Mutations and Modifications: The Cellular Fate of Thyroid Hormone Receptors

Thyroid hormone receptors (TRs), members of the nuclear receptor super family, are transcription factors responsible for binding thyroid hormone (T3) and activating or repressing the expression of target genes to regulate metabolism, cell growth, and energy expenditure. Intracellular dynamics of TR, particularly its compartmentalization and mobility within the cell, are known to regulate its transcriptional role. Three TR subtypes, TRa1, TRb1, and TRb2, are abundant differentially in human tissues, with TRa1 observed predominantly in cells of the central nervous system, intestines, bones, and cardiac tissue. Recent characterization of TRa1 involvement in Resistance to Thyroid Hormone Syndrome a (RTHa), a rare genetic disorder, has initiated research efforts to elucidate the underlying mechanism of pathogenesis. Several mutations have been identified in RTHa patients with varying degrees of symptom severity. Among others, missense mutations at alanine 263 have been observed with considerable frequency. A263V and A263S substitutions were identified in several families with the condition, with the valine mutation displaying increased severity in skeletomuscular and neuropsychological development delays. Certain RTHa mutants were previously demonstrated to have increased affinity for Nuclear Corepressor 1 (NCoR1), a coregulator of TRa1, and are proposed to form more stable repressor complexes compared to wild type TRa1. To determine whether NCoR1 promotes nuclear retention of RTHa mutants, in the first portion of this thesis, we cotransfected HeLa cells with expression plasmids for mCherry-TRa1 wild-type or RTHa mutants and GFP-NCoR1. Using fluorescence microscopy, we compared Nuclear-to- Cytoplasmic (N/C) localization ratios of wild-type TRa1 to the A263V and A263S mutants in the presence of NCoR1 and its non-TR-binding counterpart NCoRΔID, as a control. We observed a significant increase in the nuclear retention of A263V and significant decrease for A263S among other mutants with the overexpression of NCoR1 relative to wild-type TRa1. In NCoR1-knockout cells, nuclear retention of A263S and A263V was significantly decreased compared to control (wild-type) cells. Luciferase reporter gene transcription mediated by TRα1 was significantly repressed by NCoR1 overexpression. Most RTHα mutants showed minimal induction regardless of NCoR1 levels, but T3-mediated transcriptional activity was decreased for R384C and F397fs406X when NCoR1 was overexpressed. Our results suggest that the observed deviations in the localization of RTHa mutants relative to wild-type TRa1 may, in part, be caused by changes in the stability of the repressor complex, providing further insight into RTHa pathogenesis on a molecular level. The second portion of this thesis focuses on the post-translational modifications that regulate TR activity. Previous work in the lab using fluorescent tagged acetylation (KàQ) and nonacetylation (KàR) mimic constructs of key lysine residues within nuclear localization signal 1of TRa1 (K130/134/136) and TRb1 (K184/188/190) had shown that acetylation mimics have significantly lowered N/C localization ratios, and nonacetylation mimics had reduced intranuclear mobility correlated with increased nuclear retention. Pharmacological inhibition of sirtuin 1 (SIRT1) by EX527 and HDAC6 by Tubastatin-A (Tub-A) among a panel of lysine deacetylases (KDACs) from all classes showed no significant changes in localization of TRa1, TRb1, and TRb2 due to deacetylase inhibition. Coimmunoprecipitation experiments with anti- HDAC3, anti-HDAC6, and anti-SIRT1 antibodies showed no detectable interaction between TRa1 and HDAC3 or HDAC6, or between TRb1 and SIRT1. The culmination of these efforts did not identify a key deacetylase acting on TR as a substrate. Pilot studies in the lab revealed that acetylated TRa1 had increased susceptibility to proteases in the nucleus. While the ubiquitination sites of TR are unknown, we hypothesized that acetylation state may regulate ubiquitin-mediated degradation of TRa1 through cross-talk. Immunoprecipitation with ubiquitin affinity beads of HeLa cell lysates expressing HA-tagged wild-type TRa1, nonacetylation mimic, and acetylation mimic and subsequent anti-HA chemiluminescent western blot analysis showed that ubiquitination does not directly compete with acetylation at residues K130/134/136, as consistent monoubiquitination was observed in all three treatment groups. The nonacetylation mimic had significantly reduced polyubiquitination compared to the acetylation mimic, whereas the acetylation mimic had significantly elevated polyubiquitination in the first 3 out of 4 discreet polyubiquitinated states observed compared to wild-type. Results suggest that wild-type TRa1, which exists as a nonhomogeneous combination of the acetylated and nonacetylated populations within the cell, mostly remains in the nonacetylated state, and the acetylation of residues K130/134/136 prompts subsequent polyubiquitination which, consistent with pilot data, may target the receptor for ubiquitin mediated proteasomal degradation at the end of its life cycle.