Mapping of Novel Candidate Functional Elements with Bru-Seq Technology

摘要 ENCODE项目为科学家提供了巨大的资源，他们拥有来自 描述RNA表达(RNA-SEQ)、表观遗传特征(CHIP-SEQ)、 染色质结构(DNase-seq、ATAC-seq、Hi-C、Chia-PET)和特定蛋白与 DNA和RNA(CHIP-SEQ、CLIP-SEQ、RIP-SEQ)。我们最近开发了一套技术， 是基于新生RNA用溴(Bru)特异性标记，然后裂解，捕获 使用特定抗体和深度测序标记Bru标记的RNA。Bru-seq捕捉到新生的RNA 转录组，基因组中正在进行的转录的标志，其中转录的相对速率 所有的基因都可以得到。在BruChase-Seq中，细胞被Bru标记，然后在尿苷中追逐 评估了确定所有成绩单的相对稳定性的不同时间段。 最后，Bruuv-seq允许捕获通常被 RNA外体，可用于定位产生Erna全基因组的活性增强子元件。在这 UM1拨款申请我们建议使用这三种BRU-SEQ技术来获得批判性小说 关于新生的RNA转录组、RNA稳定体和活性增强的信息将补充 并丰富现有的编码数据。我们建议将我们的测绘工作集中在两个具体目标上： 目的1：获得人类和人类的新生RNA转录组、RNA稳定组和活性增强组数据 小鼠ENCODE细胞系。目标2：获得新生转录、RNA稳定性和 细胞暴露于环境扰动后全基因组增强子元件的活性。这个 在这份UM1拨款申请中提出的映射努力正在寻求确定新的候选监管 将补充和扩展现有ENCODE数据的全基因组元素。此外，动态的 将启动研究，以评估在选定的细胞过渡和 特定的细胞反应。 目标： 该建议的主要目标是使用我们最新开发的数据集生成全面的新数据集 BRU-SEQ分析平台，将补充和扩展现有的ENCODE数据。重点将放在 绘制人类基因组中的候选功能元件。

ABSTRACT The ENCODE project has provided a tremendous resource for scientists with a treasure trove of data from a large set of different cell types describing RNA expression (RNA-seq), epigenetic signatures (ChIP-seq), chromatin structure (DNase-seq, ATAC-seq, Hi-C, ChIA-PET) and binding patterns of specific proteins to both DNA and RNA (ChIP-seq, CLIP-seq, RIP-seq). We have recently developed a set of techniques that are based on the specific labeling of nascent RNA with bromouridine (Bru) followed by lysis, capturing of the Bru-labeled RNA using specific antibodies and deep sequencing. Bru-seq captures the nascent RNA transcriptome, a signature of ongoing transcription in the genome where the relative rates of transcription of all genes can be obtained. In BruChase-seq, cells are labeled with Bru and then chased in uridine for different periods of time to allow for the determination of the relative stability of all transcripts are assessed. Finally, BruUV-seq allows for the capturing of RNA species that normally are rapidly turned over by the RNA exosome and can be used to map active enhancer elements producing eRNA genome-wide. In this UM1 grant application we are proposing to use these three Bru-seq techniques to obtain critical novel information on the nascent RNA transcriptome, RNA stabilome and active enhancome that will complement and enrich existing ENCODE data. We are proposing to focus our mapping efforts on two specific Aims: Aim 1: Obtain nascent RNA transcriptome, RNA stabilome and active enhancome data for human and mouse ENCODE cell lines. Aim 2: Obtain dynamic signatures of nascent transcription, RNA stability and activity of enhancer elements genome-wide following cellular exposure to environmental perturbations. The mapping efforts proposed in this UM1 grant application are seeking to identify novel candidate regulatory elements genome-wide that will complement and extend existing ENCODE data. Furthermore, dynamic studies will be initiated to assess the activities of regulatory elements during selected cell transitions and specific cellular responses. GOALS: The main goal of this proposal is to generate comprehensive novel data sets using our newly developed Bru-seq analysis platform that will complement and extend existing ENCODE data. The emphasis will be on mapping candidate functional elements in the human genome.