Date Thesis Awarded
Honors Thesis -- Open Access
Bachelors of Science (BS)
The thyroid hormone receptor (TR) mediates gene transcription in response to thyroid hormone (T3) and significantly affects cell growth, development and metabolism. Although TR shuttles rapidly between the nucleus and cytoplasm, it is primarily nuclear localized at steady state. This state, according to previous research, is achieved by the balance of TR nuclear import, export and nuclear retention. The first focus of this thesis was on how nuclear receptor corepressor 1 (NCoR1) contributes to TR nuclear localization. Using transient transfection assays and fluorescence microscopy, we measured the fluorescent intensity of GFP or mCherry-tagged TR in the nucleus (N) and cytoplasm (C) of HeLa cells, and then calculated the N/C ratio as an indicator of intracellular localization patterns. We discovered that when NCoR1 is knocked down by shRNA, the amount of TR in the nucleus decreased significantly (lower N/C ratio). Inversely, when NCoR1 is over-expressed, the amount of TR in the nucleus significantly increased (higher N/C ratio). An even more dramatic cytosolic shift was observed for a TR mutant that cannot bind to NCoR1. Taken together, our results suggest that NCoR1 is a key regulator of TR nuclear retention. The second focus of this thesis was on developing a model to calculate protein-protein and protein-DNA binding rates using fluorescent recovery after photobleaching (FRAP). The model was designed to describe the fluorescent recovery curve for a linear photobleached region under a uniform laser profile. By fitting the model, we could then obtain binding rates for the complexes that interact with TR. These findings will contribute to the development of a dynamic mathematical model that aims to predict TR’s N/C ratio during the interaction with different complexes, and the effect of altered localization on transcriptional output of T3-dependent genes.
Sun, Xiaopeng, "Thyroid Hormone Receptor and Coregulators: A Dynamic Model" (2019). Undergraduate Honors Theses. William & Mary. Paper 1406.