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.

项目摘要： 可预测的哺乳动物细胞工程对于推进定量生物学和 意识到精确医学的希望。为了开发一个细胞工程的预测框架，我们 需要了解基因调节在动态染色质环境的背景下如何发挥作用。 虽然过去几年见证了遗传和表观遗传编辑工具的繁荣 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对细胞之间的异质性，因此许多问题很难 用平均细胞的方法回答。第二，在大多数实验设计中，染色质状态和 DNA组成同时不同，因此很难单独脱离染色质的作用。第三， 染色质调节涵盖了多个分子机制和长度尺度，因此难以整合 这些数据成一个连贯的预测模型。 为了应对这些挑战，我们将开发并结合合成生物学的新工具， 单细胞技术和数学建模。我们将通过 在定义的基因座上募集和释放染色质调节剂，然后在单一中测量结果 随着时间的流逝，使用延时显微镜，流式细胞仪和下一代测序。最后，我们会的 建立一个数学模型，该模型将特定基因座的分子细节与整体染色质状态集成 在同一细胞中。 我们将使用这些工具来回答有关染色质动态在基因上的作用的基本问题 法规：（1）为什么不同的细胞类型具有不同的沉默动态和表观遗传记忆？ （2）如何 快速，染色质修饰及其作用传播了多远？ （3）如何在 启动子和增强剂与染色质调节剂相互作用以确定基因表达？ 一起，对这些基本问题的答案以及开发用于衡量的新技术 控制和建模基因表达将促进哺乳动物合成生物学和基本 染色质和基因调节的生物学领域。