Abstract 1969 Investigating Protein Interactions Modulating Thyroid Hormone Receptor Localization

Thyroid hormone is an important component of the endocrine system that regulates growth and metabolism. The hormone circulates in the bloodstream and enters target cells, where it can then enter the nucleus and bind to tissue-specific thyroid hormone receptors (TRs) to facilitate transcription of thyroid hormone-responsive genes. Although primarily localized to the nucleus, TR is known to rapidly shuttle between the nucleus and cytoplasm. Prior research in our lab has demonstrated a more cytoplasmic localization of an acetylation mimic of TR compared to a non-acetylation mimic and wild-type TR. This implies that post-translational modification of TRs via lysine acetylation may be an additional mechanism of regulating thyroid hormone signaling. To investigate whether the lysine deacetylase HDAC2 might play a role in TR deacetylation and localization, we transfected HeLa cells with expression plasmids for GFP-tagged TR subtypes, TRα1, β1, and β2. Cells were incubated with either Santacruzamate A-an HDAC2 inhibitor-or a DMSO control post-transfection. After fluorescence imaging and nucleocytoplasmic scoring, cellular localization of TR, measured by N/C ratio, was only significantly different for inhibitor-treated TRβ1 relative to the control (TRα1, p = 0.61; TRβ1, p = 0.0014; TRβ2, p = 0.81). However, the observed shift was towards the nucleus, suggesting an indirect effect due to increased histone acetylation and chromatin accessibility to TR, which lead us to conclude that HDAC2 is likely not the primary regulator of TR deacetylation. Resistance to Thyroid Hormone Syndrome (RTH) is a known dysfunction of the thyroid hormone signaling pathway characterized by tissue-specific hypothyroidism. Patients with severe RTH, characterized by shortened stature, mental defects, and bradycardia, have mutations in TRα1, which is specific to skeletal, heart, and brain tissues. Two identified mutations, C392X and E403X, result in premature truncation of TRα1's ligand-binding domain. We hypothesized that these mutations might increase the affinity of mutant TRα1 for nuclear corepressor NCoR1 and enhance nuclear retention. Using cotransfection of expression plasmids for mCherry-tagged TRα1 variants with GFP-NCoR1, or an NCoR1 variant that does not bind TR as a control, we found that the nucleocytoplasmic localization of the mutants was not significantly different in the presence of overexpressed NCoR1 relative to the control, possibly because both mutants already show a primarily nuclear localization, so any enhanced nuclear retention may not be measurable. Broadly, these results contribute to general understanding of TR's binding partners within the nucleus and highlight the need for further characterization of protein-protein interactions within endocrine pathways. This work was supported by the National Institutes of Health [Grant # 2 R15 DK058028-06] and the William & Mary Charles Center.