Abstract
The transcriptional activation of the prostaglandin G/H synthase-2 (PGHS-2) gene in granulosa cells is required for ovulation. To directly study the ability of upstream stimulatory factor 1 (USF1) and USF2 to trans-activate the bovine PGHS-2 promoter in granulosa cells, USF1 or USF2 expression vectors were cotransfected with the PGHS-2/luciferase (LUC) chimeric construct, -149/-2PGHS-2.LUC. Results revealed that overexpression of USF1 or USF2 caused a marked and significant increase in basal and forskolin-inducible promoter activities (p<0.05), and these effects were dependent on the presence of a consensus E-box cis-element within the promoter fragment. Co-transfections with different N- and C-terminal truncated USF mutants led to significant reductions in promoter activation, as compared with full-length constructs (p<0.05), thus allowing identification of putative bovine USF functional domains. Overexpression of a USF2 truncated mutant lacking the first 220 residues (U2Delta1-220) acted as a dominant negative mutant and blocked endogenous and USF-stimulated PGHS-2 promoter activation. Interestingly, transfections with U2Delta1-220 blocked the forskolin-dependent induction of PGHS-2 mRNA in granulosa cells, whereas transfections with full-length USF2 increased PGHS-2 transcript levels. Immunoblot analyses confirmed overexpression of full-length and truncated USF proteins, and electrophoretic mobility shift assays (EMSAs) and supershift EMSAs established that the observed effects were dependent on specific interactions between USF proteins and the consensus E-box cis-element. Stimulation of cells with forskolin increased, whereas treatment of extracts with phosphatase decreased USF binding activities to the E-box. Thus, this study presents for the first time direct evidence for a role of USF proteins in the regulation of the PGHS-2 promoter in preovulatory granulosa cells.
Highlights
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AY241931 and AY239291
upstream stimulatory factor (USF)- and E-box-dependent Transactivation of the Bovine prostaglandin G/H synthase-2 (PGHS-2) Promoter in Granulosa Cells—To provide direct evidence that USF proteins transactivate the bovine PGHS-2 promoter in granulosa cells, upstream stimulatory factor 1 (USF1) or USF2 expression vectors were co-transfected with the Ϫ149/Ϫ2PGHS-2.LUC chimeric construct into primary cultures of granulosa cells
Results from the present study revealed that transfections of the PGHS-2 promoter construct generated basal activity that was significantly increased by forskolin
Summary
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AY241931 and AY239291. The cognate DNA binding cis-element for USF proteins, the E-box, contains the core nucleotide sequence CANNTG [18, 24]. Another very well conserved domain between USF1 and USF2 is a small domain. Initial studies on the regulation of the rat and bovine PGHS-2 promoter in granulosa cells revealed that the proximal 150 –200 bp immediately upstream of the transcriptional start site were sufficient to confer basal and forskolin/ gonadotropin inducible activities [37, 38]. The specific objectives of the present study were to clone and characterize the primary structure of bovine USF1 and USF2, and to provide direct evidence that USF proteins regulate PGHS-2 expression in preovulatory granulosa cells
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