Single Chromatin Fiber Dynamics Studied via Magnetic Tweezers

染色质是当今大量研究工作的焦点，因为很明显，染色质不仅是一种将长DNA分子被动包装在细胞核范围内的方式，而且是调节所有处理遗传信息的核活动的重要参与者：转录，复制，修复，重组。 染色质结构必须在时间和空间上改变，以允许所有这些过程以高度可调节的方式发生，以响应外部和内部刺激。 染色质结构的大部分变化归因于组蛋白和DNA的合成后修饰的动态变化。 这些修饰与染色质重塑因子和序列或结构特异性蛋白因子协同作用，以松散紧密的高阶染色质组织并进一步开放核小体结构，以允许庞大的细胞机器接近潜在的基因。 单分子方法的出现提供了一套强大的工具，以前所未有的方式接近染色质结构和动力学，允许实时观察单个染色质纤维的行为，并评估纤维群体的个体代表之间的变异性。 本计画是基于使用磁性镊子进行单分子染色质纤维的研究。 在这种自制的仪器装置中，单个DNA分子通过其末端连接到表面和磁珠上，染色质由纯化的组蛋白和染色质组装因子组装而成。 在外部磁体的帮助下对磁珠的操纵允许以高度受控的方式在DNA分子中引入扭转应力，以及用定义的力拉伸它。 目的是了解染色质纤维在外部施加张力和/或扭转时的行为，以模拟体内生理过程产生的类似条件。 具体的目标包括以下染色质纤维组装/拆卸在真实的时间作为超螺旋密度和张力的DNA系链的函数，并测量核小体强度作为组蛋白乙酰化和DNA甲基化的函数。 这些新的知识对于我们理解染色质的结构和动力学以及染色质在调节DNA交易中的作用至关重要。这些项目的工作将产生重大的教育影响，为研究生和本科生提供生物学、物理学和化学方面的培训。 来自代表性不足的少数民族的学生将努力获得博士学位。 教育部分还将包括为研究生开发新的单分子生物学课程。这些项目的多学科性质将促进理工大学向研究和教育的综合科学方法过渡。 该大学刚刚创建了奥特默跨学科研究所，其使命是促进跨学科科学和教育。 这项研究将成为实现这一目标的核心。

Chromatin is nowadays the focus of enormous research effort, since it is clear that chromatin is not only a way to passively pack long DNA molecules within the confines of a cell nucleus, but is an essential participant in the regulation of all nuclear activities that process genetic information: transcription, replication, repair, recombination. Chromatin structure has to change in time and space to allow all these processes to occur in a highly regulatable manner, in response to external and internal stimuli. Most of the changes in chromatin structure have been attributed to dynamic changes in the post-synthetic modifications of both the histones and the DNA. These modifications act in concert with chromatin remodeling factors and with sequence- or structure-specific protein factors, to loosen the compact higher-order chromatin organization and further open nucleosome structure to allow access of the bulky cellular machineries to the underlying genes. The emergence of single-molecule methods has provided a powerful set of tools to approach chromatin structure and dynamics in an unprecedented way, allowing real-time observations on the behavior of individual chromatin fibers and assessing the variability among individual representatives of a fiber population. This project is based on the use of magnetic tweezers for single-molecule chromatin fibers studies. In this home-made instrumental set-up, a single DNA molecule is attached by its termini to a surface and a magnetic bead, and chromatin is assembled from purified histones and chromatin assembly factors. The manipulation of the magnetic bead with the help of external magnets allows introducing torsional stress in the DNA molecule in a highly controlled way, as well as stretching it with a defined force. The goal is to understand the behavior of the chromatin fiber upon external application of tension and/or torsion, to mimic similar conditions created by physiological processes in vivo. The specific aims include following chromatin fiber assembly/disassembly in real time as a function of the superhelical density and tension in the DNA tether, and measuring nucleosome strength as a function of histone acetylation and DNA methylation. The new knowledge will be crucial to our understanding of chromatin structure and dynamics, and of the role chromatin plays in regulating DNA transactions.The work on these projects will have a significant educational impact providing training for graduate and undergraduate students at the interface of biology, physics and chemistry. Students from underrepresented minorities will work towards obtaining a doctoral degree. The educational component will also include development of a new Single-Molecule Biology course for graduate students. The multidisciplinary nature of the projects will stimulate the transition to the integrated scientific approach to research and education the Polytechnic University is aiming to achieve. The University has just created the Othmer Institute for Interdisciplinary Studies, with the mission to promote interdisciplinary science and education. This research will serve as a nucleation core towards achieving this goal.