Structural and functional analysis of myostatin-2 promoter alleles from the marine fish Sparus aurata: Evidence for strong muscle-specific promoter activity and post-transcriptional regulation
ABSTRACT Myostatin (MSTN) is a negative regulator of skeletal muscle growth. In contrast to mammals, fish possess at least two paralogs of MSTN: MSTN-1 and MSTN-2. In this study, we analyzed the structural-functional features of the four variants of Sparus aurata MSTN-2 5'-flanking region: saMSTN-2a, saMSTN-2as, saMSTN-2b and saMSTN-2c. In silico analysis revealed numerous putative cis regulatory elements including several E-boxes known as binding sites to myogenic transcription factors. Transient transfection experiments using non-muscle and muscle cell lines showed surprisingly high transcriptional activity in muscle cells, suggesting the presence of regulatory elements unique to differentiated myotubes. These observations were confirmed by in situ intramuscular injections of promoter DNA followed by reporter gene assays. Moreover, high promoter activity was found in differentiated neural cell, in agreement with MSTN-2 expression in brain. Progressive 5'-deletion analysis, using reporter gene assays, showed that the core promoter is located within the first -127 bp upstream of the ATG, and suggested the presence of regulatory elements that either repress or induce transcriptional activity. Transient transgenic zebrafish provided evidence for saMSTN-2 promoter ability to direct GFP expression to myofibers. Finally, our data shows that although no mature saMSTN-2 mRNA is observed in muscle; unspliced forms accumulate, confirming high level of transcription. In conclusion, our study shows for the first time that MSTN-2 promoter is a very robust promoter, especially in muscle cells.
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ABSTRACT: Myostatin (MSTN) is a member of the transforming growth factor-ß superfamily, known as a negative regulator of skeletal muscle development and growth in mammals. In contrast to mammals, fish possess at least two paralogs of MSTN: MSTN-1 and MSTN-2. Here we describe the cloning and sequence analysis of spliced and precursor (unspliced) transcripts as well as the 5' flanking region of MSTN-2 from the marine fish Umbrina cirrosa (ucMSTN-2). In silico analysis revealed numerous putative cis regulatory elements including several E-boxes known as binding sites to myogenic transcription factors. Transient transfection experiments using non-muscle and muscle cell lines showed high transcriptional activity in muscle cells and in differentiated neural cells, in accordance with our previous findings in MSTN-2 promoter from Sparus aurata. Comparative informatics analysis of MSTN-2 from several fish species reveals high conservation of the predicted amino acid sequence as well as the gene structure (exon length) although intron length varied between species. The proximal promoter of MSTN-2 gene was found to be conserved among Perciforms. In conclusion, this study reinforces our conclusion that MSTN-2 promoter is a very strong promoter, especially in muscle cells. In addition, we show that MSTN-2 gene structure is highly conserved among fishes as is the predicted amino acid sequence of the peptide.Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology 11/2012; DOI:10.1016/j.cbpb.2012.11.003 · 1.90 Impact Factor
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ABSTRACT: In the last decade, myostatin (MSTN), a member of the TGFβ superfamily, has emerged as a strong inhibitor of muscle growth in mammals. In fish many studies reveal a strong conservation of mstn gene organization, sequence, and protein structures. Because of ancient genome duplication, teleostei may have retained two copies of mstn genes and even up to four copies in salmonids due to additional genome duplication event. In sharp contrast to mammals, the different fish mstn orthologs are widely expressed with a tissue-specific expression pattern. Quantification of mstn mRNA in fish under different physiological conditions, demonstrates that endogenous expression of mstn paralogs is rarely related to fish muscle growth rate. In addition, attempts to inhibit MSTN activity did not consistently enhance muscle growth as in mammals. In vitro, MSTN stimulates myotube atrophy and inhibits proliferation but not differentiation of myogenic cells as in mammals. In conclusion, given the strong mstn expression non-muscle tissues of fish, we propose a new hypothesis stating that fish MSTN functions as a general inhibitors of cell proliferation and cell growth to control tissue mass but is not specialized into a strong muscle regulator.General and Comparative Endocrinology 09/2013; DOI:10.1016/j.ygcen.2013.08.012 · 2.67 Impact Factor
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ABSTRACT: Glucocorticoids (GCs) strongly regulate myostatin transcript levels in mammals via glucocorticoid response elements (GREs) in the myostatin promoter, and bioinformatics methods suggest that this regulatory mechanism is conserved among many vertebrates. However, the multiple myostatin genes found in some fishes may be an exception. In rainbow trout (Oncorhynchus mykiss), two genome duplication events have produced three putatively functional myostatin genes, myostatin-1a, -1b and -2a, which are ubiquitously and differentially expressed. In addition, in silico promoter analyses of the rainbow trout myostatin promoters have failed to identify putative GREs, suggesting a divergence in myostatin function. Therefore, we hypothesized that myostatin mRNA expression is not regulated by glucocorticoids in rainbow trout. In this study, both juvenile rainbow trout and primary trout myoblasts were treated with cortisol to examine the relationship between this glucocorticoid and myostatin mRNA expression. Results suggest that exogenous cortisol does not regulate myostatin-1a and -1b expression in vivo, as myostatin mRNA levels were not significantly affected by cortisol treatment in either red or white muscle tissue. In red muscle, myostatin-2a levels were significantly elevated in the cortisol treatment group relative to the control, but not the vehicle control, at both 12h and 24h post-injection. As such, it is unclear if cortisol was acting alone or in combination with the vehicle. Cortisol increased myostatin-1b expression in a dose-dependent manner in vitro. Further work is needed to determine if this response is the direct result of cortisol acting on the myostatin-1b promoter or through an alternative mechanism. These results suggest that regulation of myostatin by cortisol may not be as highly conserved as previously thought and support previous work that describes potential functional divergence of the multiple myostatin genes in fishes.Comparative Biochemistry and Physiology - Part A Molecular & Integrative Physiology 05/2014; 175. DOI:10.1016/j.cbpa.2014.05.015 · 2.37 Impact Factor