Tuesday, March 18, 2025
3/18/2025
PROJECT SUMMARY Stochastic fluctuations in gene expression are unavoidable at the single-cell level and affect fate decisions from HIV to embryonic development. Yet, there remain fundamental gaps in our understanding of the mechanisms generating and regulating expression fluctuations in mammalian cells. Addressing this gap in knowledge is critical to therapeutic development in systems where fluctuations and heterogeneity present treatment barriers, such as in HIV, stem-cell therapeutics, and cancer. Our long-term goal is to develop therapeutics that target mechanisms of cellular heterogeneity to overcome barriers to precise control of cell fate. During the past 5 years, our work established mechanistic roles for noise and heterogeneity, demonstrating that noise is a feature, rather than a bug, of biological systems that can be modulated for therapeutic effect. Specifically, we: (i) elucidated a viral transcriptional-feedback circuit that harnesses noise to regulate HIV latency, (ii) found this circuit to be optimized by evolution to function as a viral bet-hedging circuit, (iii) made contributions to cell biology showing that transcriptional fluctuations are, in general, amplified by nuclear export and translation, and, (iv) we discovered a novel post-transcriptional feedback architecture that efficiently suppresses noise to stabilize fate commitment. Most excitingly, and most relevant to this application, we (v) discovered noise- enhancer molecules that appear to substantially improve viral reactivation from latency and have been used by other labs to increase noise in diverse systems (e.g., circadian rhythm). The objective of this renewal is to identify molecular mechanisms of noise modulation in mammalian cell-fate circuits to enable therapeutic control of noise. Based on our findings and extensive preliminary evidence in embryonic stem cells (ESCs), our central hypothesis is that generalized `core' cellular mechanisms exist to tune expression noise and that these mechanisms can be pharmacologically perturbed. Our specific aims build off our unique tool of noise-enhancer molecules and will: (Aim 1) map the molecular mechanistic pathways of noise enhancer molecules to develop a mathematical model predictive of transcriptome-wide noise in mammalian cells; (Aim 2) map the molecular mechanisms of noise-suppressor molecules; and (Aim 3) to quantify relative contributions of stochastic vs. deterministic mechanisms underlying HIV latency in vivo and safety & efficacy of noise-modulating molecules in vivo. The proposed research has broad significance as it will determine core molecular mechanisms regulating expression in disparate fate-specification models, reveal genetic targets of noise enhancement and suppression that can lead to the development of new broad-spectrum noise modulators, and propel clinical translation of noise-modulating molecules. Ultimately, the knowledge gained will guide new therapeutic approaches to overcome barriers to precise cell-fate control across diverse mammalian systems.
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