Identification of a New Hybrid Serum Response Factor and Myocyte Enhancer Factor 2-binding Element in MyoD Enhancer Required for MyoD Expression during Myogenesis

Abstract

MyoD is a critical myogenic factor induced rapidly upon activation of quiescent satellite cells, and required for their differentiation during muscle regeneration. One of the two enhancers of MyoD, the distal regulatory region, is essential for MyoD expression in postnatal muscle. This enhancer contains a functional divergent serum response factor (SRF)-binding CArG element required for MyoD expression during myoblast growth and muscle regeneration in vivo. Electrophoretic mobility shift assay, chromatin immunoprecipitation, and microinjection analyses show this element is a hybrid SRF-and MEF2 Binding (SMB) sequence where myocyte enhancer factor 2 (MEF2) complexes can compete out binding of SRF at the onset of differentiation. As cells differentiate into postmitotic myotubes, MyoD expression no longer requires SRF but instead MEF2 binding to this dual-specificity element. As such, the MyoD enhancer SMB element is the site for a molecular relay where MyoD expression is first initiated in activated satellite cells in an SRF-dependent manner and then increased and maintained by MEF2 binding in differentiated myotubes. Therefore, SMB is a DNA element with dual and stage-specific binding activity, which modulates the effects of regulatory proteins critical in controlling the balance between proliferation and differentiation. INTRODUCTIONSkeletal muscle repair is fulfilled by satellite cells, quiescent myogenic progenitors located between the basement membrane and myofibers in both growing and mature muscle (Mauro, 1961;Schultz et al., 1978;Muir et al., 1965;Bischoff and Heintz 1994;Yablonka-Reuveni, 1995). They undergo mitogenic activation in response to exercise or damageinduced signals proliferate, differentiate, and fuse into preexisting or newly formed myofibers during muscle regeneration (Grounds and Yablonka-Reuveni, 1993;Schultz and McCormick, 1994).Among basic helix-loop-helix (bHLH) myogenic regulatory factors (MRFs), MyoD is the earliest to be induced and detected both in vivo upon muscle regeneration with activation of satellite cells and in ex vivo in cell lines derived from such precursors (Fü tchbauer and Westphal, 1992;Grounds et al., 1992). Indeed, after isolation, activated satellite cells enter the cell cycle and express MyoD concomitantly with proliferating cell nuclear antigen (Smith et al., 1994;Yablonka-Reuveni and Rivera, 1994). This finding suggested a possible role for MyoD during satellite cell activation in regeneration (Yablonka-Reuveni and Rivera, 1994;Yablonka-Reuveni et al., 1999). In agreement with this, we have reported an early induction of MyoD 4 -6 h after entry of quiescent myoblasts into the cell cycle . In addition, muscles from MyoDϪ/Ϫ mutant mice are severely deficient in regenerative capacity after injury, supporting an essential role of MyoD in adult muscle (Megeney et al., 1996). Both in vivo and ex vivo studies using isolated myofibers and primary cultured myoblasts from MyoDϪ/Ϫ mice show that without MyoD, satellite cells undergo enhanced self-renewal rather than giving ...

Satellite Cells and the Muscle Stem Cell Niche

“…In proliferating myoblasts, SRF only drives low levels of MyoD expression (286), whose activity is inhibited by cyclin D1 induced cyclin dependent kinase 4 (Cdk4) (587). However, the induction of MEF2 expression prior to differentiation enables MEF2 to out-compete SRF for the SRE binding site and leads to high levels of MyoD expression and initiation of differentiation (286). This function of MEF2 is further regulated by a member of the myocardin family of transcription factors, MASTR, whose expression is upregulated in response to muscle injury (348).…”

MASTR directs MyoD-dependent satellite cell differentiation during skeletal muscle regeneration

“…3E; Kucharczuk et al 1999;Chen et al 2001Chen et al , 2002. Interestingly, the DRR enhancer harbors one canonical MEF2-binding site and a MEF2/SRF hybrid site (L'Honore et al 2007), suggesting that MASTR and MEF2 coregulate the DRR enhancer to induce MyoD expression. To test this hypothesis, we performed luciferase assays in C2C12 myoblasts using MyoD-DRR and MyoD-core reporter constructs.…”

Acetylcholinesterase Expression in Muscle Is Specifically Controlled by a Promoter-Selective Enhancesome in the First Intron

“…This DNA sequence resembles a serum responsive element (Galvagni et al, 1997;Li et al, 2005) or more likely a hybrid serum responsive factor and MEF2 binding element (L'Honore et al, 2007). The sequence is clearly critical to AChE transcriptional activation, because scrambling the DNA sequence through this region ablates AChE activity (Fig.…”