Molecular Analysis of Genome Stability/Meiosis-Chromatin Transactions

嗜热四膜虫是一种单细胞真核纤毛虫，具有核二型性。多倍体大核(MAC)是细胞的体细胞核，无丝分裂，是营养转录的场所。含有5条中部着丝粒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。这种核在有性生命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。两个核染色质状态不同，体细胞MAC主要处于开放等染状态，MIC主要处于闭合异染状态。同一细胞质中两个功能不同的核的存在为解决核染色质配置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)以及两个核中组蛋白沉积和染色质重塑的机制(S)。四膜虫复杂的后生动物生物学、完全测序的体细胞核和生殖核、广泛的蛋白质组学分析以及对整个生命周期基因表达的微阵列和RNA序列分析使四膜虫成为这些基础和应用研究的有用和被开发的模型系统。 这项延续40多年的NSERC发现赠款支持的请求侧重于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依赖于我最近在四膜虫基因组学、蛋白质组学和表达微阵列研究方面的重大贡献，并得到了NSERC发现拨款的重大支持。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等重要和有趣的基本和应用问题，以及关于早期接合事件中的信号问题。重点关注的领域包括： 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四膜虫的蛋白质/蛋白质相互作用(交互作用组)，推测的SC蛋白或建议与SC相互作用或复合的蛋白质的同源物。这将提供关于在没有SC的情况下减数分裂染色体组织的重要信息。 2)双链断裂(DSB)修复对基因组稳定性的影响。GFP或其他标记蛋白将在减数分裂过程中定位于MIC中，并响应于DSB诱导剂的处理。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碎裂之间的关系将被处理。 3)组蛋白在MIC中的沉积主要集中在DSB修复和减数分裂。这些研究将涉及H3-H4组蛋白伴侣蛋白Asf1p。亲和纯化和质谱学已被用于鉴定一些与ASF1相互作用的蛋白质。已经确定了一些有趣的蛋白质，并将对其中一些进行功能分析。重点将放在似乎是MIC特异的、可能在MIC功能中具有特殊作用的蛋白质上，包括减数分裂。表达数据的计算网络分析将解决有关识别的蛋白质/蛋白质相互作用的问题。 4)在细胞配对和早期接合阶段诱导酪氨酸磷酸化的新信号通路。免疫共沉淀与抗磷酸化酪氨酸抗体和质谱学将允许分离和鉴定涉及的磷酸化分子(S)。

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.