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
17
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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