ANALYSIS OF THE DNA-BINDING ACTIVITIES OF MYC MAX/MAD NETWORK COMPLEXES DURING INDUCED-DIFFERENTIATION OF U-937 MONOBLASTS AND F9 TERATOCARCINOMA CELLS/

The bHLHZip protein Max interacts with both the Myc and Mad family pro teins forming heterodimers which specifically bind certain E-box DIVA recognition sequences, thereby regulating transcription, Whereas Myc p roteins actively promote cell proliferation, Mad complexes have the op posite function, Although the main regulation of this network seems to be the control of myc- and mad family gene expression, regulation at the level of DNA-binding and transactivation may also be in operation, Few studies on the DNA-binding activity of native Myc:Max or Max:Mad complexes have been reported mainly due to technical difficulties, To overcome these problems we have developed a specific and sensitive sol id phase DNA-binding assay based on partial purification of native Myc , Max and Mad1 complexes by immunological methods, Using this techniqu e we report that the DNA-binding activity of c-Myc-containing complexe s is reduced during induced differentiation of U-937 monoblasts and F9 embryonic teratocarcinoma cells. In contrast, the DNA-binding of Mad1 -containing complexes increases during monocytic differentiation, In g eneral, the DNA-binding activity of c-Myc and Mad1 correlate with thei r expression, However, our studies of early kinetics of TPA-induced di fferentiation of U-937 cells as well as of late events during F9 diffe rentiation suggest that post-translational regulation of Myc and Max D NA-binding may also occur. The solid phase DNA-binding assay may thus provide a tool to study the regulation of DNA-binding in more detail.