Mapping of Novel Candidate Functional Elements with Bru-Seq Technology

摘要 ENCODE项目为科学家提供了一个巨大的资源宝库 来自描述RNA表达的一大组不同细胞类型的数据(RNA-seq)，表观遗传学 标记(CHIP-SEQ)、染色质结构(DNase-SEQ、ATAC-SEQ、Hi-C、CHIA-PET)和结合 DNA和RNA的特定蛋白质模式(CHIP-SEQ、CLIP-SEQ、RIP-SEQ)。而RNA- SEQ数据在提供稳定状态RNA水平的签名方面提供了非常丰富的信息 特定的基因，RNA合成和降解的个体对稳态的贡献- 核糖核酸的水平无法确定。此外，这些表达数据集是静态的，并且 不提供关于特定细胞响应的动态信息。关于芯片序列分析 特异组蛋白修饰，如高H3K1me1和H3K27ac和低H3K4me3，它们是 对特定细胞类型中假定的增强子元件的鉴定有价值，但不是 告知这些功能元件是否在功能上处于活动状态。我们最近开发了 一套基于用溴(Bru)特异性标记新生RNA的技术 然后裂解，使用特定的抗体和深度捕获Bru标记的RNA 测序。Bru-seq捕捉到了“新生RNA转录组”，这是 基因组中的转录，可以获得所有基因的相对转录速率。 在BruChase-seq中，细胞被Bru标记，然后在尿苷中追逐不同时期的 允许测定“RNA稳定体”的时间，其中相对RNA降解 对全基因组的比率进行了评估。最后，Bruuv-seq允许捕获rna物种。 它们通常会被RNA外切体快速翻转，并允许从不同的角度观察 活性增强子元件和其他候选功能元件的新转录组 产生不稳定的RNA是在全基因组范围内识别的。在此UM1授权期中，我们使用了 我们在人类细胞系上的BRU-SEQ分析套件，以补充之前 是使用各种基因组分析产生的。在授权期的第五年，我们计划 为了将BRU-SEQ分析扩展到1)新冠肺炎细胞系实验，2)免疫/血细胞 样本和3)遗传扰动。

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). While RNA- seq data is very informative in providing signatures of steady-state levels of RNA expressed for particular genes, individual contributions of RNA synthesis and degradation to the steady-state- level of RNA cannot be determined. Furthermore, these expression data sets are static and do not provide dynamic information on specific cell responses. Regarding the ChIP-seq analyses of specific histone modifications, such as high H3K1me1 and H3K27ac and low H3K4me3, they are valuable for the identification of putative enhancer elements in a particular cell type but does not inform on whether such functional elements are functionally active. 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 “RNA stabilome” where the relative RNA degradation rates genome-wide are assessed. Finally, BruUV-seq allows for the capturing of RNA species that normally are rapidly turned over by the RNA exosome and allows for a different view of the nascent transcriptome where active enhancer elements and other candidate functional elements generating unstable RNAs are identified genome-wide. In this UM1 granting period we have used our Bru-seq suite of assays on human cell lines to complement existing data that have previously been generated using various genomic assays. In year 5 of this granting period, we are planning to extend the Bru-seq analysis for 1) COVID-19 cell line experiments, 2) immune/blood cell samples and 3) genetic perturbations.