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

基本信息

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
项目类别：
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

项目摘要

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.

抽象的增强子是顺式调节性DNA序列，可增加其同源靶基因的转录从其基因到巨型基础的线性距离处的发育和调节信号传导的响应针对指导关键转录程序的目标。从机械上讲，大量数据支持该模型增强子和启动子之间的相互作用是通过循环实现的。染色体构象捕获分析（3C）及其衍生技术提供了有关增强子的3D基因组结构的信息促进者的相互作用，并表明增强子功能的远程性质紧密地连接调节染色质体系结构。基因组折叠到空间域设施转录调节与观察到同一域内的启动子和增强子优先与之相互作用的观察者彼此。但是，对3D染色体组织和拓扑相关的最新见解接触域，机制和疾病风险的影响，是通过激活的增强剂仍然不完全理解。探索增强器促销的基础机制选择，我们将采用CRIS/CAS9的基因编辑来专门删除或突变雌激素的启动子调节基因。有趣的是，同源启动子的突变导致其激活增强子“切换” 靶基因启动子选择，导致替代基因靶标的激活。我们以前称这个未欣赏的现象是“增强子释放和重新定位（err）”。系统地研究错误现象并将其确立为主要的疾病风险机制，我们建议开发和应用生物信息管道来分析来自基因组学和正常人人群的几个数据集的数据例如GWAS和GTEX。因此，我们可以在计算上确定可能是的假定错误事件与人类疾病有关，包括邻近替代基因的内分泌疾病，而不是比主持突变体启动子的基因是引起疾病的。此外，我们计划在实验中证实通过使用CRISPR-KRAB介导的抑制作用，通过在系统性方法中识别假定的错误基因对促进者。我们将详细研究具有适当强大的疾病意义的错误病例当代全球基因组技术，包括多种NGS方法（例如4C，GRO-SEQ，RNA-SEQ， ATAC-SEQ等），单细胞技术（例如SCRNA测序和实时成像）以及必要的用于分析数据的生物信息学管道探索了ERR现象和增强子的基础机制启动子选择。通过对ERR的全面了解，包括识别启动子 CTCF绑定是启动子选择的关键决定者，拟议的研究可能代表一个重要的步骤在了解增强子功能和哺乳动物细胞中的增强剂促销方面以及提供新的了解许多以前混淆启动子突变或与之相关的变化的见解人类疾病。我们希望确定潜在的治疗靶标，包括各种内分泌疾病。