PROJECT SUMMARY Thyroid hormone receptors (TRs) are essential transcription factors that either activate or repress the expression of target genes in response to thyroid hormone (T3). The proposed research explores a novel facet of our overarching hypothesis that mislocalization of TR contributes to pathogenesis, by investigating the molecular mechanism by which acetylation acts as a regulatory switch for TR localization. Our prior studies supported by 2 R15 DK058028 provide a robust premise for the proposed research. Although TRs primarily reside in the nucleus, we have shown that they shuttle rapidly between the nucleus and cytoplasm. We have identified multiple nuclear localization signals and nuclear export signals within TR that interact with importins and exportins, respectively, to mediate translocation into the nucleus. Our published data show that acetylation mimetics of TR promote cytoplasmic localization of TR but at the same time enhance ligand-dependent transcriptional activity. In contrast, mutants that mimic nonacetylated TR have reduced intranuclear mobility, greater nuclear retention, and impaired ligand-dependent transcriptional activity. Based on these published data and other preliminary findings, our working model implicates acetylation as a key molecular switch for modulating TR localization, intranuclear mobility, and transcriptional activity. Here, we will address the following unanswered questions: Is there one key acetylation site that alters nucleocytoplasmic distribution, intranuclear mobility, and transcriptional activity? Is TR modification compartment-specific? Which acetyltransferase(s) and deacetylase(s) regulate TR modification? Does TR acetylation affect its protein- protein interactions, stability, or ubiquitin-mediated degradation? We will use a multi-pronged approach of transient transfection of mammalian cells followed by quantitative fluorescence microscopy, fluorescence recovery after photobleaching (FRAP), luciferase reporter assays, pharmacological inhibitor screening, and coimmunoprecipitation assays, to test our model. In addition, acetylation sites will be confirmed by MS/MS analysis, and a novel TR-based NanoBRET assay will be developed to study protein-protein interactions. Finally, we will investigate whether mutations in TR linked to Resistance to Thyroid Hormone (RTH) syndrome respond aberrantly to the acetylation switch, providing insight into the correlation between altered trafficking signals in TR and endocrine disorders. Elucidating how TR acetylation is modulated will enhance understanding of the impact this pivotal post-translational modification has upon T3 signaling. A team of talented undergraduates will be actively engaged in the proposed research, gaining hands-on experience with the scientific process, from project design and execution to publication and/or presentation.

PROJECT NARRATIVE Trafficking of regulatory proteins to their appropriate destination is fundamental to the function of all living cells. In some endocrine disorders this highly regulated traffic control may be disrupted. This proposal explores the signals regulating traffic control of the thyroid hormone receptor, an essential protein which either activates or represses the expression of its target genes in response to thyroid hormone, and plays a pivotal role in human health, development, and metabolism.