Pancreatic β Cell-specific Transcription of thepdx-1 Gene

Abstract

To identify potential transactivators of pdx-1, we sequenced approximately 4.5 kilobases of the 5 promoter region of the human and chicken homologs, assuming that sequences conserved with the mouse gene would contain critical cis-regulatory elements. The sequences associated with hypersensitive site 1 (HSS1) represented the principal area of homology within which three conserved subdomains were apparent: area I (؊2694 to ؊2561 base pairs (bp)), area II (؊2139 to ؊1958 bp), and area III (؊1879 to ؊1799 bp). The identities between the mouse and chicken/human genes are very high, ranging from 78 to 89%, although only areas I and III are present within this region in chicken. Pancreatic ␤ cell-selective expression was shown to be controlled by mouse and human area I or area II, but not area III, from an analysis of pdx-1-driven reporter activity in transfected ␤-and non-␤ cells. Mutational and functional analyses of conserved hepatic nuclear factor 3 (HNF3)-like sites located within area I and area II demonstrated that activation by these regions was mediated by HNF3␤. To determine if a similar regulatory relationship might exist within the context of the endogenous gene, pdx-1 expression was measured in embryonic stem cells in which one or both alleles of HNF3␤ were inactivated. pdx-1 mRNA levels induced upon differentiation to embryoid bodies were down-regulated in homozygous null HNF3␤ cells. Together, these results suggest that the conserved sequences represented by areas I and II define the binding sites for factors such as HNF3␤, which control islet ␤ cell-selective expression of the pdx-1 gene.The PDX-1 homeodomain transcription factor is an essential regulator of pancreatic endocrine cell development and adult islet ␤ cell function. Pancreatic development in homozygous pdx-1 Ϫ/Ϫ mice is blocked at a very early stage (1-3), and endocrine cells in the rostral duodenum (3) and stomach (4) are also reduced or absent. The finding that a human born without a pancreas was homozygous for an inactivating mutation in pdx-1 (5) highlights the critical conserved role that this factor plays in pancreas formation in vertebrates.In the adult pancreas, PDX-1 is expressed at highest levels within islet ␤ cells (6). PDX-1 can specifically stimulate reporter gene expression from transfected constructs driven by promoter sequences from several genes that are normally transcribed selectively in ␤ cells, including insulin (7-11), glucokinase (12), islet amyloid polypeptide (13-16), and glucose transporter type 2 (GLUT2) 1 (17). The importance of PDX-1 in controlling the expression of genes required for the sensing and regulation of blood glucose levels suggested that an inactivating mutation within the protein would result in dysfunctional ␤ cells. Indeed, mice that are heterozygous mutant carriers of PDX-1 exhibit symptoms of glucose intolerance, suggesting a dosage effect on insulin expression and ␤ cell function (18,19). More significantly, humans with this condition are susceptible to a form of Type II (non-insulin-dependent)...

Foxa2 (HNF3β) Controls Multiple Genes Implicated in Metabolism-Secretion Coupling of Glucose-induced Insulin Release

“…However, our results contradict previous reports suggesting that Foxa2 is the upstream transactivator of Pdx1, Hnf4␣, and Hnf1␣ (1) (36). Foxa2 may indeed transactivate these genes in early embryonic development as the authors performed the experiments in embryoid bodies differentiated from embryonic stem cells (1,36).…”

“…Foxa2 may indeed transactivate these genes in early embryonic development as the authors performed the experiments in embryoid bodies differentiated from embryonic stem cells (1,36). Sund et al (16) have shown that Hnf1␣ and Hnf1␤ mRNA levels are unchanged, whereas Hnf4␣ mRNA expression is slightly elevated in the liver-specific Foxa2 knockout mice.…”

Transcription factor regulation of pancreatic organogenesis, differentiation and maturation

“…93 The Pdx1 gene has 4 highly conserved regions viz., Areas I-II-III (the proximal enhancer region) 94 and Area IV (the distal enhancer). 95 Hnf1a, 96 Foxa2, 94 Hnf6, 69 Pax6, 97 and MafA 98 have binding sites within Areas I-II-III whereas Foxa1 and Foxa2 regulate Pdx1 expression via Area IV. 79 Pdx1 regulates b-cell identity by repressing the a-cell program via a shift in transcription profiles.…”

Dissecting the transcriptional network of pancreatic islets during development and differentiation

“…Furthermore, PDX-1 expression levels are reduced in islets of HNF-1␣ Ϫ/Ϫ mice, indicating that HNF-1␣ is required for normal PDX-1 expression in pancreatic ␤ cells (22). Similarly HNF-3␤, a member of the forkhead͞winged helix family of transcription factors, has also been shown to bind to conserved regulatory elements in the mouse and human PDX-1 promoters and to activate their transcription (21,23,24). The majority of our current understanding in the transcriptional control of islet-enriched genes involves positive regulators of gene expression.…”