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 Strongylocentrotus purpuratus. Earlier work had displayed a complex pattern of expression of this gene during embryogenesis. Endo16 transcripts are confined to the definitive vegetal plate in blastula stage embryos; at gastrula stage this gene is expressed throughout the archenteron, but later only in the midgut. In this work we exploited the exceptional experimental accessibility of the sea urchin embryo, with respect to both functional assays of gene regulatory systems and to characterization of transcription factors, in order to approach a complete description of potential Endo16 regulatory interactions. Accurate expression of an Endo16 fusion gene was obtained with a 2200-nucleotide (nt) upstream fragment of the gene. We present a map locating high specificity target sites for DNA-binding proteins within the 2200-nt Endo16 regulatory 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 initially mapped by gel shift reactions carried out on nested sets of end-labeled restriction fragments, and then to finer resolution by oligonucleotide gel shift competitions. Thirty-eight sites of high specificity DNA-protein interaction were thus identified. Appropriate oligonucleotides were then used for partial purification of the DNA-binding proteins by affinity chromatography. DNA-binding proteins specific for each target site were identified by molecular weight, using southwestern blotting procedures and two-dimensional gel shift separations, and by directly renaturing and reacting with oligonucleotide probes specific proteins that had been resolved by SDS-PAGE from selected affinity column fractions. A complete series of gel shift cross-competitions amongst the target sites was carried out. We conclude that nine different protein factors are bound at unique sites within the Endo16 regulatory domain. Multiple target sites for five other proteins account for the remaining binding site locations. The target sites appear to be organized in a sequence of clusters, focused on the unique factors. The high complexity of the Endo16 gene regulatory system may be characteristic for genes that are spatially regulated in early embryonic development.
© 1994 Elsevier Science Ireland Ltd. Received 18 April 1994; accepted 22 April 1994. Many people contributed importantly to this work. We owe the success of the essential robotic affinity chromatography procedures to the experienced advice provided by Dr. James Coffman, who also provided much additional assistance in respect to various procedures we relied heavily upon and which he has been instrumental in developing to their present form (as described by Coffman et al., 1992). The affinity column runs were carried out expertly by Nicolette O'Halloran, whose contributions to this study are a pleasure to acknowledge. The nuclear extracts used for this study were prepared earlier by Dr. Frank Calzone (then of UC Irvine; present address, Amgen, Inc.), James Coffman and others. Extremely useful reviews of drafts of this manuscript were provided by Professors Michael Levine of UC San Diego and Ellen Rothenberg of Caltech. This work was supported by NICHHD (Grant no. 05753), by a grant from the Ralph M. Parsons Foundation, and by the Caltech Beckman Institute. CHY was supported by a Gordon Ross fellowship, AR by an NIH fellowship (HD-07257) and PM by a fellowship from the Ministerio de Education y Ciencia, Spain.