MOUSE SIN3A INTERACTS WITH AND CAN FUNCTIONALLY SUBSTITUTE FOR THE AMINOTERMINAL REPRESSION DOMAIN OF THE MYC ANTAGONIST MXI1

Mxi1 is a basic region helix-loop-helix leucine zipper (bHLH/LZ) prote in that, in association with Max, antagonizes Myc oncogenic activities . A possible mechanistic basis for Mxi1-mediated repression was provid ed by the recent demonstration that the repressive potential of Mxi1 c orrelates with its ability to physically associate with mSin3B, one of two mammalian homologues of the yeast transcriptional repressor SIN3. Here, we sought to characterize more fully the physical properties of the second homologue, mSin3A and to determine whether the recruitment of mSin3A by Mxi1 is indeed required for anti-Myc activity. Transient transfection of mammalian cells showed that the mSin3A protein can as sociate with the strong repressive isoform of Mxi1 (Mxi1-SR) and that, like other Myc superfamily members, both mSin3A and Mxi1-SR localize to the nucleus. From a developmental standpoint, a comparative analysi s of Myc, Mxi1-SR and Sin3A expression during postnatal mouse developm ent and in differentiating mouse erythroleukemia (MEL) cells revealed that dramatic and reciprocal changes in Myc and Mxi1-SR mRNA levels ar e accompanied by minimal stage-specific changes in mSin3A gene express ion. This constant expression profile, coupled with the observation th at over-expression of mSin3A does not augment the anti-Myc activity of Mxi1-SR in the rat embryo fibroblast (REF) transformation assay, sugg ests that mSin3A is not a limiting factor in the regulation of Myc sup erfamily function. Finally, a mSin3A-Mxi1 fusion protein, in which the amino terminal mSin3-interacting domain of Mxi1-SR was replaced with the full-length mSin3A, exhibited a level of repression activity equiv alent to, or greater than, the level of repression obtained with Mxi1- SR. Taken together, these observations directly demonstrate that the a mino-terminal repression domain of Mxi1-SR functions solely to recruit mSin3A and possibly other proteins like mSin3A and this association i s necessary for the anti-Myc activity of Mxi1-SR.