Abstract
Transcription of the L-type pyruvate kinase (L-PK) gene is induced by glucose in the presence of insulin and repressed by glucagon via cyclic AMP. The DNA regulatory sequence responsible for mediating glucose and cyclic AMP responses, called glucose response element (GlRE), consists of two degenerated E boxes spaced by 5 base pairs and is able to bind basic helix-loop-helix/leucine zipper proteins, in particular the upstream stimulatory factors (USFs). From ex vivo and in vivo experiments, it appears that USFs are required for correct response of the L-PK gene to glucose, but their expression and binding activity are not known to be regulated by glucose. A genetic screen in yeast has allowed us to identify a novel transcriptional factor binding to the GlRE, i.e. the chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII). Binding of COUP-TFII to the GlRE was confirmed by electrophoretic mobility shift assays, and COUP-TFII-containing complexes were detectable in liver nuclear extracts. Neither abundance nor binding activity of COUP-TFII appeared to be significantly regulated by diets. In footprinting experiments, two COUP-TFII-binding sites overlapping the E boxes were detected. Overexpression of COUP-TFII abrogated the USF-dependent transactivation of an artificial GlRE-dependent promoter in COS cells and the glucose responsiveness of the L-PK promoter in hepatocytes in primary culture. In addition, a mutated GlRE with increased affinity for USF and very low affinity for COUP-TFII conferred a dramatically decreased glucose responsiveness on the L-PK promoter in hepatocytes in primary culture by increasing activity of the reporter gene in low glucose condition. We propose that COUP-TFII could be a negative regulatory component of the glucose sensor complex assembled on the GlRE of the L-PK gene and most likely of other glucose-responsive genes as well.
Highlights
Transcription of the L-type pyruvate kinase (L-PK) gene is induced by glucose in the presence of insulin and repressed by glucagon via cyclic AMP
chicken ovalbumin upstream promoter transcription factor (COUP-TF) is a member of the steroid/thyroid hormone receptor (TR) superfamily and was first identified as a homodimer that binds to a direct repeat regulatory element (DR1) in the chicken ovalbumin gene promoter (27)
We propose that the glucose responsiveness mediated by the glucose response element (GlRE) could involve a complex interaction between upstream stimulatory factors (USFs) transactivators and COUP-TFII, probably in association with other, as yet not characterized partners
Summary
Transcription of the L-type pyruvate kinase (L-PK) gene is induced by glucose in the presence of insulin and repressed by glucagon via cyclic AMP. We defined a sequence responsible for mediating the positive response to glucose and the negative responses to cAMP in the proximal L-PK promoter from the position Ϫ172 to Ϫ142 bp, ex vivo by transient expression assays in hepatocytes in primary culture and in vivo in transgenic mice (8 –11) This DNA-binding site was termed the glucose response element (GlRE). In the context of the wild-type L-PK promoter, the complex that binds to this element cooperates with an adjacent DNA-binding site, the auxiliary site L3 (8, 10), to confer a strong glucose responsiveness This auxiliary site has been shown to bind mainly hepatocyte nuclear factor 4 (HNF4) in the liver but has some affinity for chicken ovalbumin upstream promoter transcription factor (COUP-TF) (10, 14) and nuclear factor 1 (15). COUPTFII-containing dimers could be involved in a glucose sensor system abrogating transactivation by USFs in the absence of glucose
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