Functional characterization of the promoter of pp63, a gene encoding a natural inhibitor of the insulin receptor tyrosine kinase.
PP63 is a liver specific phosphorylated glycoprotein encoded by a single copy gene, which has the property of inhibiting both autophosphorylation and tyrosine kinase activity of the insulin receptor. In this study, we have analyzed the structure activity relationship of the pp63 gene promoter. Five protein binding sites were found in the proximal 5' flanking region of the gene (-223 to +4). Using oligonucleotides as competitors and purified recombinant C/EBP in footprinting and gel retardation assays, we identified two typical C/EBP sites (X1 and X3) plus a heterogenous, C/EBP-NF1 like site (X5), separated by two classical NF1 binding sites (X2 and X4). C/EBP or the related proteins were predominantly involved in supporting cell-free transcription. Occupancy of the first high affinity C/EBP site conferred almost maximal promoter efficiency, in vitro. However, this pp63 promoter activity remained very low as compared to that in intact hepatocytes. In these cells, occupancy of the first C/EBP (X1) and NF1 (X2) sites was already required for achieving a weak transcriptional activity. The use of the second C/EBP site (X3) strongly enhanced transcription, up to 60-70% of the maximum, whereas occupancy of the two more distal sites (X4 and X5) was necessary to fully activate the promoter. Thus, the strength of the promoter as well as the liver specific expression of pp63 gene appear to result from the interplay of several DNA-protein complexes involving mainly C/EBP and/or related proteins as well as the ubiquitous NF1 factor(s), rather than from the interaction of a more liver specific trans-acting factor with the promoter.
Falquerho
L
L
Centre CNRS-INSERM de Pharmacologie-Endocrinologie, Montpellier, France.
Paquereau
L
L
Vilarem
M J
MJ
Galas
S
S
Patey
G
G
Le Cam
A
A
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D022762:CCAAT-Enhancer-Binding Proteins; D002474:Cell-Free System; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000502:physiology*; D006023:Glycoproteins / Q000235:genetics*; D008297:Male; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors; D051381:Rats; D011919:Rats, Inbred Strains; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic / Q000235:genetics; D060749:alpha-2-HS-Glycoprotein
C071388:Ahsg protein, rat; D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins; D006023:Glycoproteins; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D060749:alpha-2-HS-Glycoprotein; D004247:DNA; D011505:Protein-Tyrosine Kinases