1 The MODY1 gene HNF4α and a feedback loop control COUP-TFII ...

1

The MODY1 gene HNF4αααα and a feedback loop control COUP-TFII expression in 1

pancreatic beta cells 2

3

Anaïs Perilhou1,2

, Cécile Tourrel-Cuzin1,2

, Pili Zhang3, Ilham Kharroubi

1,2, Haiyan Wang

4, 4

Véronique Fauveau1,2

, Donald K. Scott3, Claes B. Wollheim

5, and Mireille Vasseur-Cognet

1,2# 5

6

1 Institut Cochin, Université Paris Descartes, CNRS (UMR 8104), Department of 7

Endocrinology, Metabolism and Cancer Paris, France. 8

2 Inserm, U567, Paris, France. 9

3 Division of Endocrinology and Metabolism, University of Pittsburgh School of 10

Medicine, Pittsburgh, PA 15261, USA. 11

4 PRBD-Metabolic diseases, Hoffmann-La Roche, Basel, Switzerland. 12

5 Department of Cell Physiology and Metabolism, University Medical Centre, 1211 13

Geneva 4, Switzerland 14

15

Running title: COUP-TFII and HNF4α in islet regulatory network 16

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18

#corresponding author: tel (33)-1 44 41 24 20; fax (33)-1 44 41 24 21; 19

e-mail [email protected] 20

21

22

Word count for the Materials and Methods section: 1811 23

Word count for the Introduction, Results and Discussion sections: 2287 24

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Copyright © 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.Mol. Cell. Biol. doi:10.1128/MCB.01191-07 MCB Accepts, published online ahead of print on 12 May 2008

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Abstract 25

Pancreatic islet beta cell differentiation and function are dependent upon a group of 26

transcription factors that maintain the expression of key genes and suppress others. Knockout 27

mice with heterozygous deletion of the Chicken Ovalbumin Upstream Promoter- 28

Transcription Factor II (COUP-TFII) gene or complete disruption of the Hepatocyte Nuclear 29

Factor 4 α (HNF4α) gene in pancreatic beta cells have similar insulin secretion defects 30

leading us to hypothesize that there is transcriptional cross-talk between these two nuclear 31

receptors. Here we show specific HNF4α activation of a reporter plasmid containing the 32

COUP-TFII promoter region in transfected pancreatic beta cells. A stable association of the 33

endogenous HNF4α with a region of the COUP-TFII gene promoter that contains a direct 34

repeat 1 (DR-1) binding site was revealed by chromatin immunoprecipitation. Mutation 35

experiments showed that this DR-1 site is essential for HNF4α transactivation of COUP-TFII. 36

Dominant negative suppression of HNF4α function decreased endogenous COUP-TFII 37

expression and specific inactivation of COUP-TFII by short interfering (si)RNA caused 38

HNF4α mRNA levels to decrease in 832/13 INS-1 cells. This positive regulation of HNF4α 39

by COUP-TFII was confirmed by adenoviral overexpression of human (h)COUP-TFII which 40

increased HNF4α mRNA in 832/13 INS-1 cells and in mouse pancreatic islets. Finally, 41

hCOUP-TFII overexpression showed that there is direct COUP-TFII autorepression as 42

COUP-TFII occupies the proximal DR-1 binding site of its own gene in vivo. Therefore 43

COUP-TFII could contribute to the control of insulin secretion through the complex 44

HNF4α/maturity-onset diabetes of the young 1 (MODY1) transcription factor network 45

operating in beta cells. 46

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Introduction 47

Chicken Ovalbumin Upstream Promoter-Transcription Factor II (COUP-TFII, also called 48

NR2F2) is an orphan member of the steroid/thyroid hormone receptor superfamily classed in 49

the same subfamily as Hepatocyte Nuclear Factor 4 α (HNF4α)/maturity-onset diabetes of the 50

young 1 (MODY1) and retinoid X receptor (RXR) (4, 11). Several molecular mechanisms 51

have been shown by which COUP-TFII controls gene expression in pancreatic islet beta cell 52

differentiation and function. COUP-TFII binds DNA by a Zn-finger DNA binding domain on 53

a variety of hormone response elements (HRE) that contain imperfect AGGTCA direct or 54

inverted repeats with various spacings (3, 14). It can form heterodimeric complexes with 55

RXR, the universal partner of many nuclear receptors, and as such acts as a repressor (15). 56

We previously showed that COUP-TFII acts as an inhibitor of the glucose activation of the 57

liver pyruvate kinase (L-PK) gene by binding to the glucose responsive element (9). On most 58

promoters, HNF4α response elements are also bound by COUP-TFII that often behaves as a 59

transcriptional repressor antagonizing the enhancement of transcription by HNF4α (8, 12, 60

24). In a functional study, the impaired synergy between COUP-TFII and the E276Q mutant 61

of human HNF4α on the HNF1 promoter was found to be due their altered interactions (21). 62

Recently, we showed that heterozygous COUP-TFII deletion in mouse pancreatic beta cells 63

led to impaired glucose sensitivity and abnormal insulin secretion (1). These mutant mice 64

presented hyperinsulinemia in fasted and fed states and impaired glucose tolerance. 65

Interestingly, mice with complete disruption of the HNF4α gene in beta cells have a similar 66

phenotype, i.e., hyperinsulinemia in fasted and fed states, impaired glucose tolerance (6, 16) 67

and glucose-stimulated insulin secretion defects (13). These observations raised the question 68

of the possible interdependency of these two transcription factors. To address this, we 69

investigated the capacity of HNF4α to regulate COUP-TFII expression and the possible 70

cross-regulation of HNF4α and COUP-TFII. We also tested the idea that expression of 71

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COUP-TFII, like some other transcription factors, is autoregulated. 72

73

Materials and Methods 74

Cell culture. The rat insulinoma 832/13 INS-1 cell line (7), generously provided by C. 75

Newgard, was used between passages 19 and 29. Cells were cultured at 5% CO2/95% O2 at 76

37°C in INS-1 medium (RPMI 1640 medium containing 11 mM D-glucose supplemented 77

with 10% (v/v) heat inactivated fetal bovine serum, 100 U/mL penicillin, 100 U/mL 78

streptomycin, 10 mM HEPES, 1 mM sodium pyruvate (Invitrogen) and 50 µM beta-79

mercaptoethanol (Sigma)). DN-HNF4α-26 cells were maintained as described before (25). To 80

induce DN-HNF4α, cells were incubated for 24 h with 500 ng/ml doxycycline and total RNA 81

was extracted 24 h later. 82

Pancreatic islet isolation and culture. Mouse pancreatic islets were isolated from 6-week to 83

8-week-old male C57BL/6J mice using an adapted collagenase digestion method (1). Briefly, 84

mice were anesthetized with 3.5 bar isoflurane/0.5 bar oxygen (Minerve) and type V 85

collagenase P (Roche) was injected into the common bile ducts. Infused and distended 86

pancreases were then removed and left to digest for 4 minutes at 37°C with gentle mixing. 87

Islets were washed and handpicked in HEPES balanced salt solution (HBSS) (124 mM NaCl, 88

5 mM KCl, 0.8 mM MgSO4.7H2O, 1 mM NaH2PO4, 10 mM HEPES, 1.8 mM CaCl2, 14 mM 89

NaHCO3, and 0.5% bovine serum albumin (BSA) (Sigma), pH 7.4) containing 3 mM glucose 90

under an inverted light microscope and were then separated into study groups. Islets were 91

cultured overnight in RPMI 1640 medium (Invitrogen) containing 11 mM glucose 92

supplemented with 10% heat inactivated fetal bovine serum (Invitrogen), 1 mM sodium 93

pyruvate (Invitrogen), 100 U/mL penicillin, and 100 U/mL streptomycin (Invitrogen). 94

Immunostaining. Pancreases were removed from 4-month-old male C57/BL6N mice, fixed 95

overnight in 4% paraformaldehyde and embedded in paraffin. Blocks were serially sectioned 96

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(5 µm thickness). Sections were immunostained for insulin using a guinea pig anti-insulin 97

antibody (1:2000 dilution of A0564 from Dakocytomation) followed by incubation with 98

biotin-labeled goat anti-guinea pig IgG, then with peroxidase–labeled streptavidin, and 99

development in 3, 3’-diaminobenzidine-tetra-hydrochloride (DAB) (Vectastain from Vector 100

Laboratories). For COUP-TF staining, sections were incubated for 2 h with primary anti-101

human COUP-TFI (NR2F1,PP-H8132-00 at 1:100 dilution) or COUP-TFII (NR2F2, PP-102

H7147-00 at 1:50 dilution) mouse monoclonal antibodies (Perseus Proteomics) in 3% BSA, 103

0.05% Tween 20 phosphate buffered saline (PBS). An indirect peroxidase labeling technique 104

coupled with development with DAB was then used (EnVision+ System-HRP from 105

Dakocytomation). For immunofluorescent staining, 832/13 INS-1 cells were grown on glass 106

coverslips treated with poly-L-lysine (Sigma). Cells were washed and fixed in 4% 107

paraformaldehyde for 10 min, and blocked and permeabilized with 2% BSA, 0.05% Triton X-108

100 in PBS for 10 min. Then cells were incubated with the first COUP-TFII antibody (1:100) 109

followed by a FITC-conjugated goat anti-rabbit secondary antibody (1:400) (Jackson 110

ImmunoResearch). Slides were then mounted using Vectashield mounting medium with 4'-6-111

diamidino-2-phenylindole (DAPI) (Vector Laboratories). 112

Preparation of a recombinant virus expressing COUP-TFII and adenovirus infection. 113

The full-length human COUP-TFII cDNA was inserted into the KpnI/XhoI sites of the 114

pAdTrack-CMV shuttle vector which also contains a CMV/Green Fluorescent Protein (GFP) 115

reporter gene for following the efficiency of adenoviral infection (ad-hCOUP-TFII). 116

Recombinant adenoviral (ad–hCOUP-TFII) and control (pAdTrack with no exogenous gene) 117

plasmids were produced by INSERM U649, Nantes, France. 832/13 INS-1 cells were seeded 118

into 12-well tissue culture plates at a density of 0.9 x 106

cells/well in INS-1 medium and 24 h 119

later were exposed overnight to adenovirus at 2-5 plaque forming units (pfu) per cell. The 120

virus-containing medium was removed the next day and replaced by fresh medium for 36 h 121

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before extracting RNA (described below). After isolation, pancreatic islets were cultured for 2 122

hours in 11 mM glucose before exposing them to adenovirus at 200 pfu/cell for 1 h 30 min. 123

After infection, islets were cultured in fresh 11 mM glucose medium for 3 days before RNA 124

or protein extraction. 125

Nuclear extract preparation and electrophoresis mobility shift assays (EMSA). Nuclear 126

extracts were prepared from 832/13 INS-1 and mouse liver as described in (1) and (9). The 127

double-stranded oligonucleotide 5’-TGC AGC AGT CGT GTC AAA GTT CAC TAT ATA 128

GAG-3’ was used as the COUP-TFII DR-1 probe. The double-stranded M oligonucleotide 5’ 129

TGC AGC AGT CGT GAT GCA TTT CAC TAT ATA GAG-3’ was used as a COUP-TFII 130

DR-1 mutant. EMSA probe labeling, binding reactions, competitors and antibody supershift 131

were performed as reported previously (9). For supershift analysis with the HNF4α antibody 132

(kindly provided by M. Pontoglio and F. Ringeisen) and the COUP-TFII antibody (NR2F2, 133

PP-H7147-00, 1:500, Perseus Proteomics), 1 µl of antiserum was included in the binding 134

reactions. 135

Plasmids and site-directed mutagenesis. The reporter plasmid -3000/luc (-3047 to +873) 136

was subcloned into the pGL3-Basic vector (Promega) from the fragment described previously 137

(27) using BamHI/BglII restriction sites. Then fragments from –688 (EcoRI), -328 (ApaI), -138

48 (SacI), +202 (FspI), +418 (ApaI) and +639 (SacI) to +873, respectively, were subcloned 139

into the pGL3- Basic plasmid. Site-directed mutagenesis of the -328/luc reporter plasmid was 140

performed by GenScript, NJ, USA. Isolated clones were totally sequenced. We introduced 141

five point mutations at the DR-1 site as in the sequence of the double-stranded M 142

oligonucleotide (-328M/luc). The Renilla luciferase coding sequence from the pRL null 143

vector (Promega) is controlled by the RSV promoter (9). pcDNA3 HNF4α and pcDNA, a 144

dominant negative form of HNF4α, DN-HNF4α, have been described previously (22). 145

Transfection and reporter gene assay. Transient transfections were carried out with 146

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Lipofectamine 2000 reagent (Invitrogen). Transfected cells were cultured in INS-1 medium, 147

and harvested 24 h after transfection. Cell extracts were assayed for reporter enzyme activities 148

using the Dual Luciferase kit (Promega) as described (9). 149

Small interfering (si)RNA-mediated silencing and transfection. A 21-nucleotide RNA was 150

designed and synthesized by Qiagen SA (2-For-Silencing siRNA). This siRNA sequence 151

targets mouse and rat COUP-TFII (GenBankTM

accession numbers, 009697 and 080778, 152

respectively) but not human COUP-TFII (GenBankTM

accession number, 021005) or mouse 153

COUP-TFI (GenBankTM

accession number, 010151) and is from position 1023 relative to the 154

mouse gene start codon. The COUP-TFII siRNA sequence is 155

r(AGUGUGCUUUGGAAGAGUA) dTdT (sense) and r(UACUCUUCCAAAGCACACU) 156

dGdG (antisense) and the non specific control siRNA used was from Qiagen SA. 832/13 INS-157

1 cells were grown to 75-80% confluence in 10 cm diameter dishes. Then cells were 158

trypsinized and transiently transfected by electroporation with the Amaxa NucleofectorTM

II 159

device with solution T and program T20 (Amaxa Biosystems) using 68 pmol of siRNA 160

duplex per 1.2 x 106 cells. Nucleofection was done on cells in INS-1 medium in 12-well 161

plates, the culture medium was changed 24 h after electroporation, and RNA and protein 162

extractions were done 24 h later. 163

Isolation of mRNA from 832/13 INS-1 cells and mouse pancreatic islets and detection by 164

real-time quantitative PCR (RT-QPCR). Total RNA was extracted and purified from 165

cultured cells using the RNA-PLUSTM

reagent (Q-BIOgene) according to the instructions. 166

Reverse transcription was performed with 2 µg of total RNA using Superscript II Reverse 167

Transcriptase (Invitrogen) or 1 µg of total RNA using the iScriptTM

cDNA Synthesis Kit (Bio-168

Rad), depending on the gene and according to the manufacturers’ protocols. 169

Total RNA was extracted from hand-picked islets using the Absolutely RNA

Microprep kit 170

according to the instructions (Stratagene). Each RNA or nuclear extract sample was prepared 171

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from 200 to 400 islets from about three mice. RNA (0.5 µg) was reverse transcribed for RT-172

QPCR. 173

RT-QPCR was performed on 6.25 ng of reverse transcribed total RNA with 10 µM of each 174

primer (Eurogentec), 2 mM MgCl2, and 1 x Light Cycler DNA Master SYBR Green I mix in 175

a Light Cycler apparatus (Roche). The relative amounts of the different mRNAs were 176

quantified using the threshold cycle (CT) methodology. All samples were normalized to the 177

CT value of the cyclophilin reference mRNA. Forward and reverse primers used for specific 178

amplification of cDNA fragments, designed to hybridize to rat transcripts, were as follows: 179

5’-CAG AGC CAG CAG CAC ATC GAG-3’ and 5’-TTA AGT TCC TGC GGA CGC TCC 180

T-3’ for COUP-TFI (121 bp); 5’-CGC TCC TTG CCG CTG CT-3’ and 5’-AAG AGC TTT 181

CCG AAC CGT GTT-3’ for COUP-TFII (289 bp); 5’-TTG CCA TTC CTG GAC CCA AA-182

3’and 5’-ATG GCA CTG GTG GCA AGT CC-3’ for cyclophilin (325 bp); 5’-AAA TGT 183

GCA GGT GTT GAC CA-3’ and 5’-CAC GCT CCT CCT GAA GAA TC-3’ for HNF4α 184

(178 bp); 5'-AGC AGT GCT GGC TAC CTT CAA-3' and 5'-AAT ATG TAG CCA CCC 185

CCT TGG-3' for PPARα (97 bp); 5’-GCC CAG CTT AAT GCC ATC TTT-3’ and 5’- CAA 186

AAG GGC TGC CTT CTG TAA-3’ for Neuro-D1 (113 bp); and 5’-GCG CTG AGA GTC 187

CGT GAG-3’ and 5’-CCG GGG TAG GGA GCT ACA-3’ for Pdx1 (60 bp). 188

We checked that these COUP-TFII primers specifically amplify the rat (and mouse) COUP-189

TFII; they do not amplify COUP-TFII from cDNA prepared from a human cell line 190

expressing COUP-TFII. 191

Protein analysis by Western blotting and ECL detection. For total protein extraction from 192

832/13 INS-1 or INS-1 DN-HNF4α-26, 0.9 x 106 cells were washed in cold PBS. Cell pellets 193

were lysed in 100 µl of lysis buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, 194

30 mM Na4P2O7, 50 mM NaF with 1% Triton, 10 mg/ml leupeptin, 10 mg/ml pepstatin, 10 195

mg/ml aprotinin, and 1 mM phenylmethylsulfonyl fluoride). Nuclear extracts from pancreatic 196

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islets were obtained using NE-PER nuclear and cytoplasmic extraction reagents (Pierce). 197

Immunoblotting procedures were as described previously (1). Blots were developed with ECL 198

SuperSignal West Pico Chemiluminescent reagents (Pierce) and visualized using the high-end 199

CDD LAS-3000 imaging system (Fujifilm). Bands were quantified by densitometry using the 200

Multigauge 3.0 image processor program (FujiFilm) normalizing band intensities to those 201

obtained for the appropriate loading controls. Dilutions and sources of antibodies were as 202

follows: anti-myc tag (1:1000; sc40 from Santa Cruz), anti-COUP-TFII (1:500; NR2F2, PP-203

H7147-00 from Perseus Proteomics), anti-cyclophilin (1:3000; 07-313 from Upstate) and 204

anti-GAPDH (1:200; sc 25778 from Santa Cruz). 205

Chromatin immunoprecipitation (ChIP) assays. 832/13 INS-1 cells were cultured in INS-1 206

medium and ChIP was done as previously described (17). Supernatants were incubated with 207

polyclonal antibodies directed against HNF4α (Santa Cruz Biotechnologies, Santa Cruz, CA, 208

Cat#SC-8987 using 5 µl/mg protein) with normal rabbit IgG as the control (Santa Cruz, 209

Cat#sc2027), or a monoclonal antibody directed against COUP-TFII (NR2F2, PP-H7147-00, 210

Perseus Proteomics using 4µg/mg protein) with normal mouse IgG (IgG, Cat#12-371, 211

Upstate) as the control. The primer sequences used to amplify the COUP-TFII promoter 212

region by PCR were 5’-GCT AGG ACC GGG CTG TTC-3’ and 5’-TGA ACT TTG ACA 213

CGA CTG CTG-3’. The PCR primers for the COUP-TFII gene coding region were 5'-CAG 214

CAG CAG CAC ATC GAG-3' and 5'-GGC ACT ACT GGC ACT GGT TG-3'. 215

Statistical analysis. Quantitative results are expressed as the mean with the standard 216

deviation (S.D.). The Mann-Whitney test, a non parametric statistical program accepted to be 217

appropriate when the number of experiments is less than 10, was used for statistical analyses 218

and null hypotheses were rejected at P ≥ 0.05. All experiments were performed at least three 219

times. 220

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Results 221

COUP-TFII is expressed in adult mouse pancreatic islet beta cells and in the 832/13 222

INS-1 beta cell line. 223

We found previously using rabbit polyclonal COUP-TF antibodies that COUP-TFII and 224

COUP-TFI are expressed in mouse pancreatic islets (27). Here, using mouse monoclonal 225

antibodies specific to COUP-TFII and COUP-TFI, we observed that COUP-TFII protein 226

expression is restricted to mouse (Fig. 1A) and human (data not shown) pancreatic beta cells. 227

We detected COUP-TFI protein in mouse islet non-beta cells (Fig. 1C). These results suggest 228

that these two proteins have specific functions in mouse pancreatic islets. We then analysed 229

COUP-TFII protein expression in a beta cell line, 832/13 INS-1 (7), and found that COUP-230

TFII protein is expressed in the nuclei of these cells (Fig. 1E and F). This cell line was used as 231

a model to study at the molecular level whether HNF4α can regulate COUP-TFII expression. 232

233

HNF4αααα binds and transactivates the COUP-TFII gene promoter 234

We reported that the first 3000 nucleotides upstream of the transcription initiation site are 235

sufficient for transcription of the mouse COUP-TFII gene in insulin positive cells in 236

transgenic mice (27). To identify key promoter elements within this region, reporter plasmids 237

encoding the luciferase gene controlled by various portions of the mouse COUP-TFII gene 5’ 238

regulatory region were transiently transfected into 832/13 INS-1 cells (Fig. 2A). This deletion 239

analysis identified two major regions responsible for different promoter activities: a negative 240

element between nucleotides -688 and -328, the deletion of which led to increased activity, 241

and a strong positive element between nucleotides -328 and -48 relative to the transcription 242

start site. 243

Comparison of transcription factor DNA binding motifs in the latter region in different 244

species, revealed a conserved HRE, a direct repeat 1 (DR-1) with a single base pair spacer, 245

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that is known to bind nuclear hormone receptors including HNF4α (Fig. 2B) (14). So we 246

tested the ability of HNF4α to bind the DR-1 binding site of the COUP-TFII gene promoter in 247

vitro and in vivo. In antibody-mediated supershift assays using a probe containing the DR-1 248

element (Fig. 3A), incubation of 832/13 INS-1 nuclear extracts with the radiolabeled COUP-249

TFII DR-1 probe resulted in the formation of several complexes. The specificities of these 250

complexes were analyzed by competition with unrelated oligonucleotides and displacement or 251

supershifting by specific antibodies. All the complexes were specific as they could be 252

displaced by a 50 fold excess of the unlabeled COUP-TFII DR-1 oligonucleotide (Fig. 3, lane 253

5) but not by an unrelated oligonucleotide (Fig. 3, lane 6). The faster-migrating band 254

corresponds to HNF4α binding activity as it was displaced by anti-HNF4α antibodies (Fig. 3, 255

lane 2) whereas addition of anti-USF antibodies did not affect this binding activity (Fig. 3, 256

lane 3). In addition, none of the binding activities were altered by the inclusion of a 50 fold 257

excess of an unlabeled mutant version of the COUP-TFII DR-1 oligonucleotide (Fig. 3, lane 258

4). These results suggest that all the complexes could contain members of the nuclear receptor 259

superfamily including HNF4α. Liver cell nuclear extracts known to contain abundant HNF4α 260

proteins were tested by EMSA. In this case, stronger complexes were observed that could be 261

displaced with the anti-HNF4α serum. These results suggest that this DR-1 element is an 262

HNF4α DNA binding site. The in vivo relevance of the observed HNF4α binding activity was 263

analyzed in the context of chromatin in intact 832/13 INS-1 cells by chromatin 264

immunoprecipitation of endogenous HNF4α (Fig. 3B). HNF4α antibodies efficiently and 265

specifically immunoprecipitated the COUP-TFII promoter DNA, indicating there is a stable 266

association between the endogenous factor and the COUP-TFII promoter in vivo. 267

To test the involvement of HNF4α in the regulation of the –328 bp region of the COUP-TFII 268

gene, we first transfected 832/13 INS-1 cells with this reporter construct in the absence or 269

presence of a wild-type HNF4α expression plasmid. Figure 2C shows that HNF4α expression 270

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generated a 25-fold activation of the promoter but did not activate the -48 bp COUP-TFII 271

construct lacking the DR-1 DNA binding site. To further characterize the specific HNF4α-272

dependent activation, we used the selective dominant negative HNF4α (DN-HNF4α) mutant 273

which specifically forms defective heterodimers with wild-type HNF4α thereby preventing 274

DNA binding and hence transcriptional activation by HNF4α (5). DN-HNF4α had no effect 275

on the binding of PPARγ-RXRα heterodimers to a PPAR response element (22) or on the 276

binding of COUP-TFII to a DR-1 site in these cells (data not shown). Overexpression of DN-277

HNF4α results in a 40% inhibition of the –328 bp COUP-TFII basal promoter activity evoked 278

by endogenous HNF4α expression (Fig. 2C; bar 1 and bar 3 compared) and suppresses 279

transactivation by HNF4α (Fig. 2C; bar 2 and bars 4-5 compared). To definitively 280

demonstrate the implication of HNF4α in the control of the COUP-TFII -328 bp promoter 281

region, critical bases were mutated in the DR-1 site. The mutant sequence failed to bind 282

HNF4α as shown by EMSA (Fig. 3, lane 4). The activity of the mutant reporter plasmid (-283

328M/luc) was then tested in 832/13 INS-1 cells. The mutant showed a lower basal activity, 284

50% of the wild-type activity (Fig. 2C; bar 6 and bar 1 compared). In addition, the mutations 285

in the DR-1 site led to a complete loss of HNF4α transactivation (Fig. 2C; bars 7 and 8 and 286

bar 6 compared). Thus, we conclude that the COUP-TFII promoter is specifically activated 287

by HNF4α. 288

289

Mechanism of interdependency of the COUP-TFII and HNF4αααα genes 290

Given the strength of HNF4α transactivation on the –328 COUP-TFII promoter construct, we 291

wanted to assess its influence on endogenous COUP-TFII expression in beta cells. We used 292

DN-HNF4α-26 cells, a derivative of the INS-1 cell line that contains a plasmid encoding a 293

doxycycline-inducible dominant negative HNF4α (25). As shown in Fig. 4A, in the presence 294

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of doxycycline the c-Myc tagged DN-HNF4α protein is induced. As a consequence of the 295

dominant-negative suppression of HNF4α function there is a significant decrease in 296

endogenous COUP-TFII gene expression (Fig. 4B). 297

A transcriptional network with reciprocal activation of the HNF1α and HNF4α genes has 298

been described (10). These observations led us to test whether such reciprocal cross-299

regulation occurs between COUP-TFII and HNF4α in pancreatic beta cells in two ways. 300

Firstly, endogenous COUP-TFII expression was down-regulated, almost completely, by 301

electroporating 832/13 INS-1 cells with a specific COUP-TFII siRNA (Fig. 5A and B). In 302

these conditions, HNF4α gene transcript levels are decreased by 40% (Fig. 5C). Secondly, we 303

over-expressed COUP-TFII using a recombinant adenovirus encoding a human COUP-TFII 304

protein (Ad-hCOUP-TFII) in 832/13 INS-1 cells and in mouse pancreatic islets. Based on 305

GFP reporter expression, 80% of 832/13 INS-1 cells and 50% of islet beta cells were infected 306

(data not shown). EMSA using the COUP-TFII binding probe showed that COUP-TFII was 307

strongly expressed in 832/13 INS-1 cells (Fig. 6A). Similarly, after infection of pancreatic 308

islets with Ad-hCOUP-TFII, COUP-TFII was readily detected by western blot (Fig. 6A). As 309

shown in Fig. 6B, adenovirus-mediated induction of COUP-TFII resulted in a 300% increase 310

in HNF4α mRNA expression in 832/13 INS-1 cells and a 100% increase in HNF4α mRNA 311

expression in pancreatic islets. A known target of HNF4α, the nuclear receptor peroxisome 312

proliferator-activated receptor α (PPARα) expression, was also induced under these 313

conditions (Fig. 6C). Together, these results indicate there is a cross-talk between HNF4α and 314

COUP-TFII. 315

316

Autoregulation of the COUP-TFII promoter 317

Observation of a COUP-TFII DR-1 complex in Fig. 6A (left panel) led us to hypothesize that 318

COUP-TFII might control its own expression. We tested whether endogenous rat COUP-TFII 319

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mRNA expression was modified when 832/13 INS-1 cells were infected with the human 320

COUP-TFII expression adenovirus. As shown in Fig. 6D, Ad-hCOUP-TFII decreased the 321

endogenous rat COUP-TFII mRNA expression by 30%. Next, we determined in vivo 322

promoter occupancy by chromatin immunoprecipitation from 832/13 INS-1 cells. 323

Endogenous COUP-TFII occupied the region of the rat COUP-TFII promoter gene that 324

contains the DR-1 DNA binding site between nucleotides –57 and –69 (Fig. 6E). These data 325

suggest that COUP-TFII has the capacity to directly auto-repress its expression. 326

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Discussion 327

Until now no ligand has been found for COUP-TFII which exhibits a constitutive 328

transcriptional activity as an activator or a suppressor in different cell types. It is therefore of 329

interest to identify the transcription factors that modulate COUP-TFII gene expression. The 330

central issue addressed here is the transcriptional regulation of the murine COUP-TFII gene 331

by the nuclear receptor HNF4α/MODY1. Unexpectedly, HNF4α expression was found to be 332

controlled by COUP-TFII expression in pancreatic beta cells. 333

We show here that HNF4α binds to a DR-1 DNA binding site in the proximal -328 bp COUP-334

TFII promoter in vitro and in vivo and activates both reporter gene expression and 335

endogenous COUP-TFII gene expression in beta cells. When a series of promoter-reporter 336

constructs were transiently transfected, we observed that deletion of an upstream region (-337

688/-328 bp) significantly increases the activity of the -328 bp COUP-TFII promoter. The -338

688 bp construct still mediates HNF4α activation but this response is less than that with the -339

328 bp construct (data not shown). We speculate that this repressive element may modulate 340

the endogenous activation of COUP-TFII by HNF4α and could explain the lesser effect of 341

HNF4α on endogenous COUP-TFII mRNA expression. 342

In addition, we demonstrate that COUP-TFII positively regulates HNF4α expression. To 343

assess the importance of HNF4α gene modulation, we measured the expression of its target 344

gene PPARα. Strong suppression of COUP-TFII in 832/13 INS-1 cells decreased HNF4α 345

gene transcript levels by 40%. There were no changes in PPARα target gene expression 346

though, probably because of the remaining HNF4α mRNA. However, when COUP-TFII is 347

overexpressed it leads to a marked increase in HNF4α with a statistically significant elevation 348

in PPARα gene expression. 349

In addition, the 6.8 kbp P1 and 4.1 kbp P2 HNF4α promoter regions (2) are transactivated by 350

COUP-TFII in COS cells suggesting a possible transcriptional control of COUP-TFII on 351

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HNF4α gene expression (unpublished data). 352

Reciprocal cross-regulation between COUP-TFII and HNF4α raises some important 353

questions with respect to the transcriptional activities of these factors. Other regulatory events 354

in the same circuit may prevent undesirably high intracellular expression levels. In a similar 355

vein, we show that COUP-TFII negatively regulates its own expression. In addition, COUP-356

TFII binds to the same DNA binding site as HNF4α suggesting a possible competition for 357

occupancy of this site (schematically presented in Fig. 7). 358

The physiological relevance of the cross-regulation between COUP-TFII and HNF4α might 359

be in an amplification loop controlling an acute response to a specific signal such as those 360

received in the fasted state. HNF4α expression is high during fasting and decreases in liver 361

(26) and in pancreas (Perilhou A., unpublished data) upon refeeding and correlates well with 362

the variations in COUP-TFII transcript and protein levels (Perilhou A., unpublished data). 363

The pathophysiology of maturity-onset diabetes of the young (MODY) is caused by 364

mutations in five separate genes encoding transcription factors including HNF4α, the 365

MODY1 gene (Fig. 7). This factor is involved in transcriptional regulatory networks in both 366

liver and pancreas (19, 25). It has been hypothesized that MODY could result from a collapse 367

of cell-specific transcription networks due to the haplo-insufficiency of key network genes 368

(19). In this context, our genetic analysis of COUP-TFII function in pancreatic beta cells is 369

relevant in that haplo-insufficient mice have an impaired insulin secretion phenotype (1) and 370

we have observed loss of glucose-stimulated insulin secretion in the 832/13 INS-1 cells 371

treated with COUP-TFII siRNA (Perilhou A., unpublished data). 372

In conclusion, the results shown here are of particular interest in that COUP-TFII can be 373

considered as a candidate gene for MODYX. The COUP-TFII promoter contains a functional 374

DR-1 binding site for the HNF4α protein and the molecular basis of many MODY1 375

phenotypes remains to be defined (18). Future studies should therefore include the search for 376

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mutations in the human COUP-TFII gene in patients with MODYX. 377

378

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Acknowledgments 500

This work was supported by grants from Nestlé France 2007, from the Programme National 501

de Recherches sur le Diabète-INSERM/ARD (PNRD-A06064KS), from the Agence 502

Nationale pour la Recherche (ANR 2005 Cardiovasculaire Obésité et Diabète, ANR-05-503

PCOD-088-01), from the Swiss National Science Foundation (grant no. 32-66907.01 to 504

CBW) and from ADA (grant 7-04-RA-106). A. P. is the recipient of a doctoral fellowship 505

from the Ministère de l’Enseignement Supérieur et de la Recherche and from Fondation pour 506

la Recherche Médicale. 507

We are grateful to Dr C. Newgard for the 832/13 INS-1 cell line and to Dr F. Petit for the gift 508

of some of the COUP-TFII promoter constructs. We thank all the INSERM U649 team for 509

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their expertise in adenovirus preparation. We thank Dr P. Bossard for critical reading of the 510

manuscript and Dr T. Becker for helpful discussion of the siRNA experiments. We thank Drs 511

K. Kaestner and J. Ferrer for helpful discussions. 512

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Figure legends 513

Figure 1: COUP-TFII expression in adult mouse pancreas and in the 832/13 INS-1 cell 514

line. Immunostaining of pancreatic sections from adult mice using mouse monoclonal 515

antibodies against COUP-TFII (A), insulin (B and D), COUP-TFI (C) and a secondary 516

antibody as control of specificity (E and F). The sections shown are from the same experiment 517

with the same exposure time. Magnification, ×200. (G) Immunofluorescence staining for 518

COUP-TFII and (H) nuclear counterstaining using Dapi in 832/13 INS-1 cells. Magnification, 519

×1000. 520

521

Figure 2: Mapping the COUP-TFII promoter and its transactivation by HNF4αααα. (A) 522

Various lengths of the mouse COUP-TFII promoter, designated by their 5’ ends relative to the 523

defined COUP-TFII initiation transcription site (20), driving a luciferase reporter (Luc) gene 524

and a control vector expressing Renilla luciferase were transiently co-transfected into 832/13 525

INS-1 cells (0.45 µg DNA per million cells). The ratio of firefly to Renilla luciferase 526

activities is expressed in arbitrary units as fold induction relative to the activity of the 527

promoterless pGL3 expression vector. *p<0.01 compared to values for -48 and -688 528

constructs. **p<0.01 compared to value for -328 construct. (B) The DNA sequence contains 529

an HRE motif in the –328 bp COUP-TFII promoter. The HRE is a direct repeat (DR-1) 530

(arrows) with a one nucleotide spacer and is conserved in mouse, rat and human. (C) Effect of 531

co-transfecting either WT-HNF4α (bars 2, 7, 8, 10 and 11) or DN-HNF4α (bars 3 and 12) or 532

both vectors (bars 4 and 5) at the concentrations indicated on reporter plasmids encoding the 533

luciferase gene controlled by –328 bp, -328 bp DR-1 mutated and -48 bp COUP-TFII 534

constructs in 832/13 INS-1 cells. Bars 1, 6 and 9, transfections with 100 ng of expression 535

vector with no cDNA. $p < 0.01 compared to value for bar 2.

*p < 0.05 compared to value for 536

bar 3. §p < 0.05 compared to value for bar 6.

**p < 0.01 compared to values for bars 4 and 5. 537

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Means ± S.E.M. are shown for at least three separate transfections performed in triplicate. 538

539

Figure 3: HNF4αααα binds to the COUP-TFII DR-1 site in vitro and in vivo. (A) EMSA 540

using the COUP-TFII DR-1 probe is shown. The probe was incubated with 832/13 INS-1 cell 541

nuclear extracts (lanes 1-6) and mouse liver nuclear extracts (lanes 7-9). Competition 542

experiments were performed with 50 ng of unlabeled oligonucleotides. Lane 1, 832/13 INS-1 543

nuclear extracts; lane 2, as lane 1 plus 1 µl of anti-HNF4α antibody; lane 3, as lane 1 plus 1 544

µl of anti-USF antibody; lane 4, as lane 1 plus M competitor (COUP-TFII DR-1 mutant); 545

lane 5, as lane 1 plus COUP-TFII DR-1 competitor; lane 6, as lane 1 plus MLP competitor; 546

lane 7, liver nuclear extracts; lane 8, as lane 7 plus 1 µl of anti-USF antibody; lane 9, as lane 547

7 plus 1 µl of anti-HNF4α antibody. Positions of HNF4α-specific retarded bands are 548

indicated by arrows. The asterisk indicates the position of the supershifted complexes. F, free 549

probe; ns, non specific. (B) ChIP from 832/13 INS-1 cells. Targets for real time quantitative 550

PCR amplifications were the proximal COUP-TFII promoter containing the DR-1 DNA 551

binding site and a downstream coding region (negative control). The panel shows the results 552

of three independent experiments measuring the relative amount of target chromatin 553

precipitated by the antibody against HNF4α compared to that precipitated by control IgG, and 554

is presented as an average of the percentage of input normalized to the IgG coding region 555

control (+/- S. E.M., *p = 0.03). 556

557

Figure 4: COUP-TFII mRNA expression in INS-1 DN-HNF4αααα expressing cells. Cells 558

were cultured in the presence or absence of 500 ng/ml doxycycline for 24 h. (A) The top 559

panel shows DN-HNF4α expression determined by western blot analysis with antibodies 560

against the Myc tag (30 µg/lane). The lower panel shows the same blot hybridized with 561

antibodies against cyclophilin as the loading control. (B) RT-QPCR analysis of COUP-TFII 562

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mRNA in INS-1 DN-HNF4α-26 cells. Results are means ± S.E.M. of three independent 563

experiments. 564

565

Figure 5: HNF4αααα mRNA expression in siRNA COUP-TFII transfected 832/13 INS-1 566

cells. 832/13 INS-1 cells were electroporated with unrelated (control) or COUP-TFII specific 567

siRNA (siRNA) and cultured for 48 h in INS medium. Total RNA was extracted and 568

subjected to RT-QPCR to measure mRNA levels of COUP-TFII, controls Neuro-D1 and 569

Pdx1 (A), and HNF4α (C). Results are means ± S.E.M. from 8 independent experiments. (B) 570

Western blot of nuclear extracts (20 µg/lane) probed with antibodies against COUP-TFII 571

(upper panel) and against GAPDH as a loading control (lower panel). 572

573

Figure 6: Effects of adenovirus-mediated overexpression of COUP-TFII in 832/13 INS-1 574

and mouse pancreatic beta-cells. 832/13 INS-1 cells and islets were cultured in INS-1 575

medium and infected with Ad-GFP or Ad-hCOUP-TFII as indicated (2 and 5 pfu/cell) and 576

200 pfu/cell respectively. (A) Left panel. For EMSA, the oligonucleotide duplex 577

corresponding to the COUP-TFII DR-1 site was used as probe. Lane 1, ad-GFP 832/13 INS-1 578

nuclear extracts; lane 2, ad-hCOUP-TFII 832/13 INS-1 nuclear extracts; lane 3, as lane 2 plus 579

1 µl of anti-COUP-TFII antibody. Position of COUP-TFII-specific retarded band is indicated 580

on the left (arrow). The asterisk indicates the position of the supershifted complexes. (A) 581

Right panel. A representative Western blot of COUP-TFII expression; the upper panel shows 582

COUP-TFII protein; the lower panel shows the same blot incubated with antibodies against 583

GAPDH as loading control. Total RNA was extracted and subjected to RT-QPCR to measure 584

mRNA levels of HNF4α (B), PPARα (C) and endogenous rat COUP-TFII (D). (E) ChIP from 585

832/13 INS-1 cells. Targets for amplifications were the proximal COUP-TFII promoter 586

containing the DR-1 DNA binding site and a downstream coding region as a negative control. 587

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The panel shows the results of three independent experiments measuring the relative amount 588

of target chromatin precipitated by the antibody against COUP-TFII compared to control IgG. 589

The results are presented as an average of the percentage of input normalized to the IgG 590

coding region control (+/- S.E.M. *p=0.003). 591

592

Figure 7: Schematic representation of the positive (pointed arrows) and negative (flat-headed 593

arrow) effects of COUP-TFII integrated in a complex regulatory network (adapted from G. 594

Velho (23)). 595

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