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

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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.