Modulating Stochastic Gene Expression for Cell-fate Control and Therapeutics

项目总结 基因表达的随机波动在单细胞水平上是不可避免的，并影响 艾滋病毒对胚胎发育的影响。然而，我们对这些机制的理解仍然存在根本性的差距。 在哺乳动物细胞中产生和调节表达波动。解决这一知识差距的方法是 在波动和异质性存在治疗障碍的系统中，对治疗开发至关重要， 例如在艾滋病毒、干细胞疗法和癌症方面。我们的长期目标是开发针对 克服障碍，精确控制细胞命运的细胞异质性机制。 在过去的5年里，我们的工作建立了噪声和异质性的机械作用，证明了 噪声是生物系统的一个特征，而不是一个虫子，可以为了治疗效果而进行调节。 具体地说，我们：(I)阐明了一种利用噪音来调节HIV潜伏期的病毒转录反馈电路， (Ii)发现这个电路通过进化进行了优化，以发挥病毒对冲电路的功能，(Iii)做出了贡献 细胞生物学表明，转录波动通常会因核出口和翻译而放大， 并且，(Iv)我们发现了一种新的转录后反馈结构，它有效地抑制了噪声 稳定命运承诺。最令人兴奋的，也是与这一应用程序最相关的是，我们(V)发现了噪音- 似乎可以显著改善潜伏期病毒重新激活的增强剂分子，并已被使用 由其他实验室在不同的系统中增加噪音(例如，昼夜节律)。 这一更新的目的是识别哺乳动物细胞命运中噪音调制的分子机制 电路可实现对噪声的治疗性控制。基于我们的发现和广泛的初步证据 胚胎干细胞(ESCs)，我们的中心假设是普遍的“核心”细胞机制存在以调节 这些机制可能会受到药理上的干扰。我们的具体目标是建立在 我们独特的噪音增强剂分子工具将：(目标1)绘制噪音的分子机制路径图 增强子分子开发预测哺乳动物转录组范围噪音的数学模型 细胞；(目标2)绘制噪声抑制分子的分子机制图；和(目标3)量化相对分子 HIV体内潜伏期和安全性背后的随机和确定性机制的贡献 噪声调制分子在体内的有效性。拟议的研究具有广泛的意义，因为它将决定 不同命运规范模型中调控表达的核心分子机制揭示了遗传靶点 噪声增强和抑制可以导致新的广谱噪声的发展 调节器，并推动噪音调节分子的临床翻译。最终，所获得的知识将 指导新的治疗方法克服障碍，精确控制不同哺乳动物的细胞命运 系统。

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.