Non-Coding Genetic Vulnerabilities in Human Photoreceptor Function and Disease

项目概要/摘要 顺式调节元件（克雷斯）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必需的基因的表达。克雷斯内的突变可以破坏 TF结合并引起包括视力障碍在内的遗传性人类疾病。基因组位置和 人类视觉所必需的克雷斯的功能在很大程度上是未知的。这种知识差距是一个 这是理解正常人类视觉的遗传调节和识别 克雷斯的致病突变。我们研究的长期目标是了解基因是如何 克雷斯内的变异形成视网膜的结构和功能，并有助于人类视觉。的 该建议的重点目标是确定克雷斯调节必需基因的机制， 在感光细胞中的表达，并确定克雷斯内的基因突变如何导致视网膜病变。 疾病推动这项工作的中心假设是，克雷斯内的离散DNA序列需要 调节重要的光感受器基因表达，破坏进化上保守的CRE突变， TF结合位点有助于遗传性视觉障碍。为了验证这一假设，我们正在进行以下研究： 具体目的：1）使用ATAC-PCR测定人视网膜类器官中人光感受器克雷斯的活性。 Seq、ChIP-Seq和RNA-Seq，以将它们与我们先前从成人和哺乳动物中鉴定的克雷斯进行比较。 发育人类视网膜。这将证明类器官用于研究光感受器克雷斯的效用， 他们的细胞基因组背景2)检测患者源性变体在人类感光细胞中的功能 克雷斯。使用高通量的基于AAV的报告基因测定，我们将确定哪些克雷斯序列是 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)确定多个克雷斯 调节一个关键的感光细胞转录因子NRL的表达。基于CRISPR/Cas9的方法 将靶向NRL基因座上的特定克雷斯，以揭示每个CRE对这种表达的贡献。 并为其他必需基因的调控提供案例研究。这一贡献 研究将阐明克雷斯调节人类必需基因的机制， 光感受器功能和存活。这项工作将有助于系统地识别和解释 克雷斯内的遗传变异，因此改善了不明原因视网膜疾病的遗传诊断。通过 开放非编码基因组进行功能分析，将有可能首次确定 个体克雷斯调节特定基因的机制，这些基因对光感受器细胞功能至关重要， 高通量和全面的方式。这将有助于发现基因对人类的贡献。 视力和遗传性视力疾病，迄今为止还没有接触过。

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.