Differential molecular mechanism of the estrogen action that regulates lactoferrin gene in human and mouse

Mol Endocrinol. 1992 Nov;6(11):1969-81. doi: 10.1210/mend.6.11.1480183.

Abstract

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.

Publication types

  • Comparative Study
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Base Sequence
  • Consensus Sequence
  • DNA / metabolism
  • Endometrium / drug effects
  • Endometrium / metabolism
  • Endometrium / pathology
  • Enhancer Elements, Genetic*
  • Estrogens / pharmacology*
  • Estrus
  • Female
  • Gene Expression Regulation*
  • Humans
  • Hyperplasia
  • Lactoferrin / genetics*
  • Mice / genetics*
  • Mice / metabolism
  • Molecular Sequence Data
  • Promoter Regions, Genetic*
  • Receptors, Estrogen / metabolism
  • Sequence Alignment
  • Sequence Homology
  • Species Specificity

Substances

  • Estrogens
  • Receptors, Estrogen
  • DNA
  • Lactoferrin