Cloning and characterization of the human and rat islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) genes.

Islet-specific glucose-6-phosphatase (G6Pase) catalytic subunit-related protein (IGRP) is a homolog of the catalytic subunit of G6Pase, the enzyme that catalyzes the terminal step of the gluconeogenic pathway. Its catalytic activity, however, has not been defined. Since IGRP gene expression is restricted to islets, this suggests a possible role in the regulation of islet metabolism and, hence, insulin secretion induced by metabolites. We report here a comparative analysis of the human, mouse, and rat IGRP genes. These studies aimed to identify conserved sequences that may be critical for IGRP function and that specify its restricted tissue distribution. The single copy human IGRP gene has five exons of similar length and coding sequence to the mouse IGRP gene and is located on human chromosome 2q28-32 adjacent to the myosin heavy chain 1B gene. In contrast, the rat IGRP gene does not appear to encode a protein as a result of a series of deletions and insertions in the coding sequence. Moreover, rat IGRP mRNA, unlike mouse and human IGRP mRNA, is not expressed in islets or islet-derived cell lines, an observation that was traced by fusion gene analysis to a mutation of the TATA box motif in the mouse/human IGRP promoters to TGTA in the rat sequence. The results provide a framework for the further analysis of the molecular basis for the tissue-restricted expression of the IGRP gene and the identification of key amino acid sequences that determine its biological activity.

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.

Structure and promoter activity of an islet-specific glucose-6-phosphatase catalytic subunit-related gene.

In liver and kidney, the terminal step in the gluconeogenic pathway is catalyzed by glucose-6-phosphatase (G-6-Pase). This enzyme is actually a multicomponent system, the catalytic subunit of which was recently cloned. Numerous reports have also described the presence of G-6-Pase activity in islets, although the role of G-6-Pase in this tissue is unclear. Arden and associates have described the cloning of a novel cDNA that encodes an islet-specific G-6-Pase catalytic subunit-related protein (IGRP) (Arden SD, Zahn T, Steegers S, Webb S, Bergman B, O'Brien RM, Hutton JC: Molecular cloning of a pancreatic islet-specific glucose-6-phosphatase catalytic subunit related protein (IGRP). Diabetes 48:531-542, 1999). We screened a mouse BAC library with this cDNA to isolate the IGRP gene, which spans approximately 8 kbp of genomic DNA. The exon/intron structure of the IGRP gene has been mapped and, as with the gene encoding the liver/kidney G-6-Pase catalytic subunit, it is composed of five exons. The sizes of these exons are 254 (I), 110 (II), 112 (III), 116 (IV), and 1284 (V) bp, similar to those of the G-6-Pase catalytic subunit gene. Two interspecific backcross DNA mapping panels were used to unambiguously localize the IGRP gene (map symbol G6pc-rs) to the proximal portion of mouse chromosome 2. The IGRP gene transcription start site was mapped by primer extension analysis, and the activity of the IGRP gene promoter was analyzed in both the islet-derived HIT cell line and the liver-derived HepG2 cell line. The IGRP and G-6-Pase catalytic subunit gene promoters show a reciprocal pattern of activity, with the IGRP promoter being approximately 150-fold more active than the G-6-Pase promoter in HIT cells.

An endocrine-exocrine switch in the activity of the pancreatic homeodomain protein PDX1 through formation of a trimeric complex with PBX1b and MRG1 (MEIS2).

HOX proteins and some orphan homeodomain proteins form complexes with either PBX or MEIS subclasses of homeodomain proteins. This interaction can increase the binding specificity and transcriptional effectiveness of the HOX partner. Here we show that specific members of both PBX and MEIS subclasses form a multimeric complex with the pancreatic homeodomain protein PDX1 and switch the nature of its transcriptional activity. The two activities of PDX1 are exhibited through the 10-bp B element of the transcriptional enhancer of the pancreatic elastase I gene (ELA1). In pancreatic acinar cells the activity of the B element requires other elements of the ELA1 enhancer; in beta-cells the B element can activate a promoter in the absence of other enhancer elements. In acinar cell lines the activity is mediated by a complex comprising PDX1, PBX1b, and MRG1 (MEIS2). In contrast, beta-cell lines are devoid of PBX1b and MRG1, so that a trimeric complex does not form, and the beta-cell-type activity is mediated by PDX1 without PBX1b and MRG1. The presence of specific nuclear isoforms of PBX and MEIS is precisely regulated in a cell-type-specific manner. The beta-cell-type activity can be detected in acinar cells if the B element is altered to retain binding of PDX1 but prevent binding of the PDX1-PBX1b-MRG1 complex. These observations suggest that association with PBX and MEIS partners controls the nature of the transcriptional activity of the organ-specific PDX1 transcription factor in exocrine versus endocrine cells.

Members of the meis1 and pbx homeodomain protein families cooperatively bind a cAMP-responsive sequence (CRS1) from bovine CYP17.

The mammalian Pbx homeodomain proteins provide specificity and increased DNA binding affinity to other homeodomain proteins. A cAMP-responsive sequence (CRS1) from bovine CYP17 has previously been shown to be a binding site for Pbx1. A member of a second mammalian homeodomain family, Meis1, is now also demonstrated to be a CRS1-binding protein upon purification using CRS1 affinity chromatography. CRS1 binding complexes from Y1 adrenal cell nuclear extract contain both Pbx1 and Meis1. This is the first transcriptional regulatory element reported as a binding site for members of the Meis1 homeodomain family. Pbx1 and Meis1 bind cooperatively to CRS1, whereas neither protein can bind this element alone. Mutagenesis of the CRS1 element indicates a binding site for Meis1 adjacent to the Pbx site. All previously identified Pbx binding partners have Pbx interacting motifs that contain a tryptophan residue amino-terminal to the homeodomain that is required for cooperative binding to DNA with Pbx. Members of the Meis1 family contain one tryptophan residue amino-terminal to the homeodomain, but site-directed mutagenesis indicates that this residue is not required for cooperative CRS1 binding with Pbx. Thus, the Pbx-Meis1 interaction is unique among Pbx complexes. Meis1 also cooperatively binds CRS1 with the Pbx homologs extradenticle from Drosophila melanogaster and ceh-20 from Caenorhabditis elegans, indicating that this interaction is evolutionarily conserved. Thus, CYP17 CRS1 is a transcriptional regulatory element containing both Pbx and Meis1 binding sites, which permit these two homeodomain proteins to bind and potentially regulate cAMP-dependent transcription through this sequence.

The bovine CYP17 promoter contains a transcriptional regulatory element cooperatively bound by tale homeodomain proteins.

Bovine CYP17 is regulated at the transcriptional level by ACTH acting through the second messenger cAMP in adrenal fasciculata and reticularis cells. Promoter analysis has previously identified two regions, proximal and distal, within the CYP17 promoter important in the cAMP dependent transcriptional regulation of this gene. The proximal (-80 to -40) cAMP responsive sequence (CRS2) has been identified as a binding site for Steroidogenic Factor-1 (SF-1)/Ad4BP. The distal region (-243 to -100) is also important for the cAMP transcriptional response as revealed by deletion analysis. Within this distal region from -243 to -225, an independent cAMP responsive sequence referred to as CRS1 has been described. The transcription factors binding CRS1 have been identified as homeodomain transcription factors belonging to an atypical class of homeodomain proteins referred to as TALE. Two families of homeodomain proteins which bind CRS1 are the Pbx and Meis1 families. Proteins from neither of these families can bind CRS1 individually; however, members of the Pbx family interact with members of the Meis1 family to cooperatively bind this element. CRS1 was the first identified cis-acting target element for members of both the Pbx and Meis1 family. Unlike SF-1, these proteins are not expressed in a steroidogenic tissue-specific manner but rather, appear ubiquitous. A current model for the function of these proteins in CYP17 regulation is that they may enhance the cAMP response through the downstream SF-1 binding site.

Cytochromes P450 12: diversity of ACTH (cAMP)-dependent transcription of bovine steroid hydroxylase genes.

An essential role of ACTH is to assure that optimal steroidogenic capacity is maintained in the adrenal cortex throughout life. This is achieved by maintaining transcriptional pressure on the genes encoding the adrenocortical steroid hydroxylases via the second messenger, cAMP. Even though these genes respond coordinately to cAMP, it has been surprising to discover that each gene uses its own unique cAMP response system during this coordinate response. Thus, different cis elements and sets of transcription factors control the cAMP responsiveness of each different steroid hydroxylase gene. Although the physiological basis of this diversity in biochemical mechanisms of transcriptional regulation is not apparent, a portion of this signaling pathway is common to all of these genes. In particular, the action of cAMP-dependent protein kinase and an as yet uncharacterized cycloheximide-sensitive step are necessary for ACTH-mediated transcription of each gene. Biochemical characterization of these common steps in the ACTH-dependent signaling pathways is essential to an understanding of the maintenance of optimal steroidogenic capacity in the adrenal cortex.

Mechanisms of ACTH(cAMP)-dependent transcription of adrenal steroid hydroxylases.

The action of peptide hormones from the anterior pituitary regulates transcription of a large number of genes located in most, if not all, tissues. This action is mediated through regulation of steroid hormone production in the steroidogenic factories (adrenals, gonads). These steroid hormones are transported through the circulation to the peripheral tissues where they serve as ligands for the family of zinc-finger nuclear receptor transcription factors. The mechanisms by which peptide hormones regulate steroid hormone production include a chronic response mediated by elevated levels of cAMP resulting from the binding of peptide hormones to their cell surface receptors which enhances transcription of the genes encoding steroid hydroxylases required for steroid hormone biosynthesis. The action of ACTH in the adrenal cortex has been studied in greatest detail leading to identification of unique cAMP-response sequences (CRS) in the different bovine steroid hydroxylase genes. Most likely FSH and LH mediate steroid hydroxylase gene expression in the gonads via the same response elements. Unlike developmental/tissue-specific transcription of these genes which is regulated by a common transcription factor (SF-1), cAMP-dependent transcription of each steroid hydroxylase gene requires a different transcription factor.