Potential activation of Disease susceptibility genes by Enhancer Release and Retargeting

摘要 增强子是顺式调节的DNA序列，可增加其同源靶基因在 对发育和调控信号的反应在千兆碱基与其基因的线性距离处 指导关键转录程序的目标。从机制上讲，相当多的数据支持这样的模型 增强子和启动子之间的相互作用是通过环来实现的。染色体构象捕捉 分析(3C)及其衍生技术提供了关于增强子的3D基因组结构的信息- 启动子之间的相互作用，并表明增强子功能的长程性质将它们的 对染色质结构的调节。基因组折叠到空间域促进转录调控 与同一结构域中的启动子和增强子优先相互作用的观察结果一致 彼此之间。然而，尽管最近对3D染色体的组织和拓扑结构有深入的了解 选择同源启动子的联系结构域、机制和疾病风险 激活的增强子仍然不完全被理解。探讨增强子-启动子的作用机制 选择，我们将使用CRISPR/Cas9的基因编辑来特异性地删除或突变雌激素的启动子- 受调控的基因。有趣的是，同源启动子的突变导致其激活的增强子“切换”其 靶基因启动子的选择，导致选择性靶基因的激活。我们以前把这称为- 未被认可的现象，如“增强剂释放和重定向(ERR)”。系统地调查错误 并将其建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类群体的多个大型数据集的数据的管道 例如GWAs和GTEx。因此，我们可以通过计算识别可能存在的错误事件 与人类疾病相关的，包括内分泌疾病，在这些疾病中，邻近的替代基因，而不是 比拥有突变启动子的基因更能致病。此外，我们计划通过实验证实 这是通过使用CRISPR-KRAB介导的抑制在系统方法中识别可能的ERR基因对来实现的 推动者。我们将以适当的权力详细调查具有疾病意义的差错案件 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 用于分析数据的生物信息管道，以探索错误现象和增强剂的机制- 发起人选择。通过对错误的全面了解，包括对启动子的识别 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着重要的一步 了解哺乳动物细胞中增强子的功能和增强子-启动子的选择，并提供新的 对理解许多以前混淆的启动子突变或变异的见解 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。

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.