MAD PROTEINS CONTAIN A DOMINANT TRANSCRIPTION REPRESSION DOMAIN

Transcription repression by the basic region-helix-loop-helix-zipper ( bHLHZip) protein Mad1 requires DNA binding as a ternary complex with M ax and mSinSA or mSin3B, the mammalian orthologs of the Saccharomyces cerevisiae transcriptional corepressor SIN3. The interaction between M ad1 and mSin3 is mediated by three potential amphipathic alpha-helices : one in the N terminus of Mad (mSin interaction domain, or SID) and t wo within the second pail ed amphipathic helix domain (PAH2) of mSin3A . Mutations that alter the structure of the SID inhibit in vitro inter action between Mad and mSin3 and inactivate Mad's transcriptional repr ession activity, sere me show that a 35-residue region containing the SID represents a dominant repression domain whose activity can be tran sferred to a heterologous DNA binding region. A fusion protein compris ing the Mad1 SID linked to a Gal4 DNA binding domain mediates repressi on of minimal as well as complex promoters dependent on Gal4 DNA bindi ng sites, Ln addition, the SID represses the transcriptional activity of linked VP16 and c-Myc transactivation domains. When fused to a full -length c-Myc protein, the Mad1 SID specifically represses both c-Myc' s transcriptional and transforming activities. Fusions between the GAL DNA binding domain and full-length mSin3 mere also capable of repress ion. We show that the association between Mad1 and mSin3 is not only d ependent on the helical SID but is also dependent on both putative hel ices of the mSin3 PAH2 region, suggesting that stable interaction requ ires all three helices, Our results indicate that the SID is necessary and sufficient for transcriptional repression mediated by the Mad pro tein family and that SID repression is dominant over several distinct transcriptional activators.