Abstract
We have identified two regions of the mouse gonadotropin-releasing hormone (GnRH) promoter, one between -237 and -201 (distal element) and the other between -184 and -150 (proximal element), which are required for glucocorticoid repression in transiently transfected GT1-7 cells. These sequences show no similarity to known positive or negative glucocorticoid response elements (nGREs) and do not function when placed upstream of heterologous viral promoters. The glucocorticoid receptor (GR) does not bind directly to the distal or proximal promoter elements but may participate in glucocorticoid repression of GnRH gene transcription by virtue of its association within multiprotein complexes at these nGREs. Electrophoretic mobility shift assays with GT1-7 nuclear extract demonstrate the presence of GR-containing protein complexes on GnRH nGREs. One protein that co-occupies the distal nGRE in vitro along with GR is the POU domain transcription factor Oct-1. Thus, the tethering of GR to the GnRH distal nGRE, by virtue of a direct or indirect association with DNA-bound Oct-1, could play a role in hormone-dependent transcriptional repression of the GnRH gene. In contrast, Oct-1 does not appear to be a component of the GR-containing protein complex that is bound to the proximal nGRE.
Highlights
The hypothalamic decapeptide, gonadotropin-releasing hormone (GnRH),1 is at the top of the endocrine hierarchy that controls reproductive function
Glucocorticoid Repression of the Mouse GnRH Gene Is Mediated by Two Promoter Elements—In order to localize sequence elements within the GnRH promoter that are responsible for glucocorticoid receptor (GR)-mediated transcriptional repression, dexamethasone effects were assessed on a series of plasmids containing 5Ј-flanking deletions of the GnRH promoter using the luciferase gene as a reporter
We have identified two regions of the mouse GnRH promoter, one between Ϫ237 and Ϫ201 and another between Ϫ184 and Ϫ150, which mediate transcriptional repression by glucocorticoids
Summary
GnRH, gonadotropin-releasing hormone; GR, glucocorticoid receptor; GRE, glucocorticoid response element; nGRE, negative glucocorticoid response element; bp, base pair(s); EMSA, electrophoretic mobility shift assay; DTT, dithiothreitol; MMTV, mouse mammary tumor virus; LTR, long terminal repeat; TAT, tyrosine aminotransferase; PR, progesterone receptor. Such studies have become possible in vitro due to the development of immortalized, GnRH-secreting hypothalamic cell lines (GT1 cells) from a genetically engineered brain tumor in a transgenic mouse [1]. These cell lines (GT1-1, GT1-3, and GT1-7) have been used to examine the regulation of GnRH secretion and gene expression by a variety of neurotransmitters [2,3,4,5], steroid hormones [6, 7], and second messengers involved in intracellular signaling (8 –11). We have mapped two negative glucocorticoid responsive elements (nGREs) within the mouse GnRH promoter and identified a novel mechanism for GR-mediated repression, which involves the tethering of GR to nGREs via an association with other DNA-bound transcription factors
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