The Myostatin Gene Is a Downstream Target Gene of Basic Helix-Loop-Helix Transcription Factor MyoD

Abstract

Myostatin is a negative regulator of myogenesis, and inactivation of myostatin leads to heavy muscle growth. Here we have cloned and characterized the bovine myostatin gene promoter. Alignment of the upstream sequences shows that the myostatin promoter is highly conserved during evolution. Sequence analysis of 1.6 kb of the bovine myostatin gene upstream region revealed that it contains 10 E-box motifs (E1 to E10), arranged in three clusters, and a single MEF2 site. Deletion and mutation analysis of the myostatin gene promoter showed that out of three important E boxes (E3, E4, and E6) of the proximal cluster, E6 plays a significant role in the regulation of a reporter gene in C 2 C 12 cells. We also demonstrate by band shift and chromatin immunoprecipitation assay that the E6 E-box motif binds to MyoD in vitro and in vivo. Furthermore, cotransfection experiments indicate that among the myogenic regulatory factors, MyoD preferentially upregulates myostatin promoter activity. Since MyoD expression varies during the myoblast cell cycle, we analyzed the myostatin promoter activity in synchronized myoblasts and quiescent "reserve" cells. Our results suggest that myostatin promoter activity is relatively higher during the G 1 phase of the cell cycle, when MyoD expression levels are maximal. However, in the reserve cells, which lack MyoD expression, a significant reduction in the myostatin promoter activity is observed. Taken together, these results suggest that the myostatin gene is a downstream target gene of MyoD. Since the myostatin gene is implicated in controlling G 1 -to-S progression of myoblasts, MyoD could be triggering myoblast withdrawal from the cell cycle by regulating myostatin gene expression.Myostatin is a newly identified member of the transforming growth factor ␤ superfamily, and myostatin-null mice have been found to show a two-to threefold increase in skeletal muscle mass due to an increase in the number of muscle fibers (hyperplasia) and the size of the fibers (hypertrophy) (21). Subsequently, we (15) and others (9, 22) reported that Belgian Blue and Piedmontese breeds of cattle, which are characterized by heavy muscling, have mutations in the myostatin gene coding sequence. Hence, myostatin is considered a negative regulator of muscle growth.Earlier studies have indicated that myostatin gene expression appears to be transcriptionally regulated during development (15, 21). Initially myostatin gene expression is detected in myogenic precursor cells of the myotome compartment of developing somites, and the expression is continued in adult axial and paraxial muscles (21). Different axial and paraxial muscles have been shown to express different levels of myostatin (15). Recent publications have shown that myostatin protein is also detected in heart (33) and mammary gland (14). In addition, myostatin is present in human skeletal muscle, and its expression is increased in the muscles of human immunodeficiency virus-infected men with muscle wasting compared to that in healthy men (8). Recently Wehling ...

Identification, characterization, and quantitative expression analysis of rainbow trout myostatin-1a and myostatin-1b genes

“…Every promoter contained several E-box motifs and all, but the brook trout (bt) MSTN-1b promoter contained multiple Comp 1 sites and TATA boxes in close proximity to the transcription start sites. Studies with mammalian promoters indicate that MEF2, GRE (glucocorticoid-response element), and MyoD (myogenic differentiation factor) binding sites regulate myostatin promoter transactivation , Spiller et al 2002, Forbes et al 2006. These sites were also identified in the fish promoters.…”

“…Mammalian myostatin promoters contain E-boxes and other elements critical to the differentiation and maturation of skeletal muscle, including both MyoD-and MEF2-binding sites. Indeed, both these sites have been implicated in the regulation of myostatin-gene expression in different animal and cellular systems (Spiller et al 2002, Salerno et al 2004, Shyu et al 2005. Expression of both rtMSTN-1a and rtMSTN-1b genes increases as somitogenesis progresses and rapidly decreases as it ends (Fig.…”

Sulforaphane causes a major epigenetic repression of myostatin in porcine satellite cells

“…It can be suggested that MyoD could be regulating MSTN gene expression by binding to its response element in the promoter region. Spiller et al 46 reported that one of bovine MSTN gene upstream regulatory elements appears to be critical for MSTN promoter activity and that MyoD interacts with this binding motif in vitro as well as in vivo to regulate MSTN gene expression. More recently, Liu et al 42 SFN and 5-aza-dC treatments clearly result in attenuated MSTN expression.…”

Location of myostatin expression during bovine myogenesis in vivo and in vitro

“…The fall of myostatin transcripts may occur consecutively to myogenin/MRF4 up-regulation and/or MyoD/Myf5 down-regulation during terminal differentiation. Myogenic transcription factor binding sites (E-boxes) have recently been found in the bovine myostatin promoter [44] indicating that the gene is at least a downstream target of MyoD1. However, in our differentiating cultures, myostatin levels were decreased in spite of high myogenin levels.…”