Skeletal Muscle-specific Gene Expression Research Articles

Activin A affects feeding by promoting the inner diameter and muscle development of the pharynx and oesophagus in zebrafish (Danio rerio) larvae.

Activin A belongs to the superfamily of transforming growth factor-β and plays an important role in hormone regulation and tissue development. However, few research studies have been conducted on the effect of activin A on feeding organs in fish. In this study, the zebrafish (Danio rerio) larvae were treated with 1 ng ml-1 activin A for 8 days continuously. The haematoxylin and eosin (H&E) staining section results revealed that the transverse inner diameter of the pharynx and oesophagus significantly increased on the third and eighth days after treatment compared with the control group (P < 0.05). On the eighth day, the cross-sectional area of the pharyngeal muscle increased by 8638 μm2 compared to the control group (P < 0.05). The RNA in situ hybridization results also showed that the expression of skeletal muscle-specific genes (myog and myod) was significantly increased in pharyngeal muscle on the eighth day. Furthermore, the qRT-PCR results showed the expression of gh gene was significantly increased on the eighth day (P < 0.05). At the same time, more larvae in activin A group were able to feed larger brine shrimp (Artemia) than in the control group on the eighth day. In conclusion, activin A could affect feeding by promoting the inner diameter and muscle development of the pharynx and oesophagus in zebrafish larvae. This study is the first to report that the development of the pharynx and oesophagus can directly affect food intake in fish larvae, which provides a theoretical basis for the study of food intake of fish at an early stage.

Calcineurin Broadly Regulates the Initiation of Skeletal Muscle-Specific Gene Expression by Binding Target Promoters and Facilitating the Interaction of the SWI/SNF Chromatin Remodeling Enzyme.

Calcineurin (Cn) is a calcium-activated serine/threonine protein phosphatase that is broadly implicated in diverse cellular processes, including the regulation of gene expression. During skeletal muscle differentiation, Cn activates the nuclear factor of activated T-cell (NFAT) transcription factor but also promotes differentiation by counteracting the negative influences of protein kinase C beta (PKCβ) via dephosphorylation and activation of Brg1, an enzymatic subunit of the mammalian SWI/SNF ATP-dependent chromatin remodeling enzyme. Here we identified four major temporal patterns of Cn-dependent gene expression in differentiating myoblasts and determined that Cn is broadly required for the activation of the myogenic gene expression program. Mechanistically, Cn promotes gene expression through direct binding to myogenic promoter sequences and facilitating the binding of Brg1, other SWI/SNF subunit proteins, and MyoD, a critical lineage determinant for skeletal muscle differentiation. We conclude that the Cn phosphatase directly impacts the expression of myogenic genes by promoting ATP-dependent chromatin remodeling and formation of transcription-competent promoters.

A novel tetracycline-responsive transgenic mouse strain for skeletal muscle-specific gene expression

BackgroundThe tetracycline-responsive system (Tet-ON/OFF) has proven to be a valuable tool for manipulating gene expression in an inducible, temporal, and tissue-specific manner. The purpose of this study was to create and characterize a new transgenic mouse strain utilizing the human skeletal muscle α-actin (HSA) promoter to drive skeletal muscle-specific expression of the reverse tetracycline transactivator (rtTA) gene which we have designated as the HSA-rtTA mouse.MethodsTo confirm the HSA-rtTA mouse was capable of driving skeletal muscle-specific expression, we crossed the HSA-rtTA mouse with the tetracycline-responsive histone H2B-green fluorescent protein (H2B-GFP) transgenic mouse in order to label myonuclei.ResultsReverse transcription-PCR confirmed skeletal muscle-specific expression of rtTA mRNA, while single-fiber analysis showed highly effective GFP labeling of myonuclei in both fast- and slow-twitch skeletal muscles. Pax7 immunohistochemistry of skeletal muscle cross-sections revealed no appreciable GFP expression in satellite cells.ConclusionsThe HSA-rtTA transgenic mouse allows for robust, specific, and inducible gene expression across muscles of different fiber types. The HSA-rtTA mouse provides a powerful tool to manipulate gene expression in skeletal muscle.

Subcutaneous fat transplantation alleviates diet-induced glucose intolerance and inflammation in mice.

Adipose tissue (AT) distribution is a major determinant of mortality and morbidity in obesity. In mice, intra-abdominal transplantation of subcutaneous AT (SAT) protects against glucose intolerance and insulin resistance (IR), but the underlying mechanisms are not well understood. We investigated changes in adipokines, tissue-specific glucose uptake, gene expression and systemic inflammation in male C57BL6/J mice implanted intra-abdominally with either inguinal SAT or epididymal visceral AT (VAT) and fed a high-fat diet (HFD) for up to 17 weeks. Glucose tolerance was improved in mice receiving SAT after 6 weeks, and this was not attributable to differences in adiposity, tissue-specific glucose uptake, or plasma leptin or adiponectin concentrations. Instead, SAT transplantation prevented HFD-induced hepatic triacylglycerol accumulation and normalised the expression of hepatic gluconeogenic enzymes. Grafted fat displayed a significant increase in glucose uptake and unexpectedly, an induction of skeletal muscle-specific gene expression. Mice receiving subcutaneous fat also displayed a marked reduction in the plasma concentrations of several proinflammatory cytokines (TNF-α, IL-17, IL-12p70, monocyte chemoattractant protein-1 [MCP-1] and macrophage inflammatory protein-1β [ΜIP-1β]), compared with sham-operated mice. Plasma IL-17 and MIP-1β concentrations were reduced from as early as 4 weeks after transplantation, and differences in plasma TNF-α and IL-17 concentrations predicted glucose tolerance and insulinaemia in the entire cohort of mice (n = 40). In contrast, mice receiving visceral fat transplants were glucose intolerant, with increased hepatic triacylglycerol content and elevated plasma IL-6 concentrations. Intra-abdominal transplantation of subcutaneous fat reverses HFD-induced glucose intolerance, hepatic triacylglycerol accumulation and systemic inflammation in mice.

Insm1 controls development of pituitary endocrine cells and requires a SNAG domain for function and for recruitment of histone-modifying factors

The Insm1 gene encodes a zinc finger factor expressed in many endocrine organs. We show here that Insm1 is required for differentiation of all endocrine cells in the pituitary. Thus, in Insm1 mutant mice, hormones characteristic of the different pituitary cell types (thyroid-stimulating hormone, follicle-stimulating hormone, melanocyte-stimulating hormone, adrenocorticotrope hormone, growth hormone and prolactin) are absent or produced at markedly reduced levels. This differentiation deficit is accompanied by upregulated expression of components of the Notch signaling pathway, and by prolonged expression of progenitor markers, such as Sox2. Furthermore, skeletal muscle-specific genes are ectopically expressed in endocrine cells, indicating that Insm1 participates in the repression of an inappropriate gene expression program. Because Insm1 is also essential for differentiation of endocrine cells in the pancreas, intestine and adrenal gland, it is emerging as a transcription factor that acts in a pan-endocrine manner. The Insm1 factor contains a SNAG domain at its N-terminus, and we show here that the SNAG domain recruits histone-modifying factors (Kdm1a, Hdac1/2 and Rcor1-3) and other proteins implicated in transcriptional regulation (Hmg20a/b and Gse1). Deletion of sequences encoding the SNAG domain in mice disrupted differentiation of pituitary endocrine cells, and resulted in an upregulated expression of components of the Notch signaling pathway and ectopic expression of skeletal muscle-specific genes. Our work demonstrates that Insm1 acts in the epigenetic and transcriptional network that controls differentiation of endocrine cells in the anterior pituitary gland, and that it requires the SNAG domain to exert this function in vivo.

Tyk2 and Stat3 Regulate Brown Adipose Tissue Differentiation and Obesity

Mice lacking the Jak tyrosine kinase member Tyk2 become progressively obese due to aberrant development of Myf5+ brown adipose tissue (BAT). Tyk2 RNA levels in BAT and skeletal muscle, which shares a common progenitor with BAT, are dramatically decreased in mice placed on a high-fat diet and in obese humans. Expression of Tyk2 or the constitutively active form of the transcription factor Stat3 (CAStat3) restores differentiation in Tyk2(-/-) brown preadipocytes. Furthermore, Tyk2(-/-) mice expressing CAStat3 transgene in BAT also show improved BAT development, normal levels of insulin, and significantly lower body weights. Stat3 binds to PRDM16, a master regulator of BAT differentiation, and enhances the stability of PRDM16 protein. These results define Tyk2 and Stat3 as critical determinants of brown fat lineage and suggest that altered levels of Tyk2 are associated with obesity in both rodents and humans.

Enhancer of Polycomb1 Acts on Serum Response Factor to Regulate Skeletal Muscle Differentiation

Skeletal muscle differentiation is well regulated by a series of transcription factors. We reported previously that enhancer of polycomb1 (Epc1), a chromatin protein, can modulate skeletal muscle differentiation, although the mechanisms of this action have yet to be defined. Here we report that Epc1 recruits both serum response factor (SRF) and p300 to induce skeletal muscle differentiation. Epc1 interacted physically with SRF. Transfection of Epc1 to myoblast cells potentiated the SRF-induced expression of skeletal muscle-specific genes as well as multinucleation. Proximal CArG box in the skeletal alpha-actin promoter was responsible for the synergistic activation of the promoter-luciferase. Epc1 knockdown caused a decrease in the acetylation of histones associated with serum response element (SRE) of the skeletal alpha-actin promoter. The Epc1.SRF complex bound to the SRE, and the knockdown of Epc1 resulted in a decrease in SRF binding to the skeletal alpha-actin promoter. Epc1 recruited histone acetyltransferase activity, which was potentiated by cotransfection with p300 but abolished by si-p300. Epc1 directly bound to p300 in myoblast cells. Epc1+/- mice showed distortion of skeletal alpha-actin, and the isolated myoblasts from the mice had impaired muscle differentiation. These results suggest that Epc1 is required for skeletal muscle differentiation by recruiting both SRF and p300 to the SRE of muscle-specific gene promoters.

Myocardin is a bifunctional switch for smooth versus skeletal muscle differentiation

Skeletal and smooth muscle can mutually transdifferentiate, but little molecular insight exists as to how each muscle program may be subverted to the other. The myogenic basic helix-loop-helix transcription factors MyoD and myogenin (Myog) direct the development of skeletal muscle and are thought to be dominant over the program of smooth muscle cell (SMC) differentiation. Myocardin (Myocd) is a serum response factor (SRF) coactivator that promotes SMC differentiation through transcriptional stimulation of SRF-dependent smooth muscle genes. Here we show by lineage-tracing studies that Myocd is expressed transiently in skeletal muscle progenitor cells of the somite, and a majority of skeletal muscle is derived from Myocd-expressing cell lineages. However, rather than activating skeletal muscle-specific gene expression, Myocd functions as a transcriptional repressor of Myog, inhibiting skeletal muscle differentiation while activating SMC-specific genes. This repressor function of Myocd is complex, involving histone deacetylase 5 silencing of the Myog promoter and Myocd's physical contact with MyoD, which undermines MyoD DNA binding and transcriptional synergy with MEF2. These results reveal a previously unrecognized role for Myocd in repressing the skeletal muscle differentiation program and suggest that this transcriptional coregulator acts as a bifunctional molecular switch for the smooth versus skeletal muscle phenotypes.

Roles of μ-calpain in cultured L8 muscle cells: application of a skeletal muscle-specific gene expression system

The goal of this work was to characterize the roles of μ-calpain in skeletal muscle protein degradation. Three approaches were developed to alter μ-calpain activity in rat myotubes. These included over-expression of antisense μ-calpain (μ-AS), dominant negative μ-calpain (μ-DN) and the antisense 30-kDa calpain subunit (30-AS). Constructs were expressed in rat L8 myotubes, and their effects on protein degradation and on concentrations of intact and/or degraded fodrin, desmin and tropomyosin were examined. An ecdysone-inducible expression system, in which we replaced a constitutively active CMV promoter with a skeletal muscle-specific α-actin promoter, was used to drive expression. Cell lines were evaluated by expression of the gene-of-interest following addition of ponasterone A (PA; ecdysone analog) to culture medium. Changes in calpain activity were assessed by evaluating fodrin degradation. 30-AS, which should alter both μ- and m-calpain activities, increased intact fodrin concentration. μ-DN and μ-AS reduced fodrin degradation products. μ-DN reduced total protein degradation by 7.9% ( P<0.01) at 24 h and by 10.6% ( P<0.01) at 48 h. μ-AS reduced total protein degradation by 6.4% at 24 h ( P<0.05). 30-AS reduced total protein degradation by 13.4% ( P<0.05) and 7.3% ( P<0.05) following 24 and 48 h of PA administration, respectively. We assessed effects of μ-DN, μ-AS and 30-AS on concentrations of desmin and tropomyosin. Inhibition of calpains stabilized desmin, but had no effect on tropomyosin. These data indicate that fodrin and desmin are μ-calpain substrates and that μ-calpain accounts for a small proportion of total protein degradation in muscle cells. Tropomyosin is not degraded by calpain in muscle cells.

Expression of (beta)-catenin in the developing chick myotome is regulated by myogenic signals.

The developmental signals that govern cell specification and differentiation in vertebrate somites are well understood. However, little is known about the downstream signalling pathways involved. We have shown previously that a combination of Shh protein and Wnt1 or Wnt3a-expressing fibroblasts is sufficient to activate skeletal muscle-specific gene expression in somite explants. Here, we have examined the molecular mechanisms by which the Wnt-mediated signal acts on myogenic precursor cells. We show that chick frizzled 1 (Fz1), beta-catenin and Lef1 are expressed during somitogenesis. Lef1 and beta-catenin transcripts become restricted to the developing myotome. Furthermore, beta-catenin is expressed prior to the time at which MyoD transcripts can be detected. Expression of beta-catenin mRNA is regulated by positive and negative signals derived from neural tube, notochord and lateral plate mesoderm. These signals include Bmp4, Shh and Wnt1/Wnt3a itself. In somite explants, Fz1, beta-catenin and Lef1 are expressed prior to activation of myogenesis in response to Shh and Wnt signals. Thus, our data show that a combination of Shh and Wnt1 upregulates expression of Wnt pathway components in developing somites prior to myogenesis. Thus, Wnt1 could act through beta-catenin on cells in the myotome.

Vascular smooth muscle cells spontaneously adopt a skeletal muscle phenotype: a unique Myf5(-)/MyoD(+) myogenic program.

Smooth and skeletal muscle tissues are composed of distinct cell types that express related but distinct isoforms of the structural genes used for contraction. These two muscle cell types are also believed to have distinct embryological origins. Nevertheless, the phenomenon of a phenotypic switch from smooth to skeletal muscle has been demonstrated in several in vivo studies. This switch has been minimally analyzed at the cellular level, and the mechanism driving it is unknown. We used immunofluorescence and RT-PCR to demonstrate the expression of the skeletal muscle-specific regulatory genes MyoD and myogenin, and of several skeletal muscle-specific structural genes in cultures of the established rat smooth muscle cell lines PAC1, A10, and A7r5. The skeletal muscle regulatory gene Myf5 was not detected in these three cell lines. We further isolated clonal sublines from PAC1 cultures that homogeneously express smooth muscle characteristics at low density and undergo a coordinated increase in skeletal muscle-specific gene expression at high density. In some of these PAC1 sublines, this process culminates in the high-frequency formation of myotubes. As in the PAC1 parental line, Myf5 was not expressed in the PAC1 sublines. We show that the PAC1 sublines that undergo a more robust transition into the skeletal muscle phenotype also express significantly higher levels of the insulin-like growth factor (IGF1 and IGF2) genes and of FGF receptor 4 (FGFR4) gene. Our results suggest that MyoD expression in itself is not a sufficient condition to promote a coordinated program of skeletal myogenesis in the smooth muscle cells. Insulin administered at a high concentration to PAC1 cell populations with a poor capacity to undergo skeletal muscle differentiation enhances the number of cells displaying the skeletal muscle differentiated phenotype. The findings raise the possibility that the IGF signaling system is involved in the phenotypic switch from smooth to skeletal muscle. The gene expression program described here can now be used to investigate the mechanisms that may underlie the propensity of certain smooth muscle cells to adopt a skeletal muscle identity.(J Histochem Cytochem 48:1173-1193, 2000)

Loss of β1Integrin Function Results in a Retardation of Myogenic, but an Acceleration of Neuronal, Differentiation of Embryonic Stem Cellsin Vitro

Integrin cell surface receptors play an important role for cell adhesion, migration, and differentiation during embryonic development by mediating cell–cell and cell–matrix interactions. Less is known about the function of integrins during commitment and lineage determination of early embryogenesis. Homozygous inactivation of the β1integrin gene results in embryonal death in mice around the time of implantation.In vitro,differentiation of embryonic stem (ES) cells which lack β1integrin (β1−/−) into the cardiogenic lineage is delayed and results in a disordered cellular specification (Fässleret al., J. Cell Sci.109, 2989–2999, 1996). To analyze β1integrin function during myogenesis and neurogenesis we studied differentiation of β1−/−ES cells via embryoid bodies into skeletal muscle and neuronal cellsin vitro.β1−/−cells showed delayed and reduced myogenic differentiation compared to wildtype and heterozygous (β1+/−) ES cells. RT–PCR analysis demonstrated delayed expression of skeletal muscle-specific genes in the absence of β1integrin. Immunofluorescence studies with antibodies against the sarcomeric proteins myosin heavy chain, titin, nebulin, and slow C-protein showed that myotubes formed, but their number was reduced and the assembly of sarcomeric structures was retarded. In contrast, neuronal cells differentiating from β1−/−ES cells appeared earlier than wildtype and heterozygous (β1+/−) ES cells. This was shown by the accelerated expression of neuron-specific genes and an increased number of neuronal cells in β1−/−embryoid bodies. However, neuronal outgrowth was retarded in the absence of β1integrin. No functional difference between wildtype and β1−/−cells was found with respect to secretion of γ-aminobutyric acid, the main neurotransmitter of ES cell-derived neuronal cells. The lineage-specific effects of loss of β1integrin function, that is the inhibition of mesodermal and acceleration of neuroectodermal differentiation, were supported by differential expression of genes encoding lineage-specific transcription factors (Brachyury, Pax-6, Mash1) and signaling molecules (BMP-4 and Wnt-1). Because of the reduced and delayed expression of the BMP-4 encoding gene in β1−/−cells, we analyzed in wildtype and β1−/−cells the regulatory role of exogenously added BMP-4 on the expression of the mesodermal and neuronal marker genes,Brachyuryandwnt-1,respectively. The data suggest that BMP-4 plays a regulatory role during differentiation of wildtype and β1−/−cells by modifying mesodermal and neuronal pathways. The reduced expression of BMP-4 in β1−/−cells may account for the accelerated neuronal differentiation in β1−/−ES cells.

Formation of Postsynaptic-Like Membranes during Differentiation of Embryonic Stem Cellsin Vitro

To analyze the formation of neuromuscular junctions, mouse pluripotent embryonic stem (ES) cells were differentiated via embryoid bodies into skeletal muscle and neuronal cells. The developmentally controlled expression of skeletal muscle-specific genes coding for myf5, myogenin, myoD and myf6, α1subunit of the L-type calcium channel, cell adhesion molecule M-cadherin, and neuron-specific genes encoding the 68-, 160-, and 200-kDa neurofilament proteins, synaptic vesicle protein synaptophysin, brain-specific proteoglycan neurocan, and microtubule-associated protein tau was demonstrated by RT-PCR analysis. In addition, genes specifically expressed at neuromuscular junctions, the γ- and ϵ-subunits of the nicotinic acetylcholine receptor (AChR) and the extracellular matrix protein S-laminin, were found. At the terminal differentiation stage characterized by the formation of multinucleated spontaneously contracting myotubes, the myogenic regulatory gene myf6 and the AChR ϵ-subunit gene, both specifically expressed in mature adult skeletal muscle, were found to be coexpressed. Only the terminally differentiated myotubes showed a clustering of nicotinic acetylcholine receptors (AChR) and a colocalization with agrin and synaptophysin. The formation of AChRs was also demonstrated on a functional level by using the patch clamp technique. Taken together, our results showed that during ES cell differentiationin vitroneuron- and muscle-specific genes are expressed in a developmentally controlled manner, resulting in the formation of postsynaptic-like membranes. Thus, the embryonic stem cell differentiation model will be helpful for studying cellular interactions at neuromuscular junctions by “loss of function” analysisin vitro.

Developmental modulation of a β myosin heavy chain promoter-driven transgene

The molecular mechanisms underlying heart and skeletal muscle-specific gene expression during development and in response to physioloic stimuli are largely unknown. Using a novel immunohistochemical procedure to detect chloramphenicol acetyltransferase (CAT), we have investigated, in vivo at high resolution, the ability of cis-acting DNA sequences within the 5' flanking region of the mouse beta myosin heavy chain (MyHC) gene (beta-MyHC) to direct appropriate gene expression throughout development. A 5.6-kb fragment 5' to the beta-MyHC's transcriptional start site was linked to the reporter gene encoding CAT (cat) and used to generate transgenic mice. The anti-CAT in situ assay described in this report allowed us to define the ability of the promoter fragment to direct appropriate temporal, tissue- and muscle fiber type-specific gene expression throughout early development. In skeletal muscles, the transgene expression profile mimics the endogenous beta-myHC's at all developmental stages and is appropriately restricted to slow (type I) skeletal fibers in the adult. Surprisingly, transgene expression was detected in both the atria and ventricles during embryonic and fetal development, indicating that ventricular specification involves elements outside the 5.6-kb fragment. In contrast, in the adult, hypothyroid conditions led to transgene induction specifically in the ventricles, suggesting that distinct regulatory mechanisms control fetal versus adult beta-MyHC expression in the cardiac compartment.

Overexpression of Myotonic Dystrophy Kinase in BC3H1 Cells Induces the Skeletal Muscle Phenotype

Myotonic muscular dystrophy is an autosomal dominant defect that produces muscle wasting, myotonia, and cardiac conduction abnormalities. The myotonic dystrophy locus codes for a putative serine-threonine protein kinase of unknown function. We report that overexpression of human myotonic dystrophy protein kinase induces the expression of skeletal muscle-specific genes in undifferentiated BC3H1 muscle cells. BC3H1 clones expressing myotonic dystrophy kinase appear equivalent to differentiated cells with respect to expression of myogenin, retinoblastoma tumor supressor gene, M creatine kinase, beta-tropomyosin, and vimentin. In addition, differential display analysis demonstrates that the pattern of gene expression exhibited by myotonic dystrophy kinase-expressing cells is essentially identical to that of differentiated BC3H1 muscle cells. These observations suggest that myotonic dystrophy kinase may function in the myogenic pathway.

Identification and characterization of a cardiac-specific transcriptional regulatory element in the slow/cardiac troponin C gene.

The slow/cardiac troponin C (cTnC) gene has been used as a model system for defining the molecular mechanisms that regulate cardiac and skeletal muscle-specific gene expression during mammalian development. cTnC is expressed continuously in both embryonic and adult cardiac myocytes but is expressed only transiently in embryonic fast skeletal myotubes. We have reported previously that cTnC gene expression in skeletal myotubes is controlled by a developmentally regulated, skeletal muscle-specific transcriptional enhancer located within the first intron of the gene (bp 997 to 1141). In this report, we show that cTnC gene expression in cardiac myocytes both in vitro and in vivo is regulated by a distinct and independent transcriptional promoter and enhancer located within the immediate 5' flanking region of the gene (bp -124 to +32). DNase I footprint and electrophoretic mobility shift assay analyses demonstrated that this cardiac-specific promoter/enhancer contains five nuclear protein binding sites (designated CEF1, CEF-2, and CPF1-3), four of which bind novel cardiac-specific nuclear protein complexes. Functional analysis of the cardiac-specific cTnC enhancer revealed that mutation of either the CEF-1 or CEF-2 nuclear protein binding site abolished the activity of the cTnC enhancer in cardiac myocytes. Taken together, these results define a novel mechanism for developmentally regulating a single gene in multiple muscle cell lineages. In addition, they identify previously undefined cardiac-specific transcriptional regulatory motifs and trans-acting factors. Finally, they demonstrate distinct transcriptional regulatory pathways in cardiac and skeletal muscle.

Identification and characterization of a cardiac-specific transcriptional regulatory element in the slow/cardiac troponin C gene.

The slow/cardiac troponin C (cTnC) gene has been used as a model system for defining the molecular mechanisms that regulate cardiac and skeletal muscle-specific gene expression during mammalian development. cTnC is expressed continuously in both embryonic and adult cardiac myocytes but is expressed only transiently in embryonic fast skeletal myotubes. We have reported previously that cTnC gene expression in skeletal myotubes is controlled by a developmentally regulated, skeletal muscle-specific transcriptional enhancer located within the first intron of the gene (bp 997 to 1141). In this report, we show that cTnC gene expression in cardiac myocytes both in vitro and in vivo is regulated by a distinct and independent transcriptional promoter and enhancer located within the immediate 5' flanking region of the gene (bp -124 to +32). DNase I footprint and electrophoretic mobility shift assay analyses demonstrated that this cardiac-specific promoter/enhancer contains five nuclear protein binding sites (designated CEF1, CEF-2, and CPF1-3), four of which bind novel cardiac-specific nuclear protein complexes. Functional analysis of the cardiac-specific cTnC enhancer revealed that mutation of either the CEF-1 or CEF-2 nuclear protein binding site abolished the activity of the cTnC enhancer in cardiac myocytes. Taken together, these results define a novel mechanism for developmentally regulating a single gene in multiple muscle cell lineages. In addition, they identify previously undefined cardiac-specific transcriptional regulatory motifs and trans-acting factors. Finally, they demonstrate distinct transcriptional regulatory pathways in cardiac and skeletal muscle.