Analysis of E-box DNA binding during myeloid differentiation reveals complexes that contain Mad but not Max

It has been shown that during myeloid differentiation the levels of mad1 mRNA are induced before the loss of c-Myc protein. This suggests that inactivation of the differentiation-blocking activity of c-Myc might not occur primarily through the loss of Myc protein, but through an increase in the levels of its antagonist, Mad1. To investigate this question we have analysed the levels of mad1 mRNA during differentiation of myeloid leukaemic HL60 cells. Although levels of mad1 mRNA were moderately increased after induction with phorbol ester, we also found that differentiation could be achieved with other inducers without any concomitant up-regulation of mad1 mRNA. In addition, analysis of E-box DNA binding revealed that, although Myc-Max complexes were lost rapidly after differentiation induction, formation of Mad1-containing complexes only occurred during the later stages of the differentiation programme. Further analysis of these Mad-containing complexes revealed that they were also unlikely to have the capacity to antagonize c-Myc function, as they did not contain Max. Therefore these data suggest that an increase in the levels of mad1 mRNA or the formation of a Mad-Max complex are unlikely to be essential or determining events for myeloid differentiation. In addition, the discovery of DNA-binding complexes that contain Mad1, but not Max, opens up this transcription factor network to include other Max-like proteins or proteins of an unrelated nature.