Analysis of Muscle Creatine Kinase Gene Regulatory Elements in Skeletal and Cardiac Muscles of Transgenic Mice

Abstract

Regulatory regions of the mouse muscle creatine kinase (MCK) gene, previously discovered by analysis in cultured muscle cells, were analyzed in transgenic mice. The 206-bp MCK enhancer at nt ؊1256 was required for high-level expression of MCK-chloramphenicol acetyltransferase fusion genes in skeletal and cardiac muscle; however, unlike its behavior in cell culture, inclusion of the 1-kb region of DNA between the enhancer and the basal promoter produced a 100-fold increase in skeletal muscle activity. Analysis of enhancer control elements also indicated major differences between their properties in transgenic muscles and in cultured muscle cells. Transgenes in which the enhancer right E box or CArG element were mutated exhibited expression levels that were indistinguishable from the wild-type transgene. Mutation of three conserved E boxes in the MCK 1,256-bp 5 region also had no effect on transgene expression in thigh skeletal muscle expression. All of these mutations significantly reduced activity in cultured skeletal myocytes. However, the enhancer AT-rich element at nt ؊1195 was critical for expression in transgenic skeletal muscle. Mutation of this site reduced skeletal muscle expression to the same level as transgenes lacking the 206-bp enhancer, although mutation of the AT-rich site did not affect cardiac muscle expression. These results demonstrate clear differences between the activity of MCK regulatory regions in cultured muscle cells and in whole adult transgenic muscle. This suggests that there are alternative mechanisms of regulating the MCK gene in skeletal and cardiac muscle under different physiological states.The muscle creatine kinase (MCK) gene is transcriptionally activated during striated muscle differentiation and is expressed at high levels in adult heart and skeletal muscles (11,39). Previous cell culture analyses of MCK gene regulation have implicated both a 5Ј muscle-specific enhancer (bp Ϫ1256 to Ϫ1050) and the adjacent 1-kb region of DNA (bp Ϫ1049 to ϩ7) as playing important roles in expression of the MCK gene in skeletal and cardiac muscle (24,29,63). The MCK enhancer contains a number of conserved DNA motifs, which are also found in the regulatory regions of many other muscle-specific genes (reviewed in references 12, 22, and 49). These motifs bind trans-acting factors in vitro and are critical control elements in cultured muscle cells (2,3,8,9,24). The MCK enhancer sites include E boxes, which contain the core consensus binding sequence CANNTG for the myogenic basic helix-loop-helix (bHLH) proteins (MyoD, Myf-5, myogenin, and MRF-4) (for reviews, see references 17, 22, 49, and 72); a CArG element, containing the consensus serum response factor-binding sequence CC(A/T) 6 GG (68); and an AT-rich site, which has been shown in gel shift assays to bind ubiquitously expressed factors (1, 15a, 25), as well as MHox and MEF-2 (14). The 1-kb region of DNA between the enhancer and the basal promoter exhibits low-level activity in cultured skeletal myocytes and cardiomyocytes (2).Mutation of eac...

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

“…[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.…”

Mouse dystrophin enhancer preferentially targets lacZ expression in skeletal and cardiac muscle

“…Other muscle-specific enhancers that target skeletal muscle in transgenic mice include the myosin light chain (MLC) 1/3 (Rosenthal et al, 1989;Wenthworth et al, 1991), muscle creatine kinase (MCK) (Amacher et al, 1993;Shield et al, 1996;Donoviel et al, 1996), and the distal enhancer of the cardiac alpha actin (Biben et al, 1996). The MLC, MCK, and dystrophin enhancers feature an E-box consensus 5Ј-AACAc/g c/g TGC a/t that is paired to a second E-box consensus 5Ј-GG a/c CANGTGGc/gN a/g.…”

Differences in the Function of Three Conserved E-boxes of the Muscle Creatine Kinase Gene in Cultured Myocytes and in Transgenic Mouse Skeletal and Cardiac Muscle

“…3-6 and references therein) as well as the prototype E-box binding site for the myogenic regulatory factors (MRFs) MyoD, myogenin, MRF4, and Myf5 (7-11). Transgenic mouse and mouse knockout experiments, along with in situ hybridization developmental studies and transfection experiments, have shown that MyoD and Myf5 play essential roles in muscle determination and that myogenin is critical for terminal differentiation (12-14).An enhancer-promoter region of the mouse MCK gene, which extends from Ϫ1256 to ϩ7 relative to the transcription start site, exhibits tissue-specific expression similar to that of the endogenous MCK gene, as assessed by cell culture and transgenic mouse studies (6,(15)(16)(17)(18)(19). The enhancer-promoter contains seven E-box core sequences CAnnTG, but only three of these are conserved among mammals.…”

“…It was thus surprising to discover that mutation of the high affinity right E-box site did not decrease reporter gene expression in the skeletal muscle of transgenic mice (18). We speculated that other E-boxes might compensate for the loss of the right E-box, and therefore tested a construct containing mutations of both enhancer E-boxes plus a conserved promoter E-box (18,19). …”

“…An enhancer-promoter region of the mouse MCK gene, which extends from Ϫ1256 to ϩ7 relative to the transcription start site, exhibits tissue-specific expression similar to that of the endogenous MCK gene, as assessed by cell culture and transgenic mouse studies (6,(15)(16)(17)(18)(19). The enhancer-promoter contains seven E-box core sequences CAnnTG, but only three of these are conserved among mammals.…”