“Acetylation Controls Thyroid Hormone Receptor Intracellular localization and Intranuclear Mobility”

Abstract

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. Multiple amino acid sequences within TR, called nuclear localization signals (NLSs), rich in the basic residues lysine (K) and arginine (R), and nuclear export signals (NESs), rich in hydrophobic residues, are recognized by specific transport proteins called importins and exportins that direct TR nuclear entry and exit, respectively. TRα1 contains two NLSs that act together to confer strong nuclear localization, while TRβ1 only contains one NLS (NLS‐1). Our lab has revealed that TR nuclear export is mediated by multiple exportins, and that TR nuclear import is mediated by importins 7, β1, and adapter importin α1. 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 (P<0.05; n=3 replicates, ≥ 100 cells per replicate), indicating a striking decrease in nuclear localization, whereas the TR nonacetylation mimic's N/C ratio was comparable to wild‐type TR. We hypothesized that acetylation state alters either TR's ability to bind importins or its intranuclear mobility, and that inhibition of the acetyltransferase CBP/p300 would lead to enhanced TR nuclear localization. Using GFP‐Trap co‐immunoprecipitation assays, we found that importins 7, β1, and α1 interact with both the TRα1 acetylation and nonacetylation mimics, suggesting that the TR acetylation mimic's decreased nuclear localization is not due to 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 (P<0.05; n=3 replicates, 20 cells per replicate), suggesting that this reduced mobility correlates with greater nuclear retention. In contrast, the GFP‐TRα1 acetylation mimic had a similar rate of recovery as wild‐type TRα1. Lastly, pharmacological inhibition of CBP/p300 by C646 increased GFP‐TRβ1's nuclear localization in a dose‐dependent manner (P<0.05; n=3 replicates, 200 cells per replicate), providing further evidence that acetylation plays a key regulatory role in TR shuttling and retention. Taken together, these data extend our understanding of how acetylation interplays with TR‐regulated processes of growth and development.Support or Funding InformationNSF and NIH to LAAThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.