One conceptual challenge in the field of skeletal myogenesis concerns the mechanism that ensures the temporal sequence of expression of muscle genes (e.g. early and late genes) in response to one initial event. This concept applies well to MyoD-dependent myogenic conversion [24
]. Approximately 10 years ago, the prevailing model was that MyoD–E12/47 heterodimers could directly activate the transcription of all muscle genes containing E-box sequences on the regulatory sequences. This model has recently been revised, based upon discoveries that revealed the complexity of the epigenetic regulation of muscle-gene transcription. A seminal finding was the demonstration of the existence of discrete sub-programs that coordinate the temporal expression of muscle genes in fibroblasts converted by MyoD [26
]. An integrated analysis of genome-wide ChIP and expression profiling in muscle cells revealed a stepwise progression of skeletal myogenesis through a variety of events that amplify the initial induction by preexisting bHLH factors, MyoD and Myf5 [25
]. Several favorable conditions must enable transcriptional activation by MyoD and Myf5, which are otherwise inactive in myoblasts. These conditions include interactions with transcription factors that bind DNA sequences in proximity to E-box motifs, within the promoters of early muscle genes. For example, MyoD interacts with an adjacent protein complex containing the homeodomain protein Pbx/Meis, which is constitutively bound at the chromatin of the myogenin promoter [63
]. These interactions might facilitate the binding of MyoD to non-canonical E-box sequences and ‘poise’ the chromatin of target genes for rapid induction upon differentiation cues. MyoD acetylation [65
] is another important regulatory signal for the temporal pattern of gene expression in cultured myoblasts [66
] and during muscle regeneration [67
]. Other proteins that collaborate with MyoD in the activation of early muscle-gene transcription are MEF2 and Six1 [68
], which are directly induced by MyoD and can activate other muscle genes through cooperative interactions.
Activation of myogenin is an early crucial event in the differentiation program because the absence of myogenin precludes muscle development [69
]. Thus, MyoD-mediated activation of myogenin illustrates an example of ‘processive’ activation within muscle bHLH proteins that propagates the myogenic program. Despite the structural and functional similarities among muscle bHLH proteins, the degree of ‘redundancy’ with regard to their ability to activate target genes is still not completely understood. Recent studies showed that MyoD and myogenin occupy the chromatin of overlapping genes, but have distinct functions [20
]. Chromatin recruitment of MyoD at late genes initiates local epigenetic modifications that facilitate the recruitment of myogenin, which in turn is required for transcriptional activation of these genes in collaboration with MEF2D [70
], an example of co-operation between transcriptional activators. Additional targets of MyoD seem to contribute to the activation of late muscle genes [19
]. This implies that MyoD downstream genes confer on MyoD the competence to induce the transcription of genes that would otherwise be refractory to activation. One candidate mechanism by which MyoD downstream genes participate in the activation of muscle genes entails the recruitment of transcriptional co-activators, either via direct interaction with bHLH and/or MEF2 proteins or by the occupancy of adjacent chromatin domains. Thus, newly synthesized proteins might generate the ‘epigenetic variability’ that permits discrimination of different muscle genes, despite the redundant presence of E-box and MEF2 sites on their promoters. This model postulates that additional transcription factors enable bHLH and MEF2 factors to activate target genes by promoter recruitment of CMCs with complementary enzymatic activities that impart the epigenetic modifications conducive for transcription. Examples of different modalities of muscle-gene activation are illustrated in .
Figure 3 Mechanisms of activation of muscle-gene transcription. Muscle-gene transcription can be activated by different mechanisms. Cross-activation between muscle bHLH proteins, such as MyoD and myogenin, is an example of processivity by which myoblasts expand (more ...)
An additional level of control is provided by intracellular pathways, which are elicited in response to MyoD activation. MyoD-dependent expression of surface receptors, signaling components (kinases or phosphatases) and scaffold proteins can re-shape the intracellular signaling network to prepare stage-specific signal responsiveness. The same mechanism can confer on myoblasts an autonomous control of the myogenic program. One notable example is provided by the persistent induction of the p38 pathway by MyoD [21
], which extends the initial activation of p38 kinases by regeneration cues to late stages of skeletal myogenesis. It is likely that the interplay between signaling pathways and epigenetic changes induced during myogenic differentiation selects the repertoire of active loci in the genome of MSCs.