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 ␣1 (I308 and V311) and ␣2 (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.An important mechanistic link between transcriptional regulation and chromatin structure is the association of sequencespecific DNA-binding transcription factors with coactivators and corepressors (3,11,46,57). Coactivator and corepressor recruitment is generally considered to underlie the gene-specific alterations in chromatin structure that accompany changes in gene expression during differentiation, development, and oncogenesis. An emerging theme is that many different transcription factors recruit the same set of coactivators and corepressors. For example, transcriptional activation mediated by p53, c-Myb, CREB, MyoD, MEF2, and different nuclear hormone receptors, among others, involves recruitment of the coactivator p300 or CBP, each of which possesses and binds histone acetyltransferases (for a review, see reference 54). Similarly, corepressor complexes such as mSin3 and NURD have been found to interact with many DNA-binding repressors as well as with histone deacetylases (HDACs) and other factors involved in the modification of chromatin to a repressive state (for reviews, see references 4 and 28). Given this multiplicity of associations, both the nature and the specificity of the interactions between transcription factors and their coactivators and corepressors take on considerable importance.We have been studying the interactions between Mad proteins and the mSin3A corepressor complex. Mad family proteins (comprising Mad1, Mxi1, Mad3, and Mad4) belong to the ...

Repression of p53-mediated Transcription by Adenovirus E1B 55-kDa Does Not Require Corepressor mSin3A and Histone Deacetylases

“…Val markedly reduced mSin3A-Mad1 interaction with the former (i.e. V311D) being essentially inert in this interaction (38,39). As shown in Fig.…”

“…mSin3A Is Not Required for Transcriptional Repression by E1B-The mSin3A corepressor complex is involved in gene repression mediated by multiple transcriptional repressors (39,43). To assess whether mSin3A is required for E1B to repress p53 target genes, we expressed WT E1B and the L30P mutant in LN-229 cells via lentiviral vectors.…”

SHP and Sin3A expression are essential for adamantyl-substituted retinoid-related molecule–mediated nuclear factor-κB activation, c-Fos/c-Jun expression, and cellular apoptosis

“…The Sin3 proteins (A and B) bind a number of docking proteins, SAP30, SAP18, SAP30L, and SAP25, which are responsible for the tethering of HDAC-1 and HDAC-2 as well as other proteins to the Sin3 proteins (2)(3)(4). In addition, Sin3A and Sin3B contain four highly conserved paired amphipathic helix domains through which the Sin3 complexes bind specific nuclear transcription factors (2,(5)(6)(7). These include the MAD family members p53, E2F-4, p33ING1, MNFβ, Ikaros, MeCP2, and ELK1 as well as the mortality factors MORF4, MRGX, and MRG15 (2).…”

“…In addition, Sin3A and Sin3B contain four highly conserved paired amphipathic helix domains through which the Sin3 complexes bind specific nuclear transcription factors (2,(5)(6)(7). These include the MAD family members p53, E2F-4, p33ING1, MNFβ, Ikaros, MeCP2, and ELK1 as well as the mortality factors MORF4, MRGX, and MRG15 (2). The event(s) that triggers the binding of these nuclear transcription factors to the Sin3A complex and targets the repressor complex to the transcription factor consensus sequences, resulting in subsequent repression of gene transcription rather than its induction, has not been delineated.…”

Histone deacetylase (HDAC) 1 and 2 are essential for accurate cell division and the pluripotency of embryonic stem cells

“…Although there are a burgeoning number of proteins in the acetylome in addition to histones (10), the Sin3A, NuRD, and CoREST complexes contain multiple DNA/chromatin recognition motifs (11), which, in combination with transcription factors (2,12), target HDAC1/2 to chromatin. Their physiological roles should therefore be viewed within the framework of chromatin modulation.…”