title: Functional analysis of the Mad1-mSin3A repressor-corepressor interaction reveals determinants of specificity, affinity, and transcriptional response.
abstract: The recruitment of corepressors by DNA-bound repressors is likely to be a critical rate-limiting step in the transcriptional regulation of many genes. An excellent paradigm for such an interaction is the association of the basic helix-loop-helix zipper protein Mad1 with the corepressor mSin3A. When bound together, the Sin3 interaction domain (SID) of Mad1 forms extensive hydrophobic contacts with the four-helix bundle formed by the paired amphipathic helix 2 (PAH2) domain of mSin3A. Using the costructure to predict the principle residues required for binding, we have carried out an extensive mutational analysis to examine the Mad1 SID-mSin3A PAH2 interaction in vitro and in vivo. Bulky hydrophobic residues in the alpha1 (I308 and V311) and alpha2 (L329 and L332) helices of the PAH2 domain are necessary to accommodate the precise arrangement of bulky (L12) and short (A15 and A16) hydrophobic residues in the amphipathic Mad1 SID. We have also used phage display to derive an optimal SID, which shows an essentially identical arrangement of key residues. By manipulating these key residues, we have generated altered-specificity Mad1 SID mutants that bind only to a PAH2 domain with a reciprocal mutation, permitting us to demonstrate for the first time that these domains interact directly in vivo. We have also found that the integrity of the PAH1 domain affects the Mad1 SID-PAH2 interaction. It is conceivable that cross talk between different PAH domains and their binding partners helps to determine the subunit composition and order of assembly of mSin3A complexes.
authors.affiliation: Fred Hutchinson Cancer Research Center, Seattle, Washington 98109, USA.
authors.forename: Shaun M
authors.initials: SM
authors.lastname: Cowley
authors.forename: Richard S
authors.initials: RS
authors.lastname: Kang
authors.forename: John V
authors.initials: JV
authors.lastname: Frangioni
authors.forename: Jason J
authors.initials: JJ
authors.lastname: Yada
authors.forename: Alec M
authors.initials: AM
authors.lastname: DeGrand
authors.forename: Ishwar
authors.initials: I
authors.lastname: Radhakrishnan
authors.forename: Robert N
authors.initials: RN
authors.lastname: Eisenman
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D018797:Cell Cycle Proteins; D002460:Cell Line; D017930:Genes, Reporter; D006801:Humans; D008958:Models, Molecular; D009154:Mutation; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010750:Phosphoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D056264:Sin3 Histone Deacetylase and Corepressor Complex; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic
chemical_list: D018797:Cell Cycle Proteins; C501384:MAD1L1 protein, human; D009687:Nuclear Proteins; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; C099683:SIN3A transcription factor; D014157:Transcription Factors; D056264:Sin3 Histone Deacetylase and Corepressor Complex