BBSRC-NSF/BIO: PAX6 as a model for synthetic hypervariation studies

BBSRC-NSF/BIO：PAX6 作为合成超变异研究的模型

• 批准号: 1917277
• 负责人: Jef Boeke
• 金额: $ 120万
• 依托单位: New York University Medical Center
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-08-01 至 2024-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1917277&HistoricalAwards=false
• 关键词: BBSRC; NSF; BIO; PAX6; model

The human genome is made of DNA, a language of four letters A, G, C, and T, the interpretation of which is known. This project entails "writing" DNA in ways that change biological function. If the workings of genomes are known and understood, then they can be "written" from scratch. The development of the eye is controlled by special regions of DNA called enhancers, which turn on gene activity at the right time and place in the developing embryo. The project involves writing these enhancers in new ways, to test basic knowledge of how expression of the genes that control eye development are wired. As a test of eye development, the newly written DNA is transferred to the embryos of fish or mice to observe the effects on eye development. This project is a collaborative effort between a US laboratory with expertise in writing DNA and a British laboratory with expertise in analyzing eye function. The project has broader impacts through an exchange program of scientists who are learning more about DNA writing, a scientific meeting on DNA writing, a workshop on manipulating the genome of fish, and a tailored program to teach the genomics of eye disease specifically to the visually impaired. The developmental and physiological regulation of human gene expression is controlled by enhancers often located at huge (100 to 1000kb) genomic distance from their target genes. This represents a fundamental "Rule of Life" that is not very well understood. What happens if enhancers are moved around? Why are introns so huge? What happens if regulatory landscapes are pared down to a minimal size? What happens if the relative position, orientation, order, and spacing of enhancer sequences are jumbled? Can they be put altogether into one big mega-enhancer? These are all questions that currently do not have answers. To help establish a platform for systematically answering such profound basic questions about how developmental systems are "wired" in the genome, a systematic synthetic genomics-based approach called "synthetic hypervariation" is employed. This approach allows variations to be constructed with high precision in yeast cells. The variants are precisely delivered to the native locus, preserving the genomic context that is likely critical for successfully interpreting the results. As a powerful model for studying the function of non-coding regulation and impact of its perturbation, these methods are applied to one of the most complicated mammalian genes known, namely the key gene required for eye development across multicellular eukaryotes, PAX6. The PAX6 gene is a prime example of complex, long-range regulation in the mammalian genome. The PAX6 regulatory domain contains 31 known enhancers which orchestrate complex spatial and temporal PAX6 expression in the eye. Many of the enhancers were identified through mutations leading to the "aniridia" phenotype. This project aims to obtain a more comprehensive and systems-level picture of how 31 (or perhaps more to be discovered) enhancers control this single key developmental regulator. The impact of the genomic changes are assayed using three distinct readouts: 1) zebrafish embryo PAX6-GFP expression; 2) mouse ESCs differentiated into optic cup; 3) Complementation of the mouse Sey (Pax6+/?) phenotype in vivo.This collaborative US/UK project is supported by the US National Science Foundation and the UK Biotechnology and Biological Sciences Research Council.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

人类基因组是由DNA制成的，DNA是四个字母A，G，C和T的语言，其解释是已知的。该项目需要以改变生物学功能的方式“写” DNA。如果已知和理解基因组的工作，则可以从头开始“写”。眼睛的发育受到DNA的特殊区域的控制，称为增强剂，该区域在发育中的胚胎中在正确的时间和位置打开基因活性。该项目涉及以新的方式编写这些增强器，以测试如何连接控制眼睛发育的基因表达的基础知识。作为对眼睛发育的测试，新书写的DNA被转移到鱼或小鼠的胚胎中，以观察对眼睛发育的影响。该项目是在美国实验室撰写DNA的专业知识与具有分析眼功能专业知识的英国实验室方面的专业知识之间的合作努力。该项目通过科学家的交流计划产生了更大的影响，他们正在了解更多有关DNA写作的知识，关于DNA写作的科学会议，关于操纵鱼类基因组的研讨会以及针对视觉障碍的眼睛疾病基因组学的量身定制计划。人类基因表达的发育和生理调节由通常位于距其靶基因的巨大（100至1000kb）基因组距离处的增强子控制。 这代表了一个基本的“生命规则”，这不是很好的理解。 如果增强器四处移动会发生什么？ 为什么内含子这么大？ 如果将监管景观削减到最小的规模，会发生什么？ 如果增强子序列的相对位置，方向，顺序和间距发生混乱，会发生什么？ 他们可以将他们完全放入一个大型富员工中吗？ 这些都是当前没有答案的问题。 为了帮助建立一个系统地回答有关基因组中发育系统如何“有线”的深刻基本问题的平台，采用了一种系统的基于合成基因组学的方法，称为“合成过度变化”。这种方法允许在酵母细胞中以高精度构建变化。这些变体精确地传递到本地基因座，保留了可能对成功解释结果至关重要的基因组环境。作为研究非编码调节功能及其扰动影响功能的强大模型，这些方法应用于已知的最复杂的哺乳动物基因之一，即跨多细胞真核生物PAX6所需的关键基因。 PAX6基因是哺乳动物基因组中复杂的，远程调节的主要例子。 PAX6调节结构域包含31个已知增强子，这些增强子在眼中编排复杂的空间和时间PAX6表达。许多增强子是通过突变导致“ Aniridia”表型鉴定的。 该项目旨在获得更全面的系统级别的图片，即如何控制31个（或可能被发现）的增强剂控制这个单一的关键开发调节器。基因组变化的影响使用三个不同的读数进行测定：1）斑马鱼胚胎PAX6-GFP表达； 2）鼠标逃脱为视杯； 3) Complementation of the mouse Sey (Pax6+/?) phenotype in vivo.This collaborative US/UK project is supported by the US National Science Foundation and the UK Biotechnology and Biological Sciences Research Council.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.