C-MYC TARGET GENE SPECIFICITY IS DETERMINED BY A POST-DNA-BINDING MECHANISM

Uncertainty as to which member of a family of DNA-binding transcriptio n factors regulates a specific promoter in intact cells is a problem c ommon to many investigators. Determining target gene specificity requi res both an analysis of protein binding to the endogenous promoter as well as a characterization of the functional consequences of transcrip tion factor binding, By using a formaldehyde crosslinking procedure an d Gal4 fusion proteins, we have analyzed the timing and functional con sequences of binding of Myc and upstream stimulatory factor (USF)1 to endogenous cellular genes. We demonstrate that the endogenous cad prom oter can be immunoprecipitated with antibodies against Myc and USF1, W e further demonstrate that although both Myc and USF1 can bind to cad, the cad promoter can respond only to the Myc transactivation domain, We also show that the amount of Myc bound to the cad promoter fluctuat es in a growth-dependent manner. Thus, our data analyzing both DNA bin ding and promoter activity in intact cells suggest that cad is a Myc t arget gene. In addition, we show that Myc binding can occur at many si tes in vivo but that the position of the binding site determines the f unctional consequences of this binding. Our data indicate that a post- DNA-binding mechanism determines Myc target gene specificity. Importan tly; we have demonstrated the feasibility of analyzing the binding of site-specific transcription factors in vivo to single copy mammalian g enes.