Downloaded from genesdev.cshlp.org on October 5, 2021 - Published by Cold Spring Harbor Laboratory Press Mechanism of corepressor binding and release from nuclear hormone receptors Laszlo Nagy,3,5,6 Hung-Ying Kao,3,6 James D. Love,1,4 Chuan Li,3 Ester Banayo,2,3 John T. Gooch,1 V. Krishna, K. Chatterjee,4 Ronald M. Evans,2,3,7 and John W.R. Schwabe1,3,7 1Medical Research Council (MRC), Laboratory of Molecular Biology, Cambridge, CB2 2QH, UK; 2Howard Hughes Medical Institute (HHMI), 3The Salk Institute for Biological Studies, Gene Expression Laboratory, La Jolla, California 92037 USA; 4Department of Medicine, University of Cambridge, Addenbrooke’s Hospital, Cambridge, CB2 2QQ, UK The association of transcription corepressors SMRT and N-CoR with retinoid and thyroid receptors results in suppression of basal transcriptional activity. A key event in nuclear receptor signaling is the hormone-dependent release of corepressor and the recruitment of coactivator. Biochemical and structural studies have identified a universal motif in coactivator proteins that mediates association with receptor LBDs. We report here the identity of complementary acting signature motifs in SMRT and N-CoR that are sufficient for receptor binding and ligand-induced release. Interestingly, the motif contains a hydrophobic core (⌽xx⌽⌽) similar to that found in NR coactivators. Surprisingly, mutations in the amino acids that directly participate in coactivator binding disrupt the corepressor association. These results indicate a direct mechanistic link between activation and repression via competition for a common or at least partially overlapping binding site. [Key Words: Transcription corepressors; SMRT; coactivator binding; corepressor binding; nuclear hormone receptors] Received September 27, 1999; revised version accepted November 4, 1999. Members of the steroid hormone receptor superfamily tor (ACTR)/RAC3/P/CIP) (Onate et al. 1995; Hong et al. are hormone-activated transcription factors that control 1996; Kamei et al. 1996; Yao et al. 1996; Chen et al. 1997; vertebrate development, differentiation, and homeosta- Torchia et al. 1997; Blanco et al. 1998) possess intrinsic sis through regulating complex gene networks (Man- histone acetyl transferase activity and potentiate the gelsdorf and Evans 1995; Mangelsdorf et al. 1995). Re- transcriptional activity of ligand bound receptors. ceptors for thyroid hormone and retinoid acid function Nuclear receptors contain two evolutionarily con- as potent repressors in the absence of ligand and as acti- served modules, the DNA binding domain (DBD) and the vators upon ligand binding. Intensive studies on the ligand binding domain (LBD). LBDs are required for mechanisms underlying this regulation led to the iden- nuclear localization, homo- and/or heterodimerization, tification of different families of proteins that bind to the and most importantly ligand binding and ligand-induced receptors in the absence and presence of hormone. switch of the transcriptional activity. Molecular studies SMRT (for silencing mediator for retinoid and thyroid established that the LXXLL signature motif within co- hormone receptors) and N-CoR (for nuclear receptor activators confers stereospecific interaction with ligand- corepressor) are homologous proteins that mediate the activated nuclear receptors (Heery et al. 1997). Biochemi- repressive effect of unliganded nuclear receptors through cal and crystallographic analyses revealed that an LXXLL the recruitment of histone deacetylase complexes (Al- motif-containing ␣-helix from coactivators interacts land et al. 1997; Hassig et al. 1997; Heinzel et al. 1997; with a hydrophobic groove within the ligand-bound Laherty et al. 1997; Nagy et al. 1997; Zhang et al. 1997). LBDs (Darimont et al. 1998; Nolte et al. 1998; Shiau et In contrast, CBP/p300, p300/CBP-associated factor al. 1998). Importantly, the residues that comprise the (PCAF), and members of the p160 family (SRC-1; GRIP1/ hydrophobic groove are well conserved between nuclear TIF2; activator for thyroid hormones and retinoid recep- receptors and have long been recognized as an LBD sig- nature motif. Initial mapping studies with the corepressor proteins revealed that the receptor interaction and repression 5Present address: Department of Biochemistry and Molecular Biology, University Medical School of Debrecen, Debrecen, Hungary 4H-012. functions are separable (Chen and Evans 1995; Horlein et 6These authors contributed equally. al. 1995; Heinzel et al. 1997; Nagy et al. 1997), with the 7Corresponding authors. E-MAIL [email protected]; FAX (858) 455-1349. receptor interaction domains located toward the car- E-MAIL [email protected]; FAX 44 1223 213 556. boxyl terminus. Ligand binding is thought to adopt con- GENES & DEVELOPMENT 13:3209–3216 © 1999 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/99 $5.00; www.genesdev.org 3209 Downloaded from genesdev.cshlp.org on October 5, 2021 - Published by Cold Spring Harbor Laboratory Press Nagy et al. formational changes that lead to release of the corepres- based on homology between N-CoR and SMRT, as well sor and recruitment of the coactivators. However, the as proteolysis studies of receptor corepressor complexes, molecular mechanism underlying this switch remains suggested that the minimal interaction core may be unclear. smaller: 19 amino acids for ID2 and 17 amino acids for In this study we sought to characterize the molecular ID1. Two-hybrid interaction assays were established in basis for the interaction between nuclear receptors and mammalian cells in which Gal–DBD fusions of the corepressors. We have identified in both SMRT and SMRT–ID1 and SMRT-ID2 were challenged with reti- N-CoR short peptides of 19 amino acids [interaction do- noic acid receptor ␣ (RAR␣) fused to the VP16 activation main (ID) 1] and 17 amino acids (ID1), with an internal domain. As shown in Figure 1B, both the interaction signature motif (I/L)XX(I/V)I, which are sufficient for re- domains and the core motifs are sufficient to mediate ceptor interaction and ligand-induced dissociation. Se- receptor corepressor interactions and ligand-mediated re- quence analyses suggest that these motifs can adopt an lease in a fashion that mimics the full-length corepres- amphipathic ␣-helical conformation, reminiscent of the sor. These peptides also function in yeast two-hybrid as- signature motif LXXLL within the coactivators. Signifi- says (Fig. 1C) suggesting that binding does not appear to cantly, single mutations within the thyroid hormone re- require additional accessory factors. ceptor ␤ (TR␤) LBD known to be involved in coactivator binding fail to bind corepressors. These results suggest an underlying mechanistic link between coactivator and Mutations of the core hydrophobic residues corepressor binding via competition for a common or in interaction motifs abolish receptor interaction overlapping binding site. Analysis of the sequences of the core motifs reveals that each one contains a putative amphipathic ␣ helix (Gar- Results nier et al. 1978) (indicated by boldface underline, Fig. 2). To test the idea that the hydrophobic surface of this po- Mapping of the core receptor interaction motifs tential helix might form the critical surface for interac- in SMRT tion with the receptors, we mutated these residues and Previous studies have localized the receptor interaction tested, using the mammalian two-hybrid assay, their domains to the carboxyl terminus of SMRT and N-CoR. ability to interact with RAR. Figure 2 shows that any Further studies of the SMRT receptor interaction region mutation of the core hydrophobic residues, either in revealed it could be subdivided into two domains of 70 clusters (M1, M3, M10) or individually (M5–M7, M12– and 50 amino acids (ID1 and ID2) (Fig. 1A; see also M14), abolishes interaction of the corepressor with the Downes et al. 1996). Each of these domains can interact receptors. Interestingly, other mutations indicate that with receptors when isolated from the rest of the protein. the whole domain is generally sensitive to changes or Further examination of these two interacting domains further truncations (Fig. 2, M8, M9, M15, and M16), sug- Figure 1. Identification of minimal receptor in- teraction domains in SMRT. (A) A schematic rep- resentation of receptor interaction domains within the corepressor SMRT domain structure (sequence numbering corresponds to full-length human SMRT). The interaction of the different Gal–SMRT fusion constructs with RAR was evaluated using both mammalian, CV1 (B)and yeast (C) two-hybrid assays. Data were normal- ized with reference to the activity of a constitu- tive reporter. Transcriptional activity is ex- pressed either as fold activation relative to Gal–DBD alone (mammalian assay) or as report- er activity (yeast). An RAR agonist at 10 nM (TTNPB) was used in the mammalian assay to demonstrate ligand-dependent corepressor re- lease. Carboxyl-SMRT (C-SMRT) amino acids 2004–2517; (ID2) 2131–2201; (ID1) 2302–2352; (core ID2) 2131–2149; (core ID1) 2336–2352 (numbering as in the full-length human SMRT). 3210 GENES & DEVELOPMENT Downloaded from genesdev.cshlp.org on October 5, 2021 - Published by Cold Spring Harbor Laboratory Press Nuclear receptor corepressor signature motif Figure 2. Mutational analyses of the minimal interaction domains ID1, ID2, and ID(1+2). (A) Mutational analyses of SMRT–ID2; (B) mutational studies of SMRT–ID1; (C) mutation studies of SMRT ID(1+2). The bar charts show reporter activity relative to wild-type constructs. The horizontal