Figure 6: RORβ1-responsive enhancer in Ptf1a gene.

(a) Ptf1a gene; black boxes, exons; triangle, promoter; grey bars below gene, enhancer regions. Enhancer (En) fragments tested are shown as black bars with size (kb) below. Reporter constructs contained a 0.6-kb Ptf1a promoter driving luciferase without (Pr) or with enhancer fragments (Pr/En). (b) Foxn4 (F1, F2) and RORβ1 (RORE) response elements in En0.8 fragment. Arrowheads, motifs and orientation; black arrowheads, conserved between mouse and human genomes. Numbers, distances (in bp) from ATG. Vertical arrow (+3038) indicates a distant control mutation. (c) Luciferase assays in 293T cells for reporter responses to RORβ1 and Foxn4 normalized to basal expression of each reporter in absence of added factors; means±s.d. Pr/En0.8 gave >10-fold greater synergistic induction than Pr promoter alone (P<0.001). Fold induction of Pr/En0.8 by both factors together (black columns) was reduced by individual or combined mutagenesis (mut) of RORE, F1 and F2 sites (P<0.001 for each mutant compared with wild-type). Statistical analyses were performed using Student’s t-test. (d) Magnification of lower region of response scale in panel ‘c’. Note: responses to both factors (black columns) are truncated above the dashed line. (e) RORE, F1 and F2 sequences in mouse (Mm) and human (Hs) genomes; asterisks indicate identity; core motifs shaded. Mouse wild-type and mutant (mut) sequences were used as EMSA probes. (f) EMSA of RORβ1 binding to RORE using extracts from 293T cells transfected with RORβ1-expressing (β1) or empty (T) vector; probe alone (−). Competition with wild-type (wt) but not mutant (mut) unlabelled oligonucleotides abolished the shifted band (arrow). Anti-RORβ antibody (+) but not IgG control (c) supershifted the band. (g) EMSA of Foxn4 binding to F1 and F2 using extracts from 293T cells transfected with Foxn4-expressing (F4) or empty (T) vector. Competition with wild-type but not mutant oligonucleotides strongly diminished the shifted band (arrow). (h) Protein interaction between RORβ1 and Foxn4. Left, input protein from 293T cells transfected with empty vector (vec), or vectors expressing histidine-tagged Foxn4 (H.F4), RORβ1 (β1) or both (H.F4+β1) analysed by western blot (RORβ, upper; Foxn4, lower). Right, Nickel-chelate selection for H.F4 protein co-selected a weak RORβ1 band. Protein molecular size marker, kDa.