Thyroid hormone receptors (TRs) belong to a superfamily of nuclear hormone receptors including the steroid hormone, vitamin D, and retinoid receptors. They can bind as heterodimers with retinoid X receptors to hormone response elements (HREs) located in the promoter region of target genes. For positively-regulated genes, thyroid hormone (T3) binds to the receptor and increases transcription. Interestingly, in the absence of T3, TRs can still bind to HREs and repress transcription below basal level. There is emerging evidence on how the receptor mediates these effects. Recently, several groups have described co-activators and co-repressors that interact with the receptor in a ligand-dependent manner. Additionally, there are other proteins that interact with the liganded receptor/co-activator complex such as CBP and p/CAF which have intrinsic histone acetyltransferase activity. Similarly, co-repressors have been shown to complex with histone deactylases. We have several ongoing projects to examine TR action at the molecular level: 1) We have created several transgenic mouse lines in which a dominant negative form of the co-repressor, N-CoRi, has been overexpressed in liver. We observed that a number of target genes have increased basal transcription in hypothyroid transgenic mice suggesting that co-repressors can play an important modulatory role for these genes in the hypothyroid states. We also used cDNA microarray to identify genes regulated by NCoR which are involved in cell proliferation. This in vivo model of corepressor action was published in Journal of Biological Chemistry as a cover issue. We currently plan to use these mice to study the role of co-repressors in the action of steroid hormone antagonists. 2) We used green fluorescent fusion proteins (GFPs) of mutant thyroid hormone receptors to study the effect of ligand-binding, co-repressor binding, DNA-binding, and heterodimerization on nuclear/cytoplasmic distribution in living cells by confocal microscopy. We found evidence for active shuttling of TRs between the nuclear/cytoplasmic compartments in the absence and presence of T3. Additionally, interaction with RXR and N-CoR were important for nuclear retention of unliganded TR. Ligand-binding promotes nuclear reorganization of TRs with the formation of a speckling pattern. This work was published in the Journal of Biological Chemistry. We also have studied estrogen, retinoic acid, and thyroid hormone receptor shuttling and intranuclear diffusion by heterokaryon fusion experiments and fluorescence resonance after recovery (FRAP). We observed that hormone and antagonist binding, as well co-activator binding, decreased intranuclear diffusion suggesting that ligand and protein-protein interactions can modulate nuclear receptor movement. Dr. Maruvada received a New Investigator Award for her abstract describing this work at this year?s Endocrine Society Meeting. We currently are preparing a manuscript for submission. 3) We have used our expertise in GFP technology to study the localization intracellular processing of wild-type and mutant pendrins. This protein is a membrane-bound iodide transporter in which mutations have been associated with Pendred syndrome, a leading cause of congenital deafness and goiter. We found that natural pendrin mutants asosciated with the majority of cases with this syndrome, were trapped in the endoplasmic reticulum and could not target to the plasma membrane. Thus, these studies strongly suggest that Pendred syndrome is an endoplasmic storage disease. Dr. Pkielny- Rotman was awarded an Abbot Clinical Fellowship Award from the Endocrine Society for her work, and based on her performance at an oral presentation competition, I received the Abbot Thyroid Mentor Award with a $30,000 prize. We have submitted this work for publication. 4) We have identified a novel alternatively-spliced TR and a point mutant TR in TSHoma pituitary samples, and characterized their defective abilities to negatively-regulate the transcription of TSH subunits. This alternative-splicing of TR represents a novel post-transcriptional mechanism for hormone resistance and was recently reported in Molecular Endocrinology. The somatic mutation is the first example of a TR mutation described in a TSHoma and will be published in the Journal of Clinical Endocrinology and Metabolism. Dr. Ando received the Abbot Clinical Fellowship Award from the Endocrine Society for his abstract describing this work. We also have described an unusual patient with resistance to thyroid hormone (RTH) who had a novel frameshift mutation in TRb who had extreme resistance. This case will also be reported in the Journal of Clinical Endocirnology and Metabolism. We identified a patient with classical features of RTH but did not have TRb or TRa mutations. It is possible that the resistance may be due to mtuations in TR cofactors. This case will be published in Thyroid. 5) We previously have examined the effects of T3 and T4 on in vivo hepatic gene regulation in mice using cDNA microchip array analyses. In collaboration with Dr. Paul Meltzer, NHGRI, we are using similar methods to examine TR knockout mice in which individual or both TR isoforms have been selectively ablated. We have performed studies with these genetically-altered mice treated with no T3, T3, propylthiouracil, and propylthiouracil plus T3. Thus far, we have performed almost 50 microarrays, representing almost 200,000 data points. We are performing clustering analyses to characterize overall gene expression patterns as well as to identify interesting and novel target genes. These studies represent the largest and most comprehensive microarry studies of hormone-regulated gene expression to date in genetically-altered mice. We plan to submit a manuscript in the near future. 6) We have used the yeast two-hybrid system to identify novel co-factors that interact with TRs, and currently are characterizing them. 7) We currently are developing chromatin immunoprecipitation (CHIP) assays study histone acetylation, and co-factor recruitment in positively- and negatively-regulated targed genes, some of which have been identified by our microarray studies.
