Identification of a New Sea Urchin Ets Protein, SpEts4, by Yeast One-Hybrid Screening with the Hatching Enzyme Promoter

We report the use of a yeast one-hybrid system to isolate a transcriptional regulator of the sea urchin embryo hatching enzyme gene, SpHE. This gene is asymmetrically expressed along the animal-vegetal axis of sea urchin embryos under the cell-autonomous control of maternal regulatory activities and therefore provides an excellent entry point for understanding the mechanism that establishes animal-vegetal developmental polarity. To search for transcriptional regulators, we used a fragment of the SpHE promoter containing several individual elements instead of the conventional bait that contains a multimerized cis element. This screen yielded a number of positive clones that encode a new member of the Ets family, named SpEts4. This protein contains transcriptional activation activity, since expression of reporter genes in yeast does not depend on the presence of the yeast GAL4 activation domain. Sequences in the N-terminal region of SpEts4 mediate the activation activity, as shown by deletion or domain-swapping experiments. The newly identified DNA binding protein binds with a high degree of specificity to a SpHE promoter Ets element and forms a complex with a mobility identical to that obtained with 9-h sea urchin embryo nuclear extracts. SpEts4 positively regulates SpHE transcription, since mutation of the SpEts4 site in SpHE promoter transgenes reduces promoter activity in vivo while SpEts4 mRNA coinjection increases its output. As expected for a positive SpHE transcriptional regulator, the timing of SpEts4 gene expression precedes the transient expression of SpHE in the very early sea urchin blastula. The Strongylocentrotus purpuratus hatching enzyme gene, SpHE, is transcribed transiently only in nonvegetal blastomeres during the cleavage and very early blastula stages of sea urchin development (20). The activation of SpHE is early and cell autonomous and therefore is very likely to be regulated by localized maternal transcription-regulatory activities partitioned asymmetrically along the animal-vegetal (AV) axis. Consequently, the SpHE gene represents an excellent entry point for determining how the AV axis is established, through the identification of trans-acting factors that regulate its transcription. Previously, we reported that a relatively compact region of the SpHE promoter, consisting of 300 bp upstream of the transcription initiation site, is sufficient to sponsor both highlevel transcriptional activity and correct nonvegetal spatial expression (26, 27). Although this regulatory region is small, nine cis-acting elements have been defined within it, which can form complexes with at least six different proteins, as shown by in vitro DNase I footprinting and electrophoretic mobility shift assays (EMSAs) (26). Most of these cis elements were found to be occupied when the gene is active in vivo but to be unoccupied and in a nucleosome-like configuration when the gene is inactive (28). Extensive mutational dissection of the SpHE regulatory region supports a model in which all of the DNA binding proteins confer positive function in nonvegetal blastomeres (26, 27). This suggests that an important mechanism establishing AV polarity of developmental potential in the early sea urchin embryo is partitioning of positive maternal regulatory activities to nonvegetal blastomeres. To test this model, we have begun to investigate the SpHE transcriptional regulators. Here we report the cloning of an S. purpuratus egg cDNA encoding a member of the Ets family, using a nontraditional yeast one-hybrid genetic screening approach in which a large portion of the SpHE promoter sequence instead of individual multimerized cis elements was used as bait. Using this method, we isolated 13 strong positive clones containing overlapping segments of the same sequence, which encodes a member of the Ets transcription factor family. Because this protein contains a conserved Ets domain that is much more similar to that of Drosophila Ets4 (6) than to any other Ets domain, we have named it SpEts4. We demonstrate that the SpEts4 gene sequence encodes a protein that binds with the same specificity, and forms a complex of similar mobility in EMSA, as does the native protein found in nuclear protein extracts from very early blastulae. Using SpHE promoter transgenes, we show that the site to which SpEts4 binds confers positive regulatory activity and that exogeneously supplied recombinant SpEts4 augments promoter activity. Consistent with a positive function for SpEts4 in regulating SpHE transcription, SpEts4 transcripts accumulate in the egg and early embryo transiently and just prior to the burst of SpHE mRNA expression.