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First published online June 1, 2005
doi: 10.1242/10.1242/dev.01874

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The circuitry of a master switch: Myod and the regulation of skeletal muscle gene transcription

Division of Human Biology, Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue North, Seattle, WA 98109, USA

View larger version (29K): [in a new window]   Fig. 1. The functional domains of Myod. Myod (red) forms a heterodimer with an E-protein (green) through the helix-loop-helix domains (helix1 and helix2). The adjacent basic regions (also in an alpha helical conformation) contact the DNA. In Myod, the basic region also contains the `myogenic code'. This consists of three residues that are conserved in all of the myogenic bHLH proteins (Myod, Myf5, Myog and Mrf4), which do not directly affect DNA binding but are necessary to activate the transcription of specific muscle genes by either interacting with co-factors or inducing confomational change, or both. Myod has a single transcriptional activation domain (AD), and a histidine- and cysteine-rich (H/C) region that contains a tryptophan residue that is needed for Myod to interact with the Pbx/Meis complex. The helix 3 region is also required for Myod to cooperatively bind to the Pbx/Meis complex at the Myogenin (Myog) promoter. The E-protein has two independent activation domains (AD1 and AD2) and a domain that can repress the function of either activation domain (rep).

View larger version (31K): [in a new window]   Fig. 2. Epistatic relations among the myogenic bHLH factors. Shh and Wnt signaling from the notochord and dorsal neural tube, respectively, have been shown to regulate the expression of Myf5 in the epaxial dermamyotome of the somite (green). Pax3 and Myf5 independently regulate Myod expression. The factors regulating the early expression of Mrf4 are not known; however, it is likely that the same factors necessary for Myf5 expression regulate the transient expression of Mrf4 in the somite (shown as dashed lines) because these genes are physically very close together and share regulatory elements. Myod positively auto-regulates its own expression and activates the expression of Myog, and both Myod and Myog are expressed during skeletal muscle differentiation. In addition to its early and transient expression in the somite, Mrf4 is also expressed in the terminally differentiated muscle cells, and it is likely that Myod and Myog regulate this late expression of Mrf4. A transgene that drives Mrf4 expression from the Myog promoter can partly compensate for the loss of Myog (Zhu and Miller, 1997), demonstrating a partly redundant role of Mrf4 and Myog in terminal differentiation.

View larger version (13K): [in a new window]   Fig. 3. A Myod-generated feed-forward circuit temporally patterns gene expression during skeletal muscle differentiation. Myod regulates the transcription of the Mef2 isoforms, including Mef2d, and activates the p38 kinase pathway, shown here mediated by factor X. Factor X might be the Akt2 kinase, which is transcriptionally regulated by Myod and phosphorylates p38. The phosphorylated p38 becomes an active kinase and phosphorylates Mef2d, permitting it to bind and activate the myosin heavy chain (Myh3) gene together with Myod. The Myh3 gene is not activated by Myod until Mef2d is expressed and p38 is active (Penn et al., 2004). The feed-forward mechanism regulates the activity of Myod at a subset of promoters and imposes a temporal order on Myod-mediated gene expression. This diagram uses the graphical language BioD (Cook et al., 2001).

View larger version (12K): [in a new window]   Fig. 4. Evolving a feed-forward regulatory network from a single input or a simple cascade regulatory network. (A) In the feed-forward network, factor A directly regulates each gene: sequential activation is achieved by requiring both A and B to express gene C; and both A and C to express gene D. (B) In a single-input network, factor A directly regulates the three targets B, C and D and does not have temporal patterning. (C) The simple cascade accomplishes sequential gene activation with only gene B directly activated by A. It is easy to see how generating a new single-input network might generate a selectable phenotype that could evolve feed-forward regulation. Evolving a cascade motif would require a selective advantage for each stage, but once it had evolved it could be invaded by factors such as Myod to convert it into a feed-forward network.

View larger version (23K): [in a new window]   Fig. 5. Myod targets chromatin-remodeling complexes to the Myog promoter. (A) In the undifferentiated myoblast, a nucleosome (gray) is likely to be positioned over the E-box and the binding sites for Mef2 and Six factors, based on the limited access that restriction endonucleases have to this region (Gerber et al., 1997); however, Pbx/Meis is bound even in the presence of the nucleosome (Berkes et al., 2004). Chromatin immunoprecipitation (ChIP) analysis indicates that Myod (MD) recruits an Hdac to the Myog promoter in myoblasts (Mal and Harter, 2003), possibly by interacting with the Pbx/Meis complex. Id proteins are expressed in the myoblast and dimerize with E-proteins, preventing the formation of Myod/E-protein heterodimers (Jen et al., 1992). Mef2 isoforms are present but are probably not bound to the Myog promoter in the myoblast (de La Serna et al., 2005), and the same is likely to be true for the Six proteins. (B) Early on during differentiation, Id levels decrease, leading to the formation of Myod/E-protein heterodimers that interact with the Pbx/Meis complex at the Myog promoter (Berkes et al., 2004; de La Serna et al., 2005). Myod recruits HATs and the Swi/Snf complex (de La Serna et al., 2005; Simone et al., 2004), which acetylate the histones and remodel the nucleosome, respectively. (C) The remodeling of the nucleosome permits Mef2 and Six protein isoforms to access their cognate sites and the stable binding of Myod to its E-box. In this model, therefore, Myod-directed chromatin remodeling must occur before Myod and other factors can access their cognate sites in the promoter. E, E-protein; Ac, acetylation of histone tail; E-box, Myod-binding site; HATs, histone acetyltransferases; Hdac, histone deacetylase.