Single-cell analysis and synthetic control of mammalian chromatin dynamics and gene regulation

哺乳动物染色质动力学和基因调控的单细胞分析和合成控制

• 批准号: 10198945
• 负责人: Lacramioara Bintu
• 金额: $ 38.96万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-07-05 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10198945
• 关键词: Address; Biology; Cells; Chromatin; DNA; Data; Development; Disease; Enhancers; Environment; Epigenetic Process; Experimental Designs; Flow Cytometry; Gene Expression; Gene Expression Regulation; Genes; Genetic; Goals; Heterogeneity; Immune response; Length; Malignant Neoplasms; Mammalian Cell; Measures; Mediating; Methods; Microscopy; Modeling; Molecular; Outcome Measure; Regulation; Role; Techniques; Time; base; cell type; cellular engineering; chromatin modification; design; epigenetic memory; gene therapy; improved; mathematical model; next generation sequencing; precision medicine; predictive modeling; promoter; recruit; single cell analysis; synthetic biology; temporal measurement; time use; tool; transcription factor

Project Summary: Predictable mammalian cell engineering is essential both for advancing quantitative biology and realizing the promise of precision medicine. In order to develop a predictive framework for cell engineering, we need to understand how gene regulation functions in the context of a dynamic chromatin environment. While the last few years have witnessed a boom in genetic and epigenetic editing tools, there are still major challenges for measuring and predicting the effect of chromatin on gene expression. First, chromatin- mediated control results in cell-to-cell heterogeneity of gene expression, so many questions are difficult to answer with methods that average across cells. Second, in most experimental designs the chromatin state and DNA composition vary at the same time, making it difficult to disentangle the role of chromatin alone. Third, chromatin regulation spans multiple molecular mechanisms and length-scales, making it difficult to integrate these data into a coherent predictive model. In order to address these challenges, we will develop and combine new tools in synthetic biology, single-cell techniques, and mathematical modelling. We will directly manipulate the chromatin state via recruitment and release of chromatin regulators at a defined locus, and then measure the outcome in single cells over time using time-lapse microscopy, flow cytometry and next generation sequencing. Finally, we will build a mathematical model that integrates molecular details at a specific locus with the overall chromatin state in the same cells. We will use these tools to answer essential questions about the role of chromatin dynamics on gene regulation: (1) Why do different cell types have different silencing dynamics and epigenetic memory? (2) How fast and how far do chromatin modifications and their effects spread? (3) How do transcription factors at promoters and enhancers interact with chromatin regulators to determine gene expression? Together, the answer to these basic questions and the development of new techniques for measuring, controlling, and modeling gene expression will advance both mammalian synthetic biology and the basic biology fields of chromatin and gene regulation.

项目概要： 可预测的哺乳动物细胞工程对于推进定量生物学和 实现精准医疗的承诺。为了开发细胞工程的预测框架，我们 需要了解基因调控在动态染色质环境中如何发挥作用。 虽然过去几年见证了遗传和表观遗传编辑工具的蓬勃发展，但仍然存在 测量和预测染色质对基因表达的影响的主要挑战。首先，染色质- 介导的控制导致基因表达的细胞与细胞异质性，因此许多问题难以解决。 用跨单元格平均的方法来回答。其次，在大多数实验设计中，染色质状态和 DNA组成在同一时间变化，使得很难单独解开染色质的作用。第三、 染色质调控跨越多种分子机制和长度尺度，使得其难以整合 将这些数据整合到一个连贯的预测模型中。 为了应对这些挑战，我们将开发和联合收割机合成生物学的新工具， 单细胞技术和数学建模。我们将直接操纵染色质状态， 招募和释放染色质调节剂在一个确定的基因座，然后测量结果，在单一的 细胞随时间的变化，使用延时显微镜，流式细胞术和下一代测序。最后我们将 建立一个数学模型，将特定位点的分子细节与整体染色质状态相结合 在相同的细胞。 我们将使用这些工具来回答有关染色质动力学对基因表达的作用的基本问题。 调控：（1）为什么不同的细胞类型具有不同的沉默动力学和表观遗传记忆？(2)如何 染色质修饰及其影响传播的速度有多快？(3)转录因子如何在 启动子和增强子与染色质调节因子相互作用以决定基因表达？ 总之，这些基本问题的答案和新的测量技术的发展， 控制和模拟基因表达将推动哺乳动物合成生物学和基础生物学的发展。 染色质和基因调控的生物学领域。