Estrogen Regulation of Human Lactoferrin Gene Activity

Summary

Human lactoferrin gene is differentially regulated in different tissues. The 5′-flanking region of the gene contains multiple positive and negative regulatory elements that can contribute to the cell-type-specific expression of the gene. A functional estrogen response element (ERE) is located at nucleotide -354 from the initiation start site. Upstream from the ERE, a DNA sequence (-418 to -378, FP1) is selectively protected from DNase I digestion by nuclear extracts of endometrium (RL95–2) and mammary gland (HB100) cells. In the electrophoresis mobility shift assay (EMSA), nuclear proteins of the RL95–2 cells and FP1 formed three protein-DNA complexes (C1, C2, and C3). We have delineated the positions of these proteins by EMSA, site-directed mutagenesis, and DNA methylation interference analyses. Mutation at the C1-binding region, TCAAGGTCATC, abolished the formation of C1 complex and reduced the estrogen responsiveness of the reporter plasmids, which contain the intact lactoferrin ERE in RL95–2 cell. We cloned and sequenced the cDNA encoding the C1-binding protein. The nucleotide sequence of the cDNA shared 99% homology with the hERR1. As a result of several deletions in our clone, the predicated amino acid sequence was 97.5% homology between our clone and the hERR1. From these data, we have cloned a variant form of this receptor. We named its binding element estrogen receptor-related orphan receptor response element (ERRE). To examine the mechanism of hERR1 and ERRE in enhancing the estrogen response, we performed DNase I footprinting, EMSA, and far-Western analyses. The results demonstrated that the presence of hERR1 and ERRE stabilized ER binding to the ERE of human lactoferrin gene. A direct protein-protein contact between ER and hERR1 was shown by far-Western analysis. These observations suggest that hERR1 and ER acted synergistically in estrogen-stimulated lactoferrin gene activity.