Local Transcriptional Control of Utrophin Expression at the Neuromuscular Synapse

Gramolini, Anthony O.; Dennis, Carina L.; Tinsley, Jonathon M.; Robertson, George S.; Cartaud, Jean; Davies, Kay E.; Jasmin, Bernard J.

doi:10.1074/jbc.272.13.8117

Cited by 75 publications

(73 citation statements)

References 33 publications

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“…Direct gene transfer was performed on mouse soleus muscles as described in detail elsewhere (8,9,17). The soleus muscles were isolated and injected with 10 l of a solution containing the appropriate plasmids (utrophin A promoter, pCnA* and pCAT) diluted at a concentration of 2-4 g͞l.…”

Section: Methodsmentioning

confidence: 99%

“…Additionally, muscle denervation (5) as well as regeneration have been shown to also affect expression of utrophin (6,7). In this context, several laboratories have now identified specific transcription factors and promoter elements that are important for regulating the abundance and localization of utrophin transcripts within muscle fibers (8)(9)(10)(11)(12)(13)(14)(15). Of particular relevance, it was shown recently that utrophin can be transcribed from two different promoters, resulting in the expression of utrophin A and B transcripts that differ in their 5Ј end (16).…”

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confidence: 99%

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Expression of utrophin A mRNA correlates with the oxidative capacity of skeletal muscle fiber types and is regulated by calcineurin/NFAT signaling

Chakkalakal

Stocksley

Harrison

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

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139

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Utrophin levels have recently been shown to be more abundant in slow vs. fast muscles, but the nature of the molecular events underlying this difference remains to be fully elucidated. Here, we determined whether this difference is due to the expression of utrophin A or B, and examined whether transcriptional regulatory mechanisms are also involved. Immunofluorescence experiments revealed that slower fibers contain significantly more utrophin A in extrasynaptic regions as compared with fast fibers. Single-fiber RT-PCR analysis demonstrated that expression of utrophin A transcripts correlates with the oxidative capacity of muscle fibers, with cells expressing myosin heavy chain I and IIa demonstrating the highest levels. Functional muscle overload, which stimulates expression of a slower, more oxidative phenotype, induced a significant increase in utrophin A mRNA levels. Because calcineurin has been implicated in controlling this slower, high oxidative myofiber program, we examined expression of utrophin A transcripts in muscles having altered calcineurin activity. Calcineurin inhibition resulted in an 80% decrease in utrophin A mRNA levels. Conversely, muscles from transgenic mice expressing an active form of calcineurin displayed higher levels of utrophin A transcripts. Electrophoretic mobility shift and supershift assays revealed the presence of a nuclear factor of activated T cells (NFAT) binding site in the utrophin A promoter. Transfection and direct gene transfer studies showed that active forms of calcineurin or nuclear NFATc1 transactivate the utrophin A promoter. Together, these results indicate that expression of utrophin A is related to the oxidative capacity of muscle fibers, and implicate calcineurin and its effector NFAT in this mechanism.

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Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

Expression of utrophin A mRNA correlates with the oxidative capacity of skeletal muscle fiber types and is regulated by calcineurin/NFAT signaling

Chakkalakal

Stocksley

Harrison

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

122

139

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“…A better understanding of these mechanisms appears important particularly since up-regulation of utrophin is currently envisaged as a therapeutic strategy to prevent the relentless progression of DMD (24,25). In this context, we have recently shown that the nerve exerts a profound influence on utrophin gene expression (26). Since our previous experiments also demonstrated that nerve-derived electrical activity is not a key factor regulating utrophin expression (27), we postulated in these initial studies that nervederived trophic factors likely mediate the local transcriptional activation of the utrophin gene within nuclei of the postsynaptic membrane domain (26).…”

Section: Duchenne Muscular Dystrophy (Dmd)mentioning

confidence: 99%

Muscle and Neural Isoforms of Agrin Increase Utrophin Expression in Cultured Myotubes via a Transcriptional Regulatory Mechanism

Gramolini¹,

Burton²,

Tinsley³

et al. 1998

Journal of Biological Chemistry

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Duchenne muscular dystrophy is a prevalent X-linked neuromuscular disease for which there is currently no cure. Recently, it was demonstrated in a transgenic mouse model that utrophin could functionally compensate for the lack of dystrophin and alleviate the muscle pathology (Tinsley, J. M., Potter, A. C., Phelps, S. R., Fisher, R., Trickett, J. I., and Davies, K. E. (1996) Nature 384, 349 -353). In this context, it thus becomes essential to determine the cellular and molecular mechanisms presiding over utrophin expression in attempts to overexpress the endogenous gene product throughout skeletal muscle fibers. In a recent study, we showed that the nerve exerts a profound influence on utrophin gene expression and postulated that nerve-derived trophic factors mediate the local transcriptional activation of the utrophin gene within nuclei located in the postsynaptic sarcoplasm (Gramolini, A. O., Dennis, C. L., Tinsley, J. M., Robertson, G. S., Davies, K. E, Cartaud, J., and Jasmin, B. J. (1997) J. Biol. Chem. 272, 8117-8120). In the present study, we have therefore focused on the effect of agrin on utrophin expression in cultured C2 myotubes. In response to Torpedo-, muscle-, or nerve-derived agrin, we observed a significant 2-fold increase in utrophin mRNAs. By contrast, CGRP treatment failed to affect expression of utrophin transcripts. Western blotting experiments also revealed that the increase in utrophin mRNAs was accompanied by an increase in the levels of utrophin. To determine whether these changes were caused by parallel increases in the transcriptional activity of the utrophin gene, we transfected muscle cells with a 1.3-kilobase pair utrophin promoter-reporter (nlsLacZ) gene construct and treated them with agrin for 24 -48 h. Under these conditions, both muscle-and nerve-derived agrin increased the activity of ␤-galactosidase, indicating that agrin treatment led, directly or indirectly, to the transcriptional activation of the utrophin gene. Furthermore, this increase in transcriptional activity in response to agrin resulted from a greater number of myonuclei expressing the 1.3-kilobase pair utrophin promoter-nlsLacZ construct. Deletion of 800 base pairs 5 from this fragment decreased the basal levels of nlsLacZ expression and abolished the sensitivity of the utrophin promoter to exogenously applied agrin. In addition, site-directed mutagenesis of an N-box motif contained within this 800-base pair fragment demonstrated its essential contribution in this regulatory mechanism. Finally, direct gene transfer studies performed in vivo further revealed the importance of this DNA element for the synapse-specific expression of the utrophin gene along multinucleated muscle fibers. These data show that both muscle and neural isoforms of agrin can regulate expression of the utrophin gene and further indicate that agrin is not only involved in the mechanisms leading to the formation of clusters containing presynthesized synaptic molecules but that it can also participate in the local regulation of genes encoding s...

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“…Three-to 4-wk-old C57BL/6 mice were anesthetized, and anterior portions of both lower hindlimbs were shaved and disinfected. The underlying TA muscles were then directly injected with 25 l of DNA solution as described previously (Gramolini et al, 1997;Chakkalakal et al, 2003). The DNA solution contained equal amounts of the 1.3-kb human utrophin-A promoter-reporter (LacZ) construct (see Gramolini et al, 1997;Chakkalakal et al, 2003), the pPUBF⌬N-box construct (luciferase), a constitutively expressed chloramphenicol acetyl transferase (CAT) plasmid (to control for transduction efficiency), and either the ERF expression plasmid or its control (pSG5).…”

Section: Direct Gene Transfermentioning

confidence: 99%

Ets-2 Repressor Factor Silences Extrasynaptic Utrophin by N-Box–mediated Repression in Skeletal Muscle

Perkins

Basu

Budak

et al. 2007

MBoC

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Utrophin is the autosomal homologue of dystrophin, the protein product of the Duchenne's muscular dystrophy (DMD) locus. Utrophin expression is temporally and spatially regulated being developmentally down-regulated perinatally and enriched at neuromuscular junctions (NMJs) in adult muscle. Synaptic localization of utrophin occurs in part by heregulin-mediated extracellular signal-regulated kinase (ERK)-phosphorylation, leading to binding of GABP␣/␤ to the N-box/EBS and activation of the major utrophin promoter-A expressed in myofibers. However, molecular mechanisms contributing to concurrent extrasynaptic silencing that must occur to achieve NMJ localization are unknown. We demonstrate that the Ets-2 repressor factor (ERF) represses extrasynaptic utrophin-A in muscle. Gel shift and chromatin immunoprecipitation studies demonstrated physical association of ERF with the utrophin-A promoter N-box/EBS site. ERF overexpression repressed utrophin-A promoter activity; conversely, small interfering RNA-mediated ERF knockdown enhanced promoter activity as well as endogenous utrophin mRNA levels in cultured muscle cells in vitro. Laser-capture microscopy of tibialis anterior NMJ and extrasynaptic transcriptomes and gene transfer studies provide spatial and direct evidence, respectively, for ERF-mediated utrophin repression in vivo. Together, these studies suggest "repressing repressors" as a potential strategy for achieving utrophin up-regulation in DMD, and they provide a model for utrophin-A regulation in muscle. INTRODUCTIONDuchenne's muscular dystrophy (DMD) is a fatal neuromuscular disease caused by gene mutations leading to qualitative or quantitative disturbances in dystrophin expression (Hoffman et al., 1987). Dystrophin-related protein or utrophin is considered the autosomal homologue of dystrophin, because it shares extensive sequence homology as well as its size and abundance in muscle (Love et al., 1989;Khurana et al., 1990;Tinsley et al., 1992). Utrophin and dystrophin also share functional properties such as the ability to associate with the dystroglycan complex and bind F-actin (Matsumura et al., 1992;Winder and Kendrick, 1995;Rybakova et al., 2002). Direct evidence for the ability of utrophin to functionally substitute comes from experiments demonstrating that utrophin overexpression driven either by transgenic means Rafael et al., 1998;Tinsley et al., 1998;Fisher et al., 2001) or viral vectors (Gilbert et al., 1999;Wakefield et al., 2000;Cerletti et al., 2003) can rescue dystrophin-deficient muscle. Despite these functional similarities, dystrophin and utrophin exhibit distinct localization in normal adult tissues.Perinatally, utrophin is expressed throughout the sarcolemma, however, its expression declines as that of dystrophin increases (Khurana et al., 1991;Clerk et al., 1993), leading to its spatially restricted expression at neuromuscular and myotendinous junctions (NMJs and MTJs, respectively) of adult myofibers. These features and relevance toward a therapy for DMD provide a powerful impetus for b...

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Local Transcriptional Control of Utrophin Expression at the Neuromuscular Synapse

Cited by 75 publications

References 33 publications

Expression of utrophin A mRNA correlates with the oxidative capacity of skeletal muscle fiber types and is regulated by calcineurin/NFAT signaling

Expression of utrophin A mRNA correlates with the oxidative capacity of skeletal muscle fiber types and is regulated by calcineurin/NFAT signaling

Muscle and Neural Isoforms of Agrin Increase Utrophin Expression in Cultured Myotubes via a Transcriptional Regulatory Mechanism

Ets-2 Repressor Factor Silences Extrasynaptic Utrophin by N-Box–mediated Repression in Skeletal Muscle

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