title: Functional assessment of disease-associated regulatory variants in vivo using a versatile dual colour transgenesis strategy in zebrafish.
abstract: 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.
authors.affiliation: MRC Human Genetics Unit, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, United Kingdom.
authors.forename: Shipra
authors.initials: S
authors.lastname: Bhatia
authors.affiliation: INSERM U781, Hôpital Necker-Enfants Malades and Université Paris Descartes-Sorbonne Paris Cité, Institute Imagine, Paris, France.
authors.forename: Christopher T
authors.initials: CT
authors.lastname: Gordon
authors.affiliation: MRC Human Genetics Unit, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, United Kingdom.
authors.forename: Robert G
authors.initials: RG
authors.lastname: Foster
authors.affiliation: INSERM U781, Hôpital Necker-Enfants Malades and Université Paris Descartes-Sorbonne Paris Cité, Institute Imagine, Paris, France.
authors.forename: Lucie
authors.initials: L
authors.lastname: Melin
authors.affiliation: Service de Pédiatrie Générale, Université Paris Descartes, Hôpital Necker-Enfants Malades, Paris, France.
authors.forename: Véronique
authors.initials: V
authors.lastname: Abadie
authors.affiliation: Departement de Génétique, Hôpital Necker-Enfants Malades, AP-HP, Paris France.
authors.forename: Geneviève
authors.initials: G
authors.lastname: Baujat
authors.affiliation: Service de Chirurgie Maxillo-Faciale et Plastique, CRMR des Malformations de la Face et de la Cavité Buccale, Hôpital Necker-Enfants Malades, Paris, France.
authors.forename: Marie-Paule
authors.initials: MP
authors.lastname: Vazquez
authors.affiliation: INSERM U781, Hôpital Necker-Enfants Malades and Université Paris Descartes-Sorbonne Paris Cité, Institute Imagine, Paris, France; Departement de Génétique, Hôpital Necker-Enfants Malades, AP-HP, Paris France.
authors.forename: Jeanne
authors.initials: J
authors.lastname: Amiel
authors.affiliation: INSERM U781, Hôpital Necker-Enfants Malades and Université Paris Descartes-Sorbonne Paris Cité, Institute Imagine, Paris, France; Departement de Génétique, Hôpital Necker-Enfants Malades, AP-HP, Paris France.
authors.forename: Stanislas
authors.initials: S
authors.lastname: Lyonnet
authors.affiliation: MRC Human Genetics Unit, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, United Kingdom.
authors.forename: Veronica
authors.initials: V
authors.lastname: van Heyningen
authors.affiliation: MRC Human Genetics Unit, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, United Kingdom.
authors.forename: Dirk A
authors.initials: DA
authors.lastname: Kleinjan
mesh_terms: D000818:Animals; D005136:Eye Proteins / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D053823:Hedgehog Proteins / Q000235:genetics / Q000378:metabolism; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism; D050835:Interferon Regulatory Factors / Q000235:genetics / Q000378:metabolism; D000071841:PAX6 Transcription Factor; D051761:Paired Box Transcription Factors / Q000235:genetics / Q000378:metabolism; D010855:Pierre Robin Syndrome / Q000235:genetics*; D011485:Protein Binding; D050436:Regulatory Elements, Transcriptional; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism; D019076:Transgenes; D015027:Zebrafish; D029961:Zebrafish Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D005136:Eye Proteins; D053823:Hedgehog Proteins; D018398:Homeodomain Proteins; C504917:IRF6 protein, zebrafish; D050835:Interferon Regulatory Factors; D000071841:PAX6 Transcription Factor; C000605730:PAX6 protein, human; D051761:Paired Box Transcription Factors; D012097:Repressor Proteins; C524053:Shha protein, zebrafish; D029961:Zebrafish Proteins; D049452:Green Fluorescent Proteins