The genomic landscape of the ED170 insertion. (A) ED170 is an insertion in an exon of atp6v0a1b, 10 kb away from the transcription start site of its associated gene, ptrfb. This insertion is oriented with the GFP enhancer trap upstream of ptrfb. Four candidate enhancer sequences were selected, 1 to 4 (black boxes). (B) Enhancer activity assays show that sequences 1, 2, and 3 are enhancers that drive expression in the notochord, as observed by expression of GFP. (C) When performing a 3C assay comparing the levels of interaction of the enhancer 1 and two control sequences with the promoter of ptrfb, a significant difference is observed; (**) P < 0.01. This same assay was performed using homozygous embryos for ED170, and the difference in the levels of interaction of the enhancer 1 and the two control sequences is not statistically significant (ns, not significant). The primers used for the 3C experiment are represented in A as an orange triangle for the enhancer 1, blue triangles for control sequences, and a magenta triangle for the ptrfb promoter sequence.
