Organization of the proximal promoter of the hatching-enzyme gene, the earliest zygotic gene expressed in the sea urchin embryo

The hatching enzyme (HE) gene is the earliest zygotic gene expressed in the sea urchin embryo. To investigate the regulation of the HE gene activity, 5' flanking DNA and the 5' untranslated leader were inserted upstream of reporter genes whose expression was monitored in vivo during development after transfer into eggs. By deletion analysis we showed that no more than 3 kb of flanking sequence are required for correct expression of transgenes. The proximal region of 0.5 kb does not precisely control spatial restriction but drives expression at a nearly maximal level. The proximal promoter was searched extensively for sites of protein-DNA interactions by DNAse protection and gel-shift methods. The 12 sites identified form 3 groups: core promoter; central region; and distal region. The central region bears three sites that contain a direct or inverted CCAAT box. Mutation and deletion analysis showed that, in addition to the core-promoter elements, the two most-distal CCAAT-containing sites are indispensable for promoter activity. These sites bind the same set of proteins, which are abundant in the nuclei of cleavage embryos.