Systems Level Characterization of a New Epigenetic Mechanism of Gene Expression and Cellular Adaptation

基因表达和细胞适应的新表观遗传机制的系统水平表征

• 批准号: 10191180
• 负责人: Amir Momen-Roknabadi
• 金额: $ 2.31万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-07-01 至 2020-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10191180
• 关键词: Affect; Biological Assay; CRISPR interference; CRISPR/Cas technology; Cell Wall; Cells; ChIP-on-chip; Chromatin; Code; Coupled; Deoxyribonuclease I; Environment; Epigenetic Process; Feedback; Fluorescence; Frequencies; Gene Expression; Genes; Genetic Transcription; Geology; Guide RNA; Habitats; Health; Hypersensitivity; Image; Individual; Kinetics; Laboratories; Libraries; Mammalian Cell; Messenger RNA; Methods; Microscopy; Molecular; Monitor; Nature; Noise; Nucleosomes; Organism; Output; Pathway interactions; Pharmaceutical Preparations; Population; Process; RNA library; Regulator Genes; Reporter; Reporter Genes; Resistance; Resolution; Role; Saccharomyces cerevisiae; Sorting - Cell Movement; Stochastic Processes; Surveys; System; Technology; Thick; Time; Work; Yeasts; cancer cell; cancer type; chemotherapy; chromatin immunoprecipitation; chromatin modification; chromatin remodeling; cost; fitness; genome-wide; genomic locus; histone modification; insight; live cell imaging; mRNA tagging; mutant; novel; promoter; response; transcriptome; transcriptomics

Project Summary: The reprogramming of gene expression in response to changes in the external environment is critical for cellular adaptation and survival. These hard-coded responses evolve over geological time-scales and are fine- tuned to the requirements of the native habitat. However, organisms may encounter novel or extreme environments for which their gene regulatory network is inadequate for appropriate reprogramming of gene expression. Our laboratory has recently discovered a powerful new epigenetic mechanism in Saccharomyces cerevisiae that allows cells to adapt to novel extreme environments without the benefit of hardwired regulatory networks. We call this mechanism “stochastic tuning,” by which inherent transcriptional noise and fitness- driven feedback enable cells to optimize appropriate gene expression states without the need to sense the external environment directly. The aim of this study is to characterize the stochastic tuning dynamics in individual cells and to discover the underlying effectors and regulators involved. Given the inherently stochastic nature of the phenomenon, characterizing the single-cell trajectory of tuning at the molecular level is an essential step. The characterization of gene expression can be achieved at a global scale using high- throughput methods or at high resolution using microscopy to study mRNA temporal dynamics. Single-cell transcriptomics in mammalian cells is a rapidly advancing field. However, these methods are not effective in yeast due the presence of a thick cell wall. I have therefore started to develop a promising alternative technology for efficient and low-cost transcriptome profiling in yeast cells. I will also use live cell imaging of MS2/ PP7 tagged mRNAs to study transcriptional dynamics at high temporal and spatial resolution. In addition, I aim to systematically discover all the factors involved in stochastic tuning. I will use Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology to activate or repress all essential and non-essential genes in order to systematically discover loci that affect stochastic tuning. Our preliminary studies in yeast demonstrate that stochastic tuning operates locally at the level of each individual gene and that chromatin modification and remodeling machinery modulates the efficacy of this process. I will study the dynamics of local histone modifications using Chromatin Immunoprecipitation coupled with quantitative PCR along the different stages of stochastic tuning. In addition, I will study the local chromatin state using DNase-I- Hypersensitivity assays. These studies will enable us to monitor the local chromatin state during the process of stochastic tuning and match it to transcriptional output and cellular fitness. The proposed studies will reveal the molecular details of a powerful new adaptation mechanism at the single-cell level and within the local chromatin context. Our work will also reveal the key effectors and regulators of tuning, a critical step in achieving a full mechanistic understanding of this powerful new phenomenon of cellular adaptation.!

项目总结： 基因表达的重新编程以响应外部环境的变化对于 细胞适应和生存。这些硬编码的反应在地质时间尺度上演变，并且很好- 适应了当地栖息地的要求。然而，生物可能会遇到新奇的或极端的 他们的基因调控网络不足以进行适当的基因重新编程的环境 表情。我们实验室最近在酵母菌中发现了一种强大的新表观遗传机制 允许细胞适应新的极端环境的啤酒，而不需要硬线调控的好处 网络。我们把这种机制称为“随机调谐”，通过这种机制，固有的转录噪音和适应性- 驱动反馈使细胞能够优化适当的基因表达状态，而不需要感知 直接面对外部环境。这项研究的目的是刻画随机调谐动态在 并发现涉及的潜在效应器和调节器。从本质上讲， 这种现象的随机性，在分子水平上表征了单细胞的调谐轨迹 这是至关重要的一步。基因表达的表征可以在全球范围内实现，使用高密度的 方法或在高分辨率下用显微镜研究mRNA的时间动力学。单细胞 哺乳动物细胞的转录学是一个快速发展的领域。然而，这些方法在以下方面并不有效 酵母由于存在较厚的细胞壁。因此，我开始开发一种前景看好的替代方案 酵母细胞高效低成本转录组图谱技术。我还将使用活细胞成像技术 MS2/PP7标记的mRNAs用于在高时间和空间分辨率下研究转录动力学。在……里面 此外，我的目标是系统地发现随机调优涉及的所有因素。我将使用集群 规则间隔短回文重复(CRISPR)技术可激活或抑制所有必需和 非必需基因，以便系统地发现影响随机调谐的基因。我们的预赛 在酵母中的研究表明，随机调节在每个单独基因和 染色质修饰和重塑机制调节着这一过程的效果。我会研究一下 染色质免疫沉淀结合定量聚合酶链式反应对组蛋白局部修饰的动力学研究 沿着随机调谐的不同阶段。此外，我还将使用DNA ase-I-研究局部染色质状态。 超敏反应试验。这些研究将使我们能够监测过程中局部染色质的状态 并将其与转录输出和细胞适合度进行匹配。拟议的研究将揭示 在单细胞水平和局部细胞水平上强大的新适应机制的分子细节 染色质上下文。我们的工作还将揭示调整的关键影响因素和调节器，这是 实现对这一强大的细胞适应新现象的全面机械性理解。