The Orphan Receptor COUP-TF Binds to a Third Glucocorticoid Accessory Factor Element within the Phosphoenolpyruvate Carboxykinase Gene Promoter

Donald K. Scott, John A. Mitchell, Daryl K. Granner
1996 Journal of Biological Chemistry  
The phosphoenolpyruvate carboxykinase (PEPCK) gene promoter contains a glucocorticoid response unit (GRU) that includes, as a linear array, two accessory factor binding sites (AF1 and AF2) and two glucocorticoid receptor binding sites. All of these elements are required for a complete glucocorticoid response. AF1 and AF2 also partially account for the response of the PEPCK gene to retinoic acid and insulin, respectively. A second retinoic acid response element was recently located just
more » ... cated just downstream of the GRU. In this study we show that mutation of the 3 half-site of this element results in a 60% reduction of the glucocorticoid response of PEPCK promoter-chloramphenicol acetyltransferase (CAT) fusion constructs in transient transfection assays, thus the half-site is now termed AF3. A variety of assays were used to show that chicken ovalbumin upstream promoter transcription factor (COUP-TF) binds specifically to AF3 and that upstream stimulatory factor (USF) binds to an E-box motif located 2 base pairs downstream of AF3. Mutations of AF3 that diminish binding of COUP-TF reduce the glucocorticoid response, but mutation of the USF binding site has no effect. The functional roles of AF1, AF2, and AF3 in the glucocorticoid response were explored using constructs that contained combinations of mutations in all three elements. All three elements are required for a maximal glucocorticoid response, and mutation of any two abolish the response. Phosphoenolpyruvate carboxykinase (PEPCK; EC 1 catalyzes the rate-limiting step of gluconeogenesis. The activity of this enzyme is therefore tightly regulated by a variety of hormonal and dietary signals that are involved in the maintenance of appropriate plasma glucose levels (for reviews, see Refs. 1 and 2). The PEPCK gene product apparently is not allosterically or post-translationally modified. Rather, the activity of PEPCK is modulated by changes in the amount of the protein, which are achieved predominantly by alterations of the rate of the transcription of the gene (3) . PEPCK gene transcription is positively regulated by glucagon (via cAMP), glucocorticoids and retinoic acid (RA), while insulin inhibits PEPCK gene transcription and is dominant over the positive effectors (4 -8). Thus, PEPCK is an excellent model system for studies concerning the integration of multiple metabolic signals through a single gene promoter. The glucocorticoid response of the PEPCK gene is mediated by a complex glucocorticoid response unit (GRU). This region of DNA, located between positions Ϫ451 and Ϫ353 relative to the transcription start site, is composed of a tandem array of two accessory factor elements (AF1, Ϫ451 to Ϫ439 and AF2, Ϫ416 to Ϫ407) and two glucocorticoid receptor binding sites (GR1 and GR2, Ϫ386 to Ϫ353) (4). Mutation of either AF1 or AF2 results in a 50 -60% reduction of the glucocorticoid response and deletion of both AF elements abolishes activity of the GRU (4, 9). GR1 and GR2 are essentially silent in the absence of accessory factors, which distinguishes them from simple glucocorticoid response elements (GREs) that are not dependent upon accessory factors for a glucocorticoid response (10). At least one other element is also required for a complete glucocorticoid response of the PEPCK gene. Deletion of the PEPCK cAMP response element (CRE; Ϫ93 to Ϫ86) also results in a 50 -60% reduction of the glucocorticoid response (11). This effect may be mediated by protein-protein interactions, since the glucocorticoid receptor (GR) and CRE binding protein (CREB) interact in solution (11) . The accessory activity of AF1 can be attributed to the specific binding of chicken ovalbumin upstream promoter transcription factor (COUP-TF) or hepatic nuclear factor 4 (HNF-4), members of a subfamily of the steroid/thyroid/retinoid superfamily of nuclear hormone receptors (12, 13). These proteins are not known to bind ligands, hence they are called orphan receptors (14). Both HNF-4 and COUP-TF are independently capable of acting as accessory factors in the PEPCK glucocorticoid response (13). The AF2 element is bound by CCAAT enhancerbinding protein (C/EBP) family members and by hepatic nuclear factor 3 (HNF-3) (15, 16) . A detailed analysis of the AF2 element has led to the conclusion that the accessory factor activity is mediated by HNF-3 and not by C/EBP family members (17) . In addition to their involvement in the glucocorticoid response, AF1 and AF2 are also required for other hormone responses. The minimal functional boundaries of AF1 are co-
doi:10.1074/jbc.271.50.31909 pmid:8943235 fatcat:sg5s6uhpb5fixnfsckotib4jpe