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.
Date: 2015
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
MeSH Terms:
  • Animals
  • Eye Proteins/genetics
  • Eye Proteins/metabolism
  • Green Fluorescent Proteins/genetics
  • Green Fluorescent Proteins/metabolism
  • Hedgehog Proteins/genetics
  • Hedgehog Proteins/metabolism
  • Homeodomain Proteins/genetics
  • Homeodomain Proteins/metabolism
  • Interferon Regulatory Factors/genetics
  • Interferon Regulatory Factors/metabolism
  • PAX6 Transcription Factor
  • Paired Box Transcription Factors/genetics
  • Paired Box Transcription Factors/metabolism
  • Pierre Robin Syndrome/genetics*
  • Protein Binding
  • Regulatory Elements, Transcriptional*
  • Repressor Proteins/genetics
  • Repressor Proteins/metabolism
  • Transgenes*
  • Zebrafish
  • Zebrafish Proteins/genetics
  • Zebrafish Proteins/metabolism
PubMed: 26030420 Full text @ PLoS Genet.
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.