用单分子操控结合荧光成像研究染色质的组装和动力学

Single molecule manipulation and single molecule imaging are two cutting-edge techniques for modern biological physics. In this project we will make a combination of them to study the longstanding fundamental problems of nucleosome assembly and chromatin dynamics. In eukaryotic cell chromatin is hierarchically compacted with different folding levels and organized in three dimensions via interacting with other nuclear structures within the nucleus. Nucleosomes form the fundamental repeating units of eukaryotic chromatin and are thought to carry epigenetically inherited information in the form of covalent modifications of their core histones. We will first perform in situ studies on the assembly of individual nucleosomes on short DNA molecules by using magnetic tweezers which are integrated onto a fluorescence microscope. At the same time we will use single molecule spectroscopy to monitor interactions of histones with tethered DNA, and nucleosome sliding caused by chromatin remodeling complexes as well. Secondly, we will study the structural changes of pre-assembled chromatins under manipulation of magnetic tweezers. Again, single molecule spectroscopy can be used to monitor the behaviors of different nucleosome proteins accompanying the structural changes of the chromatin. In all these studies, we will investigate how the DNA methylation, histone modifications and variants and chromatin remodeling complexes are involved in regulating higher order chromatin dynamics. The results of our studies will provide important mechanistic insights into the biological function of chromatin structure and organization in epigenetic inheritance, development and cell differentiation, and epigenetic de-regulation in human diseases.

单分子操控与单分子显微成像是现代生物物理学(biological physics)中的创新技术。本项目中我们将两种技术集成在一起，并应用到核小体和染色质的组装及动力学研究中。正确的染色质结构是保证DNA复制、转录、重组和修复等正常进行的基础。染色质结构变化及调控的分子机理是理解细胞增殖发育和分化过程中重要基因的表达差异及其表观遗传学的关键。我们用单分子磁镊研究短DNA链上单个核小体的原位组装，同时用单分子光谱监测与该DNA链相互作用的蛋白质的数量与时序,以及核小体在染色质重塑因子等作用下的移位等。其次我们用单分子力谱研究预先组装好的染色质结构转变，用单分子光谱研究伴随这些结构转变的蛋白质的动力学。我们还将定量比较各种表观遗传机制（如DNA化学修饰以及组蛋白变体等）对单个核小体和染色质纤维结构和动力学的调控，为阐明各种表观遗传机制的作用机理提供物理学基础。

本项目按既定计划研究了核小体与染色质的动态结构。在项目执行过程中，我们提高了实验设备的功能，将单分子力谱技术与单分子荧光技术有机结合起来，做出了很多出色的研究成果。首先，我们研究了30nm染色纤维的动态结构，发现了一个我们命名为“四聚核小体串珠”染色质中间态；其次，我们发现组蛋白H1不仅增强核小体的稳定性，还加快核小体的折叠动力学；其三，我们研究了神经细胞中代替H1的修饰蛋白MeCP2对染色质结构的改变，发现了一个全新的结构。此外，我们还研究了与基因调控有关的几个酶，也获得了重要成果。本项目研究成员在学术杂志共发表论文5篇，培养博士5名，其中毕业4名，圆满地完成任务书的内容。