Natural and synthetic DNA elements with the CArG motif differ in expression and protein-binding properties.

Santoro, Irma M.; Walsh, Kenneth

doi:10.1128/mcb.11.12.6296

Cited by 46 publications

(31 citation statements)

References 58 publications

(76 reference statements)

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“…Based on observations that substitution of the c-fos SRE consensus CArG for the SM α-actin 5′ CArGs had no detectable effect on SMC specificity in vivo, we questioned whether SRF binding affinity was altered as significantly as would be predicted based on previous EMSA analyses of a wide range of SRE consensus CArG substitution mutants (16)(17)(18)(19). EMSAs previously done in our lab showed that the SRE consensus CArG, in the context of the c-fos promoter, bound SRF with greater activity than either CArG-A or CArG-B in the context of the SM α-actin promoter (7).…”

Section: Resultsmentioning

confidence: 99%

5′ CArG degeneracy in smooth muscle α-actin is required for injury-induced gene suppression in vivo

Hendrix¹,

Wamhoff²,

McDonald³

et al. 2005

J. Clin. Invest.

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) mechanisms are required for gene expression in smooth muscle cells (SMCs). However, an unusual feature of many SMC-selective promoter CArG elements is that they contain a conserved single G or C substitution in their central A/T-rich region, which reduces binding affinity for ubiquitously expressed SRF. We hypothesized that this CArG degeneracy contributes to cell-specific expression of smooth muscle α-actin in vivo, since substitution of c-fos consensus CArGs for the degenerate CArGs resulted in relaxed specificity in cultured cells. Surprisingly, our present results show that these substitutions have no effect on smooth muscle-specific transgene expression during normal development and maturation in transgenic mice. However, these substitutions significantly attenuated injury-induced downregulation of the mutant transgene under conditions where SRF expression was increased but expression of myocardin, a smooth muscle-selective SRF coactivator, was decreased. Finally, chromatin immunoprecipitation analyses, together with cell culture studies, suggested that myocardin selectively enhanced SRF binding to degenerate versus consensus CArG elements. Our results indicate that reductions in myocardin expression and the degeneracy of CArG elements within smooth muscle promoters play a key role in phenotypic switching of smooth muscle cells in vivo, as well as in mediating responses of CArG-dependent smooth muscle genes and growth regulatory genes under conditions in which these 2 classes of genes are differentially expressed.

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Section: Resultsmentioning

confidence: 99%

5′ CArG degeneracy in smooth muscle α-actin is required for injury-induced gene suppression in vivo

Hendrix¹,

Wamhoff²,

McDonald³

et al. 2005

J. Clin. Invest.

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“…Mutation of the MCK enhancer CArG element, a potential binding site for serum response factor (36,56,68), had no dramatic effect on transgene expression in either adult cardiac or skeletal muscle. Although it is possible that a conserved CArG consensus sequence at nt Ϫ177 compensated for the mutation of the enhancer CArG element, this compensation clearly fails to occur in cultured muscle cells, in which the same mutation in the enhancer CArG site produced a loss of transcriptional activity in both cardiomyocytes and skeletal myocytes (2).…”

Section: Discussionmentioning

confidence: 99%

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

Donoviel

Shield

Buskin

et al. 1996

Molecular and Cellular Biology

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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...

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“…This CArG sequence is completely conserved between the rabbit, mouse, rat, and human MCK genes (16,18,37,45), but the functional significance of this MCK sequence had not been documented prior to this study. The CArG motif forms the core of regulatory elements in a number of muscle-specific as well as immediate-early genes (29,38), and the proximal CArG motif of the skeletal actin gene is essential for basal expression and can confer tissue-specific regulation to heterologous promoter constructs (35,40,41). The nuclear proteins SRF and YY1 bind to many CArG elements in vitro and function as positive and negative transcription factors, respectively (13).…”

Section: Discussionmentioning

confidence: 99%

Different regulatory sequences control creatine kinase-M gene expression in directly injected skeletal and cardiac muscle.

Vincent¹,

Gualberto²,

Patel³

et al. 1993

Mol. Cell. Biol.

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Regulatory sequences of the M isozyme of the creatine kinase (MCK) gene have been extensively mapped in skeletal muscle, but little is known about the sequences that control cardiac-specific expression. The promoter and enhancer sequences required for MCK gene expression were assayed by the direct injection of plasmid DNA constructs into adult rat cardiac and skeletal muscle. A 700-nucleotide fragment containing the enhancer and promoter of the rabbit MCK gene activated the expression of a downstream reporter gene in both muscle tissues. Deletion of the enhancer significantly decreased expression in skeletal muscle but had no detectable effect on expression in cardiac muscle. Further deletions revealed a CArG sequence motif at position -179 within the promoter that was essential for cardiac-specific expression. The CArG element of the MCK promoter bound to the recombinant serum response factor and YY1, transcription factors which control expression from structurally similar elements in the skeletal actin and c-fos promoters. MCK-CArG-binding activities that were similar or identical to serum response factor and YY1 were also detected in extracts from adult cardiac muscle. These data suggest that the MCK gene is controlled by different regulatory programs in adult cardiac and skeletal muscle.Cardiac and skeletal muscle are functionally similar, highly differentiated tissues that can be distinguished by the expression of distinct tissue-specific isoforms of contractile proteins, receptors, ion channels, and cytosolic enzymes. Numerous DNA regulatory elements, transcription factors, and determination genes required for expression in skeletal muscle have been identified (17,19,29,30,36,37,40,42,45), but far less is known about the developmental molecular biology of cardiac muscle. These studies are complicated by the lack of cardiac cell lines and by the tendency of cultured cells to express the fetal isoforms of muscle-specific genes (3,8,9). Recently, adult cardiac and skeletal muscle tissues have been shown to take up directly injected plasmid DNA and to express reporter genes from viral and muscle-specific regulatory sequences (1,2,5,21,25,33,43,44). Expression from these extrachromosomal, circular plasmids is detected in a small number of myocytes at the site of injection, and expression can persist for months. The simplicity of this technique and the stability of expression has led to the belief that the direct intramuscular injection of DNA may ultimately be used to deliver vaccines, hormones, and other polypeptides to adult organisms, thereby requiring the identification of gene regulatory sequences that control expression in terminally differentiated tissues.We chose to investigate the M isozyme of the creatine kinase (MCK) gene by the direct injection method because, unlike the case for myosin and actin, the same isoform is expressed in cardiac and skeletal muscle, and because its regulatory sequences have been extensively characterized in skeletal muscle cell culture and in transgenic mice studies (12, 16...

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Natural and synthetic DNA elements with the CArG motif differ in expression and protein-binding properties.

Cited by 46 publications

References 58 publications

5′ CArG degeneracy in smooth muscle α-actin is required for injury-induced gene suppression in vivo

5′ CArG degeneracy in smooth muscle α-actin is required for injury-induced gene suppression in vivo

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

Different regulatory sequences control creatine kinase-M gene expression in directly injected skeletal and cardiac muscle.

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