Molecular Analysis of Genome Stability/Meiosis-Chromatin Transactions

单细胞真核纤毛虫原生动物嗜热四膜虫（Tetrahymena thermophila）具有核二型性。多倍体大核（MAC）作为细胞的体细胞核，无丝分裂，是营养体转录的位点。含有5条中部着丝粒染色体的二倍体微核（MIC）是细胞的萌发核，在营养生长期间转录沉默。这个细胞核在性生活周期中经历减数分裂（接合），导致程序性基因组重排和基因组不稳定。两个核的染色质状态不同，体细胞MAC主要处于开放的常染色质状态，MIC主要处于闭合的异染色质状态。两个功能不同的细胞核在同一细胞质中的存在提供了一个独特的机会，以解决核染色质配置的功能后果。这些包括转录活性、包括DNA修复的基因组稳定性以及两个核中组蛋白沉积和染色质重塑的机制。四膜虫复杂的后生动物样生物学，完整测序的体细胞和生殖细胞核，广泛的蛋白质组学分析，以及整个生命周期中基因表达的微阵列和RNA测序分析，使四膜虫成为这些基础和应用研究的有用和广泛开发的模型系统。这一要求延续40多年的NSERC发现资助支持的重点是独立细胞核的染色质状态和从基因组稳定性和基因组重排的角度进行的减数分裂的分子研究。提出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重要贡献，并得到NSERC发现资助基金的大力支持。最近NSERC资助的这一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中重要而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）四膜虫减数分裂不涉及联会复合体（SC）。我建议分析蛋白质以及蛋白质/蛋白质相互作用（相互作用组）的四膜虫直系同源物的推定SC蛋白质或蛋白质建议相互作用或复杂的SC。这将提供关于在缺乏SC的情况下减数分裂染色体的组织的重要信息。*2）通过双链断裂（DSB）修复研究基因组稳定性。GFP或其他标记的蛋白质将在减数分裂期间和响应于用DSB诱导剂处理而定位于MIC中。DSB修复、减数分裂中的DSB和发育程序性基因组重排中的染色体断裂之间的关系将得到解决。3)MIC中的组蛋白沉积集中于DSB修复和减数分裂。这些研究将涉及H3-H4组蛋白伴侣Asf 1 p。亲和纯化和质谱已被用来确定与Asf 1相互作用的蛋白质的数量。一些有趣的蛋白质已被确定，其中一些将进行功能分析。重点将放在蛋白质，似乎是MIC特异性，并可能有特定的作用，MIC功能，包括减数分裂。 表达数据的计算网络分析将解决有关蛋白质/蛋白质相互作用的问题。 *4）涉及细胞配对和结合早期诱导的酪氨酸磷酸化的新型信号通路。 抗磷酸酪氨酸抗体免疫共沉淀和质谱分析将允许分离和鉴定所涉及的磷酸化分子。

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.