AIMS/HYPOTHESIS - The upstream glucokinase (betaGK) promoter has previously been shown to contain two 9-bp sequences, termed the Pal motifs, that are conserved in humans, rats and mice. These motifs are necessary for expression of the betaGK promoter in pancreatic beta cell lines and pituitary corticotrope cell lines. DNA probes containing the Pal motifs bind cell-type-specific protein complexes, but these motifs have not been completely characterised.
METHODS - Methylation interference and ultraviolet-crosslinking analysis were utilised to characterise, at the single nucleotide level, sites of protein binding within the elements themselves. To determine the function of these elements, mutational analysis of the betaGK promoter and of multimerised GK promoter sequences was performed.
RESULTS - Both Pal elements are 14 bp in length and have dyad symmetry. However, while the Pal-1 element is a perfect inverted repeat (GTCACCA-TGGTGAC), the Pal-2 element (GTCACCA-TAGAAAC) is an imperfect repeat. Ultraviolet-crosslinking analysis using nuclear extracts prepared from hamster insulinoma tumour (HIT) cells revealed that the three resolvable complexes that bind to the Pal-1 and Pal-2 elements contain different ratios of three proteins of different size (approximately 90, 110 and 150 M(r)). Mutation of a single nucleotide binding site abrogates betaGK promoter activity. Multimerised repeats of the Pal-1 element augment transcription in HIT cells, but not in baby hamster kidney (BHK) cells.
CONCLUSIONS/INTERPRETATION - These results suggest that different combinations of three proteins of different size bind to the Pal elements, probably as homodimers and heterodimers. Together, these results indicate that the betaGK promoter contains two novel 14-bp elements that, when multimerised, exhibit enhancer activity specific to neuroendocrine cells.