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 ma d1 mRNA are induced before the loss of c-Myc protein. This suggests th at inactivation of the differentiation-blocking activity of c-Myc migh t not occur primarily through the loss of Myc protein, but through an increase in the levels of its antagonist, Mad1. To investigate this qu estion we have analysed the levels of mad1 mRNA during differentiation of myeloid leukaemic HL60 cells. Although levels of mad1 mRNA were mo derately increased after induction with phorbol ester, we also found t hat 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 rapid ly after differentiation induction, formation of Mad1-containing compl exes only occurred during the later stages of the differentiation prog ramme. Further analysis of these Mad-containing complexes revealed tha t they were also unlikely to have the capacity to antagonize c-Myc fun ction, 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 c omplex are unlikely to be essential or determining events for myeloid differentiation. In addition, the discovery of DNA-binding complexes t hat contain Mad1, but not Max, opens up this transcription factor netw ork to include other Max-like proteins or proteins of an unrelated nat ure.