Roles of Non-coding RNA, roX, in Global Chromatin Organization

智力优点：最近对各种高等真核动物（如小鼠和人类）基因组的分析表明，出乎意料的大部分转录本不编码蛋白质，而是指定功能性RNA。越来越多的证据表明，这些非编码转录本参与了包括染色质组织在内的各种调控机制，这些过程的破坏可能对生物体产生严重后果。该项目的重点是roX RNA在果蝇剂量补偿中的作用，其中雄性有一个X染色体，雌性有两个X染色体，类似于人类和其他哺乳动物中存在的XX和XY互补。 在果蝇中，剂量补偿需要上调X染色体上存在的数百个不相关基因的表达，以平衡性别之间基因产物的数量。 因此，它是一个很好的模型系统，了解真核生物基因调控的几个方面的非编码RNA。剂量补偿中的核心参与者是MSL（男性特异性致死）复合物，其包括非编码roX 1和roX 2 RNA，并仅作用于男性X染色体。该领域的一个关键进展是发现MSL复合物对X染色体的作用是由组蛋白修饰介导的（特别是组蛋白H4中赖氨酸16的乙酰化）。本研究的目标是表征roX RNA的保守功能结构域通过其调节男性X染色体上MSL复合物的酶活性以实现X连锁基因的两倍上调的分子机制。通过揭示roX RNA的结构和功能之间的关系，该项目将定义RNA-蛋白质相互作用的新方面及其在染色质重塑复合物背景下的相关性。重要的是，哺乳动物中的X染色体失活也依赖于一种复合物，该复合物包括一种称为Xist的非编码RNA。 虽然Xist复合物诱导X染色体上的基因沉默，与果蝇中MSL复合物促进的上调相反，一个共同的特征是伴随着从非编码RNA结合位点顺式扩散的染色质重塑的起始。除了参与由全局组蛋白修饰诱导的表观遗传变化之外，非编码RNA在这些过程中的作用还没有得到很好的理解。因此，从roX RNA的研究中获得的信息将有助于加深对哺乳动物X染色体失活的理解，并有可能促进对真核生物中非编码RNA依赖的基因调控的普遍理解。参加少数民族夏季学生研究计划的高中和本科生将被纳入研究活动，以教他们的基本原则和技术，科学为了传播关于RNA分析工具的信息，包括RNA结构预测、分析和搜索，将利用通过该项目产生的非编码RNA（roX）数据不断更新一个供公众用户使用的因特网设施（http：//bioinformatics.njit.edu/rna）。为了确保公开的科学交流，通过该项目开发的所有数据，协议，构建和转基因苍蝇将与其他实验室和机构共享。研究团队的成员将参加多学科和跨学科会议，并介绍从该项目中获得的研究成果。 长期目标是将研究对染色质组织领域的贡献纳入基础教育资源，如研究生和本科生的教科书。

Intellectual merit: Recent analyses of the genomes of various higher eukaryotic animals such as mice and humans have shown that an unexpectedly large fraction of transcripts do not encode proteins but rather specify functional RNAs. A growing body of evidence suggests that these non-coding transcripts are involved in a variety of regulatory mechanisms including chromatin organization, and that disruption of these processes can have drastic consequences for the organism. This project focuses on the role of the roX RNAs in dosage compensation in fruit flies, where males have a single X chromosome and females have two X chromosomes, similar to the XX and XY complement present in humans and other mammals. In flies, dosage compensation entails up-regulating the expression of hundreds of unrelated genes present on the X chromosome in order to equalize the quantities of the gene products between the sexes. Thus, it is an excellent model system to understand several aspects of eukaryotic gene regulation by non-coding RNA. The central player in dosage compensation is the MSL (Male Specific Lethal) complex, which includes the non-coding roX1 and roX2 RNAs and acts exclusively on the male X chromosome. A key development in the field was the discovery that the action of the MSL complex on the X chromosme is mediated by histone modification (specifically, acetylation of lysine 16 in histone H4). The goal of the present research is to characterize the molecular mechanism through which the conserved functional domains of the roX RNAs regulate the enzymatic activity of the MSL complex on the male X chromosome to achieve two-fold up-regulation of X-linked genes. By revealing the relationship between the structure and function of the roX RNAs, the project will define new aspects of RNA-protein interactions and their relevance in the context of chromatin remodeling complexes. Importantly, X-chromosome inactivation in mammals is also dependent on a complex that includes a non-coding RNA known as Xist. Although the Xist complex induces gene silencing on the X chromosome, in contrast to the up-regulation promoted by the MSL complex in fruit flies, a shared feature is the initiation of chromatin remodeling accompanied by spreading in cis from a non-coding RNA binding site. Beyond the involvement of epigenetic changes induced by global histone modification, the roles of non-coding RNAs in these processes are not well understood. Thus, the information obtained from studies of roX RNA will lead to enhanced understanding of X-chromosome inactivation in mammals and has the potential to advance general understanding of non-coding RNA-dependent gene regulation in eukaryotes.Broader impacts: Participants in a minority summer student research program for high school and undergraduate students will be included in the research activities in order to teach them principles and techniques of basic science. To disseminate information about RNA analysis tools including RNA structure prediction, alignmentsand searches, an internet-based facility for public users (http://bioinformatics.njit.edu/rna) will be continuously updated with non-coding RNA (roX) data generated through the project. To ensure open scientific communication, all data, protocols, constructs and transgenic flies developed through the project will be shared with other laboratories and institutions. Members of the research team will participate in multi- and interdisciplinary conferences and present research results obtained from the project. A long-term goal is for the contributions of the research to the field of chromatin organization to be integrated into basic education resources such as textbooks for graduate and undergraduate students.