The c-Myc oncoprotein, which is required for cellular proliferation, resembles in its structure a growing number of transcription factors. However, the mechanism of its action in

The M-CAT binding factor transcription enhancer factor 1 (TEF-1) has been implicated in the regulation of several cardiac and skeletal muscle genes. Previously, we identified an E-box-M-CAT hybrid (EM) motif that is responsible for the basal and cyclic AMP-inducible expression of the rat cardiac ␣-myosin heavy chain (␣-MHC) gene in cardiac myocytes. In this study, we report that two factors, TEF-1 and a basic helix-loop-helix leucine zipper protein, Max, bind to the ␣-MHC EM motif. We also found that Max was a part of the cardiac troponin T M-CAT-TEF-1 complex even when the DNA template did not contain an apparent E-box binding site. In the protein-protein interaction assay, a stable association of Max with TEF-1 was observed when glutathione S-transferase (GST)-TEF-1 or GST-Max was used to pull down in vitro-translated Max or TEF-1, respectively. In addition, Max was coimmunoprecipitated with TEF-1, thus documenting an in vivo TEF-1-Max interaction. In the transient transcription assay, overexpression of either Max or TEF-1 resulted a mild activation of the ␣-MHC-chloramphenicol acetyltransferase (CAT) reporter gene at lower concentrations and repression of this gene at higher concentrations. However, when Max and TEF-1 expression plasmids were transfected together, the repression mediated by a single expression plasmid was alleviated and a three-to fourfold transactivation of the ␣-MHC-CAT reporter gene was observed. This effect was abolished once the EM motif in the promoter-reporter construct was mutated, thus suggesting that the synergistic transactivation function of the TEF-1-Max heterotypic complex is mediated through binding of the complex to the EM motif. These results demonstrate a novel association between Max and TEF-1 and indicate a positive cooperation between these two factors in ␣-MHC gene regulation.

The c-Myc oncoprotein, which is required for cellular proliferation, resembles in its structure a growing number of transcription factors. However, the mechanism of its action in vivo is not yet clear. The discovery of the specific cognate DNA-binding site for Myc and its specific heterodimerization partner, Max, enabled the use of direct experiments to elucidate how Myc functions in vivo and how this function is modulated by Max. Here we demonstrate that exogenously expressed Myc is capable of activating transcription in vivo through its specific DNA-binding site. Moreover, transcriptional activation by Myc is dependent on the basic region, the integrity of the helix-loop-helix and leucine zipper dimerization motifs located in the carboxy-terminal portion of the protein, and the regions in the amino terminus conserved among Myc family proteins. In contrast to Myc, exogenously expressed Max elicited transcriptional repression and blocked transcriptional activation by Myc through the same DNA-binding site. Our results suggest a functional antagonism between Myc and Max which is mediated by their relative levels in the cells. A model for the activity of Myc and Max in vivo is presented.