Mediator Subunit MED1 Differentially Modulates Mutant Thyroid Hormone Receptor Intracellular Localization and Intranuclear Mobility

Abstract

Thyroid hormone receptor (TR) plays a key role in mediating the effect of thyroid hormone (T3) by controlling hundreds of genes important in growth, development and metabolism. The two major subtypes of TR, TRα1 and TRβ1 can either activate or repress transcription of target genes. In the absence of T3, TR interacts with corepressors and represses transcription. After binding T3, TR undergoes a conformational change that releases corepressors and recruits coactivators like Mediator subunit MED1. Mutations in TR lead to Resistance to Thyroid Hormone syndrome (RTH), where the thyroid gland produces enough T3, but the RTH mutants are defective in T3 binding, blocking the conformational change required to recruit coactivators. Our previous research has shown that TR is primarily nuclear localized but shuttles rapidly between the nucleus and cytoplasm and that MED1 knockout decreases the nuclear population of TR and impacts its intranuclear mobility. To investigate the effect of MED1 on mutant TR, we either cotransfected mCherry‐tagged RTH TRα1 mutant (A263V, C392X and P398R) expression plasmids into HeLa cells with GFP‐MED1, or transfected mutant TRs into MED1‐/‐ mouse embryonic fibroblasts. The nuclear/cytoplasmic (N/C) ratio of the RTH TRα1 mutants was measured by comparing the average fluorescence intensity in the nucleus and the cytoplasm. Our data show that MED1 overexpression and knockout did not change the N/C ratio of the C392X and P398R mutants at physiological T3 levels, while MED1 overexpression increased the P398R mutant’s N/C ratio in elevated T3 levels (Figure 1; p<0.05, n=3, 60 cells per replicate). A significantly greater number of cells cotransfected with A263V and MED1 had nuclear or cytoplasmic aggregates compared to wildtype (WT) TR (p<0.05, n=3, ≥60 cells per replicate), while both an increase in T3 levels and MED1 knockout significantly decreased the number of cells with aggregates (p<0.05, n=3, 60 cells per replicate). Similar aggregates were found in cells cotransfected with MED1 and RTH mutant A263S or cancer mutant K74E, A264V, indicating mutations near residue 263 may contribute to aggregation. Cotransfection of A263V and MED13, a Mediator complex subunit that does not interact with TR directly, also resulted in aggregation, indicating that nonspecific interactions may also contribute to aggregate formation. Interestingly, MED1 overexpression significantly increased the intranuclear mobility of A263V TR in fluorescence recovery after photobleaching (FRAP) assays (Figure 2; p<0.05, n=3, 20 nuclei per replicate) suggesting that MED1 interacts with A263V TR at physiological T3 levels. However, MED1 overexpression did not impact the intranuclear mobility of either C392X or P398R TR, indicating that MED1 does not interact with them at physiological T3 levels (p>0.05, n=3, 20 nuclei per replicate). Lastly, results show that MED1 knockout increased the intranuclear mobility of WT but not mutant TRs at physiological T3 levels. Clinical records show a larger number of less severe RTH cases with A263V/S mutations, a smaller number of cases with P398R mutation and both a larger number and more severe cases with C392X mutation. Together these data further our understanding of how the interaction between MED1 and RTH TRα1 mutants may contribute to RTH pathology.