Studies on the Mechanism of Glucocorticoid-Mediated Repression from a Negative Glucocorticoid Response Element from the Bovine Prolactin Gene

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Abstract
Several models for repression of transcription by glucocorticoid hormone, some of which involve so-called negative glucocorticoid response elements (nGRE), have been suggested. In the cases where nGREs are required, the glucocorticoid receptor (GR) is thought to bind to the nGRE and interfere with transcriptional activation by positively acting transactivating factors. We have studied an nGRE from the bovine prolactin gene promoter (PRL3), which increases basal expression from a heterologous promoter in rat pituitary cells (GH3) and is repressed by glucocorticoids. Two proteins in addition to the GR were identified in pituitary cells to bind specifically to the PRL3 nGRE, one of which was the pituitary-specific transcription factor Pit-1/GHF-1. A mutation in the PRL3 nGRE, which destroyed Pit-1/GHF-1 binding, totally abolished the increased basal expression as well as glucocorticoid repression in transfected GH3 cells. A mutation in the binding site for the second protein, termed XTF, partially impaired basal activity but totally abrogated glucocorticoid repression. The same mutation had no effect on GR binding to the PRL3 nGRE. Mixing experiments with whole-cell extracts containing overexpressed GR from COS cells decreased the binding of both Pit-1/GHF-1 and XTF to the PRL3 element. However, Pit-1/GHF-1 displacement from the PRL3 element by the GR required XTF binding. Furthermore, GR binding to the PRL3 nGRE was required for glucocorticoid repression to occur, because a mutation of the GR binding site abolished the glucocorticoid effect. Moreover, the PRL3 nGRE was found to contain only half a palindromic GRE, allowing only one GR moiety to contact the DNA. These data demonstrate that the PRL3 nGRE is composite in nature and that the ability of the GR to repress transactivation by displacement requires an intermediary factor, XTF.