The insulin promoter binds a number of tissue-specific and ubiquitous transcription factors. Of these, the homoeodomain protein PDX-1 (pancreatic duodenal homeobox factor-1), the basic leucine zipper protein MafA and the basic helix-loop-helix heterodimer E47/BETA2 (beta-cell E box transactivator 2; referred to here as beta2) bind to important regulatory sites. Previous studies have shown that PDX-1 can interact synergistically with E47 and beta2 to activate the rat insulin 1 promoter. The aim of the present study was to determine the relative contribution of PDX-1, MafA and E47/beta2 in regulating the human insulin promoter, and whether these factors could interact synergistically in the context of the human promoter. Mutagenesis of the PDX-1, MafA and E47/beta2 binding sites reduced promoter activity by 60, 74 and 94% respectively, in INS-1 beta-cells. In the islet glucagonoma cell line alphaTC1.6, overexpression of PDX-1 and MafA separately increased promoter activity approx. 2.5-3-fold, and in combination approx. 6-fold, indicating that their overall effect was additive. Overexpression of E47 and beta2 had no effect. In HeLa cells, PDX-1 stimulated the basal promoter by approx. 40-fold, whereas MafA, E47 and beta2 each increased activity by less than 2-fold. There was no indication of any synergistic effects on the human insulin promoter. On the other hand, the rat insulin 1 promoter and a mutated version of the human insulin promoter, in which the relevant regulatory elements were separated by the same distances as in the rat insulin 1 promoter, did exhibit synergy. PDX-1 was shown further to activate the endogenous insulin 1 gene in alphaTC1.6 cells, whereas MafA activated the insulin 2 gene. In combination, PDX-1 and MafA activated both insulin genes. Chromatin immunoprecipitation assays confirmed that PDX-1 increased the association of acetylated histones H3 and H4 with the insulin 1 gene and MafA increased the association of acetylated histone H3 with the insulin 2 gene.

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Using MIN6 b-cells and chromatin immunoprecipitation (ChIP) assays, the chronological sequence of binding of MafA, E47/b2 and PDX-1 to the insulin promoter in living b-cells were investigated. All four factors were shown to bind to the mouse insulin 2 promoter in a cyclical manner with a periodicity of approximately 10-15 min. The cyclical binding of MafA, E47 and b2 was largely unaffected by the glucose or insulin concentration in the media. However, the binding and cycling of PDX-1 was markedly abolished in low glucose (1 mM), and this was reversed in the presence of low concentrations of insulin.

The PrP gene encodes the cellular isoform of the prion protein (PrP c ) which has been shown to be crucial to the development of transmissible spongiform encephalopathies (TSEs). PrP knock-out mice, which do not express endogenous PrP c , exhibit resistance to TSE disease. The regulation of PrP gene expression represents, therefore, a crucial factor in the development of TSEs. Two sequence motifs in the PrP promoter (positions ؊287 to ؊263 from transcriptional start) were previously reported as being highly conserved, and it was suggested that they represent binding sites for as yet unidentified transcription factors. To test this hypothesis, binding of nuclear proteins was analyzed by electrophoretic mobility shift assays using ovine or murine cells and tissues with radiolabeled DNA probes containing the conserved motif sequences. Specific binding was observed to both motifs, and polymorphic variants of these motifs exhibited differential binding. Two proteins bound to these motifs were identified as the Yin Yang 1 (YY1) (motif 1) and E4BP4 (motif 2) transcription factors. Functional promoter analysis of four different promoter variants revealed that motif 1 (YY1) was associated with inhibitory activity in the context of the PrP promoter, whereas motif 2 (E4BP4) was linked to a slight enhancing activity. This represents the first demonstration of binding of nuclear factors to two highly conserved DNA sequence motifs within mammalian PrP promoters. The action of these factors on the PrP promoter is haplotype-specific, leading us to propose that the prion protein expression pattern and, with it, the distribution of TSE infectivity may be associated with PrP promoter genotype.The ovine PrP gene (PRNP in human, prn-p in mice) encodes the prion protein (PrP C ), 4 which is a crucial component of prion diseases, also known as transmissible spongiform encephalopathies (TSEs). This group of diseases include scrapie in sheep, bovine spongiform encephalopathy in cattle, and Creutzfeldt-Jakob disease in humans (1). The hallmark of TSEs is the aggregation of a pathological isoform PrP Sc of the cellular PrP C protein (2). PrP C is highly expressed in neurons of the brain and also found in many other tissues, especially the lymphoreticular system. Prn-p knock-out mice, which do not express endogenous PrP C , exhibit resistance to TSE disease (3). Transgenic mice with differing copy numbers of the prn-p gene show a clear relationship between expression level and disease progression, characterized by changes in incubation periods (4). It is assumed that the TSE agent can only replicate in cells expressing PrP C , so that up-or down-regulation of the PrP promoter can have consequences for the distribution of the agent in the host and, therefore, the risk of transmission; for example, from consumption of specific animal parts or blood transfusion in humans (5).Beside its key role in disease, the physiological function of PrP C remains elusive with signal transduction, synaptic transmission, neuroprotection, and immunoregulation...

a b s t r a c tAn engineered zinc finger protein (eZFP) was isolated from a library based on its ability to activate expression of the endogenous insulin gene in HEK-293 cells. Using a panel of insulin promoter constructs, the eZFP was shown to act through the variable number of tandem repeat (VNTR) region located 365 base pairs upstream of the transcription start site. The eZFP also activated expression of the IGF2 gene that lies close to INS on chromosome 11p15. These results demonstrate that the INS-VNTR controls expression of the insulin and IGF2 genes and provide a mechanistic explanation for previous studies that demonstrated an association between INSVNTR genotypes and placental levels of IGF2.