It is well established that estrogen-like environmental chemicals interact with the ligand-binding site of estrogen receptors (ERs) to disrupt transcriptional control of estrogen responsive targets. Here we investigate the possibility that estrogens also impact splicing decisions on estrogen responsive genes, such as that encoding ERα itself. Targeted PCR cloning was applied to identify six ERα mRNA variants in zebrafish. Sequencing revealed alternate use of transcription and translation start sites, multiple exon deletions, intron retention and alternate polyadenylation. As determined by quantitative (q)PCR, N-terminal mRNA variants predicting long (ERαA(L)) and short (ERα(S)) isoforms were differentially expressed by tissue-type, sex, stage of development and estrogen exposure. Whereas ERα(L) mRNA was diffusely distributed in liver, brain, heart, eye, and gonads, ERα(S) mRNA was preferentially expressed in liver (female>male) and ovary. Neither ERα(L) nor ERα(S) transcripts varied significantly during development, but 17β-estradiol selectively increased accumulation of ERα(S) mRNA (∼170-fold by 120 hpf), an effect mimicked by bisphenol-A and diethylstilbestrol. Significantly, a C-truncated variant (ERα(S)-Cx) lacking most of the ligand binding and AF-2 domains was transcribed exclusively from the short isoform promoter and was similar to ERα(S) in its tissue-, stage- and estrogen inducible expression. These results support the idea that promoter choice and alternative splicing of the esr1 gene of zebrafish are part of the autoregulatory mechanism by which estrogen modulates subsequent ERα expression, and further suggest that environmental estrogens could exert some of their toxic effects by altering the relative abundance of structurally and functionally distinct ERα isoforms.
Keywords: 17β-estradiol; AF; Alternative splicing; BPA; DBD; DES; DMSO; DNA binding domain; E2; EDC; ER; Endocrine disruption; Estrogen receptor alpha; LBD; RACE; TCDD; TSS; XE; Zebrafish; activation function; bisphenol A; diethylstilbestrol; dimethyl sulfoxide; endocrine disrupting chemicals; estrogen receptor; hours post fertilization; hpf; ligand binding domain; mRNA variants; qPCR; quantitative polymerase chain reaction; rapid amplification of cDNA ends; tetrachlorodibenzo-p-dioxin; transcription start site; xenoestrogens.
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