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，紧密排列在复杂的、有层次组织的结构中，称为染色体。在第一级组织中，DNA缠绕在组蛋白核心上，就像线轴上的绳子一样，形成称为核小体的结构。核小体相互作用形成阵列和更高级别的结构，紧密地压缩DNA以适应细胞核。为了正常的细胞功能，高阶染色质结构必须展开，以使DNA能够被执行DNA转录、复制、重组和修复的酶所访问。或者，染色质可以采用一种被称为异染色质的浓缩状态，这种状态在转录上是不活跃的(被抑制)。这个项目的目标是推导出核小体之间相互作用的基本原理，这种相互作用导致不同的高阶染色质结构的可逆形成，这些结构是凝聚态或解凝聚态的基础。该项目将通过实验测试这一假设，即浓缩异染色质中的核小体阵列致密是由a)核小体阵列内和核小体阵列之间的核小体间相互作用以及b)连接体DNA灵活性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”)决定的。实验设计包括两个特定的目的：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)原位测定天然间期染色质和浓缩中期染色体的核小体间相互作用模式。第二个目的是将核小体相互作用固定在活细胞中，然后分离、展开染色质，并用电子显微镜进行分析。这些实验应该阐明整体染色质的高阶组织及其在体内的转变。这项工作采用了成熟的生化和电子显微镜实验技术和新的生物化学方法来捕捉体内的染色质结构(原位电子显微镜辅助的核小体相互作用捕捉)。更广泛的影响：预计这个项目可能会极大地改变现代分子生物学和遗传学教科书中的现有模型，在这些教科书中，30 nm的染色质纤维被描述为染色体折叠的通用中间体。这些研究有望揭示间期细胞和中期染色体中存在的另一种类型的高级结构(S)，这种结构是动态染色质紧凑的基础。在该项目下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，该项目还将为参加宾夕法尼亚州立大学暑期本科生研究计划的本科生提供新的研究培训和教育机会，他们中的许多人都在宾夕法尼亚州中部的小型农村学院就读。这些学生来自代表性不足的少数群体以及农村环境，这些环境以前几乎没有接触到实验科学。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。这个项目还包括一些特别适合本科生的任务，使他们能够将生物化学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.