An array of binding sites for hepatocyte nuclear factor 4 of high and low affinities modulates the liver-specific enhancer for the human alpha1-microglobulin/bikunin precursor.
Alpha1-Microglobulin and bikunin are two plasma glycoproteins encoded by a gene for alpha1-microglobulin/bikunin precursor (AMBP). The strict liver-specific transcription of the AMBP gene is controlled by an elaborate and remote enhancer made of six clustered boxes numbered 1 to 6 (core enhancer) that are binding sites for the hepatocyte-enriched nuclear factors HNF-1, HNF-4, HNF-3, HNF-1, HNF-3 and HNF-4 respectively. Three further boxes, 7 to 9, have now been found in the enhancer area in a position 5' of box 2, 5' of box 1 and 3' of box 6, respectively. Electrophoretic mobility-shift assays with nuclear extracts from the HepG2 hepatoma cell line demonstrated that boxes 7 and 8 are both functional HNF-4-binding sites of high and low affinity respectively, whereas no binding capacity of box 9 was detected by this method. Transfection of HepG2 and Chinese hamster ovary cells with chloramphenicol acetyltransferase constructs harbouring the core or extended AMBP enhancer with wild-type or mutated boxes and co-transfection with expression plasmids for a wild-type or defective HNF-4 identified box 7 as an essential element for the basal activity of this enhancer. The response of boxes 7 and 8 varies with the level of HNF-4 in cells. Box 9 exhibits a repressor activity that can be detected when box 8 is ablated. In vivo this corresponds to conditions of low box 8 occupancy when the intracellular level of HNF-4 is limited. These results reinforce the view that the AMBP enhancer is a quite elaborate and unusual example of a modular enhancer whose activity is fine-tuned by the level of cognate nuclear factors in the cell.
Rouet
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P
INSERM Unit-78 and Institut Fédératif de Recherches Multidisciplinaires sur les Peptides, Faculté de Médecine-Pharmacie, 22 Boulevard Gambetta, 76000 Rouen, France.
Raguenez
G
G
Ruminy
P
P
Salier
J P
JP
D000510:Alpha-Globulins / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D001665:Binding Sites / Q000235:genetics; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins; D004742:Enhancer Elements, Genetic; D006023:Glycoproteins / Q000235:genetics*; D051557:Hepatocyte Nuclear Factor 4; D006801:Humans; D007700:Kinetics; D008099:Liver / Q000378:metabolism*; D008562:Membrane Glycoproteins; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000378:metabolism*; D011498:Protein Precursors / Q000235:genetics*; D014157:Transcription Factors / Q000378:metabolism*; D015533:Transcriptional Activation; D014162:Transfection; D014360:Trypsin Inhibitor, Kunitz Soybean
D000510:Alpha-Globulins; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D004268:DNA-Binding Proteins; D006023:Glycoproteins; D051557:Hepatocyte Nuclear Factor 4; C403128:MLX protein, human; D008562:Membrane Glycoproteins; D010750:Phosphoproteins; D011498:Protein Precursors; C109148:SPINT2 protein, human; D014157:Transcription Factors; C016001:alpha-1-microglobulin; D004247:DNA; D014360:Trypsin Inhibitor, Kunitz Soybean