Abstract
GPR40 is a G protein-coupled receptor expressed preferentially in pancreatic beta cells. It is activated by long-chain fatty acids and has been implicated in mediating physiological and pathological effects of long-chain fatty acids on beta cells. We mapped the GPR40 transcription start site to a location 1044 bp upstream of the translation start site. This permitted definition of the GPR40 core promoter and the organization of the gene, which comprises a 24-bp non-coding exon, a 698-bp intron and a 4402-bp second exon, containing the entire protein coding sequence. Sequence analysis of the GPR40 locus revealed three evolutionarily conserved regions upstream to the translation start site (HR1-HR3). DNase I-hypersensitive sites were present in the HR2 and HR3 regions in beta cells but not in non-beta cells. The 5'-flanking region of the GPR40 gene was capable of directing transcriptional activity selectively in beta cells. An important component of this is attributable to the HR2 region, which showed strong beta cell-specific enhancer activity. Systematic mutagenesis of HR2 revealed several important sub-regions. Mutagenesis of sub-regions 4-5, and 9 reduced transcriptional activity by approximately 60 and 40%, respectively. These sub-regions can bind the beta cell-specific transcription factors PDX1 and BETA2, respectively, both in vitro and in vivo. Thus, cell-specific expression of the GPR40 gene involves a characteristic chromatin organization of the locus and is controlled at the transcriptional level through HR2, a potent beta cell-specific enhancer.
Highlights
AUGUST 10, 2007 VOLUME 282 NUMBER 32 sues, combined with defective insulin secretion by pancreatic  cells [1]
These data establish the organization of the GPR40 gene, Fig. 6C was analyzed using the Wilcoxon two-sample test cor- which is comprised of two exons of 24 and 4402 bp separated by rected for multiple comparisons by the Holms method
Insulin secretion is regulated mainly according to fluctuating levels of blood glucose and in response to nutrients, neurotransmit
Summary
Total RNA was extracted from cell cultures using the TriReagent procedure (Molecular Research Center Inc.) according to the manufacturer’s instructions. The cDNA was purified, and PCR was performed using the Expand high fidelity PCR system (Roche Applied Science), with 5 l of cDNA and 30 pmol of a reverse primer complementary to GPR40 sequences nested to the primers used for reverse transcription and a forward primer: GAATTC(C). A fraction of the PCR product was resolved on a 1% agarose gel. Plasmids from the resulting bacterial clones were isolated, fractionated on 1% agarose gel, and analyzed by Southern blot, using as a probe, a radioactive end-labeled primer specific for GPR40 and nested to the primer used for PCR. A plasmid (pBS-RPA3-T7antisense) was constructed in which a genomic fragment, spanning the GPR40 transcription start site, was inserted antisense to the T7 promoter and sense to the T3 promoter. Samples were resolved on polyacrylamide urea gels and visualized by autoradiography
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