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

人类基因组是由DNA构成的，DNA是一种由a、G、C和T四个字母组成的语言，其解释是已知的。这个项目需要以改变生物功能的方式“书写”DNA。如果基因组的工作原理是已知和理解的，那么它们就可以从零开始“编写”。眼睛的发育是由DNA中被称为增强子的特殊区域控制的，它在发育中的胚胎中正确的时间和地点开启基因活性。该项目涉及以新的方式编写这些增强子，以测试控制眼睛发育的基因表达是如何连接的基本知识。作为眼睛发育的测试，新编写的DNA被转移到鱼或老鼠的胚胎中，观察对眼睛发育的影响。这个项目是美国一个专门研究DNA的实验室和英国一个专门研究分析眼睛功能的实验室的合作成果。该项目通过以下方式产生了更广泛的影响：更多了解DNA书写的科学家之间的交流计划、DNA书写的科学会议、操纵鱼类基因组的研讨会，以及专门向视障人士教授眼病基因组学的定制计划。人类基因表达的发育和生理调控是由增强子控制的，这些增强子通常位于与其靶基因的巨大（100至1000kb）基因组距离上。这代表了一种基本的“生命法则”，但人们对它的理解还不是很好。如果增强子被移动会发生什么？为什么内含子这么大？如果监管格局被缩减到最小规模，会发生什么？如果增强子序列的相对位置、方向、顺序和间距混乱会发生什么？它们能被放在一个巨大的增强器里吗？这些都是目前还没有答案的问题。为了帮助建立一个平台，系统地回答关于发育系统是如何在基因组中“连接”的这些深刻的基本问题，采用了一种基于系统合成基因组学的方法，称为“合成超变异”。这种方法允许在酵母细胞中以高精度构建变异。这些变异被精确地传递到原生位点，保留了可能对成功解释结果至关重要的基因组背景。作为研究非编码调控功能及其扰动影响的强大模型，这些方法被应用于已知最复杂的哺乳动物基因之一，即多细胞真核生物眼睛发育所需的关键基因PAX6。PAX6基因是哺乳动物基因组中复杂、远程调控的一个主要例子。PAX6调节结构域包含31个已知的增强子，它们在眼睛中协调复杂的空间和时间PAX6表达。许多增强子是通过导致“无虹膜”表型的突变确定的。该项目旨在获得更全面和系统级的图像，以了解31个（或可能更多的被发现）增强子如何控制这个单一的关键发育调节因子。使用三种不同的读数来分析基因组变化的影响：1)斑马鱼胚胎PAX6-GFP表达；2)小鼠ESCs分化成视杯；3)小鼠体内赛（Pax6+/?）表型的互补。这个英美合作项目由美国国家科学基金会和英国生物技术和生物科学研究委员会支持。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

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.