title: Estrogen regulation of chicken riboflavin carrier protein gene is mediated by ERE half sites without direct binding of estrogen receptor.
abstract: Estrogen is an important steroid hormone that mediates most of its effects on regulation of gene expression by binding to intracellular receptors. The consensus estrogen response element (ERE) is a 13bp palindromic inverted repeat with a three nucleotide spacer. However, several reports suggest that many estrogen target genes are regulated by diverse elements, such as imperfect EREs and ERE half sites (ERE 1/2), which are either the proximal or the distal half of the palindrome. To gain more insight into ERE half site-mediated gene regulation, we used a region from the estrogen-regulated chicken riboflavin carrier protein (RCP) gene promoter that contains ERE half sites. Using moxestrol, an analogue of estrogen and transient transfection of deletion and mutation containing RCP promoter/reporter constructs in chicken hepatoma (LMH2A) cells, we identified an estrogen response unit (ERU) composed of two consensus ERE 1/2 sites and one non-consensus ERE 1/2 site. Mutation of any of these sites within this ERU abolishes moxestrol response. Further, the ERU is able to confer moxestrol responsiveness to a heterologous promoter. Interestingly, RCP promoter is regulated by moxestrol in estrogen responsive human MCF-7 cells, but not in other cell lines such as NIH3T3 and HepG2 despite estrogen receptor-alpha (ER-alpha) co transfection. Electrophoretic mobility shift assays (EMSAs) with promoter regions encompassing the half sites and nuclear extracts from LMH2A cells show the presence of a moxestrol-induced complex that is abolished by a polyclonal anti-ERalpha antibody. Surprisingly, estrogen receptor cannot bind to these promoter elements in isolation. Thus, there appears to be a definite requirement for some other factor(s) in addition to estrogen receptor, for the generation of a suitable response of this promoter to estrogen. Our studies therefore suggest a novel mechanism of gene regulation by estrogen, involving ERE half sites without direct binding of ER to the cognate elements.
authors.affiliation: Department of Molecular Reproduction, Development and Genetics, Indian Institute of Science, Bangalore 560012, India.
authors.forename: Urvashi
authors.initials: U
authors.lastname: Bahadur
authors.forename: Goutham K
authors.initials: GK
authors.lastname: Ganjam
authors.forename: Nandini
authors.initials: N
authors.lastname: Vasudevan
authors.forename: Paturu
authors.initials: P
authors.lastname: Kondaiah
mesh_terms: D000818:Animals; D001665:Binding Sites; D045744:Cell Line, Tumor; D002645:Chickens; D004967:Estrogens / Q000502:physiology*; D004997:Ethinyl Estradiol / Q000031:analogs & derivatives / Q000494:pharmacology; D005786:Gene Expression Regulation; D026901:Membrane Transport Proteins / Q000235:genetics*; D011401:Promoter Regions, Genetic; D011960:Receptors, Estrogen / Q000378:metabolism*; D020218:Response Elements
chemical_list: D004967:Estrogens; D026901:Membrane Transport Proteins; D011960:Receptors, Estrogen; C019079:riboflavin-binding protein; D004997:Ethinyl Estradiol; C011310:moxestrol