title: 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.
abstract: 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 beta TC-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 beta TC-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/beta-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.
authors.affiliation: Department of Molecular Physiology and Biophysics, 761 PRB, Vanderbilt University Medical School, Nashville, TN 37232-0615, USA.
authors.forename: Cyrus C
authors.initials: CC
authors.lastname: Martin
authors.forename: Christina A
authors.initials: CA
authors.lastname: Svitek
authors.forename: James K
authors.initials: JK
authors.lastname: Oeser
authors.forename: Eva
authors.initials: E
authors.lastname: Henderson
authors.forename: Roland
authors.initials: R
authors.lastname: Stein
authors.forename: Richard M
authors.initials: RM
authors.lastname: O'Brien
mesh_terms: D000818:Animals; D051792:Basic Helix-Loop-Helix Proteins; D020134:Catalytic Domain; D006224:Cricetinae; D005786:Gene Expression Regulation / Q000502:physiology*; D005952:Glucose-6-Phosphatase / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D007515:Islets of Langerhans / Q000201:enzymology*; D051379:Mice; D009419:Nerve Tissue Proteins / Q000378:metabolism / Q000502:physiology*
chemical_list: D051792:Basic Helix-Loop-Helix Proteins; D005952:Glucose-6-Phosphatase; D009419:Nerve Tissue Proteins; C093564:Neurogenic differentiation factor 1