BACKGROUND: Type 1 iodothyronine deiodinase (D1), the product of the hdio1 gene, is involved in thyroid hormone activation by the deiodination of thyroxine (T4) to form 3, 5, 3'-triiodothyronine (T3). Recent studies have identified two thyroid hormone response elements (TREs) in the 5 flanking region of the hdio1 gene. TRE1, proximal to TRE in the hdio1 gene, consists of a direct repeat of thyroid hormone receptor (TR) binding octamers with 10 bp separating the two TR binding sites. The upstream TRE, TRE2, is a classical direct repeat of retinoid X receptor (RXR)/TR binding half-sites with a 4-bp separation. There are few studies clarifying the TR dynamics in the TRE of a specific gene with or without the exposure of activated thyroid hormone. We evaluated TR binding patterns in the proximal and distal TREs of the hdio1 gene before and after T3 stimulation. METHODS: We employed chromatin immunoprecipitation (ChIP) technique to investigate the TR-TRE interaction before and after T3 stimulation in human hepatocellular carcinoma HepG2 cell line.Following cross-linking and sonication of the cells, immunoprecipitation was performed overnight at 4degrees C with TR 1, TR 1 and TR 2 antibodies. We analyzed the binding patterns and amounts of TR 1, TR 1 and TR 2 to TRE1 and TRE2 before and after 12 hours stimulation with 100 nM T3 by using conventional and quantitative real-time polymerase chain reactions (RQ-PCR). Reverse transcriptional PCR (RT-PCR) and Western blot with TR 1, TR 1 and TR 2 antibodies were performed to measure the levels of hdio1 mRNA and TR 1, TR 1 and TR 2 proteins before and after 12 hours exposure to 100 nM T3. RESULTS: In TRE1, TR 1 binding was significantly decreased after 12 hours stimulation with 100nM T3 (3.74-->1.97, delta=-47.3%, p<0.05), but TR 1 and TR 2 bindings were not detected by conventional PCR and RQ-PCR. Although all TR isoforms were bound to TRE2, the binding patterns were quite different. While TRalpha1 and TR 1 bindings to TRE2 after 12 hours stimulation with 100 nM T3 were significantly decreased (10.41-->3.01, delta=-71.1%, p<0.05; 12.56 --> 2.93, delta=-76.7%, p<0.05, respectively), TR 2 binding was increased but not significantly (9.17 --> 9.84, delta=+7.3%). Total TR bindings in TRE2 were significantly decreased after 12 hours stimulation with 100 nM T3 (32.14 --> 15.78, delta=-50.9%, p<0.05). The TR bindings to TRE1 and TRE2 were not significantly different by the amounts of TR antibodies used during ChIP assays. The levels of hdio1 mRNA were significantly increased, 2.03 times, after 12 hours exposure to 100nM T3 (p<0.001). Western blot showed no significant change of the level of each TR isoform protein before and after 12 hours exposure to 100 nM T3. CONCLUSION: Our results demonstrate the dynamics of TR 1 at proximal TRE (TRE1) and the switching phenomenon of TR isoforms at distal TRE (TRE2) of the hdio1 gene after T3 stimulation. Further investigation, however, is needed to clarify the mechanisms of these observations.
Antibodies ; Binding Sites ; Blotting, Western ; Carcinoma, Hepatocellular ; Chromatin Immunoprecipitation ; Hep G2 Cells ; Humans* ; Immunoprecipitation ; Iodide Peroxidase* ; Polymerase Chain Reaction ; Protein Isoforms ; Receptors, Thyroid Hormone* ; Repetitive Sequences, Nucleic Acid ; Response Elements* ; Retinoid X Receptors ; RNA, Messenger ; Sonication ; Thyroid Gland* ; Thyroxine

BACKGROUND: The regulation of gene transcription can be controlled by both positive (enhancer) and negative (silencer) regulatory sequences. Several enhancer and silencer elements have been described in the 5' region of the chicken lysozyme gene. The silencer located at -2.4 kb upstream of the chicken lysozyme gene is composed of two separate modules (F1 and F2) that can function as silencers by themselves, but also show synergistic repression after multimerization. The F1 module is bound by a protein termed NeP1 and F2 module, a F2 thyroid hormone response element (F2-TRE), and can be bound by the thyroid hormone receptor (TR). F2-TRE has an inverted palindromic structure, with high affinity to TR. Although many current reported results have tried to explain the regulatory mechanism of chicken lysozyme gene expression due to the thyroid hormone, there have been few studies that clarify the TR dynamics in the F2-TRE of the chicken lysozyme gene, either with or without exposure of the thyroid hormone. Here, the changes in the TR binding patterns in the F2-TRE of the chicken lysozyme gene are described, both before and after T3 stimulation over time. METHODS: Using the stably transfected rat pituitary somatotroph tumor cell line, GC8 cells, with the F2-TRE inserted 5' to the thymidine kinase (TK) promoter, together with a mouse TRalpha- expressing plasmid, a chromatin immunoprecipitation (ChIP) technique was employed to reveal the TR-TRE interaction before and after T3 stimulation. Following the cross-linking and sonication of the cells, the immunoprecipitation was performed overnight, at 4 degrees C, with TRalpha1, TRbeta1 and TRbeta2 antibodies, respectively. The binding patterns and amounts of TRalpha1, TRbeta1 and TRbeta2 to the F2-TRE, before and after 12 hours of 100 nM T3 stimulation, were analyzed using conventional and quantitative real-time polymerase chain reactions (RQ-PCR). The ChIP technique was used to give a basal value for 20 minutes and 1, 2, 4, 6, 8 and 12 hours after the 100 nM T3 stimulation, and RQ-PCR was then performed. Western blot with TRalpha1, TRbeta1 and TRbeta2 antibodies were also performed. RESULTS: After 12 hours of 100 nM T3 stimulation of the GC8 cells, the TRalpha1 and TRbeta2 binding to the F2-TRE increased, but the TR 1 binding to the F2-TRE decreased, by conventional PCR. Although all the TR isoforms were bound to the F2-TRE by RQ-PCR, the TR 1 binding to the F2-TRE, after 12 hours of 100 nM T3 stimulation, was significantly increased (1.01-->2.73, delta=+170.3%, p<0.05), but the change in the amount of TRbeta2 binding was not significant (2.53-->2.98, delta=+17.8%). The TRbeta1 binding was significantly decreased compared with that of the basal level (4.59-->2.06, delta=-55.1%, p<0.05). The total TR bindings to the F2-TRE had a tendency to decrease after 12 hours of 100 nM T3 stimulation (8.13-->7.77, delta=-4.4%). The binding patterns and amounts of TRalpha1, TRbeta1 and TRbeta2, both before and after the 100 nM T3 stimulation, were also identified over time. While the TRbeta1 bindings to the F2-TRE after 1 hour of 100 nM T3 stimulation were acutely reduced, those of the TRalpha1 at 20 minutes and 6 hours were increased. The TRbeta2 bindings showed a maximal increase at 20 minutes. The directions of the TR binding patterns, between the before and after 2 hours of 100 nM T3 stimulation, were identical to those for between 4 and 6 hours of T3 stimulation. There was no significant difference in the TR bindings to the F2-TRE in relation to the amounts (1.5 vs. 4.5 microliter) of TR antibodies used during the ChIP assays. The Western blots showed no significant change of the levels of each TR isoform proteins, either before or after 12 hours of exposure to 100 nM T3. CONCLUSION: These results show the dynamic binding patterns of the TR isoforms to the F2-TRE of the chicken lysozyme gene, both before and after T3 stimulation, over time. Further investigation, however, will be needed to clarify the mechanisms of our observations. The ChIP technique may then be used to reveal the dynamic models of the cofactors, as well as TR isoforms, in the TR-regulated transcription machinery.
Animals ; Antibodies ; Blotting, Western ; Cell Line, Tumor ; Chickens* ; Chromatin Immunoprecipitation ; Gene Expression ; Immunoprecipitation ; Mice ; Muramidase* ; Plasmids ; Polymerase Chain Reaction ; Protein Isoforms ; Rats ; Receptors, Thyroid Hormone* ; Repression, Psychology ; Response Elements* ; Silencer Elements, Transcriptional ; Somatotrophs ; Sonication ; Thymidine Kinase ; Thyroid Gland*