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Molecular Endocrinology, Vol 6, 1969-1981, Copyright © 1992 by Endocrine Society
ARTICLES |
CT Teng, Y Liu, N Yang, D Walmer and T Panella
Laboratory of Reproductive and Developmental Toxicology, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709.
The 5'-flanking region of the human lactoferrin gene was isolated from a human placental genomic library. This genomic clone contains a 16- kilobase pair (kbp) insert and produces seven fragments when digested with the SacI restriction enzyme. We sequenced one of the fragments that comprises 1294 bp of the 5'-flanking sequence, 79 bp of the first exon, and 690 bp of the first intron. A major transcription start site was mapped by primer extension. The region immediately upstream from the transcription initiation site following the first exon is abundant in G and C nucleotides. In the promoter and 5'-flanking region within a 300-bp stretch (-465 to -165) of the DNA, we found a noncanonical TATA box (ATAAA), CAAT-like sequence (CAAC) and sequences homologous to the consensus SP1 binding site, Pu.1/Sp.1 binding element (PU box), two half-palindromic estrogen response elements (EREs; GGTCA), an imperfect ERE (GGTCAAGGCGATC), and a sequence resembling the chicken ovalbumin upstream promoter transcription factor (COUP-TF) binding site (GTCTCACAGGTCA). The COUP-TF binding site and the imperfect ERE shared five nucleotides (GGTCA). With the exception of the two half- palindromic EREs, the elements with very well matched sequences were also found in the corresponding positions in the mouse lactoferrin gene. The synthetic oligonucleotide, including the 26 bp of COUP/ERE sequence, was cloned before the SV40 promoter in a chloramphenicol acetyltransferase reporter construct. These chimeric plasmids were transiently transfected into human endometrium carcinoma RL95-2 cells to assess hormone responsiveness. We found that the COUP/ERE element acted as an enhancer in response to estrogen stimulation. In vitro DNase I footprinting analysis showed binding of the estrogen receptor on the imperfect ERE. In contrast to the mouse lactoferrin COUP/ERE element, COUP-TF does not interact with this element, as demonstrated by band shift assay and site-directed mutagenesis. Therefore, the molecular mechanisms of the estrogen action that govern the lactoferrin gene expression differ between mouse and human.
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