Profiling chromatin-associated long noncoding RNAs in numerous biological contexts

在多种生物环境中分析染色质相关的长非编码 RNA

• 批准号: 9395751
• 负责人: Jeffrey Jerome Quinn
• 金额: $ 5.67万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-01 至 2020-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9395751
• 关键词: Address; Animal Model; Antibodies; Binding; Biochemical; Biochemical Genetics; Biological; Biological Assay; Biological Process; Biology; CRISPR interference; Cancer cell line; Catalogs; Cell Nucleus; Cells; Chromatin; Clustered Regularly Interspaced Short Palindromic Repeats; Data Set; Databases; Development; Disease; Dosage Compensation (Genetics); Drosophila genome; Drosophila genus; Enhancers; Fluorescent in Situ Hybridization; Genes; Genetic; Genetic Techniques; Genetic Transcription; Genome; Genome Stability; Genomic Imprinting; Heterochromatin; Histones; Human; Human Cell Line; Human Development; Human Genome; Insulator Elements; Knowledge; Malignant Neoplasms; Methods; Motivation; Nature; Nuclear; Pattern; Physiological; Play; Process; RNA; RNA purification; Recovery; Research; Research Project Grants; Research Proposals; Role; Structure; System; Techniques; Testing; Untranslated RNA; Validation; Work; cell type; dosage; experimental study; fascinate; histone modification; imprint; improved; induced pluripotent stem cell; knock-down; novel; rapid technique; screening; tool; transcriptome; transcriptome sequencing

Project Summary Title: Profiling chromatin-associated lncRNAs in numerous biological contexts. The human genome encodes thousands of long noncoding RNAs (lncRNAs), some of which are known to regulate important phenomena in the nucleus, such as dosage compensation, imprinting, and genome stability. It is currently unclear how many lncRNAs act on chromatin, but certainly many other examples of this paradigm remain to be discovered. With this motivation, I propose to develop, validate, and apply a biochemical method for rapidly identifying chromatin-associated lncRNAs, called Profiling of Interacting RNAs on Chromatin and sequencing (PIRCh-seq). To demonstrate proof-of-principle of this method, I have used PIRCh-seq to positively identify two lncRNAs in Drosophila (roX1 and roX2) that are known to interact with dosage compensated chromatin (H4K16ac-marked). I propose to further test PIRCh-seq by profiling lncRNAs that are associated with specific chromatin states (e.g. general chromatin, enhancers, heterochromatin) in Drosophila, and then validate these lncRNA candidates using complementary techniques, such as chromatin isolation by RNA purification and sequencing (ChIRP-seq). Next, I aim to apply PIRCh-seq to human cell lines, thus defining the sets of developmental-stage or cancer-specific chromatin-associated lncRNAs. Finally, from these resulting lncRNA catalogs, I will select several for further functional characterization towards understanding their mechanism of interaction with chromatin and possible roles in chromatin-templated process. For these experiments, I will use complementary genetic and biochemical techniques, including ChIRP-seq, CRISPR- enabled screening and genetic perturbation, and RNA structure probing. This research proposal will make deep impacts on the fields of lncRNA and chromatin biology: first, it will provide a powerful tool for discovering functional lncRNAs; second, it will identify lncRNAs associated with specific chromatin states in an important model organism and human cell types that are relevant to development and disease; lastly, it will discover novel examples of lncRNA–chromatin interaction that will deepen our understanding of lncRNA function.

项目摘要 标题：在许多生物学背景下分析染色质相关的lncRNA。 人类基因组编码数千种长链非编码RNA（lncRNA），其中一些已知是 调节细胞核中的重要现象，如剂量补偿、印记和基因组稳定性。 目前还不清楚有多少lncRNA作用于染色质，但可以肯定的是，这种模式的许多其他例子 还有待发现。有了这个动机，我提议开发、验证和应用一种生物化学方法 用于快速鉴定染色质相关的lncRNA，称为染色质上相互作用RNA的分析， 测序（PIRCh-seq）。为了证明这种方法的原理，我使用PIRCh-seq来 阳性鉴定果蝇中已知与剂量相互作用的两种lncRNA（roX 1和roX 2） 补偿染色质（H4 K16 ac标记）。我建议通过分析lncRNA来进一步测试PIRCh-seq， 与果蝇中的特定染色质状态（例如一般染色质、增强子、异染色质）相关， 然后使用互补技术验证这些lncRNA候选物，例如染色质分离， RNA纯化和测序（ChIRP-seq）。接下来，我的目标是将PIRCh-seq应用于人类细胞系， 定义发育阶段或癌症特异性染色质相关lncRNA的集合。最后，从这些 因此lncRNA目录，我将选择几个进一步的功能表征，以了解 它们与染色质的相互作用机制以及在染色质模板化过程中可能的作用。为这些 实验中，我将使用互补的遗传和生化技术，包括ChIRP-seq，CRISPR- 使筛选和遗传扰动，和RNA结构探测成为可能。这项研究计划将使 对lncRNA和染色质生物学领域的深刻影响：首先，它将为发现 第二，它将在一个重要的细胞中识别与特定染色质状态相关的lncRNA。 与发育和疾病相关的模式生物和人类细胞类型;最后，它将发现 lncRNA-染色质相互作用的新例子，将加深我们对lncRNA功能的理解。