J Korean Soc Endocrinol.  2003 Jun;18(3):283-295.

Thyroid Hormone Receptor Dynamics in Thyroid Hormone Response Elements of the Human Type 1 Iodothyronine Deiodinase Gene

• Lee SJ
• Park CY
• Jeong IK
• Hong EG
• Choi CS
• Kim HK
• Kim DM
• Yoo JM
• Ihm SH
• Choi MG
• Yoo HJ
• Park SW
• Larsen PR

Abstract

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.

MeSH Terms

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
Antibodies
Iodide Peroxidase
Protein Isoforms
RNA, Messenger
Receptors, Thyroid Hormone
Retinoid X Receptors
Thyroxine