Composite Glucocorticoid Regulation at a Functionally Defined Negative Glucocorticoid Response Element of the Human Corticotropin-Releasing Hormone Gene

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Abstract
Glucocorticoid-dependent negative feedback of the hypothalamic-pituitary-adrenal axis is medi- ated in part through direct inhibition of hypotha- lamic CRH gene transcription. In the present study, we sought to further localize and charac- terize glucocorticoid receptor (GR) and AP-1 in- teractions at a functionally defined negative glu- cocorticoid response element (nGRE) of the CRH promoter. Transient transfection studies in mouse corticotroph AtT-20 cells demonstrated that internal deletion of the nGRE (2278 to 2249 nucleotides) within the context of 1 kb of the intact CRH promoter resulted in decreased 8-Br- cAMP stimulation and glucocorticoid-dependent repression of CRH promoter activity. The nGRE conferred transcriptional activation by both cAMP and overexpressed c-jun or c-fos AP-1 nucleoproteins as well as specific glucocorti- coid-dependent repression to a heterologous promoter. A similar profile of regulation was ob- served for the composite GRE derived from mouse proliferin promoter. The CRH nGRE was clearly distinct from the consensus cAMP re- sponse element (CRE) at 2224 nucleotides, which increased basal activity and cAMP respon- siveness of a heterologous promoter but did not confer glucocorticoid-dependent repression. High-affinity binding sites for both GR and AP-1 nucleoproteins were identified at adjacent ele- ments within the nGRE. Mutations that disrupted either GR or AP-1 binding activity were associ- ated with loss of glucocorticoid-dependent re- pression. These results are consistent with a composite mechanism of glucocorticoid-depen- dent repression involving direct DNA binding of GR and AP-1 nucleoproteins at discrete adjacent sites within the CRH promoter. (Molecular Endo- crinology 13: 1629-1644, 1999)

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