A conserved motif N-terminal to the DNA-binding domains of myogenic bHLH transcription factors mediates cooperative DNA binding with pbx-Meis1/Prep1.

The t(1;19) chromosomal translocation of pediatric pre-B cell leukemia produces chimeric oncoprotein E2a-Pbx1, which contains the N-terminal transactivation domain of the basic helix-loop-helix (bHLH) transcription factor, E2a, joined to the majority of the homeodomain protein, Pbx1. There are three Pbx family members, which bind DNA as heterodimers with both broadly expressed Meis/Prep1 homeo-domain proteins and specifically expressed Hox homeodomain proteins. These Pbx heterodimers can augment the function of transcriptional activators bound to adjacent elements. In heterodimers, a conserved tryptophan motif in Hox proteins binds a pocket on the surface of the Pbx homeodomain, while Meis/Prep1 proteins bind an N-terminal Pbx domain, raising the possibility that the tryptophan-interaction pocket of the Pbx component of a Pbx-Meis/Prep1 complex is still available to bind trypto-phan motifs of other transcription factors bound to flanking elements. Here, we report that Pbx-Meis1/Prep1 binds DNA cooperatively with heterodimers of E2a and MyoD, myogenin, Mrf-4 or Myf-5. As with Hox proteins, a highly conserved tryptophan motif N-terminal to the DNA-binding domains of each myogenic bHLH family protein is required for cooperative DNA binding with Pbx-Meis1/Prep1. In vivo, MyoD requires this tryptophan motif to evoke chromatin remodeling in the Myogenin promoter and to activate Myogenin transcription. Pbx-Meis/Prep1 complexes, therefore, have the potential to cooperate with the myogenic bHLH proteins in regulating gene transcription.

Monoclonal antimyogenin antibodies define epitopes outside the bHLH domain where binding interferes with protein-protein and protein-DNA interactions.

We have developed a panel of monoclonal antibodies against rat myogenin, a skeletal muscle regulatory protein of the bHLH family. Some of these monoclonals have been widely used by others, and details of their production are presented. Mapping the epitopes by immunoprecipitation of myogenin deletion mutants demonstrates that this panel recognizes epitopes spanning the entire molecule outside the HLH region. Four antibodies against epitopes outside the bHLH region interfere with the binding of myogenin/E-protein heterodimers to DNA sequences containing the myogenin heterodimer consensus recognition site. Three of these epitopes are partially masked in the heterodimers; antibody binding to these epitopes reduces the interactions between myogenin and E12. This suggests that surfaces outside the HLH dimerization domain may contribute to the stability of myogenin/E12 complexes. The binding of one antibody to its epitope did not appear to affect the myogenin/E12 interaction but nonetheless interfered with the binding of the complex to DNA, suggesting that this epitope lies near to a surface occupied by DNA.