Upstream stimulatory factor regulates Pdx-1 gene expression in differentiated pancreatic β-cells

Abstract

The homeobox gene Pdx-1 plays a key role in the development of the pancreas. In the adult, however, expression of the Pdx-1 gene is restricted to pancreatic beta-cells and endocrine cells of duodenal epithelium. Recently, the transcription factor, upstream stimulatory factor (USF), has been shown to bind in vitro to a mutationally sensitive E-box motif within the 5'-flanking region of the Pdx-1 gene [Sharma, Leonard, Lee, Chapman, Leiter and Montminy (1996) J. Biol. Chem. 271, 2294-2299]. In the present study, we show that USF not only binds to the Pdx-1 gene promoter but also functionally regulates the expression of the Pdx-1 gene in differentiated pancreatic beta-cells. Adenovirus-mediated overexpression of a dominant negative form of USF2 decreased binding of endogenous USF to the E-box element by approximately 90%. This reduction in endogenous USF binding led to a greater than 50% decrease in Pdx-1 gene promoter activity, which, in turn, resulted in marked reductions in Pdx-1 mRNA and protein levels. Importantly, the lower Pdx-1 protein levels led to a greater than 50% reduction in Pdx-1 binding activity to the A3 element on the insulin gene promoter, and a significant reduction in insulin mRNA levels. Overall, our results show that USF functionally regulates Pdx-1 gene expression in differentiated pancreatic beta-cells and provide the first functional data for a role of USF in the regulation of a normal cellular gene.

Differential configurations involving binding of USF transcription factors and Twist1 regulateAlx3promoter activity in mesenchymal and pancreatic cells

“…Inspection of the DNA sequence of the EB1 element revealed that it fits the preferred site for binding by the bHLH transcription factors USF1 and USF2 [29]. Expression of USF1 and USF2 in RC2.E10, MIN6 and primary MEM cells was confirmed by Western blot analysis ( Figure 3A).…”

Suppression of the Pancreatic Duodenal Homeodomain Transcription Factor-1 (Pdx-1) Promoter by Sterol Regulatory Element-binding Protein-1c (SREBP-1c)

“…10A) (32,33). Considering the critical importance of the E-box, but modest stimulation of the Pdx-1 promoter by USF1 cotransfection (data not shown), it has been speculated that the action of USFs is mediated in synergy with another factor (33). Our data indicate that PDX-1 directly binds to USF1 and contributes to the transactivation of the Pdx-1 promoter.…”

“…USFs play a key role in the transcription of pdx-1 through this E-box (Fig. 10A) (32,33). Considering the critical importance of the E-box, but modest stimulation of the Pdx-1 promoter by USF1 cotransfection (data not shown), it has been speculated that the action of USFs is mediated in synergy with another factor (33).…”

“…2A). In the proximal region, USFs bind to the E-box (32,33). In the distal enhancer region, located at approximately Ϫ1.9 to Ϫ2.7 kb, several ␤ cell-specific transcription factor-binding sites have been identified.…”

“…Various transcriptional factors involved in PDX-1 expression have been reported as follows: HNF-3␤/Foxa2 (28), PDX-1 (28), HNF-1␣ (29), SP1 (29), Pax6 (30), and MafA (31) in the distal enhancer region (Ϫ1.9 to Ϫ2.7 kb), and ubiquitous transcriptional factors (USFs) in the proximal region at Ϫ104 bp (32,33). A PDX-1-positive autoregulatory loop was suggested by studying adenovirus-mediated PDX-1 expression in the mouse pancreas, which activated the endogenous Pdx-1 and led to ductal proliferation and ␤ cell neogenesis (34).…”