Non-Coding Genetic Vulnerabilities in Human Photoreceptor Function and Disease

项目摘要/摘要 顺式调节元件(Cres)是转录因子(Tf)与基因组结合的关键部位 协调正常细胞功能所必需的基因的表达。CRE内的突变可能会扰乱 Tf结合并导致遗传性人类疾病，包括视力障碍。基因组定位和 人类视觉所必需的CRES的功能在很大程度上是未知的。这种知识上的差距是一个 理解正常人类视觉的遗传调节和识别的重大障碍 Cres基因的致病突变。我们研究的长期目标是了解基因是如何 CRES内的变化塑造了视网膜的结构和功能，并对人类的视觉做出了贡献。这个 这项建议的重点目标是确定Cres调节必需基因的机制 在光感受器细胞中的表达，并确定CRE内的基因突变如何导致视网膜 疾病。推动这项工作的中心假设是，Cre内的离散DNA序列被要求 调节必要光感受器基因表达和破坏进化保守的Cre突变 转铁蛋白结合位点与遗传性视觉障碍有关。为了验证这一假设，我们追求以下几点 具体目的：1)用ATAC-1测定人视网膜有机体中人光感受器Cres的活性 SEQ、CHIP-SEQ和RNA-SEQ将它们与我们之前从成人和 发育中的人类视网膜。这将证明有机化合物在研究光感受器Cres方面的实用性。 它们天然的细胞基因组背景。2)测试患者衍生的人类光感受器的功能 克雷斯。使用基于AAV的高通量报告程序分析，我们将确定哪些CRES序列 足以在小鼠视网膜和人视网膜器官中驱动细胞类型特异性表达，并确定 序列变异对Cre活性的影响。3)确定多个核心通过哪些机制 调节关键的光感受器转录因子NRL的表达。基于CRISPR/CAS9的方法 将针对NRL基因座的特定CRE来揭示每个CRE对这一表达的贡献 并为其他必需基因的调控提供了案例研究。这一点的贡献 研究将阐明Cre调节人类必需基因的机制 光感受器的功能和存活率。这项工作将使系统地识别和解释 Cres内的基因变异，因此改善了对不明原因视网膜疾病的基因诊断。通过 开放非编码基因组进行功能分析将第一次有可能确定 单个CRE调控对光感受器细胞功能至关重要的特定基因的机制 高通量和全面性。这将使发现对人类的遗传贡献成为可能 视力和遗传性视力疾病，到目前为止还无法接触到。

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.