Characterization of the upstream region that regulates the transcription of the gene for the precursor to EGF-related peptides,exogastrula-inducing peptides,of the sea urchin Anthocidaris crassispina

The EGIP gene for exogastrula-inducing peptides (EGIPs) of the sea urchin Anthocidaris crassispina, which are structurally related to the epidermal growth factor, is activated at the onset of gastrulation in subdomains of the embryonic ectoderm. We showed in our previous study that the spatial and temporal regulation of EGIP is conducted by the upstream region from -372 to +194, and that there is an enhancer element between -372 and -210. In this study, we introduced into sea urchin embryos PCR-amplified DNA containing differently truncated EGIP flanking region that was ligated to the GFP reporter gene, and examined the transient expression of the reporter gene, showing that both the -270/-238 and -249/-210 regions were essential for the enhancer activity. We further showed that there is another activating element between -65 and -21, and that even the region between -65 and +194 is sufficient for ectoderm-specific expression of the EGIP gene. The electrophoretic mobility shift assay showed that the -270/-210 enhancer region and the proximal -61/+30 region include specific binding sites for nuclear proteins of sea urchin embryos. Besides these unique sites, the presence of multiple binding sites for GCF1-like nuclear proteins have been revealed in the upstream DNA.