Characterization of the mouse islet-specific glucose-6-phosphatase catalytic subunit-related protein gene promoter by in situ footprinting - Correlation with fusion gene expression in the islet-derived beta TC-3 and hamster insulinoma tumor cell lines

Glucose-6-phosphatase (G6Pase) is a multicomponent system located in the en doplasmic reticulum comprising a catalytic subunit and transporters for glu cose-6-phosphate, inorganic phosphate, and glucose. We have recently cloned a novel gene that encodes an islet-specific G6Pase catalytic subunit-relat ed protein (IGRP) (Ebert et al., Diabetes 48:543-551, 1999). To begin to in vestigate the molecular basis for the islet-specific expression of the IGRP gene, a series of truncated IGRP-chloramphenicol acetyltransferase (CAT) f usion genes were transiently transfected into the islet-derived mouse beta TC-3 and hamster insulinoma tumor cell lines. In both cell lines, basal fus ion gene expression decreased upon progressive deletion of the IGRP promote r sequence between -306 and -66, indicating that multiple promoter regions are required for maximal IGRP-CAT expression. The ligation-mediated polymer ase chain reaction footprinting technique was then used to compare trans-ac ting factor binding to the IGRP promoter in situ in beta TC-3 cells, which express the endogenous IGRP gene, and adrenocortical Y1 cells, which do not . Multiple trans-acting factor binding sites were selectively identified in beta TC-3 cells that correlate with regions of the IGRP promoter identifie d as being required for basal IGRP-CAT fusion gene expression. The data sug gest that hepatocyte nuclear factor 3 may be important for basal IGRP gene expression, as it is for glucagon, GLUT2, and Pdx-1 gene expression. In add ition, binding sites for several trans-acting factors not previously associ ated with islet gene expression, as well as binding sites for potentially n ovel proteins, were identified.