Figure 2. MED-1 Binding Sites and Expression of Putative Target Genes(A) DNase I footprint analysis of
end-1 and
end-3 promoter fragments. The fragments used contained either 236 bp upstream of the
end-1 start codon or 284 bp upstream of the
end-3 start codon; in both cases, these fragments are sufficient to drive faithful E-specific expression of a reporter construct. Two sites (i-ii) are protected in
end-1, while four sites (iii-vi) are protected in
end-3.(B) Location of MED-1 sites in
end-1 and
end-3, shown as distance upstream of the start codon, and alignment of DNase I-protected sites to form a consensus with an invariant core sequence (gray shading). The MED-1 binding site does not conform to the canonical GATA binding site.(C) Gel shift with wild-type and point-mutated MED sites. Substitution of each MED-1 site in the
end-1 promoter (shown as 'X' in the diagrams) permits MED-1 binding at the other site, but mutation of both sites abolishes the MED-1 shift. Site (i) was mutated from 5'-aagtatacc-3' to 5'-aactagtcc-3' and site (ii) was mutated from 5'-aagtatacc-3' to 5'-aagtctaga-3'.(D) Fluorescence micrographs of 100-cell embryos showing expression of minimal
end-1 promoter::GFP fusions. Mutation of either MED-1 site abolishes expression.(E) Reporter expression of putative MED-1 target genes falls into three classes. The Class A genes
sox-1 and
hlh-28 are expressed in both the MS and E lineages, the genes
hlh-25 and
tbx-35 are expressed in the MS lineage (Class B), and
wee-1.1 and T07D1.2 are expressed in the early E lineage (Class C). In embryo images, anterior is to the left, and dorsal is up; the eggshell is indicated with a dotted line.