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 i s asymmetrically expressed along the animal-vegetal axis of sea urchin embr yos under the cell-autonomous control of maternal regulatory activities and therefore provides an excellent entry point for understanding the mechanis m that establishes animal-vegetal developmental polarity. To search for tra nscriptional 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 clone s that encode a new member of the Ets family, named SpEts4. This protein co ntains transcriptional activation activity, since expression of reporter ge nes in yeast does not depend on the presence of the yeast GAL4 activation d omain. Sequences in the N-terminal region of SpEts4 mediate the activation activity, as shown by deletion or domain-swapping experiments, The newly id entified DNA binding protein binds with a high degree of specificity to a S pHE promoter Ets element and forms a complex with a mobility identical to t hat obtained with 9-h sea urchin embryo nuclear extracts. SpEts4 positively regulates SpHE transcription, since mutation of the SpEts4 site in SpHE pr omoter transgenes reduces promoter activity in vivo while SpEts4 mRNA coinj ection increases its output. As expected for a positive SpHE transcriptiona l regulator, the timing of SpEts4 gene expression precedes the transient ex pression of SpHE in the very early sea urchin blastula.