IDENTIFICATION OF COUP-TF BINDING-ELEMENT IN THE HUMAN LACTOFERRIN PROMOTER

Human lactoferrin gene contains a functional, estrogen-response elemen t (ERE) that is responsible for the estrogen-stimulation of the gene i n uterine endometrium. A DNA fragment (-418 to -340), including the ER E, is selectively protected by nuclear protein of endometrial (RL95) a nd mammary gland (HBL100) cells from DNAase I digestion. This region w as divided into three footprint areas; FP1, FP2 and FP3. FP2 (-373 to -360) contains a potential GATA-3 binding element; FP3 (-355 to -340) houses the ERE. At least three nuclear proteins were involved in bindi ng to the FP1 (-418 to -378) and porduced three protein-DNA complexes in the electrophoresis mobility shift assay (EMSA). One protein-DNA co mplex was formed with the 5' end and two were formed with the 3' end o f FP1. There were three potential transcription factor binding element s located at the 3' end region (5'-ACCTTCAAGGTCATCTG-3'); a palindromi c COUP-TF binding element (5'-ACCTTCAAGGT-3'); a consensus ELF (5'-TCA AGGTCA-3') and HLH (5'-CATCTG-3') binding elements. By using the COUP- TF antibody and ov-COUP-TF binding element in EMSA, we demonstrated th at one of the protein-DNA complexes (C2) that formed with this DNA fra gment involved COUP-TF. It was also shown that COUP-TF bound to a dire ct repeat of AGGTCA at -363 to -350 (FP2 and FP3 region) of the lactof errin promoter. The GGTCA at 3' half of the direct repeat overlapped w ith ERE. Thus, the currently identified two COUP-TF binding elements w ere both overlapped by a positive transcription factor binding element . The unique organization of overlapping positive and negative transcr iption factor binding elements in human lactoferrin promoter provides an opportunity to study the complicated regulation that modulates its expression.