Higher-Order Structure Of Chromatin

该项目致力于研究使数米长的DNA（基因组）被包装成直径约为10万倍的细胞核的分子机制。DNA的这种惊人的紧密程度是通过DNA双螺旋围绕多个线轴反复卷曲来实现的，每个线轴都由蛋白质（组蛋白）核心组成。因此，DNA形成了一个“串珠状”的DNA/组蛋白颗粒阵列，称为核小体。然后，核小体阵列进一步折叠成包括多个环的结构，其中DNA被物理限制。除了对这种结构的基本理解之外，细胞核中DNA-蛋白质复合物（称为染色质）的结构组织仍然是个谜。这项研究将使用生物化学实验和计算三维建模来描述活细胞中DNA的结构相互作用，并预测结构如何影响功能。该项目将促进研究生和本科生的教育和培训，包括来自代表性不足的群体和宾夕法尼亚州农村服务不足地区的学生，他们通常很少有机会参与基础研究。在目前的大多数教科书中，DNA的高级结构是基于形成线性染色质纤维的核小体的Z字形或螺线管折叠来建模的。然而，最近的体内研究表明，在大多数细胞类型中，柔性和无序的核小体阵列而不是线性纤维的动态循环。该项目集中在一个假设上，即连接核小体珠的接头DNA的不同长度和构象施加不同的拓扑状态和核小体包装的顺序，将染色质结构域分离成促进远端DNA元件之间的功能相互作用的柔性核小体链，或者，抑制远端染色体位点之间相互作用的紧密包装的核小体。将使用独特的电子显微镜核小体相互作用捕获方法、DNA拓扑分析和计算建模来测试这一假设，以确定从酵母细胞中分离的精确定位的核小体和环形微型染色体的环形阵列的核小体相互作用和DNA拓扑结构的模式。研究结果将提供一个更详细的图片染色质组织，因为它可能存在于细胞中，这方面的知识将作为一个框架，更好地了解染色质的功能如何调节基因的表达。这个奖项是共同资助的计划遗传机制（分子和细胞生物科学部）和生命系统的物理学（物理部）。

This project addresses the molecular mechanisms that enable several meters of DNA (the genome) to be packaged into a cell nucleus which has a diameter that is approximately 100,000 times smaller than this. This amazing degree of compaction of the DNA is achieved by repeatedly coiling of the DNA double helix around multiple spools, each of which is composed of protein (histone) cores. The DNA is thus formed into a "beads-on-a-string" array of DNA/histone particles called nucleosomes. The nucleosome arrays are then further folded into structures that include multiple loops where the DNA is physically constrained. Beyond a basic understanding of this structure, the architectural organization of the DNA-protein complex in the cell nucleus (known as chromatin), remains enigmatic. This research will use biochemical experiments and computational 3-dimensionsal modeling to describe the structural interactions of DNA in living cells and to predict how the structure impacts function. The project will promote education and training for graduate students and for undergraduates, including those from underrepresented groups and underserved areas of rural Pennsylvania who typically have little opportunity to participate in fundamental research. In most current textbooks, the higher-order structure of DNA is modeled based on zigzag or solenoidal folding of nucleosomes forming linear chromatin fibers. However, recent in vivo studies suggested a dynamic looping of flexible and disordered nucleosome arrays rather than linear fibers in most cell types. This project is centered on a hypothesis that different lengths and conformations of the linker DNA connecting nucleosome beads impose distinct topological states and orders of nucleosome packing that segregate the chromatin domains into either flexible nucleosome chains facilitating functional interactions between distant DNA elements or, alternatively, tightly packed nucleosomes that inhibit interactions between distant chromosomal sites. This hypothesis will be tested using a unique electron microscopy nucleosome interaction capture method, DNA topology assays, and computational modeling to determine patterns of nucleosome interactions and DNA topology of both circular arrays of precisely positioned nucleosomes and circular minichromosomes isolated from yeast cells. The results will provide a more detailed picture of chromatin organization as it is likely to exist in the cell, and this knowledge will serve as a framework for better understanding of how chromatin features regulate gene expression.This award is co-funded by programs in Genetics Mechanism (Division of Molecular and Cellular Biosciences) and the Physics of Living Systems (Division of Physics).