Journal
PLOS GENETICS
Volume 11, Issue 6, Pages -Publisher
PUBLIC LIBRARY SCIENCE
DOI: 10.1371/journal.pgen.1005193
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Funding
- Medical Research Council
- National Institutes of Health [EY018000]
- Royal Society of Edinburgh and Caledonian Research Fund [R43399]
- Leverhulme [EM-2013-062]
- ANR (EvoDevoMut)
- CRANIRARE
- MRC [MC_U127527199, MC_PC_U127527199] Funding Source: UKRI
- Medical Research Council [MC_PC_U127527199, MC_U127527199] Funding Source: researchfish
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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.
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