ROX, A NOVEL BHLHZIP PROTEIN EXPRESSED IN QUIESCENT CELLS THAT HETERODIMERIZES WITH MAX, BINDS A NONCANONICAL E-BOX AND ACTS AS A TRANSCRIPTIONAL REPRESSOR

Proteins of the Myc and Mad family are involved in transcriptional reg ulation and mediate cell differentiation and proliferation, These mole cules share a basic-helix-loop-helix leucine zipper domain (bHLHZip) a nd bind DNA at the E box (CANNTG) consensus by forming heterodimers wi th Max, We report the isolation, characterization and mapping of a hum an gene and its mouse homolog encoding a new member of this family of proteins, named Pox, Through interaction mating and immunoprecipitatio n techniques, we demonstrate that Rox heterodimerizes with Max and wea kly homodimerizes, Interestingly, bandshift assays demonstrate that th e Fox-Max heterodimer shows a novel DNA binding specificity, having a higher affinity for the CACGCG site compared with the canonical E box CACGTG site, Transcriptional studies indicate that Fox represses trans cription in both human HEK293 cells and yeast. We demonstrate that rep ression in yeast is through interaction between the N-terminus of the protein and the Sin3 co-repressor, as previously shown for the other M ad family members, ROX is highly expressed in quiescent fibroblasts an d expression markedly decreases when cells enter the cell cycle. Moreo ver, ROX expression appears to be induced in U937 myeloid leukemia cel ls stimulated to differentiate with 12-O-tetradecanoylphorbol-13-aceta te, The identification of a novel Max-interacting protein adds an impo rtant piece to the puzzle of Myc/Max/Mad coordinated action and functi on in normal and pathological situations. Furthermore, mapping of the human gene to chromosome 17p13.3 in a region that frequently undergoes loss of heterozygosity in a number of malignancies, together with the biochemical and expression features, suggest involvement of ROX in hu man neoplasia.