MyoD Distal Regulatory Region Contains an SRF Binding CArG Element Required for MyoD Expression in Skeletal Myoblasts and during Muscle Regeneration

Abstract

We show here that the distal regulatory region (DRR) of the mouse and human MyoD gene contains a conserved SRF binding CArG-like element. In electrophoretic mobility shift assays with myoblast nuclear extracts, this CArG sequence, although slightly divergent, bound two complexes containing, respectively, the transcription factor YY1 and SRF associated with the acetyltransferase CBP and members of C/EBP family. A single nucleotide mutation in the MyoD-CArG element suppressed binding of both SRF and YY1 complexes and abolished DRR enhancer activity in stably transfected myoblasts. This MyoD-CArG sequence is active in modulating endogeneous MyoD gene expression because microinjection of oligonucleotides corresponding to the MyoDCArG sequence specifically and rapidly suppressed MyoD expression in myoblasts. In vivo, the expression of a transgenic construct comprising a minimal MyoD promoter fused to the DRR and ␤-galactosidase was induced with the same kinetics as MyoD during mouse muscle regeneration. In contrast induction of this reporter was no longer seen in regenerating muscle from transgenic mice carrying a mutated DRR-CArG. These results show that an SRF binding CArG element present in MyoD gene DRR is involved in the control of MyoD gene expression in skeletal myoblasts and in mature muscle satellite cell activation during muscle regeneration. INTRODUCTIONThe MyoD gene family, which includes MyoD, Myogenin, and MRF4, encode structurally related basic helix-loophelix (bHLH) transcription factors that are essential regulators of skeletal muscle lineage determination and differentiation in vertebrates (Davis et al., 1987;Olson et al., 1990;Weintraub et al., 1991). Myogenin is required for normal biochemical and morphological differentiation of skeletal muscle, but not for commitment of cells to the myogenic lineage (Hasty et al., 1993;Nabeshima et al., 1993), and MRF4 plays a role in muscle fiber maturation (Braun and Arnold, 1995;Patapoutian et al., 1995;Rawls et al., 1998). In contrast, MyoD and Myf5 are essential for skeletal muscle lineage determination and are expressed in proliferative myoblasts, before differentiation Rudnicki et al., 1992Rudnicki et al., , 1993. Additional studies at postnatal stages show that a MyoDϪ/Ϫ mutant mouse is severely deficient in regenerative capacity after injury, indicating that MyoD plays an essential role in regulating the myogenic program of satellite cells (Megeney et al., 1996, reviewed Tajbakhsh andCossu, 1997). Consistent with this conclusion, in many myogenic cell lines, the capacity of cells to terminally differentiate appears to be linked to the level of MyoD expression (Pinset et al., 1988;Brennan et al., 1990; reviewed Kitzmann and Fernandez, 2001). Therefore it is essential to understand the transcriptional controls regulating the MyoD gene in myoblasts in order to elucidate the molecular mechanisms by which postnatal skeletal muscle differentiation and regeneration are controlled. Myoblasts cell lines, being all derived from adult satellite cells, provide a...

MicroRNA-199a is induced in dystrophic muscle and affects WNT signaling, cell proliferation, and myogenic differentiation

“…49,50 This observation may be explained by the fact that SRF and MRTF factors have been detected in the interstitial cells that line the vasculature, which are absent in undifferentiated MSCs before myogenic differentiation. 51,52 The induction of miR199a-5p expression levels, coinciding with increased SRF expression during myogenic differentiation, further supports the hypothesis that SRF is a regulator of muscle hypertrophic growth while suppressing MSC proliferation. 25 Recently, it has been shown that induction of WNT7a can induce muscle hypertrophy by activating the PI3K/AKT pathway in myofibers.…”

Myod and H19-Igf2 locus interactions are required for diaphragm formation in the mouse

“…5C-E Δ13 muscles, compared with wt levels. With these data, and because Srf is one of the main transcription factors controlling Myod expression in muscle (Gauthier-Rouviere et al, 1996;L'honore et al, 2003), we propose as a working hypothesis that Igf2 controls Myod expression by downregulating Srf, possibly via the production of miR-483.…”

“…Recently, a microRNA, miR-483-5p, the gene for which is embedded in intron 2 of Igf2, has been shown to target the 3ЈUTR of serum response factor (Srf) (Qiao et al, 2011). The transcription factor Srf is one of the factors responsible for the activation of Myod expression (Gauthier-Rouviere et al, 1996;L'honore et al, 2003). Nevertheless, little is known in vivo about the role of Igf2 in skeletal muscle development and about potential interactions between Myod and Igf2.…”

Myocardin-related transcription factors are required for skeletal muscle development

“…Genetic deletions of Mrf4, MyoD or Myf5 alone do not produce overt defects in sarcomere formation in mice Rawls et al, 1998;Rudnicki et al, 1992), whereas genetic deletion of myogenin causes perinatal lethality due to a complete lack of myoblast fusion and myofiber formation (Hasty et al, 1993;Nabeshima et al, 1993). MyoD has also been shown to associate directly with SRF and to serve as a co-activator of SRF-dependent muscle genes (L'Honore et al, 2003). Interestingly, the MRTF dKO phenotype is more severe than the phenotypes of mice with single deletions of MyoD, Myf5 or Mrf4.…”