title: Mad proteins contain a dominant transcription repression domain.
abstract: Transcription repression by the basic region-helix-loop-helix-zipper (bHLHZip) protein Mad1 requires DNA binding as a ternary complex with Max and mSin3A or mSin3B, the mammalian orthologs of the Saccharomyces cerevisiae transcriptional corepressor SIN3. The interaction between Mad1 and mSin3 is mediated by three potential amphipathic alpha-helices: one in the N terminus of Mad (mSin interaction domain, or SID) and two within the second paired amphipathic helix domain (PAH2) of mSin3A. Mutations that alter the structure of the SID inhibit in vitro interaction between Mad and mSin3 and inactivate Mad's transcriptional repression activity. Here we show that a 35-residue region containing the SID represents a dominant repression domain whose activity can be transferred to a heterologous DNA binding region. A fusion protein comprising the Mad1 SID linked to a Ga14 DNA binding domain mediates repression of minimal as well as complex promoters dependent on Ga14 DNA binding sites. In 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 were also capable of repression. We show that the association between Mad1 and mSin3 is not only dependent on the helical SID but is also dependent on both putative helices of the mSin3 PAH2 region, suggesting that stable interaction requires all three helices. Our results indicate that the SID is necessary and sufficient for transcriptional repression mediated by the Mad protein family and that SID repression is dominant over several distinct transcriptional activators.
authors.affiliation: Division of Basic Science, Fred Hutchinson Cancer Research Center, Seattle, Washington 98014, USA. don.ayer@genetics.utah.edu
authors.forename: D E
authors.initials: DE
authors.lastname: Ayer
authors.forename: C D
authors.initials: CD
authors.lastname: Laherty
authors.forename: Q A
authors.initials: QA
authors.lastname: Lawrence
authors.forename: A P
authors.initials: AP
authors.lastname: Armstrong
authors.forename: R N
authors.initials: RN
authors.lastname: Eisenman
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D001665:Binding Sites; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D002460:Cell Line; D016384:Consensus Sequence; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D018257:Helix-Loop-Helix Motifs; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009687:Nuclear Proteins / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism; D012097:Repressor Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D017386:Sequence Homology, Amino Acid; D056264:Sin3 Histone Deacetylase and Corepressor Complex; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic
chemical_list: D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; C501382:MAD1 protein, S cerevisiae; C501384:MAD1L1 protein, human; C079439:MXD1 protein, human; D009687:Nuclear Proteins; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; C099683:SIN3A transcription factor; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D056264:Sin3 Histone Deacetylase and Corepressor Complex