Fig 4. Tcf7-binding sites are critical for genes expressed specifically in the posterior vegetal hemisphere domain (PVD).

(A–C) Analysis of a regulatory region of Tbx6.b. (A) Illustrations on the left depict the constructs. The numbers indicate the relative nucleotide positions from the transcription start site of Tbx6.b. Mutated Tcf7-binding sites are indicated by X. Graphs show the percentage of blastomeres expressing the reporter in the anterior vegetal blastomeres, in the posterior vegetal blastomeres, and in the animal blastomeres. Note that not all cells or embryos could express the reporter because of mosaic incorporation of the electroporated plasmid. (B, C) Images showing Gfp expression in embryos electroporated with the fourth and last constructs shown in (A). Scale bar, 100 μm. (D–F) Mapping of the Tcf7 and Zic-r.a ChIP data onto genomic regions consisting of the exons and upstream regions of (D) Tbx6.b, (E) Wnttun5, and (F) Admp. The ChIP-chip data are shown in bars, and the ChIP-seq data are shown as magenta lines. Each graph shows the fold enrichment (y-axis) for the chromosomal regions (x-axis). Green and yellow boxes indicate the regions essential for specific expression, which were revealed by the reporter gene assays shown in (A), and S5 Fig. Regions indicated by green boxes overlap peak regions identified by the peak caller programs for ChIP-seq and ChIP-chip, while the peak caller programs did not identify peaks within regions indicated by yellow boxes. (G) Gel-shift analysis showing that the proximal Tcf7-binding site did not bind GST protein but bound the Tcf7-GST fusion protein. The shifted band disappeared by incubation with a specific competitor, but not a competitor with a mutant Tcf7-binding site.