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Endocrinology Vol. 142, No. 12 5321-5331
Copyright © 2001 by The Endocrine Society

TRH-TSH-THYROID

Nuclear Corepressor and Silencing Mediator of Retinoic and Thyroid Hormone Receptors Corepressor Expression Is Incompatible with T3-Dependent TRH Regulation

Nathalie Becker1, Isabelle Seugnet1, Hajer Guissouma2, Sandrine M. Dupre3 and Barbara A. Demeneix

Laboratoire de Physiologie Générale et Comparée, Muséum National d’Histoire Naturelle, Centre National de la Recherche Scientifique UMR 8572, 75231 Paris Cedex 5, France

Address all correspondence and requests for reprints to: Dr. Barbara A. Demeneix, Laboratoire de Physiologie Générale et Comparée, Muséum National d’Histoire Naturelle, UMR Centre National de la Recherche Scientifique 8572, 7 rue Cuvier, 75231 Paris, Cedex 5, France. E-mail: demeneix{at}mnhn.fr


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Ligand-independent repression by thyroid hormone (T3) receptors on positive T3-responsive genes requires corepressor proteins. However, the role of corepressors in regulating genes such as hypothalamic TRH, which are under negative control by T3, is largely unknown. We examined the expression of mRNAs encoding the corepressors NCoR (nuclear corepressor) and SMRT (silencing mediator of retinoic and thyroid hormone receptors) in the TRH-producing paraventricular nucleus of the mouse hypothalamus. Further, we carried out in vivo functional studies by overexpression of both corepressors. Three lines of evidence show that NCoR and SMRT expression is incompatible with physiological regulation of TRH. First, Northern blotting revealed TRH and NCoR mRNA expressions to be inversely correlated during postnatal development and as a function of thyroid status. Second, in situ hybridization showed that NCoR and SMRT mRNA expression profiles in the paraventricular nucleus were distinct from that of TRH mRNA. Third, over-expression of full length NCoR and SMRT in the hypothalamus abolished T3-dependent repression of TRH-luciferase. However, over-expression of NCoR or SMRT did not affect either T3-independent activation of TRH-luciferase transcription, or transcription from a positively regulated T3-response element. We conclude that T3 -dependent feedback on TRH expression is unlikely to involve the corepressors NCoR or SMRT.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE THYROID HORMONES (TH) T3 and T4 exert essential pleiotropic effects during vertebrate development and on homeostasis in the adult. The production and secretion of these hormones are stimulated by pituitary TSH, the synthesis and secretion of which are activated by TRH produced in the paraventricular nucleus (PVN) of the hypothalamus. During evolution, negative feedback mechanisms have arisen that allow for increasing levels of circulating TH to down-regulate TSH and TRH production in the pituitary and hypothalamus, respectively. TH action on these negatively regulated genes, like TH action on positively regulated genes, occurs through binding of T3 to TRs on DNA response elements of target genes.

TRs are ligand-dependent and -independent transcription factors that belong to the steroid/thyroid nuclear receptor superfamily. They derive from two genes, c-erbA-{alpha} and c-erbA-ß (1, 2). TR{alpha} and -ß are similar in overall structure, being most related in the DNA-binding and C-terminal hormone-binding regions (3). Further diversity has been reported, with alternative splicing of the TR{alpha} primary transcripts generating the C-terminal TR{alpha}1 and TR{alpha}2 variants (4). The variant {alpha}2 fails to bind T3 (5). Also, three N-terminal variants of TRß have been described (6, 7, 8).

A number of in vitro (9, 10, 11) and in vivo studies (12, 13) have shown that TRß is a key player in the negative regulation of TRH transcription by T3. However, it is known that modulation of transcription by T3 involves interaction of the TRs with comodulator complexes that, in turn, affect chromatin acetylation (for review, see Ref. 14). Unlike most members of the nuclear receptor superfamily, TRs and RARs possess ligand-independent silencing activity. In the case of positively regulated genes, this transcriptional silencing in the absence of ligand involves association of the TR or RAR with nuclear corepressor proteins, such as nuclear corepressor (NCoR) (15) or silencing mediator of retinoid and thyroid hormone receptors (SMRT) (16, 17, 18). In turn, these nuclear corepressors recruit a multiprotein complex with histone deacetylase (HDAC) activity that modifies chromatin accessibility and thus prevents transcription (19, 20, 21). Both NCoR and SMRT contain two C-terminal nuclear receptor interaction domains as well as at least three independent repressor domains (for review, see Ref. 14). In the presence of their cognate ligands, TRs and RARs release nuclear corepressors and associate with histone acetyltransferase coactivators (for review, see Ref. 22). These findings have led a number of groups to test the in vitro interactions of nuclear corepressors and TRs, in particular TRß, on the promoters of negatively regulated genes such as TRH and TSH (10, 23, 24, 25). However, despite this spate of in vitro reports, there is as yet only limited data on the levels of expression of these corepressor molecules in the brain, and none on their expression in the TRH-producing PVN of the hypothalamus.

We chose to address this fundamental question using in situ hybridization (ISH) and Northern blotting. Expression of NCoR and SMRT mRNA was followed both during development and under different thyroid states. Both ISH and Northern blotting revealed an inverse correlation of NCoR mRNA levels with those of TRH. Further, using ISH to examine the expression pattern of each mRNA at the cellular level in the PVN of the hypothalamus showed NCoR, SMRT, and TRH mRNAs to have distinct distributions. Finally, using an in vivo gene transfer method to express NCoR or SMRT specifically in the hypothalamus, we found that increasing amounts of either corepressor abolished T3-dependent repression of a TRH luciferase reporter plasmid. Taken together these findings suggest that in adult mice the nuclear corepressors NCoR and SMRT are weakly expressed in the TRH-producing PVN of the hypothalamus and that their presence interferes with negative feedback of T3 on TRH transcription.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
ISHs on Vibratome sections
OF1 mice (Janvier, Le Genest St. Isle, France) at different postnatal stages were anesthetized, perfused with 5 ml PBS, then 4% paraformaldehyde (PFA) in PBS, pH 7.5. After dissection, whole brains were fixed overnight in 4% PFA in PBS and dehydrated the next day through a methanol/PBS containing 0.1% Tween 20 (PBT) series. After rehydration, whole brains were embedded in albumin/gelatin (26), and 200-µm Vibratome sections were made. Sections were placed individually in 24-well culture plates and used for in situ hybridization (27). After an additional dehydration-rehydration procedure in methanol, sections were rinsed twice in PBT, bleached for 1 h in 2% H2O2, and washed again in PBT three times. They were then treated with 10 mg/ml proteinase K for 15 min, washed with 2 mg/ml glycine in PBT, and postfixed in 4% PFA/0.02% glutaraldehyde in PBT for 20 min. Sections were then prehybridized for 1 h at 70 C in 50% formamide, 5x SSC (pH 4.5), 1% SDS, 50 µg/ml heparin, and 50 µg/ml yeast RNA. Digoxygenin-labeled RNA probe was added (1 µg/ml) to the prehybridization buffer, and hybridization was carried out overnight at 70 C. Sections were washed twice in 50% formamide/5x SSC for 30 min at 70 C, then in ribonuclease buffer [0.5 M NaCl, 10 mM Tris-HCl (pH 7.5), and 0.1% Tween 20] for 5 min and treated with 100 µg/ml ribonuclease A twice for 30 min each time at 37 C. After a high stringency wash in 50% formamide/2x SSC twice for 30 min each time at 65 C, the sections were preblocked with 10% decomplemented goat serum (Sigma, St. Quentin Fallavier, France) for 90 min and incubated overnight at 4 C with alkaline phosphatase-conjugated antidigoxygenin antibody (Roche Molecular Biochemicals, Meylan, France), and the enzyme activity was revealed by addition of 5-bromo-4-chloro-3-indoyl-phosphate/nitro blue tetrazolium chloride (Roche).

A cDNA insert of mSMRT (nucleotides 3576–5771) (17) in pEX-lox(+) vector, provided by J. Don Chen (Worcester, MA), was used for the synthesis of sense and antisense probes of SMRT. The plasmid was either digested by PstI and transcribed with SP6 RNA polymerase for the sense probe or digested by SacII and transcribed with T7 RNA polymerase for the antisense probe.

A cDNA insert of NCoR (nucleotides 5600–7525), in pSG5 vector (Stratagene, La Jolla, CA), provided by G. Muscat (St. Lucia, Australia), was digested by StuI and transcribed by T7 RNA polymerase for the synthesis of a sense probe. After cutting by EcoRI and religating the entire cDNA NCoR insert in reverse orientation, the plasmid obtained was digested by StuI and transcribed by T7 RNA polymerase for the synthesis of an antisense probe.

A cDNA insert of the mouse prepro-TRH gene (307–522), provided by M. Yamada (Maebashi, Japan), was used for the synthesis of sense and antisense probes. Probes were generated by cutting at SacI or ApaI (sites in the polylinker of the pGEM-T plasmid) and transcribing with T7 or SP6 polymerases respectively.

ISHs on cryostat sections
Twenty-two-day old OF1 mice (Janvier) were anesthetized, perfused with 5 ml PBS, then 4% PFA. After dissection, whole brains were fixed 1 h in 4% PFA at 4 C, then cryoprotected by incubation overnight in 15% sucrose-PBS at 4 C and immediately embedded in OCT (Amilabo, Chassieu, France), then frozen in liquid nitrogen. All tissues were kept at -80 C until used. Thick tissue sections (16 µm) were prepared (-24 C; Jung Frigocut, Leica Corp., Rueil-Malmaison, France) mounted on pretreated slides ready to use (SuperFrost Plus, Labo-Moderne, Paris, France), postfixed in 4% PFA 10 min at room temperature, and processed immediately for the ISH experiment. ISH was proceeded as previously described (28). Sections were incubated for two 15-min periods in PBS containing 0.1% active diethylpyrocarbonate (Sigma) and equilibrated for 15 min in 5x SSC (0.75 M NaCl and 0.075 M sodium citrate), then prehybridized for 2 h at 58 C in the hybridization mix [50% formamide (Fluka, St. Quentin Fallavier, France), 5x SSC, and 50 µg/ml tRNA (Sigma)]. The probes were denatured for 10 min at 65 C and added to the hybridization mix (1 µg/ml). The hybridization reaction was carried out at 58 C for 40 h with 25 µl hybridization mix on each section, covered by hybrislips (Biolabs, Ozyme, St. Quentin Fallavier, France). Prehybridization and hybridization were performed in a box saturated with a solution to avoid evaporation (5x SSC/50% formamide). After incubation, the sections were washed for 30 min in 2x SSC at room temperature, 1 h in 2x SSC (65 C), 1 h in 2x SSC, ribonuclease A at 50 µg/ml (Roche) at 65 C, 5 min in 2x SSC at room temperature, 1 h in 0.1x SSC at 65 C, and two 5-min periods in buffer 1 (100 mM Tris-HCl and 150 mM NaCl, pH 7.5). The sections were equilibrated for 1 h in buffer 1 containing 10% normal goat serum (Sigma), then incubated with alkaline phosphatase-coupled antidigoxigenin antibody (Roche) diluted 1:2500 in buffer 1 containing 1% normal goat serum at 4 C overnight. Excess antibody was removed by two 15-min washes in buffer 1, and the sections were equilibrated for 30 min in 100 mM Tris-HCl, 100 mM NaCl, 50 mM MgCl2, and 0.1% Tween 20, pH 9.5, containing 0.5 g/liter levamisol (Sigma). Color development was performed at room temperature in 100 mM Tris-HCl, 100 mM NaCl, 50 mM MgCl2, and 0.1% Tween 20, pH 9.5-levamisol containing PBS containing 0.1% Tween 20 and nitro blue tetrazolium chloride (Roche) for 24 h. Staining was stopped by three 15-min washes in PBS, and the sections were mounted in Mowiol.

Northern blotting
Hypothalami of mice, decapitated at different ages (from 1–22 d), were dissected on ice and immediately frozen in liquid nitrogen. From 1–7 d, one sample included three pooled hypothalami. From 15 d onward, one hypothalamus was used per sample. Subsequently, hypothalami were extracted with RNA PLUS buffer (Qbiogene, Illkirch, France), and purified according to the manufacturer’s conditions. Twenty to 30 µg total RNA of each sample were loaded on a 1% agarose/2.2 M formaldehyde gel and transferred in 10x SSC buffer overnight onto a Hybond-N nylon membrane (Amersham Pharmacia Biotech, San Francisco, CA). After transfer, lanes used for localization of 28S and 18S ribosomal RNAs were cut off and revealed in 0.02% methylene blue and 0.3 M NaOAc, pH 5.5 (29). The remaining RNAs were fixed on the membrane by baking at 80 C for 2 h before hybridization with specific probes. Radioactive probes were prepared as follows: cDNA fragments of murine NCoR (5775–6780), SMRT (5246–5771), and TRH (307–522) were purified on agarose gels and subsequently purified on Sephadex columns (Schleicher and Schuell, Ecquevilly, France). Labeling with [{alpha}-32P]ATP (ICN Biochemicals, Inc., Irvine, CA) was performed as described by Feinberg and Vogelstein (30); radioactive probes were purified on nick columns (Amersham Pharmacia Biotech). Hybridization was performed successively for each probe on the membranes. Washes included two steps in 2x SSC/0.1% SDS, then two steps in 0.2x SSC/0.1% SDS during 5 min at ambient temperature, then two steps in 0.2x SSC/0.1% SDS during 15 min at 42 C before exposition for autoradiography. Filters were stripped in boiling 0.1% SDS, allowed to reach room temperature, washed, and tested for the absence of any remaining signal. For the glyceraldehyde phosphate dehydrogenase (GAPDH) probe (plasmid provided by J. M. Blanchard, Montpellier, France), a digoxygenin-labeled antisense RNA of 1 kb of 5'-end cDNA cloned in pBluescript (HindIII digestion and T7 RNA polymerase) was used as a probe. Filters were processed for specific RNA/RNA hybridization (70 C in formamide buffer) and for revelation by chemiluminescence, using the CDP-Star system (Roche). Autoradiograms were quantified by densitometry, using NCSA GelReader 2.0.

Plasmids
Plasmids were prepared using commercial columns (QIAGEN, Courtaboeuf, France), suspended in 10 mM Tris-HCl-1 mM EDTA, pH 8, and stocked as aliquots at -20 C.

The TRH-luc construct was provided by Dr. W. Balkan (Miami, FL) and contains a rat TRH gene 5' fragment, extending from -547 to +84 bp, cloned upstream of the firefly luciferase-coding region (31).

Malic enzyme-thymidine kinase-luciferase (MAL-TK-luc) and pSG5-NCoR plasmids were provided by Dr. K. Chatterjee (Cambridge, UK) and Dr. G. Muscat (St. Lucia, Australia), respectively. SMRT cDNA EcoRV insert was subcloned from pCMX-mSMRT{alpha}, provided by Dr. M. Downes (La Jolla, CA), into pSG5 (Stratagene, La Jolla, CA).

Treatment of animals, in vivo transfection, and luciferase assay
All animal studies were conducted in accordance with the highest standards of humane care and according to the principles and procedures described in Guidelines for Care and Use of Experimental Animals.

Female OF1 mice (Janvier) were mated. To induce fetal and neonatal hypothyroidism, dams were given an iodine-deficient food containing 0.15% 6-n-propyl-2-thiouracil (PTU; Harlan, Gannat, France) on d 14 of gestation. The PTU diet was continued throughout the lactation period.

DNA/PEI (polyethylenimine) complexes and in vivo transfections were carried out as previously described (12). Pups were anesthetized by hypothermia on ice and injected on postnatal d 1. A glass micropipette was lowered 2 mm through the skull, approximately 1 mm lateral to the sagittal suture, into the hypothalamic area. Two microliters of a 5% glucose solution containing plasmid/PEI complexes were slowly injected bilaterally. For trichostatin A (TSA; Sigma) treatment, TSA (in 100% ethanol at 0.9 M) or 100% ethanol (control) were diluted 1:10 in DNA/PEI transfection solutions and injected intracerebrally into newborn mice. For evaluating the effects of T3 on reporter gene expression, hypothyroid or normal pups were injected sc with 250 µg T3/100 g BW (in 9% saline). Controls received saline (9%) injections. After 18 h, mice were anesthetized and decapitated. The hypothalami were dissected out for luciferase analyses. Animals that were treated with T3 received sc injections (see above) immediately after gene transfer for TRH-luc assays and 12 h after gene transfer for MAL-TK-luc assays.

Statistical analysis of results
In vivo gene transfer results are expressed as the mean ± SEM per group. After ANOVA where appropriate, the Mann-Whitney test was used to analyze differences between treatments. Differences were considered significant at P < 0.05. In all cases, typical experiments are shown, each experiment having been repeated at least three times (with n >= 10 newborn mice/experiment) and providing the same results.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
NCoR and SMRT corepressor mRNA expression profiles coincide largely with that of TRs in rodent brain
To characterize the expression patterns of the two key nuclear corepressor mRNAs in the rodent brain and to compare them to the already well documented distribution of TRs, we carried out ISH for NCoR and SMRT on coronal sections of 1- and 3-wk-old mice. We used digoxygenin-labeled probes corresponding to the 3'-ends of the cDNAs so as to obtain hybridization signals that include both the full-length and putative truncated forms of each corepressor mRNA (see Fig. 3Go). The regions chosen for NCoR and SMRT probes showed no significant nucleotide identity with their respective SMRT and NCoR counterparts (BLAST pairwise alignment); thus, we deduce that the signal is specific for each probe. As shown in Fig. 1Go, a–d, both NCoR and SMRT mRNAs were widely, but not ubiquitously, expressed in the mouse central nervous system. On d 1, NCoR was observed in the cerebral cortex, hippocampus, striatum, thalamus, and hypothalamus, whereas the SMRT signal was restricted to the reticular thalamus and hypothalamus, but was also faintly observed in the cerebral cortex, hippocampus, and striatum (Fig. 1Go, a and b). At 3 wk of age, areas with high levels of expression included the pyriform cortex and hippocampus for both corepressors (Fig. 1Go, c and d). The specificity of the signal was confirmed by the absence of labeling when hybridization was carried out with the control sense probes to either NCoR or SMRT (Fig. 1Go, e and f).



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  		Figure 3. NCoR, SMRT, and TRH mRNA expression in the mouse hypothalamus during postnatal development. A, Schematic representation of NCoR and SMRT cDNAs. The nucleotidic length of each cDNA is indicated. The regions used for Northern blotting probes are underlined. The regions coding for identified nuclear receptor interaction domains or repression domains are indicated (adapted from Ref. 17 ). See Materials and Methods for details. B, Different forms of NCoR and SMRT are expressed in the mouse hypothalamus. Representative Northern blot of 20 µg RNA/lane extracted from the hypothalamus of a 2-d-old mouse, hybridized successively with probes for NCoR and SMRT, showing the overriding presence of a longer form of NCoR mRNA, corresponding to 10.5 kb, and a shorter form of SMRT mRNA, estimated at 4.5 kb. The positions of rRNAs (28S and 18S) are indicated. See Results for details. C, NCoR mRNA levels are inversely correlated with TRH mRNA levels in the postnatal mouse hypothalamus. Shown is a representative Northern blot of 20 µg hypothalamic RNA/lane, extracted at different ages postnatally (1, 2, 8, 15, and 22 d). Blots were hybridized successively with probes for NCoR, SMRT, TRH, and GAPDH as an internal control. A representative blot is shown. The experiment was repeated twice with the same essential result: progressive decrease in NCoR mRNA against increasing levels of TRH mRNA (see Results for details). D, TH status regulates NCoR mRNA levels in the developing mouse hypothalamus. Left panel, RNA was extracted from the hypothalamus of 3-d-old euthyroid or hypothyroid (PTU) mice treated with saline (+vehicle) or T3 (+T3) and killed 22 h later. Blots (20 µg/lane) were hybridized successively with probes for NCoR, TRH, and GAPDH as an internal control (upper two panels) or probes for SMRT, TRH, and GAPDH (lower two panels). No change in TRH mRNA levels was seen (data not shown). Representative blots are shown. Each experiment was repeated twice, providing the same result: reduction of NCoR in hypothyroid mice and restoration of NCoR levels by T3 treatment. A quantification of each signal, normalized on those obtained for GAPDH, is represented on the right panel. Each value is the average of two separate experiments. The mean ± SD are given.

 


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  		Figure 1. NCoR and SMRT corepressor mRNA expression at 1 and 22 d postnatally in mouse brain. ISH reveals distinct expression patterns for NCoR, SMRT, and TRH mRNAs in the paraventricular nucleus of the developing mouse hypothalamus. Thick coronal Vibratome sections (200 µm) of brains from 1-d-old (a b, h, i, and j) and 22-d-old (c, d, e, f, k, l, and m) mice hybridized with antisense NCoR RNA (a, c, i, and l), sense NCoR RNA (e), antisense SMRT RNA (b, d, j, and m), sense SMRT RNA (f), or antisense TRH (h and k) probes. The sections from the older animals appear darker, as the tissue is much denser than in newborn animals. Sections displayed from a–m are at the level of the medial paraventricular nuclei of the hypothalamus. On whole coronal brain sections (from a to f), note that NCoR mRNA is observed at high levels in the cerebral cortex (CC), striatum (STR), hippocampus (HC), and thalamus (T) at 1 d and in the pyriform cortex (PC) and hippocampus (HC) at 22 d. SMRT mRNA is seen at high levels in the striatum (STR), reticular thalamus (RT), and hypothalamus (HYP) at 1 d and in the pyriform cortex (PC) and hippocampus (HC) at 22 d. The positions of described brain structures are indicated in e (for the reticular thalamus (RT), see j). A schematic representation of a coronal section of the hypothalamus, including the main hypothalamic nuclei, is presented in g. DMN, VMN, Dorso- and ventromedial nuclei; LH, lateral hypothalamus; VIII, third ventricle (at the dorsal edge). More detailed views of the hypothalamic PVNs are presented on sections h–m. On d 1 (h–j), TRH mRNA (h) is already observed in the PVN, where NCoR and SMRT mRNAs are also expressed, albeit at lower levels (i and j). NCoR expression is observed in the thalamus (T), PVN, and DMN (i). Cell-specific labeling of SMRT mRNA (j) is seen in the lateral hypothalamus (LH) and reticular thalamus (RT), as well as less distinctly in the PVN. At 22 d (k), TRH mRNA is strongly expressed in the PVN, particularly in the peripheral neurons. NCoR mRNA expression appears, but only faintly, in the PVN (l), whereas SMRT mRNA expression (m) is detected in the PVN as well as in the DMN (l). Bars, 1 mm (a, b, and c–f), 0.5 mm (h–j and k–m).

 
TRH, SMRT, and NCoR mRNAs are found in distinct cell populations
We next used ISH to examine more closely the profiles of TRH, SMRT, and NCoR mRNA expression in the hypothalamus (schematized in Fig. 1gGo), concentrating on the PVN at 1 and 22 d postnatally. Median sections of the PVN from such animals are shown in Fig. 1Go (h–m). As shown in Fig. 1Go (h and k), TRH expression increased in the PVN over this period. However, at the same time its expression became progressively spatially restricted, changing from a general distribution of labeling throughout the whole PVN at 1 d postnatally (Fig. 1hGo) to a more intense labeling of the peripheral PVN at 22 d (Fig. 1kGo). This is particularly marked in the rostral PVN (data not shown).

Turning to the expression of corepressor mRNAs, ISH on brain sections from 1-d-old animals showed the distribution of NCoR in the PVN to be more disperse than that of TRH. In contrast to the TRH signal, the cell bodies were not easily distinguishable (Fig. 1iGo). Similarly, in 1-d-old mice, SMRT mRNA (Fig. 1jGo) expression extended beyond that of TRH, but in this case (in contrast the NCoR signal) distinct cell bodies expressing this mRNA could be seen beyond the limits of the PVN.

At 22 d, corepressor mRNA expression patterns had changed, but were still distinctive. In the PVN, NCoR expression appeared to be faintly localized in the center of this nucleus, contrasting to the more peripheral localization of TRH-expressing neurons (Fig. 1Go, k and l). The expression pattern of SMRT mRNA showed a very faint, but much wider, distribution than that of the other two signals, including the PVN and dorsomedial nuclei of the hypothalamus (Fig. 1mGo).

The nonoverlapping distribution patterns of NCoR, SMRT, and TRH mRNAs seen in thick (200-µm) sections (Fig. 1Go) led us to carry out a finer analysis of their localization on thin (16-µm) sections of the medial PVN of 22-d-old mice (Fig. 2Go). Figure 2Go, a and b, shows two adjacent sections respectively labeled for TRH and SMRT. The strongest TRH signal was detected in a few peripheral neurons of the PVN (Fig. 2aGo), whereas the SMRT signal was much less distinct at the cellular level and more widely distributed (Fig. 2bGo). As a control, we used the SMRT sense probe, which gave no signal (Fig. 2cGo). Given that the SMRT signal was not strong in the hypothalamus (Fig. 2bGo), we ensured the specificity of the signal by examining extrahypothalamic areas. As shown in Fig. 2dGo, the hippocampus was negative for TRH, as expected, but showed a strong signal for SMRT (Fig. 2eGo). Again the SMRT sense probe was negative (Fig. 2fGo). Similarly, two adjacent sections were labeled for TRH (Fig. 2gGo) and NCoR (Fig. 2hGo). Once more, the peripheral distinct labeling for TRH contrasted with the more diffuse and central signal for NCoR. The NCoR signal was even fainter than that of SMRT in the hypothalamus (Fig. 2Go, compare sections h and b). As a control, the NCoR sense probe again showed no signal (Fig. 2iGo). Moreover, as a positive control for NCoR, we looked at the pyriform cortex, where no signal was seen for TRH (Fig. 2jGo) or the sense NCoR probe (Fig. 2lGo), whereas a strong signal was seen for NCoR antisense probe (Fig. 2kGo).



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  		Figure 2. ISH of adjacent sections reveals faint expression patterns for SMRT and NCoR in the PVN of the 22-d-old mouse hypothalamus. Thin coronal cryostat sections (16 µm) of brains from 22-d-old mice hybridized with antisense RNA probes for TRH (a, d, g, and j), SMRT (b and e), and NCoR (h and k). Sense probes for SMRT (c and f) and NCoR (i and l) are also shown. Arrowheads indicate the dorsal limit of the third ventricle. Arrows indicate examples of labeled cells. Whereas TRH mRNA (a) is strongly expressed in a subset of medial and lateral cells of the PVN, SMRT mRNA expression is detected sparsely throughout the whole periventricular area, as shown on the adjacent section b. The specificity of expression in the hypothalamus was controlled with a sense RNA probe for SMRT (c). Other areas of the brain, such as the hippocampus, were used as controls for the absence of labeling with TRH antisense RNA probe (d), strong labeling for SMRT antisense (e), and no labeling for the SMRT sense probe (f). Adjacent to g, labeled for TRH mRNA, NCoR mRNA expression (h) seems faint and restricted to the center of the PVN, partially excluding the TRH-expressing domain. The specificity of expression in the hypothalamus was controlled with sense RNA probes for NCoR (i). Areas of strong expression for NCoR in the cortex (k) showed no labeling for TRH antisense RNA probes in adjacent sections (j) or for NCoR RNA sense probes (l). Bars, 50 µm (a–l). as, Antisense probe; s, sense probe.

 
Different forms of NCoR and SMRT are found in the developing hypothalamus
To examine the expression of NCoR and SMRT mRNAs during the development of the hypothalamus, Northern blots were hybridized successively with the TRH probe and then with specific cDNA probes for either NCoR or SMRT; these probes are represented schematically in Fig. 3AGo. Typical signals obtained in Northern blotting with these probes are shown in Fig. 3BGo. The main band for NCoR corresponded to a mRNA of 10.5 kb in size, which was in accordance with previous data (15), as well as a minor form of 4.5 kb. For SMRT, two different forms were found, with sizes approximating those seen for NCoR (i.e. 4.5 and 10.5 kb). A faint, additional form of SMRT was found at 1.5 kb. Northern blot quantification (n = 2) indicated that the signal for the 4.5-kb form of SMRT was 3–10 times more intense than that for the 10.5-kb form (data not shown). In contrast, for NCoR, the signal corresponding to the 4.5-kb form was roughly 10 times less intense than that of the 10.5-kb form (data not shown). We next analyzed the developmental profiles and physiological regulation of corepressor expression by following the predominant form of each mRNA, i.e. the full-length 10.5-kb form of NCoR and the 4.5-kb form of that encoding SMRT.

NCoR mRNA levels are inversely correlated with those of TRH in the postnatal mouse hypothalamus
As shown in Fig. 3CGo, full-length NCoR mRNAs are detected at relatively high levels in the newborn mouse hypothalamus and decrease during development to become virtually undetectable by 22 d postnatally. In contrast, the levels of both the predominant and less represented forms of SMRT seem unchanged throughout this period, whereas those of TRH increase steadily (Fig. 3CGo and data not shown). Quantification and normalization of the predominant corepressor signals against GAPDH showed that the decrease in NCoR expression between 8 and 22 d postnatally was 83.7 ± 4.5% (n = 2), and that of SMRT was 37.4 ± 7.1% (n = 2).

TH status regulates NCoR mRNA levels in the developing mouse hypothalamus
Given the marked decrease in NCoR mRNAs during postnatal development, over a period in which circulating hormones are reaching their adult levels (32), we analyzed the effects of modulating thyroid status on NCoR and SMRT expression in the hypothalamus. Using Northern blotting, we found that rendering pups hypothyroid decreased NCoR mRNA levels. Normalization of the signal against GADPH showed a decrease of 42 ± 18% (n = 2). In line with this observation, we found that treating the hypothyroid pups with 2 mg T3/g animal 24 h before death restored NCoR mRNA levels to 93 ± 11% of the control levels, always compared with GAPDH standard levels (Fig. 3DGo). SMRT levels showed much more variability between samples of each test group (see high SDs in Fig. 3DGo histograms). We attribute this high interassay variability to the fact that the dissection of the PVN of the hypothalamus probably included some of the lateral hypothalamic area that shows high SMRT expression in newborn animals (see Fig. 1jGo)

SMRT and NCoR overexpression interferes with transcriptional regulation of TRH in the newborn mouse hypothalamus
The low expression levels of SMRT and NCoR in the TRH-positive areas of the PVN raised the question of what effect the overexpression of these corepressors would have on TRH transcription. To this end, we carried out cotransfections of a TRH-luciferase reporter plasmid (hereafter, TRH-luc) into newborn mice hypothalami, with increasing amounts of a plasmid expressing SMRT or NCoR or a control plasmid bearing the same promoter sequence but no coding region.

The TRH-luc plasmid contains the regulatory regions necessary and sufficient to observe a T3-dependent transcriptional repression when expressed from transfected DNA introduced into hypothalami of newborn mice (Fig. 4Go, A and B, left columns; see Materials and Methods for details). As shown in Fig. 4AGo, overexpression of full-length SMRT significantly reduced the T3-dependent repression of TRH-luc transcription at both low (10 ng) and higher (200 ng) amounts. Most importantly, SMRT overexpression had no effect on T3-independent TRH activation (white columns in Fig. 4AGo). As seen for SMRT expression, overexpression of full-length NCoR abolished the T3-dependent repression, again at both 10 and 200 ng, without altering T3-independent TRH-luc transcription (Fig. 4BGo). Expressing control plasmid at any amount (up to 200 ng) had no statistically significant effect on either T3-independent TRH activation or T3-dependent repression of TRH-luc transcription (Figs. 4Go, A and B, left columns).



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  		Figure 4. SMRT and NCoR abrogate T3-dependent repression of TRH-luciferase in vivo. A, TRH-luciferase transcription was measured in hypothyroid 1-d-old mice treated with T3 (2.5 µg/g BW) or saline, 18 h after hypothalamic injection of 1 µg TRH-luciferase construct and 10 or 200 ng expression vector (pSG5-SMRT; see Materials and Methods). One control group only (cotransfection of empty pSG5) is shown (ct), as all groups equivalent to 10 or 200 ng pSG5 vector gave similar results (data not shown). The mean ± SEM are given (n >= 10/point). In each case the experiment was repeated three times, with similar results. **, P < 0.001. B, Same experiments as described in A, performed with pSG5-NCoR. The mean ± SEM are given (n >= 10/point). In each case the experiment was repeated three times, with similar results. **, P < 0.001.

 
To evaluate whether these effects were specific to the TRH gene, we transfected SMRT or NCoR corepressor plasmids with a reporter gene positively regulated by THs. We used a MAL-TK-luc construct [containing the positive TRE (TR response element) of the malic enzyme promoter] with the maximum amounts of SMRT or NCoR used in the TRH experiments (i.e. 200 ng). A significant T3-induced activation was observed in vivo for the control group (Fig. 5Go, left column), and no change in this effect was seen when SMRT or NCoR was overexpressed (Fig. 5Go, center and right columns).



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  		Figure 5. SMRT and NCoR do not modify T3-dependent activation of Mal-TK-luc in vivo. MAL-TK-luc transcription was measured in hypothyroid 1-d-old mice treated with T3 (2.5 µg/g BW) or saline 18 h after hypothalamic injection of 1 µg MAL-TK-luc construct and 200 ng expression vector (pSG5-NCoR or SMRT; see Materials and Methods). The control consists of equivalent amounts of an empty expression vector (pSG5). The mean ± SEM are given (n >= 10/point). In each case the experiment was repeated three times, with similar results. *, P < 0.05; **, P < 0.001.

 
T3-dependent transcriptional repression of TRH-luc in the newborn mouse hypothalamus involves HDAC
HDAC are known to mediate transcriptional repression via chromatin accessibility modifications on positively T3-regulated genes (for review, see Ref. 33). We evaluated the activity of HDAC in TRH-luc T3-dependent repression in vivo by cotransfection of the TRH-luc plasmid with a HDAC inhibitor, TSA. We show that addition of TSA relieves T3-dependent repression of TRH (Fig. 6Go, black columns) without significantly modifying T3-independent activation of transcription (Fig. 6Go, white columns).



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  		Figure 6. The HDAC inhibitor TSA alters T3-dependent repression of TRH-luc in vivo. TRH-luc transcription was measured in the hypothalami of hypothyroid 1-d-old mice treated with T3 (2.5 µg/g BW) or saline, 18 h after hypothalamic injection of 1 µg TRH-luciferase construct, in 90 nM TSA solution or in vehicle (see Materials and Methods). The mean ± SEM are given (n >= 12/point). Transfecting the vehicle alone had no effect on TRH-luc transcription in the same experiment (data not shown). In each case the experiment was repeated three times, with similar results. *, P < 0.05; ***, P < 0.0001.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The isolation of the corepressors NCoR and SMRT provoked a spate of studies on their interactions with TR and retinoic acid signaling. Such studies led to a model in which these molecules interact with unliganded TRs and recruit a protein complex that includes HDAC resulting in transcriptional repression (35). The models are based on in vitro results on consensus positive TREs in promoters of genes activated by T3 (for review, see Refs. 33 and 36). However, there is a distinct paucity of data on corepressor function on ligand-dependent transcriptional repression, particularly in in vivo models.

We chose to analyze, first, the physiological and developmental regulations exerted on mRNA expression for NCoR and SMRT in the mouse hypothalamus and, second, their function on TRH transcription. The data provide three lines of evidence indicating that expression of NCoR and SMRT is incompatible with physiological regulation of TRH. First, the ISH studies showed that expression profiles of NCoR and SMRT mRNAs in the PVN were weak and distinct from the intense TRH labeling. Second, Northern blotting showed TRH and NCoR mRNA expressions to be inversely correlated during postnatal development and after T3 treatment. Third, in vivo functional studies using somatic gene transfer to overexpress full-length NCoR and SMRT in the hypothalamus demonstrated that NCoR and SMRT expression abolished T3-dependent repression of TRH-luc. However, these functional studies also showed that effects of overexpressing NCoR and SMRT in the hypothalamus were limited to repression of TRH-luc and did not affect either T3-independent activation of TRH-luc transcription or transcription from a positively regulated T3-response element.

The ISH data showed distinct and dynamic expression profiles for NCoR and SMRT. Interestingly, areas showing the strongest corepressor expression (hippocampus and cortex) also displayed high levels of mRNA encoding TRs (37). Immunocytochemistry (38) demonstrated high levels of expression of TR{alpha} and TRß isoforms in the hippocampus, cerebral cortex, and pyriform cortex. The correlation of corepressor expression profiles with that of TRs suggests that corepressors could be implicated in mediating TR signaling in these brain areas.

We next examined NCoR and SMRT expression in the hypothalamus during development. Between 1 and 22 d, TRH expression increased, whereas that of NCoR mRNA decreased. At weaning (3 wk) circulating TH levels stabilize at adult levels (32), and the TRH expression pattern is similar to that seen in adult mice (data not shown). More strikingly, at 3 wk, mRNA expression in the PVN for NCoR was limited to the core of the PVN, whereas neurons expressing the strongest TRH signal were clearly peripheral. SMRT and TRH expression patterns were not so sharply distinguishable as those for NCoR and TRH. Expression of both corepressors was extremely weak in the PVN compared with other brain areas, (e.g. pyriform cortex and hippocampus for NCoR and SMRT, respectively). Thus, sites of high expression for TRH in the PVN, where a T3-dependent repression takes place, are not correlated with sites of high expression for corepressors. Interestingly, in the hippocampus and pyriform cortex (where the corepressors are strongly expressed), TRH gene expression can be induced (by amygdala kindling). However, in these locations, as opposed to the PVN, TRH cannot be down-regulated by T3 (39).

Northern blotting results bolster the concept of distinct expressions and inverse regulations for corepressor and TRH mRNAs. Down-regulation of NCoR mRNA was seen in the hypothalamus during postnatal development concomitant with increased TRH mRNA. The fact that NCoR expression is high at birth, when TRH expression is low, could indicate a repressive role for NCoR during early development. Similarly, hypothyroidism and T3 treatment of 3-d-old animals caused, respectively, down- and up-regulation of NCoR mRNA expression. These treatments should have inverse effects on TRH expression over the longer term. We did not see any effect of T3 on TRH mRNA expression (data not shown). This is due to the short time periods of T3 treatment used (<24 h); many days of T3 treatment are needed to quantify reductions in TRH mRNA levels (40).

The question arises as to the levels of the respective proteins in TRH neurons and whether truncated or full-length repressors are produced. Park et al. (17) detected truncated forms of SMRT by Western blotting performed on HeLa nuclear extracts, with antibodies directed against the C-terminal part of SMRT. We tried to carry out double immunocytochemistry with antibodies that have been used in Western blotting, but we could not obtain satisfactory signals on cryostat or paraffin sections. However, the Northern results shed some light on the problem. Of the two forms of SMRT mRNA observed, the majority is the shorter form (4.5 kb). This truncated form of SMRT was detected with a probe corresponding to the C-terminal part of the protein. The predicted translation product of this shorter mRNA will be capable of interacting with TRs, as it will possess the nuclear receptor interaction domains (see Fig. 3Go). However, it should not interfere with TRH transcription, as it does not possess the repression domains for interaction with the histone deacetylation complex (Sin3/HDAC). A probe corresponding to N-terminal repression domains of SMRT was used by Ordentlich et al. (18) to detect the extended form of SMRT (>10 kb), which is most related to NCoR, in Northern studies. The fact that this probe does not reveal any shorter forms in Northern blotting (18) suggests that the truncated mRNA we detect may correspond to a C-terminal form of SMRT, lacking the repression domains. Turning to the forms of NCoR seen in our Northern studies, we find that the predominant form is the full-length version, as has also been observed in cultured CV1 cells (41).

Thus, putative full-length NCoR, but not truncated SMRT mRNA, is modulated by thyroid status. This could have functional consequences. For instance, in hypothyroid animals, regulation of TRH may be dependent on T3-induced repression of hypothalamic NCoR. To test this hypothesis, we examined the functional effects of corepressor expression by direct production of the proteins in the hypothalamus of newborn mice. This is a validated, physiological, in vivo assay (12). We have shown that hypothyroidism and T3 up- and down-regulate the TRH-luc construct to the same extent (40%) as the endogenous TRH gene (40).

The results show that increased corepressor proteins abrogate TRH-luc repression in the presence of T3. Interestingly, in the absence of added T3, no effect of NCoR or SMRT overexpression was seen on TRH-luc transcription. As predicted by structural studies, corepressors should interact with TRs without T3. In vitro data suggest that this is also the case in a negative response element such as site 4, the predominant negative TRE (nTRE) of the TRH promoter and the TRH-luc construct (10, 41). Indeed, the NCoR interaction domains are able to associate with TRs on site 4 in vitro in the absence of T3 only; the addition of T3 promotes their dissociation from TRs on site 4 (41). Similar results have been observed with SMRT (42). Thus, the EMSA results show that the predominant nTRE of the TRH promoter used here permits classical TR-corepressor interactions.

Two nonexclusive hypotheses may explain the absence of corepressor effect on T3-independent activation of TRH-luc. The binding site on DNA could be inaccessible to corepressors in the absence of T3, thus precluding any effect on TRH-luc transcription. Considering this first scenario, a potential cAMP-responsive element-binding site is juxtaposed to site 4. The cAMP-responsive element-binding protein activation pathway could be dominant in the absence of T3, impairing TR binding by steric hindrance (43). Alternatively, the effect of TR-corepresssor complexes bound to the TRH promoter in the absence of T3 could be prevented by a conformational modification. Such a mechanism has been proposed for the TRß2 isoform, which by specific interactions with its N-terminus prevents formation of a functional corepressor complex on TREs (25). As TRß2 is expressed in TRH-producing neurons (44), it could contribute to hinder the function of corepressors if bound to the TRH promoter in the absence of T3.

In contrast to the lack of effect seen in the absence of T3, overexpression of NCoR or SMRT totally impaired T3-dependent TRH-luc repression. Given that liganded TRs do not interact with corepressors, one explanation could be that the corepressors antagonize T3-dependent repression by titrating key molecules needed for this particular mechanism, such as a HDAC. Specific observations support the hypothesis of a specific HDAC being needed for TRH repression. First, our experimental data using TSA indicate that HDAC are necessary for T3-dependent TRH-luc repression, but not for T3-independent TRH-luc activation. Second, in vitro data (45) support the idea that a specific HDAC would be involved in ligand-dependent repression of the TSH gene. Indeed, DNA affinity binding assays showed that the negative TR binding sequence (nTRE) in the TSHß promoter can bind, via TRs, HDAC2 in a ligand-dependent manner, thus suggesting a mechanism by which T3 can negatively regulate gene transcription without NCoR or SMRT (45).

In summary, our data show functional impairment of T3-dependent TRH regulation by NCoR and SMRT. During development, a gradual reduction in NCoR levels is followed by the acquisition of adult TRH expression, and facilitation of this expression in hypothyroidism is preceded by a further reduction in NCoR levels. As for SMRT, our hypothesis is that normally a truncated form is produced that does not interact with a repressor complex and therefore does not interfere with TRH transcription. In the TRH-producing paraventricular region of the hypothalamus, the overall level of the mRNA encoding these corepressors is generally lower than in other brain areas. Thus alternative mechanisms are needed to account for ligand-dependent repression of the TRH gene in the PVN.


   Acknowledgments
 
We thank S. Garel, O. Almeida, T. Michaelidis, Z. Liposits, H. Gronemeyer, and members of the Centre National de la Recherche Scientifique UMR 8572 for discussions and comments on the manuscript. We are grateful to Drs. Balkan, Chatterjee, Muscat, Yamada, Chen, Blanchard, and Downes for providing plasmids.


   Footnotes
 
This work was supported by the Association pour la Recherche contre le Cancer and European Grant QL 93 CT 2000 00 844.

1 N.B. and I.S. contributed equally to this work. Back

2 Fellow of the Ligue contre le Cancer. Back

3 Fellow of the Ministère de la Recherche. Back

Abbreviations: GAPDH, Glyceraldehyde phosphate dehydrogenase; HDAC, histone deactetylases; ISH, in situ hybridization; MAL-TK-luc, malic enzyme-thymidine kinase-luciferase; NCoR, nuclear corepressor; nTRE, negative TR response element; PBT, PBS containing 0.1% Tween 20; PEI, polyethylenimine; PFA, paraformaldehyde; PTU, 6-n-propyl-2-thiouracil; PVN, paraventricular nucleus; SMRT, silencing mediator of retinoic and thyroid hormone receptors; TRE, TR response element; TSA, trichostatin A.

Received June 26, 2001.

Accepted for publication August 30, 2001.


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 Introduction
 Materials and Methods
 Results
 Discussion
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