Molecular and structural determinants of heterochromatin

异染色质的分子和结构决定因素

• 批准号: 0615536
• 负责人: Sergei Grigoryev
• 金额: --
• 依托单位: Pennsylvania State Univ University Park
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-09-01 至 2010-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0615536&HistoricalAwards=false
• 关键词: Molecular; structural; determinants; heterochromatin

In the chromosomes within the cell nuclei of eukaryotic organisms, DNA is periodically coiled around histone protein cores to form nucleosome arrays. These arrays are further folded in a zig-zag manner into higher order chromatin structures. For proper cell functioning, the higher order chromatin structures must unfold to make DNA accessible to transcription, replication, recombination, and repair. Alternatively, it can adopt a condensed and repressed state known as heterochromatin. The objective of this project is to deduce the basic principles of nucleosome array architecture that support the condensed heterochromatin state and may prevent chromatin unfolding. This project is aimed to test the hypothesis that nucleosome array compaction in condensed heterochromatin is determined by a) nucleosome orientation in zig-zag chromatin arrays and b) protein factors that promote inter-array nucleosome bridging (nucleosome array self association caused by a protein factor connecting two or more separate arrays) over intra-array nucleosome folding (compaction within a single nucleosome array). The experimental design includes two specific aims. One specific aim is to determine the internucleosome orientations and the architectural protein factors that promote either folding or bridging of the nucleosome arrays. The second specific aim is to determine the topography of nucleosome interactions in compact chromatin. This work combines the utilization of established biochemical and electron microscopic experimental techniques with the development of novel approaches to assess chromatin structure, such as electron microscopy and polymerase chain reaction-assisted nucleosome interaction capture techniques. Broader Impacts: More than 50 years since the discovery of DNA double helix, its higher order packing in condensed chromatin and chromosomes is still unknown. This work should help to solve the long-standing problem of spatial organization of higher-order chromatin and uncover the nature of chromatin structural transitions involved in heterochromatin formation and chromatin condensation. The new information and methodology that is being developed under this project is crucial for understanding spatial organization of DNA in chromatin and its relationship to fundamental mechanisms of heterochromatin formation, gene silencing, and cell differentiation and thus should significantly contribute to basic science education in molecular biology and genetics. In addition to the general scientific knowledge, this project is aimed to provide new research training and education opportunities for undergraduate students from the Penn State Summer Undergraduate Research program and other colleges in central Pennsylvania. Many of these students represent minority groups and come from environments that have little previous exposure to experimental science. This research will be also essential for developing educational infrastructure to facilitate graduate student training in molecular imaging and electron microscopy. This project includes a number of tasks (such as chromatin template "construction kit", histone isolation, and nucleosome reconstitution) especially suitable for undergraduates and entry-level graduate students, that will allow them to relate biochemical experiments to visual changes in chromatin structure as observed by electron microscopy and to develop the initial confidence necessary for them to promote their interest and motivation for scientific research.

在真核生物的细胞核内的染色体中，DNA周期性地盘绕在组蛋白核心周围以形成核小体阵列。这些阵列进一步以Z字形方式折叠成更高阶的染色质结构。为了正常的细胞功能，更高级的染色质结构必须展开，使DNA能够转录、复制、重组和修复。或者，它可以采取浓缩和抑制状态称为异染色质。本计画的目的是推导出核小体阵列架构的基本原理，以支持异染色质的凝聚状态，并可能阻止染色质的展开。本项目旨在检验以下假设：凝聚异染色质中的核小体阵列压实由a）Z字形染色质阵列中的核小体方向和B）促进阵列间核小体桥接（由连接两个或多个单独阵列的蛋白质因子引起的核小体阵列自缔合）超过阵列内核小体折叠（单个核小体阵列内的压实）的蛋白质因子决定。实验设计包括两个具体目标。一个具体的目标是确定核小体间的方向和建筑蛋白质的因素，促进折叠或桥接的核小体阵列。 第二个具体目标是确定紧凑的染色质中的核小体相互作用的拓扑结构。 这项工作结合了利用已建立的生化和电子显微镜实验技术的发展，新的方法来评估染色质结构，如电子显微镜和聚合酶链反应辅助核小体相互作用捕获技术。更广泛的影响：DNA双螺旋结构被发现已有50多年的历史，但其在凝聚染色质和染色体中的高级排列仍是未知数。这项工作应有助于解决长期存在的问题，空间组织的高阶染色质和发现的性质，染色质结构的转变参与异染色质形成和染色质凝聚。该项目正在开发的新信息和方法对于了解染色质中DNA的空间组织及其与异染色质形成、基因沉默和细胞分化的基本机制的关系至关重要，因此应该为分子生物学和遗传学的基础科学教育做出重大贡献。除了一般的科学知识，该项目的目的是提供新的研究培训和教育机会，本科生从宾夕法尼亚州立大学夏季本科研究计划和其他学院在宾夕法尼亚州中部。这些学生中的许多人代表少数群体，来自以前很少接触实验科学的环境。这项研究也将是必不可少的发展教育基础设施，以促进研究生在分子成像和电子显微镜的培训。该项目包括一些特别适合本科生和入门级研究生的任务（如染色质模板“构建工具包”，组蛋白分离和核小体重建），这将使他们能够将生化实验与电子显微镜观察到的染色质结构的视觉变化联系起来，并培养他们对科学研究的兴趣和动机所需的初步信心。