Abstract
Estrogen receptor-alpha (ERa) mediates diverse estrogen functions through transactivation of target gene expression and the membrane-associated signaling pathways. Recently, we identified and cloned a novel variant of ERa, ERa36, which lacks API and AF2 domains of the original ERa (ERa66). It functions mainly as a membrane-associated estrogen receptor. Thus, ERa36 is a novel and potentially important player in estrogen and antiestrogen signaling. To elucidate the molecular mechanisms underlying the regulation of this biologically important gene, we have cloned the 751bp 5’ flanking region of ERa36 from the first intron of the ERa66 gene using a genomic PCR method. Based on computer analyses of the DNA sequence, we found many putative regulatory elements for various transcription factors such as Spl, API, AhR, WT1 and V2 ERE within this region. We also found that the cloned 5’ Hanking sequence of ERa36 exhibits strong promoter activity. The transcription initiation site in this promoter was mapped by rapid amplification of cDNA 5’ end (5’ RACE). We further generated seven 5’ deletion mutants of the ERa36 promoter and analyzed their promoter activities in three different cell lines: HEK293 cells, MCF-7 cells and MDA-MB-231 cells. Transient cotransfection experiments demonstrate that ERa66 down-regulates ERa36 promoter activity in an estrogen-independent manner through binding to the ERE half site in the promoter, which can be released by ERa46 or ERa36 itself. Wilm’s tumor suppressor (WT1) inhibited the promoter activity of ERa36 by binding to the WT1 binding site in the promoter. In conclusion, we successfully cloned the ERa36 promoter and generally characterized this promoter.