Abstract
The cAMP responsiveness of the phosphoenolpyruvate carboxykinase (PEPCK) gene promoter is mediated by a cAMP response unit, which includes three CCAAT/enhancer-binding protein (C/EBPs) sites, and a cAMP response element (CRE). Because both the CRE-binding protein and several C/EBP isoforms can to bind to the CRE with similar affinity, a variety of transcription factor bindings arrays in the cAMP response unit are possible that may affect the protein kinase A (PKA) responsivity of the promoter. To explore this issue, we have designed PEPCK promoter variants that have the native cis-elements within the cAMP response unit replaced with one or more LexA- and/or GAL4-binding sites. We also engineered the corresponding C/EBP and CRE-binding protein chimeras, which have their basic region leucine zipper domains replaced with LexA or GAL4 DNA-binding domains. Using this approach, we have reconstituted the PKA responsiveness of permissive PEPCK promoters in hepatoma cells and have characterized the PKA responsivity of the promoter under defined transcription factor occupancy patterns. Furthermore, analysis of deletion mutants of C/EBPalpha indicated that the domains that mediate its constitutive and PKA-inducible activities vary depending on which cis-element it occupies on the PEPCK promoter. These results suggest that promoter context may influence which domains within a transcription factor are employed to mediate transactivation.
Highlights
The gene coding for phosphoenolpyruvate carboxykinase (PEPCK), an enzyme involved in gluconeogenesis, is an archetype for transcriptional regulation and has been used extensively to study hormonal regulation of gene transcription [1]
It is interesting to note that CCAAT/enhancer binding proteins (C/EBPs) can compete with CRE-binding protein (CREB) for binding to the cAMP response element (CRE) in the PEPCK promoter, and a significant level of cAMP responsiveness is maintained when C/EBPα replaces CREB at the CRE [9]
The data indicate that the PKA-inducible activities of these two fusion proteins were similar, while the constitutive activity of LexA-C/EBPα was consistently greater than its GAL4 counterpart
Summary
The gene coding for phosphoenolpyruvate carboxykinase (PEPCK), an enzyme involved in gluconeogenesis, is an archetype for transcriptional regulation and has been used extensively to study hormonal regulation of gene transcription [1]. Its promoter is composed of a complex array of cis-elements, and different combinations of these cis-elements appear to functionally cooperate to form "hormone response units" that mediate the effects of thyroid hormone, glucocorticoids, and cAMP in liver [2]. A mutation in any one of the five cis-elements results in a severe abrogation of cAMP responsiveness in liver-derived cells [6]. Inhibition of C/EBPα expression in hepatoma cells by antisense methodology impairs the ability of the PEPCK gene to respond to cAMP [8]. Structure/function analysis of C/EBPα identified domains which mediate the constitutive activity of C/EBP as well as the cAMP-inducible activity, and while the domains which mediate these two activities overlap to some degree, mutational analysis suggests that they are distinct from one another [9,10]. The presence of CREB on the PEPCK promoter does not appear to be an absolute requirement for cAMP responsiveness
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