The estrogen receptor gene : promoter organization and expression

The estrogen receptor (ER) is a ligand-activated transcription factor and a member of a large family of nuclear hormone receptors. As a mediator of estrogen hormone action, the ER is involved in many important physiological processes. ER gene expression has been demonstrated to be restricted to certain tissues and under complex hormonal control. However, the molecular mechanisms involved have remained largely unknown.

Due to this lack of knowledge the work presented in this thesis was undertaken to characterize the promoter organization of the ER gene and investigate its expression. Approximately 3 kb of the 5' flanking region of the human ER (hER) gene were isolated and sequenced. By performing RT-PCR and RACE experiments it was shown that the hER gene is transcribed from three different promoters, the proximal A promoter and a more distal B promoter, both located within the sequenced 5' flank, and promoter C, located further upstream.

Transcription of the hER gene from these promoters yields three different mRNA isoforms with unique 5' untranslated regions (5'UTRs), but identical coding regions. DNase I hypersensitive sites were mapped in the vicinity of the A and B promoters and it was shown that the presence of hypersensitive sites correlated with transcriptional activity of these two promoters. The expression pattern of the hER mRNA isoforms was investigated by RT-PCR. Both the A- and B- mRNA isoforms were found to be expressed in breast and uterus, whereas expression of the C-transcript was predominantly detected in liver. In bone cells only expression of the B-mRNA be could detected. Taken together this indicates a tissue-specific control of hER expression by alterative promoters.

The steady-state levels of the A- and B-transcripts in normal breast and uterus were quantified and compared with the hER mRNA levels in established cancer cell lines derived from the same tissues. This demonstrated approximately equal levels of the two transcripts in normal tissues whereas the A-mRNA was the most abundant isoform in the cancer cell lines investigated. This suggests that cell transformation might be associated with an alteration of the relative expression levels of the A- and B-mRNA isoforms.

Approximately 4.5 kb of the 5' flanking region of the rat ER (rER) gene were sequenced. Sequence analysis and PCR experiments suggested that the rER gene is not transcribed from a homologue of the A promoter of the hER gene. In contrast, the promoter region of the B promoter was found to be well conserved between the species. Additionally it was found that the rER gene is transcribed from a second, more distal promoter C. Expression of the rER C-mRNA isoform was predominantly detected in liver, in similarity to the hER C-mRNA. However, the sequence of the 5'UTR of the rat C-mRNA isoform was not homologous to the human C-mRNA. It thus appears as if the promoter organization of the rat and human ER genes is only partially conserved which might indicate species-specific differences in the regulation of ER expression.

Finally, as the hER mRNA isoforms have different 5'UTRs it was investigated if the 5'UTRs might affect the translational control of hER expression. The hER mRNA leader sequences had a negative effect on translational efficiency in vitro, suggesting that hER expression is under translational control. The low translational efficiency in vitro appeared to be caused by the presence of upstream open reading frames (uORFs) and secondary structures in the 5'UTRs. The negative effects of uORFs were confirmed in transfection experiments with an uterine adenocarcinoma cell line but an effect of secondary structures was not observed. This could indicate the presence of a cellular factor(s) in these cells that relives the inhibitory effects on translation caused by secondary structures in the 5'UTRs of mRNAs.

In conclusion, this work suggests tissue-specific alternative promoter usage as a mechanism in the regulation of human and rat ER gene expression. In addition, translational control of hER expression appears to be influenced by the 5'UTRs of the hER mRNA isoforms.