Single Chromatin Fiber Dynamics Studied via Magnetic Tweezers

染色质是目前大量研究工作的焦点，因为很明显，染色质不仅是在细胞核范围内被动地包装长DNA分子的一种方式，而且是处理遗传信息的所有核活动的调节的重要参与者：转录，复制，修复，重组。染色质结构必须在时间和空间上发生变化，以使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。大多数染色质结构的变化归因于组蛋白和DNA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，使致密的高阶染色质组织松散，并进一步打开核小体结构，使庞大的细胞机器能够接近潜在的基因。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，并评估纤维群体个体代表之间的可变性。这个项目是基于使用磁镊子对单分子染色质纤维的研究。在这个自制的仪器装置中，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。在外部磁铁的帮助下操纵磁珠，可以以高度可控的方式在DNA分子中引入扭转应力，并以确定的力拉伸它。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。具体目标包括实时跟踪染色质纤维的组装/拆卸，作为DNA系链中超螺旋密度和张力的函数，以及测量核小体强度作为组蛋白乙酰化和DNA甲基化的函数。新的知识将是至关重要的，我们的染色质结构和动力学的理解，以及染色质在调节DNA交易中的作用。这些项目的工作将对教育产生重大影响，为研究生和本科生提供生物学、物理学和化学界面方面的培训。来自未被充分代表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设新的单分子生物学课程。这些项目的多学科性质将促进向综合科学方法的过渡，以研究和教育理工大学的目标是实现。牛津大学刚刚成立了奥特曼跨学科研究所，其使命是促进跨学科的科学和教育。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.