Characterizing Thyroid Hormone Mediated Action on Gene Expression in Mice: Mechanistic Insight into Thyroid Hormone Response Elements, Thyroid Hormone Receptor-binding Sites, and MicroRNAs

Thyroid hormone (TH) exerts its effects by binding to the TH receptor (TR), which binds to TH response elements (TREs) to regulate target gene expression. Disruption of TH action can have detrimental health effects. The precise molecular mechanisms involved in TH mediated gene expression remain unclear. The overall objectives of this thesis were to: i) characterize global gene and microRNA (miRNA) expression in early response to TH perturbation in mouse liver; ii) identify TREs and TR-binding sites found throughout the mouse genome; and iii) compare TRE half-site organizations and their ability to drive gene expression. Transcriptional profiling of mRNA liver samples from TH disrupted mice enabled the identification of genes that were under direct TH-regulation. TREs in the promoter region of Tor1a, Hectd3, Slc25a45 and 2310003H01Rik were validated in vitro, adding four genes to the battery of only 13 known TRE-containing mouse genes. Hepatic miRNAs were also found to be significantly altered following perturbations in TH levels. In vitro analyses confirmed TH regulation of miR-206. Moreover, Mup1 and Gpd2 were confirmed to be targeted by miR-206 in response to TH, demonstrating that miRNAs can act as master regulators of the TH response pathway. ChIP-chip analysis identified TR-DNA interactions in juvenile mouse liver revealing only a few TR-binding sites consistent between all analyzed samples, suggesting that relatively few genes are under direct TH/TR control. Reporter assays confirmed the presence of TREs in the promoter regions of Ddx54 and Thrsp, thus validating two additional functional mouse TREs. Finally, we investigated the relative ability of liganded homodimers of TR and retinoid X receptor (RXR), and the heterodimer TR/RXR, to regulate gene expression for three TRE half-site organizations. We found that there were fundamental differences between TRE configurations that affect nuclear receptor interactions with the response element and the ability to specifically bind their ligands. These studies provide mechanistic insight into TREs, TR-binding sites and TH action. Collectively this thesis increases our understanding of how TH operates to control genome function and provides a basis to develop appropriate testing strategies for environmental chemicals that may disrupt TH-associated genes expression by interaction with TR.