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式生物，已被证明对解决基本生物学问题的细胞和分子研究非常有用。获得诺贝尔奖的关于端粒和端粒酶以及催化RNA和核酶的研究都是用四膜虫完成的。其他基础研究包括组蛋白修饰酶作为转录因子的初步描述，打开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都是可用的，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，在一个共同的细胞质中既有转录活跃的多倍体体细胞核（MAC），也有转录沉默的二倍体生发核（MIC）。这为解决基因表达调控的问题提供了一个强大的模型，这些问题集中在两个结构和功能不同的细胞核中不同的染色质配置上。广泛的RNA定向基因组重组发生在体细胞核发育过程中，涉及不可逆的基因组沉默，是RNA参与基因沉默的一种模式。***在NRC/NSERC连续资助的50年里，我的实验室的研究已经为利用四膜虫的分子、细胞和遗传方法解决基本生物学问题做出了贡献，为这些研究开发了重要的技术，并为HQP的培训做出了贡献。我的研究计划的长期目标是通过利用共同细胞质中两个核的不同染色质状态来解决基因表达的表观遗传调控和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集中于三个目标；1)染色质动力学和表观遗传学与tChd染色质重塑蛋白家族成员（SNF2超家族的染色体结构解旋酶-DNA结合；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性RNAi途径。我们将使用基因敲除、RNAi基因敲除、CRISPR/Cas和RNAi筛选冠心病编码基因、冠心病蛋白的结构分析、冠心病蛋白与组蛋白翻译后修饰结合的分析以及冠心病蛋白的RNA结合特性；2)另一个目标是比较MAC和MIC蛋白的蛋白质组学分析；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和影响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力学的重要基本问题，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. **