Date Awarded

2022

Document Type

Thesis

Degree Name

Master of Science (M.Sc.)

Department

Biology

Advisor

Lizabeth A Allison

Committee Member

Shantá D Hinton

Committee Member

Matthew J Wawersik

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 the 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 TRα1 is primarily nuclear localized but shuttles rapidly between the nucleus and cytoplasm and that MED1 knockout decreases the nuclear population of TRα1, but overexpression of MED1 does not impact TRα1’s intranuclear mobility. To investigate the effect of MED1 on mutant TRα1, we either cotransfected mCherry-tagged RTH TRα1 mutant (A263V, C392X, and P398R) expression plasmids into HeLa cells with GFP-MED1 or transfected TRα1 mutants 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 at elevated T3 levels. A significantly greater number of cells transfected with A263V had nuclear or cytoplasmic aggregates compared to wildtype (WT) TRα1, while an even greater number of cells cotransfected with A263V and MED1 had aggregates. Both an increase in T3 levels and MED1 knockout significantly decreased the number of cells with aggregates. Interestingly, MED1 overexpression significantly increased the impaired intranuclear mobility of A263V TRα1 in fluorescence recovery after photobleaching (FRAP) assays, suggesting that MED1 overexpression may be able to rescue the nonfunctional A263V TRα1 aggregates. However, MED1 overexpression did not impact the intranuclear mobility of either C392X or P398R TRα1, indicating that MED1 does not interact with them at physiological T3 levels. Although our previous research has shown that MED1 overexpression does not impact WT TRα1’s intranuclear mobility, our recent results show that MED1 knockout increased the intranuclear mobility of WT but not mutant TRs at physiological T3 levels. When provided with T3 supplement, MED1 knockout significantly decreased the t1/2 for P398R and A263V mutants while not impacting the C392X mutant. Clinical records show a larger number of less severe RTH cases with A263V/S mutations, a smaller number of cases with the P398R mutation, and both a larger number and more severe cases with the C392X mutation. Together, these data further our understanding of how the interaction between MED1 and RTH TRα1 mutants may contribute to RTH pathology.

DOI

https://dx.doi.org/10.21220/s2-48e5-7804

Rights

© The Author

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