Multiple regulatory elements contribute differentially to muscle creatine kinase enhancer activity in skeletal and cardiac muscle.

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 A/T-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 10T1/2 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.     

Analysis of muscle creatine kinase gene regulatory elements in skeletal and cardiac muscles of transgenic mice.

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 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 muscles cells and in whole adult transgenic muscle. This suggests that there are alternative mechanism of regulating the MCK gene in skeletal and cardiac muscle under different physiological states.     

Expression of a transfected mouse muscle-creatine kinase gene is induced upon growth factor deprivation of myogenic but not of nonmyogenic cells

To determine whether mitogen-regulated expression of skeletal muscle genes is independent of cell type, muscle and nonmuscle cells were transfected with cloned 5'-flanking sequences of muscle creatine kinase (MCK) fused to a heterologous reporter gene and tested for expression in high and low mitogen culture conditions. Consistent with the behavior of endogenous MCK, a -3300MCK-CAT gene is expressed at high levels in differentiated muscle cells but at low to undetectable levels in proliferating myoblasts and in either mitogen-deprived or stimulated nonmuscle cells of mesodermal, ectodermal, or endodermal origin. A -776MCK-CAT gene behaves similarly with respect to its cell type specificity but it supports only an intermediate expression level in response to mitogen deprivation in skeletal muscle cells. These data suggest that the -3300 to +7 nucleotide region of mouse MCK contains one or more elements which are activable by mitogen deprivation only in myogenic cells.     

Transgenic and tissue culture analyses of the muscle creatine kinase enhancer Trex control element in skeletal and cardiac muscle indicate differences in gene expression between muscle types

The muscle creatine kinase (MCK) gene is expressed at high levels only in differentiated skeletal and cardiac muscle. The activity of the cloned enhancer–promoter has previously been shown to be dependent on the Trex element which is specifically bound by a yet unidentified nuclear factor, TrexBF. We have further characterized the function of the Trex site by comparing wild-type and Trex-mutated MCK transgenes in five mouse skeletal muscles: quadriceps, extensor digitorum longus (EDL), soleus, diaphragm, and distal tongue, as well as in heart ventricular muscle. Several types of statistical analysis including analysis of variance (ANOVA) and rank sum tests were used to compare expression between muscle types and between constructs. Upon mutation of the Trex site, median transgene expression levels decreased 3- to 120-fold in the muscles examined, with statistically significant differences in all muscles except the EDL. Expression in the largely slow soleus muscle was more affected than in the EDL, and expression in the distal tongue and diaphragm muscles was affected more than in soleus. Median expression of the transgene in ventricle decreased about 18-fold upon Trex mutation. Transfections into neonatal rat myocardiocytes confirmed the importance of the Trex site for MCK enhancer activity in heart muscle, but the effect is larger in transgenic mice than in cultured cells.     

Chicken cardiac tropomyosin and a low-molecular-weight nonmuscle tropomyosin are related by alternative splicing

We have isolated and characterized complementary DNAs (cDNAs) encoding chicken cardiac muscle tropomyosin and a low-molecular-weight nonmuscle tropomyosin. The cardiac muscle cDNA (pCHT-4) encodes a 284-amino acid protein that differs from chicken skeletal muscle alpha- and beta-tropomyosins throughout its length. The nonmuscle cDNA (pFT-C) encodes a 248-amino acid protein that is most similar (93-94%) to the tropomyosin class including rat fibroblast TM-4, equine platelet tropomyosin, and human fibroblast TM30pl. The nucleotide sequences of the cardiac and nonmuscle cDNAs are identical from the position encoding cardiac amino acid 81 (nonmuscle amino acid 45) through cardiac amino acid 257 (nonmuscle amino acid 221). The sequences differ both 5' and 3' of this region of identity. These comparisons suggest that the chicken cardiac tropomyosin and low-molecular-weight "platelet-like" tropomyosin are derived from the same genomic locus by alternative splicing. S1 analysis suggests that this locus encodes at least one other tropomyosin isoform.     

Smooth muscle myosin light chain kinase expression in cardiac and skeletal muscle

The purpose of this study was to characterize myosin light chain kinase (MLCK) expression in cardiac and skeletal muscle. The only classic MLCK detected in cardiac tissue, purified cardiac myocytes, and in a cardiac myocyte cell line (AT1) was identical to the 130-kDa smooth muscle MLCK (smMLCK). A complex pattern of MLCK expression was observed during differentiation of skeletal muscle in which the 220-kDa-long or "nonmuscle" form of MLCK is expressed in undifferentiated myoblasts. Subsequently, during myoblast differentiation, expression of the 220-kDa MLCK declines and expression of this form is replaced by the 130-kDa smMLCK and a skeletal muscle-specific isoform, skMLCK in adult skeletal muscle. These results demonstrate that the skMLCK is the only tissue-specific MLCK, being expressed in adult skeletal muscle but not in cardiac, smooth, or nonmuscle tissues. In contrast, the 130-kDa smMLCK is ubiquitous in all adult tissues, including skeletal and cardiac muscle, demonstrating that, although the 130-kDa smMLCK is expressed at highest levels in smooth muscle tissues, it is not a smooth muscle-specific protein.     

Brain and muscle creatine kinase genes contain common TA-rich recognition protein-binding regulatory elements.

We have previously reported that the rat brain creatine kinase (ckb) gene promoter contains an AT-rich sequence that is a binding site for a protein called TARP (TA-rich recognition protein). This AT-rich segment is a positively acting regulatory element for the ckb promoter. A similar AT-rich DNA segment is found at the 3' end of the 5' muscle-specific enhancer of the rat muscle creatine kinase (ckm) gene and has been shown to be necessary for full muscle-specific enhancer activity. In this report, we show that TARP binds not only to the ckb promoter but also to the AT-rich segment at the 3' end of the muscle-specific ckm enhancer. A second, weaker TARP-binding site was identified in the ckm enhancer and lies at the 5' end of the minimal enhancer segment. TARP was found in both muscle cells (C2 and L6 myotubes) and nonmuscle (HeLa) cells and appeared to be indistinguishable from both sources, as judged by gel retardation and footprinting assays. The TARP-binding sites in the ckm enhancer and the ckb promoter were found to be functionally interchangeable. We propose that TARP is active in both muscle and nonmuscle cells and that it is one of many potential activators that may interact with muscle-specific regulators to determine the myogenic phenotype.     

The myosin light chain enhancer and the skeletal actin promoter share a binding site for factors involved in muscle-specific gene expression.

The myosin light chain (MLC) 1/3 enhancer (MLC enhancer), identified at the 3' end of the skeletal MLC1/3 locus, contains a sequence motif that is homologous to a protein-binding site of the skeletal muscle alpha-actin promoter. Gel shift, competition, and footprint assays demonstrated that a CArG motif in the MLC enhancer binds the proteins MAPF1 and MAPF2, previously identified as factors interacting with the muscle regulatory element of the skeletal alpha-actin promoter. Transient transfection assays with constructs containing the chloramphenicol acetyltransferase reporter gene demonstrated that a 115-bp subfragment of the MLC enhancer is able to exert promoter activity when provided with a silent nonmuscle TATA box. A point mutation at the MAPF1/2-binding site interferes with factor binding and abolishes the promoter activity of the 115-bp fragment. The observation that an oligonucleotide encompassing the MAPF1/2 site of the MLC enhancer alone cannot serve as a promoter element suggests that additional factor-binding sites are necessary for this function. The finding that MAPF1 and MAPF2 recognize similar sequence motifs in two muscle genes, simultaneously activated during muscle differentiation, implies that these factors may have a role in coordinating the activation of contractile protein gene expression during myogenesis.     

Two distinct nonmuscle myosin-heavy-chain mRNAs are differentially expressed in various chicken tissues. Identification of a novel gene family of vertebrate non-sarcomeric myosin heavy chains

Two distinct cDNA clones for nonmuscle myosin heavy chain (MHC) were isolated from a chicken fibroblast cDNA library by cross-hydridization under a moderate stringency with chicken gizzard smooth muscle MHC cDNA. These two fibroblast MHC and the gizzard MHC are each encoded in different genes in the chicken genome. Northern blot analysis showed that both of the nonmuscle MHC mRNAs were expressed not only in fibroblasts but also in a variety of tissues including brain, lung, kidney, spleen, and skeletal, cardiac and smooth muscles. However, the relative contents of the two nonmuscle MHC mRNAs varied greatly among tissues. The encoded amino acid sequences of the nonmuscle MHCs were highly similar to each other (81% identity) and to the smooth muscle MHC (81-84%), but much less similar to vertebrate skeletal muscle MHCs (38-41%) or to protista nonmuscle MHCs (35-36%). A phylogenic tree of MHC isoforms was constructed by calculating the similarity scores between these MHC sequences. An examination of the tree showed that the vertebrate sarcomeric (skeletal and cardiac) MHC isoforms are encoded in a very closely related multigene family, and that the vertebrate non-sarcomeric (smooth muscle and nonmuscle) MHC isoforms define a distinct, less conserved MHC gene family.     

Lower skeletal muscle mass in male transgenic mice with muscle-specific overexpression of myostatin

Mutations in the myostatin gene are associated with hypermuscularity, suggesting that myostatin inhibits skeletal muscle growth. We postulated that increased tissue-specific expression of myostatin protein in skeletal muscle would induce muscle loss. To investigate this hypothesis, we generated transgenic mice that overexpress myostatin protein selectively in the skeletal muscle, with or without ancillary expression in the heart, utilizing cDNA constructs in which a wild-type (MCK/Mst) or mutated muscle creatine kinase (MCK-3E/Mst) promoter was placed upstream of mouse myostatin cDNA. Transgenic mice harboring these MCK promoters linked to enhanced green fluorescent protein (EGFP) expressed the reporter protein only in skeletal and cardiac muscles (MCK) or in skeletal muscle alone (MCK-3E). Seven-week-old animals were genotyped by PCR of tail DNA or by Southern blot analysis of liver DNA. Myostatin mRNA and protein, measured by RT-PCR and Western blot, respectively, were significantly higher in gastrocnemius, quadriceps, and tibialis anterior of MCK/Mst-transgenic mice compared with wild-type mice. Male MCK/Mst-transgenic mice had 18-24% lower hind- and forelimb muscle weight and 18% reduction in quadriceps and gastrocnemius fiber cross-sectional area and myonuclear number (immunohistochemistry) than wild-type male mice. Male transgenic mice with mutated MCK-3E promoter showed similar effects on muscle mass. However, female transgenic mice with either type of MCK promoter did not differ from wild-type controls in either body weight or skeletal muscle mass. In conclusion, increased expression of myostatin in skeletal muscle is associated with lower muscle mass and decreased fiber size and myonuclear number, decreased cardiac muscle mass, and increased fat mass in male mice, consistent with its role as an inhibitor of skeletal muscle mass. The mechanism of gender specificity remains to be clarified.