Determinants of Myogenic Specificity within MyoD Are Required for Noncanonical E Box Binding

Abstract

The MyoD family of basic helix-loop-helix (bHLH) transcription factors has the remarkable ability to induce myogenesis in vitro and in vivo. This myogenic specificity has been mapped to two amino acids in the basic domain, an alanine and threonine, referred to as the myogenic code. These essential determinants of myogenic specificity are conserved in all MyoD family members from worms to humans, yet their function in myogenesis is unclear. Induction of the muscle transcriptional program requires that MyoD be able to locate and stably bind to sequences present in the promoter regions of critical muscle genes. Recent studies have shown that MyoD binds to noncanonical E boxes in the myogenin gene, a critical locus required for myogenesis, through interactions with resident heterodimers of the HOX-TALE transcription factors Pbx1A and Meis1. In the present study, we show that the myogenic code is required for MyoD to bind to noncanonical E boxes in the myogenin promoter and for the formation of a tetrameric complex with Pbx/Meis. We also show that these essential determinants of myogenesis are sufficient to confer noncanonical E box binding to the E12 basic domain. Thus, these data show that noncanonical E box binding correlates with myogenic potential, and we speculate that the myogenic code residues in MyoD function as myogenic determinants via their role in noncanonical E box binding and recognition.

The methyl-CpG-binding protein CIBZ suppresses myogenic differentiation by directly inhibiting myogenin expression

“…PCR primers for the ChIP assay were designed to amplify the promoter region (−185 to −1) or a more distally located sequence (−1 565 to −1 447) in the 5′-regulatory region. As expected, MyoD bound to the promoter region containing its binding sites [9,26], but not to the distal sequence, which lacks a binding site ( Figure 4A and 4B). Similarly, strong binding of CIBZ was observed at the promoter region but not at the distal region.…”

“…Immunoblot data showed that the expression of either CIBZ or MyoD was not greatly changed after the 5-aza-dC treatment (Supplementary information, Figure S3). ChIP analysis revealed that 5-aza-dC treatment almost abolished the binding of CIBZ to the Myog promoter region, but did not weaken the binding of MyoD, which binds to CpG-free E-box motifs at the Myog promoter [9], to this region ( Figure 4C). These results strongly suggest that binding of CIBZ to the Myog promoter is methylationdependent.…”

“…Cumulative evidence indicates that the promoter region (−184 to +18) is indispensable for Myog expression [8,9]. Transcription of Myog is stimulated mainly by MRFs or by members of the myocyte enhancer factor 2 (MEF2) family, through binding to the E-box elements or to the MEF2-binding site of the Myog promoter, respectively [5,10].…”

MEF2 Transcription Factors Regulate Distinct Gene Programs in Mammalian Skeletal Muscle Differentiation

“…Meis1 and Pknox2 are members of a small family of transcription factors that function in skeletal muscle as pioneer transcription factors to stabilize the MyoD/E12 DNA interaction on suboptimal E box sites, such as the E box found on the myogenin promoter (32)(33)(34). Although little is known about the function of Pknox2, it is similar in homology to Meis1 and is highly expressed in skeletal muscle tissue (35).…”

MyoD Directly Up-regulates Premyogenic Mesoderm Factors during Induction of Skeletal Myogenesis in Stem Cells

“…Furthermore, studies illustrate that two amino acids in the basic domain of MyoD, an alanine and threonine, are essential to activate the transcription of target genes (18,19). Finally, it was shown that the histidine/cysteine-rich and the amphipathic ␣-helix (helix III) domains of MyoD can regulate a subset of genes, distinct from the genes regulated by the classical N-terminal activation domain, through an interaction with the Pbx and Meis homeodomain proteins located adjacent to the myogenin promoter (20,21).…”