Genome wide identification and functional analysis of chromatin regulatory RNAs

除了基因组中蛋白质编码基因的转录外， 转录编码不产生mRNA的RNA分子。这些非编码RNA 在细胞中的重要作用，包括调节剂量补偿，控制基因组 印记和调节转录。然而，人类细胞转录数千种 非编码RNA，我们只将功能归于少数。的一个主要 理解非编码RNA功能的挑战是， 缺乏鉴定和表征非编码RNA。在这个建议中，我们开发了一种新的 这种方法可以在任何细胞类型中识别所有非编码RNA， 与染色体相互作用，同时绘制这些RNA结合的位点， 染色质我们的方法包括直接连接非编码RNA的基础DNA， 在染色体结合的RNA和DNA之间产生共价嵌合体。使用下 通过一代测序，我们可以识别细胞中可能调节 染色体结构或功能，并确定其在染色体上的作用位点。在我们 第一个目的我们使用果蝇细胞来开发这种方法，利用了这样一个事实， 已知已建立的染色体RNA roX1和roX2包覆X染色体， 在飞行中完成剂量补偿。然后，我们在目标2中扩展了这种方法， 确定在整个人类基因组中结合染色质的RNA，并开发新的 分析基础设施来分类和功能分配这些RNA。在目标3中， 干扰实验来测试非编码RNA和RNA基序的功能， 对局部染色体可及性、组蛋白修饰状态和转录输出的影响。 我们应用一个系统将非编码RNA重定向到新的基因组区域，以测试它们的功能。 影响染色体并通过RNA依赖性调节不同的基因组区域 控制通过定义人类染色质相关RNA的景观以及 它们在细胞中调节我们提出的这些RNA如何发挥作用以及缺陷的影响， 在RNA依赖性控制中导致细胞功能障碍。

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.