Myc and Mad bHLHZ domains possess identical DNA-binding specificities but only partially overlapping functions in vivo

The opposing transcriptional activities of the basic-helix–loop–helix-leucine zipper proteins Myc and Mad, taken together with information related to their expression patterns and biological effects, have led to a model of the Myc/Max/Mad network in which Myc and Mad proteins function as antagonists. This antagonism is presumed to operate at the level of genes targeted by these complexes, where Myc:Max activates and Mad:Max represses expression of the same set of genes. However, a detailed analysis of the DNA-binding preferences for Mad proteins has not been performed. Furthermore, the model does not address the findings that Myc:Max indirectly represses transcription of several regulatory genes. To examine these issues relating to DNA-binding specificity and biological responses, we have determined the DNA-binding preferences of Mad1 by using selection and amplification of randomized oligonucleotides and demonstrated that its intrinsic specificity is identical with that of c-Myc. We have also used a chimeric Myc protein, containing a substitution of the entire Mad basic-helix–loop–helix-leucine zipper motif, and shown that it can reproduce the growth-promoting activities of Myc, but not its apoptotic function. Our results suggest that Myc and Mad, although possessing identical in vitro DNA-binding specificities, do not have an identical set of target genes in vivo, and that apoptosis is one biological outcome in which the transcriptional effects of Myc are not directly antagonized by those of Mad.