QUANTITATIVE FUNCTIONAL INTERRELATIONS WITHIN THE CIS-REGULATORY SYSTEM STRONGYLOCENTROTUS-PURPURATUS ENDO16 GENE

Embryonic expression of the Endo16 gene of Strongylocentrotus purpurat us is controlled by interactions with at least 13 different DNA-bindin g factors. These interactions occur within a cis-regulatory domain tha t extends about 2300 bp upstream from the transcription start site. A recent functional characterization of this domain reveals six differen t subregions, or cis-regulatory modules, each of which displays a spec ific regulatory subfunction when linked with the basal promoter and in some cases various other modules (C.-H. Yuh and E. Davidson (1996) De velopment 122, 1069-1082). In the present work, we analyzed quantitati ve time-course measurements of the CAT enzyme output of embryos bearin g expression constructs controlled by various Endo16 regulatory module s, either singly or in combination. Three of these modules function po sitively in that, in isolation, each is capable of promoting expressio n in vegetal plate and adjacent cell lineages, though with different t emporal profiles of activity. Models for the mode of interaction of th e three positive modules with one another were tested by assuming math ematical relations that would generate, from the measured single modul e time courses, the experimentally observed profiles of activity obtai ned when the relevant modules are physically linked in the same constr uct. The generated and observed time functions were compared, and the differences were minimized by least squares adjustment of a scale para meter. When the modules were tested in context of the endogenous promo ter region, one of the positive modules (A) was found to increase the output of the others (B and G), by a constant factor. In contrast, a s olution in which the time-course data of modules A and B are multiplie d by one another was required for the interrelations of the positive m odules when a minimal SV40 promoter was used. One interpretation is th at, in this construct, each module independently stimulates the basal transcription complex. We used a similar approach to analyze the repre ssive activity of the three Erzdo16 cis-regulatory modules that act ne gatively in controlling spatial expression. The evidence obtained conf irms that the repressive modules act only by affecting the output of m odule A (C.-H. Yuh and E. Davidson (1996) Development 122, 1069-1082). A new hierarchical model of the cisregulatory system was formulated i n which module A plays a central integrating role, and which also impl ies specific functions for certain DNA-binding sites within the basal promoter fragment of the gene. Additional kinetic experiments were the n carried out, and key aspects of the model were confirmed.