Chromatin Dynamics and Transcription Regulation in the Animal Model Eukaryote Tetrahymena

单细胞纤毛虫原生动物嗜热四膜虫是一种动物模型生物，已被证明对于解决基本生物学问题的细胞和分子研究非常有用。荣获诺贝尔奖的关于端粒和端粒酶以及催化 RNA 和核酶的研究是用四膜虫完成的。其他基础研究包括对组蛋白修饰酶作为转录因子的初步描述，开启表观遗传学的分子分析，建立连接组蛋白 H1 的作用等等。体细胞和生发基因组序列均可用，并促进了基本生物过程的分子和遗传分析。 ***四膜虫的一个独特的生物学特征是核二态性，在共同的细胞质中既有转录活跃的多倍体体细胞核（MAC），又有转录沉默的二倍体生发核（MIC）。这为解决有关基因表达调控的问题提供了一个强大的模型，重点关注两个结构和功能不同的细胞核中的不同染色质配置。广泛的RNA指导的基因组重组发生在体细胞核的发育过程中，涉及不可逆的基因组沉默，并且是RNA涉及的基因沉默的模型。 ***我实验室的研究在 50 年来的 NRC/NSERC 持续资助下，为使用四膜虫的分子、细胞和遗传方法解决基本的生物学问题做出了贡献，开发了对这些研究和 HQP 培训至关重要的技术。我的研究计划的长期目标是通过利用共同细胞质中两个细胞核的不同染色质状态来解决基因表达的表观遗传调控、RNA 介导的不可逆基因沉默和发育基因组重组的问题。短期目标将按优先顺序重点关注三个目标； 1) 染色质动力学和表观遗传学，涉及 tChd 染色质重塑蛋白家族成员（染色质结构域解旋酶-DNA 结合；SNF2 超家族的 ATP 依赖性解旋酶）、候选 DNA 和 RNA 结合蛋白 (DRBP)，参与不可逆基因沉默和基因组重组的发育 RNAi 途径。我们将利用基因敲除、RNAi基因敲低、Chd编码基因的CRISPR/Cas和RNAi筛选、Chd蛋白的结构分析、Chd蛋白与组蛋白翻译后修饰的结合分析以及Chd蛋白的RNA结合特性； 2) 另一个目标是对 MAC 和 MIC 蛋白质进行比较蛋白质组分析； 3) 组蛋白、染色质重塑蛋白和参与转录及其调控的蛋白的相互作用组研究。解决这些明确的目标将提供重要的见解，并影响 RNA 和表观遗传学如何在复杂的真核调节机制中协调染色质动态的重要基本问题，并为 HQP 提供良好的培训环境。 **

The unicellular ciliate protozoan Tetrahymena thermophila is an animal model organism that has proven very useful for cell and molecular studies addressing basic biological questions. Nobel Prize winning studies on telomeres and telomerase and on catalytic RNA and ribozymes were done with Tetrahymena. Additional fundamental studies include the initial description of a histone modifying enzyme as a transcription factor opening molecular analysis of epigenetics, establishing the role of the linker histone H1, and much more. Both the somatic and germinal genome sequences are available and have facilitated molecular and genetic analysis of fundamental biological processes. ***A unique biological feature of Tetrahymena, is nuclear dimorphism with both a transcriptionally active polyploid somatic nucleus (MAC) and a mostly transcriptionally silent diploid germinal nucleus (MIC) in a common cytoplasm. This provides a powerful model for addressing questions about regulation of gene expression focusing on the different chromatin configuration in the two structurally and functionally distinct nuclei. Extensive RNA-directed genome reorganization occurs during the development of the somatic nucleus involving irreversible genome silencing and is a model for RNA involved gene silencing. ***Research in my lab in 50 years of continuous NRC/NSERC funding has contributed to addressing fundamental biological questions using molecular, cell, and genetic approaches with Tetrahymena, the development of technologies important for these studies, and for the training of HQP. The long term goal of my research program is to address questions about epigenetic regulation of gene expression and RNA-mediated irreversible gene silencing and developmental genome reorganization by exploiting the different chromatin states of the two nuclei in a common cytoplasm. Short term goals will focus on three aims in order of priority; 1) chromatin dynamics and epigenetics with a tChd chromatin remodeler protein family member (chromodomain helicase-DNA-binding; ATP-dependent helicases of the SNF2 superfamily), candidate DNA and RNA binding proteins (DRBP), involved in the developmental RNAi pathway of irreversible gene silencing and genome reorganization. We will use gene knockouts, RNAi gene knockdowns, and CRISPR/Cas and RNAi screens of Chd encoding genes, structural analysis of Chd proteins, analysis of Chd proteins binding to histone post translational modifications, and RNA binding properties of Chd proteins; 2) an additional aim will be comparative proteomic analysis of MAC and MIC proteins; and 3) interactome studies of histones, chromatin remodeling proteins, and proteins involved in transcription and its regulation. Addressing these defined aims will provide significant insights and impact the important fundamental question of how RNAs and epigenetics orchestrate chromatin dynamics in complex eukaryotic regulatory mechanisms and provide an excellent training environment for HQP. **