MULTIPLE OTX BINDING-SITES REQUIRED FOR EXPRESSION OF THE STRONGYLOCENTROTUS-PURPURATUS SPEC2A GENE

The control region of the aboral ectoderm-specific Spec2a gene of Stro ngylocentrotus purpuratus contains a 188-bp enhancer element, the RSR enhancer, required for temporal activation and aboral ectoderm/mesench yme cell expression. Within the enhancer is a positive cis-regulatory element with the core consensus sequence TAATCC, which is capable of b inding the sea urchin orthodenticle-related homeobox protein SpOtx. In this report, we extend our analysis of the RSR enhancer by dissecting it into smaller pieces and testing these pieces in an enhancer activa tion assay. The 188-bp enhancer region could not be divided without pa rtial loss of activity, and two of the three pieces tested exhibited s ome activity. Using site-directed mutagenesis, we showed that three Ot x consensus binding sites were responsible for the activity of the enh ancer, acting in a noncooperative manner to yield full activity. Mutag enizing the three Otx sites and a fourth one just upstream abolished a ll activity in the context of the complete Spec2a control region. Band shift analysis revealed that the Otx sites were able to bind SpOtx, su ggesting that this transcription factor mediates positive control at t hese sites. Non-SpOtx binding sites overlapping two of the Otx sites m ay also play a role in Spec2a expression. Using a lacZ reporter gene, we showed that a 76-bp DNA fragment containing two of the Otx sites wa s sufficient for aboral ectoderm/mesenchyme cell expression. These res ults suggest that the RSR enhancer plus an upstream DNA element requir ed for mesenchyme cell repression are necessary and sufficient for the proper temporal activation and aboral ectoderm expression of the Spec 2a gene and that the Otx elements play a positive role in this process . (C) 1994 Academic Press, Inc.