Genetic Analysis of the Nucleosome and RNA Polymerase II: A Systems Approach

描述（由申请人提供）：我们最近开发了一种遗传筛选方法，称为E-MAP（上位微型阵列技术分析），可以量化系统产生的成对遗传相互作用的强度。该方法确定了负双突变相互作用，其中突变组合导致缺陷导致生长缺陷增强或致命性。这种相互作用通常指定平行生化途径的成员关系。此外，E-MAP还发现了积极的相互作用，其中突变体的组合表现出相互抑制或缺乏任何加性缺陷，这些缺陷在物理相互作用的基因产物中丰富。利用非必需基因的基因缺失和必需基因的次胚等位基因，我们最近在酿酒酵母中生成了E-MAPs，主要关注1)早期分泌途径和2)染色体生物学，包括转录调控、染色质重塑和DNA修复。我们现在提出第二代E-MAP分析，通过检查多功能和必需基因的点突变，使我们能够解决下一个复杂水平。具体来说，我们将从遗传学上剖析基因表达和染色质结构核心的两个重要的多亚基多功能复合物：RNA聚合酶II （RNAPII）和核小体。这种方法将使我们能够A)将这些复合物的结构特征映射到它们的功能作用上，B)表征RNAPII与核小体和更广泛的基因表达装置之间的功能关系。在目标#1中，我们将使用染色体生物学E-MAP对一组大约450个组蛋白H3和H4突变体进行遗传检查，包括a)完整的丙氨酸（或丝氨酸）扫描，B)可修饰残基的全面替代，以及C) n端尾部的半系统缺失。这项工作是与NIH赖氨酸修饰路线图TCNP（网络和途径技术中心）（PI Jef Boeke）合作完成的，将有助于揭示组蛋白-组蛋白和组蛋白- dna接触和组蛋白修饰如何影响转录和染色质调控的步骤。在Aim #2中，我们将筛选与Craig Kaplan和Roger Kornberg合作分离的几个基本RNAPII亚基的大约100个不同和多样的点突变体。在Aim #3中，我们将对这些数据进行分层聚类和我们最近开发的指标（S-和cop -评分系统），以帮助识别使用E-MAP数据的功能关系。我们还将采用新开发的算法，从大规模交互数据集中识别功能相关的基因（或模块）集，并允许多功能基因成为多个模块的成员。我们预计，这里描述的系统遗传方法将提供真核细胞中染色质功能和转录调控的更全面的观点。

DESCRIPTION (provided by applicant): We have recently developed a genetic screening approach, termed E-MAP (Epistatic MiniArray Technology Profiling), that can quantify the strength of systematically generated pair-wise genetic interactions. The method identifies negative double mutant interactions, where combination of mutations causes defects leads to enhancement of growth defects or lethality. Such interactions often specify membership of parallel biochemical pathways. Additionally, E-MAP also identifies positive interactions, where combination of mutants show mutual suppression or lack any additive defects, which are enriched among physically interacting gene products. Using gene deletions of non-essential genes and hypomorphic alleles of essential genes, we have recently generated E-MAPs in S. cerevisiae that have focused on 1) the early secretory pathway and 2) chromosome biology, which includes transcriptional regulation, chromatin remodeling and DNA repair. We now propose the second generation of E-MAP analysis, allowing us to address the next level of complexity, via examination of point mutants of multifunctional and essential genes. Specifically, we will genetically dissect two essential, multisubunit, multifunctional complexes at the heart of gene expression and chromatin structure: RNA polymerase II (RNAPII) and the nucleosome. This approach will allow us to A) map the structural features of these complexes onto their functional roles, and B) characterize the functional relationships between RNAPII and the nucleosome and the wider gene expression apparatus. In Aim #1, we will use the chromosome biology E-MAP to genetically examine a set of approximately 450 histone H3 and H4 mutants including A) complete alanine (or serine)-scans, B) comprehensive substitution of modifiable residues, and C) semisystematic deletions of the N-terminal tails. The work, which is being done collaboratively with NIH Roadmap TCNP (Technology Center for Networks and Pathways) of Lysine Modification (PI Jef Boeke), will help reveal how histone-histone and histone-DNA contacts and histone modifications influence the steps of transcription and chromatin regulation. In Aim #2, we will screen approximately 100 distinct and diverse point mutants of several essential RNAPII subunits isolated in collaboration with Craig Kaplan and Roger Kornberg. In Aim #3, we will subject these data to hierarchical clustering and our recently developed metrics (S- and COP-scoring systems) to help identify functional relationships using the E-MAP data. We will also employ newly developed algorithms that identify functionally related sets of genes (or modules) from large-scale interaction datasets and allows for multi-functional genes to be members of more than one module. We anticipate that a systematic genetic approach described here will provide a more holistic view of chromatin function and transcriptional regulation in eukaryotic cells.