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.

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.

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

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

1996 ◽  
Vol 16 (4) ◽  
pp. 1649-1658 ◽  
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.

scholarly journals Muscle creatine kinase sequence elements regulating skeletal and cardiac muscle expression in transgenic mice.

1989 ◽  
Vol 9 (8) ◽  
pp. 3393-3399 ◽  
Author(s):  
J E Johnson ◽  
B J Wold ◽  
S D Hauschka
Keyword(s):  
Skeletal Muscle ◽  
Creatine Kinase ◽  
Transgenic Mice ◽  
Cardiac Muscle ◽  
Fusion Gene ◽  
Proximal Promoter ◽  
Muscle Creatine Kinase ◽  
Specific Expression ◽  
Muscle Creatine

Muscle creatine kinase (MCK) is expressed at high levels only in skeletal and cardiac muscle tissues. Previous in vitro transfection studies of skeletal muscle myoblasts and fibroblasts had identified two MCK enhancer elements and one proximal promoter element, each of which exhibited expression only in differentiated skeletal muscle. In this study, we have identified several regions of the mouse MCK gene that are responsible for tissue-specific expression in transgenic mice. A fusion gene containing 3,300 nucleotides of MCK 5' sequence exhibited chloramphenicol acetyltransferase activity levels that were more than 10(4)-fold higher in skeletal muscle than in other, nonmuscle tissues such as kidney, liver, and spleen. Expression in cardiac muscle was also greater than in these nonmuscle tissues by 2 to 3 orders of magnitude. Progressive 5' deletions from nucleotide -3300 resulted in reduced expression of the transgene, and one of these resulted in a preferential decrease in expression in cardiac tissue relative to that in skeletal muscle. Of the two enhancer sequences analyzed, only one directed high-level expression in both skeletal and cardiac muscle. The other enhancer activated expression only in skeletal muscle. These data reveal a complex set of cis-acting sequences that have differential effects on MCK expression in skeletal and cardiac muscle.

scholarly journals Muscle creatine kinase sequence elements regulating skeletal and cardiac muscle expression in transgenic mice

1989 ◽  
Vol 9 (8) ◽  
pp. 3393-3399
Author(s):  
J E Johnson ◽  
B J Wold ◽  
S D Hauschka
Keyword(s):  
Skeletal Muscle ◽  
Creatine Kinase ◽  
Transgenic Mice ◽  
Cardiac Muscle ◽  
Fusion Gene ◽  
Proximal Promoter ◽  
Muscle Creatine Kinase ◽  
Specific Expression ◽  
Muscle Creatine

Muscle creatine kinase (MCK) is expressed at high levels only in skeletal and cardiac muscle tissues. Previous in vitro transfection studies of skeletal muscle myoblasts and fibroblasts had identified two MCK enhancer elements and one proximal promoter element, each of which exhibited expression only in differentiated skeletal muscle. In this study, we have identified several regions of the mouse MCK gene that are responsible for tissue-specific expression in transgenic mice. A fusion gene containing 3,300 nucleotides of MCK 5' sequence exhibited chloramphenicol acetyltransferase activity levels that were more than 10(4)-fold higher in skeletal muscle than in other, nonmuscle tissues such as kidney, liver, and spleen. Expression in cardiac muscle was also greater than in these nonmuscle tissues by 2 to 3 orders of magnitude. Progressive 5' deletions from nucleotide -3300 resulted in reduced expression of the transgene, and one of these resulted in a preferential decrease in expression in cardiac tissue relative to that in skeletal muscle. Of the two enhancer sequences analyzed, only one directed high-level expression in both skeletal and cardiac muscle. The other enhancer activated expression only in skeletal muscle. These data reveal a complex set of cis-acting sequences that have differential effects on MCK expression in skeletal and cardiac muscle.

Insulin alters cardiac muscle creatine kinase activity

2000 ◽  
Vol 15 (1) ◽  
pp. 23-29 ◽  
Author(s):  
S. Mitani ◽  
Kenji Okumura ◽  
Hideo Matsui ◽  
Yukio Toki ◽  
Hidekazu Hashimoto ◽  
...  
Keyword(s):  
Creatine Kinase ◽  
Cardiac Muscle ◽  
Kinase Activity ◽  
Creatine Kinase Activity ◽  
Muscle Creatine Kinase ◽  
Muscle Creatine

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.

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