Internucleosomal Interactions in Chromatin Fibers and Metaphase Chromosomes

智力优势：包括人类细胞在内的真核细胞的DNA存在于细胞核中，细胞核紧密地包裹在复杂的、有层次组织的结构中，称为染色体。在组织的第一级，DNA像线轴上的线一样缠绕在组蛋白核心周围，形成称为核小体的结构。核小体相互作用，形成阵列和高阶结构，将DNA紧密压缩，使其适合细胞核。为了正常的细胞功能，高阶染色质结构必须展开，使DNA能够被进行转录、复制、重组和DNA修复的酶所接触。另外，染色质可以采用一种被称为异染色质的浓缩状态，这种状态是转录非活性的（抑制的）。该项目的目的是推断核小体之间相互作用的基本原理，核小体导致不同高阶染色质结构的可逆形成，这些结构是凝聚或去凝聚状态的基础。该项目将通过实验验证以下假设，即核小体阵列在凝聚异染色质中的压缩是由a)核小体阵列内部和之间的核小体相互作用和b)连接子DNA的灵活性决定的，这种灵活性可以促进阵列内相互作用（染色质“二级结构”）或阵列间相互作用（染色质“三级结构”）。实验设计包括两个特定目的：1)鉴定重组核小体阵列中调节连接体DNA构象和核小体间相互作用的连接体DNA序列基序。2)确定原生间期染色质和浓缩中期染色体原位核小体间相互作用的模式。在第二个目标中，核小体相互作用将固定在活细胞中，然后分离染色质，展开并通过电子显微镜进行分析。这些实验将阐明整体染色质高阶组织及其在体内的转变。这项工作采用已建立的生化和电子显微镜实验技术以及新的生化方法来捕获体内染色质结构（“原位电子显微镜辅助核小体相互作用捕获”）。更广泛的影响：预计该项目可能会大大改变现代分子生物学和遗传学教科书中目前的模型，其中30纳米染色质纤维被描述为染色体折叠的普遍中间体。这些研究有望揭示间期细胞和中期染色体中动态染色质压实的另一种高阶结构类型。该项目正在开发的新信息和方法对于理解染色质中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。除了一般的科学知识外，该项目还将为参加宾夕法尼亚州立大学暑期本科生研究项目的本科生提供新的研究培训和教育机会，其中许多人就读于宾夕法尼亚州中部的小型农村学院。这些学生来自代表性不足的少数群体，以及农村环境，他们以前很少接触实验科学。该项目将在支持分子成像和电子显微镜研究和培训的教育基础设施方面发挥关键作用。该项目还包括一些特别适合本科生学员的任务，这些任务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，并培养他们促进科学研究兴趣和动机所需的初步信心。

Intellectual Merit: The DNA of eukaryotic cells, including human cells, is found in the nucleus tightly packed in complex, hierarchically organized structures called chromosomes. At the first level of organization, the DNA is coiled around histone protein cores like string on a spool to form structures called nucleosomes. The nucleosomes interact with each other to form arrays and higher-order structures, tightly compacting the DNA to fit it in the nucleus. For proper cell functioning, the higher-order chromatin structures must unfold to make the DNA accessible to enzymes that carry out transcription, replication, recombination, and repair of DNA. Alternatively, chromatin can adopt a condensed state known as heterochromatin that is transcriptionally inactive (repressed). The objective of this project is to deduce the basic principles of interaction between nucleosomes that leads to reversible formation of different higher-order chromatin structures that underlie the condensed or decondensed states. This project will experimentally test the hypothesis that nucleosome array compaction in condensed heterochromatin is determined by a) inter-nucleosomal interactions within and between nucleosomal arrays and b) linker DNA flexibility that promotes either intra-array interactions (chromatin "secondary structure") or inter-array interactions (chromatin "tertiary structure"). The experimental design includes two specific aims: 1) To identify linker DNA sequence motifs modulating linker DNA conformation and inter-nucleosomal interactions in reconstituted nucleosome arrays. 2) To determine patterns of inter-nucleosomal interactions in native interphase chromatin and condensed metaphase chromosomes in situ. In the second aim, the nucleosome interactions will be fixed in living cells and then chromatin isolated, unfolded, and analyzed by Electron Microscopy. These experiments should elucidate global chromatin higher-order organization and its transitions in vivo. This work employs established biochemical and electron microscopic experimental techniques and novel biochemical approaches to capture in vivo chromatin structure ("in-situ electron microscopy-assisted nucleosome interaction capture"). Broader Impacts: It is anticipated that this project may substantially change the current models featured in modern molecular biology and genetics textbooks in which the 30 nm chromatin fiber is depicted as a universal intermediate in chromosome folding. These studies are expected to reveal alternative type(s) of higher order structures underlying dynamic chromatin compaction in interphase cells and in metaphase chromosomes. 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. In addition to the general scientific knowledge, this project will provide new research training and education opportunities to undergraduate students participating in the Penn State Summer Undergraduate Research program, many of whom attend small rural colleges in central Pennsylvania. The students come from under-represented minority groups as well as rural environments which provide little previous exposure to experimental science. The project will play a crucial role in supporting the educational infrastructure for research and training in molecular imaging and electron microscopy. This project also includes a number of tasks especially suitable for undergraduate trainees 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.