The Nuclear Corepressor, Ncor, Regulates Thyroid Hormone Action in Vivo

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 hypothyroid 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 between 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. mRNA levels are expressed as fold change compared with WT hepatic T3 targets. The data presented here were obtained by group. All values are expressed as meanϮ SEM. Signiﬁcant differences com- using female hepatic mRNA but we have also analyzed expres- pared with WT group: ***, P Ͻ 0.001. PGF-perigonadal fat pad. (D) Western sion of a number of these targets in male mice and obtained blot analysis of NCoR in the liver in a variety of genotypes. RNA pol II is used similar results (data not shown). Expression of dio1, bcl3, gpd2, as a loading control. (E) Co-IPs were performed on extracts from control and ⌬ idh3 and cyp27a was significantly repressed in control animals in L-NCoR ID livers by using 2 anti-TR antibodies (see SI Text). The protein ⌬ complexes were resolved on NuPAGE Tris-acetate gel, and analyzed by West- the hypothyroid state and strongly derepressed in L-NCoR ID ern blot using anti-NCoR antibody. The blots were scanned and quantiﬁed by animals by 2- to 3-fold. Expression of dio1, cyp27a and idh3 was using Image J software. also significantly activated in euthyroid L-NCoR⌬ID animals when examined in a greater number of animals than used in the microarray studies (Fig. 3A). Other classic T3 targets, thrsp, fasn equal amounts of NCoR and NCoR⌬ID (Fig. 1D). Importantly, and mod1, were not significantly repressed in control hypothy- only full-length NCoR protein is present in the heart and muscle of roid mice explaining why they were not present in the first set of L-NCoR⌬ID mice (Fig. S1D). Finally, there is no compensatory genes in our microarray analysis. However, expression of each of up-regulation of SMRT mRNA or protein levels in L-NCoR⌬ID these genes was significantly elevated (2- to 4-fold) in L- mice (Fig. S1E), as was the case in a previous transgenic mouse NCoR⌬ID mice in the hypothyroid state and exceeded their model that overexpressed an NCoR inhibitor (14). expression levels in euthyroid control mice. In addition, these To demonstrate that NCoR⌬ID localized to the nucleus in a targets were strongly activated in the euthyroid state confirming similar fashion to NCoR we performed immunocytochemistry our microarray data and demonstrating the role of NCoR in on HepG2 cells transfected with plasmids expressing Flag- mediating T3 sensitivity. tagged NCoR or NCoR⌬ID, as well as immunohistochemical We next looked at the role of NCoR in the regulation of negative ⌬ staining of control and L-NCoR ID liver sections (Fig. S2 A and B). To T3 targets including fbxo21, a target identified in our array analysis, confirm that NCoR⌬ID is defective in its ability to be recruited to the and previously described targets gsta2 and st3gal4. In contrast to TR we used coimmunoprecipitation experiments using protein extracts positive T3 targets, the regulation of these genes was not altered in from livers of hypothyroid NCoRlox/lox and L-NCoR⌬ID mice as well hypothyroid L- NCoR⌬ID mice. Thus, NCoR appears to play little as 293T cells transfected with expression plasmids for NCoR, role in negative regulation in the liver (Fig. 3C). NCoR⌬ID and TR␤1. As shown in Fig. 1E and Fig. S3A TR␤1 strongly recruits WT NCoR both in vivo and in mammalian cells but cannot Role of NCoR in TR-Mediated Regulation of Serum Cholesterol Levels. recruit NCoR⌬ID. This was confirmed with 2 separate antibodies that Hypothyroidism is known to cause an elevation of serum cho- recognize TR␤1. Thus, L-NCoR⌬ID mice provide a unique in vivo lesterol levels, through a rise in LDL cholesterol due to repres- model to explore the role of NCoR in TR action. sion of cyp7a1 by unliganded TR␤1 (27). Surprisingly, despite significant derepression of cyp7a1 expression in hypothyroid NCoR Regulates Expression of TR-Target Genes in the Liver. To (and euthyroid) L-NCoR⌬ID mice (Fig. 4A) there was only a determine the role of NCoR in vivo we studied male and female small decrease in LDL-C levels and no change in total choles- WT, NCoRlox/lox and L-NCoR⌬ID mice at 9 weeks of age that terol levels between hypothyroid L-NCoR⌬ID and control an- were euthyroid, hypothyroid or hyperthyroid. Total T4 and total imals (Fig. 4C and Table S2). In addition, hepatic cholesterol T3 levels in female mice in all states showed no difference among content was not changed (Fig. 4D). Since HMG-CoA reductase

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Fig. 2. Microarray analysis of hepatic gene expression in euthyroid and hypothyroid lox/lox and L-NCoR⌬ID female mice (n ϭ 3 per group). (A) Scatter plot representing genes that are differentially expressed in hypothyroid compared with euthyroid control animals. Genes that are expressed differentlyin hypothyroid L-NCoR⌬ID animals as compared with hypothyroid controls are shown in red. (B) Heat map showing expression levels of the genes that are down-regulated in the hypothyroid state and expressed differently in hypothyroid L-NCoR⌬ID and control animals. (C) Heat map showing expression levels of the genes that are up-regulated in the hypothyroid state and expressed differently in hypothyroid L-NCoR⌬ID and control animals. (D) Hierarchical clustering of the genes differentially expressed in euthyroid L-NCoR⌬ID mice compared with euthyroid lox/lox controls. All of the heat maps show color-coded expression levels (red, high expression; black, medium expression; and green, low expression). Each column represents data for 1 animal.

(hmgcr), a rate-limiting enzyme in cholesterol biosynthesis, is absence of ligand. In the presence of T0901317, which should also known to be a T3-target we examined its expression in dissociate NCoR form LXR, scd1 expression was significantly L-NCoR⌬ID mice and found that it is significantly elevated by up-regulated in both control and L-NCoR⌬ID hepatocytes and the close to 2-fold in both the hypothyroid and euthyroid states in 7-fold derepression seen in NCoR⌬ID cells in the absence of ligand, L-NCoR⌬ID animals (Fig. 4B). Further examination of our had become much less pronounced. In contrast, T0901317 had no array data demonstrates that expression of other enzymes in- effect on thrsp or dio1 and derepression of these targets was volved in cholesterol biosynthesis such as sqle, mvd, pmvk and maintained in L-NCoR⌬ID hepatocytes consistent with a specific fdps is elevated in euthyroid and/or hypothyroid L-NCoR⌬ID role for the TR in mediating repression of these targets via NCoR. animals. This increase in cholesterogenic enzyme mRNA levels likely leads to enhanced cholesterol synthesis and offsets the Discussion increase in cholesterol elimination mediated by elevated cyp7a1 Since their identification, NCoR and SMRT have been shown to expression. play a key role in nuclear receptor signaling. In particular, it has been assumed that either or both are responsible for the potent NCoR Regulates Signaling by LXR in the Liver. As our microarray effects of the unliganded TR. However, because global deletion analysis also showed significantly enhanced expression of a of both NCoR and SMRT is embryonic lethal, this hypothesis number of genes previously reported to be regulated by the LXR has not been tested in vivo before this report (12, 13). isoforms, we hypothesized that NCoR⌬ID might also be defi- To analyze NCoR function in vivo, we took advantage of the cient in its ability to interact with LXR␣, the principal isoform unique role that RIDs play in its interaction with TR and other in the liver (28, 29). Indeed, co-IP experiments using extracts NRs. The NCoR⌬ID mutant protein lacks both the N3 and N2 from euthyroid control and L-NCoR⌬ID mice show that LXR RIDs and thus cannot be recruited to the TR. Importantly, the recruits NCoR well, while its interaction with NCoR⌬ID is rest of the protein is produced, thereby preventing any detri- substantially reduced (Fig. 5A). Similar results were also seen in mental effects to the animal. Thus, L-NCoR⌬ID mice provide a 293T cells cotransfected with expression plasmids for LXR␣, loss of function model to assess the role of NCoR in thyroid PHYSIOLOGY NCoR⌬ID and NCoR (Fig. S3B). Thus, the N3 and N2 RIDs are hormone action in vivo. important for LXR–NCoR interaction. QPCR analysis of the Herein, we demonstrate that NCoR mediates repression of expression of the LXR targets abca1, srebp1c, scd1 and pltp1 positively regulated T3 target genes by the unliganded TR. confirmed that expression of each was significantly activated in Indeed, expression of dio1, bcl3, fasn, mod1, cyp27a, cyp7a and L-NCoR⌬ID mice, and induction of hypothyroidism did not thrsp is significantly elevated in hypothyroid L-NCoR⌬ID mice change the level of enhancement (Fig. 5B). Despite activation of as compared with hypothyroid control animals confirming the LXR targets in L-NCoR⌬ID mice, serum and hepatic triglyc- key role of NCoR in ligand-independent action by the TR. eride levels in the euthyroid and hypothyroid state were similar Importantly, the activation seen on some of these targets (fasn, to controls but did trend higher (Fig. 5C). mod1 and thrsp) is almost identical to that seen in hypothyroid Because of the potential overlap between TR and LXR signaling TR␤Ϫ/Ϫ mice confirming that TR-mediated repression of these pathways in the regulation of hepatic target genes such as thrsp, targets is due to NCoR (30). While the reversal of repression of mod1 and fasn we wanted to ensure that the effect of NCoR loss was dio1 is markedly less than the other targets, this is not unexpected specific for the TR. To accomplish this we isolated primary as corepressors may not play a significant role in repression of hepatocytes from control and NCoR⌬ID mice and examined this gene (31). Our data do not rule out a role for SMRT in expression of these genes in the absence and presence of the LXR mediating ligand-independent repression by the TR on certain ligand T0901317. As shown in Fig. 6, expression of thrsp, dio1 and targets, but clearly NCoR is sufficient to mediate this key scd1 was increased in hepatocytes from L-NCoR⌬ID mice in the function of the TR.

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C D

B Fig. 4. Hypothyroidism-associated hypercholesterolemia is not reversed in L-NCoR⌬ID mice. (A and B) Hepatic cyp7a1 (A) and hmgcr (B) mRNA levels are elevated in euthyroid and hypothyroid L-NCoR⌬ID animals. mRNA levels are expressed as fold change compared with controls. (C) Serum cholesterol is significantly increased by hypothyroidism in all groups of animals, while hepatic cholesterol content is not changed (D). n ϭ 5–7 per group. All values are expressed as mean Ϯ SEM. Significant differences compared with control C mice on the same diet: *, P Ͻ 0.05; **, P Ͻ 0.01; ***, P Ͻ 0.001. Significant differences compared with the same genotype on chow: ###, P Ͻ 0.001.

expression of 1 of them, fbxo21, confirmed it to be a negative target, but showed that it was activated equally well in hypothyroid L-NCoR⌬ID and control mice. Analysis of other well- known negative T -targets in the liver showed that the loss of N3 Fig. 3. Changes in hepatic expression of TR target genes in L-NCoR⌬ID animals. 3 (A) Expression of genes that are repressed in the hypothyroid state in control and N2 in NCoR has no influence on their expression, although animals is derepressed in the livers of hypothyroid L-NCoR⌬ID animals. (B) Ex- it remains possible that NCoR⌬ID can still interact with the TR pression of positively regulated T3-target genes that are not signiﬁcantly repressed in the hypothyroid state in control animals is activated in the livers of L-NCoR⌬ID mice in both the hypo and euthyroid state. (C) Expression of negatively regulated T3-target genes is not affected in L-NCoR⌬ID animals. n ϭ 5–7 A animals. mRNA levels are expressed as fold change compared with lox/lox chow group. All values are expressed as mean Ϯ SEM. Signiﬁcant differences compared with control mice on the same diet: *, P Ͻ 0.05; **, P Ͻ 0.01; ***, P Ͻ 0.001.

We also demonstrate that NCoR plays a key role in determining the response to T3 in the euthyroid state. Most positively regulated B targets examined showed significantly enhanced mRNA expression in euthyroid L-NCoR⌬ID mice. This is consistent with data demonstrating that only 50% of hepatic TRs are bound to T3 in the euthyroid state (32). Thus, in the absence of recruited NCoR, total TR-responsive gene expression is enhanced as has been seen previously in vitro (33). Furthermore, these data strongly suggest that levels of NCoR can determine individual responses to set levels of T3 and could explain why tissue-specific responses to identical C levels of T3 can vary in individuals. Interestingly, the response of certain targets is also enhanced in the hyperthyroid state but to a lesser extent than in the euthyroid state. It is possible that this reflects the persistence of unliganded TRs, even in the setting of excess ligand. However, in certain cases, such as in the regulation of fasn, this may also be explained by cross-talk with other NR signaling pathways such as LXR (34–36). Fig. 5. NCoR modulates LXR signaling in the liver. (A) Co-IPs were performed We also examined the role of NCoR in the regulation of on extracts from control and L-NCoR⌬ID livers by using an anti-LXR antibody expression of genes known to be negatively regulated by T3 (37, that recognizes both LXR isoforms. The protein complexes were resolved on 38). It has been speculated that corepressors may be paradoxi- NuPAGE Tris-acetate gel, and analyzed by Western blot using anti-NCoR cally responsible for ligand-independent activation on negative antibody. The blots were scanned and quantified by using Image J software. targets (14, 39). If this were the case, the activation of TR-targets (B) mRNA expression levels of LXR target genes are elevated in the livers of L-NCoR⌬ID mice. mRNA levels are expressed as fold change compared with in hypothyroidism would be reversed in L-NCoR⌬ID mice. Ͼ lox/lox chow controls. (C) Serum and liver triglyceride levels are not signifi- While we identified 300 genes that were activated in hypothy- cantly increased in L-NCoR⌬ID animals. n ϭ 5–7. All values are expressed as roid control mice, only 3 of these were significantly deactivated mean Ϯ SEM. Significant differences compared with control mice on the same or repressed in hypothyroid L-NCoR⌬ID animals. Analysis of diet: *, P Ͻ 0.05; **, P Ͻ 0.01; ***, P Ͻ 0.001.

19546 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0804604105 Astapova et al. Downloaded by guest on September 28, 2021 implies in particular that thyroid status of a particular tissue would depended on both tissue-specific NCoR and T3 levels. Materials and Methods Generation of NCoR⌬ID Mice. The targeting vector was constructed in the pZErO-1 plasmid (Invitrogen) by using a BAC clone containing a fragment of mouse NCoR gene derived from the 129S6 mouse strain. All cloning manipu- lations were performed by using homologous recombination in EL250 and EL350 Escherichia coli strains as described by Lee et al. (42). For more details and breeding strategies used to obtain L-NCoR⌬ID mice see SI Text. Fig. 6. NCoR specifically regulates TR-target genes. mRNA levels of TR and LXR target genes were determined from primary hepatocytes isolated from Animal Experiments. All experiments described in SI Text were approved by the ⌬ ␮ control and L-NCoR ID animals cultured in the presence of 1 M LXR ligand Beth Israel Deaconess Medical Center Institutional Animal Care and Use T0901317 where indicated. Relative mRNA levels are shown. All values are Committee. expressed as mean Ϯ SEM. Significant differences compared with controls on the same treatment: , P Ͻ 0.001. Significant differences compared *** Blood Chemistry and Hormonal Analysis. Total T4 and T3 levels were measured with the same genotype with no treatment: #, P Ͻ 0.05; ###, P Ͻ 0.001. by solid-phase RIA (Coat-a-Count; Diagnostic Products) in 25 and 50 ␮lof serum, respectively. Enzymatic-colorimetric assays for total cholesterol, triglycerides and alkaline phosphatase activity were purchased form Stanbio on negative TREs. Thus, recruitment of NCoR through N3 and Laboratory. Number and size of lipoprotein particles were measured by NMR N2 appears to play a preferential role on positively regulated spectroscopy, and the amount of HDL cholesterol was calculated based on targets. The mechanism by which the majority of genes are these results by LipoScience. activated by the TR in the hypothyroid state remains unclear. Given that the rise in serum cholesterol levels seen in hypo- Liver Triglycerides and Cholesterol. The lipids were extracted by the method of thyroidism have been shown to be mediated by the action of Folch with modifications as described in SI Text (43). unliganded TR␤ in the liver, we expected hypothyroid L-NCoR⌬ID mice to be resistant to the development of hyper- Coimmunoprecipitation and Western Blot Analysis. Coimmunoprecipitations cholesterolemia. Despite derepression of cyp7a expression, se- (Co-IP) were performed as described in SI Text using protein extracts from livers of lox/lox and L-NCoR⌬ID euthyroid or hypothyroid mice or 293T cells rum cholesterol levels were similar in hypothyroid control and ⌬ ⌬ transfected with expression plasmids for murine full-length and ID NCoR, L-NCoR ID mice and LDL-C levels were only slightly reduced. human TR␤1 and human LXR␣ as indicated (44). This is likely explained by the elevated mRNA expression of hmgcr and other enzymes in the cholesterol biosynthetic pathway Immunocytochemistry and Immunohistochemistry. Immunostaining of HepG2 in hypothyroid L-NCoR⌬ID mice. Indeed, deletion of hdac3 cells transfected with either Flag-tagged NCoR or Flag-tagged NCoR⌬ID ex- from the liver also activates similar genes in the cholesterol pression plasmid, and frozen liver sections from lox/lox and L-NCoR⌬ID mice synthesis pathway demonstrating that the recruitment of was performed as described in SI Text. HDAC3 by NCoR plays a key role in regulating cholesterol biosynthesis (40). Gene Expression Profiling. Three female mice from the NCoRlox/lox and ⌬ In addition to its role in TR action, NCoR also regulates the L-NCoR ID euthyroid and hypothyroid groups described above were chosen for microarray analysis. Isolated mRNA was further purified with the RNeasy action of LXR isoforms (29, 41). Our data demonstrate that Mini kit per manufacturer’s instructions (Qiagen). Total RNA (200 ng) was then recruitment of NCoR to LXR requires 2 NCoR RIDs in vivo. used for GeneChip analysis. Preparation of terminal-labeled cDNA, hybridiza- Furthermore, known LXR targets are clearly activated in tion to genome-wide murine Gene Level 1.0 ST GeneChips (Affymetrix) and L-NCoR⌬ID mice. A role for NCoR in LXR signaling is further scanning of the arrays were carried out according to manufacturer’s protocols supported by the finding that the derepression of scd1 seen in (https://www.affymetrix.com). L-NCoR⌬ID primary hepatocytes as compared with control hepatocytes is almost lost in the presence of LXR ligand, consistent with Real-Time Quantitative (Q)PCR. Total RNA extraction and real-time QPCR were dismissal of NCoR and recruitment of coactivators. performed by using standard techniques as described in SI Text. Overall the results of our microarray analysis show activation of TR and LXR signaling pathways that are both known to positively Bioinformatic Analysis. RMA Signal extraction, normalization and filtering was performed as described (www.bioconductor.org/) (45, 46). All calculations regulate expression of lipogenic enzymes and enzymes involved in were performed in ‘‘R.’’ Heat maps were generated by using EPCLUST (http:// cholesterol catabolism, and share a number of target genes. While ep.ebi.ac.uk/EP/EPCLUST/). The details are described in SI Text. Complete our data do not exclude the possibility that other nuclear receptor GeneChip datasets are available online as GEO entry GSE10001 (www.ncbi. nlm.nih.gov/projects/geo/query/acc.cgi?accϭGSE10001).

signaling pathways might be affected, it clearly demonstrates that PHYSIOLOGY NCoR plays an important role in TR-signaling in vivo. In summary, we have generated mice that express only in the Primary Hepatocytes. Hepatocytes were isolated from 8- to 10-week-old mice liver a modified NCoR protein that is defective in its ability to by using a 2-step collagenase perfusion procedure as described in SI Text. The interact with TR and LXR. Using these mice we demonstrate cells were cultured in RPMI medium 1640 supplemented with 10% steroid- depleted FBS (HyClone), 100 nM dexamethasone, 10 ␮g/ml insulin and 1 ␮M that NCoR specifically regulates repression of positively regu- LXR ligand T090137 (Sigma) or DMSO. After 16–18 h hepatocytes were har- lated TR-target genes in the hypothyroid state. NCoR also vested and RNA was prepared for Q-PCR analysis as above. N ϭ 3 per treatment modulates expression of positive TR targets in the euthyroid group. state and regulates the response to set levels of T3. In contrast, NCoR appears to play little or no role in negative regulation by ACKNOWLEDGMENTS. We thank Evan Rosen for helpful discussions and the T3 in the liver. NCoR recruitment also regulates expression of Gene Manipulation Facility of the Mental Retardation and Developmental LXR targets in the liver. Taken together these observations Disabilities Research Center at Children’s Hospital in Boston [National Insti- tutes of Health (NIH) Grant P30-HD18655] for ES cell work. This work was suggest that nuclear receptor action on target genes could be supported by NIH Grants DK056123 and DK078090 (to A.N.H.) and T32 altered in vivo by the relative amount of NCoR present. This DK07516 (to I.A.).

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