COMPLEXITY AND ORGANIZATION OF DNA-PROTEIN INTERACTIONS IN THE 5'-REGULATORY REGION OF AN ENDODERM-SPECIFIC MARKER GENE IN THE SEA-URCHIN EMBRYO

This study concerns the organization of sites of specific DNA/protein interaction within the regulatory domain of the Endo16 gene of Strongy locentrotus purpuratus. Earlier work had displayed a complex pattern o f expression of this gene during embryogenesis. Endo16 transcripts are confined to the definitive vegetal plate in blastula stage embryos; a t gastrula stage this gene is expressed throughout the archenteron, bu t later only in the midgut. In this work we exploited the exceptional experimental accessibility of the sea urchin embryo, with respect to b oth functional assays of gene regulatory systems and to characterizati on of transcription factors, in order to approach a complete descripti on of potential Endo16 regulatory interactions. Accurate expression of an Endo16 fusion gene was obtained with a 2200-nucleotide (nt) upstre am fragment of the gene. We present a map locating high specificity ta rget sites for DNA-binding proteins within the 2200-nt Endo16 regulato ry domain, and an assessment of the complexity of the set of putative Endo16 transcription factors that we have been able to recover from 24 -h (blastula stage) nuclear extract. Protein binding sites were initia lly mapped by gel shift reactions carried out on nested sets of end-la beled restriction fragments, and then to finer resolution by oligonucl eotide gel shift competitions. Thirty-eight sites of high specificity DNA-protein interaction were thus identified. Appropriate oligonucleot ides were then used for partial purification of the DNA-binding protei ns by affinity chromatography. DNA-binding proteins specific for each target site were identified by molecular weight, using southwestern bl otting procedures and two-dimensional gel shift separations, and by di rectly renaturing and reacting with oligonucleotide probes specific pr oteins that had been resolved by SDS-PAGE from selected affinity colum n fractions. A complete series of gel shift cross-competitions amongst the target sites was carried out. We conclude that nine different pro tein factors are bound at unique sites within the Endo16 regulatory do main. Multiple target sites for five other proteins account for the re maining binding site locations. The target sites appear to be organize d in a sequence of clusters, focused on the unique factors. The high c omplexity of the Endo16 gene regulatory system may be characteristic f or genes that are spatially regulated in early embryonic development.