Death-Seq, a Method for Genome-wide Identification of Functional Silencer Elements

一种全基因组鉴定功能性沉默因子的方法--Death-Seq 元素 摘要 人类基因组估计含有98.5%的非编码DNA1。人们对其功能知之甚少 非编码区DNA，检测功能性非编码区的工具有限。这些非编码 区域包含重要的监管元素(例如，增强剂、消音器、绝缘体)，这些元素在 人类正常发育，其失调与许多人类疾病有关。映射 我们基因组中的调控元件目前依赖于结构分析、转录因子的定位 结合部位或染色质标记和一些功能分析，包括Starr-Seq(一种自我转录报告 测定增强子功能)。尽管人们对其功能和位置的了解越来越多 增强剂，不存在从功能上分析消音器元件的工具。 我们提出了一种新的方法，称为Death-Seq，用于询问全基因组DNA的功能沉默能力 通过负选择。简而言之，用于询问的基因组文库将调节自杀基因在 一个可转化的载体。在增强子元件的控制下，自杀基因将表达并诱导细胞 死亡，导致病媒枯竭。沉默元件将抑制自杀基因的表达，导致细胞(和 载体)生存。在选择之后，来自存活细胞的质粒将被测序，并预计包含 只有正常工作的消音器元件。目标1建议使用基于Caspase 9的方法识别构成消音器 自杀基因诱导细胞凋亡。目标2描述了该方法的一种变体，允许在 用户定义的时间点(用于检测依赖于诱导抑制器的消音器)。此工具解决了一个 基因调节界未得到满足的需求，因为目前的技术不允许对消音器进行全基因组研究 功能。此外，死亡序列可能被用于未来的研究，以检验各种假说；它的多功能性 允许询问不同的输入库(例如基因组或ATAC/芯片-序列)及其可转染性 允许研究不同细胞类型中的消音器。 Death-Seq将允许对基因组沉默元件进行编目，既有细胞类型特有的，也有通用的。这个 功能沉默分子的位置将通过以下方式加强对转录和染色质调节的理解 转录因子。识别新的消音器基序以及消音器的全基因组特征 地点，将使基因组研究界广泛受益。确定消声器元件在非 全基因组关联研究的编码结果将为未来的合理研究提供方向 人类疾病的机制。我们相信，通过这项提议开发的工具Death-Seq将 为研究消音器生物学提供了一种创新而可行的解决方案。

Title: Death-Seq, a Method for Genome-wide Identification of Functional Silencer Elements Summary The human genome contains an estimated 98.5% of non-coding DNA 1. Little is known about the function of this non-coding DNA, and limited tools are available to assay for functional non-coding regions. These non-coding regions contain essential regulatory elements (e.g., enhancers, silencers, insulators) that are paramount during normal human development and whose dysregulation is implicated in numerous human diseases. Mapping regulatory elements in our genome currently relies upon structural assays, location of transcription factor binding sites or chromatin marks and a few functional assays, including STARR-Seq (a self-transcribing reporter assay to measure enhancer functionality). Despite increasing knowledge of the function and location of enhancers, no tool exists to functionally assay silencer elements. We propose a novel method, termed Death-Seq, to interrogate genome-wide DNA for functional silencer ability by negative selection. Briefly, genomic libraries for interrogation will regulate expression of a suicide gene within a transfectable vector. Under the control of an enhancer element, the suicide gene will express and induce cell death, leading to vector depletion. A silencer element will repress suicide gene expression, leading to cell (and vector) survival. After selection, plasmids from the surviving cells will be sequenced and are expected to contain only functional silencer elements. Aim 1 proposes to identify constitutive silencers using a Caspase 9- based suicide gene to induce apoptosis. Aim 2 describes a variation the approach, allowing for silencer interrogation at user-defined timepoints (for detection of silencers dependent on inducible repressors). This tool addresses an unmet need in the gene regulation community, as no current technique allows for genome-wide study of silencer function. Additionally, Death-Seq may be used in future investigations to test diverse hypotheses; its versatility allows for interrogation of varied input libraries (e.g., genomic or ATAC/ChIP-Seq), and its transfectability allows for study of silencers in diverse cell types. Death-Seq will allow for the cataloguing of genomic silencer elements, both cell-type specific and universal. The location of a functional silencer will enhance understanding of transcriptional and chromatin regulation by transcription factors. Identifying novel silencer motifs, as well as genome-wide characteristics of silencer locations, will benefit the community of genomic research broadly. Identifying silencer element locations in non- coding results of genome-wide association studies (GWAS) will yield directions for future rational research into the mechanisms of human disease. We believe that the tool developed through this proposal, Death-Seq, will provide an innovative yet feasible solution to investigating silencer biology.