Hormonal regulation of assembly of the pepck gene transcription complex

Abstract

Phosphoenolpyruvate carboxykinase (PEPCK) gene transcription is activated in response to glucocorticoids, such as dexamethasone (Dex). Previous studies, using a variety of techniques, including reporter gene assays, showed that the induction of PEPCK gene transcription by Dex requires a complex array of DNA accessory elements and associated DNA-binding transcription factors. These, in turn, bind a set of transcription coactivators, including CREB binding protein (CBP) and p300. We have now used the chromatin immunoprecipitation assay (ChIP) to assess promoter occupancy of these proteins, in vivo, in the presence and absence of the ligand, Dex. Dex causes a remarkable increase in the binding of glucocorticoid receptor, polymerase II, CBP and p300 to the promoter. However, even though both CBP and p300 are expressed in the nucleus of H4IIE cells, they do not co-exist in the same transcription complex on the PEPCK gene promoter. Furthermore, the selective reduction of either p300 or CBP, using small interfering RNAs (siRNAs), partially abolishes the Dex response of the PEPCK gene. Reduction of both CBP and p300 totally abolishes the Dex response of the PEPCK gene. Although endogenous p300 and CBP are not capable of compensating for the loss of CBP and p300, respectively, the overexpression of either of these proteins compensates for the loss of the other, and both coactivators support the dominant suppression of the gene by insulin.