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 of estrogen are increased, the lives-specific estrogen-dependent very low density apolipoprotein II (apoVLDLII) gene is expressed. This expression takes place primarily at the level of transcription, driven by two estrogen response elements that reside in the promoter. In transient 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. The functional significance of these binding sites was evaluated by mutation and transient transfection. Two of the protein-binding regions were shown to increase transcription. Site-specific mutations introduced at either the +66 to +86 or +112 to +129 sites disrupted trans-factor 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 regions are not additive. Competition with known sequences in electrophoretic mobility shift assays suggested that the +66 to +86 site binds a chicken member of the nuclear receptor transcription factor family.