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

智力优势：动物、植物和真菌细胞都是通过将DNA以规则的间隔缠绕在蛋白质核心上来包装它们的DNA，形成DNA线轴（核小体）的连接链。假脱机使DNA不易接近，从而干扰其遗传信息的阅读。那么DNA缠绕是如何影响基因的活性和调控的呢？本研究通过研究单基因分子的结构和单细胞水平的基因表达来解决这个问题。这项研究的基本工具是电子和荧光显微镜，结合数学建模和酵母遗传学。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数量随时间波动。这些波动可以让我们了解基因是如何被激活的，但是开/关切换的分子基础还不清楚。一种可能性是DNA的随机缠绕和解开-类似于在随机选择的时间点继续打开和关闭一扇门-可能是活性基因的ON/OFF切换的基础。检验这一假设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酵母单细胞内特定基因的结构。这些分子将从细胞中分离出来，这些细胞在DNA线轴的关键生化成分中发生突变，包括构成DNA线轴核心的蛋白质。这些结构研究将与荧光显微镜和数学建模的单细胞基因表达分析相结合，以揭示DNA缠绕对基因表达和调控的动力学和功能意义。更广泛的影响：在这个项目中，将培养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募。这些学生将直接在实验室和双周联合小组会议上由PI监督，并由博士后研究员和本项目的研究生监督。学生将接受荧光和电子显微镜，酵母遗传学的培训，重要的是，通过定量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监督使本科生受益，并使研究生和博士后学员有机会监督初学者。研究生和博士后实习生将有机会在当地和国家会议上介绍他们的工作，该项目由分子和细胞生物科学司的遗传机制小组和数学科学司的应用数学和数学生物学方案共同资助。

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.