basic helix-loop-helix (bHLH) proteins (MyoD, Myf-5, myogenin, and MRF-4) (for reviews, see references 17, 22, 49, and 72); a CArG element, containing the consensus serum response factor-binding sequence CC(A/T) 6 GG (68); and an AT-rich site, which has been shown in gel shift assays to bind ubiquitously expressed factors (1, 15a, 25), as well as MHox and MEF-2 (14). The 1-kb region of DNA between the enhancer and the basal promoter exhibits low-level activity in cultured skeletal myocytes and cardiomyocytes (2).Mutation of eac...

Adenoviral gene transfer holds promise for gene therapy, but effective transduction of a large and distributed tissue such as muscle will almost certainly require systemic delivery. In this context, the use of muscle-specific regulatory elements such as the muscle creatine kinase (MCK) promoter and enhancer will avoid potentially harmful ectopic expression of transgenes. We describe here the development and testing of adenoviral vectors containing small, striated muscle-specific, highly active MCK expression cassettes. One of these regulatory elements (CK6) is less than 600 bp in length and is 12% as active as the CMV promoter/enhancer in muscle. A recombinant adenoviral vector containing this regulatory element retains very high muscle specificity, expressing 600-fold higher levels of transgene in muscle than in liver. Muscle-specific regulatory elements may also increase persistence of transduced muscle cells. Adenoviral transduction of dendritic cells has been shown to stimulate cytotoxic T-lymphocyte (CTL) responses directed against transgene epitopes. We show that human dendritic cells infected in vitro with MCK-containing adenoviruses do not express significant levels of transgene. Furthermore, while adenoviral vectors containing nonspecific promoters are normally cleared from muscle tissue within 1 month, we show that MCK-containing vectors express significant levels of transgene 4 months after intramuscular injection.