Figure 5.

ZIC2 binds to the Nodal locus and promotes transcriptional activation. (a) ChIP-seq data to show ZIC2 binding at the Nodal locus in embryonic stem cells (ESC)²⁷ and in epiblast stem cells (EpiSC)²⁶. The five previously-characterised enhancer elements are indicated by yellow boxes in the cartoon above, while exons are indicated by blue boxes. Coloured dots in the cartoon indicate putative ZIC2 binding sites. In ESCs (upper trace), two high-affinity binding sites are seen, located within the PEE and HBE enhancers (green dots). In EpiSCs (lower trace), four high-affinity sites are observed which includes the two bound in ESCs (green dots) as well as two additional sites located within the NDE and HBE enhancers (red dots). Several lower-affinity binding sites map to ASE, HBE and exon 1, the site located within HBE is indicated by a blue dot. (b) Luciferase assays performed using reporter constructs consisting of each of the known Nodal enhancers linked to a viral E1b promoter and luciferase, as shown in the cartoon. Blue circles indicate control transfections, orange triangles ZIC2 transfections. Luciferase activity is plotted relative to control. The data show that the HBE reporter is activated by ZIC2 above background. (c) Luciferase assays using the native HBE reporter, performed as in b. Cells were transfected with the reporter and an expression plasmid for ZIC3, ZIC2 or ZIC2-ISO, or a control empty vector (pcDNA). (d) Luciferase assays using modified HBE reporters, performed as in b. HBE contains 3 putative ZIC2 binding sites, indicated by the coloured dots in the cartoon, deleted binding sites are indicated by an “X”. Deletion of sites ZBS1 and ZBS2 (red and green dots in the cartoon) has no effect on the ability of ZIC2 to activate the reporter, while deletion of site ZBS3 (blue dot) eliminates this activity. (e) Gel shift (EMSA) assays to study the binding of ZIC2 to HBE. P = probe only, Z = probe + HA-ZIC2, ZC = probe + HA-ZIC2 + unlabelled competitor, ZA = probe + HA-ZIC2 + αHA antibody. Red arrow = gel shift, blue arrow = supershift. Images show cropped gel pictures, uncropped images are shown in the supplemental data.