Inslien gene transcription is a unique feature of the pancreatic 1 cells and Is increased In response to glucose.The recent cloning of inin promoter factor 1 (IPF1) and somatosatin ascription factor 1 (STF1) unexpectedly revealed that these are mouse and rat homologues of the same protein mediating trctivation through binding of CT boxlike elements in rat insulin 1 and somatostatin promoter/enhancer egions, respectively. By using oligonuceotides Early gene transfection analysis demonstrated the presence oftranscriptional control elements within the 5' flanking region of the rat insulin I gene (3). Although not fully conserved in sequence among rodents and man, the 5' flanking region is functionally conserved, allowing (-cellspecific transcription of the human insulin (HI) gene in transgenic mice (4-6) and in pluripotent rat islet tumors (7). The control of tissue-specific expression involves the interaction of specific DNA-binding factors with the 5' flanking region of the gene. Two types of sequence motifs, the E and the CT boxes, have been shown to be important for transcriptional regulation ofthe HI gene (8). The E box (positions -110 to -104) binds IEF1 which is composed of helix-loophelix proteins such as Panl or Pan2 [the human homologues are E47 and E12, respectively (9)] interacting with an unidentified protein of "'25 kDa (10,11). In contrast to the rat insulin I enhancer, the HI enhancer does not contain a second E box, but the corresponding sequence (-237 to -232) binds the helix-loop-helix protein USF (12). A negative regulatory element has been identified that decreases the transcriptional activity in both insulin-producing and non-insulin-producing cells (13,14).

Glucose-stimulated expression of the insulin gene in L L cells is mediated by the PDX-1 transcription factor. In this report, we show that stimulation results from effects on activation and DNA-binding potential. Thus, glucose specifically stimulated expression in MIN6 L L cells from chimeras of PDX-1 and the GAL4 DNA-binding domain which spanned the Nterminal PDX-1 activation domain located between amino acids 1 to 79. GAL4:PDX activity was induced over physiological glucose concentrations and was also regulated by effectors of this response. The level of endogenous PDX-1 binding and phosphorylation were also induced under these conditions. We discuss how changes in PDX-1 phosphorylation may influence activity in glucose-treated L L cells. z 1998 Federation of European Biochemical Societies.

The mouse homeodomain protein insulin promoter factor-1 (IPF-1) and the rat homologue somatostatin transactivating factor-1 (STF-1) are involved in early pancreatic development and have been implicated in the cell-specific regulation of insulin- and somatostatin-gene expression in mature islet beta- and delta-cells. The cell specificity of IPF-1/STF-1 expression in mature islets is, however, still unclear. Using antisera against recombinant IPF-1 and STF-1 in combination with antisera against islet hormones we find that all beta-cells in monolayers of newborn rat islet cells express STF-1, as do a fraction of the delta-cells. In adult rat and mouse pancreas we find a similar distribution. IPF-1/STF-1 expression was not detected in glucagon-producing alpha-cells. In islet cell tumour models we found that a glucagon/islet amyloid polypeptide (IAPP)-producing pluripotent rat islet cell line (NHI-6F-GLU) expresses STF-1 in all cells prior to insulin gene activation induced by in vivo culture. In contrast, a mouse alpha-cell line (alpha TC1) exclusively expressed IPF-1 in a small subset of insulin-producing cells while an insulin-negative subclone (alpha TC1.9) was negative for IPF-1. In transfection experiments using alpha TC1.9 cells STF-1 activated a rat insulin 1 reporter gene dependent not only on both STF-1-binding sites, but also on the E1-binding site for the helix-loop-helix factor IEF-1. However, the endogenous mouse insulin genes remained inactive in these cells. These results suggest that the insulin promoter acquires its very high, yet cell-specific, activity at least partly through the action of IPF-1/STF-1. This action is dependent on helix-loop-helix factors bound to the E1 element.

The helix^loop^helix transcription factor NeuroD1 (also known as Beta2) is involved in L L-cell survival during development and insulin gene transcription in adults. Here we show NeuroD1 is primarily cytoplasmic at non-stimulating glucose concentrations (i.e. 3 mM) in MIN6 L L-cells and nuclear under stimulating conditions (i.e. 20 mM). Quanti¢cation revealed that NeuroD1 was in 40^45% of the nuclei at 3 mM and 809 0% at 20 mM. Treatment with the MEK inhibitor PD98059 or substitution of a serine for an alanine at a potential mitogenactivated protein kinase phosphorylation site (S274) in NeuroD1 signi¢cantly increased the cytoplasmic level at 20 mM glucose. The rise in NeuroD1-mediated transcription in response to glucose also correlated with the change in sub-cellular localization, a response attenuated by PD98059. The data strongly suggest that glucose-stimulation of the MEK^ERK signalling pathway in£uences NeuroD1 activity at least partially through e¡ects on sub-cellular localization. ß

The homeodomain (HD) protein Nkx6