The two amino acids of the myogenic code, an alanine and threonine in the basic domain, are essential determinants of myogenic specificity and provide the critical distinction between the MRF family of bHLH transcription factors and all other bHLH family members. Mutation of these amino acids results in a bHLH protein that is competent to activate transcription but that is no longer capable of converting nonmuscle cells into muscle. It has remained unclear what properties these amino acids afford to the MRF family for conferring myogenic-inducing ability. In the current study, we presented work that establishes the myogenic code as a critical determinant that allows MyoD to bind DNA with high affinity. We showed that these amino acid determinants of myogenic specificity within MyoD are required for efficient dimerization with E proteins and are required for high-affinity DNA binding. The requirement of the myogenic code for DNA binding is especially pronounced at low-affinity sites within the myogenin promoter, where the myogenic code is completely required for MyoD to bind directly to noncanonical E boxes and to form a tetrameric DNA-bound complex with Pbx/Meis.
Previous studies have come to different conclusions about the function of the myogenic code in transcriptional activation and the induction of myogenesis. Some studies have suggested that the primary role of the myogenic code is for proper display of the transcriptional activation domains of the MRFs (5
). These models suggest that the activation domain of MyoD is masked or the signal to activate is not transmitted properly to the activation domain in the absence of an intact myogenic code. By contrast, the initial work on the myogenic code suggested that it functioned to recruit a “recognition factor” that was essential for myogenic activation (40
). The existence of this factor was based on the observation that myogenic code mutants for several of the MRFs activated transcription in certain cell types better than in others (40
). Our data presented here suggest that the primary requirement of the myogenic code is for DNA binding and dimerization, but the data do not preclude the possibility that an intact myogenic code may result in a conformation of MyoD that enables interaction with a factor that aids in activation, or perhaps more likely, a factor that helps to stabilize DNA binding. Indeed, our data support a model in which Pbx/Meis serves as a myogenic pioneer factor that functions to stabilize the initial binding of a small amount of MyoD to myogenin
and possibly other essential targets, which would then allow more robust binding subsequently to canonical E boxes, as has been suggested previously (3
MyoD mutants that lack a region that is enriched for cysteines and histidines, or a C-terminal region known as helix III, were originally shown to be defective in chromatin remodeling at the myogenin
). Subsequently, it was shown that the chromatin remodeling domains of MyoD are also required for efficient binding to noncanonical E boxes, suggesting that the original stabilization of binding at the promoter through interaction with Pbx/Meis is a critical step in the formation of a stably remodeled locus and transcriptional activation (3
). An important distinction between the chromatin remodeling domains of MyoD and the myogenic code is that the residues of the myogenic code are essential for myogenesis, whereas the chromatin remodeling domains are not (16
). Chromatin remodeling domain mutants retain the ability to induce myogenesis, albeit less potently than wild-type MyoD (16
). We predict that this retained ability to induce myogenesis is the result of the residual capacity to bind noncanonical E boxes, which is in contrast to the complete inability of MyoD(NN) to bind to noncanonical E boxes and to induce myogenesis.
The existence of distinct mutations in diverse regions of MyoD that affect interaction with Pbx/Meis further supports the notion that there is a particular conformation that is critical to inducing the muscle transcriptional program. Indeed, recent work has shown that the carboxy- and amino-terminal portions of MyoD function together to activate transcription, suggesting that the positioning of these domains in relation to one another is a critical aspect of gene activation (19
). In addition, mutation of the critical alanine of the myogenic code in MyoD is known to cause the protein to have an altered conformation when bound to DNA, as shown by increased sensitivity to protease cleavage (18
). It is possible that a DNA binding defect could contribute, at least in part, to the increased protease sensitivity of the myogenic code mutants since stable binding to DNA by wild-type MyoD might provide some protection against protease cleavage.
A conformation role for the myogenic code is appealing, since the MyoD crystal structure shows that the two residues of the myogenic code do not make direct contact with DNA (25
). The crystal structure also suggests that replacing the AT with bulkier amino acids, such as the asparagines found in E12, would displace an arginine slightly C-terminal to the myogenic code (25
). However, a second site suppressor screen to identify substitutions at the position of this arginine that could restore myogenic activity did not yield any proteins with dominant inducing activity (18
). This, and altered protease sensitivity in myogenic code mutants bound to DNA, suggests that the AT may be required to set the basic domain in a particular conformation specific to the MRF family of bHLH transcription factors (18
). The crystal structure of MyoD further indicates that the basic domain forms an α-helix when bound to DNA, and the stability of this helix is a determinant in the binding affinity for a particular site (25
). The myogenic code affects not only the DNA binding affinity of MyoD but also the binding specificity, as substitution of AT into the E12 basic domain confers the DNA binding specificity of MyoD (22
). A role in establishing proper conformation of the basic domain would explain how seemingly subtle mutations in the MyoD basic domain have such drastic effects on numerous aspects of MyoD function, including myogenic specificity.
The ability of MyoD to induce myogenesis in cell culture and to initiate muscle specification in vivo requires that MyoD bind to select, immediate early targets. The ability to bind stably at critical muscle loci, such as myogenin
, requires the ability to interact with Pbx/Meis and to bind to noncanonical E boxes (3
). We hypothesize that the myogenic code is the critical determinant for optimal dimerization and DNA binding, which are required to stabilize MyoD on noncanonical binding sites in critical muscle loci to allow for subsequent chromatin remodeling, transcriptional activation, and myogenic induction. Examples such as this, of highly fine-tuned binding specificity, are likely to be a common mechanism by which individual members of large families of transcription factors are able to discriminate appropriate binding sites in the context of the genome and activate distinct transcriptional programs.