Potential activation of Disease susceptibility genes by Enhancer Release and Retargeting

摘要 增强子是顺式调控 DNA 序列，可增加其同源靶基因的转录 对距其基因数千至兆碱基线性距离处的发育和调节信号作出反应 指导关键转录程序的目标。从机制上讲，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 3D 基因组结构的信息 启动子相互作用并表明增强子功能的远程性质将它们紧密地联系在一起 染色质结构的调节。基因组折叠成空间域促进转录调控 与同一结构域内的启动子和增强子优先相互作用的观察结果一致 彼此。然而，尽管最近对 3D 染色体组织和拓扑相关的见解 接触域、同源启动子选择的机制和疾病风险影响 激活的增强子仍不完全清楚。探索增强子-启动子的潜在机制 选择，我们将采用 CRISPR/Cas9 进行基因编辑，特异性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 靶基因启动子选择，导致替代靶基因的激活。我们之前称之为—— 未被重视的现象为“增强子释放和重定向（ERR）”。系统地调查 ERR 现象并将其确立为重大疾病风险机制，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多个大型数据集的数据的管道 例如 GWAS 和 GTEx。因此，我们可以通过计算来识别可能的 ERR 事件 与人类疾病相关，包括内分泌疾病，其中邻近的替代基因，而不是 比托管突变启动子的基因更能引起疾病。此外，我们计划通过实验证实 通过使用 CRISPR-KRAB 介导的抑制以系统方法识别假定的 ERR 基因对 的发起人。我们将通过适当的有力措施详细调查具有疾病意义的 ERR 病例 当代全球基因组技术，包括各种 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 用于分析数据以探索 ERR 现象和增强剂背后机制的生物信息学管道 启动子选择。通过全面了解 ERR，包括启动子的识别 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 了解哺乳动物细胞中的增强子功能和增强子-启动子选择，并提供新的 深入了解许多先前令人困惑的启动子突变或变异 人类疾病。我们希望找到潜在的治疗靶点，包括多种内分泌疾病。

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.