RESUMO
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.
Assuntos
Proteínas de Transporte , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Sequência de Aminoácidos , Sequência de Bases , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos , Sítios de Ligação , Proteínas de Ciclo Celular , Linhagem Celular , Sequência Consenso , Proteínas de Ligação a DNA/química , Genes Reporter , Sequências Hélice-Alça-Hélice , Humanos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Estrutura Secundária de Proteína , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Repressoras/biossíntese , Proteínas Repressoras/química , Proteínas de Saccharomyces cerevisiae , Homologia de Sequência de Aminoácidos , Complexo Correpressor Histona Desacetilase e Sin3RESUMO
The bHLH-ZIP protein Mad heterodimerizes with Max as a sequence-specific transcriptional repressor. Mad is rapidly induced upon differentiation, and the associated switch from Myc-Max to Mad-Max heterocomplexes seem to repress genes normally activated by Myc-Max. We have identified two related mammalian cDNAs that encode Mad-binding proteins. Both possess sequence homology with the yeast transcription repressor Sin3, including four conserved paired amphipathic helix (PAH) domains. mSin3A and mSin3B bind specifically to Mad and the related protein Mxi1. Mad-Max and mSin3 form ternary complexes in solution that specifically recognize the Mad-Max E box-binding site. Mad-mSin3 association requires PAH2 of mSin3A/mSin3B and the first 25 residues of Mad, which contains a putative amphipathic alpha-helical region. Point mutations in this region eliminate interaction with mSin3 proteins and block Mad transcriptional repression. We suggest that Mad-Max represses transcription by tethering mSin3 to DNA as corepressors and that a transcriptional repression mechanism is conserved from yeast to mammals.