Non-Coding Genetic Vulnerabilities in Human Photoreceptor Function and Disease

人类感光功能和疾病中的非编码遗传漏洞

• 批准号: 10372058
• 负责人: TIMOTHY JOEL CHERRY
• 金额: $ 45.66万
• 依托单位: SEATTLE CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-04-01 至 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10372058
• 关键词: ATAC-seq; Address; Adult; Affect; Age; Automobile Driving; Binding; Binding Sites; Biological Assay; Biological Process; CRISPR/Cas technology; Case Study; Cell physiology; ChIP-seq; Chromatin; Complex; Conserved Sequence; DNA; DNA Sequence; DNA Sequence Alteration; Data; Development; Diagnosis; Diagnostic; Disease; Elements; Enhancers; Essential Genes; Gene Expression; Gene Expression Regulation; Genes; Genetic; Genetic Predisposition to Disease; Genetic Variation; Genome; Genomics; Goals; Human; Human Activities; Human Genome; In Vitro; Individual; Inherited; Knowledge; Lead; Location; Maps; Methods; Mus; Mutation; Nucleic Acid Regulatory Sequences; Organoids; Pathogenicity; Patients; Persons; Photoreceptors; Public Health; Regulation; Regulatory Element; Reporter; Research; Resources; Retina; Retinal Diseases; Role; Shapes; Site; Structure; System; Testing; Time; Untranslated RNA; Variant; Vision; Vision Disorders; Visual; Work; base; cell type; disease-causing mutation; gene therapy; genetic disorder diagnosis; genetic variant; genomic locus; human disease; improved; in vivo; insight; novel; promoter; success; targeted treatment; therapy development; transcription factor; transcriptome sequencing

PROJECT SUMMARY/ABSTRACT Cis-regulatory elements (CREs) are critical sites of transcription factor (TF) binding to the genome that orchestrate the expression of genes necessary for normal cellular function. Mutations within CREs can disrupt TF binding and cause inherited human diseases including disorders of vision. The genomic location and function of CREs that are necessary for human vision is largely unknown. This gap in knowledge is a significant obstacle toward understanding the genetic regulation of normal human vision and to identifying disease-causing mutations with CREs. The long-term goal for our research is to understand how genetic variation within CREs shapes the structure and function of the retina and contributes to human vision. The focused objective of this proposal is to determine the mechanisms by which CREs regulate essential gene expression in photoreceptor cells and to determine how genetic mutations within CREs lead to retinal disease. The central hypothesis driving this work is that discrete DNA sequences within CREs are required to regulate essential photoreceptor gene expression and that CRE mutations that disrupt evolutionarily conserved TF binding sites contribute to inherited visual disorders. To test this hypothesis we are pursuing the following specific aims: 1) Determine the activity of human photoreceptor CREs in human retinal organoids using ATAC- Seq, ChIP-Seq and RNA-Seq to compare them to CREs we have previously identified from adult and developing human retinas. This will demonstrate the utility of organoids for studying photoreceptor CREs in their native cellular-genomic context. 2) Test the function of patient-derived variants in human photoreceptor CREs. Using high-throughput AAV-based reporter assays we will determine which CREs sequences are sufficient to drive cell-type-specific expression in the mouse retina and human retinal organoids and determine the consequence of sequence variants on CRE activity. 3) Determine the mechanisms by which multiple CREs regulate the expression of a critical photoreceptor transcription factor, NRL. CRISPR/Cas9-based approaches will target specific CREs at the NRL locus to reveal the contribution of each CRE to the expression of this essential gene and to serve as a case study for the regulation of other essential genes. The contribution of this research will be to elucidate the mechanisms by which CREs regulate genes that are necessary for human photoreceptor function and survival. This work will enable the systematic identification and interpretation of genetic variants within CREs and therefore improve genetic diagnostics for unexplained retinal disease. By opening up the non-coding genome to functional analyses it will be possible for the first time to determine the mechanisms by which individual CREs regulate specific genes that are critical for photoreceptor cell function in a high-throughput and comprehensive manner. This will enable discovery of genetic contributions to human vision and inherited visual diseases that have thus far been inaccessible.

项目概要/摘要 顺式调控元件 (CRE) 是转录因子 (TF) 与基因组结合的关键位点， 协调正常细胞功能所需基因的表达。 CRE 内的突变可能会破坏 TF 结合会导致遗传性人类疾病，包括视力障碍。基因组位置和 人类视觉所必需的 CRE 的功能在很大程度上尚不清楚。这种知识差距是 理解正常人类视觉的基因调控和识别的重大障碍 CRE 的致病突变。我们研究的长期目标是了解遗传如何 CRE 内的变异塑造了视网膜的结构和功能，并有助于人类视觉。这 该提案的重点目标是确定 CRE 调节必需基因的机制 感光细胞中的表达并确定 CRE 内的基因突变如何导致视网膜 疾病。推动这项工作的核心假设是 CRE 中离散的 DNA 序列需要 调节重要的光感受器基因表达以及破坏进化保守性的 CRE 突变 TF 结合位点会导致遗传性视觉障碍。为了检验这个假设，我们正在追求以下内容 具体目标： 1) 使用 ATAC- 确定人类视网膜类器官中人类光感受器 CRE 的活性 Seq、ChIP-Seq 和 RNA-Seq，将它们与我们之前从成人和成人中鉴定出的 CRE 进行比较 人类视网膜正在发育。这将证明类器官在研究光感受器 CRE 方面的实用性 他们的原生细胞基因组背景。 2) 测试人类光感受器中源自患者的变异的功能 商业地产 (CRE)。使用基于 AAV 的高通量报告分析，我们将确定哪些 CRE 序列是 足以驱动小鼠视网膜和人视网膜类器官中的细胞类型特异性表达并确定 序列变异对 CRE 活性的影响。 3) 确定多个 CRE 的机制 调节关键光感受器转录因子 NRL 的表达。基于 CRISPR/Cas9 的方法 将针对 NRL 基因座的特定 CRE，以揭示每个 CRE 对此表达的贡献 必需基因并作为其他必需基因调控的案例研究。本次活动的贡献 研究将阐明 CRE 调节人类必需基因的机制 光感受器功能和存活。这项工作将能够系统地识别和解释 CRE 内的遗传变异，从而改善不明原因视网膜疾病的遗传诊断。经过 开放非编码基因组进行功能分析将有可能第一次确定 单个 CRE 调节对感光细胞功能至关重要的特定基因的机制 高通量、综合性的方式。这将使发现基因对人类的贡献成为可能 视力和遗传性视力疾病是迄今为止无法治愈的。