Functional characterization of the L-type pyruvate kinase gene glucose response complex.
L-type pyruvate kinase (L-PK) gene expression is modulated by hormonal and nutritional conditions. We have previously shown that the glucose/insulin response element (GlRE) of the L-PK gene is built around two noncanonical E boxes (element L4) that cooperate closely with a contiguous binding site (element L3). We present in this report the identification of proteins that interact with both elements. The L3 site binds hepatocyte nuclear factor 4 (HNF4)- and COUP/TF-related proteins. In fibroblasts, the overexpression of HNF4 transactivates the L-PK promoter. On the contrary, COUP/TF strongly inhibits the active promoter in hepatocytes. The L4 site binds the major late transcription factor (MLTF) in vitro and ex vivo; mutations that suppress this binding activity also inactivated the GlRE function. Mutations transforming one or two noncanonical E boxes of element L4 into consensus MLTF/USF binding sites strongly increase the affinity for MLTF/USF and do not impair the glucose responsiveness. However, merely the ability to bind MLTF/USF does not seem to be sufficient to confer a GlRE activity: those elements in which one E box has been destroyed and the other has been transformed into a consensus MLTF/USF sequence bind MLTF/USF efficiently but do not confer a high glucose responsiveness on the L-PK gene promoter. Consequently, the full activity of the L-PK GlRE seems to require the cooperation between two putative MLTF/USF binding sites located in the vicinity of an HNF4 binding site.
Diaz Guerra
M J
MJ
Laboratoire de Recherches en Génétique et Pathologie Moléculaire, Institut Cochin de Génétique Moléculaire, INSERM U-129, CHU Cochin, Paris, France.
Bergot
M O
MO
Martinez
A
A
Cuif
M H
MH
Kahn
A
A
Raymondjean
M
M
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000235:genetics; D004268:DNA-Binding Proteins / Q000378:metabolism; D005947:Glucose / Q000494:pharmacology*; D051557:Hepatocyte Nuclear Factor 4; D007328:Insulin / Q000494:pharmacology; D008099:Liver / Q000187:drug effects / Q000378:metabolism; D008297:Male; D008969:Molecular Sequence Data; D010750:Phosphoproteins; D011401:Promoter Regions, Genetic; D011770:Pyruvate Kinase / Q000235:genetics* / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D014157:Transcription Factors / Q000378:metabolism; D051720:Upstream Stimulatory Factors
D004268:DNA-Binding Proteins; D051557:Hepatocyte Nuclear Factor 4; D007328:Insulin; D010750:Phosphoproteins; D014157:Transcription Factors; D051720:Upstream Stimulatory Factors; C497182:Usf1 protein, rat; D004247:DNA; D011770:Pyruvate Kinase; D005947:Glucose