The nuclear orphan receptors COUP-TFII and Ear-2 act as silencers of the human oxytocin gene promoter.
We have previously shown that COUP-TFII and Ear-2, two members of the nuclear orphan receptor family, are able to repress oestrogen-stimulated transcriptional activity of the human oxytocin (OT) gene promoter by binding to a site that overlaps with the oestrogen response element (ERE) present in the 5' flanking region of the gene. Although most nuclear receptor-mediated transcriptional repression conforms with the paradigm of passive repression and involves competitive binding to an activator site, active repression, i.e. silencing of basal promoter activity, has been observed in a limited number of cases. Here we show by co-transfection experiments using COUP-TFII and Ear-2 expression vectors and reporter constructs containing OT gene promoter fragments linked to the chloramphenicol acetyltransferase gene that both COUP-TFII and Ear-2 are capable of silencing basal OT gene promoter activity by 54 and 75% respectively. 5' Deletion and footprint analyses revealed two areas of functionally important interaction sites: (1) a direct TGACC(T/C) repeat overlapping the ERE and (2) a more promoter-proximal area centred at - 90 containing three imperfect direct repeats (R1-R3) spaced by four nucleotides each. Mutagenesis of reporter constructs as well as electrophoretic mobility-shift assays demonstrated that each of the three proximal repeats R1-R3 contributed to orphan receptor binding and the silencing effect. Inasmuch as the orphan receptor-binding sites are not involved in mediating basal transcriptional activity of the OT gene promoter, the observed effects are best interpreted as active repression or promoter silencing. Moreover, since COUP-TFII and Ear-2 are both co-expressed in OT-expressing uterine epithelial cells, the novel transcriptional effects described here are likely to be of functional importance in the fine-tuning of uterine OT gene expression in vivo.
Chu
K
K
Laboratory of Molecular Endocrinology, Royal Victoria Hospital Research Institute, McGill University, Montreal, Quebec, Canada.
Zingg
H H
HH
D000818:Animals; D051837:COUP Transcription Factor II; D051836:COUP Transcription Factors; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D051379:Mice; D009447:Neuroblastoma; D010121:Oxytocin / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic; D051381:Rats; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics; D011987:Receptors, Steroid / Q000235:genetics* / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics* / Q000502:physiology; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism; D014407:Tumor Cells, Cultured
D051837:COUP Transcription Factor II; D051836:COUP Transcription Factors; D004268:DNA-Binding Proteins; D010121:Oxytocin; D018160:Receptors, Cytoplasmic and Nuclear; D011987:Receptors, Steroid; D012097:Repressor Proteins; D014157:Transcription Factors; C075835:NR2F6 protein, human; C497587:Nr2f2 protein, mouse; C497588:Nr2f2 protein, rat; C489584:Nr2f6 protein, rat; C095569:NR2F2 protein, human