In vivo competition identifies positive cis-regulatory elements required for lineage-specific gene expression in the sea urchin embryo

Abstract

Several cis-regulatory elements within the 5' regulatory region of the lineage-specific CyIIIa actin gene have been identified by in vivo competition. Sea urchin eggs were coinjected with a fusion construct in which the bacterial chloramphenicol acetyltransferase (CAT) gene is controlled by the CyIIIa regulatory domain, together with molar excesses of various DNA subfragments that are derived from this region. Each subfragment studied includes one or several known sites where highly specific interactions occur in vitro with nuclear DNA-binding proteins. Coinjection of excess molecules of some of these subregions results in a decrease in the activity of the CyIIIa-CAT fusion gene, as a function of the molar subfragment: CyIIIa-CAT ratio. This result implies that these sites complete with cis sequences linked to the CAT reporter gene for limited factors that positively regulate CyIIIa transcription in the embryo, and demonstrates the functional importance of a number of the DNA-protein interactions that have been observed in vitro.