High resolution chromatin structure of purified eukaryotic genes

项目摘要/摘要 在真核生物中，基因组DNA通过与组蛋白结合而浓缩成染色质，并且 染色质的折叠在细胞核内组织基因组，在调节基因方面起着至关重要的作用 抄写。染色质功能障碍和由此导致的基因表达失调是公认的 是许多人类疾病的贡献者，包括癌症和神经疾病。尽管进行了数十年的研究 以及它在基因表达、编码的天然染色质结构和调节区中的关键作用 基因组仍然没有得到很好的解析。虽然传统的结构生物学方法已经产生了许多高质量的 关于染色质组分的分辨结构，这些研究的一个普遍限制是所使用的条件 体外制备样品不能如实概括体内天然染色质环境，因其复杂 以及天然染色质的可变组成。另一方面，一直致力于 原位探测天然染色质景观非常适合提供大范围的信息(例如 全基因组)，但在分辨率或维度上有限。我的目标是弥合这些传统的 通过分析天然组装的、但纯化的特定的染色质的组成和结构的方法 酵母基因座。这项工作的初始阶段将旨在优化分离的染色体的制备 片段，以便最大限度地保存其原始组成和结构，使用 电子显微镜(EM)和质谱仪蛋白质组学作为质量控制分析方法。定量质量 同位素标记样品的光谱学也将被用于通过分析 微染色体基因诱导型和抑制型之间染色质组成的变化。 在优化纯化条件后，将使用负染和旋转阴影EM来产生 特定纯化酵母种群间核小体定位的高分辨率二维图谱 处于活跃或受抑制状态的基因。最后，利用约翰霍普金斯大学提供的最先进的仪器 大学，现代冷冻电磁和冷冻电子断层扫描技术将被用来分析三个- 纯化基因的染色质空间结构，包括对染色质变异性的评估 被抑制或诱导的基因群体之间的结构。最终，这种方法应该提供一种 一个基因在抑制和激活状态下的三维“结构”的概述，以及 特定基因片段的放大视图，例如显示组蛋白和非组蛋白之间的相互作用 基因启动子区域的组蛋白。这项研究的结果将大大推进我们的 了解通过染色质进行转录调控背后的机制，并可以为 转录失调导致疾病的机制。

PROJECT SUMMARY/ABSTRACT In eukaryotes, genomic DNA is condensed into chromatin by associating with histone proteins, and the folding of chromatin organizes the genome within the cell nucleus and plays an essential role in regulating gene transcription. Chromatin dysfunction and the resulting dysregulation of gene expression is a well-recognized contributor to many human diseases, including cancers and neurological disorders. Despite decades of research and its crucial role in the expression of genes, the native chromatin structure of coding and regulatory regions of the genome remains poorly resolved. While conventional structural biology methods have yielded many high- resolution structures of chromatin components, a general limitation of these studies is that the conditions used to prepare samples in vitro do not faithfully recapitulate the native chromatin environment in vivo, due to complex and variable composition of native chromatin. On the other hand, methods that have been geared towards probing the native chromatin landscape in situ are well-suited to provide information of a broad scale (e.g. genome wide) but are limited in resolution or dimensionality. I aim to bridge the gap between these traditional approaches by analyzing the composition and structure of natively assembled, yet purified, chromatin of specific yeast gene loci. The initial phase of this work will be aimed at optimizing the preparation of isolated chromosomal fragments in order to maximize preservation of their native composition and structure, using a combination of electron microscopy (EM) and mass spectrometry proteomics as quality control assays. Quantitative mass spectrometry of isotope-labeled samples will also be used to perform “chromatin proteomics” by analyzing changes in chromatin composition between the induced and repressed forms of the minichromosome genes. Following optimization of purification conditions, negative stain and rotary shadowing EM will be used to generate high-resolution two-dimensional maps of nucleosome positioning across populations of a given purified yeast gene in its active or repressed state. Finally, utilizing state-of-the-art instrumentation available at Johns Hopkins University, modern cryo-EM and cryo-electron tomography techniques will be used to analyze the three- dimensional chromatin structure of the purified gene, including an assessment of the variability in chromatin structure amongst populations of repressed or induced genes. Ultimately, this approach should provide an overview of the three-dimensional “structure” of a gene in both repressed and activated states, as well as “zoomed-in” views of specific gene fragments, showing for instance the interplay between histones and non- histone proteins at the promoter region of genes. The results of this study will significantly advance our understanding of the mechanisms behind transcriptional regulation via chromatin, and could provide insights into mechanisms of transcriptional dysregulation that cause disease.