Thyroid Hormone Receptor Acetylation and Nucleocytoplasmic Shuttling

Human thyroid hormone receptor α1 (TRα1) modulates target gene expression in response to thyroid hormone (T3), but also undergoes post-translational modification by acetylation and nucleocytoplasmic shuttling. The current model for TR shuttling predicts that acetylation occurs in the nucleus, but it remains unclear whether TRα1's nuclear transport is influenced by its acetylation status. Here, we aimed to determine whether acetylation of TRα1 at the K130, K134, and K136 residues which reside in one of its nuclear localization signals, NLS-1, affects its ability to shuttle between the nucleus and cytoplasm. Using fluorescence microscopy, we investigated the intracellular localization of GFP-tagged TRα1 mutants mimicking acetylation (K→Q) and non-acetylation (K→R), alongside wild-type TRα1 in a human-mouse heterokaryon model, using the Hoechst 33342 DNA stain to differentiate between human and mouse nuclei and TRITC-Phalloidin to visualize the actin cytoskeleton and confirm heterokaryon formation. Our findings revealed evidence of TRα1 export from the transfected human nucleus and import to the mouse nucleus, suggesting that TRα1 can be imported to and exported from the nucleus in both its acetylated and non-acetylated states. We also conducted a pilot study using importazole, a small-molecule reversible inhibitor of importin β, to test the viability of using nuclear import inhibition and washout with live cell imaging to compare the import kinetics of the acetylation and non-acetylation mimics. Using fluorescence imaging, we concluded that at non-toxic concentrations, importazole is not suitable for live-cell imaging due to a lack of sufficiently visualizable changes in TRα1 localization.