SINGLE-CELL QUANTIFICATION AND STOCHASTIC MODELING OF GLUCOCORTICOID RECEPTOR TRANSPORT AND DUSP1 MRNA DYNAMICS

Abstract

The work presented in this dissertation combines quantitative single-cell experimentation with mathematical modeling to investigate glucocorticoid receptor (GR) signaling and the transcriptional regulation of the glucocorticoid-responsive gene Dual-Specificity Phosphatase 1 (DUSP1). The overall objective is to establish experimentally constrained, predictive models that describe how GR localization dynamics are linked to transcriptional activation and mRNA abundance at thesingle-cell level. First, quantitative immunocytochemistry (ICC) and single-cell image analysis are used to characterize GR nucleocytoplasmic transport following glucocorticoid (GC) stimulation. Analysis of GR localization dynamics across individual cells indicates that nuclear clearance processes, rather than sustained cytoplasmic recycling, modulate nuclear GR levels. These observations restrict the class of GR transport and degradation models that are consistent with the data and emphasize the importance of nuclear turnover in shaping GR localization. Second, endogenous DUSP1 transcriptional activity is quantified using single-molecule inexpensive RNA fluorescence in situ hybridization (smiFISH), enabling direct measurement of transcription site activity and mRNA distributions across single cells. Utilizing a chemical master equation framework, we have developed a discrete stochastic model that accurately reproduces the time-varying probability distributions of GR and DUSP1 spatiotemporal dynamics – at all times and across multiple Dex concentrations. Stochastic two-state promoter models are developed to interpret these data. Model comparisons indicate that the observed transcriptional activation of DUSP1 can be reproduced by changes in promoter switching dynamics, specifically through increased promoter activation rates, decreased promoter deactivation rates, or combinations of both. These results suggest that modulation of transcriptional bursting provides a mechanistic explanation for the observed transcriptional response. Third, cytoplasmic DUSP1 mRNA accumulation is examined in the context of transcription, nuclear export, and mRNA decay. Quantitative analysis supports a model in which cytoplasmic mRNA levels reflect a balance between mRNA production and export and AU-rich element-mediated decay processes. Incorporating these regulatory steps into a unified modeling framework improves agreement with experimentally observed cytoplasmic mRNA distributions and temporal dynamics. Together, this work provides a quantitative description of GR localization dynamics, DUSP1 transcriptional activation, and post-transcriptional regulation using endogenous single-cell measurements. The resulting models establish a foundation for future studies aimed at extending these approaches to more complex regulatory interactions and perturbations in GC signaling.