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Department of Medicine and Molecular Science (S.I., M.Y., T.S., T.M., K.H., N.S., M.M.) and Department of Pediatrics and Developmental Medicine (K.O., A.M.), Gunma University Graduate School of Medicine, Maebashi 371-8511, Japan
Address all correspondence and requests for reprints to: Masanobu Yamada, M.D., Ph.D., Department of Medicine and Molecular Science, Gunma University Graduate School of Medicine 3-39-15 Showa-machi, Maebashi, Gunma 371-8511, Japan. E-mail: myamada{at}med.gunma-u.ac.jp.
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ABSTRACT |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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On the genes positively regulated by thyroid hormone, TR binds to target promoters as a homodimer or a heterodimer with the retinoid X receptor (RXR) and regulates promoter activity by recruiting specific coregulatory protein complexes (3, 4). In the unliganded state, TR assumes a conformation that stably interacts with corepressor molecules such as nuclear receptor corepressor (NCoR) and silencing mediator of retinoic and thyroid hormone receptors (SMRT). Numerous histone deacetylases (HDACs), including HDAC-1, -2, -3, -4, -5, -7, and -9, have been shown to interact with NCoR and SMRT in one context or another, and then repress basal transcriptional activity. Recent chromatin immunoprecipitation (ChIP) experiments have demonstrated that HDAC3 on NCoR, not on SMRT, is most important for the repression by unliganded TR (5).
Stimulation with T3 leads to the dissociation of corepressors and recruitment of coactivators including members of the p160/steroid receptor coactivator (SRC) family and TR-associated protein/vitamin D receptor-interacting protein mediators. These proteins are thought to function in part by associating with potent histone acetylytransferases (HATs) such as p300/cAMP response element binding protein-binding protein and ultimately import the HAT activity to promoter-bound TR, resulting in the acetylation of nucleosome histones. Additionally, some p160/SRC family members have intrinsic HAT activity, further supporting a functional role for these factors in chromatin modification (6, 7). It was reported that the ordered recruitment and release of coactivators are important for transcriptional activation (8).
In contrast to the mechanism of positive regulation, the mechanism of trans-repression of the hypothalamic TRH and pituitary TSH subunit genes remains poorly understood. It remains to be elucidated whether direct binding of TR to DNA is necessary for the negative regulation. A detailed analysis of TR knockout mice demonstrated that at least the ß isoform of the TR (TRß) has a key role in the negative feedback regulation of the hypothalamic-pituitarythyroid axis (9). Although a number of distinct mechanisms for TRß-mediated negative regulation by thyroid hormone have been proposed, several investigators reported that coregulators including NCoR, SMRT, and SRC-1 are involved in the negative regulation by thyroid hormone and the dominant-negative effect of the mutant TR observed in RTH patients. It is of interest how such cofactors affect histone acetylation status and chromatin structure in the negative gene regulation by thyroid hormone.
In the present study, we first report a novel RTH mutant, F455S, characterized using conventional molecular methods including transient transfection analysis, glutathione-S-transferase (GST) pull-down assay, and EMSA. In addition, to investigate the chromatin structure, we established cell lines stably expressing the TRH gene, a typical gene negatively regulated by thyroid hormone, together with the wild-type or mutant TR, and then performed ChIP analysis. We found that transcriptional repression by thyroid hormone of the TRH gene is associated with rapid local histone deacetylation. The dynamics were significantly impaired in the presence of the F455S mutant in vivo.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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). There was no family history of thyroid disease. She was admitted to the hospital to examine responses to thyroid hormone. After 7 d of treatment with 160 µg T3, a TRH test showed that the response of serum TSH was not completely depressed, indicating severe RTH (data not shown).
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). Both parents had a normal TRß sequence, indicating that the mutation in the patient was sporadic.
F455S TR Mutant Showed Normal T3 Binding, DNA Binding, and Homo- and Heterodimerization on DNA
The T3 binding affinity was determined for the F455S mutant and compared with that of the wild-type TR. As shown in Fig. 2A, the T3 binding affinity of F455S was 86.4% of that of the wild type. Homodimers and heterodimers with RXR of the wild type and F455S were observed in a similar manner in the EMSA study (Fig. 2B).
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In the first instance, we assayed the ability of mutant receptors to activate transcription of PAL-TK-Luc containing a palindromic TRE (thyroid hormone response element). The wild-type receptor activated PAL-Luc activity in a T3 dose-dependent manner. Whereas E457A showed almost a complete loss of ability to activate transcription, the stimulation by F455S was significantly impaired at 10 nM and 100 nM T3 but comparable with that of the wild type in the presence of 1 µM T3. The ranking in order of activation potency at 1 µM T3 was F455S>P214R>AHT>E457A (Fig. 3A).
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). As previously reported, the promoter region of the TRH gene used in this study contains a typical element negatively regulated by thyroid hormone (10, 11). Expression of the wild-type TR induced a ligand-independent activation that was approximately 2- to 3-fold the basal level (data not shown). Addition of T3 depressed the activity in a dose-dependent manner with maximal inhibition, 10% of that without T3, at a concentration of 1 µM. The strongest ligand-independent activation was observed, unexpectedly, when the F455S mutant was expressed, resulting in a 1.5-fold activation compared with the wild type. Furthermore, the inhibition by T3 was significantly impaired. E457A also exhibited strong ligand-independent activation and impairment of repression by T3, but the activation was significantly stronger with F455S than with E457A. In contrast, AHT and P214R showed almost no ligand-independent activation and no repression by T3.
Dominant-Negative Effect of F455S on Positively and Negatively Regulated Promoters
Because a dominant-negative effect is critical to the phenotype and autosomal dominant inheritance of RTH, we investigated the ability to inhibit wild-type receptor action in a dominant-negative manner. In these experiments, equal amounts of wild-type and mutant receptors were coexpressed, and reporter gene activities were assayed at various hormone concentrations. The mutant receptors exhibited a variable spectrum of dominant-negative properties when investigated using PAL-TK-Luc. The greatest dominant-negative effect was observed with E457A, which inhibited the wild-type receptor activation by approximately 80% at 100 nM T3. The F455S mutant also has a clear dominant effect, showing 40% of inhibition; the AHT mutant did not affect the activation by the wild-type TR, and P214R acted like a wild-type TR (Fig. 4A).
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NCoR and SRC-1 Binding Properties of Mutant Receptors
We next examined the binding of each mutant to a corepressor, NCoR, and a coactivator, SRC-1. The GST pull-down assay showed that F455S exhibited stronger interaction with NCoR than the wild type, whereas E457A showed a mildly attenuated association. Addition of 1 µM T3 failed to dissociate NCoR from F455S and led to about 70% dissociation from E457A. EMSA revealed that addition of 10 nM T3 led to a complete dissociation of NCoR from the wild-type and E457A mutant TRs, but almost no dissociation from the F455S mutant. There was apparent interaction of NCoR with the F455S mutant even at 100 nM T3. As previously reported, no significant interaction with NCoR was observed in the AHT and P214R mutants with either the GST pull-down assay or EMSA (Fig. 5, A and B).
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, A and B).
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). Northern blot analysis showed equal expression of the endogenous and exogenous wild-type or F455S TR mRNAs (Fig. 7B), reflecting the pathophysiology of the heterozygote for the F455S mutant. After this Northern blot analysis, we further confirmed by Western blot analysis that equal amounts of TR, including endogenous and exogenous TRs, were expressed in different cell lines (Fig. 7C). Furthermore, the TRH promoter stably expressed in the established cell lines was functionally suppressed by T3 (Fig. 7D), as observed in the transient transfection analysis shown in Fig. 3B.
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). Unexpectedly, acetylated histones H3 and H4 were detected in the TRH promoter without stimulation by T3. Treatment with T3 caused a significant loss of both acetylated histones H3 and H4 within 15 min. The acetylation level of H3 declined by about 30% within 30 min and recovered to up to 80% of the basal level 120 min after addition of T3. Histone H4 reached a minimum acetylation level within 15 min (
45% on average) and reacetylation began at 30 min. Histone 4 was completely reacetylated within 120 min. The deacetylation of histones at the TRH promoter suggested that deacetylase complexes should be recruited to this site. Therefore, we performed ChIP analysis with antibodies against HDACs 1, 2, and 3. The results demonstrated that no HDACs were coprecipitated with the TRH promoter in the absence of T3. However, addition of T3 induced a dramatic recruitment of HDACs 3 and 2, particularly HDAC3, on the TRH promoter within 15 min. HDAC3 gradually dissociated and disappeared within 60 min. In contrast, HDAC2 rapidly disappeared from the promoter within 30 min. Interestingly, the dynamics of histone H3 deacetylation and reacetylation correlated well with the recruitment and release of HDAC3, and histone H4 acetylation status seems to be related to the dynamics of HDAC2. These results suggest that nucleosomal changes mediated by transient histone deacetylation with specific histone deacetylases play an important role in mediating the transcriptional repression of the TRH gene by thyroid hormone. Furthermore, the recruitment of HDACs was specific, and HDAC1 was not detected in the absence or presence of T3.
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). Treatment of the cells with 10 nM TSA did not affect the basal level of the TRH gene promoter activity at all. However, this concentration of TSA completely abolished the repression of the gene induced by thyroid hormone. Furthermore, a higher concentration of TSA (100 nM) induced a nonspecific stimulation of the basal promoter activity probably due to the global inhibition of histone deacetylases. Even at this concentration, the repression of the gene by thyroid hormone was lost. These findings strongly suggested that histone deacetylation is not involved in ligand-independent stimulation by unliganded TR but essential for the TRH gene repression by thyroid hormone.
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As on the positively regulated gene, NCoR was also associated with the TRH promoter in the absence of T3. However, HDACs 1–3 were not detected, suggesting that NCoR was located in the complex on the TRH promoter without the association of HDACs. It is of interest to note that NCoR remained on the promoter even 15 min after addition of T3, when HDACs were recruited. NCoR then began to dissociate from the TRH promoter at 30 min in parallel with the release of HDAC2 and -3 and disappeared completely in 120 min.
In contrast, SRC-1 was not associated with the promoter in the absence of T3 but the recruitment of SRC-1 started 30 min after addition of T3. A significant amount of SRC-1 was detected within 90 min, and then it dissociated again within 120 min (Fig. 9B).
Hyperacetylation and Impaired Deacetylation of Histones at the TRH Gene in Cells Expressing the F455S Mutant TR
To analyze how the chromatin structure is affected on the TRH gene by the presence of the F455S mutant, we also established a cell line stably expressing the mutant. As expected from the strong ligand-independent activation of the TRH promoter by the F455S mutant, the degree of histone H3 acetylation at the TRH promoter was significantly higher than that in cells expressing wild-type TR (140% of the wild-type control) (Fig. 10A). The deacetylation of H3 induced by T3 was also diminished and delayed: even 60 min after addition of T3 the acetylation level reached only 80% of the basal level with wild-type TR. In contrast, although the acetylation status of histone H4 in the absence of T3 was similar to that with the wild-type TR, the deacetylation induced by addition of T3 was almost completely abolished (Fig. 10B). These results suggested that the hyperacetylation and diminished deacetylation of the histones on the TRH gene are responsible for the impairment of the negative regulation by T3 in the RTH patient with F455S.
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, C and D). Again, of note is the correlation of the dynamics between HDAC3 and the deacetylation of histone H3, and between HDAC2 and the deacetylation of histone H4.
We finally examined the kinetics of NCoR and SRC-1 on the TRH promoter. Reflecting the results obtained by EMSA and GST pull-down assays, in cells expressing F455S, the occupancy of the TRH promoter by NCoR in the absence of T3 was markedly increased (200% of the control), and the maximum dissociation from the gene was also impaired, reaching only the same level as the wild-type TR without T3. In contrast, the recruitment of SRC-1 was observed in a similar manner as in cells expressing the wild-type TR (Fig. 10, E and F).
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DISCUSSION |
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, computer analysis revealed that the residue of F455S examined in the present study is localized inside of helix 12 (16, 17). Furthermore, it has been suggested that the presence of helix 12 inhibits NCoR binding to TR, estrogen receptor, and RXR (18, 19, 20). On the basis of these observations, it is suggested that helix 12 of F455S cannot be in the position required for suppression of NCoR binding, resulting in the increased interaction with NCoR.
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and -ß gene promoters. Using ChIP analyses, we found that lysine residues of histone H3 and H4 at the TRH promoter were acetylated in the absence of T3. After addition of T3, histone tails were rapidly deacetylated within 30 min, and this was correlated with the simultaneous recruitment of HDACs, particularly HDACs 3 and 2. Thus, it is speculated that recruited HDACs deacetylate histone tails on the TRH gene promoter, resulting in repression of the gene. The precise mechanism by which a transient histone deacetylation upon T3 treatment led to continuous repression over a few days remains to be elucidated. It is possible that cyclic turnover of transcription complex may occur every several hours as recently reported for estrogen receptor (21), or other histone configuration such as methylation, phosphorylation, or chromatin remodeling may be involved in the late stage of the repression. However, our results showing that treatment of a HDAC inhibitor, TSA, completely abolished the repression of the TRH gene indicated that rapid deacetylation of the histone tails of the gene is critical for the transcriptional repression of the TRH gene by thyroid hormone.
One of the remaining questions is how HDACs are recruited to the TRH gene. One possibility is that HDACs are corecruited with NCoR because NCoR has been shown to bind HDAC or Sin3A/HDACs. However, to our surprise, NCoR did not bind HDAC 1–3 on the TRH promoter in the absence of T3. After addition of T3, NCoR remained on the gene for approximately 15 min before dissociating, and in this period HDACs were recruited to the gene. Therefore, T3 may be a trigger for recruitment of HDACs to NCoR on the TRH promoter. Alternatively, HDACs could be recruited by another complex containing specific transcriptional factors, although our attempts to identify the specific recruitment factor are still in progress. Indeed, this is the first demonstration of the presence of NCoR without binding HDACs on the TRH promoter in the absence of T3 in vivo.
After NCoR began to dissociate, HAT SRC-1 was recruited to the TRH promoter. The appearance of SRC-1 correlated well with the reacetylation of histones, suggesting that the reacetylation was induced, at least in part, by the recruitment of SRC-1. In addition, it is of interest that the recruited SRC-1 started to dissociate again after 90 min. Recent analyses on positively regulated genes (dio1 and SERCA gene) revealed the same dissociation, and acetylated H4 was observed even after the dissociation (8). On the basis of these findings and our results, SRC-1 is thought to be necessary for the initiation of histone acetylation but not for the maintenance of acetylated histones on the TRH promoter.
To explain the negative regulation of the gene expression by thyroid hormone, both DNA bindingdependent and -independent mechanisms have been proposed (22, 23, 24, 25). Recent analysis demonstrated that the negative thyroid hormone receptor response element (TRE) on the GH promoter bound to TR, and that T3 causes the release of TR as well as disappearance of acetylated histone from the promoter (26). They also demonstrated the importance of HDAC1 for this repression. Therefore, the mechanism of the negative regulation of the GH gene by thyroid hormone is completely different from that of the TRH promoter gene observed in this study, suggesting that there would be several distinct mechanisms involved in the negative regulation of each gene by thyroid hormone.
Previous studies revealed that NCoR acted as a coactivator on the negative TRE (22, 23, 27). Reflecting the nature of F455S, which strongly interacts with and is hard to dissociate from NCoR, our ChIP analyses demonstrated that the occupancy of the TRH promoter by NCoR was significantly increased in the presence of F455S. These observations suggested that the increased amount of NCoR may affect the histone hyperacetylation at the TRH promoter in the absence of T3, and that dissociation of NCoR may contribute to the transcriptional repression. Furthermore, these results suggested direct interaction of TR and NCoR in the complex on the TRH promoter in vivo. Taken together, these observations indicate that the abnormal histone acetylation status of the TRH promoter in the presence of F455S is responsible for the impairment of the negative regulation of TRH expression by thyroid hormone.
Another example of a disorder related to aberrant interaction with NCoR is acute promyelocytic leukemia (PML). In this case, key molecular events are caused by chromosomal rearrangements of the retinoic acid receptor (RAR), resulting in the fusion gene transcript PML-RAR (28). RAR in PML-RAR shows stronger interaction with NCoR, but the target genes of PML-RAR remain obscure. Therefore, our present study on RTH demonstrated the first direct involvement of the disturbance of the chromatin structure on the targeted gene with the human disorder in vivo. We propose a new category of diseases: RTH is a chromatin structure disease as well as a genetic disease.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Mammalian Cell Culture and Transfection
CV-1 cells and GH4C1 cells were grown in DMEM supplemented with 10% fetal bovine serum (FBS), as described previously (29). Cells were split into six-well plates at subconfluency 24 h before transfection. The transient transfection was performed using a calcium phosphate precipitation method, as described previously (29). The total amount of transfected plasmid was adjusted by adding an empty expression vector in all experiments. After transfection (16 h), the medium was changed to DMEM supplemented with 10% FBS and treated with AG1-X8 resin (Bio-Rad Laboratories, Inc., Hercules, CA) and activated charcoal (Sigma Chemical Co., St. Louis, MO) to remove thyroid hormones. Cells were further incubated in the absence or presence of T3 (Sigma).
For stable transfection, cells were grown in 100-mm diameter dishes and cotransfected with 6.7 µg of each linearized plasmid (TRH promoter (–790 +54) luciferase reporter, pKCR2-wild type TR, or pKCR2-F455S TR mutant and pKJ2-Neo) for 24 h using calcium-phosphate methods. The cells were cultured in complete medium for 48 h before selection using 0.7 mg/ml G-418 sulfate (Life Technologies, Inc., Gaithersburg, MD). In 20 clones tested for reporter activity, integration of the TR or TR F455S gene was examined by extracting genomic DNA and PCR. Southern blot analysis was performed to confirm the single integration of all constructs into the genomic DNA. Luciferase (1–259) and full-length TRß cDNA fragments were used for probes. Expression levels of each mRNA and protein were examined by Northern blot and Western blot analysis as previously described (29, 30). A cRNA fragment for TRß (499–835) was used for probes.
Plasmid Constructions
The mutant human TRß1 cDNAs (F455S, E457A, P214R, and AHT, which contains three amino acid substitutions in the hinge region) were prepared by PCR mutagenesis and verified by sequencing the DNA. Mutant and wild-type receptor cDNAs were subcloned into the vector pKCR2 for in vitro transcription/translation and for transient expression analysis. Firefly luciferase reporter plasmids (pA3Luc) carrying the palindromic (PAL-TK-Luc) or direct repeat-type (DR4) TRE were prepared as previously described (30). TRH-Luc contains 790 bp of the 5'-flanking sequence and 54 bp of exon 1 from the human TRH gene in pA3-Luc (TRH-Luc).
T3 Binding Experiments
Mutant or wild-type TR was transcribed and translated using a TNT-coupled reticulolysate system (Promega Corp., Madison, WI). T3-binding affinity was determined using a filter-binding assay as reported previously (31). Fitting with lines in Scatchard plots was done with Cricket Graph (Computer Associates Ltd., Islandia, NY).
EMSA
The EMSA was performed using radiolabeled TRE DR4 or TRE palindrome fragments as described previously (29). The consensus sequences used as TRE DR4 and palindrome were 5'-agcttcaggtcacaggaggtcagagag-3', and 5'-aagattaaggtcatgacctgaggaga-3', respectively. Double-stranded oligonucleotides were labeled with [32P]dCTP by a fill-in reaction using a Klenow fragment of DNA polymerase I. The binding reaction, gel electrophoreses, and autoradiographies were performed under conditions described previously (29).
GST Pull-Down Assay
[35S] methionine-labeled wild-type and mutant TRß were synthesized by in vitro transcription/translation from pKCR2-TR, F455S, E457A, R214R, and AHT using T7 RNA polymerase and the TNT-coupled reticulocyte lysate system (Promega Corp.). The synthesis of proteins of expected molecular weights was confirmed by SDS-PAGE). A cDNA fragment encoding the receptor interaction domain of NCoR and SRC-1 was amplified by PCR using pKCR2-NCoRI and pKCR2-SRC-1 as a template and subcloned in frame into pGEX4T1 to yield GST fusion proteins in Escherichia coli DH5. The GST fusion proteins were purified on glutathione-agarose beads (Sigma) and analyzed by SDS-PAGE. Interaction assays and autoradiographies were performed as described previously (30). Bound protein was quantified using a Molecular Imager FX (Bio-Rad).
Luciferase Assay
To determine the luciferase (Luc) activity, cell monolayers were rinsed twice with PBS, and then lysed with 300 µl of 25 mM glycylglycine (pH 7.8) containing 15 mM MgSO4, 4 mM EGTA, 1 mM dithiothreitol, and 1% (vol/vol) Triton X-100. Cells were scraped from the dishes and centrifuged at 12,000 x g for 5 min at 4 C. Assays for Luc activity were performed using 150 µl aliquots of cell lysate and 210 µl of 25 mM glycylglycine (pH 7.8) containing 15 mM MgSO4, 4 mM EGTA, 3.3 mM KPO4, 1 mM dithiothreitol, and 0.45 mM ATP. The reaction was initiated by addition of 200 µl of 0.2 mM D-luciferin, and light emission was measured for 10 sec using a luminometer. Luc activity was expressed as arbitrary light units per µg of cellular protein. All the transfection experiments were repeated at least twice with triplicate determinants.
Antibodies
Antibodies against TRß (no. 06-539), acetylated Histone H3 (no. 06-599) and H4 (no. 06-866) were obtained from Upstate Biotechnology, Inc. (Lake Placid, NY). Antibodies against HDAC1 (H-51), HDAC2 (H-54), HDAC3 (H-99), NCoR (C-20), and SRC-1(N-19) were obtained form Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).
ChIP Assay
ChIP analyses were performed according to the manufacturer’s instructions (Upstate Biotechnology) with some modifications. Cells (2 x107) were grown in DMEM supplemented with 10% FBS treated with AG1-X8 resin and activated charcoal for 24 h. After addition of T3, cells were washed with PBS and treated with the cross-linking reagent formaldehyde (final concentration, 1%) for 10 min at 37 C. They were then rinsed twice with cold PBS containing 0.5 mM phenylmethylsulfonyfluoride and 1 µg/ml of aprotinin. Cells were collected by centrifugation for 4 min at 4 C and resuspended in 150 µl of sodium dodecyl sulfate (SDS) lysis buffer (1% SDS/10 mM EDTA/50 mM Tris-HCl, pH 8.1) with proteinase inhibitors and incubated for 10 min on ice. Samples were sonicated on ice five times for 8 sec each (i.e. until the average length of the sheared genomic DNA was 0.2–1.0 kbp) and centrifuged for 10 min. One percent of the supernatant was used as input, and the remaining amount was subjected to the ChIP procedure. Next, 40 µl of salmon sperm DNA/Protein A Agarose-50% slurry were added to reduce the nonspecific background and incubated for 30 min at 4 C with agitation. The solution was then incubated with 1–3 µg of specific antibody or normal IgG and rotated at 4 C overnight. Immunoprecipitated chromatin complexes were isolated by adding 50 µl of salmon sperm DNA/Protein A Agarose-50% slurry and rotating the reactions for 1 h at 4 C. Immunoprecipitates were sequentially washed with low-salt immune complex wash buffer [20 mM Tris-HCl (pH 8.1)/2 mM EDTA/0.1% SDS/1% Triton X-100], followed by high-salt wash buffer [20 mM Tris-HCl (pH 8.1)/2 mM EDTA/0.1% SDS/1% Triton X-100/500 mM NaCl], LiCl Immune complex wash buffer (10 mM Tris-HCl (pH 8.1)/0.25 M LiCl/1% Nonidet P-40/1% deoxycholate/1 mM EDTA) and twice in 1x Tris-EDTA, pH 8.0. To elute the immunoprecipitated chromatin complexes from the resin, 100 µl of elution buffer (1% SDS/0.1 M NaHCO3) were added to the beads, and the tubes were vortexed and incubated at room temperature for 15 min with rotation. The supernatant was then collected, and the elution was repeated with a fresh 100 µl of elution buffer. After combining the eluants in one tube, the protein-DNA cross-linking was reversed by adding 5 M NaCl to a final concentration of 200 mM and heating at 65 C for 4 h. Inputs were diluted to 200 µl and subjected to the same procedure. Each sample was added to 8 µl of 1 M Tris-Cl (pH 6.5), 4 µl of 0.5 M EDTA, and 5 µg of proteinase K (Life Technologies) and subsequently incubated at 45 C for 1 h. Samples were then extracted with phenol/chloroform/isoamylalcohol (25:24:1), and the DNA was precipitated with ethanol and subsequently resuspended in 50 µl H2O. PCR was performed with 5 µl of immunoprecipitate or input (see above), 0.5 µM of each primer, 1.5 mM MgCl2, 0.2 mM deoxynucleotide triphosphate mixture, 1x thermophilic buffer, and 2.5 U of AmpliTaq DNA polymerase (Applied Biosystems) in a total volume of 50 µl. The primers for the human TRH promoter were: forward, 5'-ctgagcgctgcagactcctgacct-3'; and reverse, 5'-tgttcacctcgatatgtgcatctgt-3'. Initially, PCR was performed with a serial dilution of input DNA to determine the linear range of the amplification for each gene. PCR conditions were 25 cycles of 45 sec at 94 C, 45 sec at 60 C, and 1 min at 72 C. All PCR signals were visualized by Southern blot analysis with the fragment of the TRH-luciferase cDNA (pA3TRH-Luc) as a probe and quantified with the Molecular Imager FX (Bio-Rad). The probe was labeled by the random-priming method with Ready-To-Go DNA Labeling Kit (Pharmacia Biotech, Piscataway, NJ) and [32P]dCTP. The values were corrected using the input values, and those obtained with IgG were used as background values.
Statistical Analysis
Statistical analysis was performed using ANOVA and Student’s t test or Duncan’s multiple range test. The level of significance was set at P < 0.05.
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FOOTNOTES |
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Abbreviations: ChIP, Chromatin immunoprecipitation; FBS, fetal bovine serum; GST, glutathione-S-transferase; HAT, histone acetylytransferase; HDAC, histone deacetylase; NCoR, nuclear receptor corepressor; PML, promyelocytic leukemia; RAR, retinoic acid receptor; RTH, resistance to thyroid hormone; RXR, retinoid X receptor; SDS, sodium dodecyl sulfate; SMRT, silencing mediator of retinoic and thyroid hormone receptor; SRC, steroid receptor coactivator; TR, thyroid hormone receptor; TRE, thyroid hormone receptor response element; TSA, trichostatin A.
Received for publication February 16, 2004. Accepted for publication April 14, 2004.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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ligand-binding domain bound to all-trans retinoic acid. Nature 378:681–689[CrossRef][Medline]
gene by the thyroid hormone receptor. J Biol Chem 274:22345–22353 from a negative hormone response element. J Biol Chem 277:49517–49522NURSA Molecule Pages Link:
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