Identification of an AP-1-like Sequence as the Glucocorticoid Response Element in the Rat Tyrosine Hydroxylase Gene

Glucocorticoids (GC) generally stimulate gene transcription via the consensus glucocorticoid response elements (GRE) located in the promoter region. To identify the GRE in the rat tyrosine hydroxylase (TH) gene promoter, we transiently transfected PC12 cells with a 9 kb TH promoter-luciferase (Luc) construct. Dexamethasone (Dex) stimulated Luc activity, which was abolished by RU-486. Serial deletion mutations revealed a Dex-responsive 7-bp sequence TGACTAA, located at -5734 to -5728. Deletion of just these 7 nucleotides from the 9 kb promoter completely abolished the Dex response and partially reduced the response to phorbol ester, but not to forskolin. The Dex response was fully retained in a construct in which most of the 9 kb promoter was deleted, except for 100 bp around the -5.7 kb region, clearly identifying this 7-bp sequence as solely responsible for GC responsiveness. Conversely, deletion of the proximal CRE (-45/-38) or AP-1 (-207/-201) sites in the 9 kb promoter did not affect Dex and phorbol ester responses. A radiolabeled 25 bp promoter fragment bearing the 7-bp TH-GRE/AP-1 showed specific binding to PC12 nuclear proteins. Using antibodies against GR and AP-1 family of proteins and primers for the TH-GRE/AP-1 region, we detected a specific DNA amplicon in a ChIP assay. This 7-bp TH-GRE/AP-1 sequence (TGACTAA) does not bear similarity to any known GRE, but closely resembles the consensus AP-1 binding site, TGACTCA. Our studies describe for the first time a novel GRE/AP-1 site present in the TH gene promoter that is critical for glucocorticoid regulation of the TH gene. 3 This article has not been copyedited and formatted. The final version may differ from this version. Molecular Pharmacology Fast Forward. Published on December 5, 2008 as DOI: 10.1124/mol.108.051219 at A PE T Jornals on Sptem er 9, 2016 m oharm .aspeurnals.org D ow nladed from