Abstract 2107 Examining Factors Affecting Nuclear Retention of the Thyroid Hormone Receptor

The thyroid hormone receptors, TRα1, TRβ1, and TRβ2, are nuclear receptors that regulate many genes involved in development, growth, and homeostasis in response to thyroid hormone (T3). TRs undergo nucleocytoplasmic shuttling and mutations in TR can lead to altered intracellular localization and diseases such as Resistance to Thyroid Hormone (RTH). RTH is caused by mutations in TR leading to a decreased response to T3. Previous studies in our lab have established, using acetylation and non-acetylation mimics, that the acetylation of TR at specific sites leads to a more cytosolic localization and non-acetylation leads to a more nuclear localization, indicating that the deacetylation of TR plays an important role in modulating nucleocytoplasmic shuttling. To determine if histone deacetylases (HDAC) 1 and 3 play a role in the deacetylation of TR, we transfected HeLa cells with GFP-tagged TRα1, TRβ1, or TRβ2 and added an HDAC 1/3 inhibitor at 1μM and 10μM concentrations. Analysis of average relative nucleocytoplasmic (N/C) ratios revealed no significant change in localization of any TR variant when compared to the solvent control (TRα1: 1μM, p = 0.17; 10μM, p = 0.15; TRβ1: 1μM, p = 0.47; 10μM, p = 0.26; TRβ2: 1μM, p = 0.44; 10μM, p = 0.07). These data suggest that HDAC 1 and HDAC 3 are not directly responsible for the localization patterns or deacetylation of TR, so other members of the deacetylase family are now under investigation. While in the nucleus, TR binds to nuclear receptor corepressor 1 (NCoR1), silencing gene expression in the absence of T3. In our investigation of RTHα mutations and NCoR1 binding, we hypothesized that nuclear retention of RTHα mutants would be increased in the presence of overexpressed NCoR1, since some RTHα mutants have been reported to have increased affinity for NCoR1. We selected RTHα mutants with substitution of arginine for either a cysteine or histidine at position 384 (R384C or R384H) for analysis, because we had previously shown that these TR variants have a more cytosolic population than wild-type TRα1. We transfected HeLa cells with mCherry-tagged wild type TRα1, R384C, or R384H, as well as GFP-tagged wild type NCoR1 or an NCoR1 mutant that does not bind TR, as a control. N/C ratios for wild-type TRα1 and the RTHα mutants were not significantly different in the presence of overexpressed NCoR1 compared with the non-binding NCoR1 mutant (TRα1: p = 0.61, R384C: p = 0.45, R384H: p = 0.06), suggesting that interaction with NCoR1 is not a key factor affecting localization patterns of these RTHα mutants. Overall, these findings provide further insight into TR's interactions with other proteins and the resulting impacts on nuclear retention. This research was made possible through the generous support of the Charles Center at William & Mary and National Institutes of Health Research Grant # 2 R15 DK058028-06.