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

项目概要： 基因表达的重编程以响应外部环境的变化， 细胞的适应和存活这些硬编码的响应在地质时间尺度上演变，并且是精细的- 适应了当地环境的要求。然而，生物体可能会遇到新的或极端的 环境，其基因调控网络不足以进行适当的基因重编程 表情我们的实验室最近在酵母中发现了一种强大的新表观遗传机制 酿酒酵母，允许细胞适应新的极端环境，而无需硬连线调节的益处。 网络.我们称这种机制为"随机调谐"，通过这种机制，固有的转录噪音和适应性- 受驱动的反馈使细胞能够优化适当的基因表达状态，而不需要感测基因的表达。 外部环境直接影响。本研究的目的是描述随机调谐动力学， 单个细胞，并发现潜在的效应和监管机构参与。鉴于固有的 随机性质的现象，表征单细胞轨迹的调谐在分子水平上是 一个重要的步骤。基因表达的表征可以在全球范围内使用高- 通量方法或使用显微镜以高分辨率研究mRNA时间动态。单细胞 哺乳动物细胞中的转录组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： 酵母由于厚的细胞壁的存在。因此，我开始研究一种有前途的替代方案， 在酵母细胞中进行高效和低成本转录组分析的技术。我还会用活细胞成像技术 MS2/PP7标记的mRNA，以高时间和空间分辨率研究转录动力学。在 此外，我的目标是系统地发现所有的因素参与随机调整。我将使用 定期间隔短回文重复序列（CRISPR）技术，以激活或抑制所有必要的， 非必需基因，以便系统地发现影响随机调谐的基因座。我们的初步 在酵母中的研究表明，随机调谐在每个单独基因的水平上局部地操作， 染色质修饰和重塑机制调节这一过程的功效。我会研究 染色质免疫沉淀结合定量PCR技术研究组蛋白局部修饰的动态变化 沿着随机调谐的不同阶段。此外，我将使用DNase-I研究局部染色质状态， 超敏反应测定。这些研究将使我们能够监测过程中的局部染色质状态 并将其与转录输出和细胞适应性相匹配。这些研究将揭示 一个强大的新的适应机制的分子细节在单细胞水平和局部 染色质背景。我们的工作还将揭示调节的关键效应器和调节器，这是调节的关键步骤。 实现对这种强大的细胞适应新现象的全面机械理解。

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.!