of Work: Thyroid hormone receptors (TRs) belong to a superfamily of nuclear hormone receptors that include the steroid hormone, vitamin D, and retinoid receptors TRs bind as heterodimers with retinoid X receptors to thyroid hormone response elements (TREs) located in the promoter region of target genes. In positively-regulated target genes, thyroid hormone (TH; T4 and T3) binds to TRs and increases transcription. In contrast, in the absence of T3, TRs repress transcription below basal level. There is emerging evidence on how the receptor mediates these effects. Recently, co-activators and co-repressors have been described that interact with the receptor in a ligand-dependent manner. In the presence of T3, TR interacts with the steroid receptor coactivator (SRC)/CBP-p300/p/CAF complex which has histone acetyltransferase activity. Other complexes such as the vitamin D receptor interacting protein/TR associated protein (DRIP/TRAP) complexes also can bind to liganded TR, and may stimulate transcription via shared components with RNA polymerase II. Additionally, a number of other co-activators also have been described but their precise interplay with these other co-activators remains to be elucidated. In the absence of T3, TR binds to co-repressors such as nuclear receptor co-repressor (NCoR) and silencing mediator for retinoic acid receptor and TR (SMRT). These, in turn, can recruit other repressors such as sin3 and histone deacetylases which can maintain local chromatin structure of target genes in a repressed state. We have several ongoing projects to examine TR action at the molecular level: 1) Microarray Studies of TR Action: We previously used cDNA microarray technology to identify and characterize genes that are regulated by (TH) in liver. In collaboration with Dr. Jacques Samarut (l'Ecole Normale Superieure, Lyons, France) and Dr. Samuel Refetoff (Univ. Chicago, Chicago, IL), we have performed cDNA microarrays to study hepatic target gene regulation in knockout (KO) mice that lack either one TR isoform (TRa or TRb), or both TR isoforms under hypo-, eu-, and hyperthyroid states. Expression patterns of target genes were determined by clustering analyses from 48 microarrays totaling over 190,000 data points. Positively-regulated genes fit into three major expression patterns with target genes differentially activated in the hypothyroid, euthyroid, and hyperthyroid states. Additionally, only a subpopulation of target genes exhibited a decrease in transcription in the hypothyroid state, suggesting that not all target genes exhibited basal repression of transcription in the hypothyroid state. Interestingly, TRaKO and TRbKO mice followed a similar gene expression pattern as WT mice, demonstrating these isoforms co-regulated most target genes in the liver. Differences between the gene expression patterns of the TRa/TRb_double KO and hypothyroid WT mice, suggest that absence of receptor can have different transcriptional effects on target genes than absence of hormone. This large-scale study of hormonal regulation in genetically-engineered mice by cDNA microarray, demonstrates the variety and extent of gene profile patterns by TRs. These findings were reported at an oral session at the most recent Endocrine Society Meeting in San Francisco, and a manuscript is in preparation. 2) Green Fluorescent Protein (GFP) Studies of TR Action: We previously used GFP-TRb and GFP-TRb mutants to show that that TR shuttles between the nucleus and cytoplasm, and is retained in the nucleus by protein-protein interactions with NCoR and RXR, rather than DNA-binding. Additionally, T3-binding causes a nuclear reorganization of GFP-TRb [Baumann, 2001 #1068]. We have extended these studies to estrogenic (ER) and retinoic acid (RAR) and have observed similar nuclear/cytoplasmic shuttling and intranuclear reorganization upon ligand-binding. Moreover, using fluorescent resonance after photobleaching (FRAP), we observed that estrogen and tamoxifen binding to ER deceases the intranuclear diffusion rate of ER, and may be a mechanism for agonist and antagonist activity. A manuscript describing this work is in final revision at JBC. We also have studied GFP-TRb and WT-TRb distribution during the cell cycle and have shown that both are most highly expressed in the nucleus at G2/M. Using cell cycle inhibitors, we have observed that transcriptional activity is highest when cells are arrested at this stage. These findings suggest that cell cycle stage may be an important determinant of transcriptional activity by TRs. These latter findings were reported at an oral session at the most recent Endocrine Society Meeting in San Francisco. 3) P62, a subunit of TFIIH, is a co-activator for nuclear hormone receptors: We used a reverse yeast two-hybrid system to identify, p62, a component of TFIIH, as a protein that interacts with TRb in a ligand-dependent manner. TFIIH is a multi-subunit complex required for both transcription and DNA repair. Recent studies have shown that components of TFIIH can interact with estrogen receptors (ERs) and regulate their transcriptional activation. We used the yeast-two hybrid system to investigate interactions between the thyroid hormone receptor (TR) and individual TFIIH subunits and found that TR_ interacted only with p62 in a ligand-dependent-manner. ER, vitamin D receptor, and retinoid X acid receptor also interacted with p62 in a similar manner. Glutathione-S-transferase (GST) pull-down studies confirmed that TR_ interacted with p62 in a T3-dependent-manner. Co-transfection studies showed that p62 could enhance T3-mediated transcription, which could be further enhanced when co-transfected with p44, another component of TFIIH. Chromatin immunoprecipitation (CHIP) assays showed that p62 and p44 were rapidly recruited to TREs of several target genes within the same time frame as p160 co-activators and histone acetylation near the TRE. These findings show that p62 acts as a novel coactivator in TR-mediated transactivation, and suggest that TFIIH may also serve as a transcription complex for nuclear hormone receptors. These findings were reported at an oral session at the most recent Endocrine Society Meeting in San Francisco, and the most recent CHIP will be presented as an oral hot topic at the upcoming American Thyroid Association Meeting in Los Angeles.
