Abstract
The PDX-1 transcription factor plays a key role in pancreas development. Although expressed in all cells at the early stages, in the adult it is mainly restricted to the beta-cell. To characterize the regulatory elements and potential transcription factors necessary for human PDX-1 gene expression in beta-cells, we constructed a series of 5' and 3' deletion fragments of the 5'-flanking region of the gene, fused to the luciferase reporter gene. In this report, we identify by transient transfections in beta- and non-beta-cells a novel beta-cell-specific distal enhancer element located between -3.7 and -3.45 kilobases. DNase I footprinting analysis revealed two protected regions, one binding the transcription factors SP1 and SP3 and the other hepatocyte nuclear factor 3beta (HNF-3beta) and HNF-1alpha. Cotransfection experiments suggest that HNF-3beta, HNF-1alpha, and SP1 are positive regulators of the herein-described human PDX-1 enhancer element. Furthermore, mutations within each motif abolished the binding of the corresponding factor(s) and dramatically impaired the enhancer activity, therefore suggesting cooperativity between these factors.
Highlights
The mammalian pancreas develops by fusion of dorsal and ventral buds which form as evaginations of the upper duodenal part of the gut
The gene is expressed both in endocrine and exocrine cells of the developing pancreas; in the adult islet, its expression is predominantly restricted to the -cell [1,2,3], where it acts as the mediator of glucose action on insulin gene expression (6 –9)
DNase I footprinting and binding analyses revealed that both sequences bind and are transactivated by HNF-3; this is in accordance with the fact that its absence in mouse embryonic stem cells had a dramatic effect on pdx-1 gene expression [13]
Summary
The mammalian pancreas develops by fusion of dorsal and ventral buds which form as evaginations of the upper duodenal part of the gut. To characterize the regulatory elements and potential transcription factors necessary for human PDX-1 gene expression in -cells, we constructed a series of 5 and 3 deletion fragments of the 5-flanking region of the gene, fused to the luciferase reporter gene.
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