SINGLE-MOLECULE APPROACHES TO CHROMATIN STRUCTURE AND DYNAMICS

我们的研究是通过单分子方法研究染色质的结构和功能。单分子方法(例如原子力显微镜、原子力显微镜、光学镊子)可以回答通过总体总体实验(例如凝胶电泳法等)难以(如果不是不可能)回答的特定问题。我们对染色质的兴趣是：(I)连接体和核心组蛋白(变体，化学计量)及其对纤维结构的贡献(Ii)翻译后修饰(乙酰化、磷酸化、ADP-核糖化等)。对于AFM，我们的方法是双重的：一方面是蛋白质/DNA复合体的成像(不同组成的染色质纤维的组成、化学计量比和翻译后修饰)，另一方面是通过AFM尖端的直接操作来探测将染色质纤维结合在一起的力。我们已经开始应用一种新技术来研究染色质纤维--用原子力显微镜直接测量单个染色质纤维的拉伸力。在任何真核细胞的生命中，力是涉及染色质和染色体结构重组的许多过程中的一个因素。此外，还需要强制清除DNA中的组蛋白，以进行生物过程，如转录、复制和修复。力的产生和对生物结构的应用是动态生物过程的一个重要组成部分，但到目前为止，控制染色质结构和功能的力还没有得到实验研究。拉动合成的染色质纤维会导致连接物DNA的拉伸，但即使施加数百皮牛顿的力，核小体也会留在纤维上。这种作用力远远超出了已知的分子马达的能力，这意味着在核小体被移除之前，染色质结构必须被削弱，可能是由于染色质重塑因素。我们对染色质纤维作用力效应的兴趣使我们与荷兰特温特大学的研究人员合作，将光学镊子应用于染色质纤维。使用光镊子，我们可以探测1-150皮牛顿的下力区，而使用原子力显微镜，我们可以探测100皮牛顿以上的力。在这些光钳实验中，我们首先在两个珠子之间连接一段DNA，证明它是一个完整的DNA单分子，可以经历众所周知的B-DNA到Z-DNA的转变，然后通过将核组装提取物注入仪器的液体细胞中，将组蛋白组装到DNA上。这些类型的实验开启了一种全新的方法，因为有了核提取液，就有可能将染色质与组蛋白的各种互补成分(即仅核心组蛋白H3/H4四聚体、荧光修饰的组蛋白、带有或不带有连接子的组蛋白亚型等)组装在一起。初步结果表明，该系统具有足够的灵敏度来检测组装在lambda DNA 48,502bp上的~250个核小体中的单个核小体的破坏。-原子力显微镜、单分子检测和操纵、染色质结构、功能和动力学，

Our research is investigating chromatin structure and function as revealed by single molecule approaches. Single molecule approaches (e.g. atomic force microscopy AFM, optical tweezers) can answer particular questions that are difficult (if not impossible) to answer by population-ensemble experiments (e.g. gel electrophoresis etc.). Our interests in chromatin are (i) linker and core histones (variants, stoichiometry) and their contributions to fiber structure (ii) post- translational modifications (acetylation, phosphorylation, ADP- ribosylation etc.) of histones affecting structure, (iii) effect of DNA methylation on fiber structure and (iv) interactions of sequence specific chromatin fibers or single nucleosomes with biological processes (e.g. polymerases and other chromatin remodeling factors).With AFM our approach is twofold: on one side there is the imaging of protein/DNA complexes (chromatin fibers of various composition, stoichiometry and post-translational modifications), and on the other side there is the direct manipulation with the AFM tip to probe the forces holding the chromatin fiber together. We have initiated the application of a new technique to study chromatin fibers - directly measuring the force of stretching single chromatin fibers with the atomic force microscope. Force is a factor in many processes involving chromatin and chromosome structural reorganizations during the life of any eukaryotic cell. Additionally, there is a need for force to clear histones from the DNA for biological processes such as transcription, replication and repair. Force generation and application to biological structure is a major component of dynamic biological processes, but forces governing chromatin structure and function have not been experimentally approached up to now. Pulling of synthetic chromatin fiber causes stretching of the linker DNA, but the nucleosomes stay on the fiber, even when forces of several hundred piconewtons are applied. Such forces are well beyond the capability of known molecular motors, which implies that chromatin structure must be weakened, possibly by chromatin remodeling factors, before nucleosomes can be removed. Our interests in the effect of force applied to chromatin fibers has lead us to collaborate with researchers at the University of Twente (The Netherlands) on applying optical tweezers to chromatin fibers. With the optical tweezers we can probe the lower region of forces 1-150 piconewtons whereas with the AFM we can probe forces above 100 piconewtons. In these optical tweezers experiments, we first attach a piece of DNA between two beads, demonstrate it is an intact single molecule of DNA that can undergo the well known B-DNA to Z-DNA transition, and then assemble histones onto DNA by injecting a nuclear assembly extract into the liquid cell of the instrument. These kinds of experiments open a whole new approach because with the nuclear extract it is possible to assemble chromatin with various complements of histones (i.e., only core histone H3/H4 tetramers, fluorescently modified histones, with or without linker histone subtypes, etc.). Preliminary results suggest that this system is sensitive enough to detect the disruption of single nucleosomes among the ~250 nucleosomes assembled on the 48,502 bp of lambda DNA. - Atomic force microscopy, single molecule detection and manipulation, chromatin structure, function and dynamics,