Identification of estrogen-responsive genes in chick liver

Identification of targets of estrogen is an important step in understanding the mechanisms of estrogen action. A two-step strategy was developed to identify estrogen-responsive genes (ERGs) in chick liver. Initially, differential-display, reverse transcription polymerase chain reaction (DDRT-PCR) was introduced to isolate ERGs. Isolated ERGs were then analyzed using Northern blot hybridization. A number of differentially expressed cDNA fragments were isolated following estrogen exposure. Four cDNA fragments that displayed dramatic change after estrogen administration were identified as liver adenylosuccinate lyase (ADL), phenobarbital-inducible cytochrome P-450 (CYP450), ovoinhibitor, and glutathione-dependent prostaglandin D-2 synthase (PGDS). Time sequence analysis showed that estrogen-induced alteration occurred as early as 0.5 h and peaked between 1 and 4 h after estrogen exposure. Nuclear run-off assay indicated that estrogen significantly increased the transcription rate of these genes. To determine whether the observed alteration was due to the direct effect of estrogen, protein synthesis was inhibited by cycloheximide (CHX) during stimulation by estradiol. Estrogen-mediated upregulation of PGDS was completely abolished by a concurrent treatment with CHX, suggesting that its activation requires the participation of some newly synthesized factor(s). In contrast, CHX did not affect the expression of other genes, indicating the alteration is a direct response to estrogen. In conclusion, ADL, CYP450, ovoinhibitor, and PGDS represent the novel targets of estrogen, which regulates the transcriptional activity of these genes.