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

Death-Seq，一种全基因组识别功能性沉默元件的方法

• 批准号: 9979291
• 负责人: Artem Barski
• 金额: $ 15.9万
• 依托单位: CINCINNATI CHILDRENS HOSP MED CTR
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-06-01 至 2022-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9979291
• 关键词: ATAC-seq; Address; Apoptosis; Binding; Binding Proteins; Binding Sites; Biological Assay; Biology; CASP9 gene; Cataloging; Catalogs; Cell Culture Techniques; Cell Death; Cells; Cessation of life; ChIP-seq; Characteristics; Chromatin; Communities; DNA; DNase I hypersensitive sites sequencing; Data; Detection; Development; Disease; Elements; Enhancers; Future; Gene Expression; Gene Expression Regulation; Genetic Enhancer Element; Genetic Transcription; Genome; Genomic Library; Genomics; Goals; Half-Life; Harvest; Histones; Human; Human Development; Human Genome; Individual; Infusion procedures; Investigation; Jurkat Cells; Knowledge; Laboratories; Libraries; Location; Luciferases; Measures; Mediating; Methodology; Methods; Pattern; Pharmacology; Plasmids; Regulatory Element; Reporter; Reporter Genes; Repressor Proteins; Research; Research Personnel; Single Nucleotide Polymorphism; Structure; T-Lymphocyte; Tacrolimus Binding Proteins; Techniques; Technology; Testing; Time; Transcriptional Regulation; Transcriptional Silencer Elements; Transfection; Untranslated RNA; Variant; XCL1 gene; base; cell type; design; dimer; disorder risk; epigenome; fusion gene; gene induction; genome wide association study; genome-wide; genome-wide analysis; human disease; innovation; next generation sequencing; novel; programs; promoter; suicide gene; temporal measurement; therapy design; tool; transcription factor; vector

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.

Death-Seq，一种全基因组鉴定功能沉默子的方法 元件 总结 人类基因组包含估计98.5%的非编码DNA 1。人们对这一功能知之甚少 非编码DNA，并且有限的工具可用于测定功能性非编码区。这些非编码 区域包含必需的调节元件（例如，增强器、消音器、绝缘体）， 正常的人类发育，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， 结合位点或染色质标记和一些功能测定，包括STARR-Seq（自转录报告基因 测定增强子功能性）。尽管人们越来越了解 增强子，没有工具存在功能测定沉默元件。 我们提出了一种新的方法，称为死亡测序，询问全基因组DNA的功能沉默能力 负选择。简而言之，用于询问的基因组文库将调节细胞内自杀基因的表达。 可转染载体。在增强子元件的控制下，自杀基因将表达并诱导细胞凋亡。 死亡，导致载体耗竭。沉默元件将抑制自杀基因的表达，导致细胞（和 载体）存活。选择后，将对来自存活细胞的质粒进行测序，并预期含有 只有功能性消音器元件。目的1提出使用基于半胱天冬酶9的 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型阻遏物的沉默子）。该工具解决了 基因调控领域的需求未得到满足，因为目前没有技术允许对沉默者进行全基因组研究 功能此外，Death-Seq可用于未来的调查，以测试各种假设;其多功能性 允许询问不同的输入库（例如，基因组或ATAC/ChIP-Seq）及其转染能力 允许研究不同细胞类型中的沉默器。 Death-Seq将允许对细胞类型特异性和通用性的基因组沉默元件进行编目。的 功能性沉默子的定位将增强对转录和染色质调控的理解， 转录因子鉴定新的沉默子基序，以及沉默子的全基因组特征 地点，将有利于社区的基因组研究广泛。确定非- 全基因组关联研究（GWAS）的编码结果将为未来的理性研究提供方向， 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 为研究沉默者生物学提供了一个创新而可行的解决方案。