CIS-ACTING ELEMENTS REINFORCING THE ACTIVITY OF THE ESTROGEN-RESPONSE ELEMENT IN THE VERY-LOW-DENSITY APOLIPOPROTEIN-II GENE PROMOTER

The gene coding for chicken very low density apolipoprotein II (apoVLDLII) is expressed exclusively in liver in response to estrogen. Previous work in our laboratory identified several protein binding sites, identified by the letters A to F, and their cognate factors within the first 300 bp flanking the gene. Here we present an extensive functional analysis of the apoVLDLII promoter by gene transfer experiments using a chicken hepatoma cell line and cultured non-hepatic cells. Deletion analysis revealed that the -301 to -163-bp promoter region, comprising elements E1, E2 and F, is sufficient for strong estrogen-dependent expression. Mutation analysis demonstrated that efficient transcription requires the interplay of the major estrogen response element El with several other cis-acting elements. Analysis of individual protein binding sites showed that element El is sufficient by itself to confer weak estrogen-induced transcription from the apoVLDLII promoter, and that additional promoter elements are required for full estrogen-responsiveness. Elements F and B1 were capable of strongly potentiating the activity of element E1. In general, the activity of certain cis-acting elements appeared to be strongly promoter-context dependent. Cultured non-liver cells expressed transfected VLDL-CAT reporter plasmids in the presence of cotransfected estrogen receptor expression vector in a hormone-dependent way, indicating that for the control of tissue specificity the 5'-proximal promoter region is not sufficient