MAD3 AND MAD4 - NOVEL MAX-INTERACTING TRANSCRIPTIONAL REPRESSORS THATSUPPRESS C-MYC DEPENDENT TRANSFORMATION AND ARE EXPRESSED DURING NEURAL AND EPIDERMAL DIFFERENTIATION

The basic helix-loop-helix-leucine zipper (bHLHZip) protein Max associ ates with members of the Myc family, as well as with the related prote ins Mad (Mad1) and Mxi1, Whereas both Myc:Max and Mad:Max heterodimers bind related E-box sequences, Myc:Max activates transcription and pro motes proliferation while Mad:Max represses transcription and suppress es Myc dependent transformation, Here we report the identification and characterization of two novel Mad1-and Mxi1-related proteins, Mad3 an d Mad4. Mad3 and Mad4 interact with both Max and mSin3 and repress tra nscription from a promoter containing CACGTG binding sites, Using a ra t embryo fibroblast transformation assay, we show that both Mad3 and M ad4 inhibit c-Myc dependent cell transformation, An examination of the expression patterns of all mad genes during murine embryogenesis reve als that mad1, mad3 and mad4 are expressed primarily in growth-arreste d differentiating cells. mxi1 is also expressed in differentiating cel ls, but is co-expressed with either c-myc, N-myc, or both in prolifera ting cells of the developing central nervous system and the epidermis, In the developing central nervous system and epidermis, downregulatio n of myc genes occurs concomitant with upregulation of mad family gene s, These expression patterns, together with the demonstrated ability o f Mad family proteins to interfere with the proliferation promoting ac tivities of Myc, suggest that the regulated expression of Myc and Mad family proteins function in a concerted fashion to regulate cell growt h in differentiating tissues.