Previously, we found that MRFs (myogenic regulatory factors) regulated the expression of PGC-1alpha (peroxisome-proliferator-activated receptor gamma co-activator 1alpha) by targeting a short region, from nt -49 to +2 adjacent to the transcription initiation site, that contained two E-boxes. However, only the E2-box had significant affinity for MRFs, and the E1-box was predicted to be the target of Bhlhe40 (basic helix-loop-helix family, member e40, also known as Stra13, Bhlhb2, DEC1 and Sharp2), a transcriptional repressor implicated in the regulation of several physiological processes. In the present study, by using EMSA (electrophoresis mobility-shift assay), we confirmed that Bhlhe40 targeted the E1-box and formed a complex with the basic helix-loop-helix transcription factor MyoD (myogenic differentiation factor D) on the PGC-1alpha core promoter. We demonstrate that Bhlhe40 binds to the promoters of PGC-1alpha and myogenic genes in vivo and that Bhlhe40 represses the MyoD-mediated transactivation of these promoters. Furthermore, we found that this repression could be relieved by P/CAF (p300/CBP-associated factor) in a dose-dependent manner, but not by CBP [CREB (cAMP-response-element-binding protein)-binding protein]. Bhlhe40 interacted with P/CAF and this interaction disrupted the interaction between P/CAF and MyoD. These results suggest that Bhlhe40 functions as a repressor of MyoD by binding to adjacent E-boxes and sequestering P/CAF from MyoD.

c PGC-1␣ is a transcriptional coactivator promoting oxidative metabolism in many tissues. Its expression in skeletal muscle (SKM) is induced by hypoxia and reactive oxidative species (ROS) generated during exercise, suggesting that PGC-1␣ might mediate the cross talk between oxidative metabolism and cellular responses to hypoxia and ROS. Here we found that PGC-1␣ directly interacted with Bhlhe40, a basic helix-loop-helix (bHLH) transcriptional repressor induced by hypoxia, and protects SKM from ROS damage, and they cooccupied PGC-1␣-targeted gene promoters/enhancers, which in turn repressed PGC-1␣ transactivational activity. Bhlhe40 repressed PGC-1␣ activity through recruiting histone deacetylases (HDACs) and preventing the relief of PGC-1␣ intramolecular repression caused by its own intrinsic suppressor domain. Knockdown of Bhlhe40 mRNA increased levels of ROS, fatty acid oxidation, mitochondrial DNA, and expression of PGC-1␣ target genes. Similar effects were also observed when the Bhlhe40-mediated repression was rescued by a dominantly active form of the PGC-1␣-interacting domain (PID) from Bhlhe40. We further found that Bhlhe40-mediated repression can be largely relieved by exercise, in which its recruitment to PGC-1␣-targeted cis elements was significantly reduced. These observations suggest that Bhlhe40 is a novel regulator of PGC-1␣ activity repressing oxidative metabolism gene expression and mitochondrion biogenesis in sedentary SKM. Skeletal muscle (SKM) is one of the major metabolic organs, and it has the ability to adapt to various physiological conditions, such as cold, exercise, and sedentary life, by adjusting the balance between glycolytic and oxidative metabolism to meet the energy requirement under these conditions (1). Recent studies suggest that this adaptation relies much on the expression and activity of PGC-1␣, a transcriptional coactivator highly expressed in tissues with high energy metabolism (1, 2). PGC-1␣ enhances the activity of many nuclear receptors (NR), including peroxisome proliferator-activated receptor ␥ (PPAR␥), estrogen receptor (ER), and glucocorticoid receptor (GR), and non-NR transcription factors, such as MEF2, Sox9, FoxO1, and SREBP1 (reviewed in reference 3 and references therein), to promote oxidative metabolism, metabolic thermogenesis adaptation, biogenesis of mitochondria, gluconeogenesis, and fatty acid oxidation in various tissues (4-6). Overexpression of PGC-1␣ can promote metabolic switch from anaerobic glycolysis to oxidative metabolism (7,8). Current studies also show that PGC-1␣ plays critical roles in orchestrating the cellular response to hypoxia (9).In SKM, PGC-1␣ is preferentially expressed in oxidative metabolism-dependent slow-twitch fibers (10), and its overexpression can convert putative fast-twitch fibers into slow-twitch fibers (10). The activation of oxidative and slow-twitch muscle-specific genes by PGC-1␣ is mediated through its coactivation of Mef2 and PPAR binding to the upstream regulatory sites of these target genes (11). PGC-1␣ null mice sho...

M- and N-cadherin are members of the Ca(2+)-dependent cell-cell adhesion molecule family. M-cadherin is expressed predominantly in developing skeletal muscles and has been implicated in terminal myogenic differentiation, particularly in myoblast fusion. N-cadherin-mediated cell-cell adhesion also plays an important role in skeletal myogenesis. In the present study, we found that both genes were differentially expressed in C2C12 and Sol8 myoblasts during myogenic differentiation and that the expression of M-cadherin was preferentially enhanced in slow-twitch muscle. Interestingly, most MRFs (myogenic regulatory factors) significantly activated the promoter of M-cadherin, but not that of N-cadherin. In line with this, overexpression of MyoD in C3H10T1/2 fibroblasts strongly induced endogenous M-cadherin expression. Promoter analysis in silico and in vitro identified an E-box (from -2 to +4) abutting the transcription initiation site within the M-cadherin promoter that is bound and differentially activated by different MRFs. The activation of the M-cadherin promoter by MRFs was also modulated by Bhlhe40 (basic helix-loop-helix family member e40). Finally, chromatin immunoprecipitation proved that MyoD as well as myogenin binds to the M-cadherin promoter in vivo. Taken together, these observations identify a molecular mechanism by which MRFs regulate M-cadherin expression directly to ensure the terminal differentiation of myoblasts.

M-cadherin is a skeletal muscle-specific transmembrane protein mediating the cell-cell adhesion of myoblasts during myogenesis.  It is expressed in the proliferating satellite cells and highly induced by myogenic regulatory factors (MRFs) during terminal myogenic differentiation.  Several conserved cis-elements, including 5 E-boxes, 2 GC boxes, and 1 conserved downstream element (CDE) were identified in the M-cadherin proximal promoter.  We found that E-box-3 and -4 close to the transcription initiation site (TIS) mediated most of its transactivation by MyoD, the strongest myogenic MRF.  Including of any one of the other E-boxes restored the full activation by MyoD, suggesting an essential collaboration between E-boxes.  Stronger activation of M-cadherin promoter than that of muscle creatine kinase (MCK) by MyoD was observed regardless of culture conditions and the presence of E47.  Furthermore, MyoD/E47 heterodimer and MyoD~E47 fusion protein achieved similar levels of activation in differentiation medium (DM), suggesting high affinity of MyoD/E47 to E-boxes 3/4 under DM.  We also found that GC boxes and CDE positively affected MyoD mediated activation.  The CDE element was predicted to be the target of the chromatin-modifying factor Meis1/Pbx1 heterodimer.  Knockdown of Pbx1 significantly reduced the expression level of M-cadherin, but increased that of N-cadherin.  Using ChIP assay, we further found significant reduction of MyoD recruitment to M-cadherin promoter when CDE was deleted.  Taken together, these observations suggest that the chromatin-modifying function of Pbx1/Meis1 is critical to M-cadherin promoter activation before MyoD is recruited to E-boxes to trigger transcription.