PAX6 as a model for synthetic hypervariation studies

Lead Research Organisation: University of Edinburgh
Department Name: MRC Human Genetics Unit

Abstract

The regulation of human gene expression during development is controlled by genetic switch elements called enhancers. Enhancers are often located at huge (100 to 1000kb) genomic distance from their target genes and genes important in development have many tens of such elements controlling gene expression in different cell types, different tissues and at different times. This represents a fundamental "Rule of Life" that we do not understand well at all.

To help establish a platform to systematically address such a profound basic question about how our genome controls development, we will use a systematic synthetic genomic approach. This allows multiple variations of a large portion of the human genome (five hundred thousand base pairs) to be constructed with great precision in yeast cells. Then the variants will be precisely delivered to the vertebrate genome to test whether and how the engineered synthetic variants work during development.

For our study, we have chosen the key gene required for eye development - PAX6.

Technical Summary

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 we do not understand well at all. What happens if we move enhancers around? Why are introns so huge? What happens if we pare regulatory landscapes down to a minimal size? What happens if we jumble the relative position, orientation, order and spacing of enhancer sequences? Can we put them 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 our genome, we will employ a systematic synthetic genomics-based approach that we call "synthetic hypervariation" and that allows such variations to be constructed with great precision in yeast cells. Then the mammalian genome variants will be precisely delivered to the native locus, preserving the genomic context that is likely to be critical for successfully interpreting their functional capability. We have chosen for study one of the most complicated mammalian genes known, namely the key gene required for eye development across multicellular eukaryotes, PAX6. Function in driving correct PAX6 expression in early embryonic development will be tested initially by electroporation/random integration of the engineered YAC in zebrafish embryos. Subsequently, selected constructs will be used to "ectopically" replace the native mouse PAX6 locus in mouse embryonic stem cells. Regulated PAX6 expression from such synthetic loci will be assessed in optic cup organoids and also in mouse development.

Planned Impact

There are several major components to our research that will have broad impacts Our research findings will benefit the developmental genetics community and those studying how gene expression is regulated by enhancers - one of the most important topics in contemporary molecular genetics and development.

By developing a systematic and large-scale synthetic approach, our research will impact the way in which others do and design their experiments in the future. The development of approaches for the precise targeting of very large DNA constructs to specific sites in the vertebrate genome for functional testing will drive transgenic technology in fish and mice and will improve their value as models of human disease - especially disease caused by mutations in enhancers.

Our research will also benefit the general human genetic community by providing a tractable synthetic platform upon which regulatory variants implicated in human disease can be functionally tested. More specifically our research will benefit those working on eye-related genetic disease.

By sharing best practice regarding automation and laboratory information management systems, our research will benefit those working in other genome foundries and the wider synthetic biology community.

Publications

10 25 50
 
Description Synthetic variation at the PAX6 locus 
Organisation NYU Langone Medical Center
Country United States 
Sector Academic/University 
PI Contribution Functional testing of engineered PAX6 loci in vertebrate cell and organism systems
Collaborator Contribution Joint NSF/BBSRC grant. NYU collaborator is providing the expertise in synthetic biology using S.cerevisiae
Impact Too early for outputs
Start Year 2020