The Nuclear Corepressors Recognize Distinct Nuclear Receptor Complexes

The Nuclear Corepressors Recognize Distinct Nuclear Receptor Complexes Ronald N. Cohen, Andrew Putney, Fredric E. Wondisford, and Anthony N. Hollenberg Thyroid Unit Department of Medicine Beth Israel Deaconess Medical Center and Harvard Medical School Boston, Massachusetts 02215 been shown to be due to the recruitment of at least two nuclear corepressor proteins, nuclear receptor corepressor (NCoR) and silencing mediator of retinoid and thyroid hormone receptors (SMRT) (2–7), which, in turn, recruit a multiprotein complex with histone deacetylase activity that appears to modify chromatin to prevent transcription (8–10). In the presence of their cognate ligands, the TR and RAR isoforms release the nuclear corepressors and recruit members of the coactivator family, which include the p160 family members [steroid receptor coactivator-1 (SRC-1), TIF II, and ACTR], CREB-binding protein (CBP) and p300, pCAF (11–18) (reviewed in Ref. 19), and other coactivators such as p120 (20). Unlike the nuclear corepressors, the coactivator complex possesses histone acetyl transferase activity, which allows for transcriptional activation. NCoR and SMRT are modular proteins (see Fig. 1) that contain at least three repressing domains in their N termini, and two domains that mediate interactions with the TR and RAR isoforms in their C termini (21– 24). In addition to mediating interactions with mSin3, Sun-CoR (25), and other members of the corepressor complex, the central domains of NCoR and SMRT also appear to mediate interactions with the AML-ETO product, which may prevent normal differentiation and lead to acute myelogenous leukemia in patients with this chromosomal translocation (26). In addition, the extreme amino-terminal domain of NCoR appears to be important in the regulation of the mature protein through its interaction with mSIAH2, which allows for proteolytic degradation of NCoR (27). Both NCoR and SMRT contain two C-terminal interacting domains that mediate interactions with both the TR isoforms and the RAR isoforms. While similar in structure, the more proximal of the interacting domains (ID 2) shares only 23% amino acid homology, while the more distal interacting domains (ID 1) are 53% homologous. Consistent with these differences in structure are results from several different groups, including our own, which suggest that NCoR and SMRT interact differently with nuclear receptors and that specificity may exist in the recruitment of nuclear The thyroid hormone receptor (TR) and retinoic acid receptor (RAR) isoforms have the capacity to silence gene expression in the absence of their ligands on target response elements. This active repression is mediated by the ability of the corepressors, nuclear receptor corepressor (NCoR) and silencing mediator of retinoid and thyroid hormone receptors (SMRT), to recruit a complex containing histone deacetylase activity. Interestingly, NCoR and SMRT share significant differences in the their two nuclear receptor-interacting domains (IDs), suggesting that they may recruit receptors with different affinities. In addition, the role of the receptor complex bound to a response element has not been fully evaluated in its ability to recruit separate corepressors. We demonstrate in this report that the proximal ID in NCoR and SMRT, which share only 23% homology, allows preferential recognition of nuclear receptors, such that TR prefers to recruit NCoR, and RAR prefers to recruit SMRT, to DNA response elements. However, mutations in the TR found in the syndromes of resistance to thyroid hormone can change the corepressor recruited by changing the complex (homodimer or heterodimer) formed on the TRE. These results demonstrate that the corepressor complex recruited can be both nuclear receptor- and receptor complex-specific. (Molecular Endocrinology 14: 900–914, 2000) INTRODUCTION The thyroid hormone (TR) and retinoic acid receptor (RAR) isoforms are members of the nuclear receptor superfamily (1). Unlike the majority of the members of this family, the TR and RAR possess ligand-independent activity that leads to the silencing of positively regulated target genes. This silencing activity has 0888-8809/00/$3.00/0 Molecular Endocrinology 14(6): 900–914 Copyright © 2000 by The Endocrine Society Printed in U.S.A. 900 Corepressors and Nuclear Receptor Complexes 901 Fig. 1. The Nuclear Corepressor Family Amino acid sequences of NCoR and SMRTe are indicated. The NCoR interacting domains are outlined and numbered (22). Corresponding portions of the homologous regions of SMRTe are also identified. corepressors by nuclear complexes. For example, it has been demonstrated by Zamir et al. (22) that the orphan receptor RevErb can interact only with NCoR on its DNA response element and is unable to recruit SMRT. We have demonstrated that the TR␤1 isoform preferentially recruits NCoR rather than SMRT to a DR ⫹ 4 response element (24). In addition, using glutathione-S-transferase (GST) pull-down assays and the mammalian two-hybrid system, Wong and Privalsky (28) have shown that separate RAR isoforms can recruit SMRT with different affinities. Taken together, these data indicate that the polypeptides present in the interacting domains of NCoR and SMRT allow for specific interactions with nuclear receptors that may allow for separate biological actions in vivo. The interactions between the corepressors and the TR isoforms are also influenced by complex formation. We and others have demonstrated that the TR␤1 isoform recruits NCoR preferentially as a homodimer on DNA and that the addition of retinoid X receptor (RXR) causes a diminution in corepressor binding. Indeed, NCoR appears to stabilize the homodimer complex in solution where it normally does not form (24). In contrast, work using the mammalian two-hybrid system and the TR␣1 ligand-binding domain (LBD) has demonstrated that RXR can enhance interactions with nuclear corepressors (29). However, this study investigated TR-corepressor interactions in the absence of an underlying thyroid hormone response element, and did not utilize full-length TRs. To address these issues, we studied TR␤1 and RAR␣ and examined their ability to recruit either NCoR or SMRT to their cognate response elements. In addition, we examined the ability of RXR to influence corepressor recruitment in the context of specific nuclear receptors. By using a TR mutant that is defective in its ability to homodimerize, we demonstrate that the TR complex present on a native response element determines the nature of the corepressor recruited. Our data demonstrate that the polypeptides that represent the corepressor interacting domains appear to recognize both specific nuclear receptors and the complexes that they form. RESULTS The Proximal Corepressor Interacting Domains Allow for Specific Interactions with TRs and RARs To delineate the specificity of NCoR and SMRT for the nuclear hormone receptors, TR and RAR, we constructed plasmids that expr (...truncated)