Mechanistic studies of heterochromatin mesoscale structural dynamics with DNA origami nanotechnology

基因组的物理性质在控制基本过程中起着核心作用，包括基因表达，DNA修复和DNA复制。所有的真核生物，从酵母到人类，都是通过重复地将它们的基因组DNA包裹在DNA相关的蛋白质（称为组蛋白）周围，形成称为核小体的小线轴来组织它们的基因组DNA。这些核小体的大小约为百万分之一厘米（10纳米），它们像珠子一样串在一起，形成长的染色质纤维，最终构成染色体。这些核小体在染色质纤维内的空间排列经常通过协调运动而不断变化。然而，目前的技术在量化这些结构重排方面受到限制，这种限制反过来又限制了我们对染色质如何控制基因表达的理解。该项目将通过开发基于DNA的纳米尺寸卡尺来监测染色质中的结构变化，因为基因表达在“开”和“关”状态之间切换，以检测10纳米至100纳米尺度的染色质结构变化，这涵盖了基因表达调控过程中事件的关键范围。研究生和本科生将接受分子生物学，生物化学，DNA纳米技术和单分子检测方面的培训，这将使他们成为下一代生物技术科学家的重要贡献者。PI还将与外展计划合作，包括OSU少数民族工程计划，工程计划中的妇女和硕士到博士。桥梁计划，以提高生物技术和生物物理学领域的多样性和兴趣。染色质在开放常染色质和紧密异染色质之间的转换对于转录和细胞分化的调控至关重要。这种转换是由组蛋白翻译后修饰（PTM）和染色质结构蛋白，识别这些PTM动态控制染色质结构。然而，调控转录的动态染色质结构特性目前还不清楚。这些性质包括核小体之间距离的变化、核小体方向的变化以及这些结构变化发生的时间。为了直接研究染色质结构动力学，该项目正在使用DNA折纸纳米技术开发基于DNA的纳米卡尺，以量化大分子复合物内10 nm至100 nm的距离变化。该项目将使用这种DNA纳米卡尺设计：（i）确定异染色质蛋白1与组蛋白H3 PTM，赖氨酸9三甲基化相互作用时形成的异染色质的长程结构动力学，以及（ii）确定压实异染色质对转录激活复合物结合的长程结构和动力学响应。这种方法涉及将含有PTM的染色质分子整合到DNA纳米卡尺中，其中每个末端连接到相对的纳米卡尺臂。然后分别用透射电子显微镜检测DNA纳米卡尺构象变化，以确定染色质构象的分布，并用单分子荧光检测不同染色质结构状态之间的转换速率。这些研究将提供关键的距离和时间测量的结构动态过程中发生的异染色质形成和异染色质解压缩的转录激活剂结合到它们的靶位点。更广泛地说，这种DNA折纸纳米技术的发展将广泛适用于研究许多在10 nm至100 nm长度尺度上发生结构变化的生物复合物。该项目由生物科学理事会分子和细胞生物科学部的遗传机制和分子生物物理学项目联合支持，以及物理和数学科学理事会材料研究部的生物材料计划。

The physical properties of the genome play a central role in controlling essential processes, including gene expression, DNA repair, and DNA replication. All eukaryotic organisms, from yeast to humans, organize their genomic DNA by repeatedly wrapping it around DNA-associated proteins, called histones, into small spools known as nucleosomes. These nucleosomes, which are about a millionth of a centimeter (10 nanometers) in size, are strung like beads on a string to form long chromatin fibers that ultimately make up chromosomes. The spatial arrangement of these nucleosomes within the chromatin fibers continually changes, often through coordinated movements. However, current technologies are limited in quantifying these structural rearrangements, and this limitation has in turn limited our understanding of how chromatin controls gene expression. This project will monitor structural changes in chromatin as gene expression is switched between the 'on' and 'off' states by developing DNA-based nanometer-sized calipers to detect structural changes in chromatin at the 10 nanometer to 100 nanometer scale, which covers a critical range for events during the regulation of gene expression. Graduate and undergraduate students will receive training in molecular biology, biochemistry, DNA nanotechnology, and single molecule detection, which will position them to become significant contributors as the next generation of biotechnology scientists. The PIs will also work with outreach programs, including the OSU Minority Engineering Program, the Women in Engineering Program, and the Masters to Ph.D. Bridge Program, to improve diversity and interest in the fields of biotechnology and biophysics.The conversion of chromatin between open euchromatin and compact heterochromatin is essential for the regulation of transcription and cellular differentiation. This conversion is regulated by histone post-translational modifications (PTMs) and chromatin architectural proteins that recognize these PTMs to dynamically control chromatin structure. However, the dynamic chromatin structural properties that regulate transcription are currently not well understood. These properties includes changes in the distance between nucleosomes, changes in nucleosome orientation, and the time for these structural changes to occur. To directly investigate chromatin structural dynamics, this project is using DNA origami nanotechnology to develop DNA-based nano-calipers that quantify 10 nm to 100 nm distance changes within large macromolecular complexes. The project will use this DNA nano-caliper design to: (i) determine the long range structural dynamics of heterochromatin that is formed by Heterochromatin Protein 1 as it interacts with the histone H3 PTM, lysine 9 trimethylation, and (ii) determine the long range structural and dynamic response of compacted heterochromatin to the binding of transcription activating complexes. This approach involves integrating PTM-containing chromatin molecules into DNA nano-calipers where each end is attached to opposite nano-caliper arms. The DNA nano-caliper conformational changes will then be detected separately with transmission electron microscopy to determine the distribution of chromatin conformations and single molecule fluorescence to determine the transition rates between distinct chromatin structural states. These studies will provide key distance and time measurements of the structural dynamics that occur during heterochromatin formation and heterochromatin decompaction as transcription activators bind to their target sites. More broadly, the development of this DNA origami nanotechnology will be widely applicable to the study of numerous biological complexes that undergo structural changes on the 10 nm to 100 nm length scale.This project is jointly supported by the Genetic Mechanisms and the Molecular Biophysics Programs of the Molecular and Cellular Biosciences Division in the Directorate for Biological Sciences, and by the Biomaterials Program of the Division of Materials Research in the Directorate for Physical and Mathematical Sciences.