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

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.

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

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.

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

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.

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

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.

scholarly journals Brain and muscle creatine kinase genes contain common TA-rich recognition protein-binding regulatory elements.

1990 ◽  
Vol 10 (9) ◽  
pp. 4826-4836 ◽  
Author(s):  
R A Horlick ◽  
G M Hobson ◽  
J H Patterson ◽  
M T Mitchell ◽  
P A Benfield
Keyword(s):  
Creatine Kinase ◽  
Binding Site ◽  
Regulatory Element ◽  
Gene Promoter ◽  
Regulatory Elements ◽  
Muscle Creatine Kinase ◽  
Enhancer Activity ◽  
L6 Myotubes ◽  
Recognition Protein ◽  
Muscle Creatine

We have previously reported that the rat brain creatine kinase (ckb) gene promoter contains an AT-rich sequence that is a binding site for a protein called TARP (TA-rich recognition protein). This AT-rich segment is a positively acting regulatory element for the ckb promoter. A similar AT-rich DNA segment is found at the 3' end of the 5' muscle-specific enhancer of the rat muscle creatine kinase (ckm) gene and has been shown to be necessary for full muscle-specific enhancer activity. In this report, we show that TARP binds not only to the ckb promoter but also to the AT-rich segment at the 3' end of the muscle-specific ckm enhancer. A second, weaker TARP-binding site was identified in the ckm enhancer and lies at the 5' end of the minimal enhancer segment. TARP was found in both muscle cells (C2 and L6 myotubes) and nonmuscle (HeLa) cells and appeared to be indistinguishable from both sources, as judged by gel retardation and footprinting assays. The TARP-binding sites in the ckm enhancer and the ckb promoter were found to be functionally interchangeable. We propose that TARP is active in both muscle and nonmuscle cells and that it is one of many potential activators that may interact with muscle-specific regulators to determine the myogenic phenotype.

The muscle creatine kinase gene is regulated by multiple upstream elements, including a muscle-specific enhancer

1988 ◽  
Vol 8 (1) ◽  
pp. 62-70
Author(s):  
J B Jaynes ◽  
J E Johnson ◽  
J N Buskin ◽  
C L Gartside ◽  
S D Hauschka
Keyword(s):  
Creatine Kinase ◽  
Cultured Cells ◽  
Simian Virus 40 ◽  
Regulatory Elements ◽  
Simian Virus ◽  
Major Influence ◽  
Transcriptional Enhancer ◽  
Muscle Creatine Kinase ◽  
Kinase Gene ◽  
Muscle Creatine

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.

Genotypes Of The Skeletal Muscle Creatine Kinase Gene And Exertional Rhabdomyolyis

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

scholarly journals 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

2003 ◽  
Vol 278 (47) ◽  
pp. 46494-46505 ◽  
Author(s):  
Quynh-Giao V. Nguyen ◽  
Jean N. Buskin ◽  
Charis L. Himeda ◽  
Margaret A. Shield ◽  
Stephen D. Hauschka
Keyword(s):  
Creatine Kinase ◽  
Cardiac Muscle ◽  
Transgenic Mouse ◽  
Muscle Creatine Kinase ◽  
Kinase Gene ◽  
Muscle Creatine ◽  
E Boxes

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

2004 ◽  
Vol 24 (5) ◽  
pp. 2132-2143 ◽  
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.

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