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Neonatal diabetes mellitus can be transient or permanent. The severe form of permanent neonatal diabetes mellitus can be associated with pancreas agenesis. Normal pancreas development is controlled by a cascade of transcription factors, where insulin promoter factor 1 (IPF1) plays a crucial role. Here, we describe two novel mutations in the IPF1 gene leading to pancreas agenesis. Direct sequence analysis of exons 1 and 2 of the IPF1 gene revealed two point mutations within the homeobox in exon 2. Genetic analysis of the parents showed that each mutation was inherited from one parent. Mutations localized in helices 1 and 2, respectively, of the homeodomain, decreased the protein half-life significantly, leading to intracellular IPF1 levels of 36% and 27% of wild-type levels. Both mutant forms of IPF1 were normally translocated to the nucleus, and their DNA binding activity on different known target promoters was similar to that of the wild-type protein. However, transcriptional activity of both mutant IPF1 proteins, alone or in combination with HNF3 beta/Foxa2, Pbx1, or the heterodimer E47-beta 2 was reduced, findings accounted for by decreased IPF1 steady state levels and not by impaired protein-protein interactions. We conclude that the IPF1 level is critical for human pancreas formation.

The promoter element G 1 , critical for ␣-cell-specific expression of the glucagon gene, contains two AT-rich sequences important for transcriptional activity. Pax-6, a paired homeodomain protein previously shown to be required for normal ␣-cell development and to interact with the enhancer element G 3 of the glucagon gene, binds as a monomer to the distal AT-rich site of G 1 . However, although the paired domain of Pax-6 is sufficient for interaction with the G 3 element, the paired domain and the homeodomain are required for high affinity binding to G 1 . In addition to monomer formation, Pax-6 interacts with Cdx-2/3, a caudal-related ho-

p8 is a nuclear DNA-binding protein, which was identified because its expression is strongly activated in response to several stresses. Biochemical and biophysical studies revealed that despite a weak sequence homology p8 is an HMG-I/Y-like protein, suggesting that p8 may be involved in transcription regulation. Results reported here strongly support this hypothesis. Using a pull-down approach, we found that p8 interacts with the general co-activator p300. We also found that, similar to the HMG proteins, p300 was able to acetylate recombinant p8 in vitro, although the significance of such modification remains to be determined. Then a screening by the twohybrid system, using p8 as bait, allowed us to identify the Pax2 trans-activation domain-interacting protein (PTIP) as another partner of p8. Transient transfection studies revealed that PTIP is a strong inhibitor of the trans-activation activities of Pax2A and Pax2B on the glucagon gene promoter, which was chosen as a model because it is a target of the Pax2A and Pax2B transcription factors. This effect is completely abolished by co-transfection of p8 in glucagon-producing InRIG9 cells, indicating that p8 binding to PTIP prevents inhibition of the glucagon gene promoter. This was not observed in NIH3T3 fibroblasts that do not express glucagon. Finally, expression of p8 enhances the effect of p300 on Pax2A and Pax2B transactivation of the glucagon gene promoter. These observations suggest that in glucagon-producing cells p8 is a positive cofactor of the activation of the glucagon gene promoter by Pax2A and Pax2B, both by recruiting the p300 cofactor to increase the Pax2A and Pax2B activities and by binding the Pax2-interacting protein PTIP to suppress its inhibition.

The endocrine pancreas is organised as islets of Langerhans embedded in the exocrine tissue and is composed of four cell types: beta, a, d, and g cells producing insulin, glucagon, somatostatin and the pancreatic polypeptide (PP), respectively. Gene knockout studies have shown the involvement of a variety of transcription factors in pancreas development. Null mutations of the homeodomain proteins pdx-1 and isl-1 result in pancreatic agenesis and the absence of the islets of Langerhans, respectively [1,2]. The lack of Beta2, Pax-6, Pax-4, Nk 2.2, and Nk 6.1 affects the organisation of the endocrine pancreas and the number of endocrine cells or both [3±9]. The formation of specific islet cell types can be affected by the absence of any of these factors: no or few glucagon-producing a cells are observed in homozygous pax-6 mutants, whereas pax-4 knockout mice lack beta and d cells; nk 2.2 and nk 6.1 mu-The pancreatic beta-cell-specific transcription factor Pax-4 inhibits glucagon gene expression through Pax-6 Abstract Aims/hypothesis. The paired-homeobox genes pax-4 and pax-6 are crucial for islet development; whereas the null mutation of pax-6 results in the nearly absence of glucagon-producing a cells, pax-4 homozygous mutant mice lack insulin and somatostatin-producing beta and d cells but contain an increased number of a cells suggesting that a cells could develop by a default mechanism. Methods. To investigate whether beta-cell specific factors act negatively on glucagon gene transcription, we ectopically expressed pax-4 in glucagon producing InR1G9 cells; Pax-4 inhibited basal transcription of the glucagon gene promoter by 60 %. To assess the mechanism of this inhibition, we cotransfected the non-islet cell line BHK-21 with Pax-4 and various transcription factors present in a cells.Results. In addition to a general repressor activity on basal glucagon gene promoter activity of 30±50 %, a specific 90 % inhibition of Pax-6 mediated transactivation was observed. In contrast, Pax-4 had no effect on Cdx-2/3 or HNF3a mediated transcriptional activation. Pax-4 showed similar affinity to the Pax-6 binding sites on the glucagon gene promoter compared to Pax-6, but varying with KCl concentrations. Conclusion/interpretation. Pax-4 impairs glucagon gene transcription specifically through inhibition of Pax-6 mediated transactivation. Transcriptional inhibition seems to be mediated by direct DNA binding competition with Pax-6 and potentially additional mechanisms such as protein-protein interactions and a general repressor activity of Pax-4. Glucagon gene expression in a cells could thus result from both the presence of islet cell specific transcription factors and the absence of Pax-4. [Diabetologia (2002) 45: 97±107]