Abstract
The cyclic AMP response element (CRE) of the rat phosphoenolpyruvate carboxykinase (PEPCK) gene promoter is required for a complete glucocorticoid response. Proteins known to bind the PEPCK CRE include the CRE-binding protein (CREB) and members of the CCAAT/enhancer-binding protein (C/EBP) family. We took two different approaches to determine which of these proteins provides the accessory factor activity for the glucocorticoid response from the PEPCK CRE. The first strategy involved replacing the CRE of the PEPCK promoter/chloramphenicol acetyltransferase reporter plasmid (pPL32) with a consensus C/EBP-binding sequence. This construct, termed pDeltaCREC/EBP, binds C/EBPalpha and beta but not CREB, yet it confers a nearly complete glucocorticoid response when transiently transfected into H4IIE rat hepatoma cells. These results suggest that one of the C/EBP family members may be the accessory factor. The second strategy involved co-transfecting H4IIE cells with a pPL32 mutant, in which the CRE was replaced with a GAL4-binding sequence (pDeltaCREGAL4), and various GAL4 DNA-binding domain (DBD) fusion protein expression vectors. Although chimeric proteins consisting of the GAL4 DBD fused to either CREB or C/EBPalpha are able to confer an increase in basal transcription, they do not facilitate the glucocorticoid response. In contrast, a fusion protein consisting of the GAL4 DBD and amino acids 1-118 of C/EBPbeta provides a significant glucocorticoid response. Additional GAL4 fusion studies were done to map the minimal domain of C/EBPbeta needed for accessory factor activity to the glucocorticoid response. Chimeric proteins containing amino acid regions 1-84, 52-118, or 85-118 of C/EBPbeta fused to the GAL4 DBD do not mediate a glucocorticoid response. We conclude that the amino terminus of C/EBPbeta contains a multicomponent domain necessary to confer accessory factor activity to the glucocorticoid response from the CRE of the PEPCK gene promoter.
Highlights
A rate-controlling gluconeogenic enzyme, is expressed in a tissue-specific manner and is regulated at the transcriptional level by various hormones and nutrients [1, 2]
When the cyclic AMP response element (CRE) was replaced with a consensus CCAAT/ enhancer-binding protein (C/EBP)-binding site (p⌬CREC/EBP) (Fig. 1, bottom row), basal transcription increased 2-fold, and the absolute dexamethasone response was substantially greater than that conferred by the wild type promoter
Four accessory factor elements, including the CRE, and two GR-binding sites are required for a complete glucocorticoid response, and the proper order and placement of these elements is necessary for this effect [35]
Summary
A rate-controlling gluconeogenic enzyme, is expressed in a tissue-specific manner and is regulated at the transcriptional level by various hormones and nutrients [1, 2]. Members of the CCAAT/ enhancer-binding protein (C/EBP) and hepatocyte nuclear factor 3 (HNF3) families bind to AF2, but only the latter provide accessory factor activity for the glucocorticoid response from this element [10, 11]. GR, glucocorticoid receptor; CRE, cyclic AMP response element; HRUs, hormone response units; HNF, hepatocyte nuclear factor; COUP-TF, chicken ovalbumin upstream promoter-transcription factor; C/EBP, CCAAT/enhancer-binding protein; CREB, cyclic AMP response element-binding protein; CAT, chloramphenicol acetyltransferase; DBD, DNA-binding domain; EMSA, electrophoretic mobility shift assay; PCR, polymerase chain reaction; bZIP, basic and leucine zipper; PKA, cyclic AMP-dependent protein kinase A; PKI, heat-stable inhibitor of the cyclic AMP-dependent protein kinase; PKImut, mutant of PKI; CREwt, CRE wild type; USF, upstream stimulatory factor. A very good glucocorticoid response was retained when the CRE was replaced with a consensus
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
