Genome wide identification and functional analysis of chromatin regulatory RNAs

染色质调节 RNA 的全基因组鉴定和功能分析

基本信息

批准号：
10062511
负责人：
William James Greenleaf
金额：
$ 61.6万
依托单位：
STANFORD UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-12-01 至 2022-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10062511
关键词：
Binding Proteins Binding Sites Biology Cell Maintenance Cell physiology Cells Chimera organism Chromatin Chromosomal RNA Chromosome Mapping Chromosome Structures Chromosomes Clustered Regularly Interspaced Short Palindromic Repeats Code DNA DNA Sequence Data Defect Development Dosage Compensation (Genetics)Drosophila genus Drosophila melanogaster Female Functional disorder Gene Expression Genes Genetic Transcription Genome Genomic Imprinting Genomic Segment Human Human Chromosomes Human Development Human Genome Infrastructure Libraries Link Maps Messenger RNA Metabolic Methods Mouse Cell Line Nuclear Output Phenotype Play Process Proteins RNA RNA Binding Regulation Resources Role Route Salvelinus Sampling Site Stress Structure System Technology Testing Time Transcriptional Regulation Untranslated RNA Validation X Chromosome base cell type comparative developmental disease ds-DNA experimental study fly gene repression genome-wide genome-wide analysis genomic locus histone modification human disease human tissue knock-down male next generation sequencing novel novel strategies protein expression response single cell analysis stressor transcriptome sequencing tumor progression

项目摘要

In addition to the transcription of protein coding genes in the genome, a large amount of transcription encodes RNA molecules that do not generate mRNA. These noncoding RNAs play important roles in the cell that include regulating dosage compensation, controlling genomic imprinting and regulating transcription. However, human cells transcribe thousands of noncoding RNAs and we have only ascribed functions to a small number. One of the main challenges to understanding the functions of noncoding RNAs is that technologies to rapidly identify and characterize noncoding RNAs are lacking. In this proposal, we develop a novel method that makes it possible to identify, in any cell type, all of the noncoding RNAs that interact with chromosomes and at the same time map the sites where those RNAs bind chromatin. Our approach involves directly linking noncoding RNAs to the underlying DNA by generating a covalent chimera between a chromosome bound RNA and DNA. Using next generation sequencing, we can identify the RNAs in the cell that are likely to regulate chromosome structure or function and define their sites of action on the chromosome. In our first Aim we use Drosophila cells to develop this approach, taking advantage of the fact that established chromosomal RNAs, roX1 and roX2, are known to coat the X chromosome to accomplish dosage compensation in the fly. We then broaden this approach in Aim 2 and identify the RNAs that bind chromatin throughout the human genome and develop a new analytical infrastructure to classify and functionally assign these RNAs. In Aim 3 develop perturbation experiments to test the functions of noncoding RNAs and RNA motifs for their impact on local chromosome accessibility, histone modification state and transcriptional output. We apply a system to redirect noncoding RNAs to new genomic regions to test their functional impact on chromosomes and to regulate different genomic regions through RNA dependent control. By defining the landscape of chromatin associated RNAs in humans and the sites that they regulate in the cell our proposal how these RNAs function as well as the impacts of defects in RNA dependent control that result in cellular dysfunction.

除了基因组中蛋白质编码基因的转录外，大量转录编码不产生mRNA的RNA分子。这些不编码的RNA播放细胞中的重要作用，包括调节剂量补偿，控制基因组印迹和调节转录。但是，人类细胞会抄录数千个非编码RNA，我们只归因于少数数字。主要之一理解非编码RNA功能的挑战是快速的技术缺乏识别和表征非编码RNA。在这一提议中，我们开发了一本小说可以在任何单元格中识别所有非编码RNA的方法与染色体相互作用，同时映射这些RNA结合的位点染色质。我们的方法涉及通过在染色体结合的RNA和DNA之间产生共价嵌合体。使用下一步生成测序，我们可以识别细胞中可能调节的RNA 染色体结构或功能，并定义其在染色体上的作用位点。在我们的首先，我们使用果蝇细胞来开发这种方法，利用已知已建立的染色体RNA，ROX1和ROX2将X染色体涂覆到即时完成剂量补偿。然后，我们在AIM 2和识别在整个人类基因组中结合染色质的RNA并发展新的RNA 分析基础架构分类并在功能上分配这些RNA。在AIM 3发展扰动实验以测试非编码RNA和RNA图案的功能对局部染色体可及性，组蛋白修饰状态和转录输出的影响。我们应用系统将非编码RNA重定向到新的基因组区域以测试其功能对染色体的影响并通过依赖RNA调节不同的基因组区域控制。通过定义与人类染色质相关的RNA的景观以及他们在单元中调节这些RNA的功能以及缺陷的影响在RNA依赖控制中，导致细胞功能障碍。