Single-Molecule Approaches to Chromatin Structure and Dynamics

我们的研究是通过单分子方法来研究染色质的结构和功能。单分子方法（例如原子力显微镜AFM，光学镊子）可以回答特定的问题，这些问题很难（如果不是不可能的话）通过群体系综实验（例如凝胶电泳等）来回答。我们对染色质的兴趣是（i）接头和核心组蛋白（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-核糖基化等）影响结构的组蛋白，（iii）DNA甲基化对纤维结构的影响，以及（iv）序列特异性染色质纤维或单个核小体与生物过程的相互作用（例如聚合酶和其他染色质重塑因子）。使用AFM，我们的方法是双重的：一方面是蛋白质/DNA复合物的成像（各种组成、化学计量和翻译后修饰的染色质纤维），另一方面是用AFM针尖直接操作，以探测将染色质纤维保持在一起的力。我们开创了一种研究染色质纤维的新技术--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过程中，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，需要力来从DNA中清除组蛋白，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，但是到目前为止，控制染色质结构和功能的力还没有被实验研究。拉扯合成染色质纤维会导致连接DNA的拉伸，但核小体会留在纤维上，即使施加数百微微牛顿的力。这种力远远超出了已知分子马达的能力，这意味着在核小体被去除之前，染色质结构必须被削弱，可能是通过染色质重塑因子。我们对施加于染色质纤维的力的影响的兴趣使我们与Twente大学（荷兰）的研究人员合作，将光镊应用于染色质纤维。使用光镊，我们可以探测1-150微微牛顿的力的较低区域，而使用AFM，我们可以探测100微微牛顿以上的力。在这些光镊实验中，我们首先将一段DNA连接在两个珠子之间，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单元中将组蛋白组装到DNA上。这些类型的实验开启了一种全新的方法，因为使用核提取物，可以将染色质与组蛋白的各种互补物组装在一起（即，仅核心组蛋白H3/H4四聚体、荧光修饰的组蛋白、具有或不具有接头组蛋白亚型等）。初步结果表明，该系统足够灵敏，可以检测到在48，502 bp的λ DNA上组装的~250个核小体中单个核小体的破坏。- 原子力显微镜，单分子检测和操作，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,