Abstract
Members of the Mad family of bHLHZip proteins heterodimerize with Max and function to repress the transcriptional and transforming activities of the Myc proto-oncogene. Mad:Max heterodimers repress transcription by recruiting a large multi-protein complex containing the histone deacetylases, HDAC1 and HDAC2, to DNA. The interaction between Mad proteins and HDAC1/2 is mediated by the corepressor mSin3A and requires sequences at the amino terminus of the Mad proteins, termed the SID, for Sin3 interaction domain, and the second of four paired amphipathic alpha-helices (PAH2) in mSin3A. To better understand the requirements for the interaction between the SID and PAH2, we have performed mutagenesis and structural studies on the SID. These studies show that amino acids 8-20 of Mad1 are sufficient for SID:PAH2 interaction. Further, this minimal 13-residue SID peptide forms an amphipathic alpha-helix in solution, and residues on the hydrophobic face of the SID helix are required for interaction with PAH2. Finally, the minimal SID can function as an autonomous and portable repression domain, demonstrating that it is sufficient to target a functional mSin3A/HDAC corepressor complex.
Highlights
Transcriptional regulation depends on the assembly of large multiprotein complexes
The recent discovery that several transcriptional co-activators are histone acetyltransferases and that co-repressor complexes contain histone deacetylases as active components has provided a mechanistic basis for this correlation [22,23,24,25,26]. mSin3A and mSin3B were identified as corepressors required for the transcriptional and biological activities of the Mad proteins [27, 28]. mSin3A has recently been shown to be a component of a large multi-protein complex(s) that contains the histone deacetylases HDAC11 and HDAC2 in apparently stoichiometric amounts
To understand the structural basis for the direct interaction between the transcriptional repressor Mad1 and its corepressors mSin3A and mSin3B, we defined the minimal sequence of Mad1 required for interaction with PAH2, showed that this minimal interaction domain can adopt an amphipathic ␣-helical structure in solution, and determined that the hydrophobic face of this helix makes key contacts with mSin3A
Summary
Transcriptional regulation depends on the assembly of large multiprotein complexes. For example, the preinitiation complex [1], chromatin remodeling complexes [2, 3], and histone deacetylase-containing corepressor complexes [4, 5] have been shown to be in the 1–2 ϫ 106 dalton size range. In order to better understand the interaction between Mad family members and their corepressor mSin3A we have delineated the minimal functional SID, determined key contact residues required for interaction with PAH2 and demonstrated that the minimal SID domain is helical in solution.
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