Mediator subunit MED1 modulates intranuclear dynamics of the thyroid hormone receptor.

The thyroid hormone receptors (TRs) mediate thyroid hormone (T3 )-dependent gene expression. The nuclear import and export signals that direct TR shuttling are well characterized, but little is known about factors modulating nuclear retention. We used fluorescence-based nucleocytoplasmic scoring and fluorescence recovery after photobleaching in transfected cells to investigate whether Mediator subunits MED1 and MED13 play a role in nuclear retention of TR. When MED1 was overexpressed, there was a striking shift towards a greater nuclear localization of TRß1 and the oncoprotein v-ErbA, subtypes with cytosolic populations at steady-state, and TRß1 intranuclear mobility was reduced. For TRα1, there was no observable change in its predominantly nuclear distribution pattern or mobility. Consistent with a role for MED1 in nuclear retention, the cytosolic TRα1 and TRß1 population were significantly greater in MED1-/- cells, compared with MED1+/+ cells. Exposure to T3 and epidermal growth factor, which induces MED1 phosphorylation, also altered TR intranuclear dynamics. Overexpression of miR-208a, which downregulates MED13, led to a more cytosolic distribution of nuclear-localized TRα1; however, overexpression of MED13 had no effect on TRß1 localization. The known binding site of MED1 overlaps with a transactivation domain and nuclear export signal in helix 12 of TR's ligand-binding domain (LBD). Coimmunoprecipitation assays demonstrated that TR's LBD interacts directly with exportins 5 and 7, suggesting that binding of exportins and MED1 to TR may be mutually exclusive. Collectively, our data provide evidence that MED1 promotes nuclear retention of TR, and highlight the dual functionality of helix 12 in TR transactivation and nuclear export.

Acetylation modulates thyroid hormone receptor intracellular localization and intranuclear mobility.

The thyroid hormone receptor (TR) undergoes nucleocytoplasmic shuttling, but is primarily nuclear-localized and mediates expression of genes involved in development and homeostasis. Given the proximity of TR acetylation and sumoylation sites to nuclear localization (NLS) and nuclear export signals, we investigated their role in regulating intracellular localization. The nuclear/cytosolic fluorescence ratio (N/C) of fluorescent protein-tagged acetylation mimic, nonacetylation mimic, and sumoylation-deficient TR was quantified in transfected mammalian cells. While nonacetylation mimic and sumoylation-deficient TRs displayed wild-type N/C, the acetylation mimic's N/C was significantly lower. Importins that interact with wild-type TR also interact with acetylation and nonacetylation mimics, suggesting factors other than reduced importin binding alter nuclear localization. FRAP analysis showed wild-type intranuclear dynamics of acetylation mimic and sumoylation-deficient TRs, whereas the nonacetylation mimic had significantly reduced mobility and transcriptional activity. Acetyltransferase CBP/p300 inhibition enhanced TR's nuclear localization, further suggesting that nonacetylation correlates with nuclear retention, while acetylation promotes cytosolic localization.