|ZFIN ID: ZDB-PUB-150602-2|
Functional Assessment of Disease-Associated Regulatory Variants In Vivo Using a Versatile Dual Colour Transgenesis Strategy in Zebrafish
Bhatia, S., Gordon, C.T., Foster, R.G., Melin, L., Abadie, V., Baujat, G., Vazquez, M.P., Amiel, J., Lyonnet, S., Heyningen, V.V., Kleinjan, D.A.
|Source:||PLoS Genetics 11: e1005193 (Journal)|
|Registered Authors:||Bhatia, Shipra, Gordon, Chris, Lyonnet, Stanislas|
|Keywords:||Zebrafish, Embryos, Hypothalamus, Morpholino, Gene expression, Transcription factors, Enhancer elements, Point mutation|
|PubMed:||26030420 Full text @ PLoS Genet.|
Bhatia, S., Gordon, C.T., Foster, R.G., Melin, L., Abadie, V., Baujat, G., Vazquez, M.P., Amiel, J., Lyonnet, S., Heyningen, V.V., Kleinjan, D.A. (2015) Functional Assessment of Disease-Associated Regulatory Variants In Vivo Using a Versatile Dual Colour Transgenesis Strategy in Zebrafish. PLoS Genetics. 11:e1005193.
ABSTRACTDisruption 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.