Multiscale tools and approaches for understanding and engineering cell-fate transitions

项目总结 合成生物学的目标是利用生物系统的力量动态获取信息 细胞，使合成的生物医学任务，如肿瘤监测，病原体鉴定，或细胞命运 重新编程。细胞工程中的这种任务依赖于强大的机制来调节转基因 酶的传递、基因矫正和细胞疗法。为了释放它的全部潜力，哺乳动物 合成生物学需要基础工具来实现对原代细胞中基因表达的可靠控制。 例如，转基因沉默(例如，表达丧失)仍然是有效地 设计原代细胞。一系列长度和时间尺度的监管层级协调事件 分子与细胞信号的结合调节基因表达，从而调节细胞命运。多尺度方法包括 需要整合控制细胞命运转变的不同过程。细胞命运转变代表着关键 需要协调从表观遗传和细胞骨架重塑到增殖的多个过程的事件 和抄写。了解这些转变可能会解释癌基因如何协同这些过程来驱动 细胞转化。在这里，我们提出了一个多尺度的方法来理解和设计细胞命运。 过渡(例如，重新编程、差异化)。 从我们之前确定细胞命运转换原理的工作中，我们确定了系统级的 限制重新编程的限制，并开发了一种鸡尾酒，使鼠标的重新编程次数增加了100倍 细胞。比较人类和小鼠对重新编程的反应，我们确定了物种特定的差异 在重新编程过程中的增殖、信号和先天免疫反应，这可能有助于降低 人类细胞的重新编程率。我们建议研究这些分子关联，以确定每一个 影响重新编制方案的进程和结果。我们将利用这些洞察力来设计遗传控制器来 引导细胞通过重新编程。我们已经确定了一种通过诱导来优化重新编程的策略 一个瞬时的“擦除”阶段，然后是一个“写入”阶段，以建立新的单元命运。我们建议开发 能够自主地引导细胞通过这些相互竞争的目标以提高效率的控制器 重新编程。遗传控制器是由与天然基因相连的合成基因电路组成的。 监管网络。虽然大量的努力已经投入到增强型合成材料的逻辑设计上 电路(例如用于同步法定数检测、边缘检测的电路)，关于如何 细胞硬件和遗传元素的新兴三维结构会影响电路。在这里，我们 建议提高我们对转录如何重塑DNA以及它如何影响 基因电路。明确染色质结构在细胞识别中的作用将指导分子工程工作 构建能够调节行为的基因控制器。我们预计我们开发的原则和工具 将广泛适用于细胞工程应用以及研究细胞命运转变。

Project summary Synthetic biology aims to harness the power of biological systems to dynamically access information in the cell, enabling synthetic biomedical tasks such as tumor surveillance, pathogen identification, or cell-fate reprogramming. Such tasks in cellular engineering rely on robust mechanisms to regulate transgenes for the delivery of enzymes, genetic corrections, and cellular therapies. To unleash its full potential, mammalian synthetic biology requires foundational tools for implementing reliable control of gene expression in primary cells. For example, transgene silencing (e.g. loss of expression) remains a common challenge to effectively engineering primary cells. Layers of regulation across a range of length- and time-scales coordinate events from molecular binding to cell signaling regulate gene expression and thus cell fate. Multiscale approaches are needed to integrate the diverse processes that control cell-fate transitions. Cell-fate transitions represent pivotal events requiring coordination of multiple processes from epigenetic and cytoskeletal remodeling to proliferation and transcription. Understanding these transitions may illuminate how oncogenes coopt these processes to drive cellular transformation. Here, we propose a multiscale approach for understanding and engineering cell-fate transitions (e.g. reprogramming, differentiation). From our previous work to identify principles of cell-fate transitions, we identified systems-level constraints that limited reprogramming and developed a cocktail that increased reprograming 100-fold in mouse cells. Comparing the human and mouse response to reprogramming, we identified species-specific differences in proliferation, signaling, and the innate immune response during reprogramming that may contribute to lower reprogramming rates for human cells. We propose to examine these molecular correlates to determine how each impacts the reprogramming process and outcomes. We will use these insights to design genetic controllers to guide cells through reprogramming. Already we have identified a strategy to optimize reprogramming by inducing a transient “erase” phase followed by a “write” phase to establish the new cell fate. We propose to develop controllers capable of autonomously guiding cells through these competing objectives to enhance the efficiency of reprogramming. Genetic controllers are composed from synthetic gene circuits connected to native gene regulatory networks. While significant efforts have been devoted to the logical design of enhanced synthetic circuitry (e.g. circuits for synchronized quorum sensing, edge-detection), less is understood regarding how cellular hardware and the emergent three-dimensional structure of genetic elements affect circuits. Here, we propose to improve our understanding how transcription reshapes DNA and how it impacts the performance of gene circuits. Defining the role of chromatin structure in cellular identity will guide molecular engineering efforts to build genetic controllers capable of regulating behavior. We anticipate that the principles and tools we develop will be broadly applicable across cellular engineering applications as well as for investigating cell-fate transitions.