Regulation of pluripotent differentiation by gene circuit interactions.

描述（由申请人提供）：细胞过程由基因和蛋白质之间的相互作用组成的基因调控回路控制。从细菌到哺乳动物干细胞的生物体都有一个这样的过程，即多能分化，细胞可以分化成几种可能的命运之一。我们建议在分子水平上研究遗传回路内部和之间的相互作用如何在细胞水平上决定多能分化。我们将研究枯草芽孢杆菌（Bacillus subtilis）的多能分化，这是一种简单、特征明确、实验可及的系统，可作为细胞命运选择的遗传控制模型。环境胁迫诱导枯草芽孢杆菌细胞产生孢子或分化为胜任状态，并吸收细胞外DNA并将其整合到染色体中。我们最近确定了核心能力回路，并表明它表现出由噪声触发的可兴奋动态。许多调节枯草芽孢杆菌能力和产孢的分子成分是已知的。然而，能力和孢子回路内部和之间的相互作用如何调节适当分化程序的选择和执行，人们知之甚少。我们将在单细胞水平上使用定量荧光延时显微镜来研究这个问题，以确定分子相互作用的动力学如何调节这一过程。利用可用于枯草芽孢杆菌的遗传操作技术，我们将测量如何系统地重新设计电路相互作用来控制分化。利用已建立的能力和孢子循环的连通性图，我们还将构建数学框架来生成预测和分析结果。具体而言，我们将应用这些方法：(1)通过将其与硅和体内的工程替代拓扑进行比较，确定能力电路架构的功能重要性。(2)确定交叉调控在细胞命运选择中的功能重要性。(3)确定能力回路的瞬时活动如何改变产孢的进程和执行。这种综合研究对于确定遗传回路内部和之间的分子相互作用如何在细胞水平上控制多能分化是必要的。确定这种模式生物中决定细胞命运选择和执行的机制可能与包括哺乳动物系统在内的多种生物的多能分化有关。公共卫生相关性：该提案将建立细菌如何进行分化的全面描述。所得到的数据将与以下方面有关：1)控制细菌的分化以防止食物中细菌孢子的形成。2)防止细菌自然产生对抗生素的耐药性。3)开发控制细胞分化的新技术，未来可用于控制哺乳动物干细胞的分化，以替代任何缺失或病变的细胞类型。

DESCRIPTION (provided by applicant): Cellular processes are controlled by gene regulatory circuits that are comprised of interactions among genes and proteins. One such process common to organisms ranging from bacteria to mammalian stem cells is pluripotent differentiation, where cells can differentiate into one of several possible fates. We propose to investigate how interactions at the molecular level within and across genetic circuits determine pluripotent differentiation at the cellular level. We will study pluripotent differentiation in Bacillus subtilis, a simple, well characterized and experimentally accessible system as a model for genetic control of cell fate choice. Environmental stress induces B. subtilis cells to undergo sporulation or differentiate into the competence state and take up extracellular DNA and incorporate it into their chromosome. We have recently identified the core competence circuit and showed that it exhibits excitable dynamics triggered by noise. Many of the molecular components that regulate competence and sporulation in B. subtilis are known. How interactions within and across competence and sporulation circuits regulate the choice and execution of appropriate differentiation programs is, however, poorly understood. We will study this problem using quantitative fluorescence time- lapse microscopy at the single-cell level to establish how the dynamics of molecular interactions regulate this process. Exploiting genetic manipulation techniques available for B. subtilis, we will measure how systematic re-engineering of circuit interactions control differentiation. Utilizing established connectivity maps of competence and sporulation circuits, we will also construct mathematical frameworks to generate predictions and analyze results. Specifically, we will apply these methods to: (1) Determine the functional importance of competence circuit architecture by comparing it to engineered alternative topologies in silico and in vivo. (2) Determine the functional importance of cross-regulation in cell fate choice. (3) Determine how the transient activity of the competence circuit alters the progression and execution of sporulation. This integrative research is necessary to determine how molecular interactions within and across genetic circuits control pluripotent differentiation at the cellular level. Identification of the mechanics that dictate the choice and execution of cell fate in this model organism are likely to be relevant to pluripotent differentiation in diverse organisms including mammalian systems. PUBLIC HEALTH RELEVANCE: This proposal will establish a comprehensive description of how bacteria undergo differentiation. The resulting data will be relevant to: 1) Controlling differentiation in bacteria to prevent bacterial spore formation in foods. 2) Preventing the ability of bacteria to naturally become resistant to antibiotics. 3) Developing new techniques to control how cells differentiate, which can be applied in the future to control differentiation of mammalian stem cell to substitute for any missing or diseased cell types.