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An Estrogen Receptor- Splicing Variant Mediates Both Positive and Negative Effects on Gene Transcription

Michigan State University Department of Physiology East Lansing, Michigan 48824-1101

Analysis of mRNA prepared from a variety of estrogen-responsive cell lines, breast tumor specimens, and normal breast tissue have established that estrogen receptor- (ER) mRNA is typically expressed as a mixture of transcripts. Using PCR amplification, this heterogeneity has been shown to result largely from an imprecise pattern of mRNA splicing that gives rise to a family of correctly processed and exon-skipped ER transcripts. We have reconstructed ER cDNAs representing the single exon-skipped variants ERE2 through ERE7 to enable their functional characterization in a well defined cell transfection system. All six of the ER splicing variants support the efficient expression of stable proteins in Cos7 cells, and each shows a characteristic pattern of subcellular distribution. Each of the variants displays a dramatic reduction in DNA-binding activity with a consensus estrogen response element (ERE) in an in vitro gel mobility shift assay. While this DNA-binding defect appears to be complete for ERE2, ERE3, ERE4, and ERE6, weak DNA binding is observed for ERE5 and ERE7. Scatchard analysis of hormone binding demonstrates that among the variants, only ERE3 binds 17ß-estradiol (E₂) and does so with an affinity similar to wild-type ER (wt ER). Individual variants cotransfected with the pERE-TK-CAT reporter plasmid [a consensus ERE-driven chloramphenicol acetyltransferase (CAT) reporter gene that is highly responsive to E₂-liganded wt ER] were ineffective at inducing CAT expression in ER-negative HeLa cells. Only ERE5 showed indications of positive transcriptional activity on the pERE-TK-CAT reporter, but this activity was limited to approximately 5% of the activity of wt ER. When variants were expressed simultaneously with wt ER, ERE3 and ERE5 were observed to have a dominant negative effect on wt ER transcriptional activity. Like the wild-type receptor, both ERE3 and ERE5 interact with steroid receptor coactivator-1e (SRC-1e) in vitro; however, only ERE3 retained the ability to dimerize with wt ER. Transcription from a region of the ovalbumin promoter, which contains an ERE half-site and an AP-1 motif, is positively regulated by liganded wt ER and ERE3 in phorbol ester-treated, transiently transfected HeLa cells. In both cases, this activity was enhanced by cotransfected cJun. These observations suggest that selected ER splicing variants are likely to exert important transcriptional effects, especially on genes that are regulated by nonconsensus EREs and subject to complex hormonal control.

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