INTRODUCTION
Extracellular signals received by eucaryotic cells are transferred to the nucleus where the transcription of specific sets of genes is modulated The final targets of these signaling pathways are different combinations of transcription factors that bind to DNA-response elements and form various protein-protein interactions on gene promoters and thereby either activate or repress gene transcription. DNA-response elements in gene promoters often overlap or are adjacent to one another This provides various possibilities for the recruitment of transcription factors and their cognate coregulators in response to distinct signals and creates the potential for considerable flexibility in gene regulation. Regulation of the phosphoenolpyruvate carboxykinase (PEPCK) gene provides an excellent model system for exploring the mechanisms of signal integration in the context of gluconeogenesis, an important physiologic process The expression of hepatic PEPCK is primarily accomplished at the transcriptional level Glucagon (acting through cAMP), glucocorticoids, all-trans-retinoic acid (RA), and thyroid hormone activate PEPCK gene expression, whereas insulin and glucose are inhibitory .
Activation of PEPCK gene transcription by RA occurs through a multicomponent retinoic acid-response unit (RARU) that consists of two separate retinoic acid-response elements, RARE1 and RARE2, each of which binds an RAR/RXR heterodimer . Activation of PEPCK gene transcription by Dex also requires a multicomponent glucocorticoid-response unit (GRU), which consists of two nonconsensus glucocorticoid receptor-binding sites (GR1 and GR2) and four accessory factor-binding sites (gAF1, gAF2, gAF3, and the cAMP-response element (CRE)), which bind COUP-TF/HNF4, HNF3, COUP-TF, and C/EBP, respectively ). A mutation of any one of the accessory elements results in a 50–60% reduction of Dex-induced PEPCK gene transcription in H4IIE hepatoma cellsAny combination of the mutation of two elements nearly abolishes this response .
Most interesting, RARE1 and RARE2 in the RARU are coincident with gAF1 and gAF3 in the GRU The respective transcription factors are present in about the same abundance in the H4IIE cell nucleus, and each binds with approximately the same affinity to these elements. Thus, it is of interest to understand how a cell responds to either of these inducers, whether different constellations of accessory factors and coregulators are used for each response and whether there is any functional interaction between the two units in the presence of both ligands.
In this study, we demonstrate that the Dex/RA combination has a synergistic effect on endogenous PEPCK gene expression in both primary hepatocytes and H4IIE hepatoma cells. This response is most dependent on the gAF3 and GR1 elements. Upon treatment with Dex/RA, ligand-activated RAR/RXR heterodimers, GR, HNF3, HNF4, p/CIP, SRC-1, CBP, p300, and RNA polymerase II (pol II) are present on the PEPCK gene promoter. Notably, the recruitment of p300 and pol II on the PEPCK promoter is increased upon Dex/RA treatment. The important role of p300 is illustrated by the fact that its reduction results in a selective loss of the synergistic effect of Dex/RA, whereas a similar reduction of CBP has no effect.
Extracellular signals received by eucaryotic cells are transferred to the nucleus where the transcription of specific sets of genes is modulated The final targets of these signaling pathways are different combinations of transcription factors that bind to DNA-response elements and form various protein-protein interactions on gene promoters and thereby either activate or repress gene transcription. DNA-response elements in gene promoters often overlap or are adjacent to one another This provides various possibilities for the recruitment of transcription factors and their cognate coregulators in response to distinct signals and creates the potential for considerable flexibility in gene regulation. Regulation of the phosphoenolpyruvate carboxykinase (PEPCK) gene provides an excellent model system for exploring the mechanisms of signal integration in the context of gluconeogenesis, an important physiologic process The expression of hepatic PEPCK is primarily accomplished at the transcriptional level Glucagon (acting through cAMP), glucocorticoids, all-trans-retinoic acid (RA), and thyroid hormone activate PEPCK gene expression, whereas insulin and glucose are inhibitory .
Activation of PEPCK gene transcription by RA occurs through a multicomponent retinoic acid-response unit (RARU) that consists of two separate retinoic acid-response elements, RARE1 and RARE2, each of which binds an RAR/RXR heterodimer . Activation of PEPCK gene transcription by Dex also requires a multicomponent glucocorticoid-response unit (GRU), which consists of two nonconsensus glucocorticoid receptor-binding sites (GR1 and GR2) and four accessory factor-binding sites (gAF1, gAF2, gAF3, and the cAMP-response element (CRE)), which bind COUP-TF/HNF4, HNF3, COUP-TF, and C/EBP, respectively ). A mutation of any one of the accessory elements results in a 50–60% reduction of Dex-induced PEPCK gene transcription in H4IIE hepatoma cellsAny combination of the mutation of two elements nearly abolishes this response .
Most interesting, RARE1 and RARE2 in the RARU are coincident with gAF1 and gAF3 in the GRU The respective transcription factors are present in about the same abundance in the H4IIE cell nucleus, and each binds with approximately the same affinity to these elements. Thus, it is of interest to understand how a cell responds to either of these inducers, whether different constellations of accessory factors and coregulators are used for each response and whether there is any functional interaction between the two units in the presence of both ligands.
In this study, we demonstrate that the Dex/RA combination has a synergistic effect on endogenous PEPCK gene expression in both primary hepatocytes and H4IIE hepatoma cells. This response is most dependent on the gAF3 and GR1 elements. Upon treatment with Dex/RA, ligand-activated RAR/RXR heterodimers, GR, HNF3, HNF4, p/CIP, SRC-1, CBP, p300, and RNA polymerase II (pol II) are present on the PEPCK gene promoter. Notably, the recruitment of p300 and pol II on the PEPCK promoter is increased upon Dex/RA treatment. The important role of p300 is illustrated by the fact that its reduction results in a selective loss of the synergistic effect of Dex/RA, whereas a similar reduction of CBP has no effect.
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