An estrogen receptor-alpha splicing variant mediates both positive and negative effects on gene transcription

Abstract

Analysis of mRNA prepared from a variety of estrogen-responsive cell lines, breast tumor specimens, and normal breast tissue have established that estrogen receptor-alpha (ER alpha) 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 alpha transcripts. We have reconstructed ER alpha cDNAs representing the single exon-skipped variants ERdeltaE2 through ERdeltaE7 to enable their functional characterization in a well defined cell transfection system. All six of the ER alpha 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 ERdeltaE2, ERdeltaE3, ERdeltaE4, and ERdeltaE6, weak DNA binding is observed for ERdeltaE5 and ERdeltaE7. Scatchard analysis of hormone binding demonstrates that among the variants, only ERdeltaE3 binds 17beta-estradiol (E2) and does so with an affinity similar to wild-type ER alpha (wt ER alpha). 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 alpha] were ineffective at inducing CAT expression in ER-negative HeLa cells. Only ERdeltaE5 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 alpha. When variants were expressed simultaneously with wt ER alpha, ERdeltaE3 and ERdeltaE5 were observed to have a dominant negative effect on wt ER alpha transcriptional activity. Like the wild-type receptor, both ERdeltaE3 and ERdeltaE5 interact with steroid receptor coactivator-1e (SRC-1e) in vitro; however, only ERdeltaE3 retained the ability to dimerize with wt ER alpha. 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 alpha and ERdeltaE3 in phorbol ester-treated, transiently transfected HeLa cells. In both cases, this activity was enhanced by cotransfected cJun. These observations suggest that selected ER alpha splicing variants are likely to exert important transcriptional effects, especially on genes that are regulated by nonconsensus EREs and subject to complex hormonal control.