RNA-mediated Chromatin Regulation and Epigenetic Inheritance in C. elegans

描述（由申请人提供）：RNA干扰（RNAi）是一种进化上保守的、基于同源性的基因沉默机制，其中小干扰RNA （sirna）导致宿主细胞中mRNA降解和/或异染色质形成。这种分子途径对于发育转变和细胞对致病挑战的反应至关重要。sirna介导的异染色质反应，在本提案中被称为核RNAi，作为一种表观遗传机制，RNA水平的事件可以“反馈”调节染色质的结构和功能，引起了极大的兴趣。这种现象最令人兴奋的方面是它通过有丝分裂或减数分裂细胞周期传递表观遗传信息的非凡能力。这在植物rna介导的DNA甲基化和裂变酵母rna介导的转录沉默中得到了最好的研究。然而，这一过程在多大程度上导致动物的跨代反应仍然是未知的。最明确的证据来自对秀丽隐杆线虫种系核RNAi的研究。尽管在哺乳动物中的相关研究还远未得出结论，但秀丽隐杆线虫的异染色质反应和许多RNAi成分也可以在人类中发现。在高度可处理的模式生物秀丽隐杆线虫中定义种系核RNAi途径的分子细节将为染色质基因组调控和表观遗传的保守机制提供重要见解。为了使该领域向前发展，必须通过实验解决该模型中的以下基本问题。种系核RNAi的天然靶点是什么？在种系发育过程中靶基因的转录和沉默是如何协调的？在连续生殖周期中维持靶基因沉默的表观遗传机制是什么？解决这些基本问题将有助于我们预测染色质位点对靶向调节RNA的敏感性，这对于我们将靶向染色质干预作为治疗工具的长期目标至关重要。我们将采用实验和计算相结合的方法来解决这些问题，具体目标有三个：(1)线虫种系中核RNAi靶点的全基因组鉴定、表征和分类。(2)确定启动子在种系核RNAi调控中的作用。(3)确定异染色质在种系核RNAi介导的跨代基因沉默中的作用。这些提出的研究，探索了基础但未知的领域，将对理解与人类发育和疾病相关的基因组-环境相互作用和表达状态的遗传具有深远的意义。

DESCRIPTION (provided by applicant): RNA interference (RNAi) is an evolutionarily conserved, homology-based gene silencing mechanism, in which small interfering RNAs (siRNAs) lead to mRNA degradation and/or heterochromatin formation in host cells. This molecular pathway is fundamentally important for development transition and cellular responses to pathogenic challenges. The siRNA-mediated heterochromatic response, which is referred to as nuclear RNAi in this proposal, has been of great interest as an epigenetic mechanism by which events at the RNA level can "feed back" to modulate chromatin structure and function. The most exciting aspect of this phenomenon is its remarkable ability to transmit epigenetic information through mitotic or meiotic cell cycles. This was best studied in RNA-mediated DNA methylation in plants and RNA-mediated transcriptional silencing in fission yeast. However, the extent to which this process can lead to a trans-generational response in animals remains largely unknown. The most definitive evidence came from the study of germline nuclear RNAi in C. elegans. Although related studies in mammals are far from conclusive, both the heterochromatin response and many of the C. elegans RNAi components can be found in humans as well. Defining molecular details of the germline nuclear RNAi pathway in a highly tractable model organism C. elegans will provide important insights into conserved mechanisms of chromatin-based genome regulation and epigenetic inheritance. The following fundamental questions in this model must be experimentally addressed for the field to move forward. What are the natural targets of germline nuclear RNAi? How are transcription and silencing of target genes coordinated during germline development? What are the mechanisms of epigenetic inheritance that maintain the silencing of target genes in successive reproductive cycles? Addressing these fundamental questions will help us to predict the sensitivity of a chromatin locus to a targeting regulatory RNA, which is critically important to our long-term goal of targete chromatin intervention as a therapeutic tool. We will employ a combination of experimental and computational approaches to address these questions in three specific aims: (1) Genome-wide identification, characterization, and classification of nuclear RNAi targets in the C. elegans germline. (2) Determine the role of promoters in regulating germline nuclear RNAi. (3) Determine the role of heterochromatin in trans- generational gene silencing mediated by germline nuclear RNAi. The proposed studies, which probe fundamental yet uncharted areas, will hold profound implications for understanding of genome-environment interaction and inheritance of expression states relevant to human development and disease.