Transcriptional regulation of BEST1 in retina pigment epithelium.

视网膜色素上皮细胞中 BEST1 的转录调控。

• 批准号: 10200053
• 负责人: Yin Shen
• 金额: $ 39.91万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-30 至 2022-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10200053
• 关键词: Affect; Age of Onset; Alleles; Biological Process; Biological Testing; Blindness; CISH gene; CRISPR/Cas technology; Cell Line; ChIP-seq; Chromatin; Chromatin Conformation Capture and Sequencing; Code; Disease; Disease Progression; Distal; Dominant-Negative Mutation; Elements; Enhancers; Equilibrium; Eye diseases; Family member; Frequencies; Gene Expression; Gene Expression Regulation; Gene Mutation; Gene Proteins; Gene Silencing; Genes; Genetic; Genetic Enhancer Element; Genetic Transcription; Genetic Variation; Genome; Genomic approach; Goals; Homo; Human; Individual; Ion Channel; Knowledge; Link; Lipofuscin; Location; Maps; Mediating; Methods; Modeling; Molecular; Mutation; Nonhomologous DNA End Joining; Nucleic Acid Regulatory Sequences; Ophthalmic examination and evaluation; Outcome Study; Patients; Phase; Phenotype; Pigments; Point Mutation; Regulatory Element; Reporter; Research; Resolution; Retina; Site; Structure of retinal pigment epithelium; Synapses; System; Testing; Transcription Initiation Site; Transcriptional Regulation; Transplantation; Untranslated RNA; Validation; Vision; Vitelliform macular dystrophy; base; causal variant; cell type; clinical phenotype; density; design; effectiveness testing; epigenomics; functional genomics; genome editing; genomic locus; human embryonic stem cell; in vivo; induced pluripotent stem cell; insight; mutant; new therapeutic target; novel; promoter; therapeutic genome editing; tool

Abstract The overarching goal of this study is to understand how BEST1 expression is modulated by cis-regulatory elements in the retinal pigment epithelium (RPE). The variability of retinal phenotypes and age of onset of visual loss, even in individuals who carry the same causative mutation, is one of the biggest mysteries for BEST1 related diseases. Genetic variations in non-coding regulatory elements can serve as an attractive model to provide the molecular basis for the observed various onsets of BEST1 vitelliform macular dystrophy (BVMD) in patients. Therefore understanding the transcriptional control mediated by cis-regulatory elements in the RPE will give us insight into novel target regions for therapeutic editing of the enhancer elements identified by this study. We are utilizing human induced pluripotent stem cells (iPSCs)-derived RPEs as a model to understand cell type-specific transcriptional controls of BEST1. We are also utilizing integrative, unbiased, and high throughput epigenomic and genetic tools to achieve the following aims: (1) We will use a high resolution 4C-seq analysis to identify potential regulatory elements that interact with the BEST1 promoter in human primary RPE. (2) In Aim 2, we will generate BEST1 dual allele reporter lines and use these lines to interrogate cis-regulatory elements of BEST1 in their native chromatin contexts utilizing high throughput CRIPSR/Cas9 mediated genome editing approach. (3) Finally, we will test the utilities of allelic specific enhancer deletion to modulate allelic expression of BEST1. We expect these analyses will significantly advance our knowledge of the precise control of BEST1 transcription in RPEs.

抽象的 这项研究的总体目标是了解如何通过顺式调节调节最佳表达 视网膜色素上皮（RPE）中的元素。视网膜表型和发作年龄的变异性 视觉丧失，即使在带有相同因果突变的个人中，视觉丧失也是最大的奥秘之一 Best1相关疾病。非编码调节元件中的遗传变异可以作为有吸引力的 模型为观察到的最佳1叶片黄斑营养不良的各种on的分子基础提供分子基础 （BVMD）患者。因此，了解由顺式调节元件介导的转录控制 RPE将使我们深入了解已确定的增强子元素的治疗编辑的新型目标区域 通过这项研究。我们利用人类诱导的多能干细胞（IPSC）衍生的RPE作为模型 了解BEST1的细胞类型特异性转录控制。我们还利用综合性，公正和 高吞吐量的表观基因组和遗传工具以实现以下目的：（1）我们将使用高分辨率 4C-seq分析以确定与人类最佳1启动子相互作用的潜在调节元件 主RPE。 （2）在AIM 2中，我们将生成Best1双重等位基因记者行并使用这些行询问 使用高吞吐量cripsr/cas9的本机染色质环境中最佳1的顺式调节元素 介导的基因组编辑方法。 （3）最后，我们将测试等位基因特定增强子删除的实用程序 调节BEST1的等位基因表达。我们希望这些分析将大大提高我们对 RPE中最佳1转录的精确控制。