E-Box Sites and a Proximal Regulatory Region of the Muscle Creatine Kinase Gene Differentially Regulate Expression in Diverse Skeletal Muscles and Cardiac Muscle of Transgenic Mice

Abstract

Previous analysis of the muscle creatine kinase (MCK) gene indicated that control elements required for transcription in adult mouse muscle differed from those required in cell culture, suggesting that distinct modes of muscle gene regulation occur in vivo. To examine this further, we measured the activity of MCK transgenes containing E-box and promoter deletions in a variety of striated muscles. Simultaneous mutation of three E boxes in the 1,256-bp MCK 5 region, which abolished transcription in muscle cultures, had strikingly different effects in mice. The mutations abolished transgene expression in cardiac and tongue muscle and caused a reduction in expression in the soleus muscle (a muscle with many slow fibers) but did not affect expression in predominantly fast muscles: quadriceps, abdominals, and extensor digitorum longus. Other regulatory sequences with muscle-type-specific activities were found within the 358-bp 5-flanking region. This proximal region conferred relatively strong expression in limb and abdominal skeletal muscles but was inactive in cardiac and tongue muscles. However, when the 206-bp 5 enhancer was ligated to the 358-bp region, high levels of tissue-specific expression were restored in all muscle types. These results indicate that E boxes and a proximal regulatory region are differentially required for maximal MCK transgene expression in different striated muscles. The overall results also imply that within skeletal muscles, the steady-state expression of the MCK gene and possibly other muscle genes depends on transcriptional mechanisms that differ between fast and slow fibers as well as between the anatomical and physiological attributes of each specific muscle.Creatine kinase catalyzes the regeneration of ATP from creatine phosphate and provides energy for contraction in all striated muscle types. The muscle creatine kinase (MCK) gene is transcriptionally activated during differentiation of myoblasts to myocytes (8, 31) and encodes the predominant creatine kinase isoform expressed in mammalian skeletal and cardiac muscle. While it seems likely that intrinsic differences in striated muscle anatomy and physiology would affect MCK gene expression, little is known concerning the steady-state mechanisms of MCK transcription in diverse muscle types. This transgenic study seeks to identify regulatory regions and elements within the MCK gene that are involved in controlling muscle-type-specific expression.Several regions within the mouse MCK gene 5Ј-flanking sequence are required for muscle-specific expression in cultured myocytes and cardiomyocytes (1, 32, 64). Of particular interest is a 206-bp enhancer located approximately 1 kb upstream of the transcription start site. This region functions in an orientation-independent manner and drives the expression of heterologous promoters in skeletal myocytes (32). The 5Ј MCK enhancer contains a number of sequence motifs-including the E box, CArG, and AT-rich sites-which are recognized by tissue-specific and ubiquitous transcription factors. Mutation...

Construction and analysis of compact muscle-specific promoters for AAV vectors

“…In our previous studies, we used a truncated penh358MCK promoter 9 to drive the human minidystrophin genes in the AAV vectors. Those results showed muscle-specific expression and functional recovery of dystrophic skeletal muscle, but the expression was lower than that of the CMV promoter.…”

“…[6][7][8] The viral promoters, such as human cytomegalovirus (CMV) immediate-early gene promoter, achieve high levels of nonspecific gene expression; in contrast, the use of the muscle creatine kinase (MCK) promoter is specific to muscle tissue and can avoid the potentially harmful effects of ectopic transgene expression. [9][10][11][12] However, the MCK promoter is less active than viral promoters, and its large size (6.5 kb) makes it incompatible with adeno-associated viral (AAV) vectors, a noteworthy gene delivery vehicle with a 4.5-kb capacity for the treatment of muscular dystrophies and other degenerative disorders. [13][14][15] We have shown previously that a truncated MCK promoter (enh358MCK, 584-bp) could achieve musclespecific expression of the human minidystrophin genes (3.8-4.2 kb) in dystrophin-deficient mdx mice, ameliorate pathologies and improve muscle contractile function in mdx mice.…”

“…19,[20][21][22][23] However, most comprehensive work has been done by Hauschka's laboratory on the modification and optimization of MCK-based muscle-specific promoters. 9,[24][25][26][27] We have made our own efforts to further reduce the size and retain/improve the strength and specificity of the truncated MCK promoters, as the minidystrophin genes we tested for gene therapy have the coding sequences around 4.0 kb, whereas the AAV vectors could only afford less than 4.7 kb of foreign gene cassette. This has left little room to accommodate the promoter and polyA sequences in addition to the minidystrophin gene.…”

Adeno-associated virus vector-mediated gene transfer into dystrophin-deficient skeletal muscles evokes enhanced immune response against the transgene product

“…The truncated MCK promoter was shown to drive muscle-specific expression of the reporter gene 38 and constructed by PCR from the mouse genomic DNA. Briefly, 206 bp of the enhancer region (from Ϫ1256 to Ϫ1050 relative to the transcriptional start site, Accession No.…”

“…M21390) and 358 bp of the proximal regulatory region (from Ϫ358 to +7) were amplified separately by PCR reactions, subcloned into pCR2.1 (Invitrogen, Groningen, The Netherlands), sequenced and connected directly, based on the report described. 38 The recombinant viral vector stocks were produced by cotransfection methods as already described. 39 The AAV virions were purified twice by CsCl density gradient centrifugation, dialyzed against sterile phosphate-buffered saline (PBS) and titrated by a quantitative DNA dot blot assay, according to the previously published protocol.…”