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Hepatocyte nuclear factor 3beta is involved in pancreatic beta-cell-specific transcription of the pdx-1 gene
K L Wu1, M Gannon, M Peshavaria
1Department of Molecular Physiology and Biophysics, Vanderbilt Medical Center, Nashville, Tennessee 37232, USA.
Insights
Researchers identified key DNA sequences controlling pdx-1 gene expression in pancreatic islet beta cells. Hepatocyte nuclear factor 3beta (HNF3beta) binding to a specific site is crucial for this cell-type-specific transcription.
Area of Science:
- Developmental biology
- Genetics
- Endocrinology
Background:
- The pdx-1 homeobox gene is vital for pancreas and duodenum development.
- In adults, pdx-1 is primarily found in pancreatic islet beta cells and duodenal epithelium.
Purpose of the Study:
- To pinpoint the DNA sequences responsible for regulating pdx-1 expression specifically in pancreatic islets.
- To elucidate the molecular mechanisms underlying beta-cell-specific pdx-1 transcription.
Main Methods:
- Transgenic mouse models with beta-galactosidase reporter constructs.
- Analysis of nuclease-hypersensitive sites in the pdx-1 gene's 5'-flanking region.
- In vitro transfection assays using beta-cell and non-beta-cell lines.
- Gel shift analysis and immunohistochemistry to study transcription factor binding.
Main Results:
- A genomic fragment (-4.5 to +8.2 kb) of the pdx-1 gene contains regulatory elements for correct expression.
- Nuclease-hypersensitive sites 1, 2, and 3 were identified in the 5'-flanking region.
- Site 1 sequences are critical for pancreatic beta-cell-specific expression, confirmed in vivo and in vitro.
- A specific HNF3beta binding site within Site 1 is essential for pdx-1 transcription, with HNF3beta present in islet beta cells.
Conclusions:
- The pdx-1 gene's cell-type-specific expression in pancreatic islets is mediated by regulatory sequences within its 5'-flanking region.
- Hepatocyte nuclear factor 3beta (HNF3beta) plays a crucial role in regulating pdx-1 transcription in islet beta cells.
- HNF3beta's interaction with a specific DNA element is essential for pdx-1 gene activation in the pancreas.
Abstract:
The mammalian homeobox gene pdx-1 is expressed in pluripotent precursor cells in the dorsal and ventral pancreatic bud and duodenal endoderm, which will produce the pancreas and the rostral duodenum. In the adult, pdr-1 is expressed principally within insulin-secreting pancreatic islet beta cells and cells of the duodenal epithelium. Our objective in this study was to localize sequences within the mouse pdx-1 gene mediating selective expression within the islet. Studies of transgenic mice in which a genomic fragment of the mouse pdx-1 gene from kb -4.5 to +8.2 was used to drive a beta-galactosidase reporter showed that the control sequences sufficient for appropriate developmental and adult specific expression were contained within this region. Three nuclease-hypersensitive sites, located between bp -2560 and -1880 (site 1), bp -1330 and -800 (site 2), and bp -260 and +180 (site 3), were identified within the 5'-flanking region of the endogenous pdx-1 gene. Pancreatic beta-cell-specific expression was shown to be controlled by sequences within site 1 from an analysis of the expression pattern of various pdr-1-herpes simplex virus thymidine kinase promoter expression constructs in transfected beta-cell and non-beta-cell lines. Furthermore, we also established that this region was important in vivo by demonstrating that expression from a site 1-driven beta-galactosidase reporter construct was directed to islet beta-cells in transgenic mice. The activity of the site 1-driven constructs was reduced substantially in beta-cell lines by mutating a hepatocyte nuclear factor 3 (HNF3)-like site located between nucleotides -2007 and -1996. Gel shift analysis indicated that HNF3beta present in islet beta cells binds to this element. Immunohistochemical studies revealed that HNF3beta was present within the nuclei of almost all islet beta cells and subsets of pancreatic acinar cells. Together, these results suggest that HNF3beta, a key regulator of endodermal cell lineage development, plays an essential role in the cell-type-specific transcription of the pdx-1 gene in the pancreas.