Structural and functional characterizations of the 5'-flanking region of the mouse glucagon receptor gene: comparison with the rat gene.

A putative proximal promoter was defined previously for the mouse glucagon receptor (GR) gene. In the present study, a distal promoter was characterized upstream from a novel non-coding exon revealed by the 5'-rapid amplification of cDNA ends from mouse liver tissue. The 5'-flanking region of the mouse GR gene was cloned up to 6 kb and the structural organization was compared to the 5' untranslated region of the rat gene cloned up to 7 kb. The novel exon, separated by an intron of 3.8 kb from the first coding exon, displayed a high homology (80%) with the most distal of the two untranslated exons found in the 5' region of the rat GR gene. The mouse distal promoter region, extending up to -1 kb from the novel exon, displayed 85% identity with the rat promoter. Both contain a highly GC-rich sequence with five putative binding sites for Sp1, but no consensus TATA or CAAT elements. To evaluate basal promoter activities, 5'-flanking sequences of mouse or rat GR genes were fused to a luciferase reporter gene and transiently expressed in a mouse and in a rat cell line, respectively or in rat hepatocytes. Both mouse and rat distal promoter regions directed a high level of reporter gene activity. Deletion of the Sp1 binding sites region or mutation of the second proximal Sp1 sequence markedly reduced the distal promoter activity of the reporter gene. The mouse proximal promoter activity was 2- to 3-fold less than the distal promoter, for which no functional counterpart was observed in the similar region of the rat gene.

Geiger A ，Decaux JF ，Burcelin R ，Le Cam A ，Salazar G ，Charron MJ ，Girard J ，Kervran A ... -  《BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS》

Role of the Sp family of transcription factors on glucagon receptor gene expression.

The glucagon receptor mediates the actions of glucagon on carbohydrate metabolism by the liver and on insulin release by the pancreatic beta-cell, which are key processes in the control of glucose homeostasis. The 5'-region of the mouse glucagon receptor gene has been recently cloned and two functional promoters were characterized. In the present study, we show that most of the glucagon receptor mRNA was transcribed from the distal promoter, in the mouse liver. In the distal promoter region, a GC-rich sequence with five putative binding sites for the Sp family of transcription factors was localized. To elucidate the role of these Sp1-binding sites in the mouse MIN6 beta-cell line, the expression of reporter gene constructs containing deletion or point mutation of each site was carried out. Selective mutation of the second Sp1-binding site decreased the activity of the distal promoter. Electrophoretic mobility shift assay with a DNA fragment spanning the three first Sp1 sites confirmed that the second site bound specifically MIN6 nuclear proteins, and supershift using specific Sp antibodies demonstrated that it interacted with Sp3 but not Sp1 transcription factors. These data illustrate that the basal expression of the mouse glucagon receptor gene, driven by the distal promoter, requires an Sp1-binding site that binds Sp3 proteins.

Geiger A ，Salazar G ，Kervran A 《BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS》

Genomic cloning and characterization of the rat glutathione S-transferase-A3-subunit gene.

Fotouhi-Ardakani N ，Batist G 《-》

Structure of the 5'-flanking region of the rat prostaglandin F(2alpha) receptor gene and its transcriptional control functions in hepatocytes.

Prostaglandin F(2alpha) (PGF(2alpha)), modulates hepatocyte functions via a heptahelical G(q)-coupled PGF(2alpha)-receptor (FP-R) which in liver is expressed exclusively in hepatocytes. The aim of the present study was to isolate the 5'-flanking region of the rat FP-R gene and to elucidate its basal and IL-6-modulated transcription control function in rat hepatocytes. The 5'-non-translated region of the rat hepatocyte FP-R mRNA differed from the corresponding region in rat fetal astrocyte or corpus luteum. It was encoded by exons 1a and 2 which were separated by a 1. 4 kb intron containing the exons 1b and 1c coding for the 5'-untranslated region of rat fetal astrocyte and corpus luteum FP-R mRNA, respectively. The transcription initiation site in hepatocytes was localized 263 bp upstream of the start ATG by 5'-RACE. A DNA-fragment covering the 5'-flanking region of the rFP-R gene from -1 of the transcription initiation site to -2590 bp was cloned and sequenced. Its 3'-two thirds had a 65% sequence identity to the mouse FP-R promoter however no homology to the bovine FP-R promoter. In the overlapping sequence most of the putative transcription factor binding sites were conserved between mouse and rat. The rat promoter contained no classical TATA- or CAAT-boxes but putative binding sites for the transcription factors C/EBP, GATA-1, HNF-1, HNF-3beta, SP-1, and USF. Luciferase reporter gene constructs containing portions of the 5'-flanking region were transfected into rat hepatocytes. Luciferase expression ranked -181 >/= -608 < -1418 > -1821 >/= -2590. The strongest transcriptional activity was conferred by the region between -608 and -1418 containing a cluster of potential HNF-1 and HNF-3beta binding sites that might allow the exclusive expression of FP-R mRNA in hepatocytes. The amount of FP-R mRNA and the luciferase expression under control of the -2590 promoter fragment were reduced by IL-6 in hepatocytes.

Neuschäfer-Rube F ，Möller U ，Püschel GP 《BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS》

Genomic organization and expression of the mouse equilibrative, nitrobenzylthioinosine-sensitive nucleoside transporter 1 (ENT1) gene.

We have cloned and characterized the genomic structure of the mouse gene for the NBMPR-sensitive equilibrative nucleoside transporter (mENT1), which is located on chromosome 17C. About 12-kb of genomic DNA was sequenced including the promoter region, 12 exons, 11 introns, and the 3'-untranslated region. All exon-intron junction sequences conform to the GT/AG rule. Primer extension analysis demonstrated a transcription initiation site located 252 bp upstream of the translation start site. Analysis of the 2.5-kb 5'-flanking sequence shows putative binding sites for several transcription factors, including GATA-1, IRF-2, Pit-1, myogenin, CREB, Sp-1, Ap-2, MAZ, and GR. We demonstrated that mouse ENT1 mRNA was highly expressed in heart, spleen, lung, liver, and testis. Lower levels of expression were detected in brain and kidney. Functional analysis of the 5'-flanking region showed that the nucleotide sequence from -652 to -111 contains cis-regulatory elements that promote gene expression. We found two Sp-1 binding sites (-296/-303, -307/-313) and two MAZ binding sites (-353/-359, -522/-528) in this region. Luciferase assay results suggest that MAZ and Sp-1 transcription factors are important positive regulators of transcription for the ENT1 gene in NG108-15 cells.

Choi DS ，Handa M ，Young H ，Gordon AS ，Diamond I ，Messing RO ... -  《BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS》