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，一种全基因组鉴定功能消音器的方法元素概括人类基因组估计有98.5％的非编码DNA 1。非编码DNA和有限的工具可用于分析功能性非编码区域。这些非编码区域包含必不正常的人类发展且其失调与许多人类疾病有关。映射我们基因组中的调节元素目前依赖于结构性测定，转录因子的位置绑定位点或染色质标记和一些功能性测定，包括starr-seq（一个自转录的记者测量增强子功能的测定）。尽管越来越了解增强剂，没有任何工具可以在功能上测定消音器元素。我们提出了一种新的方法，称为“死亡序言”，以询问全基因组DNA的功能消音器能力通过否定选择。简而言之，询问的基因组文库将调节自杀基因的表达可转染的向量。在增强子元件的控制下，自杀基因将表达并诱导细胞死亡，导致矢量耗竭。消音器元素将抑制自杀基因表达，导致细胞（和矢量）生存。选择后，将测序来自幸存细胞的质粒，并预计将包含仅功能消音器元素。 AIM 1提议使用caspase 9基识别组成型消音器自杀基因诱导凋亡。 AIM 2描述了方法的变化，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）。该工具解决了一个基因调节社区中未满足的需求，因为当前的技术没有允许对消音器进行全基因组的研究功能。此外，在未来的研究中可以使用死亡史来检验多种假设。它的多功能性允许询问各种输入库（例如基因组或ATAC/CHIP-SEQ）及其可转录性允许研究各种细胞类型中的消音器。死亡季节将允许将基因组消音器元素分类，包括细胞类型的特异性和通用。这功能消音器的位置将通过转录因子。识别新颖的消音器基序以及消音器的全基因组特征位置，将广泛地使基因组研究社区受益。识别非 - 中的消音器元素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指导人类疾病的机制。我们认为，通过该提案开发的工具，死亡seq，将为调查消音器生物学提供了创新但可行的解决方案。