Investigation of the Causal Relationship Between Chromatin Structure Fluctuations and Gene Expression Noise by Electron and Fluorescence Microscopy

智力优势：动物、植物和真菌细胞一样，通过在蛋白质核心周围有规律的间隔缠绕DNA来包装它们的DNA，形成一个连接的DNA假脱氧核小体链。假脱机使DNA更难获得，从而干扰了对其遗传信息的读取。那么，DNA假脱机是如何影响基因活性和调控的呢？这项研究通过研究分离的单基因分子的结构和在单细胞水平上的基因表达来解决这个问题。这项研究的基本工具是电子显微镜和荧光显微镜，结合数学模型，以及酵母遗传学。令人惊讶的是，最近的研究提供了证据，表明活跃的基因在基因开启或关闭的状态之间随机切换，而不是一直处于开启状态。这种随机切换导致单个基因产生的基因产物的数量随着时间的推移而波动。这些波动可以提供对基因如何激活的洞察，但开启/关闭切换的分子基础尚不清楚。一种可能性是，DNA的随机假脱机--类似于在随机选择的时间点继续打开和关闭一扇门--可能是活性基因开关的基础。对这一假设的检验需要在单基因分子水平上分析DNA假脱机。为此，本研究将通过分离基因分子和随后的电子显微镜分析来研究面包师酵母单细胞内特定基因的结构。这些分子将从携带DNA假脱机关键生化成分突变的细胞中分离出来，其中包括组成DNA假脱机核心的蛋白质。这些结构研究将结合荧光显微镜和数学模型对单细胞基因表达的分析，揭示DNA假脱机在基因表达和调控中的动力学和功能意义。更广泛的影响：在这个项目中，将培训一名博士后研究员和一名研究生。此外，本科生将通过学年或暑期实习进行培训。学生实习生将通过现有的校园计划从代表性不足的群体中招聘。这些学生将由PI直接在实验室和每两周一次的联合小组会议上监督，以及由博士后研究员和该项目的研究生监督。学生将接受荧光和电子显微镜、酵母菌遗传学方面的培训，更重要的是，通过量化手段解决生物学问题。与处于职业生涯不同阶段的科学家的日常互动将通过多层次的监督使本科生受益，并将使研究生和博士后实习生有机会监督初学者。研究生和博士后实习生将有机会在当地和全国会议上介绍他们的工作。这个项目由分子和细胞生物科学系的遗传机制组和数学科学系的应用数学和数学生物学项目共同资助。

Intellectual Merit: Animal, plant and fungal cells alike package their DNA by spooling it in regular intervals about a protein core, forming a jointed chain of DNA spools (nucleosomes). The spooling renders the DNA less accessible and thus interferes with the reading of its genetic information. How then does DNA spooling affect gene activity and regulation? This research addresses this question by investigating the structure of isolated single-gene molecules and gene expression at the single cell level. The essential tools for this research are electron and fluorescence microscopy, combined with mathematical modeling, and yeast genetics. Surprisingly, recent studies have provided evidence that active genes randomly toggle between states during which the gene is either ON or OFF, rather than being on all the time. This random toggling causes the number of gene products produced from a single gene to fluctuate over time. These fluctuations can provide insight into how the gene is activated, but the molecular basis of ON/Off toggling is not understood. One possibility is that random spooling and unspooling of DNA--analogous to the continued opening and closing of a door at randomly chosen time points--may underlie the ON/OFF toggling of active genes. Testing of this hypothesis requires analysis of DNA spooling at the level of single gene molecules. To this end, this research will investigate the structure of specific genes within single cells of baker's yeast by isolation of the gene molecules and subsequent analysis by electron microscopy. The molecules will be isolated from cells that bear mutations in critical biochemical components for DNA spooling, including the proteins that make up the core of the DNA spool. These structural investigations will be combined with analysis of gene expression of single cells by fluorescence microscopy and mathematical modeling to reveal the dynamics and functional significance of DNA spooling for gene expression and regulation. Broader Impacts: In this project a postdoctoral fellow and one graduate student will be trained. In addition, undergraduate students will be trained through academic-year or summer internships. Student interns will be recruited from underrepresented groups through existing campus programs. These students will be overseen by the PIs, both directly in the lab and in biweekly joint group meetings, and by the post-doctoral fellow and the graduate students of this project. Students will be trained in fluorescence and electron microscopy, yeast genetics and, importantly, in approaching biological problems by quantitative means. Daily interactions with scientists at different stages of their careers will benefit the undergraduates through multiple layers of supervision and will allow the graduate student and post-doctoral trainees to have the opportunity to supervise beginning students. The graduate student and post-doctoral trainee will be given the opportunity to present their work locally and at national meetings.This project is co-funded by the Genetic Mechanisms Cluster in the Division of Molecular and Cellular Biosciences and by the Applied Mathematics and Mathematical Biology Programs in the Division of Mathematical Sciences.