[Placental function test].

We have previously identified a silencer element (S1) that is situated between promoters I.3 and II of the human aromatase gene and that down-regulates the action of these promoters. We recently applied the yeast one-hybrid approach to screen a human breast tissue hybrid cDNA expression library for genes encoding the proteins binding to the silencer region. Most proteins identified from this approach belong to the nuclear receptor superfamily. Fifty % of the positive clones encode for ERR alpha-1, and other positive clones include EAR-2, EAR-3 (COUP-TF1), RAR gamma, and p120E4F. Because ERR alpha-1 was found to be the major protein interacting with S1, we decided to examine the regulatory action of ERR alpha-1 on promoter I.3 of the human aromatase gene. Using a reporter plasmid that includes the aromatase genomic fragment containing promoter I.3 and S1, ERR alpha-1 was found to have a positive regulatory function in breast cancer SK-BR-3 cells. Gel mobility shift assays 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, despite the fact that the nuclear receptor SF1 was shown previously to bind to the same site and to mediate a cAMP response in ovary, our yeast one-hybrid screening did not find any SF-1 clones. Gel mobility shift assays further revealed that SF-1 can bind to the silencer element with an affinity comparable 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, it 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 cells as well as in a breast tumor fibroblast line WS3TF. The results suggest that ERR alpha-1 is one of the nuclear proteins interacting with S1 in breast cancer tissue. It is thought that the silencer element in the human aromatase gene may function differently in different tissues because of distinct expression patterns of transcription factors.

Aromatase↗

PubMed1998

Characterization of a silencer element in the human aromatase gene.

Aromatase, a cytochrome P450, catalyzes three consecutive hydroxylation reactions converting C19 androgens to aromatic C18 estrogens. The control of human aromatase expression is complex in that several promoters drive aromatase expression in a tissue-specific manner. Promoters I.3 and II are situated within the 700-bp region immediately upstream of exon II of the human aromatase gene, and these promoters have been shown to drive aromatase expression in breast tumors. This paper reports the characterization of a negative regulatory element that is situated between promoters I.3 and II and down regulates the action of these promoters. This negative regulatory element is thought to be a silencer element (S1) because it acts in an orientation- and promoter-independent manner. The position of S1 (5'-CCAAGGTCAGAAATGCTGCAATTCAAGCCA-3') was mapped by DNase I footprinting and DNA deletion analyses. The region contains three pairs of inverted repeats, as indicated by an underline, probably explaining why S1 functions in both orientations. Chloramphenicol acetyltransferase functional analyses indicated that the transcriptional activity of promoter I.3 was suppressed two- to three-fold by S1. Mutations of two inverted repeat segments (i.e., TTC and CC) in S1 destroyed its silencing action and modified the protein binding patterns in DNA mobility shift analysis. UV cross-linking experiments with 32P-labeled bromodeoxyuridine-substituted S1 as the probe and nuclear extracts from MCF-7 breast cancer cells and skin fibroblasts revealed that four major nuclear proteins, with molecular masses of approximately 150, 45, 30, and 25 kDa, bound to this element. Interestingly, two smaller proteins could be competed by an unlabeled fragment which contains promoter I.3. In addition, mutation of S1 at the region CC destroyed the ability to compete with the wild-type S1 for the binding of 30- and 25-kDa proteins. These results led us to propose that S1 down regulates the action of promoter I.3, and the 30- and 25-kDa proteins present in the nuclear extract of MCF-7 cells and skin fibroblasts are involved in the silencer action.

Aromatase↗