Two orphan receptors binding to a common site areinvolved in the regulation of the oxytocin gene in the bovineovary.

Although an increasing number of nuclear orphan receptors have recently been identified, the number of known naturally occurring genes that are directly regulated by orphan receptors is still small. We have shown previously that the gene encoding the neuropeptide oxytocin (OT) is negatively regulated by the orphan receptors chicken ovalbumin upstream transcription factor I (COUP-TFI) and II. Here we show that the mouse OT gene promoter is activated by ROR , a representative of the ROR/RZR orphan receptor subfamily. Using promoter/chloramphenicol acetyltransferase reporter constructs in heterologous transfection assays, we determined that ROR action induces a <6-fold increase in promoter activity. By 5 and 3 deletion analysis, DNase footprint analysis and electrophoretic mobility shift assays, we found that ROR action is mediated by two 14 bp regions centered at 160 and 180 nucleotides upstream of the transcriptional initiation site. Both sites contain significant sequence identities with an established ROR recognition sequence. Mutations in either or both of these sites reduce significantly RORinduced activation of the OT promoter. In view of the strong transcriptional activation exerted by ROR on the OT gene promoter and the widespread distribution of different members of the ROR/RZR family, interactions between ROR/RZR isoforms and the OT gene may form part of the multifactorial regulatory mechanisms that control OT gene expression in different tissues.

Section: Discussionmentioning

confidence: 98%

Section: Discussionmentioning

confidence: 99%

Activation of the mouse oxytocin promoter by the orphan receptor RORalpha

Chu

Zingg

1999

“…7), which does not include an SF-1 binding element, suggesting that SF-1 may not be absolutely required to mediate the stimulatory effect of LH. Other genes have also been described in the same or related cell types, for example, P450 SCC in luteal cells (Liu & Simpson 1997) or oxytocin in granulosa cells (Wehrenberg et al 1994), which also depend upon SF-1 interaction, and respond to LH stimulation. These responses, however, have a quite different time kinetic and cell specificity.…”

Section: Discussionmentioning

confidence: 99%

Acute regulation of the bovine gene for the steroidogenic acute regulatory protein in ovarian theca and adrenocortical cells

Ivell¹,

Tillmann²,

Wang³

et al. 2000

Self Cite

Upregulation of the steroidogenic acute regulatory protein (StAR) is implicated in the rapid synthesis and secretion of steroidogenic cells to produce steroids in response to stimulation by trophic hormones of the gonadal and stress axes. In the present study, we have assessed the kinetics of both StAR gene transcription and protein biosynthesis in primary cell cultures of bovine adrenocortical and ovarian theca cells, under conditions of acute stimulation by corticotrophin (ACTH) and luteinizing hormone (LH), respectively. In both cell systems, detectable upregulation of StAR gene transcription occurred within 1-2 h, reaching maxima at 4 h (theca cells) or 6 h (adrenocortical cells). mRNA levels returned rapidly to baseline, by 12 h or 24 h, respectively. Specific StAR protein levels were assessed by western blotting using a novel antibody raised against a bovine StAR peptide, and showed a similar fast upregulation, albeit delayed by 1-2 h compared with the mRNA. The response of the cultured theca cells was more acute than that of the adrenocortical cells, possibly reflecting the propensity of the LH receptor to desensitize rapidly, unlike the ACTH receptor. The primary bovine theca cell cultures were also used for fully homologous transfection studies using various deletion promoter-reporter constructs of the bovine StAR gene. Kinetic analysis of the results indicated that the acute transcriptional response resides within the proximal (-315 bp) promoter region, which includes two putative responsive elements for the steroidogenic factor-1. More distal promoter regions may be involved in modulating the specificity of expression by combining enhancer and inhibitory functions.

“…In the human and rat genes, the repression of oestrogenic induction by COUP-TFs involves direct competition of binding to an oestrogen response element (ERE) centred around 160 bp upstream of the transcriptional initiation site (Burbach et al 1994, Chu et al 1997. In the bovine corpus luteum, the OT gene is induced by steroidogenic factor 1, and this induction is suppressed by COUP-TFI by competition for a common binding site (Wehrenberg et al 1994).…”

Section: Introductionmentioning

confidence: 99%

The nuclear orphan receptors COUP-TFII and Ear-2 act as silencers of the human oxytocin gene promoter

Chu

Hh²

1997

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 promoterproximal 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.