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

基本信息

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
项目类别：
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

项目摘要

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.

标题：Death-Seq，一种全基因组识别功能沉默子的方法元素概括人类基因组包含估计 98.5% 的非编码 DNA 1。人们对其功能知之甚少非编码 DNA，可用于检测功能性非编码区的工具有限。这些非编码区域包含必要的调节元件（例如增强子、消音器、绝缘子），这些元件在正常的人类发育，其失调与许多人类疾病有关。测绘我们基因组中的调控元件目前依赖于结构分析、转录因子的位置结合位点或染色质标记以及一些功能测定，包括 STARR-Seq（一种自转录报告基因）测定增强子功能）。尽管对功能和位置的了解不断增加增强剂，不存在用于功能分析消音器元件的工具。我们提出了一种称为 Death-Seq 的新方法，用于询问全基因组 DNA 的功能性沉默能力通过负选择。简而言之，用于审讯的基因组文库将调节自杀基因的表达可转染的载体。在增强子元件的控制下，自杀基因会表达并诱导细胞死亡，导致载体耗尽。沉默元件将抑制自杀基因的表达，导致细胞（和矢量）生存。选择后，将对来自存活细胞的质粒进行测序，并预计包含仅功能性消音器元件。目标 1 建议使用基于 Caspase 9 的方法来识别组成型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阻遏物的消音器）。该工具解决了基因调控领域的需求未得到满足，因为目前的技术还没有允许对沉默子进行全基因组研究功能。此外，Death-Seq 可能会在未来的研究中用于测试不同的假设；它的多功能性允许询问各种输入文库（例如基因组或 ATAC/ChIP-Seq）及其转染性允许研究不同细胞类型中的沉默子。 Death-Seq 将允许对基因组沉默元件进行编目，包括细胞类型特异性的和通用的。这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。识别新的沉默子基序以及沉默子的全基因组特征地点，将使基因组研究界广泛受益。识别非消音器元件位置全基因组关联研究（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，通过该提案开发的工具 Death-Seq 将为研究消音器生物学提供创新且可行的解决方案。