Acetylation Controls Thyroid Hormone Receptor Intracellular Localization and Intranuclear Mobility

Thyroid hormone receptor (TR) is responsible for mediating the expression of genes involved in growth, development, and homeostatic regulation, in response to thyroid hormone. There are two main subtypes of TR, TRα1 and TRβ1, that mediate these physiological processes. Dysregulation of TR mediated processes is a contributing factor in disease pathology. Our prior studies show that TR is shuttled rapidly between the nucleus and cytosol, while localizing primarily to the nucleus. TRα1 contains two nuclear localization signals (NLSs) that act together to confer strong nuclear localization, while TRβ1 only contains one NLS (NLS-1). Recently, we found that the intracellular localization of TR is regulated, in part, by post-translational modification of lysines within NLS-1 by acetylation. We constructed GFP or mCherry-tagged TRα1 and TRβ1 acetylation-mimic and nonacetylation-mimic (unable to be endogenously acetylated) expression plasmids, transfected them into HeLa cells, and used fluorescence microscopy to determine the nuclear/cytosolic (N/C) ratio of the mutant receptors by measuring fluorescence intensity. Data show that the TR acetylation mimic has a significantly lower N/C ratio compared to wild-type TR, indicating a striking decrease in nuclear localization, whereas the TR nonacetylation mimic\u27s N/C ratio was the same as wild-type TR. Previously, we showed that TR nuclear import is mediated by importins 7, β1, and α1. We hypothesized that acetylation state alters either TR\u27s ability to bind importins or its intranuclear mobility, and that inhibition of the acetyltransferase CBP/p300 would lead to enhanced TR nuclear localization, while inhibition of the deacetylase SIRT1 would lead to TR cytoplasmic localization. Using GFP-Trap co-immunoprecipitation, we found that importins 7, β1, and α1 interact with both the TRα1 acetylation and nonacetylation mimics, suggesting that the TRα1 acetylation mimic\u27s decreased nuclear localization is due to factors other than reduced importin binding. Intriguingly, fluorescence recovery after photobleaching (FRAP) revealed that the GFP-TRα1 nonacetylation mimic has a significantly slower rate of recovery than wild-type TRα1, suggesting that this reduced mobility correlates with greater nuclear retention. in contrast, the GFP-TRα1 acetylation mimic had the same rate of recovery as wild-type TRα1. Lastly, pharmacological inhibition of CBP/p300 by C646 increased GFP-TRβ1\u27s nuclear localization in a dose-dependent manner (GFP-TRβ1 has a slight cytosolic population due in part to having one NLS); however, inhibition of SIRT1 by EX-527 did not alter GFP-TRα1\u27s localization. Taken together, these data provide further evidence that acetylation plays a key regulatory role in TR shuttling and retention and extends our understanding of how acetylation interplays with TR-regulated processes of growth and development