Molecular Analysis of Genome Stability/Meiosis-Chromatin Transactions

基因组稳定性/减数分裂-染色质交易的分子分析

• 批准号: RGPIN-2014-03739
• 负责人: Pearlman, Ronald
• 金额: $ 2.99万
• 依托单位: York University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2014
• 资助国家: 加拿大
• 起止时间: 2014-01-01 至 2015-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=566063
• 关键词: Molecular; Analysis; Genome; Stability; Meiosis

The unicellular eukaryotic ciliated protozoa Tetrahymena thermophila is characterized by nuclear dimorphism. The polyploid macronucleus (MAC) functions as the cell’s somatic nucleus, divides amitotically and is the site of vegetative transcription. The diploid micronucleus (MIC) containing 5 metacentric chromosomes is the cell’s germinal nucleus, transcriptionally silent during vegetative growth. This nucleus undergoes meiosis in the the sexual life cycle (conjugation) leading to programmed genome rearrangement and genome instability. The chromatin state of the two nuclei differ with the somatic MAC being mainly in an open euchromatic state and the MIC being mainly in a closed heterochromatic state. The presence of the two functionally distinct nuclei in the same cytoplasm provides a unique opportunity to address functional consequences of nuclear chromatin configurations. These include transcriptional activity, genome stability including DNA repair as well as the mechanism(s) of histone deposition and chromatin remodeling in the two nuclei. Tetrahymena’s complex metazoan-like biology, the completely sequenced somatic and germinal nuclei, extensive proteomic analysis, and microarray and RNA seq analysis of gene expression throughout the life cycle make Tetrahymena a useful and much exploited model system for these basic and applied studies. This request for renewal of 40+ years of NSERC Discovery Grant support focuses on the chromatin state of the independent nuclei and on molecular studies of meiosis approached from the perspective of genome stability and genome rearrangements. Experiments proposed will depend heavily on my very significant recent contributions to Tetrahymena genomics, proteomics, and expression microarray studies with significant support from NSERC Discovery Grant funding. This focus of recent NSERC funding allows the exploitation of these tools in functional analysis to address important and interesting basic and applied questions in meiosis, genome stability, histone deposition and chromatin remodeling, as well as questions about signaling in early conjugation events. Areas of focus are: 1) Meiosis in Tetrahymena does not involve a synaptonemal complex (SC). I propose analysis of proteins as well as protein/protein interactions (the Interactome) of Tetrahymena orthologues of putative SC proteins or of proteins suggested to interact or complex with SCs. This will provide important information about the organization of meiotic chromosomes in the absence of an SC. 2) Studies of genome stability through double strand break (DSB) repair. GFP or other tagged proteins will be localized in MIC during meiosis and in response to treatment with inducers of DSB. Relationships between DSB repair, DSBs in meiosis, and chromosome fragmentation in developmentally programmed genome rearrangements will be addressed. 3) Histone deposition in the MIC focusing on DSB repair and on meiosis. These studies will involve the H3-H4 histone chaperone Asf1p. Affinity purification and mass spectrometry have been used to identify a number of proteins interacting with Asf1. A number of interesting proteins have been identified and functional analysis of some of these will be pursued. Focus will be on proteins that appear to be MIC-specific and may have particular roles in MIC function including meiosis. Computational network analysis of expression data will address questions about protein/protein interactions identified. 4) A novel signaling pathway involving tyrosine phosphorylation induced during cell pairing and early stages of conjugation. Co-immunoprecipitation with anti phosphtyrosine antibodies and mass spectrometry will allow isolation and identification of the phosphorylated molecule(s) involved.

单细胞真核纤毛原生动物嗜热四膜虫具有核二态性的特征。多倍体大核（MAC）充当细胞的体细胞核，进行无丝分裂，并且是营养转录的位点。含有 5 条偏心染色体的二倍体微核 (MIC) 是细胞的生发核，在营养生长过程中转录沉默。该核在性生命周期（接合）中经历减数分裂，导致程序性基因组重排和基因组不稳定。两个细胞核的染色质状态不同，体细胞MAC主要处于开放常染色质状态，而MIC主要处于闭合异染色质状态。同一细胞质中两个功能不同的细胞核的存在为解决核染色质构型的功能后果提供了独特的机会。这些包括转录活性、基因组稳定性（包括 DNA 修复）以及两个细胞核中组蛋白沉积和染色质重塑的机制。四膜虫具有复杂的后生动物样生物学特性、完全测序的体细胞和生发细胞核、广泛的蛋白质组分析以及整个生命周期基因表达的微阵列和 RNA seq 分析，使四膜虫成为这些基础和应用研究中有用且得到广泛利用的模型系统。此次请求续签 40 多年的 NSERC 发现资助支持，重点关注独立细胞核的染色质状态以及从基因组稳定性和基因组重排角度进行的减数分裂分子研究。拟议的实验将在很大程度上取决于我最近对四膜虫基因组学、蛋白质组学和表达微阵列研究的非常重要的贡献，并得到了 NSERC 发现拨款的大力支持。最近 NSERC 资助的重点允许在功能分析中利用这些工具来解决减数分裂、基因组稳定性、组蛋白沉积和染色质重塑方面重要且有趣的基本和应用问题，以及有关早期接合事件中信号传导的问题。重点领域是： 1）四膜虫的减数分裂不涉及联会复合体（SC）。我建议对假定的 SC 蛋白或与 SC 相互作用或复合的蛋白质的四膜虫直系同源物的蛋白质以及蛋白质/蛋白质相互作用（相互作用组）进行分析。这将提供有关在没有 SC 的情况下减数分裂染色体组织的重要信息。 2) 通过双链断裂（DSB）修复研究基因组稳定性。 GFP 或其他标记蛋白将在减数分裂期间定位于 MIC，并对 DSB 诱导剂的治疗做出反应。 DSB 修复、减数分裂中的 DSB 和发育程序基因组重排中的染色体碎片之间的关系将得到解决。 3) MIC 中的组蛋白沉积，重点关注 DSB 修复和减数分裂。这些研究将涉及 H3-H4 组蛋白伴侣 Asf1p。亲和纯化和质谱已用于鉴定许多与 Asf1 相互作用的蛋白质。已经鉴定了许多有趣的蛋白质，并将对其中一些蛋白质进行功能分析。重点将放在似乎具有 MIC 特异性并且可能在 MIC 功能（包括减数分裂）中具有特殊作用的蛋白质上。表达数据的计算网络分析将解决有关已识别的蛋白质/蛋白质相互作用的问题。 4）一种新的信号通路，涉及细胞配对和接合早期阶段诱导的酪氨酸磷酸化。与抗磷酸酪氨酸抗体的免疫共沉淀和质谱分析将能够分离和鉴定所涉及的磷酸化分子。