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 betaTC-3 and hamster insulinoma tumor cell lines.
Glucose-6-phosphatase (G6Pase) is a multicomponent system located in the endoplasmic reticulum comprising a catalytic subunit and transporters for glucose-6-phosphate, inorganic phosphate, and glucose. We have recently cloned a novel gene that encodes an islet-specific G6Pase catalytic subunit-related protein (IGRP) (Ebert et al., Diabetes 48:543-551, 1999). To begin to investigate the molecular basis for the islet-specific expression of the IGRP gene, a series of truncated IGRP-chloramphenicol acetyltransferase (CAT) fusion genes were transiently transfected into the islet-derived mouse betaTC-3 and hamster insulinoma tumor cell lines. In both cell lines, basal fusion gene expression decreased upon progressive deletion of the IGRP promoter sequence between -306 and -66, indicating that multiple promoter regions are required for maximal IGRP-CAT expression. The ligation-mediated polymerase chain reaction footprinting technique was then used to compare trans-acting factor binding to the IGRP promoter in situ in betaTC-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 betaTC-3 cells that correlate with regions of the IGRP promoter identified as being required for basal IGRP-CAT fusion gene expression. The data suggest 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 addition, binding sites for several trans-acting factors not previously associated with islet gene expression, as well as binding sites for potentially novel proteins, were identified.
Bischof
L J
LJ
Department of Molecular Physiology and Biophysics, Vanderbilt University Medical School, Nashville, Tennessee 37232-0615, USA.
Martin
C C
CC
Svitek
C A
CA
Stadelmaier
B T
BT
Hornbuckle
L A
LA
Goldman
J K
JK
Oeser
J K
JK
Hutton
J C
JC
O'Brien
R M
RM
D000818:Animals; D019677:Artificial Gene Fusion; D001483:Base Sequence / Q000235:genetics; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D006224:Cricetinae; D004268:DNA-Binding Proteins / Q000378:metabolism; D015870:Gene Expression; D017930:Genes, Reporter; D005952:Glucose-6-Phosphatase; D051541:Hepatocyte Nuclear Factor 3-alpha; D051542:Hepatocyte Nuclear Factor 3-beta; D007340:Insulinoma / Q000235:genetics / Q000473:pathology; D007515:Islets of Langerhans / Q000166:cytology / Q000502:physiology; D051379:Mice; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism; D010446:Peptide Fragments / Q000502:physiology; D011401:Promoter Regions, Genetic / Q000235:genetics* / Q000502:physiology; D023302:Protein Footprinting; D011506:Proteins / Q000737:chemistry / Q000235:genetics*; D013237:Stereoisomerism; D014157:Transcription Factors
D004268:DNA-Binding Proteins; C496500:Foxa1 protein, mouse; C496503:Foxa2 protein, mouse; D051541:Hepatocyte Nuclear Factor 3-alpha; D009687:Nuclear Proteins; D010446:Peptide Fragments; D011506:Proteins; D014157:Transcription Factors; D051542:Hepatocyte Nuclear Factor 3-beta; D015500:Chloramphenicol O-Acetyltransferase; D005952:Glucose-6-Phosphatase; C117983:G6pc2 protein, mouse