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Originally published In Press as doi:10.1074/jbc.M702115200 on May 24, 2007

J. Biol. Chem., Vol. 282, Issue 32, 23561-23571, August 10, 2007
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Regulation of the Gene Encoding GPR40, a Fatty Acid Receptor Expressed Selectively in Pancreatic beta Cells*Formula

Reut Bartoov-Shifman, Gabriela Ridner, Keren Bahar, Nir Rubins1, and Michael D. Walker2

From the Department of Biological Chemistry, Weizmann Institute of Science, P. O. Box 26, Rehovot 76100, Israel

Received for publication, March 12, 2007 , and in revised form, May 21, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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 ~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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Type 2 diabetes mellitus (T2DM)3 is a serious metabolic disease characterized by impaired insulin action on peripheral tissues, combined with defective insulin secretion by pancreatic beta cells (1). The incidence of T2DM worldwide is increasing dramatically, driven by a sedentary lifestyle and the associated obesity in the population (2). Obesity and T2DM are associated with elevated levels of lipids such as LCFAs in the serum (3, 4). LCFAs have complex, divergent effects on beta cells: whereas acute exposure leads to enhanced glucose-stimulated insulin secretion, prolonged exposure results in impaired beta cell function ("lipotoxicity") (5, 6). It has been proposed that this impairment may contribute to the defective beta cell function associated with T2DM (7). Yet the precise mechanisms linking obesity and T2DM remain incompletely understood.

The mouse GPR40 gene is a member of a small gene family encoding G protein-coupled receptors (8). These receptors are activated by fatty acids: GPR40 responds to medium- and long-chain fatty acids (9-11), whereas the other two members of the family GPR41 and GPR43 respond to short-chain fatty acids (12, 13). The gene family is clustered in a region of ~40 kb on mouse chromosome 7, downstream to the gene encoding CD22, a receptor involved in modulation of B lymphocyte function (14).

GPR40 is expressed in highly selective manner. In rodents, GPR40 mRNA is present at high levels in pancreatic islets and beta cell lines, but at very low levels in all other tissues examined (9, 15). In humans, it has been reported that GPR40 is expressed not only in pancreas, but also in brain and monocytes (11). A recent report suggests that GPR40 protein is expressed in mouse pancreatic {alpha} cells (16). Several studies have examined the role of GPR40 in beta cells. In cultured beta cells, down-regulation of GPR40 inhibits LCFA-dependent increases in intracellular [Ca2+] and insulin secretion (9, 17-19). Consistent with this, islets isolated from mice lacking GPR40 show impaired insulin secretion in response to short term exposure to LCFAs (15). Interestingly, these mice display significant protection from obesity-induced hyperglycemia and fatty liver (15): on the other hand, overexpression of GPR40 in beta cells leads to impaired beta cell function and diabetes (15). Taken together, these results indicate that GPR40 plays a role in mediating both acute and chronic effects of LCFAs on beta cells, and may therefore help explain the link between obesity and T2DM.

The important physiological role of GPR40 prompted us to study the regulation of the gene. We identified the GPR40 transcription start site (TSS) and showed that the promoter is selectively active in beta cells. The GPR40 5'-flanking region contains two highly conserved regions that display DNase I hypersensitivity in beta cells: one of these, HR3, contains the GPR40 core promoter, whereas the other, HR2, is a strong transcriptional enhancer that plays an important role in directing GPR40 expression selectively to beta cells.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
RNA Preparation
Total RNA was extracted from cell cultures using the Tri-Reagent procedure (Molecular Research Center Inc.) according to the manufacturer's instructions. Where indicated, RNA was treated with DNase I (RQ1, Promega, Madison, WI) to remove traces of genomic DNA.

5'-Rapid Amplification of cDNA Ends (5'-RACE)
First strand cDNA was synthesized from 5 µg of DNase I-treated betaTC1 RNA, using reverse transcriptase (SuperScript II, Invitrogen) according to the manufacturer's instructions. Primers used were complementary to a specific sequence located either within the GPR40 coding region or upstream to it. The cDNA was purified and a poly-G tail was added to the cDNA 3'-end using terminal deoxynucleotidyl transferase (Promega) by incubating 80% of the cDNA purified above with 1x terminal deoxynucleotidyl transferase buffer (Promega), 0.83 mM dGTP and 20 units of enzyme in a final volume of 60 µl at 37 °C for 1 h. The reaction was stopped by heating to 65 °C for 15 min. The cDNA was purified, and PCR was performed using the Expand high fidelity PCR system (Roche Applied Science), with 5 µl 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)24. A fraction of the PCR product was resolved on a 1% agarose gel. When a band appeared, it was excised from the gel, purified, and sequenced. When a smear appeared, the PCR product was purified using the Roche Applied Science purification kit and subcloned into pGEM T-Easy vector (Promega). 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. Three of the relevant plasmids were sequenced.

RNase Protection Assay (RPA)
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. The insert contained 133 bp of the GPR40 second exon, the entire first exon (24 bp), and 52 bp upstream to the transcription start site. In vitro transcription reaction was carried out as described (20). The resulting radioactive probe was 278 bases, of which 209 bases are derived from the GPR40 genomic sequence. RPAs were performed using 50 µg of betaTC1 (21) RNA, {alpha}TC1 (22) RNA, yeast RNA, or 25 pg of positive control RNA (prepared by incubating pBS-RPA3-T7antisense in the presence of T3 RNA polymerase) (20). Samples were resolved on polyacrylamide urea gels and visualized by autoradiography.

Cell Culture
The following established cell lines were used in this study: betaTC1 (mouse beta cells) (21), HIT M2.2.2 (hamster beta cells) (23, 24), CHO (Chinese hamster ovary cells), 293T (human embryonic kidney cells), and NIH-3T3 (mouse fibroblast cells). betaTC1, HIT, CHO, NIH-3T3, and 293T cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum (or 15% horse serum, 2.5% fetal calf serum for betaTC1 cells), penicillin (200 IU/ml), and streptomycin (100 µg/ml).

Genomic Library Screening
A DNA fragment spanning the GPR40 gene was isolated from a mouse genomic library using a 32P-labeled probe containing the 900-bp GPR40 open reading frame. The clone obtained is 16,195 bp long, begins in the tenth intron of the CD22 gene, contains the entire GPR40 gene, and ends 3 kb downstream to the GPR41 open reading frame. The DNA fragment was characterized by restriction enzyme analysis and partial sequencing.

Transient Transfections
Transfections were carried out by the calcium phosphate co-precipitation technique (25). CHO and HIT transfections consisted of a mixture of 2 µg of reporter construct and 250 ng internal control plasmid. When expression plasmids were used, 1-2 µg of each was added. The total amount of expression plasmid DNA was equalized by addition of the empty expression plasmid pcDNA3. Transfection of 293T cells utilized 4 µg of expression plasmid and 0.5 µg of plasmid pCMV-GFP. Total DNA amount was adjusted to 10 µg by addition of pUC18 DNA. Glycerol shock was applied to HIT cells (20% glycerol) and CHO cells (10% glycerol) 4-7 h after transfection. Cells were harvested 40-48 h after transfection, and whole cell extracts were prepared as described below.

Cell Extracts
Whole Cell Extracts—Cells were washed with PBS, collected, and pelleted by centrifugation for 30 s in a microcentrifuge. The pellet was resuspended in KPi buffer (0.1 M potassium phosphate, pH 7.8, 1 mM dithiothreitol), and extracted by 3 cycles of freezing in liquid nitrogen and thawing at room temperature. Cell debris was removed by 5 min centrifugation in a microcentrifuge at 4 °C.

Nuclear Extracts—Cells were washed with PBS and collected by centrifugation for 30 s in a microcentrifuge. Nuclear extracts were prepared as described (26). Protein concentration of cell extracts was determined by the Bradford method (27) using bovine serum albumin as a standard.

Luciferase Assays
Firefly luciferase and Renilla luciferase assays were carried out as follows: whole cell extracts containing 5-50 µg (1-5 µl) of protein were added to 100 µl of either firefly luciferase assay buffer (20 mM Tricine, 0.1 mM EDTA, 1.07 mM (MgCO3)4Mg(OH)2*5H2O, 2.67 mM MgSO4, 3.3 mM dithiothreitol, 270 µM coenzyme A, 470 µM luciferin (Promega #E1602), and 530 µM ATP, pH 7.8) or Renilla luciferase assay buffer (0.1 M potassium phosphate, pH 7.4, and 0.5 µM coelenterazine (Calbiochem)). The light output was determined over a 10-s interval using a LUMAC Biocounter M2500 luminometer. Firefly luciferase activity was normalized according to the activity of Renilla luciferase.

Mutagenesis of GPR40 Promoter
Site-directed mutagenesis was carried out using the QuikChange system (Stratagene) according to the manufacturer's protocol. Oligonucleotides designed for mutagenesis of the HR2 region of GPR40 are indicated in the supplemental information.

Bioinformatics
Sequence alignment of genomic DNA sequences was performed using NCBI-BLAST (www.ncbi.nlm.nih.gov/BLAST) and software from GCG. Identification of transcription factor binding sites was performed using rVista program and Geno-matix suite programs MatInspector and FrameWorker.

Electrophoretic Mobility Shift Assay (EMSA)
Probe Labeling—The double-stranded oligonucleotides from the HR2 region of the mouse GPR40 promoter, which were used as probes are described in the supplemental materials. Radioactive probes were generated as described (28). Specific activity obtained was typically ~2,000 cpm/fmol.

Binding Reaction—Nuclear extracts of betaTC1, NIH-3T3, 293T, and transfected 293T cells were prepared as described above. Binding reactions and electrophoresis conditions used for PDX1 were as follows: protein extracts (0.5-2.5 µg) were incubated for 10 min on ice in binding buffer (20 mM Hepes, pH 7.9, 1 mM EDTA, 70 mM KCl, 10% glycerol, 0.5 mM dithiothreitol, 5 mM MgCl2, and 0.2% Nonidet P-40) with addition of 900 ng of poly(dI·dC) (Sigma) in a final assay volume of 14 µl, in the presence or absence of antibody, as specified in the figure legends. Competition experiments included 10-, 50-, 100-, or 200-fold molar excesses of the following three unlabeled oligonucleotides: HR2-2-6, HR2-M4-5, and the E1 element from the rat insulin gene promoter (29). Following this incubation, 1 µl of 32P-labeled probe was added (~25 fmol), and incubation was allowed to continue on ice for an additional 20 min. Samples were subsequently resolved on 5.5% polyacrylamide gels (37.5:1 acrylamide:bisacrylamide) in 45 mM Tris borate, 1 mM EDTA at 184 V for 80 min at 4 °C.

Reactions to test BETA2 binding were carried out in the presence or absence of appropriate antibodies as described (28). Competition experiments included 10-, 50-, or 100-fold molar excesses of unlabeled oligonucleotide (HR2-7-10, HR2-EM, and P1 (from the rat insulin promoter (30)). HR2-EM contains two point mutations (underlined) in the E-box element: 5'-CCTGTTCCCAAGCCTGGCTGCTGATATAAAGCTAC-3'. Following electrophoresis (28), gels were dried, and labeled DNA-protein complexes were visualized by autoradiography and quantitated by using a PhosphorImager and ImageGauge software.

DNase I Hypersensitivity Assay
Preparation of Nuclei—Cells were washed with PBS, trypsinized, and resuspended in growth medium. They were pelleted at 1000 rpm for 5 min, washed twice in cold PBS, and incubated on ice for 10 min in Dounce buffer (20 mM Tris-HCl, pH 7.0, 3 mM CaCl2, 2 mM MgCl2, and 0.3% Nonidet P-40). Cells were then homogenized on ice using a Dounce homogenizer (20 strokes, pestle B (loose)), and nuclei were pelleted at 1000 rpm for 7 min at 4 °C. Nuclei were then resuspended in cold RSB buffer (10 mM Tris-HCl, pH 7.0, 10 mM NaCl, and 3 mM MgCl2), counted in a hemocytometer, pelleted again, and resuspended in 1x RQ1 DNase buffer at concentration of 108 nuclei/ml.

DNase I Treatment—A series of digestions using DNase I (RQ1) was performed in the presence of 107 nuclei and increasing concentrations of DNase (0-20 units/ml) for 15 min at 37 °C. Reactions were terminated by addition of 1 volume of 2x TNESK (20 mM Tris-HCl, pH 7.0, 200 mM NaCl, 2 mM EDTA, 2% SDS, and 200 µg/ml proteinase K). Samples were incubated overnight at 37 °C and then phenol-chloroform-extracted, ethanol-precipitated, and resuspended in 100 µl of TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.8). The concentration of purified genomic DNA was determined using a NanoDrop spectrophotometer.

Southern Blot—Purified DNase I-treated genomic DNA (10 µg) was digested to completion at 37 °C overnight with KpnI and resolved on 1% agarose gel in 1x TAE buffer (40 mM Tris acetate, 1 mM EDTA) at 20 V overnight. Southern blot analysis was performed as described (20).

Chromatin Immunoprecipitation (ChIP)
Soluble chromatin was prepared as described (31) from 2 x 107 betaTC1 cells. For each immunoprecipitation reaction, 25 µg of pre-cleared chromatin was incubated with anti-PDX1 or anti-BETA2 or control antiserum overnight at 4 °C on a rotator wheel. Following centrifugation, the extracts were incubated with 20-30 µl of 50% (w/v) protein A-Sepharose suspension at 4 °C for 2 h on a rotator wheel, and the beads were then washed. The immune complexes were then eluted by incubating the beads three times with 100 µl of elution buffer (1% SDS, 0.1 M NaHCO3, 20 µg/ml glycogen) for 10 min each. The combined eluates were heated at 65 °C overnight to reverse the formaldehyde cross-links. The eluates were extracted once with phenolchloroform and once with chloroform, precipitated with ethanol, and resuspended in 20 µl of TE buffer. A 2-µl sample was used for amplification by quantitative real-time PCR.

The real-time PCR reactions were performed in an ABI 7300 real-time PCR system using the "absolute quantification method" option with SYBR-Green PCR master Mix (ABI) and 333 nM primers. The primers used are described in the supplemental materials. In each experiment, a standard curve for each chromatin was plotted using serial dilutions of chromatin input. The PCR program employed 40 cycles of denaturation (95 °C for 10 s), annealing, elongation, and fluorescence detection (60 °C for 1 min). At the end of the final cycle, a dissociation curve of all samples was performed. The DNA recovered from each immunoprecipitate was calculated as a percentage of the total input chromatin.

Statistical Analysis
Statistical significance between two groups was assessed using unpaired two-tailed t test. The data in Fig. 5 was analyzed by two-factor analysis of variance using experiment and mutant as the factors. Means for each mutant were compared with wild-type mean using the Dunnett test ({alpha} = 0.05). The data in Fig. 6C was analyzed using the Wilcoxon two-sample test corrected for multiple comparisons by the Holms method. Values are shown as mean ± S.E.


Figure 1
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  		FIGURE 1.
Mapping of the transcription start site of the GPR40 gene by 5'-RACE and RPA. A, primers used for 5'-RACE. Primers E2bot4 and E2bot5 match the 5'-end of the GPR40 open reading frame, and primer 44413 matches 5'-untranslated region sequences in exon 2. Orientation of primers is indicated. B, 5'-RACE results. DNase I-treated RNA from betaTC1 cells was reverse transcribed using primer E2bot4. The cDNA was poly(dG)-tailed and amplified by PCR using a poly-C primer and primer E2bot5 (lane 1). As a positive control, the cDNA was amplified using primers E2bot5 and 44413 (lane 2). As a negative control PCR was carried out with RNA that was not reverse-transcribed (-RT, lane 3) or without RNA (-RNA, lane 4). C, vector used for generation of the RPA probe and its predicted protected products. The RNA probe is complementary to genomic sequence -52 to +24, containing 52 bp of the proximal 5'-flank and exon 1, and +723 to +856, containing 133 bp of the 5'-end of exon 2, according to the 5'-RACE result. D, RPA results. 50 µg of betaTC1 RNA (lane 1) or {alpha}TC1 RNA (lane 2) was hybridized to the 32P-labeled RNA probe, then subjected to RNase digestion and separated on urea-polyacrylamide gel. The assay was also performed using 25 pg in vitro transcribed sense RNA as a positive control (lane 3) or 50 µg of yeast RNA as a negative control (lane 4). M indicates molecular weight marker. Full protection band (209 bases) corresponds to protection of the entire probe sequence derived from the GPR40 gene, whereas the positive control band (239 bases) corresponds to this full protection and an additional 30 bases derived from the vector used for in vitro transcription.

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Characterization of the GPR40 Transcription Start Site and Intron-Exon Organization—As a first step toward understanding the molecular mechanisms controlling cell-specific regulation of the mouse GPR40 gene, we identified the 5'-end of the GPR40 mRNA. 5'-RACE experiments using betaTC1 RNA and primers complementary to the open reading frame of GPR40 (Fig. 1A) produced a major product of ~400 bp (Fig. 1B, lane 1). Comparison of its sequence with that of mouse genomic DNA revealed the presence of a 698-bp intron beginning 321 bp upstream of the ATG initiation codon, and a 24-bp first exon. Thus, the major TSS is located 1044 bp upstream of the translation initiation site. Additional weaker bands, suggested the existence of minor upstream transcription start sites (Fig. 1B, lane 1). Indeed, a 5'-RACE experiment performed with a primer starting 4 bases downstream to the major transcription start site gave rise to several products: sequencing of three of these showed distinct 5'-ends mapping 100-150 bp upstream of the major transcription start site (data not shown).

To confirm this result, RPAs were performed using a 278-base RNA probe comprising sequences corresponding to the 52 bp immediately upstream of the putative transcription start site, the 24 bp of exon 1 and the 133 bp at the 5'-end of exon 2 (Fig. 1C). A protected band of ~157 bases was observed using betaTC1 RNA, but not {alpha}TC1 RNA (Fig. 1D, lanes 1 and 2), confirming the location of the start site indicated by the 5'-RACE result. An additional weak full protection band (209 bases) was also seen, consistent with the existence of the additional minor transcription start sites previously indicated by 5'-RACE.

Additional 5'-RACE experiments performed using primers distributed across the GPR40 gene (data not shown), and examination of expressed sequence tag (EST) sequences present in public data-bases, suggest that no additional introns are present in the gene. Taken together, these data establish the organization of the GPR40 gene, which is comprised of two exons of 24 and 4402 bp separated by a 698-bp intron. This arrangement of a short, non-coding first exon, followed by a long exon containing the complete protein coding information (Fig. 1A), is common in G protein-coupled receptors (32).

Characterization of the GPR40 Gene Promoter and Identification of Conserved Regions—Comparison of genomic DNA sequences of different species can often assist in identifying important control elements (33). We therefore compared the intergenic region between the CD22 gene and the GPR40 gene in mouse, rat, and human. The alignments revealed three conserved regions, which we named HR1-HR3 (Fig. 2A). HR1 is a part of an Alu repeat; it is 38 bp long, is 91% identical between mouse and human, and is located 2126 bp upstream from the transcription start site. HR2 is 194 bp long; it is 82% identical between mouse and human and 93% identical between mouse and rat (Fig. 2B). HR3 is 185 bp long; it is 81% identical between mouse and human and 89% identical between mouse and rat (Fig. 2C).


Figure 2
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  		FIGURE 2.
Conserved regions HR2 and HR3 in the 5' flank of the GPR40 gene. A, rVista plot of sequence conservation between mouse and human 5'-flank of the GPR40 gene. The conserved regions HR2 and HR3, and the GPR40 protein coding region are shown. B, the sequence of HR2 in mouse, rat, and human. The E-boxes and the putative HNF4{alpha} binding sites are indicated. The sequence duplicated in human is underlined. The recognition site for the restriction enzyme BspHI is shown. Numbers indicate nucleotides relative to the transcription start site. C, the sequence of HR3 in mouse, rat, and human. Transcription start site (+1) is indicated by an arrow, and exon 1 is shown. The following are indicated by gray shading: Int element at the start site, the DPE element at position +28 to +32, the G at position +24, and the A-element at position -89 to -86. Also indicated are two putative Sp1 binding sites. Numbers indicate nucleotides relative to the transcription start site.

 
HR2 is located 1060 bp upstream of HR3. Sequence analysis of this region revealed two conserved E-boxes at -1110 and -1032, the latter being duplicated in human (Fig. 2B). These sequences can serve as binding sites for basic helix-loop-helix (bHLH) transcription factors (34). Also present in the region is a sequence that is similar to the binding site for HNF4{alpha} (35) at -1017. Analysis of conserved sequences in the HR2 region of human, chimpanzee, rat, and mouse, performed using the rVista program and Genomatix suite programs MatInspector and FrameWorker, revealed in addition the binding sites for the transcription factors AP4, ETS family, RFX1, PBX1, MEIS1, Evi1, Bel1, and homeodomain proteins of the HOX family.

Transcription start sites and promoter elements are often conserved among species. Indeed, the HR3 homology region (Fig. 2C) spans the major transcription start site; hence it is expected to contain at least part of the promoter. Inspection of HR3 revealed characteristic features of a TATA-less promoter, including a GC-rich region (64% in mouse and 63% in human) and two putative Sp1 binding sites (Fig. 2C). The sequence at the GPR40 TSS matches the consensus sequence for the initiator (Int) element (PyPyA + 1NT/APyPy) (36) except for the last two pyrimidines. The rest of the sequence, including the A at the +1 position, matches in all three species (Fig. 2C). In addition, the sequence between +28 to +32 matches the DPE consensus sequence (36) in all three species. Indeed, DPE-containing promoters typically possess also the Int element, and the DPE element is located exactly at positions +28 to +32 relative to the A at +1 of the Int (36). In DPE-Int promoters there is a preference for G at position +24, and in fact G is present at position +24 in the GPR40 promoter of all three species. Thus, the GPR40 core promoter displays the typical features of a TATA-less, Int-DPE core promoter.

We also identified in HR3 a conserved A-element (TAAT) at position -89 to -86, which may serve as a binding site for transcription factors of the homeodomain family (Fig. 2C). Other conserved potential transcription factor binding sites found in HR3 using the rVista program include AP2, AP4, ELK1, GATA family, IK2, MZF1, PEA3, and STAT family.


Figure 3
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  		FIGURE 3.
Analysis of chromatin structure of GPR40 5'-flanking region. A, map illustrating strategy used to analyze DNase I sensitivity. The gray blocks represent exons of the CD22 gene and the GPR40 gene. The conserved regions HR1, HR2, and HR3 are shown by black boxes. The black bar indicates the sequences used as probe for Southern blot. B, nuclei isolated from betaTC1, and NIH-3T3 cells were digested with the indicated amounts of DNase I. DNA was then isolated, digested with KpnI, Southern blotted, and hybridized with the radiolabeled probe. The 8.8-kb KpnI genomic fragment and the bands resulting from digestion at hypersensitive sites are shown. C, quantitation of the disappearance of the KpnI genomic fragment with increasing DNase I concentration.

 
Chromatin Accessibility at the 5'-Flank of the GPR40 Gene—To compare the chromatin structure at the GPR40 locus in beta versus non-beta cells, we tested the DNase I sensitivity of nuclei from betaTC1 and NIH-3T3 cells. We focused on an 8.8-kb KpnI genomic fragment spanning exon 9 of CD22 to the middle of the GPR40 coding sequence, including ~7.5 kb upstream to the GPR40 TSS and the GPR40 intron (Fig. 3A). A radiolabeled probe of 870 bp corresponding to the 3'-end of the region (Fig. 3A) was used for identification of the genomic band and sub-bands.

The analysis showed that in betaTC1 cells, which express GPR40, the accessibility of chromatin to DNase I was higher than in the non-expressing cell line NIH-3T3, as indicated by the greater sensitivity of the genomic fragment to nuclease digestion (Fig. 3, B and C). Furthermore, although in NIH-3T3 cells no sub-bands appeared following DNase I digestion, clear sub-bands of ~2.5 kb and 1.5 kb were seen in betaTC1 cells (Fig. 3B), indicating sites of DNase I hypersensitivity. Because the radiolabeled probe we used corresponds to the terminus of the examined genomic region, we were able to determine the location of these hypersensitive sites. Interestingly, they correlated extremely well with the HR2 and HR3 homology regions. Because the GPR40 core promoter is located in the HR3 region, it is expected that in beta cells this region will be highly accessible. In addition, the hypersensitivity of the HR2 region is consistent with an important function for this region.

Transcription Regulation Conferred by Sequences from the 5'-Flank of the GPR40 Gene—To determine whether the 5'-flanking region of the mouse GPR40 gene is capable of directing transcriptional activity, the sequence -3315 to +844 relative to the TSS was fused to the firefly luciferase reporter gene, and transfected into the beta cell line HIT and the non-beta cell line CHO. Activity of this construct, 4155-GPR40P-LUC, was 17.7-fold higher than the parental promoterless vector pGL3-basic in HIT cells (Fig. 4B). In contrast, the activity of 4155-GPR40P-LUC was only 1.3-fold higher than pGL3-basic in CHO cells, suggesting that this fragment has beta cell-specific promoter activity. A 5' deletion of 4155-GPR40P-LUC from -3315 to -1257, yielding the construct 2095-GPR40P-LUC, did not affect the activity in HIT cells, suggesting that homology region HR1 is not required for activity of the promoter. However, deletion of an additional 159 bp, including the 5' portion of the HR2 region (1936-GPR40P-LUC), led to a dramatic 10-fold reduction in activity in HIT cells. No reduction in activity was seen on comparing 2095-GPR40P-LUC and 1936-GPR40P-LUC in CHO cells, indicating that the activity conferred by the deleted 159 bp is beta cell-specific. The data therefore suggest that an important transcriptional element lies between HinDIII and BspH1 (Fig. 4A). In addition, 3' deletion of 816 bp, including the entire intron, from 2095-GPR40P-LUC (1279-GPR40P-LUC), led to decreased activity both in HIT and CHO cells (Fig. 4B), probably due to the removal of the DPE element (see Fig. 2C).


Figure 4
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  		FIGURE 4.
Functional analysis of GPR40 promoter in beta and non-beta cells. A, the 5'-flanking region of the GPR40 gene showing the fragments used to generate reporter gene constructs. B, promoter fragments were ligated upstream to the promoterless luciferase gene in the vector pGL3-basic. Promoter activity of each construct was determined in the beta cell line HIT and the non-beta cell line CHO. Values are normalized for transfection efficiency and expressed relative to the activity of pGL3-basic. Each data point represents mean ± S.E. of 3-16 independent experiments. The ratio of transcriptional activity (HIT/CHO) is indicated. C, transcriptional activity of conserved region HR2 in beta and non-beta cells. A fragment of 258 bp from HinDIII to the end of the conserved region HR2 (Fig. 4A) was ligated upstream to the herpes simplex virus TK promoter controlling the luciferase gene (HR2-TK-LUC). Activity of this construct was determined in HIT and CHO cells. Values are normalized for transfection efficiency and expressed relative to the activity of TK-LUC. The value in HIT cells represents the mean ± S.E. of 12 independent experiments. Transfection in CHO cells represents the mean ± S.E. of five independent experiments. The asterisk indicates statistical significance (p < 0.001) compared with the values obtained for TK-LUC.

 
The ability of conserved region HR2 to activate transcription from a heterologous promoter activity was also examined. For this purpose, a 258-bp fragment spanning HR2 was fused to a herpes simplex virus thymidine kinase (TK) promoter controlling the firefly luciferase reporter gene. HR2-TK-LUC had 11.4-fold higher activity than TK-LUC in the beta cell line HIT, whereas in the non-beta cell line CHO, the HR2 region had little or no effect on the TK promoter (Fig. 4C). The HR2 region was able to activate the TK promoter in an orientation-independent fashion (data not shown). Thus, HR2 contains a potent beta-cell-specific enhancer.

Systematic Mutagenesis of the HR2 Region—Based on the 5' deletion analysis, and the evolutionary conservation of the HR2 element, it is likely that the 5' 100 bp of the HR2 element (-1191 to -1091) contains a critical control sequence. This 100-bp region was therefore systematically mutated to define key transcriptional control elements. Accordingly, a series of ten substitution mutations (M1-M10), each spanning ~10 bp, was generated in 2095-GPR40P-LUC (Fig. 5A), and their transcriptional activity following transfection to HIT cells was compared with the wild-type promoter (Fig. 5B). Striking reductions in activity were observed with the adjacent mutations M4 and M5 (remaining activity, 38 and 40%, respectively). Several mutations showed smaller, yet statistically significant reduction in activity. The observed activities were: M1, 59%; M8, 74%; and M9, 59%. Thus, the analysis defines three sub-regions within HR2 that are important for transcription activation: sub-region 1, sub-region 4-5, and sub-region 8-9.


Figure 5
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  		FIGURE 5.
Systematic mutational analysis of the 5' 100bp of HR2 in mouse. A, the location of mutations M1-M10 in conserved region HR2. Mutations were generated as follows: A {leftrightarrow} C and G {leftrightarrow} T. Putative binding sites for HOX family, RFX1, PBX1, and MEIS1 are indicated. B, HIT cells were transfected with 2 µg of either WT-HR2-2095-LUC or one of its replacement mutants M1-M10. Values are normalized for transfection efficiency and expressed relative to the activity of WT-HR2-2095-LUC wild-type vector. C, HIT cells were transfected with 2 µg of either WT-HR2-TK-LUC or one of its replacement mutants M1-M10. Values are normalized for transfection efficiency and expressed relative to the activity of WT-HR2-TK-LUC wild-type vector. For B and C, values are shown as mean ± S.E. (n = 3-7) after normalizing the wild-type mean to 1.0. Asterisks denote means differing from the wild-type by the Dunnett test ({alpha} = 0.05).

 
To examine the transcriptional activity of the cis-elements in the HR2 region independently of the remainder of the promoter, the same series of substitution mutations of HR2 was introduced to HR2-TK-LUC and tested in the beta cell line HIT. The results obtained were similar to those of 2095-GPR40 mutants (Fig. 5C).

Binding of PDX1 to HR2 in Vitro and in Vivo—Bioinformatic analysis identified within sub-region 4-5 potential binding sites for transcription factors of the homeodomain family (Fig. 5A). We therefore tested whether PDX1, an important beta cell-specific homeodomain protein can bind this sequence in vitro using EMSA. We used a radioactive probe (designated HR2-2-6) that contains the wild-type sequence of region HR2 from the middle of sub-region 2 to the middle of sub-region 6. Incubation with nuclear extracts from 293T cells transfected with a PDX1 expression vector, generated a strong band (Fig. 6A, lane 2). Incubation of the probe with betaTC1 cell nuclear extracts yielded a band of similar mobility, whereas incubation with NIH-3T3 cell extracts did not show this band (Fig. 6A, lanes 3 and 4). Addition of anti-PDX1 anti-serum abolished the band (Fig. 6A, lane 6), whereas addition of PBS, pre-immune serum (anti-P33) or irrelevant anti-serum (anti-N22) had no effect (Fig. 6A, lanes 5, 7, and 8). To examine the specificity of binding, we performed quantitative competition experiments using increasing concentrations of unlabeled oligonucleotides (Fig. 6B). The oligonucleotides used were HR2-2-6, HR2-M4-5 (which contains HR2-2-6 sequence mutated at sub-regions 4 and 5), and E1 (which contains an E-box element from the rat insulin I promoter, and thus is not expected to bind PDX1). HR2-M4-5 and E1 oligonucleotides showed significantly reduced ability to compete for binding (Fig. 6B), indicating that PDX1 binds in a sequence-specific manner and with highest affinity to the wild-type sequence of sub-region 4 to 5 within HR2.

To determine whether PDX1 binds to the HR2 enhancer in vivo, we performed ChIP experiments using anti-PDX1 or control antibody. PDX1 occupancy of the HR2 region in betaTC1 cells was significantly higher than its occupancy of GPR40 or CD22 coding sequences (CDS), which reside within the vicinity of HR2, but are not expected to bind PDX1 (Fig. 6C). In contrast, PDX1 occupancy on HR2 was similar to its occupancy on the promoter of the mouse insulin II gene, a bona fide PDX1 target gene (data not shown). As control, we used pre-immune serum and observed significantly lower binding to HR2 and the insulin II gene promoter as compared with anti-PDX1 antibody (Fig. 6C and data not shown).

Binding of BETA2 to HR2 in Vitro and in Vivo—Mutation M9 overlaps the 5' E-box of the HR2 region. Because E-box elements represent potential binding sites for transcription factors of the bHLH family, we examined whether BETA2 (NeuroD1), an important beta cell-specific bHLH protein (37), can bind this sequence in vitro using EMSA. BETA2 typically binds E-box sequences as a heterodimeric complex with products of the ubiquitously expressed E2A gene (38). We used a radioactive probe (designated HR2-7-10) that contains the wild-type sequence of HR2 from sub-region 7 to the end of sub-region 10. Incubation of the probe with betaTC1 cell nuclear extract yielded a slow migrating complex (Fig. 7A, lane 1). Addition of anti-BETA2 antibody to the incubation mixture resulted in a super-shift of the complex, whereas addition of pre-immune serum (anti-P33) did not (Fig. 7A, lanes 2 and 3). Incubation of the probe with nuclear extracts from 293T cells did not generate this complex (Fig. 7A, lane 4), whereas 293T cells transfected with a BETA2 expression vector generated a clear band whose mobility is similar to the complex observed with betaTC1 extract (Fig. 7A, lane 5). The complex was supershifted upon addition of anti-BETA2 antibody (Fig. 7A, lane 6).


Figure 6
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  		FIGURE 6.
PDX1 binds the HR2 region of the GPR40 flank in vitro and in vivo. A, EMSA analysis. Nuclear extracts from untransfected 293T cells (lane 1), 293T transfected with PDX1 expression vector (lane 2), NIH-3T3 (lane 3), or betaTC1 (lanes 4-8) were incubated with 32P-labeled HR2-2-6 probe and resolved on a non-denaturing polyacrylamide gel. In lanes 5-8 the incubation reaction was performed in the presence of the following: PBS (lane 5), anti-PDX1 antibodies (lane 6), pre-immune serum anti-P33 (lane 7), and irrelevant anti-serum anti-N22 (lane 8). B, competition experiments were performed by incubation of nuclear extracts from betaTC1 cells with 32P-labeled HR2-2-6 probe, and increasing concentrations of unlabeled E1, HR2-2-6, or HR2-M4-5 oligonucleotides. Extent of binding is expressed relative to that observed in the absence of unlabeled oligonucleotides. C, ChIP. Cross-linked chromatin from betaTC1 cells was immunoprecipitated using either anti-PDX1 antibodies or pre-immune serum. DNA fragments recovered from immunoprecipitations were amplified using specific sets of primers corresponding to the beta cell-specific enhancer HR2, and the coding sequence (CDS) of GPR40 and CD22 genes. Binding of antisera to the tested regions was analyzed using quantitative real-time PCR and presented as percentage recovery of input chromatin. Results are expressed as the mean ± S.E. of three or more independent ChIP experiments. Asterisks indicate statistical significance (p < 0.04 for anti-PDX1 compared with GPR40 CDS, p < 0.02 for anti-PDX1 compared with CD22 CDS, and p = 0.05 for anti-PDX1/HR2 compared with pre-immune/HR2). Statistical significance involving more than two groups in the experiment was determined by comparing the occupancy of PDX1 on HR2 region to each of the other regions using the Wilcoxon 2-sample test corrected for multiple comparisons by the Holms method.

 
To examine the specificity of binding, we performed quantitative competition experiments using increasing concentrations of unlabeled oligonucleotides (Fig. 7B). The oligonucleotides used were HR2-7-10, HR2-EM (which contains a mutated sequence of the E-box element), and P1 (which contains an A-element from the rat insulin I gene promoter, and thus is not expected to bind BETA2). P1 and HR2-EM oligonucleotides showed significantly reduced ability to compete for binding (Fig. 7B), indicating that BETA2 is capable of binding to sub-region 8-9 within HR2 with high affinity and in a sequence-specific manner.

To determine whether BETA2 binds to the HR2 enhancer in vivo, we performed ChIP experiments using anti-BETA2 antibody. BETA2 occupancy of the HR2 region in betaTC1 cells was significantly higher than its occupancy of the CDS of GPR40, CD22, or the HR3 region, none of which were expected to bind BETA2 (Fig. 7C). The signal observed with control antibody was low for all regions tested (Fig. 7C).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The pancreatic beta cell plays a crucial role in maintaining metabolic homeostasis through the production and regulated secretion of the key metabolic hormone insulin in response to physiological needs (39). Inadequate production of insulin is a hallmark of both major forms of diabetes mellitus. Insulin secretion is regulated mainly according to fluctuating levels of blood glucose but also in response to nutrients, neurotransmitters, and hormones (40). To fulfill their complex role, beta cells express a characteristic profile of genes, including many that are expressed selectively in beta cells. Regulation of these genes is mediated in large part through transcriptional control mechanisms (29) involving the combinatorial actions of ubiquitous and cell-specific transcription factors (28, 41). It has recently been shown that the GPR40 gene is expressed selectively in beta cells, where it appears to play an important role in mediating the effects of fatty acids on insulin secretion (5, 42).


Figure 7
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  		FIGURE 7.
BETA2 binds the HR2 region of the GPR40 promoter in vitro and in vivo. A, EMSA analysis. Nuclear extracts from betaTC1 cells (lanes 1-3), untransfected 293T cells (lane 4), and 293T cells transfected with BETA2 expression vector (lanes 5 and 6) were incubated with 32P-labeled HR2-7-10 probe and resolved on a non-denaturing polyacrylamide gel. The incubation reactions were performed in the presence of anti-BETA2 antibody (lanes 2 and 6) or pre-immune (lane 3). B, competition experiments were performed by incubation of nuclear extracts from betaTC1 cells with 32P-labeled HR2-7-10 probe and increasing concentrations of unlabeled P1, HR2-EM, or HR2-7-10 oligonucleotides. Binding is expressed relative to that observed in the absence of unlabeled oligonucleotides. C, ChIP. Cross-linked chromatin from betaTC1 cells was immunoprecipitated using either anti-BETA2 antibodies or pre-immune serum. DNA fragments recovered from immunoprecipitations were amplified using specific sets of primers corresponding to the beta cell-specific enhancer HR2, the coding sequence (CDS) of GPR40 and CD22 genes, and the GPR40 core promoter HR3. Binding of antisera to the tested regions was analyzed using quantitative real-time PCR and presented as percentage recovery of input chromatin. Results are expressed as the mean ± S.E. of three or more independent ChIP experiments. Asterisks indicate statistical significance (p < 0.05) compared with control antibody and compared with GPR40 CDS, CD22 CDS, and HR3.

 
In this study, we have examined the molecular basis for the selectivity of GPR40 expression in the beta cell. We have defined the organization of the gene and the location of the core promoter, which lacks a TATA element but contains an Int-like element and a DPE sequence. Reporter gene experiments demonstrated that beta cell-specific expression of the GPR40 gene is mediated through selective transcription via the 5'-flanking region of the gene. Cell-specific transcription of mammalian genes generally involves evolutionarily conserved sequences located upstream of the core promoter. Indeed, we demonstrated that the conserved HR2 element, which coincides with a nuclease hypersensitive site, functions as a strong beta cell-specific transcriptional enhancer.

Systematic mutagenesis of the HR2 enhancer, revealed three important domains. One of these (sub-region 8-9) contains an E-box element that can bind transcription factors of the bHLH family. Indeed, BETA2, a beta cell-specific bHLH protein, implicated in regulation of several beta cell genes (28, 37), was shown to bind in vitro to sub-region 8-9 of HR2 in a sequence-specific manner, and was found to occupy the HR2 region in vivo.A second domain (sub-region 3-5) contains an A-element that can bind transcription factors of the homeodomain family. We were able to demonstrate that PDX1, a crucial beta cell homeodomain transcription factor, binds to this region both in vitro and in vivo.

These results represent the first characterization of the GPR40 gene promoter. Like other beta cell-specific genes, cell specificity is controlled at the transcriptional level through enhancer elements located in the 5' flank. This may be mediated by interactions among PDX1, BETA2, and additional transcription factors. Accumulating evidence has indicated a critical role for GPR40 in mediating the responsiveness of beta cells to LCFAs (9, 17, 19); furthermore, persistent activation of GPR40 in obesity has been proposed as a mechanism to explain the well established link between obesity and type 2 diabetes (15). The important physiological role proposed for GPR40 emphasizes the likely need for maintaining appropriate intracellular concentrations of the protein. Thus, regulated transcription of the gene is probably essential for maintaining beta cell function, and defects could lead to perturbed insulin secretion. The present study has elucidated the molecular basis for understanding beta cell-specific transcription of GPR40 gene expression. Future studies will be aimed to determining the detailed mechanisms modulating GPR40 gene transcription in normal beta cells and to investigate possible defects in diabetes.


   FOOTNOTES
 
* This work was supported in part by grants from the Russell Berrie Foundation and D CURE Diabetes Care in Israel, the Israel Academy of Sciences and Humanities, and the Mitchell Caplan Fund for Diabetes Research (to M. D. W.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains a supplemental list of sequences. Back

1 Current address: Dept. of Genetics, University of Pennsylvania, School of Medicine, Philadelphia, PA 19104-6145. Back

2 Incumbent of the Marvin Meyer and Jenny Cyker Chair of Diabetes Research at the Weizmann Institute. To whom correspondence should be addressed: Tel.: 972-8-934-3597; Fax: 972-8-934-4118; E-mail: m.walker{at}weizmann.ac.il.

3 The abbreviations used are: T2DM, type 2 diabetes mellitus; RACE, rapid amplification of cDNA ends; RPA, RNase protection assay; ChIP, chromatin immunoprecipitation; LCFA, long-chain fatty acid; TSS, transcription start site; CHO, Chinese hamster ovary; HIT, hamster beta cell; PBS, phosphate-buffered saline; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; EMSA, electrophoretic mobility shift assay; bHLH, basic helix-loop-helix; Int, initiator element; STAT, signal transducers and activators of transcription; TK, thymidine kinase; CDS, coding sequences. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Dena Leshkowitz and Dr. Shifra Ben-Dor for valuable bioinformatics advice, Dr. Hillary Voet for statistical consultation, and Sara Weiss for expert technical assistance. Dr. Shimon Efrat kindly provided {alpha}TC1 and betaTC1 cells. We gratefully acknowledge a gift of anti-PDX1 antibodies from Dr. Michael German. We thank Dr. Haim Garty for advice on genomic library screening.



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