Structural and functional analysis of the 5'-flanking region of the rat ceruloplasmin gene.
To characterize the mechanisms determining tissue-specific ceruloplasmin gene expression during development, the rat ceruloplasmin gene was isolated in a series of overlapping phage clones. The 5'-flanking region was characterized and the transcription initiation site was identified by primer extension and RNase protection. Nucleotide sequence analysis of this region revealed a typical eukaryotic promotor structure, but no obvious homology with cis-acting elements previously characterized as determining tissue-specific gene expression. Transient expression of chimeric ceruloplasmin-reporter gene constructs containing up to 5200 base pairs (bp) of the 5'-flanking region revealed that sequences 732 bp upstream of the start nucleotide were sufficient to confer hepatocyte-specific expression. The region from -393 to -348 was determined by deletion analysis to contain a positive-acting element, and includes sequence partially homologous to the rat albumin D site. Mobility shift analysis revealed that this region specifically binds a heat-labile nuclear protein from rat liver and from newborn but not adult rat lung. Binding to this region was competed by oligonucleotides corresponding to the albumin D site, but not by oligonucleotides corresponding to binding sites for the hepatocyte transcription factors HNF-1, HNF-3, HNF-4, and C/EBP. These data indicate that ceruloplasmin gene expression is determined in part by a cis-acting region 393 bp upstream of the transcription start site, which binds a previously uncharacterized nuclear protein. The tissue distribution of this nuclear protein suggests that it plays a role in directing ceruloplasmin gene expression in lung and liver during development.
Fleming
R E
RE
Edward Mallinckrodt Department of Pediatrics, Washington University School of Medicine, St. Louis, Missouri 63110.
Gitlin
J D
JD
D000418:Albumins / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002570:Ceruloplasmin / Q000235:genetics*; D003001:Cloning, Molecular; D005786:Gene Expression Regulation; D008099:Liver / Q000378:metabolism; D008168:Lung / Q000378:metabolism; D008969:Molecular Sequence Data; D051381:Rats; D011919:Rats, Inbred Strains; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid; D014158:Transcription, Genetic
D000418:Albumins; D002570:Ceruloplasmin