Higher-Order Structure Of Chromatin

这个项目解决了分子机制，使几米长的DNA（基因组）能够被包装成细胞核，而细胞核的直径大约比细胞核小10万倍。DNA的这种惊人的紧实程度是通过DNA双螺旋绕多个线轴反复盘绕而实现的，每个线轴都由蛋白质（组蛋白）核心组成。这样，DNA就形成了一种被称为核小体的DNA/组蛋白颗粒的“串珠”阵列。然后，核小体阵列进一步折叠成包含多个环的结构，其中DNA受到物理约束。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）的结构组织仍然是一个谜。本研究将使用生化实验和计算三维模型来描述活细胞中DNA的结构相互作用，并预测结构如何影响功能。该项目将促进对研究生和本科生的教育和培训，包括那些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人，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的高阶结构是基于核小体形成线状染色质纤维的锯齿状或线状折叠来建模的。然而，最近的体内研究表明，在大多数细胞类型中，柔性和无序核小体阵列的动态环而不是线性纤维。该项目的核心假设是，连接核小体珠的连接体DNA的不同长度和构象施加了不同的拓扑状态和核小体包装顺序，这些结构域将染色质结构域分离成灵活的核小体链，促进远端DNA元件之间的功能相互作用，或者紧密排列的核小体抑制远端染色体位点之间的相互作用。这一假设将通过一种独特的电子显微镜核小体相互作用捕获方法、DNA拓扑分析和计算模型来验证，以确定精确定位的核小体和从酵母细胞中分离的圆形小染色体的核小体相互作用模式和DNA拓扑结构。这些结果将提供更详细的染色质组织图，因为它可能存在于细胞中，并且这些知识将作为更好地理解染色质特征如何调节基因表达的框架。该奖项由遗传学机制（分子和细胞生物科学部）和生命系统物理学（物理部）的项目共同资助。

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).