Identification of upstream and intragenic regulatory elements that confer cell-type-restricted and differentiation-specific expression on the muscle creatine kinase gene.

Abstract

Terminal differentiation of skeletal myoblasts is accompanied by induction of a series of tissue-specific gene products, which includes the muscle isoenzyme of creatine kinase (MCK). To begin to define the sequences and signals involved in MCK regulation in developing muscle cells, the mouse MCK gene has been isolated. Sequence analysis of 4,147 bases of DNA surrounding the transcription initiation site revealed several interesting structural features, some of which are common to other muscle-specific genes and to cellular and viral enhancers. To test for sequences required for regulated expression, a region upstream of the MCK gene from -4800 to +1 base pairs, relative to the transcription initiation site, was linked to the coding sequences of the bacterial chloramphenicol acetyltransferase (CAT) gene. Introduction of this MCK-CAT fusion gene into C2 muscle cells resulted in high-level expression of CAT activity in differentiated myotubes and no detectable expression in proliferating undifferentiated myoblasts or in nonmyogenic cell lines. Deletion mutagenesis of sequences between -4800 and the transcription start site showed that the region between -1351 and -1050 was sufficient to confer cell type-specific and developmentally regulated expression on the MCK promoter. This upstream regulatory element functioned independently of position, orientation, or distance from the promoter and therefore exhibited the properties of a classical enhancer. This upstream enhancer also was able to confer muscle-specific regulation on the simian virus 40 promoter, although it exhibited a 3- to 5-fold preference for its own promoter. In contrast to the cell type- and differentiation-specific expression of the upstream enhancer, the MCK promoter was able to function in myoblasts and myotubes and in nonmyogenic cell lines when combined with the simian virus 40 enhancer. An additional positive regulatory element was identified within the first intron of the MCK gene. Like the upstream enhancer, this intragenic element functioned independently of position, orientation, and distance with respect to the MCK promoter and was active in differentiated myotubes but not in myoblasts. These results demonstrate that expression of the MCK gene in developing muscle cells is controlled by complex interactions among multiple upstream and intragenic regulatory elements that are functional only in the appropriate cellular context.

Multiple Changes in E2F Function and Regulation Occur upon Muscle Differentiation

“…For these studies, the C2 muscle cell line was selected because differentiation proceeded efficiently and completely in culture (71). The standard conditions for growth were 20% FCS with subsequent exposure to 2% FCS to initiate differentiation (64). Full differentiation is observed within 48 h and is characterized by the appearance of multinucleated myotubes and expression of several muscle-specific markers.…”

“…Transfections. Transfections into C2 cells were done by the BES method (58), and expression in undifferentiated and differentiated C2 cells proceeded exactly as described by Olson and colleagues (64). Thirty micrograms of reporter plasmid and 3 g of a reference luciferase plasmid (pGLC-luciferase [Promega] or SV-luciferase) were added per 100-mm-diameter plate.…”

“…The reference plasmid SVluciferase was used to normalize for transfection efficiency. SV40 promoter activity does not vary with C2 cell differentiation (64). Relative CAT expression was expressed as a CAT/ luciferase ratio and was determined by quantitative assays (CAT ELISA and luciferase enzymatic assay).…”

Cross-binding of factors to functionally different promoter elements in c-fos and skeletal actin genes.

“…Though a number of muscle promoters have been characterized, this element is the smallest sequence identified to contain sufficient information for muscle-specific expression. Other cis-acting regions that have been shown to confer muscle-specific expression in heterologous promoter constructs include the creatine kinase (M) gene enhancer (257 to 352 bp) (23,45), the myosin light chain 1/3 gene enhancer (0.9 kilobase-pair) (10), and a 67-bp segment of the cardiac troponin T promoter (27). The troponin gene segment contains a sequence motif that is present in other muscle genes but appears to be different from the skeletal actin MRE.…”

Analysis of the myogenin promoter reveals an indirect pathway for positive autoregulation mediated by the muscle-specific enhancer factor MEF-2.

“…Transient transfections were performed by calcium phosphate precipitation as described previously (67). Forty-eight hours after transfection, cultures were transferred from GM to DM for an additional 48 h. Cell extracts were then prepared, and levels of chloramphenicol acetyltransferase (CAT) activity were determined by using aliquots of extract containing equivalent quantities of protein or aliquots normalized to 0-galactosidase activity generated from cotransfected RSV-lacZ, which contains the Rous sarcoma virus (RSV) long terminal repeat linked to lacZ and is expressed constitutively.…”