Figure 1. Foxn1 binds to β5t-proximal site #13.

(a) Schematic diagram of the locations of 18 sites that contain the Foxn1-binding invariant core ACGC tetranucleotide within the 14-kb region proximal to β5t-encoding gene between two neighbouring genes in the mouse genome. Arrows indicate the orientation of the transcription. (b) Distances of the 18 sites from β5t translation initiation site are listed. The nucleotide sequences of those sites and their mismatches from the Foxn1-binding consensus 11-bp sequence previously reported²¹ are also listed. (c,d) HEK293T cells were transfected with a vector that expressed Foxn1 and a plasmid that contained mouse genomic DNA fragment proximal to β5t-encoding gene, as illustrated schematically (c). Forty-eight hours after the transfection, formaldehyde-fixed cell lysates that contained protein–DNA complexes were immunoprecipitated with either goat anti-Foxn1 antibody (filled bars) or control IgG (open bars) and PCR-amplified for the indicated candidate sites of the Foxn1-binding sequences. Graphs show the frequency of immunoprecipitated DNA in input DNA (mean±s.e.m., n=5), which was sonicated at mild or strong amplitude (d). *P<0.05; NS, not significant; ND, not detectable. (e,f) A plasmid that contained the 3 kb DNA fragment upstream of β5t-encoding gene or its variants mutating at the indicated site was used for immunoprecipitation (e). A control plasmid that contained no genomic DNA fragment was used where indicated (mock). Graphs show the frequency of immunoprecipitated DNA in input DNA (mean±s.e.m., n=3) (f). *P<0.05; NS, not significant; ND, not detectable. All statistical analyses were performed by student's t-test.