The nuclear corepressor, NCoR, regulates thyroid hormone action in vivo Inna Astapovaa, Larissa J. Leea, Crystal Moralesa, Stefanie Tauberb,c, Martin Bilbanb, and Anthony N. Hollenberga,1 aDivision of Endocrinology, Metabolism and Diabetes, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA 02215; bDepartment of Laboratory Medicine, Medical University of Vienna and Ludwig Boltzmann Institute for Clinical and Experimental Oncology, A-1090 Vienna, Austria; and cDepartment of Statistics and Probability Theory, University of Technology A-1040 Vienna, Austria Edited by John D. Baxter, Methodist Hospital Research Institute, Houston, TX, and approved October 16, 2008 (received for review May 12, 2008) The thyroid hormone receptor (TR) has been proposed to regulate shown to preferably bind NCoR both in vitro and in mammalian expression of target genes in the absence of triiodothyronine (T3) cell lines (22, 23) via the most 5Ј of its RIDs, N3, which is through the recruitment of the corepressors, NCoR and SMRT. required for strong interactions with DNA- bound TR (18, 21, Thus, NCoR and SMRT may play an essential role in thyroid 24). Importantly, N3 must cooperate with another RID, pref- hormone action, although this has never been tested in vivo. To erably N2, to interact with a TR homodimer on DNA as each TR accomplish this, we developed mice that express in the liver a binds to 1 of the RIDs (18, 21–25). mutant NCoR protein (L-NCoR⌬ID) that cannot interact with the TR. Given the preference of NCoR for the TR, we took advantage L-NCoR⌬ID mice appear grossly normal, however, when made of the role that the RIDs play in TR recruitment to develop an hypothyroid the repression of many positively regulated T3-target approach to test the role of NCoR in T3- action in the liver. Using genes is abrogated, demonstrating that NCoR plays a specific and this model we show that NCoR mediates both repression by the sufficient role in repression by TR in the absence of T3. Remarkably, TR in the hypothyroid state and modulates the response to T3 in in the euthyroid state, expression of many T3-targets is also the euthyroid state on positively regulated T3-target genes. In up-regulated in L-NCoR⌬ID mice, demonstrating that NCoR also addition, we show the key role that NCoR plays in LXR-signaling determines the magnitude of the response to T3 in euthyroid in the liver demonstrating the ability of this model to address the animals. Although positive T3 targets were up-regulated in role of NCoR in other NR-signaling pathways in vivo. L-NCoR⌬ID mice in the hypo- and euthyroid state, there was little effect seen on negatively regulated T3 target genes. Thus, NCoR is Results a specific regulator of T3-action in vivo and mediates repression by Generation and Characterization of L-NCoR⌬ID Mice. We created a the unliganded TR in hypothyroidism. Furthermore, NCoR appears conditional NCoR allele by inserting loxP sites around the exons to play a key role in determining the tissue-specific responses to coding for the 2 most N-terminal RIDs, termed N3 and N2 similar levels of circulating T3. Interestingly, NCoR recruitment to [supporting information (SI) Fig. S1 A–C]. Upon Cre-mediated LXR is also impaired in this model, leading to activation of LXR- recombination, the targeted NCoR locus would encode a protein target genes, further demonstrating that NCoR recruitment regu- that contains only 1 RID–N1 (NCoR⌬ID) and thus would be lates multiple nuclear receptor signaling pathways. unable to interact with the TR (Fig. 1A). Using targeted ES cells we generated NCoRloxϪneo/ϩ animals, which were then crossed gene expression ͉ thyroid hormone receptor with mice ubiquitously expressing the flp recombinase from the Rosa26 locus to remove the Neo cassette. he nuclear receptor corepressor (NCoR) and the silencing Given the paramount role the TRs play in the liver we developed Tmediator of retinoic acid and thyroid hormone receptors mice that only expressed the NCoR⌬ID allele in hepatocytes by (SMRT) are key regulators of nuclear receptor signaling (1, 2). crossing NCoRlox/lox mice with an albumin-Cre transgenic strain Among the first interaction partners of NCoR and SMRT (26). NCoRlox/lox-Cre (L-NCoR⌬ID) mice were born at the ex- identified were the thyroid hormone receptor (TR) isoforms. pected frequency and developed normally. Expression of the 5Ј NCoR and SMRT have been postulated to mediate the ability of region of NCoR mRNA common to both NCoR and NCoR⌬ID lox/lox ⌬ the TR to repress transcription of positively regulated T3-target was similar in the livers of WT, NCoR and L-NCoR ID mice genes in the absence of ligand (T3) by providing a platform for (Fig. 1B). However, expression levels of the mRNA region encom- a multiprotein complex that mediates histone deacetylation passing N3 and N2 in L-NCoR⌬ID livers were Ͻ10% of those (3–7). Because of this property the corepressors have been found in NCoRlox/lox and WT animals (Fig. 1C). We found no differences in the expression levels of this region in muscle, heart implicated in the pathophysiology of hypothyroidism and resis- PHYSIOLOGY tance to thyroid hormone (8, 9). This putative role of the and adipose tissue of control and L-NCoR⌬ID mice confirming corepressors in vivo is further substantiated by the fact that mice selective expression of NCoR⌬ID in the liver (Fig. 1C). This was that lack all TR-isoforms are viable, while neonatal hypothy- confirmed by analysis of hepatic protein extracts which demon- roidism is uniformly fatal in mice, consistent with a detrimental strates that NCoR⌬ID is present in L-NCoR⌬ID mice and mice role of the unliganded TR bound to corepressors (10, 11). heterozygous for the NCoR⌬ID allele (L-NCoR⌬ID/ϩ) express Despite the perceived roles of the corepressors in TR action, in vivo data are lacking as deletion of NCoR or SMRT is lethal late in embryogenesis, although transgenic overexpression of a Author contributions: I.A. and A.N.H. designed research; I.A., L.J.L., C.M., S.T., and M.B. performed research; I.A., S.T., M.B., and A.N.H. analyzed data; and I.A. and A.N.H. wrote the NCoR inhibitor in liver did suggest a role for NCoR/SMRT in paper. ligand-independent repression by the TR (12–14). The authors declare no conflict of interest. Both NCoR and SMRT are recruited to nuclear receptors via This article is a PNAS Direct Submission. C-terminal receptor interacting domains (RIDs), which are 1To whom correspondence should be addressed at: Division of Endocrinology, Metabolism characterized by the presence of an isoleucine rich motif termed and Diabetes, Beth Israel Deaconess Medical Center, 330 Brookline Avenue, Boston, a CoRNR box (15–17). Extensive analysis has disclosed the MA 02215. E-mail: [email protected]. presence in both molecules of 3 RIDs that can be expressed This article contains supporting information online at www.pnas.org/cgi/content/full/ alternatively in a tissue-specific manner (18–21). Despite their 0804604105/DCSupplemental. similarity the RIDs do not function equivalently. TR has been © 2008 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0804604105 PNAS ͉ December 9, 2008 ͉ vol. 105 ͉ no. 49 ͉ 19543–19548 Downloaded by guest on September 28, 2021 genotypes (Table S1). Male L-NCoR⌬ID mice had a slight A lox/lox decrease in T4 levels compared with NCoR mice only, but otherwise showed no difference in thyroid hormone levels across genotypes. To assess the role of the NCoR⌬ID on T3 action in the liver, B C we performed microarray analysis of hepatic gene expression. We identified 173 targets that were significantly repressed (representing positively regulated TR/T3-target genes) in hypo- thyroid control animals versus euthyroid control animals (Fig. 2A). Of these, 27 (16%) were significantly derepressed or activated in hypothyroid L-NCoR⌬ID mice and are shown in red in the upper left quadrant of Fig. 2A, with the heat map representing these genes shown in Fig. 2B. In contrast, 326 genes were activated in hypothyroidism (representing negatively reg- D ulated TR/T3-target genes) in control mice, and only 3 of these were significantly repressed (Ͻ1%) in hypothyroid L-NCoR⌬ID mice. These genes are shown in red in the right lower quadrant of Fig. 2A with the corresponding heat map in Fig. 2C. Thus, NCoR recruitment via N3 and N2 plays an important role in E mediating ligand-independent repression of positive T3 targets in the liver, while its role in ligand-independent activation by the TR appears very limited. We also identified 39 genes whose expression was significantly altered in the euthyroid state be- tween control and L-NCoR⌬ID mice. Of these 39 genes, 26 were activated in L-NCoR⌬ID mice and included known classic positive T3 targets such as thrsp, fasn and mod1 (group 1; Fig. 2D) suggesting that NCoR recruitment may play a role in transcrip- Fig. 1. Expression of the mutant NCoR⌬ID in L-NCoR⌬ID mice. (A) Schematic tional activation by T3. Interestingly, 13 genes were repressed in representation of the approach used to generate a mouse strain that ex- ⌬ presses mutant NCoR lacking N3 and N2 RIDs in the liver (L-NCoR⌬ID). (B) euthyroid L-NCoR ID mice when compared with controls Hepatic expression of NCoR mRNA in L-NCoR⌬ID and control mice as assessed (group 2; Fig. 2D). by Q-PCR directed against the 5Ј region of the mRNA. (C) Expression of NCoR To validate these data we performed QPCR analysis on a mRNA in different tissues as assessed by Q-PCR directed against the 3Ј region number of genes identified in the array, and on other known of the mRNA.