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

Muscle creatine kinase (MCK) is induced to high levels during skeletal muscle differentiation. We have examined the upstream regulatory elements of the mouse MCK gene which specify its activation during myogenesis in culture. Fusion genes containing up to 3,300 nucleotides (nt) of MCK 5' flanking DNA in various positions and orientations relative to the bacterial chloramphenicol acetyltransferase (CAT) structural gene were transfected into cultured cells. Transient expression of CAT was compared between proliferating and differentiated MM14 mouse myoblasts and with nonmyogenic mouse L cells. The major effector of high-level expression was found to have the properties of a transcriptional enhancer. This element, located between 1,050 and 1,256 nt upstream of the transcription start site, was also found to have a major influence on the tissue and differentiation specificity of MCK expression; it activated either the MCK promoter or heterologous promoters only in differentiated muscle cells. Comparisons of viral and cellular enhancer sequences with the MCK enhancer revealed some similarities to essential regions of the simian virus 40 enhancer as well as to a region of the immunoglobulin heavy-chain enhancer, which has been implicated in tissue-specific protein binding. Even in the absence of the enhancer, low-level expression from a 776-nt MCK promoter retained differentiation specificity. In addition to positive regulatory elements, our data provide some evidence for negative regulatory elements with activity in myoblasts. These may contribute to the cell type and differentiation specificity of MCK expression.

Download Full-text

A ras-dependent pathway abolishes activity of a muscle-specific enhancer upstream from the muscle creatine kinase gene

Molecular and Cellular Biology ◽

10.1128/mcb.9.2.594-601.1989 ◽

1989 ◽

Vol 9 (2) ◽

pp. 594-601

Author(s):

E A Sternberg ◽

G Spizz ◽

M E Perry ◽

E N Olson

Keyword(s):

Creatine Kinase ◽

Growth Factors ◽

Myogenic Differentiation ◽

Regulatory Elements ◽

Negative Control ◽

Proximal Promoter ◽

Muscle Creatine Kinase ◽

Kinase Gene ◽

Muscle Creatine ◽

Ras Oncogenes

Differentiation of skeletal myoblasts is accompanied by induction of a series of tissue-specific genes whose products are required for the specialized functions of the mature muscle fiber. The program for myogenic differentiation is subject to negative control by several peptide growth factors and by the products of mutationally activated ras oncogenes, which persistently activate intracellular cascades normally triggered by specific growth factors. Previously, we reported that induction of the muscle creatine kinase (mck) gene during myogenesis was dependent on a distal upstream enhancer that cooperated with a proximal promoter to direct high levels of expression in developing muscle cells (E. A. Sternberg, G. Spizz, W. M. Perry, D. Vizard, T. Weil, and E. N. Olson, Mol. Cell. Biol. 8:2896-2909). To investigate the mechanisms whereby ras blocks the induction of muscle-specific genes, we have examined the ability of mck 5' regulatory elements to direct expression of the linked reporter gene for chloramphenicol acetyltransferase (cat) in C2 myoblasts bearing mutant N-ras and H-ras oncogenes. In this paper we report that expression of activated ras alleles abolishes activity of the mck upstream enhancer but does not affect the activity of the mck promoter. The ability of ras to repress the expression of mck-cat fusion genes that have been transfected either transiently or stably into myoblasts suggests that ras may exert its effects on muscle-specific genes through mechanisms independent of chromatin configurations or DNA methylation. These results also suggest that ras blocks establishment of the myogenic phenotype by preventing the accumulation of regulatory factors required for transcriptional induction of muscle-specific genes.

Download Full-text

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.

Molecular and Cellular Biology ◽

10.1128/mcb.16.9.5058 ◽

1996 ◽

Vol 16 (9) ◽

pp. 5058-5068 ◽

Cited By ~ 86

Author(s):

M A Shield ◽

H S Haugen ◽

C H Clegg ◽

S D Hauschka

Keyword(s):

Creatine Kinase ◽

Skeletal Muscles ◽

Transgene Expression ◽

Regulatory Region ◽

Regulatory Sequences ◽

Striated Muscles ◽

Muscle Creatine Kinase ◽

E Box ◽

Muscle Creatine ◽

E Boxes

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.

Download Full-text

Genotypes Of The Skeletal Muscle Creatine Kinase Gene And Exertional Rhabdomyolyis

Medicine & Science in Sports & Exercise ◽

10.1249/01.mss.0000354149.69365.b8 ◽

2009 ◽

Vol 41 ◽

pp. 164

Author(s):

Yuval Heled ◽

Patricia A. Deuster ◽

Sheila Muldoon ◽

Carmen Sesvold-Contreras ◽

Kimbra Kenny ◽

...

Keyword(s):

Skeletal Muscle ◽

Creatine Kinase ◽

Muscle Creatine Kinase ◽

Kinase Gene ◽

Muscle Creatine

Download Full-text

Quantitative Proteomic Identification of Six4 as the Trex-Binding Factor in the Muscle Creatine Kinase Enhancer

Molecular and Cellular Biology ◽

10.1128/mcb.24.5.2132-2143.2004 ◽

2004 ◽

Vol 24 (5) ◽

pp. 2132-2143 ◽

Cited By ~ 64

Author(s):

Charis L. Himeda ◽

Jeffrey A. Ranish ◽

John C. Angello ◽

Pascal Maire ◽

Ruedi Aebersold ◽

...

Keyword(s):

Creatine Kinase ◽

Cardiac Muscle ◽

Magnetic Beads ◽

Striated Muscle ◽

Regulatory Element ◽

Mouse Heart ◽

Muscle Creatine Kinase ◽

Adult Skeletal Muscle ◽

Binding Factor ◽

Muscle Creatine

ABSTRACT Transcriptional regulatory element X (Trex) is a positive control site within the Muscle creatine kinase (MCK) enhancer. Cell culture and transgenic studies indicate that the Trex site is important for MCK expression in skeletal and cardiac muscle. After selectively enriching for the Trex-binding factor (TrexBF) using magnetic beads coupled to oligonucleotides containing either wild-type or mutant Trex sites, quantitative proteomics was used to identify TrexBF as Six4, a homeodomain transcription factor of the Six/sine oculis family, from a background of ∼900 copurifying proteins. Using gel shift assays and Six-specific antisera, we demonstrated that Six4 is TrexBF in mouse skeletal myocytes and embryonic day 10 chick skeletal and cardiac muscle, while Six5 is the major TrexBF in adult mouse heart. In cotransfection studies, Six4 transactivates the MCK enhancer as well as muscle-specific regulatory regions of Aldolase A and Cardiac troponin C via Trex/MEF3 sites. Our results are consistent with Six4 being a key regulator of muscle gene expression in adult skeletal muscle and in developing striated muscle. The Trex/MEF3 composite sequence ([C/A]ACC[C/T]GA) allowed us to identify novel putative Six-binding sites in six other muscle genes. Our proteomics strategy will be useful for identifying transcription factors from complex mixtures using only defined DNA fragments for purification.

Download Full-text

Expression of a transfected mouse muscle-creatine kinase gene is induced upon growth factor deprivation of myogenic but not of nonmyogenic cells

Developmental Biology ◽

10.1016/0012-1606(89)90095-x ◽

1989 ◽

Vol 134 (1) ◽

pp. 258-262 ◽

Cited By ~ 6

Author(s):

Jane E. Johnson ◽

Cyndy L. Gartside ◽

James B. Jaynes ◽

Stephen D. Hauschka

Keyword(s):

Creatine Kinase ◽

Growth Factor ◽

Muscle Creatine Kinase ◽

Kinase Gene ◽

Mouse Muscle ◽

Growth Factor Deprivation ◽

Muscle Creatine

Download Full-text

The upstream muscle-specific enhancer of the rat muscle creatine kinase gene is composed of multiple elements

Molecular and Cellular Biology ◽

10.1128/mcb.9.6.2396-2413.1989 ◽

1989 ◽

Vol 9 (6) ◽

pp. 2396-2413

Author(s):

R A Horlick ◽

P A Benfield

Keyword(s):

Creatine Kinase ◽

Transient Expression ◽

Myogenic Differentiation ◽

Regulatory Element ◽

Chloramphenicol Acetyltransferase ◽

Muscle Creatine Kinase ◽

Kinase Gene ◽

Enhancer Element ◽

Rat Muscle ◽

Muscle Creatine

A series of constructs that links the rat muscle creatine kinase promoter to the bacterial chloramphenicol acetyltransferase gene was generated. These constructs were introduced into differentiating mouse C2C12 myogenic cells to localize sequences that are important for up-regulation of the creatine kinase gene during myogenic differentiation. A muscle-specific enhancer element responsible for induction of chloramphenicol acetyltransferase expression during myogenesis was localized to a 159-base-pair region from 1,031 to 1,190 base pairs upstream of the transcription start site. Analysis of transient expression experiments using promoters mutated by deletion indicated the presence of multiple functional domains within this muscle-specific regulatory element. A DNA fragment spanning this region was used in DNase I protection experiments. Nuclear extracts derived from C2 myotubes protected three regions (designated E1, E2, and E3) on this fragment from digestion, which indicated there may be three or more trans-acting factors that interact with the creatine kinase muscle enhancer. Gel retardation assays revealed that factors able to bind specifically to E1, E2, and E3 are present in a wide variety of tissues and cell types. Transient expression assays demonstrated that elements in regions E1 and E3, but not necessarily E2, are required for full enhancer activity.

Download Full-text

Analysis of muscle creatine kinase gene regulatory elements in skeletal and cardiac muscles of transgenic mice.

Molecular and Cellular Biology ◽

10.1128/mcb.16.4.1649 ◽

1996 ◽

Vol 16 (4) ◽

pp. 1649-1658 ◽

Cited By ~ 67

Author(s):

D B Donoviel ◽

M A Shield ◽

J N Buskin ◽

H S Haugen ◽

C H Clegg ◽

...

Keyword(s):

Skeletal Muscle ◽

Creatine Kinase ◽

Transgenic Mice ◽

Cardiac Muscle ◽

Muscle Cells ◽

Regulatory Elements ◽

Muscle Creatine Kinase ◽

Regulatory Regions ◽

Mouse Muscle ◽

Muscle Creatine

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 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 muscles cells and in whole adult transgenic muscle. This suggests that there are alternative mechanism of regulating the MCK gene in skeletal and cardiac muscle under different physiological states.

Download Full-text