Functional characterization of Ets-binding sites in the sea urchin embryo: three base pair conversions redirect expression from mesoderm to ectoderm and endoderm

Abstract

Because of the limited knowledge of target genes for the ets family of transcription factors, it is yet unclear how specificity of biological function among different members is achieved in this class of proteins. In the present study, we compared two Ets-binding sites in two differentially expressed genes of the sea urchin embryo. The first gene examined is the cytoskeletal actin CyIIa, which is transiently expressed in skeletogenic and secondary mesenchyme and in its terminal and permanent phase in the gut. The second one encodes the hatching enzyme gene of Strongylocentrotus purpuratus, and is regulated cell-autonomously and asymmetrically along the maternally determined animal–vegetal axis. The Ets sites within the regulatory regions of these two genes interact and form different binding complexes with proteins present in the nuclei of mesenchyme blastula embryos. We also demonstrated that the DNA binding specificity of the CyIIa Ets-binding site can be converted to the other type of Ets site, as in the hatching enzyme promoter, by changing only three base pairs near the Ets core sequence. Switching of these three base pairs near the central GGA trinucleotide motif characteristic of all Ets-binding targets was also sufficient to redirect expression of a reporter gene construct containing a heterologous basal promoter from mesenchyme to non-mesenchyme cell type in transgenic sea urchin embryos. These observations suggest that binding affinity of ets transcription factors plays an important role in determining cell type-specific gene expression.