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.