Functional analysis of estrogen receptor-alpha mRNA splicing variants

Analysis of mRNA prepared from a variety of estrogen responsive cells and tissues has established that ERα mRNA is expressed as a mixture of transcripts. This heterogeneity results largely from a pattern of alternative mRNA splicing that gives rise to a family of correctly processed and exon-skipped ERα mRNAs. Although variant forms of ERα derived by alternative splicing have been identified in all ERα expressing tissues and cell lines examined and their expression levels are well documented in published reports, little is understood regarding their function. We have reconstructed ERα cDNAs representing the single exon-skipped variants ERΔE2 through ERΔE7 in order to analyze their activities in a well defined cell transfection system.; The aims of this thesis are two-fold. First, it addresses the hypothesis that certain ERα mRNA splicing variants retain many aspects of wt ERα biochemical function; and secondly it addresses the hypothesis that ERα splicing variants alter gene expression.; Biochemical analysis of ERα mRNA splicing variants has identified two exon-skipped variants that share significant residual function compared to their full-length counterpart, wt ERα. The variants with sequence deletions corresponding to exon 3 (ERΔE3) or exon 5 (ERΔE5) are observed to display normal nuclear uptake and retain the ability to interact with transcriptional co-regulators. Furthermore, the variant ERΔE3, like wt ERα, binds ligand. These properties confer on ERΔE3 and ERΔE5 the ability to inhibit the activity of some ERα-responsive promoters through an interplay with wt ERα signaling, and potentially to stimulate the transcription of other genes. Results from this study suggest that potential targets for transcriptional activation by these ERα splicing variants include the extracellular matrix protease, human collagenase gene, and the human IGF-1 gene. The promoters of these genes lack consensus DNA-binding sites for ERα and appear to be regulated indirectly by ERα isoforms through interactions with other transcription factors such as AP-1. That these genes are implicated in tumorigenesis suggests that ERα splicing variants may have an influence on cell growth and cancer progression.; The initial discovery of ERα mRNA splicing variants challenged any previous understanding of ER function in gene transcription and cell signal transduction. That understanding is further complicated by the present study which contributes to evidence suggesting that ERα splicing variants may have a significant impact on the ability of the breast and other tissues to respond to estrogens and other regulatory signals.