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Cyclin D1 Is a Ligand-independent Co-repressor for Thyroid Hormone Receptors

Cyclin D1 Is a Ligand-independent Co-repressor for Thyroid Hormone Receptors THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 277, No. 32, Issue of August 9, pp. 28733–28741, 2002 Printed in U.S.A. Cyclin D1 Is a Ligand-independent Co-repressor for Thyroid Hormone Receptors* Received for publication, April 8, 2002, and in revised form, May 23, 2002 Published, JBC Papers in Press, June 4, 2002, DOI 10.1074/jbc.M203380200 Huei-min Lin, Li Zhao, and Sheue-yann Cheng‡ From the Gene Regulation Section, Laboratory of Molecular Biology, Center for Cancer Research, NCI, National Institutes of Health, Bethesda, Maryland 20892-4264 Thyroid hormone receptors (TRs) are critical regula- terminus of the core histones of chromatins. Several co-activa- tors such as steroid hormone receptor co-activator (SRC) family tors of growth, differentiation, and homeostasis. TRs function by regulating the expression of thyroid hor- members pCBP/300 and p/CAF have been show to harbor in- mone (T3) target genes in both ligand-dependent and trinsic histone acetyltransferase activity (3–5). In contrast, -independent pathways. Distinct classes of co-regula- co-repressors such as N-CoR and SMRT have been shown to be tory proteins modulate these two pathways. We show associated with histone deacetylases (HDAC) and mediate the here a novel role of cyclin D1 as a T3-independent co- transcriptional silencing by TR (6 –9). repressor for TRs. Cyclin D1 interacted with TR in vitro Dynamic changes in the state of acetylation of core histones, and in cells in a ligand-independent manner. Cyclin D1 which are controlled by the opposing actions of histone acetyl- acted to repress both the silencing activity of the unli- transferases and HDACs, lead to transcriptional activation or ganded TR and the transcriptional activity of the ligan- repression of genes (10). Hyperacetylation of the histone tail ded TR. The repression was not due to the inhibition of relaxes the chromatin structure and leads to transcriptional the binding of TR to the thyroid hormone response ele- activation. Conversely, deacetylation condenses chromatin and ment but by serving as a ligand-independent bridging results in gene repression. In humans and mice, nine HDACs factor to selectively recruit HDAC3 to form ternary com- have been cloned and identified to date. They are divided into plexes. The repression was augmented by increasing two distinct classes based on their size and sequence homology expression of HDAC3 but not by HDAC1 and was allevi- to yeast HDACs. The class I enzymes such as HDAC1 (11), ated by trichostatin A. Thus, cyclin D1 is a novel ligand- HDAC2 (12), HDAC3 (13, 14), and HDAC8 (15) consist of independent co-repressor that opens a new paradigm to 400 –500 amino acids and are homologous to yeast Rpd3. In understand the molecular basis of the silencing action contrast, class II HDACs, including HDAC4, HDAC5, HDAC6 of TR. (16), HDAC7 (17), and HDAC9 (18), are large in size (1000 amino acids) and possess domains similar to the deacetylase domain of yeast Hda1. Different HDACs are recruited by dif- Thyroid hormone receptors (TRs) belong to a steroid hor- ferent transcriptional regulators to regulate the expression of mone/retinoic acid nuclear receptor superfamily that plays an different genes (16, 17, 19, 20). important role in cell proliferation, differentiation, and home- Various cyclin-dependent kinases are sequentially activated ostasis (1, 2). Four different thyroid hormone (T3) binding TR during cell cycle progression. During the transition of cell cycle, isoforms deriving from alternative splicing of two separate the activity of these kinase complexes is activated by specific genes are identified. They are expressed in tissue-dependent cyclins and inactivated by cyclin-dependent kinase inhibitors. and developmentally regulated fashion (1, 2). TR functions not Cyclin Ds complexing with CDK4 or CDK6 are strong key only as a ligand-dependent transcription factor in the presence regulators of progression through the G phase of the cell cycle of ligand but also acts as a constitutive repressor for their 1 (21, 22). Recently, it has been reported that cyclin D1 also target genes in the absence of ligand. A network of associated harbors activities that are independent of its role as a cyclin D proteins identified as co-repressors and co-activators has been kinase regulatory subunit (23). Cyclin D1 was found to activate shown to facilitate TR in gene regulation. One mechanism for the estrogen receptor (ER)-mediated transcription in breast the action of co-regulator proteins has been shown to involve tumors by direct binding to the hormone binding domain of the acetylation/deacetylation of the lysine residues on the amino ER and by enhancing the binding of ER to their target genes (24). It was further demonstrated that cyclin D1 interacts with * The costs of publication of this article were defrayed in part by the members of the SRC family and p/CAF through a motif that is payment of page charges. This article must therefore be hereby marked similar to the co-activator binding domain of nuclear receptors “advertisement” in accordance with 18 U.S.C. Section 1734 solely to in a ligand-independent manner (25, 26). It is suggested that indicate this fact. cyclin D1 serves as a bridging factor to recruit SRC and p/CAF ‡ To whom correspondence and reprint requests should be addressed: co-activators to ER, thereby facilitating the acetylation of his- Laboratory of Molecular Biology, 37 Convent Dr., Rm. 5128A2, MSC 4264, NCI, National Institutes of Health, Bethesda, MD 20892-4264. tones and promoting the ER-mediated transcription (25, 26). Tel.: 301-496-4280; Fax: 301-480-9676; E-mail: [email protected]. Previously we demonstrated that T3 stimulates the prolifer- The abbreviations used are: TR, thyroid hormone nuclear receptor; ation of growth hormone producing GC cells (27). The stimula- TRE, thyroid hormone response element: SRC, steroid hormone recep- tory effect is mainly due to a shortening of G /G phase. The tor co-activator; TSA, trichostatin; HA, hemagglutinin; AD1-HA, cyclin 0 1 D1 tagged with hemagglutinin; ER, estrogen receptor; FBS, fetal bovine shortening in G /G phase correlated with T3-induced in- 0 1 serum; GST, glutathione S-transferase; HDAC, histone deacetylase; creases in the mRNA and protein levels of two key regulators of Lys-TRE, TRE of the chicken lysozyme gene that is an everted repeat of G progression, cyclins D1 and E (27), suggesting a potential the half-site binding motifs separated by six nucleotides; mAbC4, mono- direct or indirect functional link of these two cyclins with TRs. clonal anti-TR1 antibody; RXR, retinoic X receptor ; F-HDAC, FLAG-tagged HDAC. Because ER and TR share many co-regulatory proteins in their This paper is available on line at http://www.jbc.org 28733 This is an Open Access article under the CC BY license. 28734 Repression of TR Activity by Cyclin D1 with lysis buffer and boiled in 2 Laemmli sample buffer. The immu- signaling pathways (28), we sought to understand whether noprecipitates were separated by SDS-PAGE and transferred to poly- cyclin D1 also acted to stimulate the transcriptional activity of vinylidene difluoride membranes (Millipore). The blots were blocked in TRs. We found that indeed, cyclin D1 physically interacted Tris-buffered saline containing 0.2% Tween 20 and 5% skim milk and with the hormone binding domain of TR. However, unexpect- probed with the primarily antibodies anti-TR, anti-cyclin D1 (Santa edly we discovered that in contrast to ER, this interaction led to Cruz Biotechnology, Inc.), anti-FLAG M2 (Sigma), or anti-HA (Santa a repression of both T3-dependent and -independent transac- Cruz Biotechnology, Inc.) followed by horseradish peroxidase-conju- gated secondary antibodies. The blots were washed 5 times in Tris- tivation activities of TRs. The repression was mediated by buffered saline with 0.2% Tween 20 and developed in ECL reagent cyclin D1-dependent recruitment of HDAC3 to cyclin D1-TR (Pierce). complexes. These findings expand the role of cyclin D1 in Mobility Shift Assay—The double-stranded oligonucleotide contain- transcriptional regulation. Significantly, our studies highlight 32 ing the Lys-TRE was labeled with [- P]dCTP similarly as described a novel regulatory pathway in mediating both T3-dependent by Zhu et al. (32). About 0.2 ng of probe (3–5  10 cpm) was added to and -independent repression of the transcriptional activity of M HEPES pH 8.0, 2 mM MgCl the binding buffer (25 m , 0.01 mM ZnCl , 2 2 M dithiothreitol, 6% glycerol, 0.01% TritionX-100) and in vitro 5m TRs. translated proteins, and 0.2 g of sheared salmon sperm DNA. Binding EXPERIMENTAL PROCEDURES reactions were carried out at room temperature for 30 min, and com- plexes were resolved on 6% polyacrylamide gels in 0.5 TBE (45 mM Cell Culture and Reagents—CV-1, GC, and MCF7 cells were main- Tris-HCl, 45 mM boric acid, 0.5 mM EDTA) at 150 V for 2 h. After drying tained in Dulbecco’s modified Eagle’s medium with 10% fetal bovine of the gel, the DNA-bound proteins were detected by autoradiography. serum (FBS), 50 g/ml penicillin, 0.25 g/ml streptomycin, and 2 mM L-glutamine at 37 °C under 5% CO . T3-depleted FBS was prepared by RESULTS incubating FBS with 20 mg/ml activated charcoal and 50 mg/ml anion exchange resin (AGX-8, Bio-Rad) for 24 h followed by centrifugation and Cyclin D1 Binds Specifically to TR1—We utilized the GST/ ultrafiltration. T3 (Sigma) was dissolved in 0.1 N NaOH. Trichostatin A glutathione (GSH) binding system to assess whether TR1 (TSA) was obtained from Sigma and dissolved in dimethyl sulfoxide. physically interacted with cyclin D1. GST alone was used as a Both reagents were stored at 20 °C and diluted immediately before negative control. Fig. 1A shows that TR1 bound to GST-D1 in use. the absence (lane 3) or presence of T3 (lane 4), but not GST Plasmids—The cyclin D1 expression vectors, pCMV-cyclin D1, pCMV-D1-HA, and pGST-cyclin D1 were generous gifts from M. E. (lanes 1 and 2), indicating the specific physical interaction of Ewen. The reporter pTK-Pal-Luc contains two copies of palindrome TR TR1 with cyclin D1. To map the region of TR1 to which cyclin response element (TRE) in tandem and a luciferase gene under the D1 bound, we prepared S-labeled truncated TR1 in which control of thymidine kinase promoter (a gift from J. L. Jameson). domains were systematically deleted (Fig. 1B). Deletion of do- pCEP4F-HDAC3 expressing an amino terminus FLAG-tagged HDAC3 main A/B (lanes 5 and 6, Fig. 1A), domains A/BC(lanes 7 and and pBJ5-HD-1 encoding a carboxyl terminus FLAG-tagged HDAC1 8, Fig. 1A), or domains A/BCD(lanes 9 and 10, Fig. 1A) did were described previously (14) (generous gifts of E. Seto). The pCLC51, pCLC61, and pCMV-TR2 express human TR1, -1, and -2 cDNA, not significantly affect the binding of cyclin D1 to TR1 regard- driven by cytomegalovirus promoter, respectively (29). For in vitro less of whether T3 was present or not. These results indicate transcription/translation, pCJ3, pJL8, pJL5, and pCJ4 encode full- that domain E was the essential binding region for cyclin D1 length and truncated TR1 under the control of the T7 promoter (30). and that this physical interaction was independent of T3. Transient Transfection—Cells (3  10 cells) were seeded into 6-well To demonstrate the interaction of cyclin D1 with TR1 in plates 1 day before transfection. The reporter plasmid pTK-Pal-Luc (1 vivo, CV1 cells were transfected with expression vector of TR1 g) was transfected with 0.2 g of TR expression vector (pCLC51) and cyclin expression vectors (pCMV-cyclin D1 or pCMV-D1-HA) into cells. or cyclin D1 tagged with hemagglutinin (D1-HA) or transfected Empty vectors were used to supplement equal amounts of DNA in each with both expression vectors. Fig. 1C shows the results of such transfection. Transfection was carried out using FuGENE 6 (Roche experiments from cells cultured in the absence of T3. Western Molecular Biochemicals) according to the manufacturer’s protocol. After blot analysis of cell lysates using mAbC4 (31) detected the overnight incubation, cultures were replaced with fresh Dulbecco’s expression of TR1(lanes 1 and 3, Fig. 1C, a) but not in cells modified Eagle’s medium with 10% T3-depleted FBS and induced with transfected only with D1-HA expression vector (lane 2). When 100 nM T3 for 24 h before harvest. Cells were rinsed with phosphate- buffered saline and lysed in reporter lysis buffer (Promega). Lysates cell lysates were first immunoprecipitated with anti-HA anti- were assayed for luciferase activity and normalized to total protein bodies followed by Western blot analysis using mAbC4, TR1 concentration. All experiments were performed in triplicate and re- was detected in cells transfected with TR1 and D1-HA as peated 3–5 times. The results shown are the mean  S.E. shown in lane 6 (Fig. 1C, a) but not in cells transfected with In Vitro Binding Assay—Glutathione S-transferase (GST) fusion pro- either TR1(lane 4) or D1-HA alone (lane 5), indicating an tein was expressed in Escherichia coli BL21 strain, induced by 1 mM association of TR1 with cyclin D1 in vivo. isopropyl--D-thiogalactopyranoside, and affinity-purified by glutathi- one-Sepharose 4B beads (Amersham Biosciences). [ S]Methionine-la- The association of TR1 with cyclin D1 in vivo was further beled TR proteins were prepared by TNT-coupled reticulocyte lysate demonstrated by a reverse experiment in which cell lysates system (Promega) and incubated with 1 g of GST-cyclin D1or GST in were first immunoprecipitated with anti-TR1 (mAbC4) fol- buffer (20 mM Tris-HCl, pH 7.5, 100 mM NaCl, 2 mM EDTA, 0.1% lowed by Western blot analysis using anti-HA. As shown in Lubrol, 2 mM dithiothreitol, 0.05% BSA, 5% glycerol protease inhibi- lane 6 of Fig. 1C, b, cyclin D1-HA was detected in cells trans- tors) at 4 °C overnight with constant rotation. Subsequently, beads fected with both TR1 and cyclin D1-HA expression plasmids were washed 4 times with 25 mM Tris-HCl, pH 7.4, 2.5% sucrose, 2.5 mM EDTA, 250 mM NaCl, and 1% Lubrol. The bound proteins were ex- but not in cells transfected only with TR1(lane 4, Fig. 1C, b) tracted by boiling the beads in SDS sample buffer for 5 min and or with cyclin D1-HA (lane 5, Fig. 1C, b). The identity of the separated on SDS-polyacrylamide gel and detected by autoradiography. co-immunoprecipitated cyclin D1-H1 was confirmed by direct Immunoprecipitation and Western Blot Analysis—CV1 cells were Western analysis of cell lysates with anti-HA as shown in lanes transfected with the indicated combinations of plasmids by Lipo- 2 and 3 of Fig. 1C, b. fectAMINE (Invitrogen). GC cells were transfected with FuGENE 6 Similar results were obtained from cells cultured in the (Roche Molecular Biochemicals). Cells were harvested after 48 h and lysed on ice in lysis buffer (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM presence of T3 (Fig. 1D). TR1 was detected in cells co-trans- EDTA, 0.5% Nonidet P-40) with protease inhibitor mixture (Roche fected with both D1-HA and TR1 after first immunoprecipi- Molecular Biochemicals). Cell lysates were clarified by centrifugation at tation of lysates with anti-HA antibodies followed by Western 11,000  g for 5 min at 4 °C. For immunoprecipitation, 300 g of cell blot analysis using anti-TR1 antibody (mAbC4; lane 6, Fig. extract was incubated with anti-HA (Santa Cruz Biotechnology, Inc.) or 1D, a). Using the same protocol, no TR1 was detected in cells monoclonal anti-TR, monoclonal anti-TR1 antibody (mAbC4) (31) for transfected only with either TR1(lane 4, Fig. 1D, a) or D1-HA 2hat4 °C, and followed by incubation with protein G-agarose beads (Roche Molecular Biochemicals) overnight. Beads were washed 5 times (lane 5, Fig. 1D, a). The association of D1-HA with TR1 was Repression of TR Activity by Cyclin D1 28735 FIG. 1. Cyclin D1 complexes with TR1 in vitro and in vivo. A, pull-down assay of TR1 by cyclin D1. Immobilized GST or GST-cyclin D1 fusion protein was incubated with in vitro translated S-labeled truncated TR1 in the absence (odd-numbered lanes) or presence of 100 nM T3 (even- numbered lanes) as described under “Experimental Procedures.” B, schematic representation of full-length and TR1-truncated proteins used in the binding assay. The amino-terminal domain, DNA binding domain, hinge region, and part of the hormone binding domain of TR1 are indicated as A/B, C, D, and E, respectively. The numbers indicate the position of amino acids. Association of TR1 with cyclin D1 in vivo without T3 (C) or with T3 (D) is shown. CV1 cells were transfected with the indicated combination of TR1 and D1-HA expression plasmids (pCLC51 and pCMV-D1-HA). Whole-cell lysates were immunoprecipitated (IP) with anti-HA or anti-TR1 as indicated. Immunoprecipitates were separated by SDS-PAGE followed by Western blotting with anti-TR and anti-HA antibodies. Input represents 10% of the total lysate used in the immunoprecipitation. Proteins were detected by Western blot analysis using antibody against TR (mAbC4) or HA epitope. E, GC cells were transfected with pCMV-D1-HA (10 g) or empty vector using FuGENE 6. Cell lysates (500 g) were immunoprecipitated with anti-HA followed by Western blot analysis using mAbC4 (E, a). For E, b and c, direct Western blot analysis was performed on lysates (50 g) using anti-TR (mAbC4) or anti-HA, respectively. 28736 Repression of TR Activity by Cyclin D1 further confirmed by reversing the antibodies in that anti- Cyclin D1 Interacts with Other TR Isoforms and Differen- TR1 antibody was first used to immunoprecipitate TR1 fol- tially Represses Their Silencing and T3-dependent Transacti- vation Activities—We further evaluated whether cyclin D1 also lowed by Western blot using anti-HA (lane 6, Fig. 1D, b). Lanes 1 and 3, Fig. 1D, a, and lanes 2 and 3 of Fig. 1D, b, show the interacted with other TR isoforms in cells by carrying out the expression of TR1 and D1-HA, respectively, by direct Western co-immunoprecipitation assay (Fig. 3, A–C). CV1 cells were transfected with TR1orTR2 with or without cyclin D1-HA blot analysis. Taken together, these data indicate that the in the absence of T3 (lanes 1–5, Fig. 3, A–C). When lysates from association of D1 with TR1 in cells was T3-independent. cells transfected with cyclin D1-HA and TR1orTR2 expres- Cyclin D1 not only associated with the transfected TR but sion plasmids were first immunoprecipitated with anti-TR also with the endogenous TR in GC cells (Fig. 1E). As shown in (mAbC4) followed by Western analysis using anti-HA, D1-HA Fig. 1E, a,TR1 was detected in GC cells transfected with was detected as shown in lanes 2 and 3 of Fig. 3A, indicating cyclin D1 (lane 2 of Fig. 1E) by co-immunoprecipitation assay the association of TR1orTR2 with cyclin D1-HA, respec- but not in cells without the transfected cyclin D1 (lane 1 of Fig. tively. This association is further supported by a negative con- 1E, a). Fig. 1E, b and c, show the controls by direct Western blot trol in which no D1-HA was detected in cells without the analysis to indicate the expression of endogenous TR1 and the co-transfection of either TR1orTR2(lane 1, Fig. 3A). The transfected cyclin D1 in GC cells, respectively, (lane 2 of Fig. lack of detection of D1-HA was not due to the lack of expression 1E, c). These results indicate that cyclin D1 associates not only because direct Western blot analysis of the same lysates indi- with the transfected TR1 but, importantly, with the endoge- cates that D1-HA was similarly expressed (lane 1, Fig. 3B)asin nous TR1 in GC cells. cells co-transfected with either TR1orTR2(lanes 2 and 3, Cyclin D1 Represses the T3-dependent Transactivation Activ- Fig. 3B). Furthermore, direct Western blot analysis using an- ity of TR1—To assess the functional consequences of the phys- ti-TR antibody shows that TR1 and TR2 were expressed ical interaction of TR1 with cyclin D1, we used TRE-contain- (lanes 2–5, Fig. 3C). These results indicate that cyclin D1 also ing reporter systems (Fig. 2). CV1 cells were transfected with a physically interacted with TR1orTR2 in the absence of T3. constant amount of TR1 expression plasmid in the absence or Similar association of cyclin D1 with TR1orTR2 was also presence of an increasing amount of expression plasmid of detected in cells cultured in the presence of T3 (lanes 7 and 8, cyclin D1. Fig. 2A shows that the transactivation activity of Fig. 3A, respectively). Again, this physical association in cells TR1 was activated 20-fold by T3 (bar 4, Fig. 2A). Cyclin D1 was further confirmed by the negative controls in that when repressed the T3-dependent transactivation activity of TR1in cells were not transfected with either TR1orTR2, no D1-HA a concentration-dependent manner. The extent of repression was detected (lane 6, Fig. 3A). The data shown in Fig. 3 indicate was 55% (bar 6 versus bar 4) and 80% (bar 8 versus bar 4) that TR1 and TR2 were associated with D1-HA in cells at the cyclin D1:TR1 plasmid ratio of 1 and 2, respectively. independent of T3. These results indicate that cyclin D1 negatively regulates the The functional consequences of the physical interaction of T3-dependent transactivation activity of TR1. cyclin D1 with each of the TR isoforms were evaluated in the Consistent with the T3-independent binding of cyclin D1 to presence or absence of T3 by using TRE-containing reporter TR1 shown in Fig. 1, cyclin D1 also augmented the silencing system (Fig. 3D). In the presence of T3, the interaction of cyclin effect of the unliganded TR1 in a concentration-dependent D1 with each of the three TR isoforms led to the repression of manner (Fig. 2A). The enhancement in the silencing effect was T3-dependent transactivation of TRs. However, the extent of 1.6- and 2-fold when the ratio of the cyclin D1:TR1 was in- the repression is TR isoform-dependent in that 60, 80, and 65% creased from 1 to 2 (bars 5 and 7, respectively). Taken together, repression was observed for TR1, TR1, and TR2, these results indicate that cyclin D1 not only repressed the respectively. T3-dependent transactivation activity but also augmented the Fig. 3D also compares the extent of the effect of cyclin D1 on T3-independent silencing activity of TR. the silencing effect of the un-liganded TR1 and TR2 with The cyclin D1-mediated repression effect was further dem- that of TR1. The augmentation of the silencing effect by cyclin onstrated by the transactivation activity of endogenous TRs in D1 also differed among the TR isoforms in that the extent of the GC cells. Fig. 2B shows that transfection of increasing concen- enhancement of the silencing effect was 1.6-, 2.0-, and 1.2-fold trations of cyclin D1 expression plasmid (0.1, 0.2, and 0.5 g; for TR1(bar 5 versus 3), TR1(bar 9 versus 7), and TR2(bar bars 4, 6, and 8, respectively) into GC cells led to a concentration- 13 versus 11). dependent repression of the T3-dependent transactivation ac- Cyclin D1 Does Not Affect the Binding of TR1toTRE—As a tivity of the endogenous TRs (50%, 70, and 85% repres- first step to understand the mechanisms by which cyclin D1 sion for bars 4, 6, and 8, respectively). The silencing effect of the repressed the TR-mediated transactivation, we first addressed unliganded endogenous TR was also augmented up to 1.7-fold the question as to whether the binding of TR1 to TRE was at the highest concentration of cyclin D1 (bar 7 versus bar 1). affected by cyclin D1. We carried out electrophoretic gel mobil- Taken together, these data indicate that cyclin D1 not only ity shift assay by using the in vitro translated TR1inthe repressed the transactivation activity of the transfected TRs absence or presence of in vitro translated cyclin D1. Lane 2 of but also the endogenous TRs. Fig. 4 was the control, which indicated that no nonspecific band To further evaluate the functional consequences of the inter- was detected in the unprogrammed lysates. Lanes 1 and 8 were action of cyclin D1 with TR, we co-transfected cyclin D1 and the positive controls to show that TR1 bound to TRE as TR1 in to a cancer cell line, MCF7 cells (Fig. 2C). Similar to that homodimers and heterodimers with the retinoic X receptor found in CV1 and GC cells, the T3-dependent transactivation (RXR), respectively. Clearly, cyclin D1 itself did not bind to activity of TR1 was repressed by cyclin D1 in a concentration- TRE, as no TRE bound was detected in the presence of increas- dependent manner (compared bars 6 and 8 with bar 4, Fig. 2C). ing concentrations of the in vitro translated cyclin D1 (Fig. 4, Furthermore, the silencing effect was also further augmented by lanes 3 and 4) nor in the presence of RXR (lane 7). The binding cyclin D1 in a concentration-dependent manner (bars 5 and 7 of TR1 to TRE as homodimers (compare lanes 5 and 6 with versus bar 3, Fig. 2C). Thus, the repression of the transcriptional lane 1) or as heterodimers with RXR (compare lanes 9 and 10 activity by cyclin D1 not only occurs in CV1 and GC cells but also with lane 8) was clearly not affected by the presence of in vitro in MCF7 cells. translated cyclin D1. These data indicate that the repression of Repression of TR Activity by Cyclin D1 28737 FIG.2. Cyclin D1 represses of the transcriptional activity of transfected (A and C) and endogenous TR (B) in cells. A reporter construct containing TR response element pTK-Pal-Luc (1 g) together with cyclin D1 (0.2, 0.5 g) and TR1 (0.2 g) expression vectors were transiently transfected into CV1 cells (A) and MCF7 cells (C). The reporter activity was determined both in the absence (open bar) and in the presence (solid bar) of 100 nM T3. Activities were calculated relative to the TRE-luciferase activity in the absence of TR1, cyclin D1, and T3, which was assigned as 1. B, TR-expressing GC cells was transiently transfected with pTK-Pal-Luc (1 g) and increasing amounts of cyclin D1 expression vector (0.1, 0.2, and 0.5 g). After transfection, cells were treated with 100 nM T3 for 24 h before assay. The relative activity is calculated relative to the luciferase activity in the absence of exogenous cyclins and ligand, which was calculated as 1. Data are expressed as mean  S.D. (n  3). the transactivation activity of TR by cyclin D1 was not due to Fig. 5, the T3-dependent transactivation activity of endogenous the inhibition of the binding of TR1 to TRE. TRs was activated 12-fold by T3, which was repressed by cyclin The Repression of TR-mediated Transactivation Is Reversed D1 (70% repression; bar 4 versus bar 3). In the presence of by Histone Deacetylase Inhibitor, Trichostatin A—To investi- 330 nM TSA, the T3-dependent transactivation activity of TR gate whether the cyclin D1-mediated repression of TR involves was enhanced (bars 7 versus 3). Importantly, the extent of HDACs, we transfected TRE-containing reporter with or with- repression effect on the T3-dependent transactivation activa- out cyclin D1 into GC cells. The TRE-mediated transactivation tion of TRs by cyclin D1 was reduced from 70 to 20% (bar 8 activity of the endogenous TR was evaluated in the presence of versus 7). When the concentration of TSA was further in- increasing concentrations of TSA (Fig. 5). As shown in bar 3 of creased to 660 nM (2-fold), the repression effect on the T3-de- 28738 Repression of TR Activity by Cyclin D1 FIG.4. Cyclin D1 has no effect on the binding of TR1toTRE either as homodimers or heterodimers with the retinoid X re- ceptor. The P-labeled Lys-TRE was incubated with in vitro trans- lated TR1(lanes 1, 5, 6, 8 –10;1 l), unprogrammed lysate (lane 2,4 l), or cyclin D1 (lanes 3 and 4;1 l and 3 l, respectively) or TR1(1 l) in the presence of increasing concentrations of cyclin D1 (lanes 5 and 6; 1 and 3 l, respectively) or RXR alone (lane 7), or TR1(1 l) and RXR in the presence of increasing cyclin D1 (lanes 10 and 11, 1 and 3 l, respectively) and subjected to electrophoretic gel mobility shift assay as described under “Experimental Procedures.” Unprogrammed lysates were added to make sure all lanes had the same amounts of lysates. TR1 homodimeric and heterodimeric complexes are indicated by arrows. FIG.5. Involvement of HDAC in cyclin D1-mediated repres- sion. The pTK-Pal-Luc reporter (1 g) was co-transfected with pCMV- cyclin D1 (0.5 g) or empty vector into GC cells. After overnight incu- bation, cells were treated with 0, 330, or 660 nM HDAC inhibitor TSA FIG.3. Association of cyclin D1 with TR1orTR2 in vivo. A, for 24 h before harvest. Fold activation is calculated relative to lucifer- co-immunoprecipitation (IP) of cyclin D1 with TR1orTR2. CV1 cells ase activity of cells transfected with empty vector in the absence of T3 were transfected with TR2orTR1 expression vector and pCMV- and TSA, which was set to 1. The results are the mean  S.D. from D1-HA or empty vector. Cell extracts were immunoprecipitated with three separate experiments, each performed in triplicate. anti-TR antibodies (mAbC4) followed by Western blot analysis using anti-HA. Lanes 1–5 were from cells cultured in the absence of T3, and lanes 6 –10 were from cells cultured in the presence of 100 nM T3. The pendent transactivation activation of TRs by D1 was totally corresponding cell lysates were analyzed by Western blot analysis using abrogated (bar 11 versus 12), indicating the TSA reversed the anti-HA antibodies (B) or by anti-TR antibodies (C). D, repression of repression effect mediated by D1. transcriptional activity of TR isoforms by cyclin D1. TR1, TR1, or TR2 expression plasmid (0.2 g) was co-transfected with pTK-Pal-Luc A similar reversal of the repression effect of cyclin D1 on the (1 g) and pCMV-cyclin D1 (0.5 g) or empty vector into CV1 cells. After silencing activity of endogenous unliganded TR by TSA was overnight incubation, cells were induced with 100 nM T3 for 24 h before also observed (Fig. 5). Bar 2 (versus bar 1) shows that cyclin D1 harvesting. The fold activation is calculated relative to luciferase activ- augmented the silencing activity of unliganded TR, which was ity in the absence of exogenous cyclin D1, TR, and T3, which was set to alleviated by the increasing concentrations of TSA (bar 6 versus 1. Data are expressed as mean  S.D. (n  3). Repression of TR Activity by Cyclin D1 28739 FIG.6. Association of cyclin D1 with TR and HDAC3 in cells. A, in vivo as- sociation of cyclin D1TR complex with HDAC3 but not with HDAC1. CV1 cells were transfected with the indicated com- bination of TR1, cyclin D1-HA, FLAG- HDAC3, and FLAG-HDAC1 expression vectors. Immunoprecipitation (IP) was carried out with anti-HA antibody and immunoblotted with anti-FLAG (lanes 1– 8) or anti-TR (lanes 9 –11) as indicated. B, the expression levels of TR, FLAG- HDAC1, FLAG-HDAC3, and D1-HA in ly- sates. Whole-cell lysate equivalent to 10% of the input for immunoprecipitation was immunoblotted with anti-TR (B, a), anti- FLAG (B, b), and anti-HA (B, c). 5 and 10 versus 9). These findings indicate the involvement of similar extent of expression of cyclin D1 was detected when the HDACs in the repression effect of transactivation activation of cells were co-transfected with either F-HDAC3 (lanes 2 and 3, TR by cyclin D1. These data further suggest that the recruit- Fig. 6B, c) or with F-HDAC1 (lanes 4 and 5, Fig. 6B, c). In ment of HDACs by TRD1 complex was T3-independent. addition, this was not due to the lack of expression of F-HDAC1 Cyclin D1-dependent Recruitment of HDAC3 by TR—The because F-HDAC1was expressed as shown by the direct West- above results prompted us to ask the question as to whether ern blot analysis (lanes 4 and 5, Fig. 6B, b). These results show HDACs are physically associated with TRD1 complexes. Re- that the formation of HDAC3TR1D1 complexes requires the cently, it has been reported that HDAC3 is more distantly presence of cyclin D1 and that cyclin D1 selectively interacted related to HDAC1/HDAC2 (13, 33, 34) and, moreover, that with HDAC3. HDAC3 has been found in large complexes containing TR (35); We further determined whether the enhanced expression of we therefore focused our studies on HDAC1 and HDAC3. We HDAC3 in cells could lead to increases in the cyclin D1-medi- transfected FLAG-tagged HDAC (F-HDAC1 or F-HDAC3) to- ated repression of the transactivation activity of endogenous gether with or without cyclin D1-HA and/or TR1 into CV1 TR. To this end, we transfected TRE reporter, the expression cells and analyzed the physical interaction of these proteins by plasmids of cyclin D1 and HDAC3 (or with HDAC1), into GC co-immunoprecipitation assay (Fig. 6). Lanes 3 and 9 of Fig. 6A cells in the presence or absence of T3 (Fig. 7). Comparing bars show that when lysates from cells transfected with F-HDAC3, 2 and 4 of Fig. 7 shows that cyclin D1 repressed the T3-depend- cyclin D1-HA, and TR1 were first immunoprecipitated with ent transactivation activity of endogenous TR (70%). This anti-HA followed by Western blot analysis using anti-FLAG extent of repression of T3-dependent transactivation activity of (lane 3) or anti-TR1(lane 9), F-HDAC3 and TR1 were de- endogenous TR was not affected by the co-expression of HDAC1 tected, respectively, indicating the association of both (no significant differences between bar 8 versus 4, p  0.01, Fig. F-HDAC3 and TR1 with D1. However, TR1 was not required 7). Importantly, cyclin D1-mediated repression of the T3-de- for the recruitment of F-HDAC3 by cyclin D1 (lane 2, Fig. 6A). pendent transactivation activity of endogenous TR was further Lanes 6 – 8 were negative controls to show that in the absence intensified by the co-expression of HDAC3 (bars 12 versus 10). of transfected cyclin D1-HA, neither F-HDAC3 (lane 6, Fig. 6A) Similarly, cyclin D1-mediated augmentation of the silencing nor TR1(lane 11, Fig. 6A) was detected, indicating the re- effect of the endogenous unliganded TR in GC cells (bar 3 quirement of cyclin D1 to recruit F-HDAC3 and TR1 to form versus 1) was not intensified by the co-expression of HDAC1 complexes. (bars 7 versus 3, Fig. 7). Significantly, co-expression of HDAC3 Significantly, HDAC1 did not associate with cyclin D1 either further intensified the cyclin D1-mediated augmentation of the in the absence or presence of TR1(lanes 4 and 5, Fig. 6A, silencing effect of the unliganded TR (bar 11 versus 9). In respectively). Furthermore, the interaction of cyclin D1 with contrast, HDAC1 had no effect on the cyclin D1-mediated aug- TR1 was not affected by the expression of F-HDAC1 (lane 10, mentation of the silencing effect of the unliganded TR (bar 7 Fig. 6A). The lack of interaction shown in lanes 4 and 5 (Fig. versus 3). Taken together, these data indicate that cyclin D1 6A) is not due to the lack of expression of cyclin D1 because a recruited HDAC3 to D1TR complexes, thereby augmenting the 28740 Repression of TR Activity by Cyclin D1 T3, whereas the liganded TR is in a conformation that can no longer bind to N-CoR/SMRT, cyclin D1 assumes a critical role to recruit HDAC3 to mediate the repression of T3-dependent transcriptional activity. Thus, cyclin D1 mediates the repres- sion by recruiting HDAC3 via T3-indpendent binding to TR. Our data clearly show that the recruitment of HDAC3, but not the more distantly related HDAC1, is preferred by cyclin D1 to mediate the T3-independent repression. The basis for this preference is not entirely clear. The preferential recruit- ment of different classes of HDACs by bridging factors is not without precedents. Recently, it has been shown that large complexes containing N-CoR/SMRT and unliganded TR are enriched with HDAC3 (33–36). These findings suggest that the substrate specificity on the chromatins may dictate the pre- ferred subclass of HDACs in the context of the promoter. Al- ternatively, the preferential recruitment of HDACs may also depend on the structural requirements of the bridging factors for being able to bind physically. Recently, it was reported that cyclin D1 acts as a co-activator for the estrogen receptor (24, 26) in a ligand-independent fash- ion. Cyclin D1 functions as a bridging factor between ER and the SRCs to recruit the SRC-family co-activators to ER in the absence of ligand. In contrast, the present studies show that cyclin D1 functions as a bridging factor between TR and HDAC3 to mediate repression of the transcriptional activity of FIG.7. HDAC3, but not HDAC1, augments the cyclin D1-medi- TR. It is remarkable that cyclin D1 has the capacity to bridge ated repression by endogenous TR in GC cells. Cells were trans- different receptors and co-regulatory proteins to mediate dif- fected with pTK-pal-Luc (1 g), pCMV-cyclin D1 (0.2 g), or empty ferent functions. It is conceivable that the receptor cyclin D1 vector and FLAG-tagged HDAC3 (0.4 g) or FLAG-tagged HDAC1 (0.4 g) and pCMV-cyclin D1 (0.2 g) or empty vector. After overnight interacts with dictates which co-regulate protein that cyclin D1 incubation, cells were treated with 100 nM T3 for 24 h before assay. Fold recruits. The ER binding site for cyclin D1 is located in the activation is calculated relative to luciferase activity of cells transfected carboxyl-terminal EF region (amino acids 340 –595) (24). The with empty vector in the absence of T3, which was assigned as 1. The binding of cyclin D1 with the carboxyl-terminal EF region of results are the mean  S.D. from three separate experiments each with triplicates. ER leads to the enhancement of the binding of ER to the estrogen response element. The TR binding site for cyclin D1 is located in the second half of the thyroid hormone binding silencing effect of the unliganded TR and the T3-dependent domain. However, cyclin D1 had no effect on the binding of TR transactivation activity of TR. to TRE. Together with the findings that cyclin D1 does not activate the progesterone receptor nor a number of other ste- DISCUSSION roid hormone receptors (26), these observations suggest a lim- The present study identifies cyclin D1 as a novel co-repressor ited structural requirement of cyclin D1 to bind to receptors. for TR. Cyclin D1 acts as a bridging factor to recruit HDAC3 to Thus, it is reasonable to propose that the interaction of cyclin augment the silencing activity of the unliganded TR and to D1 with ER or TR leads to a conformation in which cyclin D1 mediate the repression of the T3-dependent transcriptional either recruits an SRC-family member or HDAC3, respectively. activity. In this regard, cyclin D1 is different from other known Therefore, the receptor that cyclin D1 binds to dictates the co-repressors, such as N-CoR/SMRT, because cyclin D1 inter- selectivity of the co-regulatory proteins by the binding surface acts with TR in a T3-indpendent manner. In vitro pull-down that cyclin D1 presents to the members of SRC/160 family or assays indicate that the binding of TR1 to cyclin D1 was HDAC3. independent of T3. Furthermore, the association of cyclin D1 in The relative contributions of the N-CoR/SMRT pathway and cells with TR was also T3-independent. Consistent with the the cyclin D1 pathway in mediating the transcription repres- findings from the in vitro and in vivo binding assays, cyclin D1 sion of the unliganded TR are unclear. One of the deciding not only repressed the T3-dependent transactivation activity of factors could be the relative tissue distributions and abundance TR but also augmented the silencing activity of unliganded TR. of cyclin D1 and N-CoR/SMRT co-repressors. Because redun- This is in contrast to the other known co-repressors such as dancy of functions is common in biology, these two pathways N-CoR/SMRT, which associate with TR and silence the TR are less likely to be mutually exclusive. Therefore, one would transcriptional activity only in the absence of T3 (6, 9). Thus, expect that these pathways could act in conjunction or syner- cyclin D1 is a novel co-repressor for TR, which acts in a T3- gistically, depending on the cellular context. indpendent manner. A distinct picture emerges in the repression of T3-dependent The present studies indicate that the augmentation of the transcriptional activity. Cyclin D1 plays a unique role in pref- silencing effect of the unliganded TR was mediated by the recruitment of HDAC3. This conclusion was supported by the erentially recruiting HDAC3 to mediate the repression not only on the exogenously transfected TR but also on the endogenous demonstration of the direct interaction of cyclin D1 with HDAC3 in cells (Fig. 6), augmentation of the unliganded TR TR in GC cells. The relevance of this cyclin D1-dependent when HDAC3 was overexpressed (Fig. 7), and the alleviation of pathway is exemplified by identification of the T3 response the repression by TSA (Fig. 5). In this regard, cyclin D1 acted genes in GC cells. Using cDNA microarrays, we have recently similarly as N-CoR/SMRT in that these two co-repressors have identified 358 distinct genes in response to T3-induced prolif- recently been shown to bind directly to HDAC3 to mediate the eration of GC cells. Among the 228 named genes, 155 genes silencing effect of the unliganded TR (33, 35). In the presence of (43%) are down-regulated by T3 (37). Twenty-six of the down- Repression of TR Activity by Cyclin D1 28741 11. Taunton, J., Hassig, C. A., and Schreiber, S. L. (1996) Science 272, 408 – 411 regulated genes (17%) are early response genes of which the 12. Yang, W. M., Inouye, C., Zeng, Y., Bearss, D., and Seto, E. (1996) Proc. Natl. repression was detected within less than 6 h after T3 treat- Acad. Sci. U. S. A. 93, 12845–12850 13. Emiliani, S., Fischle, W., Van Lint, C., Al-Abed, Y., and Verdin, E. (1998) Proc. ment. Because treatment of GC cells increases the expression Natl. Acad. Sci. U. S. 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Cyclin D1 Is a Ligand-independent Co-repressor for Thyroid Hormone Receptors

Lin, Huei-min; Zhao, Li; Cheng, Sheue-yann
Journal of Biological Chemistry – Aug 1, 2002
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Abstract

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 277, No. 32, Issue of August 9, pp. 28733–28741, 2002 Printed in U.S.A. Cyclin D1 Is a Ligand-independent Co-repressor for Thyroid Hormone Receptors* Received for publication, April 8, 2002, and in revised form, May 23, 2002 Published, JBC Papers in Press, June 4, 2002, DOI 10.1074/jbc.M203380200 Huei-min Lin, Li Zhao, and Sheue-yann Cheng‡ From the Gene Regulation Section, Laboratory of Molecular Biology, Center for Cancer Research, NCI, National Institutes of Health, Bethesda, Maryland 20892-4264 Thyroid hormone receptors (TRs) are critical regula- terminus of the core histones of chromatins. Several co-activa- tors such as steroid hormone receptor co-activator (SRC) family tors of growth, differentiation, and homeostasis. TRs function by regulating the expression of thyroid hor- members pCBP/300 and p/CAF have been show to harbor in- mone (T3) target genes in both ligand-dependent and trinsic histone acetyltransferase activity (3–5). In contrast, -independent pathways. Distinct classes of co-regula- co-repressors such as N-CoR and SMRT have been shown to be tory proteins modulate these two pathways. We show associated with histone deacetylases (HDAC) and mediate the here a novel role of cyclin D1 as a T3-independent co- transcriptional silencing by TR (6 –9). repressor for TRs. Cyclin D1 interacted with TR in vitro Dynamic changes in the state of acetylation of core histones, and in cells in a ligand-independent manner. Cyclin D1 which are controlled by the opposing actions of histone acetyl- acted to repress both the silencing activity of the unli- transferases and HDACs, lead to transcriptional activation or ganded TR and the transcriptional activity of the ligan- repression of genes (10). Hyperacetylation of the histone tail ded TR. The repression was not due to the inhibition of relaxes the chromatin structure and leads to transcriptional the binding of TR to the thyroid hormone response ele- activation. Conversely, deacetylation condenses chromatin and ment but by serving as a ligand-independent bridging results in gene repression. In humans and mice, nine HDACs factor to selectively recruit HDAC3 to form ternary com- have been cloned and identified to date. They are divided into plexes. The repression was augmented by increasing two distinct classes based on their size and sequence homology expression of HDAC3 but not by HDAC1 and was allevi- to yeast HDACs. The class I enzymes such as HDAC1 (11), ated by trichostatin A. Thus, cyclin D1 is a novel ligand- HDAC2 (12), HDAC3 (13, 14), and HDAC8 (15) consist of independent co-repressor that opens a new paradigm to 400 –500 amino acids and are homologous to yeast Rpd3. In understand the molecular basis of the silencing action contrast, class II HDACs, including HDAC4, HDAC5, HDAC6 of TR. (16), HDAC7 (17), and HDAC9 (18), are large in size (1000 amino acids) and possess domains similar to the deacetylase domain of yeast Hda1. Different HDACs are recruited by dif- Thyroid hormone receptors (TRs) belong to a steroid hor- ferent transcriptional regulators to regulate the expression of mone/retinoic acid nuclear receptor superfamily that plays an different genes (16, 17, 19, 20). important role in cell proliferation, differentiation, and home- Various cyclin-dependent kinases are sequentially activated ostasis (1, 2). Four different thyroid hormone (T3) binding TR during cell cycle progression. During the transition of cell cycle, isoforms deriving from alternative splicing of two separate the activity of these kinase complexes is activated by specific genes are identified. They are expressed in tissue-dependent cyclins and inactivated by cyclin-dependent kinase inhibitors. and developmentally regulated fashion (1, 2). TR functions not Cyclin Ds complexing with CDK4 or CDK6 are strong key only as a ligand-dependent transcription factor in the presence regulators of progression through the G phase of the cell cycle of ligand but also acts as a constitutive repressor for their 1 (21, 22). Recently, it has been reported that cyclin D1 also target genes in the absence of ligand. A network of associated harbors activities that are independent of its role as a cyclin D proteins identified as co-repressors and co-activators has been kinase regulatory subunit (23). Cyclin D1 was found to activate shown to facilitate TR in gene regulation. One mechanism for the estrogen receptor (ER)-mediated transcription in breast the action of co-regulator proteins has been shown to involve tumors by direct binding to the hormone binding domain of the acetylation/deacetylation of the lysine residues on the amino ER and by enhancing the binding of ER to their target genes (24). It was further demonstrated that cyclin D1 interacts with * The costs of publication of this article were defrayed in part by the members of the SRC family and p/CAF through a motif that is payment of page charges. This article must therefore be hereby marked similar to the co-activator binding domain of nuclear receptors “advertisement” in accordance with 18 U.S.C. Section 1734 solely to in a ligand-independent manner (25, 26). It is suggested that indicate this fact. cyclin D1 serves as a bridging factor to recruit SRC and p/CAF ‡ To whom correspondence and reprint requests should be addressed: co-activators to ER, thereby facilitating the acetylation of his- Laboratory of Molecular Biology, 37 Convent Dr., Rm. 5128A2, MSC 4264, NCI, National Institutes of Health, Bethesda, MD 20892-4264. tones and promoting the ER-mediated transcription (25, 26). Tel.: 301-496-4280; Fax: 301-480-9676; E-mail: [email protected]. Previously we demonstrated that T3 stimulates the prolifer- The abbreviations used are: TR, thyroid hormone nuclear receptor; ation of growth hormone producing GC cells (27). The stimula- TRE, thyroid hormone response element: SRC, steroid hormone recep- tory effect is mainly due to a shortening of G /G phase. The tor co-activator; TSA, trichostatin; HA, hemagglutinin; AD1-HA, cyclin 0 1 D1 tagged with hemagglutinin; ER, estrogen receptor; FBS, fetal bovine shortening in G /G phase correlated with T3-induced in- 0 1 serum; GST, glutathione S-transferase; HDAC, histone deacetylase; creases in the mRNA and protein levels of two key regulators of Lys-TRE, TRE of the chicken lysozyme gene that is an everted repeat of G progression, cyclins D1 and E (27), suggesting a potential the half-site binding motifs separated by six nucleotides; mAbC4, mono- direct or indirect functional link of these two cyclins with TRs. clonal anti-TR1 antibody; RXR, retinoic X receptor ; F-HDAC, FLAG-tagged HDAC. Because ER and TR share many co-regulatory proteins in their This paper is available on line at http://www.jbc.org 28733 This is an Open Access article under the CC BY license. 28734 Repression of TR Activity by Cyclin D1 with lysis buffer and boiled in 2 Laemmli sample buffer. The immu- signaling pathways (28), we sought to understand whether noprecipitates were separated by SDS-PAGE and transferred to poly- cyclin D1 also acted to stimulate the transcriptional activity of vinylidene difluoride membranes (Millipore). The blots were blocked in TRs. We found that indeed, cyclin D1 physically interacted Tris-buffered saline containing 0.2% Tween 20 and 5% skim milk and with the hormone binding domain of TR. However, unexpect- probed with the primarily antibodies anti-TR, anti-cyclin D1 (Santa edly we discovered that in contrast to ER, this interaction led to Cruz Biotechnology, Inc.), anti-FLAG M2 (Sigma), or anti-HA (Santa a repression of both T3-dependent and -independent transac- Cruz Biotechnology, Inc.) followed by horseradish peroxidase-conju- gated secondary antibodies. The blots were washed 5 times in Tris- tivation activities of TRs. The repression was mediated by buffered saline with 0.2% Tween 20 and developed in ECL reagent cyclin D1-dependent recruitment of HDAC3 to cyclin D1-TR (Pierce). complexes. These findings expand the role of cyclin D1 in Mobility Shift Assay—The double-stranded oligonucleotide contain- transcriptional regulation. Significantly, our studies highlight 32 ing the Lys-TRE was labeled with [- P]dCTP similarly as described a novel regulatory pathway in mediating both T3-dependent by Zhu et al. (32). About 0.2 ng of probe (3–5  10 cpm) was added to and -independent repression of the transcriptional activity of M HEPES pH 8.0, 2 mM MgCl the binding buffer (25 m , 0.01 mM ZnCl , 2 2 M dithiothreitol, 6% glycerol, 0.01% TritionX-100) and in vitro 5m TRs. translated proteins, and 0.2 g of sheared salmon sperm DNA. Binding EXPERIMENTAL PROCEDURES reactions were carried out at room temperature for 30 min, and com- plexes were resolved on 6% polyacrylamide gels in 0.5 TBE (45 mM Cell Culture and Reagents—CV-1, GC, and MCF7 cells were main- Tris-HCl, 45 mM boric acid, 0.5 mM EDTA) at 150 V for 2 h. After drying tained in Dulbecco’s modified Eagle’s medium with 10% fetal bovine of the gel, the DNA-bound proteins were detected by autoradiography. serum (FBS), 50 g/ml penicillin, 0.25 g/ml streptomycin, and 2 mM L-glutamine at 37 °C under 5% CO . T3-depleted FBS was prepared by RESULTS incubating FBS with 20 mg/ml activated charcoal and 50 mg/ml anion exchange resin (AGX-8, Bio-Rad) for 24 h followed by centrifugation and Cyclin D1 Binds Specifically to TR1—We utilized the GST/ ultrafiltration. T3 (Sigma) was dissolved in 0.1 N NaOH. Trichostatin A glutathione (GSH) binding system to assess whether TR1 (TSA) was obtained from Sigma and dissolved in dimethyl sulfoxide. physically interacted with cyclin D1. GST alone was used as a Both reagents were stored at 20 °C and diluted immediately before negative control. Fig. 1A shows that TR1 bound to GST-D1 in use. the absence (lane 3) or presence of T3 (lane 4), but not GST Plasmids—The cyclin D1 expression vectors, pCMV-cyclin D1, pCMV-D1-HA, and pGST-cyclin D1 were generous gifts from M. E. (lanes 1 and 2), indicating the specific physical interaction of Ewen. The reporter pTK-Pal-Luc contains two copies of palindrome TR TR1 with cyclin D1. To map the region of TR1 to which cyclin response element (TRE) in tandem and a luciferase gene under the D1 bound, we prepared S-labeled truncated TR1 in which control of thymidine kinase promoter (a gift from J. L. Jameson). domains were systematically deleted (Fig. 1B). Deletion of do- pCEP4F-HDAC3 expressing an amino terminus FLAG-tagged HDAC3 main A/B (lanes 5 and 6, Fig. 1A), domains A/BC(lanes 7 and and pBJ5-HD-1 encoding a carboxyl terminus FLAG-tagged HDAC1 8, Fig. 1A), or domains A/BCD(lanes 9 and 10, Fig. 1A) did were described previously (14) (generous gifts of E. Seto). The pCLC51, pCLC61, and pCMV-TR2 express human TR1, -1, and -2 cDNA, not significantly affect the binding of cyclin D1 to TR1 regard- driven by cytomegalovirus promoter, respectively (29). For in vitro less of whether T3 was present or not. These results indicate transcription/translation, pCJ3, pJL8, pJL5, and pCJ4 encode full- that domain E was the essential binding region for cyclin D1 length and truncated TR1 under the control of the T7 promoter (30). and that this physical interaction was independent of T3. Transient Transfection—Cells (3  10 cells) were seeded into 6-well To demonstrate the interaction of cyclin D1 with TR1 in plates 1 day before transfection. The reporter plasmid pTK-Pal-Luc (1 vivo, CV1 cells were transfected with expression vector of TR1 g) was transfected with 0.2 g of TR expression vector (pCLC51) and cyclin expression vectors (pCMV-cyclin D1 or pCMV-D1-HA) into cells. or cyclin D1 tagged with hemagglutinin (D1-HA) or transfected Empty vectors were used to supplement equal amounts of DNA in each with both expression vectors. Fig. 1C shows the results of such transfection. Transfection was carried out using FuGENE 6 (Roche experiments from cells cultured in the absence of T3. Western Molecular Biochemicals) according to the manufacturer’s protocol. After blot analysis of cell lysates using mAbC4 (31) detected the overnight incubation, cultures were replaced with fresh Dulbecco’s expression of TR1(lanes 1 and 3, Fig. 1C, a) but not in cells modified Eagle’s medium with 10% T3-depleted FBS and induced with transfected only with D1-HA expression vector (lane 2). When 100 nM T3 for 24 h before harvest. Cells were rinsed with phosphate- buffered saline and lysed in reporter lysis buffer (Promega). Lysates cell lysates were first immunoprecipitated with anti-HA anti- were assayed for luciferase activity and normalized to total protein bodies followed by Western blot analysis using mAbC4, TR1 concentration. All experiments were performed in triplicate and re- was detected in cells transfected with TR1 and D1-HA as peated 3–5 times. The results shown are the mean  S.E. shown in lane 6 (Fig. 1C, a) but not in cells transfected with In Vitro Binding Assay—Glutathione S-transferase (GST) fusion pro- either TR1(lane 4) or D1-HA alone (lane 5), indicating an tein was expressed in Escherichia coli BL21 strain, induced by 1 mM association of TR1 with cyclin D1 in vivo. isopropyl--D-thiogalactopyranoside, and affinity-purified by glutathi- one-Sepharose 4B beads (Amersham Biosciences). [ S]Methionine-la- The association of TR1 with cyclin D1 in vivo was further beled TR proteins were prepared by TNT-coupled reticulocyte lysate demonstrated by a reverse experiment in which cell lysates system (Promega) and incubated with 1 g of GST-cyclin D1or GST in were first immunoprecipitated with anti-TR1 (mAbC4) fol- buffer (20 mM Tris-HCl, pH 7.5, 100 mM NaCl, 2 mM EDTA, 0.1% lowed by Western blot analysis using anti-HA. As shown in Lubrol, 2 mM dithiothreitol, 0.05% BSA, 5% glycerol protease inhibi- lane 6 of Fig. 1C, b, cyclin D1-HA was detected in cells trans- tors) at 4 °C overnight with constant rotation. Subsequently, beads fected with both TR1 and cyclin D1-HA expression plasmids were washed 4 times with 25 mM Tris-HCl, pH 7.4, 2.5% sucrose, 2.5 mM EDTA, 250 mM NaCl, and 1% Lubrol. The bound proteins were ex- but not in cells transfected only with TR1(lane 4, Fig. 1C, b) tracted by boiling the beads in SDS sample buffer for 5 min and or with cyclin D1-HA (lane 5, Fig. 1C, b). The identity of the separated on SDS-polyacrylamide gel and detected by autoradiography. co-immunoprecipitated cyclin D1-H1 was confirmed by direct Immunoprecipitation and Western Blot Analysis—CV1 cells were Western analysis of cell lysates with anti-HA as shown in lanes transfected with the indicated combinations of plasmids by Lipo- 2 and 3 of Fig. 1C, b. fectAMINE (Invitrogen). GC cells were transfected with FuGENE 6 Similar results were obtained from cells cultured in the (Roche Molecular Biochemicals). Cells were harvested after 48 h and lysed on ice in lysis buffer (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM presence of T3 (Fig. 1D). TR1 was detected in cells co-trans- EDTA, 0.5% Nonidet P-40) with protease inhibitor mixture (Roche fected with both D1-HA and TR1 after first immunoprecipi- Molecular Biochemicals). Cell lysates were clarified by centrifugation at tation of lysates with anti-HA antibodies followed by Western 11,000  g for 5 min at 4 °C. For immunoprecipitation, 300 g of cell blot analysis using anti-TR1 antibody (mAbC4; lane 6, Fig. extract was incubated with anti-HA (Santa Cruz Biotechnology, Inc.) or 1D, a). Using the same protocol, no TR1 was detected in cells monoclonal anti-TR, monoclonal anti-TR1 antibody (mAbC4) (31) for transfected only with either TR1(lane 4, Fig. 1D, a) or D1-HA 2hat4 °C, and followed by incubation with protein G-agarose beads (Roche Molecular Biochemicals) overnight. Beads were washed 5 times (lane 5, Fig. 1D, a). The association of D1-HA with TR1 was Repression of TR Activity by Cyclin D1 28735 FIG. 1. Cyclin D1 complexes with TR1 in vitro and in vivo. A, pull-down assay of TR1 by cyclin D1. Immobilized GST or GST-cyclin D1 fusion protein was incubated with in vitro translated S-labeled truncated TR1 in the absence (odd-numbered lanes) or presence of 100 nM T3 (even- numbered lanes) as described under “Experimental Procedures.” B, schematic representation of full-length and TR1-truncated proteins used in the binding assay. The amino-terminal domain, DNA binding domain, hinge region, and part of the hormone binding domain of TR1 are indicated as A/B, C, D, and E, respectively. The numbers indicate the position of amino acids. Association of TR1 with cyclin D1 in vivo without T3 (C) or with T3 (D) is shown. CV1 cells were transfected with the indicated combination of TR1 and D1-HA expression plasmids (pCLC51 and pCMV-D1-HA). Whole-cell lysates were immunoprecipitated (IP) with anti-HA or anti-TR1 as indicated. Immunoprecipitates were separated by SDS-PAGE followed by Western blotting with anti-TR and anti-HA antibodies. Input represents 10% of the total lysate used in the immunoprecipitation. Proteins were detected by Western blot analysis using antibody against TR (mAbC4) or HA epitope. E, GC cells were transfected with pCMV-D1-HA (10 g) or empty vector using FuGENE 6. Cell lysates (500 g) were immunoprecipitated with anti-HA followed by Western blot analysis using mAbC4 (E, a). For E, b and c, direct Western blot analysis was performed on lysates (50 g) using anti-TR (mAbC4) or anti-HA, respectively. 28736 Repression of TR Activity by Cyclin D1 further confirmed by reversing the antibodies in that anti- Cyclin D1 Interacts with Other TR Isoforms and Differen- TR1 antibody was first used to immunoprecipitate TR1 fol- tially Represses Their Silencing and T3-dependent Transacti- vation Activities—We further evaluated whether cyclin D1 also lowed by Western blot using anti-HA (lane 6, Fig. 1D, b). Lanes 1 and 3, Fig. 1D, a, and lanes 2 and 3 of Fig. 1D, b, show the interacted with other TR isoforms in cells by carrying out the expression of TR1 and D1-HA, respectively, by direct Western co-immunoprecipitation assay (Fig. 3, A–C). CV1 cells were transfected with TR1orTR2 with or without cyclin D1-HA blot analysis. Taken together, these data indicate that the in the absence of T3 (lanes 1–5, Fig. 3, A–C). When lysates from association of D1 with TR1 in cells was T3-independent. cells transfected with cyclin D1-HA and TR1orTR2 expres- Cyclin D1 not only associated with the transfected TR but sion plasmids were first immunoprecipitated with anti-TR also with the endogenous TR in GC cells (Fig. 1E). As shown in (mAbC4) followed by Western analysis using anti-HA, D1-HA Fig. 1E, a,TR1 was detected in GC cells transfected with was detected as shown in lanes 2 and 3 of Fig. 3A, indicating cyclin D1 (lane 2 of Fig. 1E) by co-immunoprecipitation assay the association of TR1orTR2 with cyclin D1-HA, respec- but not in cells without the transfected cyclin D1 (lane 1 of Fig. tively. This association is further supported by a negative con- 1E, a). Fig. 1E, b and c, show the controls by direct Western blot trol in which no D1-HA was detected in cells without the analysis to indicate the expression of endogenous TR1 and the co-transfection of either TR1orTR2(lane 1, Fig. 3A). The transfected cyclin D1 in GC cells, respectively, (lane 2 of Fig. lack of detection of D1-HA was not due to the lack of expression 1E, c). These results indicate that cyclin D1 associates not only because direct Western blot analysis of the same lysates indi- with the transfected TR1 but, importantly, with the endoge- cates that D1-HA was similarly expressed (lane 1, Fig. 3B)asin nous TR1 in GC cells. cells co-transfected with either TR1orTR2(lanes 2 and 3, Cyclin D1 Represses the T3-dependent Transactivation Activ- Fig. 3B). Furthermore, direct Western blot analysis using an- ity of TR1—To assess the functional consequences of the phys- ti-TR antibody shows that TR1 and TR2 were expressed ical interaction of TR1 with cyclin D1, we used TRE-contain- (lanes 2–5, Fig. 3C). These results indicate that cyclin D1 also ing reporter systems (Fig. 2). CV1 cells were transfected with a physically interacted with TR1orTR2 in the absence of T3. constant amount of TR1 expression plasmid in the absence or Similar association of cyclin D1 with TR1orTR2 was also presence of an increasing amount of expression plasmid of detected in cells cultured in the presence of T3 (lanes 7 and 8, cyclin D1. Fig. 2A shows that the transactivation activity of Fig. 3A, respectively). Again, this physical association in cells TR1 was activated 20-fold by T3 (bar 4, Fig. 2A). Cyclin D1 was further confirmed by the negative controls in that when repressed the T3-dependent transactivation activity of TR1in cells were not transfected with either TR1orTR2, no D1-HA a concentration-dependent manner. The extent of repression was detected (lane 6, Fig. 3A). The data shown in Fig. 3 indicate was 55% (bar 6 versus bar 4) and 80% (bar 8 versus bar 4) that TR1 and TR2 were associated with D1-HA in cells at the cyclin D1:TR1 plasmid ratio of 1 and 2, respectively. independent of T3. These results indicate that cyclin D1 negatively regulates the The functional consequences of the physical interaction of T3-dependent transactivation activity of TR1. cyclin D1 with each of the TR isoforms were evaluated in the Consistent with the T3-independent binding of cyclin D1 to presence or absence of T3 by using TRE-containing reporter TR1 shown in Fig. 1, cyclin D1 also augmented the silencing system (Fig. 3D). In the presence of T3, the interaction of cyclin effect of the unliganded TR1 in a concentration-dependent D1 with each of the three TR isoforms led to the repression of manner (Fig. 2A). The enhancement in the silencing effect was T3-dependent transactivation of TRs. However, the extent of 1.6- and 2-fold when the ratio of the cyclin D1:TR1 was in- the repression is TR isoform-dependent in that 60, 80, and 65% creased from 1 to 2 (bars 5 and 7, respectively). Taken together, repression was observed for TR1, TR1, and TR2, these results indicate that cyclin D1 not only repressed the respectively. T3-dependent transactivation activity but also augmented the Fig. 3D also compares the extent of the effect of cyclin D1 on T3-independent silencing activity of TR. the silencing effect of the un-liganded TR1 and TR2 with The cyclin D1-mediated repression effect was further dem- that of TR1. The augmentation of the silencing effect by cyclin onstrated by the transactivation activity of endogenous TRs in D1 also differed among the TR isoforms in that the extent of the GC cells. Fig. 2B shows that transfection of increasing concen- enhancement of the silencing effect was 1.6-, 2.0-, and 1.2-fold trations of cyclin D1 expression plasmid (0.1, 0.2, and 0.5 g; for TR1(bar 5 versus 3), TR1(bar 9 versus 7), and TR2(bar bars 4, 6, and 8, respectively) into GC cells led to a concentration- 13 versus 11). dependent repression of the T3-dependent transactivation ac- Cyclin D1 Does Not Affect the Binding of TR1toTRE—As a tivity of the endogenous TRs (50%, 70, and 85% repres- first step to understand the mechanisms by which cyclin D1 sion for bars 4, 6, and 8, respectively). The silencing effect of the repressed the TR-mediated transactivation, we first addressed unliganded endogenous TR was also augmented up to 1.7-fold the question as to whether the binding of TR1 to TRE was at the highest concentration of cyclin D1 (bar 7 versus bar 1). affected by cyclin D1. We carried out electrophoretic gel mobil- Taken together, these data indicate that cyclin D1 not only ity shift assay by using the in vitro translated TR1inthe repressed the transactivation activity of the transfected TRs absence or presence of in vitro translated cyclin D1. Lane 2 of but also the endogenous TRs. Fig. 4 was the control, which indicated that no nonspecific band To further evaluate the functional consequences of the inter- was detected in the unprogrammed lysates. Lanes 1 and 8 were action of cyclin D1 with TR, we co-transfected cyclin D1 and the positive controls to show that TR1 bound to TRE as TR1 in to a cancer cell line, MCF7 cells (Fig. 2C). Similar to that homodimers and heterodimers with the retinoic X receptor found in CV1 and GC cells, the T3-dependent transactivation (RXR), respectively. Clearly, cyclin D1 itself did not bind to activity of TR1 was repressed by cyclin D1 in a concentration- TRE, as no TRE bound was detected in the presence of increas- dependent manner (compared bars 6 and 8 with bar 4, Fig. 2C). ing concentrations of the in vitro translated cyclin D1 (Fig. 4, Furthermore, the silencing effect was also further augmented by lanes 3 and 4) nor in the presence of RXR (lane 7). The binding cyclin D1 in a concentration-dependent manner (bars 5 and 7 of TR1 to TRE as homodimers (compare lanes 5 and 6 with versus bar 3, Fig. 2C). Thus, the repression of the transcriptional lane 1) or as heterodimers with RXR (compare lanes 9 and 10 activity by cyclin D1 not only occurs in CV1 and GC cells but also with lane 8) was clearly not affected by the presence of in vitro in MCF7 cells. translated cyclin D1. These data indicate that the repression of Repression of TR Activity by Cyclin D1 28737 FIG.2. Cyclin D1 represses of the transcriptional activity of transfected (A and C) and endogenous TR (B) in cells. A reporter construct containing TR response element pTK-Pal-Luc (1 g) together with cyclin D1 (0.2, 0.5 g) and TR1 (0.2 g) expression vectors were transiently transfected into CV1 cells (A) and MCF7 cells (C). The reporter activity was determined both in the absence (open bar) and in the presence (solid bar) of 100 nM T3. Activities were calculated relative to the TRE-luciferase activity in the absence of TR1, cyclin D1, and T3, which was assigned as 1. B, TR-expressing GC cells was transiently transfected with pTK-Pal-Luc (1 g) and increasing amounts of cyclin D1 expression vector (0.1, 0.2, and 0.5 g). After transfection, cells were treated with 100 nM T3 for 24 h before assay. The relative activity is calculated relative to the luciferase activity in the absence of exogenous cyclins and ligand, which was calculated as 1. Data are expressed as mean  S.D. (n  3). the transactivation activity of TR by cyclin D1 was not due to Fig. 5, the T3-dependent transactivation activity of endogenous the inhibition of the binding of TR1 to TRE. TRs was activated 12-fold by T3, which was repressed by cyclin The Repression of TR-mediated Transactivation Is Reversed D1 (70% repression; bar 4 versus bar 3). In the presence of by Histone Deacetylase Inhibitor, Trichostatin A—To investi- 330 nM TSA, the T3-dependent transactivation activity of TR gate whether the cyclin D1-mediated repression of TR involves was enhanced (bars 7 versus 3). Importantly, the extent of HDACs, we transfected TRE-containing reporter with or with- repression effect on the T3-dependent transactivation activa- out cyclin D1 into GC cells. The TRE-mediated transactivation tion of TRs by cyclin D1 was reduced from 70 to 20% (bar 8 activity of the endogenous TR was evaluated in the presence of versus 7). When the concentration of TSA was further in- increasing concentrations of TSA (Fig. 5). As shown in bar 3 of creased to 660 nM (2-fold), the repression effect on the T3-de- 28738 Repression of TR Activity by Cyclin D1 FIG.4. Cyclin D1 has no effect on the binding of TR1toTRE either as homodimers or heterodimers with the retinoid X re- ceptor. The P-labeled Lys-TRE was incubated with in vitro trans- lated TR1(lanes 1, 5, 6, 8 –10;1 l), unprogrammed lysate (lane 2,4 l), or cyclin D1 (lanes 3 and 4;1 l and 3 l, respectively) or TR1(1 l) in the presence of increasing concentrations of cyclin D1 (lanes 5 and 6; 1 and 3 l, respectively) or RXR alone (lane 7), or TR1(1 l) and RXR in the presence of increasing cyclin D1 (lanes 10 and 11, 1 and 3 l, respectively) and subjected to electrophoretic gel mobility shift assay as described under “Experimental Procedures.” Unprogrammed lysates were added to make sure all lanes had the same amounts of lysates. TR1 homodimeric and heterodimeric complexes are indicated by arrows. FIG.5. Involvement of HDAC in cyclin D1-mediated repres- sion. The pTK-Pal-Luc reporter (1 g) was co-transfected with pCMV- cyclin D1 (0.5 g) or empty vector into GC cells. After overnight incu- bation, cells were treated with 0, 330, or 660 nM HDAC inhibitor TSA FIG.3. Association of cyclin D1 with TR1orTR2 in vivo. A, for 24 h before harvest. Fold activation is calculated relative to lucifer- co-immunoprecipitation (IP) of cyclin D1 with TR1orTR2. CV1 cells ase activity of cells transfected with empty vector in the absence of T3 were transfected with TR2orTR1 expression vector and pCMV- and TSA, which was set to 1. The results are the mean  S.D. from D1-HA or empty vector. Cell extracts were immunoprecipitated with three separate experiments, each performed in triplicate. anti-TR antibodies (mAbC4) followed by Western blot analysis using anti-HA. Lanes 1–5 were from cells cultured in the absence of T3, and lanes 6 –10 were from cells cultured in the presence of 100 nM T3. The pendent transactivation activation of TRs by D1 was totally corresponding cell lysates were analyzed by Western blot analysis using abrogated (bar 11 versus 12), indicating the TSA reversed the anti-HA antibodies (B) or by anti-TR antibodies (C). D, repression of repression effect mediated by D1. transcriptional activity of TR isoforms by cyclin D1. TR1, TR1, or TR2 expression plasmid (0.2 g) was co-transfected with pTK-Pal-Luc A similar reversal of the repression effect of cyclin D1 on the (1 g) and pCMV-cyclin D1 (0.5 g) or empty vector into CV1 cells. After silencing activity of endogenous unliganded TR by TSA was overnight incubation, cells were induced with 100 nM T3 for 24 h before also observed (Fig. 5). Bar 2 (versus bar 1) shows that cyclin D1 harvesting. The fold activation is calculated relative to luciferase activ- augmented the silencing activity of unliganded TR, which was ity in the absence of exogenous cyclin D1, TR, and T3, which was set to alleviated by the increasing concentrations of TSA (bar 6 versus 1. Data are expressed as mean  S.D. (n  3). Repression of TR Activity by Cyclin D1 28739 FIG.6. Association of cyclin D1 with TR and HDAC3 in cells. A, in vivo as- sociation of cyclin D1TR complex with HDAC3 but not with HDAC1. CV1 cells were transfected with the indicated com- bination of TR1, cyclin D1-HA, FLAG- HDAC3, and FLAG-HDAC1 expression vectors. Immunoprecipitation (IP) was carried out with anti-HA antibody and immunoblotted with anti-FLAG (lanes 1– 8) or anti-TR (lanes 9 –11) as indicated. B, the expression levels of TR, FLAG- HDAC1, FLAG-HDAC3, and D1-HA in ly- sates. Whole-cell lysate equivalent to 10% of the input for immunoprecipitation was immunoblotted with anti-TR (B, a), anti- FLAG (B, b), and anti-HA (B, c). 5 and 10 versus 9). These findings indicate the involvement of similar extent of expression of cyclin D1 was detected when the HDACs in the repression effect of transactivation activation of cells were co-transfected with either F-HDAC3 (lanes 2 and 3, TR by cyclin D1. These data further suggest that the recruit- Fig. 6B, c) or with F-HDAC1 (lanes 4 and 5, Fig. 6B, c). In ment of HDACs by TRD1 complex was T3-independent. addition, this was not due to the lack of expression of F-HDAC1 Cyclin D1-dependent Recruitment of HDAC3 by TR—The because F-HDAC1was expressed as shown by the direct West- above results prompted us to ask the question as to whether ern blot analysis (lanes 4 and 5, Fig. 6B, b). These results show HDACs are physically associated with TRD1 complexes. Re- that the formation of HDAC3TR1D1 complexes requires the cently, it has been reported that HDAC3 is more distantly presence of cyclin D1 and that cyclin D1 selectively interacted related to HDAC1/HDAC2 (13, 33, 34) and, moreover, that with HDAC3. HDAC3 has been found in large complexes containing TR (35); We further determined whether the enhanced expression of we therefore focused our studies on HDAC1 and HDAC3. We HDAC3 in cells could lead to increases in the cyclin D1-medi- transfected FLAG-tagged HDAC (F-HDAC1 or F-HDAC3) to- ated repression of the transactivation activity of endogenous gether with or without cyclin D1-HA and/or TR1 into CV1 TR. To this end, we transfected TRE reporter, the expression cells and analyzed the physical interaction of these proteins by plasmids of cyclin D1 and HDAC3 (or with HDAC1), into GC co-immunoprecipitation assay (Fig. 6). Lanes 3 and 9 of Fig. 6A cells in the presence or absence of T3 (Fig. 7). Comparing bars show that when lysates from cells transfected with F-HDAC3, 2 and 4 of Fig. 7 shows that cyclin D1 repressed the T3-depend- cyclin D1-HA, and TR1 were first immunoprecipitated with ent transactivation activity of endogenous TR (70%). This anti-HA followed by Western blot analysis using anti-FLAG extent of repression of T3-dependent transactivation activity of (lane 3) or anti-TR1(lane 9), F-HDAC3 and TR1 were de- endogenous TR was not affected by the co-expression of HDAC1 tected, respectively, indicating the association of both (no significant differences between bar 8 versus 4, p  0.01, Fig. F-HDAC3 and TR1 with D1. However, TR1 was not required 7). Importantly, cyclin D1-mediated repression of the T3-de- for the recruitment of F-HDAC3 by cyclin D1 (lane 2, Fig. 6A). pendent transactivation activity of endogenous TR was further Lanes 6 – 8 were negative controls to show that in the absence intensified by the co-expression of HDAC3 (bars 12 versus 10). of transfected cyclin D1-HA, neither F-HDAC3 (lane 6, Fig. 6A) Similarly, cyclin D1-mediated augmentation of the silencing nor TR1(lane 11, Fig. 6A) was detected, indicating the re- effect of the endogenous unliganded TR in GC cells (bar 3 quirement of cyclin D1 to recruit F-HDAC3 and TR1 to form versus 1) was not intensified by the co-expression of HDAC1 complexes. (bars 7 versus 3, Fig. 7). Significantly, co-expression of HDAC3 Significantly, HDAC1 did not associate with cyclin D1 either further intensified the cyclin D1-mediated augmentation of the in the absence or presence of TR1(lanes 4 and 5, Fig. 6A, silencing effect of the unliganded TR (bar 11 versus 9). In respectively). Furthermore, the interaction of cyclin D1 with contrast, HDAC1 had no effect on the cyclin D1-mediated aug- TR1 was not affected by the expression of F-HDAC1 (lane 10, mentation of the silencing effect of the unliganded TR (bar 7 Fig. 6A). The lack of interaction shown in lanes 4 and 5 (Fig. versus 3). Taken together, these data indicate that cyclin D1 6A) is not due to the lack of expression of cyclin D1 because a recruited HDAC3 to D1TR complexes, thereby augmenting the 28740 Repression of TR Activity by Cyclin D1 T3, whereas the liganded TR is in a conformation that can no longer bind to N-CoR/SMRT, cyclin D1 assumes a critical role to recruit HDAC3 to mediate the repression of T3-dependent transcriptional activity. Thus, cyclin D1 mediates the repres- sion by recruiting HDAC3 via T3-indpendent binding to TR. Our data clearly show that the recruitment of HDAC3, but not the more distantly related HDAC1, is preferred by cyclin D1 to mediate the T3-independent repression. The basis for this preference is not entirely clear. The preferential recruit- ment of different classes of HDACs by bridging factors is not without precedents. Recently, it has been shown that large complexes containing N-CoR/SMRT and unliganded TR are enriched with HDAC3 (33–36). These findings suggest that the substrate specificity on the chromatins may dictate the pre- ferred subclass of HDACs in the context of the promoter. Al- ternatively, the preferential recruitment of HDACs may also depend on the structural requirements of the bridging factors for being able to bind physically. Recently, it was reported that cyclin D1 acts as a co-activator for the estrogen receptor (24, 26) in a ligand-independent fash- ion. Cyclin D1 functions as a bridging factor between ER and the SRCs to recruit the SRC-family co-activators to ER in the absence of ligand. In contrast, the present studies show that cyclin D1 functions as a bridging factor between TR and HDAC3 to mediate repression of the transcriptional activity of FIG.7. HDAC3, but not HDAC1, augments the cyclin D1-medi- TR. It is remarkable that cyclin D1 has the capacity to bridge ated repression by endogenous TR in GC cells. Cells were trans- different receptors and co-regulatory proteins to mediate dif- fected with pTK-pal-Luc (1 g), pCMV-cyclin D1 (0.2 g), or empty ferent functions. It is conceivable that the receptor cyclin D1 vector and FLAG-tagged HDAC3 (0.4 g) or FLAG-tagged HDAC1 (0.4 g) and pCMV-cyclin D1 (0.2 g) or empty vector. After overnight interacts with dictates which co-regulate protein that cyclin D1 incubation, cells were treated with 100 nM T3 for 24 h before assay. Fold recruits. The ER binding site for cyclin D1 is located in the activation is calculated relative to luciferase activity of cells transfected carboxyl-terminal EF region (amino acids 340 –595) (24). The with empty vector in the absence of T3, which was assigned as 1. The binding of cyclin D1 with the carboxyl-terminal EF region of results are the mean  S.D. from three separate experiments each with triplicates. ER leads to the enhancement of the binding of ER to the estrogen response element. The TR binding site for cyclin D1 is located in the second half of the thyroid hormone binding silencing effect of the unliganded TR and the T3-dependent domain. However, cyclin D1 had no effect on the binding of TR transactivation activity of TR. to TRE. Together with the findings that cyclin D1 does not activate the progesterone receptor nor a number of other ste- DISCUSSION roid hormone receptors (26), these observations suggest a lim- The present study identifies cyclin D1 as a novel co-repressor ited structural requirement of cyclin D1 to bind to receptors. for TR. Cyclin D1 acts as a bridging factor to recruit HDAC3 to Thus, it is reasonable to propose that the interaction of cyclin augment the silencing activity of the unliganded TR and to D1 with ER or TR leads to a conformation in which cyclin D1 mediate the repression of the T3-dependent transcriptional either recruits an SRC-family member or HDAC3, respectively. activity. In this regard, cyclin D1 is different from other known Therefore, the receptor that cyclin D1 binds to dictates the co-repressors, such as N-CoR/SMRT, because cyclin D1 inter- selectivity of the co-regulatory proteins by the binding surface acts with TR in a T3-indpendent manner. In vitro pull-down that cyclin D1 presents to the members of SRC/160 family or assays indicate that the binding of TR1 to cyclin D1 was HDAC3. independent of T3. Furthermore, the association of cyclin D1 in The relative contributions of the N-CoR/SMRT pathway and cells with TR was also T3-independent. Consistent with the the cyclin D1 pathway in mediating the transcription repres- findings from the in vitro and in vivo binding assays, cyclin D1 sion of the unliganded TR are unclear. One of the deciding not only repressed the T3-dependent transactivation activity of factors could be the relative tissue distributions and abundance TR but also augmented the silencing activity of unliganded TR. of cyclin D1 and N-CoR/SMRT co-repressors. Because redun- This is in contrast to the other known co-repressors such as dancy of functions is common in biology, these two pathways N-CoR/SMRT, which associate with TR and silence the TR are less likely to be mutually exclusive. Therefore, one would transcriptional activity only in the absence of T3 (6, 9). Thus, expect that these pathways could act in conjunction or syner- cyclin D1 is a novel co-repressor for TR, which acts in a T3- gistically, depending on the cellular context. indpendent manner. A distinct picture emerges in the repression of T3-dependent The present studies indicate that the augmentation of the transcriptional activity. Cyclin D1 plays a unique role in pref- silencing effect of the unliganded TR was mediated by the recruitment of HDAC3. This conclusion was supported by the erentially recruiting HDAC3 to mediate the repression not only on the exogenously transfected TR but also on the endogenous demonstration of the direct interaction of cyclin D1 with HDAC3 in cells (Fig. 6), augmentation of the unliganded TR TR in GC cells. The relevance of this cyclin D1-dependent when HDAC3 was overexpressed (Fig. 7), and the alleviation of pathway is exemplified by identification of the T3 response the repression by TSA (Fig. 5). In this regard, cyclin D1 acted genes in GC cells. Using cDNA microarrays, we have recently similarly as N-CoR/SMRT in that these two co-repressors have identified 358 distinct genes in response to T3-induced prolif- recently been shown to bind directly to HDAC3 to mediate the eration of GC cells. Among the 228 named genes, 155 genes silencing effect of the unliganded TR (33, 35). In the presence of (43%) are down-regulated by T3 (37). Twenty-six of the down- Repression of TR Activity by Cyclin D1 28741 11. Taunton, J., Hassig, C. A., and Schreiber, S. L. (1996) Science 272, 408 – 411 regulated genes (17%) are early response genes of which the 12. Yang, W. M., Inouye, C., Zeng, Y., Bearss, D., and Seto, E. (1996) Proc. Natl. repression was detected within less than 6 h after T3 treat- Acad. Sci. U. S. A. 93, 12845–12850 13. Emiliani, S., Fischle, W., Van Lint, C., Al-Abed, Y., and Verdin, E. (1998) Proc. ment. Because treatment of GC cells increases the expression Natl. Acad. Sci. U. S. 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Published: Aug 1, 2002

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