Functional assessment of disease-associated regulatory variants in vivo using a versatile dual colour transgenesis strategy in zebrafish.

• Main Authors: Shipra Bhatia, Christopher T Gordon, Robert G Foster, Lucie Melin, Véronique Abadie, Geneviève Baujat, Marie-Paule Vazquez, Jeanne Amiel, Stanislas Lyonnet, Veronica van Heyningen, Dirk A Kleinjan
• Format: Article
• Language: English
• Published: Public Library of Science (PLoS) 2015-06-01
• Series: PLoS Genetics
• Online Access: http://europepmc.org/articles/PMC4452300?pdf=render
• ISSN: 1553-7390; 1553-7404

Disruption of gene regulation by sequence variation in non-coding regions of the genome is now recognised as a significant cause of human disease and disease susceptibility. Sequence variants in cis-regulatory elements (CREs), the primary determinants of spatio-temporal gene regulation, can alter transcription factor binding sites. While technological advances have led to easy identification of disease-associated CRE variants, robust methods for discerning functional CRE variants from background variation are lacking. Here we describe an efficient dual-colour reporter transgenesis approach in zebrafish, simultaneously allowing detailed in vivo comparison of spatio-temporal differences in regulatory activity between putative CRE variants and assessment of altered transcription factor binding potential of the variant. We validate the method on known disease-associated elements regulating SHH, PAX6 and IRF6 and subsequently characterise novel, ultra-long-range SOX9 enhancers implicated in the craniofacial abnormality Pierre Robin Sequence. The method provides a highly cost-effective, fast and robust approach for simultaneously unravelling in a single assay whether, where and when in embryonic development a disease-associated CRE-variant is affecting its regulatory function.