Estrogen-mediated induction of rat prolactin gene transcription requires the formation of a chromatin loop between the distal enhancer and proximal promoter regions

Rat PRL (rPRL) gene transcription is controlled by proteins that interact with two DNA elements located in the 5'-upstream regulatory region. These elements, the distal enhancer and the proximal promoter, are separated by nearly 1500 bp of DNA. Induction of rPRL transcription by the estrogen 17 beta-estradiol (E(2)) is conferred through the estrogen response element located in the distal enhancer. To activate transcription, the estrogen-estrogen receptor complex must ''communicate'' with RNA polymerase II located at the proximal promoter. Using a novel nuclear ligation assay, it is shown that estrogen treatment of rat pituitary GH3 cells stimulates the formation of chromatin loops between the distal enhancer and proximal promoter of the rPRL gene, juxtaposing these two transcriptional control regions. The formation of these chromatin loops was observed in both the endogenous rPRL gene and in a rPRL-Tn5 minichromosome. Induction of rPRL and rPRL-Tn5 expression by E(2) was reduced by coincubation of cells with the antiestrogen 4-hydroxytamoxifen Likewise, 4-hydroxytamoxifen was found to block the E(2)-induced formation of chromatin loops between the distal and proximal regions. Concurrent treatment with the synthetic glucocorticoid dexamethasone reduced the E(2)-induced transcription rate of the rPRL and rPRL-Tn5 gene to near basal levels. Similarly, dexamethasone treatment inhibited the ability of E(2) to enhance the formation of the chromatin loops. These data suggest that the activation of rPRL gene transcription by E(2) involves the stabilization of a chromatin loop that facilitates protein-protein interactions between transcription factors that are associated with the distal enhancer and the proximal promoter.