Electronic Thesis/Dissertation
Differential Regulation of the Estrogen Responsive Gene TFF1 by Cofactor Inhibition and Growth in 3D Culture Open Access
Transcription of DNA into messenger RNA (mRNA) requires the coordination of many enzymatic processes that are catalyzed by very large protein complexes. The nuclear hormone receptors are a family of ligand-regulated proteins that catalyze the transcription of specific genes. For example, estrogen receptor alpha (ERα) when bound by its ligand, estrogen, acts in combination with other coactivator proteins to activate transcription. In this thesis, an estrogen responsive gene, TFF1, was used as a model to study the dynamics of transcription mediated by ERα. Here, single-molecule imaging of living and fixed cells was used to elucidate transcription dynamics in cells cultured in three dimensions that were exposed to inhibitors, and oscillating levels of estrogen. Fluorescence in situ hybridization (FISH) was used to show that TFF1 mRNA decreased ~three-fold in the presence of the ERα inhibitors Tamoxifen or MD9571. The dynamics of TFF1 gene expression were monitored in single cells using the MS2 stem loop system. The MS2 stem loops were bound by (eGFP)-labeled MS2 phage capsid protein to enable the direct visualization of transcription sites by live-cell imaging. Time-lapse imaging of the diffraction-limited transcription sites showed that MD9571 decreased the number of TFF1 mRNA produced in each transcription event. Methods were also developed to study transcription dynamics under conditions that closely replicate normal physiology. First, a three-dimensional cell culture model using MCF7 breast cancer cells on Matrigel was developed to obtain spheroids with characteristics similar to normal tissue. Then, a method to oscillate estrogen concentrations in culture was developed using a pump system to mimic the changing circulating levels of estrogen in the human body.
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Day_gwu_0075M_13625.pdf | 2018-01-15 | Open Access |
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