Identification of Three Required Positive Cis-Regulated Inputs of the Sea Urchin Pigment Cell Gene Polyketide Synthase 1

Sea urchin pigment cells are single cells of mesodermal origin embedded in the aboral ectoderm. Strongylocentrotus purpuratus polyketide synthase 1 (Sp-PKS1) is required for the biosynthesis of the echinochrome pigment. Evidence suggests that pigment cells are immune cells. In order to reconstruct the gene regulatory network of pigment cells a bottom-up approach combined with comparative genomics has been used in this study. We compared the cis-regulatory regions of five pigment cell genes, Sp-Pks1, flavin monooxygenase 1, 2, and 3 (Sp-Fmo) and sulfotransferase (Sp-Sult), across three different species, Strongylocentrotus purpuratus, Mesocentrotus franciscanus, and Strongylocentrotus fragilis. The computational tool used was multiple expectation maximization motif elicitation analysis. Thirty cis-regulatory motif candidates were identified, three of which were considered for further analysis. The functionality of these motifs was tested by injecting embryos with a -2KbPks-Gfp DNA construct having one of the three motifs mutagenized. All three motifs resulted to be functional cis-regulatory sequences. Specifically, they contained DNA-binding sites for transcriptional activators of Sp-Pks1