A New Myocyte-Specific Enhancer-Binding Factor That Recognizes a Conserved Element Associated with Multiple Muscle-Specific Genes

Abstract

Exposure of skeletal myoblasts to growth factor-deficient medium results in transcriptional activation of muscle-specific genes, including the muscle creatine kinase gene (mck). Tissue specificity, developmental regulation, and high-level expression of mck are conferred primarily by a muscle-specific enhancer located between base pairs (bp) -1350 and -1048 relative to the transcription initiation site (E. A. Sternberg, G. Spizz, W. M. Perry, D. Vizard, T. Weil, and E. N. Olson, Mol. Cell. Biol. 8:2896-2909, 1988). To begin to define the regulatory mechanisms that mediate the selective activation of the mck enhancer in differentiating muscle cells, we have further delimited the boundaries of this enhancer and analyzed its interactions with nuclear factors from a variety of myogenic and nonmyogenic cell types. Deletion mutagenesis showed that the region between 1,204 and 1,095 bp upstream of mck functions as a weak muscle-specific enhancer that is dependent on an adjacent enhancer element for strong activity. This adjacent activating element does not exhibit enhancer activity in single copy but acts as a strong enhancer when multimerized. Gel retardation assays combined with DNase I footprinting and diethyl pyrocarbonate interference showed that a nuclear factor from differentiated C2 myotubes and BC3H1 myocytes recognized a conserved A + T-rich sequence within the peripheral activating region. This myocyte-specific enhancer-binding factor, designated MEF-2, was undetectable in nuclear extracts from C2 or BC3H1 myoblasts or several nonmyogenic cell lines. MEF-2 was first detectable within 2 h after exposure of myoblasts to mitogen-deficient medium and increased in abundance for 24 to 48 h thereafter. The appearance of MEF-2 required ongoing protein synthesis and was prevented by fibroblast growth factor and type beta transforming growth factor, which block the induction of muscle-specific genes. A myoblast-specific factor that is down regulated within 4 h after removal of growth factors was also found to bind to the MEF-2 recognition site. A 10-bp sequence, which was shown by DNase I footprinting and diethyl pyrocarbonate interference to interact directly with MEF-2, was identified within the rat and human mck enhancers, the rat myosin light-chain (mlc)-1/3 enhancer, and the chicken cardiac mlc-2A promoter. Oligomers corresponding to the region of the mlc-1/3 enhancer, which encompasses this conserved sequence, bound MEF-2 and competed for its binding to the mck enhancer. These results thus provide evidence for a novel myocyte-specific enhancer-binding factor, MEF-2, that is expressed early in the differentiation program and is suppressed by specific polypeptide growth factors. The ability of MEF-2 to recognize conserved activating elements associated with multiple-specific genes suggests that this factor may participate in the coordinate regulation of genes during myogenesis.

Immortalized human myoblast cell lines for the delivery of therapeutic proteins using encapsulated cell technology

“…MyHC is expressed only in terminally differentiated myotubes. 47 We observed that most cells fused into myotubes (MyHC-positive cells) expressing MEF2, confirming that transduction of primary myoblasts with lentiviral vectors encoding the CDK4 and hTERT proteins can efficiently immortalize cells without affecting their myogenicity ( Figure 1 B).

Figure 1 Primary human myoblasts are efficiently immortalized after transduction with lentiviruses encoding for CDK4 and hTERT (A) Primary myoblast cells were transduced with lentiviral vectors encoding the CDK4 and hTERT proteins.

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Refining the Camelus dromedarius Myostatin Gene Polymorphism through Worldwide Whole-Genome Sequencing

“…By using the two alternative alleles for each variant, a total of 213 differential putative TF-binding sites (TFBS) (out of which 82 were unique) were observed ( Table S7, Supplementary Materials ); out of which 41 (14 being unique) relative, overall, to ten TFs with known literature evidence supporting their involvement in muscle metabolism and/or muscle development, i.e., COMP1 (cooperates with myogenic proteins 1, 2), C/EBP (CCAAT/enhancer binding protein, 12), MYOG (Myogenin, 2), COUP (Chicken Ovalbumin Upstream Promoter-transcription factor II, 2), MEF2 (Myocyte Enhancer Factor-2, 16), SRF (Serum Response Factor, 1), DELTAEF1 (Zinc Finger E-Box Binding Homeobox 1, 1), PBX1 (PBX Homeobox 1, 3), MYOD (Myoblast Determination Protein 1, 1) and E2F (Retinoblastoma-Binding Protein, 1). Out of them, MEF2, DELTAEF1 and MYOG are involved in promoting and regulating the skeletal muscle cell differentiation program during myogenesis [ 41 , 42 , 43 ]. Moreover, MYOG and MYOD belong to a family of proteins known as myogenic regulatory factor (MRF) that plays a major role in regulating the skeletal muscle differentiation [ 44 ].…”

Systematic Transfer of Prokaryotic Sensors and Circuits to Mammalian Cells

“…Not only the spacing but also the content of cis -regulatory elements have been shown to have a dramatic effect on biological processes (such as development) in eukaryotes . While enhancer elements alone can lack discernible activity, in concert with other elements they typically evoke robust expression patterns upon associated genes . Recent efforts have been dedicated to identifying mammalian enhancer elements, where naturally occurring sequences were assessed in parallel to identify the essential elements of transcriptional networks. , …”

“…101 While enhancer elements alone can lack discernible activity, in concert with other elements they typically evoke robust expression patterns upon associated genes. 102 Recent efforts have been dedicated to identifying mammalian enhancer elements, where naturally occurring sequences were assessed in parallel to identify the essential elements of transcriptional networks. 103,104 Although much work has been done to characterize the behavior and identity of naturally occurring enhancers, and to develop synthetic tools to control mammalian gene expression, issues persist in the implementation of such components toward broader applications.…”