A COMPOSITE REGULATORY ELEMENT IN THE FIRST INTRON OF THE ESTROGEN-RESPONSIVE VERY-LOW-DENSITY APOLIPOPROTEIN-II GENE

During periods of egg laying in the chicken, when circulating levels o f estrogen are increased, the lives-specific estrogen-dependent very l ow density apolipoprotein II (apoVLDLII) gene is expressed. This expre ssion takes place primarily at the level of transcription, driven by t wo estrogen response elements that reside in the promoter. In transien t transfection assays, expression is increased fourfold when the first intron is added to the promoter construct, indicating that 75% of the regulation comes from intron A. Using in vitro DNase I footprinting, six protein-binding sites were revealed throughout the first intron. T he functional significance of these binding sites was evaluated by mut ation and transient transfection. Two of the protein-binding regions w ere shown to increase transcription. Site-specific mutations introduce d at either the +66 to +86 or +112 to +129 sites disrupted trans-facto r binding and reduced the estrogen-dependent expression by 45% and 34% , respectively. A plasmid containing both mutations resulted in a 43% decrease in expression, indicating that the contributions of these reg ions are not additive. Competition with known sequences in electrophor etic mobility shift assays suggested that the +66 to +86 site binds a chicken member of the nuclear receptor transcription factor family.