Biochemical Journal

Research article

Upstream stimulatory factor (USF) and neurogenic differentiation/beta-cell E box transactivator 2 (NeuroD/BETA2) contribute to islet-specific glucose-6-phosphatase catalytic-subunit-related protein (IGRP) gene expression

Cyrus C. MARTIN, Christina A. SVITEK, James K. OESER, Eva HENDERSON, Roland STEIN, Richard M. O'BRIEN


Islet-specific glucose-6-phosphatase (G6Pase) catalytic-subunit-related protein (IGRP) is a homologue of the catalytic subunit of G6Pase, the enzyme that catalyses the final step of the gluconeogenic pathway. The analysis of IGRP-chloramphenicol acetyltransferase (CAT) fusion-gene expression through transient transfection of islet-derived βTC-3 cells revealed that multiple promoter regions, located between −306 and −97, are required for maximal IGRP-CAT fusion-gene expression. These regions correlated with trans-acting factor-binding sites in the IGRP promoter that were identified in βTC-3 cells in situ using the ligation-mediated PCR (LMPCR) footprinting technique. However, the LMPCR data also revealed additional trans-acting factor-binding sites located between −97 and +1 that overlap two E-box motifs, even though this region by itself conferred minimal fusion-gene expression. The data presented here show that these E-box motifs are important for IGRP promoter activity, but that their action is only manifest in the presence of distal promoter elements. Thus mutation of either E-box motif in the context of the −306 to +3 IGRP promoter region reduces fusion-gene expression. These two E-box motifs have distinct sequences and preferentially bind NeuroD/BETA2 neurogenic differentiation/β-cell E box transactivator 2 and upstream stimulatory factor (USF) in vitro, consistent with the binding of both factors to the IGRP promoter in situ, as determined using the chromatin-immunoprecipitation (ChIP) assay. Based on experiments using mutated IGRP promoter constructs, we propose a model to explain how the ubiquitously expressed USF could contribute to islet-specific IGRP gene expression.

  • diabetes
  • pancreas
  • promoter
  • transcription


  • Abbreviations used: G6Pase, glucose-6-phosphatase; IGRP, islet-specific G6Pase catalytic-subunit-related protein; LMPCR, ligation-mediated PCR; CAT, chloramphenicol acetyltransferase; bHLH, basic helix-loop-helix; USF, upstream stimulatory factor; ChIP, chromatin immunoprecipitation; HIT, hamster insulinoma tumour; DMEM, Dulbecco's modified Eagle's medium; SV40, simian virus 40; PDX-1, pancreatic duodenal homeobox-1; NeuroD, neurogenic differentiation; BETA2, β-cell E box transactivator 2.