MyoD Binding Research Articles

Positive control mutations in the MyoD basic region fail to show cooperative DNA binding and transcriptional activation in vitro.

An in vitro transcription system from HeLa cells has been established in which MyoD and E47 proteins activate transcription both as homodimers and heterodimers. However, heterodimers activate transcription more efficiently than homodimers, and function synergistically from multiple binding sites. Positive control mutants in the basic region of MyoD that have previously been shown to be defective in initiating the myogenic program, can bind DNA but have lost their ability to function as transcriptional activators in vitro. Additionally, positive control mutants, unlike wild-type MyoD, fail to bind cooperatively to DNA. We propose that binding of MyoD complexes to high affinity MyoD binding sites induces conformational changes that facilitate cooperative binding to multiple sites and promote transcriptional activation.

In vivo and in vitro analysis of electricalactivity-dependent expression of muscle acetylcholine receptor genes usingadenovirus.

Acetylcholine receptor (AChR) genes are repressed in extrajunctional domains of adult muscle fiber by neurally evoked electrical activity. Denervation elicits upregulation of AChR gene transcription in extrasynaptic areas. We have used an adenovirus (Ad)-based strategy to analyze in vitro and in vivo the electrical activity-dependent transcription of the chicken AChR alpha 1 subunit gene. The luciferase gene placed under the control of wild-type and mutated fragments of the alpha 1 subunit promoter was inserted in a defective Ad vector designed for the study of transcriptional regulation. Animals were infected by intramuscular injection and in vivo luciferase levels were normalized by coinfection with an Ad vector containing the chloramphenicol acetyltransferase gene driven by an electrical activity-insensitive promoter. Our results demonstrate that although both proximal MyoD binding sites of the alpha 1 promoter are required for muscle-specific expression of the alpha 1 gene, only one is necessary, albeit insufficient, to enhance alpha 1 promoter activity after denervation. Parallel results were obtained with cultured muscle cells in vitro following tetrodotoxin blocking of spontaneous electrical activity. These results substantiate a direct contribution of MyoD factors in electrical activity-dependent regulation of AChR expression and further indicate that Ad-based vectors constitute a powerful tool in the field of transcriptional regulation.

Structural and functional aspects of basic helix-loop-helix protein folding by heat-shock protein 90.

The assembly and folding of nascent polypeptides are mediated in vivo by molecular chaperones, many of which are members of the heat-shock family of proteins. We have shown previously that heat-shock protein 90 (HSP90) folds an inactive fraction of a recombinant basic helix-loop-helix (bHLH) protein generated in Escherichia coli (MyoD) into its active conformation. We show here that HSP90 also folds another bHLH protein (E12) and heterodimers of E12/MyoD into their active conformations. By purifying inactive heterodimers of E12/MyoD and subsequently rendering them active in binding DNA by treatment with HSP90, we show that one folding step mediated by HSP90 occurs after oligomerization of the bHLH protein monomers. A series of deletion mutants is used to identify the 48-amino acid region of HSP90 that confers bHLH folding activity, which lies near the COOH terminus. This region is required for activation of DNA binding of MyoD and E12 homodimers and E12/MyoD heterodimers.

A proximal promoter element in the hamster desmin upstream regulatory region is responsible for activation by myogenic determination factors.

The muscle-specific intermediate filament protein desmin is up-regulated during skeletal muscle differentiation. When myoblasts leave the cell cycle and fusion into multinucleated myotubes starts, genes associated with myogenesis become activated. The activation is believed to be mediated by the muscle-specific determination factors. We present evidence that both MyoD and myogenin are able to activate the transcription of the hamster desmin gene. A proximal promoter fragment of 89 base pairs is sufficient for this transactivation process. The single E-box in this region is essential for desmin promoter activity in mouse C2 skeletal muscle cells and upon co-transfection of a myogenin expression vector also in human primary fibroblasts. Mutation of this MyoD binding site abrogates desmin transcription, and transactivation of the promoter no longer occurs. By using gel electrophoretic mobility shift assays, we were able to demonstrate that nuclear proteins from C2 muscle cells and myogenin/E12 glutathione S-transferase fusion proteins are able to bind to the functional E-box consensus sequence. A second E-box, situated in a more upstream regulatory region, which also binds to purified Helix-Loop-Helix proteins in vitro is only moderately affected by site-directed in vitro mutagenesis.

Molecular Cloning and Functional Analysis of the Promoter of Rat Skeletal Muscle Voltage-Sensitive Sodium Channel Subtype 2 (rSkM2): Evidence for Muscle-Specific Nuclear Protein Binding to the Core Promoter

rSkM2 is a tetrodotoxin-resistant rat skeletal muscle voltage-sensitive sodium channel that is expressed in immature and denervated skeletal muscle and in adult heart. We have isolated a 3.7-kb gene segment that contains the first exon, multiple transcription initiation sites, the core promoter (nt -102 to +1), GC-rich elements (Sp1 recognition sites), three overlapping C-rich motifs (important for muscle-specific expression of some muscle genes), and multiple CANNTG (E-box) motifs (MyoD binding sites). A deletion analysis of the 5' upstream 2.8-kb segment, driving the rSkM2 core promoter, has localized a muscle-restrictive enhancer element (MRSE) at least 2 kb upstream from the core promoter. The core promoter is silenced by an additional cis element (-645/-506). The positive and negative cis-elements together drive transcription of the chloramphenicol acetyltransferase (CAT) reporter gene from the core promoter at about the same level as does the core promoter alone in a skeletal muscle differentiation stage-specific manner. Gel-shift assays have identified sequence- and cell-type-specific proteins that bind to a 16-bp region (-44/-29) containing C-rich motifs. Muscle-specific complexes formed from muscle cell nuclear extracts and a 16-bp element (-44/-29) are competed by unlabeled -44/-29 oligonucleotide but not by several mutant oligonucleotides that implicate nucleotides -40 to -38 and -34 to -32 in the binding of a nuclear protein (designated SkM2 transcription factor 1, SkM2-TF1). We conclude that rSkM2 gene expression depends on the interactions of positive and negative transcriptional regulators with tissue- and developmental stage-specific core promoter elements.

The MyoD binding site is dispensable for cardiac actin gene expression in the somites of later stage Xenopus embryos

We have studied the transcription directed by the promoter of a Xenopus borealis cardiac actin gene fused to a globin reporter sequence after injection into X.laevis embryos. This promoter is known to be effective specifically in somites of neurula stage embryos, and work on a very similar X. laevis gene has indicated the importance of an upstream MyoD binding site (E box) in this process. We show that, although in the absence of the MyoD site there is a small amount of tissue-specific expression in neurulae, transcription is abundant in the tailbud embryo a few hours later. Thus, although the E box is initially essential, other sequences must adopt the same role soon afterwards.

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.

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.

An 83-nucleotide promoter of the acetylcholine receptor epsilon-subunit gene confers preferential synaptic expression in mouse muscle.

The expression of the acetylcholine receptor epsilon-subunit gene is restricted to the endplate of adult muscle fibers. We have started to study the regulatory elements of the epsilon-subunit gene promoter that are important for its synaptic expression. We used, for this purpose, a rapid method of in vivo expression after DNA injection into the muscle tissue [Wolff, J. A., Malone, R. W., Williams, P., Chong, W., Acsadi, G., Jani, A. & Felgner, P. L. (1990) Science 247, 1465-1468]. Our results show that a construction containing 83 nucleotides upstream from the transcription start site is sufficient to obtain preferential endplate expression. Moreover, mutation of a MyoD binding site located around position-70 does not alter this synaptic expression. We also studied the expression of this promoter in vitro in muscle primary cultures and showed the presence of a positive element between positions -122 and -83. Comparison of in vivo and in vitro results reveals that the elements important for in vivo localization at the synapse and in vitro expression in cultured muscle cells may differ.

Regulation of the mouse desmin gene: transactivated by MyoD, myogenin, MRF4 and Myf5.

Desmin, the muscle specific intermediate filament (IF) protein, is expressed at low levels in myoblasts and at the onset of differentiation its expression increases several fold. In an effort to explore the mechanism involved in the tissue-specific and developmentally regulated expression of desmin, we have isolated the mouse desmin gene. Sequence analysis of 976 bp 5' flanking region revealed several potential cis-acting elements: 1) Three E boxes (MyoD binding sites), namely, E1, E2 and E3, located at -79, -832 and -936, respectively; 2) one MEF2 binding site at -864; 3) a region with homology to M-CAT motif at -587; 4) several GC boxes. Transient transfections with various 5' flank deletion mutants into C2C12 muscle cells have revealed both positive and negative elements that seem to be involved in the expression of desmin. The first 81 bp upstream of the transcription initiation site, including E1 box, were sufficient to confer muscle specific expression of the desmin gene. The maximal level of expression was achieved by the construct containing up to -897 base pairs. The region between -578 to -976 behaves as a classical enhancer in the absence of which the region between -578 and -81 suppresses CAT activity. Gel electrophoretic mobility shift assays using both C2C12 muscle cell nuclear extracts as well as in vitro translated myoD/E12 and myogenin/E12 heterodimers, showed that both myoD and myogenin bind to the proximal E1 and the distal E2 boxes of the desmin promoter and enhancer respectively. Co-transfection of myoD, myogenin, MRF4 and Myf5, with the desmin-CAT construct into 10T-1/2 cells demonstrated that all these factors could transactivate desmin gene expression.

Characterization of the human pleiotrophin gene. Promoter region and chromosomal localization.

The protein (PTN) encoded by the pleiotrophin (PTN) gene belongs to a recently described family of heparin-binding cytokines whose expression is temporally and spatially regulated during development. We have now isolated genomic clones of the human PTN gene, characterized its promoter region, determined its transcription initiation site(s), and established functional activity of the PTN promoter. A fragment -550/+191 that contains a CAAT box, no apparent TATA box, and four consensus sites for the binding of MyoD is sufficient to provide optimal promoter activity. A serum response element is found at -559 to -568. We also have identified the human PTN gene on chromosome 7, band q33 and the mouse Ptn gene on chromosome 6, respectively. The data thus identify and characterize the 5' end of the PTN gene and its promoter region, suggest potential regions that may contribute to the regulation of its transcriptional activity, and localize the PTN gene in human and mouse chromosomes.

Inhibition of protein phosphatases blocks myogenesis by first altering MyoD binding activity.

To examine the role of protein phosphatases in skeletal muscle differentiation, C2C12 myoblasts were treated with okadaic acid, a potent in vitro inhibitor of protein phosphatases 1 and 2A which regulate various cellular events in intact cells. We now show that okadaic acid treatment of the mouse myoblast C2C12 cell line reversibly altered the morphology of the cells and blocked differentiation. At a molecular level, it extinguished expression of the myogenic determination genes, MyoD1 and myogenin, but induced the expression of an inhibitor of differentiation, Id. Analysis of the MyoD1 promoter showed that inhibition of MyoD1 expression by okadaic acid occurs at the transcriptional level. These changes occur 10-20 h after okadaic acid treatment. However, within 1 h of treatment the ability of muscle extracts to support a specific MyoD-dependent gel mobility shift using a MyoD DNA binding site is lost. These data suggest that protein phosphatases play an important role during myogenic differentiation.

Acquisition of myogenic specificity by replacement of three amino acid residues from MyoD into E12.

The basic helix-loop-helix (bHLH) protein MyoD is a transcription factor that is important for the induction of the myogenic phenotype. The DNA binding basic region (13 amino acids) is necessary for recognition of the consensus MyoD binding site, for transcriptional activation, and for conversion of fibroblasts to muscle. In contrast, the non-tissue-specific bHLH protein E12 can bind to the MyoD binding site but does not induce myogenesis. Here, it is shown that only two amino acids in the MyoD basic region and a single amino acid from the junction, which separates the basic region and helix 1, are sufficient for myogenic specificity when substituted into the corresponding region of E12. These findings suggest that the recognition of particular determinants in the basic region is required for conversion of fibroblasts to muscle.

Autonomous activity of the alternate aldolase A muscle promoter is maintained by a sequestering mechanism

The mouse aldolase A gene contains two closely-spaced alternate promoter/first exons. The more distal of the two, the M promoter, is muscle-specific while the 3' promoter, the H promoter, is expressed constitutively. Various segments from these promoter regions were linked to a reporter gene and used to transfect the myogenic cell line C2C12 and the hepatoma cell line BWTG3. A muscle-specific enhancer, MEN1, responsible for 80% of promoter M activity and containing 4 consensus MyoD binding sites was localized between -2578 to -2723 of the M promoter. Another muscle-specific enhancer and a restrictive element, MEN2/MSE, were found in the interval -1100 to -350. The MSE restrictive element was found to prohibit inappropriate up-regulation of the M promoter by selectively sequestering it from H promoter elements in both myoblasts and myotubes. Among the H promoter elements was found an enhancer, HEN, situated between -533 and -200 which did not function in myotubes. These studies also show that H promoter elements can act synergistically with a non-specific element, MAE, located between -350 and -130 of the M cap site greatly stimulating M promoter transcription in all cell types when the MSE restrictive element was absent. Through the analysis of interactions between these elements and the aldolase A and HSV-TK promoters we showed that neither the enhancers nor the promoter proximal sequences by themselves contain adequate information to reproduce the native pattern of aldolase A promoter modulation. Rather, the sequestering of the M promoter by the MSE restrictive element and the relative positioning and context of promoters M and H appear critical to the regulated expression of aldolase A.

The human M creatine kinase gene enhancer contains multiple functional interacting domains

Cis-elements (-933 to -641) upstream of the human M creatine kinase gene cap site contain an enhancer that confers developmental and tissue-specific expression to the chloramphenicol acetyltransferase gene in C2C12 myogenic cells transfected in culture. Division of the enhancer at -770 into a 5' fragment that includes the MyoD binding sites (-933 to -770) and a 3' fragment that includes the MEF-2 binding site (-770 to -641) resulted in two subfragments that showed minimal activity but in combination interacted in a position- and orientation-independent fashion to enhance activity of the SV40 promoter in transient transfection experiments. A 5' enhancer construct (-877 to -832) including only one (the low affinity) MyoD binding site was active when present in multiple copies. In contrast, a 3' enhancer construct (-749 to -732) including the MEF-2 binding site was inactive even when present in multiple copies. However, if the 5' construct was extended to include the high-affinity MyoD binding site (-877 to -803) the 5' and 3' constructs interacted in a position- and orientation-independent fashion to activate the SV40 promoter. Thus, the human M creatine kinase enhancer comprises multiple functional interacting domains.

Phorbol esters inhibit the activity of the chicken acetylcholine receptor α‐subunit gene promoter

Protein kinase C has previously been implicated in the regulation of chicken acetylcholine receptor (AChR) gene expression. To investigate the molecular basis of this regulation, the promoter of the AChR alpha-subunit (alpha AChR) gene was linked to a reporter gene and introduced into cultured chick myotubes by transient transfection. Treatment of myotubes with protein-kinase-C-activating phorbol esters was found to inhibit promoter activity. These inhibitory actions were mediated by promoter sequences between nucleotides -110 and -45, relative to the start point of transcription of the alpha AChR gene. In particular, phorbol-ester responsiveness could be conferred by a short DNA sequence that contains one of the two MyoD binding sites of the alpha AChR gene muscle-specific enhancer. 12-O-Tetradecanoylphorbol 13-acetate was found to inhibit rapidly and potently the expression of mRNAs coding for the myogenic regulators CMD1 and myogenin. Moreover, its inhibitory effect on the alpha AChR gene promoter could be attenuated by cotransfection of a MyoD1 expression vector. These results provide a molecular basis for the previously demonstrated involvement of protein kinase C in the regulation of alpha AChR biosynthesis. In addition, they lend further support to the notion that myogenic proteins play an important role in the control of alpha AChR gene expression.

The MCK enhancer contains a p53 responsive element.

p53 is an antioncogene that is defective or absent in a large number of human tumors. Its function in normal cells is not known. We show that co-transfection of mouse p53 with muscle-specific creatine kinase-chloramphenicol acetyltransferase reporter gene, containing 3.3 kilobase of upstream control sequence for the muscle-specific creatine kinase gene, results in a 10- to 80-fold activation. The p53 responsive element maps to a region distinguished from the known MyoD binding region. Identification of a p53 responsive element should allow a more focused analysis of the effects of p53 in controlling gene activity.

CAIII a marker for early myogenesis: Analysis of expression in cultured myogenic cells

Carbonic anhydrase III (CAIII) is an abundant muscle protein characteristic of adult type-1, slow-twitch, muscle fibers. We demonstrate that CAIII is not confined to mature muscle but is also expressed in cultured myogenic cells that were originally derived from adult and fetal limb muscle (G8 and C2C12) and by azacytidine treatment of 10T1/2 fibroblasts (23A2). Transcripts may accumulate in these cells to levels that correspond to 6.5% of that found in mature muscle. CAIII is expressed in mononucleate myoblasts and is abundant in those that preferentially fuse to form myotubes, and these findings contrast with those for many other muscle genes whose transcripts only accumulate on or after terminal differentiation. Preliminary promoter-function assays by transfection shows that 2.8 kb of sequence flanking the 5' end of the human CAIII gene efficiently promotes transcription of the bacterial chloramphenicol acetyltransferase gene in myogenic cells. However, none of the sequences within this region are sufficient to confer muscle-specific expression. Removal of sequences 5' to -715 bp leads to a major loss of transcriptional activity of the CAIII promoter. These results imply that the proximal CAIII promoter, which includes a putative CArG box and four potential MyoD binding sites, is not adequate for either myoblast-specific or maximal transcription.

Paired MyoD-binding sites regulate myosin light chain gene expression.

The muscle-specific enhancer element located downstream of the myosin light chain (MLC) locus encoding MLC1 and MLC3 contains three binding sites (A, B, and C) for the myogenic determination factor MyoD. A 173-base-pair region of the MLC gene enhancer, including these three sites, retains full enhancer function when transfected into muscle cells. Whereas mutation of either upstream MyoD binding site (A or B) has a mild effect on muscle-specific enhancer activity, mutation of the third MyoD binding site (C) substantially weakens the enhancer, both in muscle cells or in nonmuscle cells cotransfected with a MyoD, myogenin, or myf5 expression vector. Site C is necessary but insufficient, since double mutation of two MyoD binding sites (A plus B) abrogates enhancer activity. Thus, site C requires either site A or B for enhancer function. This study shows a hierarchy of function among the three MyoD binding sites in the MLC enhancer. We propose that a protein-DNA complex is formed with at least two of these sites (A and C or B and C) to effect activation of the locus encoding MLC1/3 during myogenesis.

Rabbit muscle creatine kinase: genomic cloning, sequencing, and analysis of upstream sequences important for expression in myocytes

Muscle creatine kinase (MCK) is a major enzyme of cellular energy metabolism that is expressed upon differentiation of myoblasts into myotubes. Previously we cloned and sequenced the entire rabbit enzyme cDNA which was used as a probe in these studies to obtain a genomic clone from a rabbit library. The transcription start site was identified by primer extension analysis and over 800 bp of 5' flanking DNA was sequenced. Comparison of this sequence with the published sequences from the upstream regions of the mouse MCK gene and the human MCK gene showed two conserved regions and a large intervening block of non-conserved sequence. The conserved regions are separated by about 800 bp in the mouse and by about 400 bp in the human, but are much closer (200 bp) in the rabbit. The upstream conserved region of the mouse gene encompasses a region possessing the properties of an enhancer and containing two MyoD binding sites; the downstream element is adjacent to the start of transcription. A set of of overlapping deletions of the 5' upstream DNA was fused to the CAT gene and transfected into mouse C2 myocytes, chick primary myocytes, and chick primary liver cells. Constructs which contained both conserved 5' regions were strongly expressed in C2 and chick myocytes, but were not expressed (above background) in primary liver cells. Surprisingly, while the upstream enhancer element was required for strong expression in C2 myocytes, it was less important for expression in chick myocytes. This suggests that there are important muscle-specific transcriptional signals in the proximal promoter region of mammalian MCK genes.