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.
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Department of Biology, University of Rochester, Rochester, New York 14627, USA.
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D005260:Female; D018507:Gene Expression Regulation, Developmental; D008666:Metalloendopeptidases / Q000235:genetics*; D008969:Molecular Sequence Data; D009865:Oocytes / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011518:Proto-Oncogene Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D050783:Proto-Oncogene Proteins c-ets; D012441:Saccharomyces cerevisiae / Q000235:genetics; D012617:Sea Urchins / Q000196:embryology / Q000235:genetics* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D015533:Transcriptional Activation
D018076:DNA, Complementary; D011518:Proto-Oncogene Proteins; D050783:Proto-Oncogene Proteins c-ets; D014157:Transcription Factors; D008666:Metalloendopeptidases; C024344:envelysin