Identification of a liver preference enhancer element of the rat hemopexin gene and its interaction with nuclear factors.
Transcription of hemopexin (Hx) occurs predominantly in the liver. To investigate the contribution of the cis-acting enhancer element to the hepatocyte preferential expression, we performed chloramphenicol acetyl-transferase (CAT) assays in HepG2 cells. A strong enhancer element was identified by successive truncation to reside in the region -157/-104 from the cap site. The Hx region -145/-125 interacts with rat liver nuclear proteins, as shown by standard DNA-protein binding assays. The nucleotide sequence -141AGACTTTGACCT-130 includes, in reverse orientation, a direct repeat of the imperfect AGGTCA sequence, one of the recognition motifs of the steroid-thyroid hormone receptor superfamily. That this AGGTCA repeat is an enhancer core of the Hx element was affirmed by mutational analyses. In electrophoretic mobility shift assays, oligonucleotides, corresponding to binding regions of chicken ovalbumin upstream promoter transcription factor (COUP-TF) and apolipoprotein AI regulatory protein 1 (ARP-1) but not of hepatocyte nuclear factor-4 (HNF-4), competed with the Hx element for binding sites. Co-transfection analyses indicated that HNF-4 does not affect CAT expression whereas ARP-1 and COUP-TF repress it. Antibody supershift analyses suggested that HNF-4 and COUP-TF may not be major factors binding to the Hx element.
Satoh
H
H
Department of Pediatrics, Cornell University Medical College, New York, New York 10021.
Nagae
Y
Y
Immenschuh
S
S
Satoh
T
T
Muller-Eberhard
U
U
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D006528:Carcinoma, Hepatocellular; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis / Q000378:metabolism; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004742:Enhancer Elements, Genetic; D015870:Gene Expression; D006466:Hemopexin / Q000096:biosynthesis / Q000235:genetics*; D006801:Humans; D007700:Kinetics; D008099:Liver / Q000378:metabolism*; D008113:Liver Neoplasms; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism*; D009841:Oligonucleotides / Q000494:pharmacology; D051381:Rats; D011987:Receptors, Steroid / Q000235:genetics; D011988:Receptors, Thyroid Hormone / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured
D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D009841:Oligonucleotides; D011987:Receptors, Steroid; D011988:Receptors, Thyroid Hormone; D011994:Recombinant Proteins; D004247:DNA; D006466:Hemopexin; D015500:Chloramphenicol O-Acetyltransferase