Characterization of a SpAN promoter sufficient to mediate correct spatial regulation along the animal-vegetal axis of the sea urchin embryo.

摘要

Members of the Very Early Blastula (VEB) gene set represent the earliest zygotically expressed and spatially restricted mRNAs in the sea urchin embryo. VEB message accumulation is transient during early embryogenesis and is spatially restricted such that mRNAs are undetectable at the vegetal pole, a pattern that reflects the maternally derived animal-vegetal axis. Several lines of evidence suggest that these genes are regulated by maternal activities that are differentially arrayed along the animal-vegetal axis of the egg. This work characterizes the regulatory apparatus of the VEB gene SpAN and identifies several proteins that may participate in the developmental regulation of SpAN gene expression.; A short region (375 base pairs) of the SpAN promoter is sufficient to confer correct temporal activation and spatial expression to a reporter gene in vivo. This 'minimal' SpAN promoter contains at least seven regions of protein-DNA interaction that contribute positively, to varying degrees, towards promoter activity in vivo. These cis elements are serviced by at least five different protein complexes, two members of which have been identified as a CCAAT binding protein and the Sp1-like factor, GCF1.; The lack of detectable negative spatial regulatory elements suggests that spatial regulation of SpAN expression may be mediated by the localization of positive factor(s) or activities that are excluded from the vegetal pole. SpAN Site V is the major positive regulatory element of the promoter and it is required for detectable levels of transcriptional activity. Vegetal exclusion of the factor(s) that bind Site V could be sufficient to generate the VEB pattern of message accumulation. A putative recognition sequence for the Sox family of transcription factors is located in, and can compete for, complexes at Site V, suggesting that related factors may be involved in SpAN regulation. Several Sox cDNAs were cloned and their expression was found to be also regulated along the A-V axis of the embryo. At least one of the encoded protein products was shown to bind specifically to Site V of the SpAN promoter in vitro.