Date of Graduation


Document Type



During periods of egg laying in the chicken, when circulating levels of estrogen are increased, the liver-specific estrogen-dependent very low density apolipoprotein II (apoVLDL II) gene is expressed. Transcription is accompanied by estrogen-dependent, liver-specific changes in the chromatin conformation within and surrounding the gene. One of the regions that changes in conformation is in the promoter where the estrogen receptor along with multiple liver-specific and ubiquitous transcription factors interact in a cooperative manner to assemble a functional transcriptional initiation complex. However, the apoVLDL II gene is expressed at higher levels when the first intron is added to the promoter. Since most transcriptional regulatory events involve the interaction between multiple cis-acting regulatory elements and their cognate protein factors, DNase I footprinting was used to locate potential regulatory regions within the 1378 bp first intron. Footprinting studies identified six binding sites spaced throughout the first intron. The regions of protection are located from nucleotide position 66 to 86, 112 to 129, 535 to 566, 646 to 685, 1096 to 1116, and 1340 to 1355. The functional significance of the two 5{dollar}\\sp\\prime{dollar} most intronic binding sites was studied. Site-specific mutations introduced at either the 66 to 86 or 112 to 129 sites disrupted trans-factor binding. The regulatory contribution of each of these regions was assessed by transient transfection using the estrogen-responsive LMH cell line. Results show that mutation of either the 66 to 86 or the 112 to 129 binding site 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. These data confirm that regulatory regions which control expression of the apoVLDL II gene extend into the first intron and demonstrate that protein binding at the two 5{dollar}\\sp\\prime{dollar} most intronic binding sites results in an increase in gene expression.