Potential activation of Disease susceptibility genes by Enhancer Release and Retargeting

通过增强子释放和重新定位潜在激活疾病易感基因

• 批准号: 10301867
• 负责人: Soohwan Oh
• 金额: $ 11.51万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-01 至 2022-06-01
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10301867
• 关键词: 3-Dimensional; ATAC-seq; Appointment; Architecture; Area; Award; Binding; Bioinformatics; Biological; Biomedical Research; Breast; CRISPR/Cas technology; Cells; Chromatin; Chromosome Structures; Clustered Regularly Interspaced Short Palindromic Repeats; Code; Computer Analysis; DNA Sequence; Data; Development; Diagnostic; Disease; Disease susceptibility; Dreams; Endocrine System Diseases; Endocrine system; Enhancers; Epigenetic Process; Estradiol; Estrogen Receptor alpha; Estrogens; Event; Gene Activation; Gene Expression Regulation; Gene Targeting; Genes; Genetic; Genetic Enhancer Element; Genetic Transcription; Genome; Genomics; Genotype-Tissue Expression Project; Glucocorticoids; Goals; Hormonal; Hormones; Human; Image; Influentials; Informatics; Institution; Investigation; Knowledge; LNCaP; Laboratories; Licensing; Ligands; MCF7 cell; Mammalian Cell; Mediating; Mentored Research Scientist Development Award; Mentors; Modeling; Molecular; Mutate; Mutation; National Institute of Diabetes and Digestive and Kidney Diseases; Nature; Outcome; Population; Principal Investigator; Promoter Regions; Proteomics; Regulation; Regulatory Element; Research; Research Proposals; Signal Transduction; Stimulus; Susceptibility Gene; Techniques; Technology; Therapeutic; Time; Training; Transcriptional Regulation; Untranslated RNA; Validation; Variant; Work; androgen sensitive; base; bioinformatics pipeline; career; chromosome conformation capture; cohort; data analysis pipeline; disorder risk; genome wide association study; genome-wide; genomic data; global run on sequencing; human disease; imaging approach; insight; mutant; next generation sequencing; novel; professor; programs; promoter; real-time images; response; risk variant; single cell technology; skills; tenure track; therapeutic target; tool; transcription factor; transcriptome sequencing

ABSTRACT Enhancers are cis-regulatory DNA sequences that increase the transcription of its cognate target genes in response to developmental and regulatory signaling at linear distances of kilo- to mega bases from their gene targets directing critical transcriptional programs. Mechanistically, considerable data support the model that interactions between enhancer and promoter are achieved by looping. Chromosome Conformation Capture Analysis (3C) and its derivative techniques provide information about 3D genomic architecture of enhancer- promoter interactions and suggest that the long-range nature of enhancer functions tightly connects their regulation to chromatin architecture. Genome folding into spatial domains facilitates transcriptional regulation consistent with the observation that promoters and enhancers within the same domain preferentially interact with each other. However, despite recent insights into 3D chromosomal organization and topologically associated contact domains, the mechanisms and disease risk implications underlying cognate promoter choice by an activated enhancer remain incompletely understood. To explore the mechanism underlying enhancer-promoter choice, we will employ gene editing by CRISPR/Cas9 to specifically delete or mutate a promoter of the estrogen- regulated gene. Intriguingly, the mutation of the cognate promoter causes its activating enhancer to “switch” its target gene promoter choice, resulting in activation of the alternative gene target. We term this previously- unappreciated phenomenon as “enhancer release and retargeting (ERR).” To systematically investigate the ERR phenomenon and establish it as a major disease risk mechanism, we propose to develop and apply bioinformatic pipelines to analyze data from several large cohorts of datasets from genomics and normal human population such as GWAS and GTEx. Therefore, we can computationally identify putative ERR events that may be associated with human disease including endocrine diseases in which the neighboring alternative genes, rather than the gene hosting the mutant promoter, are disease-causing. Further, we plan to experimentally corroborate this by identifying the putative ERR gene pairs in systemic approach by using CRISPR-KRAB mediated inhibition of promoters. We will investigate the ERR cases of disease significance in detail with appropriate powerful contemporary global genomic technologies, including a variety NGS methods (e.g. 4C, GRO-seq, RNA-seq, ATAC-seq, etc.), single-cell technologies (e.g. scRNA sequencing and live imaging), and the requisite bioinformatic pipelines for analyzing data to explore the mechanism underlying ERR phenomenon and enhancer- promoter choice. By gaining a comprehensive understanding of ERR, including the identification of promoter CTCF binding as a key determinant of promoter choice, the proposed research may represent an important step in understanding enhancer function and enhancer-promoter choice in mammalian cells as well as provide new insights into understanding many previously confounding promoter mutations or variations associated with human disease. We hope to identify potential therapeutic targets including a variety of endocrine diseases.

摘要 增强子是顺式调节DNA序列，其增加其同源靶基因的转录， 对发育和调控信号的反应，从基因的线性距离为千至百万碱基 指导关键转录程序的靶点。从机制上讲，大量数据支持这一模型， 增强子和启动子之间的相互作用通过成环实现。染色体构象捕获 分析（3C）及其衍生技术提供了关于增强子- 启动子的相互作用，并表明增强子功能的长程性质紧密连接其 调节染色质结构。基因组折叠成空间结构域促进转录调控 这与相同结构域内的启动子和增强子优先与 对方.然而，尽管最近对3D染色体组织和拓扑相关的认识， 接触域，机制和疾病风险的影响，潜在的同源启动子的选择， 激活的增强子仍然不完全理解。探讨增强子-启动子的作用机制 选择，我们将采用CRISPR/Cas9的基因编辑来特异性地删除或突变雌激素的启动子- 调控基因有趣的是，同源启动子的突变导致其激活增强子“切换”其启动子。 靶基因启动子选择，导致替代靶基因的激活。我们以前称之为- 这种现象被称为“增强子释放和重靶向（ERR）”。系统地调查ERR 现象，并建立它作为一个主要的疾病风险机制，我们建议开发和应用生物信息学 用于分析来自基因组学和正常人群的几个大型数据集队列的数据的管道 例如GWAS和GTEx。因此，我们可以通过计算确定可能是 与包括内分泌疾病在内的人类疾病相关，其中邻近的替代基因， 而不是携带突变启动子的基因，都是致病的。此外，我们计划通过实验证实 这通过使用CRISPR-KRAB介导的抑制在系统方法中鉴定推定的ERR基因对来实现 的推动者。我们将详细调查ERR病例的疾病意义，并提供适当的有力证据。 当代全球基因组技术，包括各种NGS方法（例如4C，GR 0-seq，RNA-seq， ATAC-seq等），单细胞技术（例如scRNA测序和活体成像），以及必要的 用于分析数据的生物信息学管道，以探索ERR现象和增强子的潜在机制， 启动子选择通过对ERR的全面了解，包括启动子的鉴定， CTCF结合作为启动子选择的关键决定因素，拟议的研究可能代表了重要的一步 在理解哺乳动物细胞中增强子功能和增强子-启动子选择方面， 深入了解许多以前混淆的启动子突变或变异相关的 人类疾病我们希望确定潜在的治疗靶点，包括各种内分泌疾病。