Modulation of aromatase expression in the breast tissue by ERR alpha-1 orphan receptor

We have previously identified a silencer element (S1) that is situated betw een promoters I.3 and II of the human aromatase gene and that dean-regulate s the action of these promoters. We recently applied the yeast one-hybrid a pproach to screen a human breast tissue hybrid cDNA expression library for genes encoding the proteins binding to the silencer region. Most proteins i dentified from this approach belong to the nuclear receptor superfamily. Fi fty % of the positive clones encode for ERR alpha-1, and other positive clo nes include EAR-2, EAR-3 (COUP-TF1), RAR gamma, and p120E4F. Because ERR al pha-1 was found to be the major protein interacting with S1, we decided to examine the regulatory action of ERR alpha-1 on promoter IJ of the human ar omatase gene. Using a reporter plasmid that includes the aromatase genomic fragment containing promoter I.3 and S1, ERRa-1 was found to have a positiv e regulatory function in breast cancer SR-BR-3 cells, Gel mobility shift as says have confirmed that ERR alpha-1 binds to S1 in a dose-dependent manner , and DNase I footprinting analysis has revealed that ERR alpha-1 binds to a region, 5'-AAGGTCAGAAAT-3', which is within S1 and between 96 and 107 bp relative to the transcriptional start site of promoter I.3, In addition, de spite the fact that the nuclear receptor SF1 was shown previously to bind t o the same site and to mediate a cAMP response in ovary, our yeast one-hybr id screening did not find any SF-1 clones. Gel mobility shift assays furthe r revealed that SF-I can bind to the silencer element with an affinity comp arable with ERR alpha-1. Because our reverse transcription-PCR analysis was not able to detect SF1 mRNA in breast cancer tissue or in SK-BR-3 cells, i t is thought that SF1 protein is not expressed in breast cancer tissue. Two ERR alpha-1 RNA variants with differences at the 5'-end have been reported . Our reverse transcription-PCR analysis identified the shorter variant in 28 of 32 breast tumor specimens and the longer variant in only 1 specimen. In addition, the shorter variant was detected in breast cancer SK-BR-3 cell s as well as in a breast tumor fibroblast line WS3TF. The results suggest t hat ERR alpha-1 is one of the nuclear proteins interacting with S1 in breas t cancer tissue. It is thought that the silencer element in the human aroma tase gene may function differently in different tissues because of distinct expression patterns of transcription factors.