Cloning of the ovine estrogen receptor-alpha promoter and functional regulation by ovine interferon-tau.

Interferon-tau (IFNtau), the ruminant pregnancy recognition signal, inhibits transcription of the estrogen receptor alpha (ERalpha) gene in the endometrial lumenal epithelium of the sheep uterus, thereby abrogating production of luteolytic PGF(2alpha) pulses. The effects of IFNtau are mediated in part by IFN-stimulated response elements (ISREs) and IFN regulatory factor elements (IRFEs). The promoter/enhancer region of the ovine ERalpha gene was cloned, sequenced, and predicted to contain four IRFEs and one ISRE. Electrophoretic mobility shift assays indicated that the -2110 IRFE bound only IRF-1, whereas the -1877 IRFE and the -1284 ISRE were functional in binding IRF-1 and IRF-2. IFNtau inhibited transcriptional activity of the 2.7-kb ovine ERalpha promoter in transfection assays using ovine lumenal epithelium cells. Analyses of sequential 5'-deletion mutants of the ovine ERalpha promoter indicated that the effects of IFNtau may be mediated by IRFEs as well as other elements. Overexpression of ovine IRF-2, but not IRF-1, inhibited transcriptional activity of several regions of the ovine ERalpha promoter containing an IRFE or an ISRE as well as some, but not all, regions lacking these elements.

A sucrose repression element in the Phaseolus vulgaris rbcS2 gene promoter resembles elements responsible for sugar stimulation of plant and mammalian genes.

Protoplasts isolated from the primary leaves of Phaseolus vulgaris L. were used in transient expression experiments to identify promoter sequences of the P. vulgaris rbcS2 gene, encoding ribulose 1,5-bisphosphate carboxylase/oxygenase small subunit, concerned with sucrose repression. The protoplasts supported high rates of expression of the chloramphenicol acetyl transferase reporter gene fused to 1433 bp of the rbcS2 5' flanking sequences. Expression was repressed by 50 mM sucrose whereas that driven by control promoters was not. Assays of promoter deletions revealed that 203 bp 5' to the transcription start site were sufficient for high rates of sucrose-repressible expression. A -187 bp deletion supported much lower rates of expression and was not subject to sucrose repression. The -203 to -187 bp region contains sequences resembling elements involved in the sugar stimulation of transcription of other genes: the SURE (sucrose response element) of plant genes and the ChoRE (carbohydrate response element) of mammalian genes. A G-box (CACGTG) located at -200 to -205 was important for high levels of sucrose-repressible expression, since deletion of a nucleotide from this element in the context of the 1433 bp promoter gave much reduced expression. However, a modified G-box (CcCGTG) in the -203 bp fusion and adjacent vector sequences remained functional. Measurements of rbcS and chalcone synthase (CHS) transcript levels in the protoplasts indicated that 4 mM sucrose was sufficient to repress or stimulate the respective genes. Further experiments suggested that metabolism of 6-carbon sugars is the signal for rbcS repression and CHS stimulation.