The rat pancreatic β-cell line, INS-1 (24), which had kindly been provided by Dr C.B. Wollheim and Dr N. Sekine (University of Geneva, Geneva, Switzerland), was cultured at 37°C in a 5% CO₂ atmosphere in RPMI-1640 medium which was supplemented with 10% fetal bovine serum, 10 mM HEPES, 1 mM sodium pyruvate and 50 mM 2-mercaptoeth-anol. KP-3 (human pancreatic cancer), KP-4 (pancreatic ductal cell carcinoma), QGP-1 (human pancreatic endocrine) and SUIT-2 (human pancreatic cancer) cells were obtained from the Health Science Research Resources Bank (Osaka, Japan). AsPC-1 (human pancreatic adenocarcinoma), Capan-1 (human pancreatic ductal adenocarcinoma), 293T and HeLa cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). The KP-3, QGP-1, SUIT-2 and AsPC-1 cells were cultured in RPMI-1640 medium which was supplemented with 10% fetal bovine serum. The KP-4 and Capan-1 cells were cultured in Iscove’s modified Dulbecco’s medium (IMDM) supplemented with 10% fetal bovine serum. The 293T and HeLa cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) which was supplemented with 10% fetal bovine serum.

The oligonucleotides used in the present study were synthesized at Japan BioServices Co. Ltd. (Saitama, Japan). These oligonucleotides contained either risk or non-risk alleles through single-nucleotide substitution (Table I). DNA-immobilized nanobeads were prepared as previously described with slight modifications (21,25). Two complementary oligonucleotides were phosphorylated, annealed and then ligated to produce oligomers that ranged mainly from 100 to 3,000 bp. These oligomers containing tandemly repeated SNP regions were applied to the NICK Column according to the manufacturer’s instructions (GE Healthcare, Buckinghamshire, UK). The second fractions of the NICK Column were ready for coupling onto FG beads (Tamagawa Seiki Co., Ltd., Nagano, Japan) (23). One milligram of FG beads was then mixed with 100 µg of prepared DNA, and the coupling reaction was carried out at 50°C for 24 h.

Nuclear extracts were prepared from the INS-1 cells according to the method described in the study by Dignam et al (26). DNA-immobilized nanobeads (200 µg) were equilibrated with binding buffer (20 mM HEPES-NaOH, pH 7.9, 10% glycerol, 100 mM KCl, 1 mM MgCl₂, 0.2 mM CaCl₂, 0.2 mM EDTA, 1 mM DTT and 0.2 mM PMSF). INS-1 nuclear extracts (200 µg) were mixed with 100 µg of single-stranded DNA (Ambion/Life Technologies, Austin, TX, USA) and 10 µg of poly(dI-dC):poly(dI-dC) (Sigma-Aldrich, St. Louis, MO, USA), and then incubated with equilibrated DNA-immobilized nanobeads for 4 h at 4°C using a RT-50 rotator (Taitec Corp., Saitama, Japan). After washing with binding buffer 3 times, the bound proteins were eluted with binding buffer supplemented with 1 M KCl (final concentration).

Affinity-purified proteins were separated using SDS-PAGE and subjected to silver staining as described in the study by Shevchenko et al (27). The specific protein bands were excised and mass spectrometry was then performed as previously described (28). Band slices were reduced with 10 mM dithiothreitol, alkylated with 55 mM iodoacetamide and digested with sequencing grade modified trypsin (12 µg/ml; Promega Corp., Madison, WI, USA) overnight. The extracted peptides were desalted with ZipTip C18 (Millipore, Billerica, MA, USA) and separated by nano-flow liquid chromatography (LC) (Paradigm MS4; Michrom BioResources, Inc., Auburn, CA, USA) using a reverse phase C18 column (Magic C18, 0.2 ×50mm; Michrom BioResources, Inc.). The LC eluent was coupled to a micro-ionspray source attached to an LCQ Advantage MAX mass spectrometer (Thermo Fisher Scientific Inc., Waltham, MA, USA). All MS/MS spectra were searched using the TurboSEQUEST algorithm within the BioWorks 3.2 software (Thermo Fisher Scientific Inc.).

Nuclear extracts and affinity-purified fractions were fractionated by SDS-PAGE and electrotransferred onto a polyvinylidene difluoride membrane (Millipore). The membrane was incubated with 5% non-fat milk for 1 h and incubated with antibody against nuclear transcription factor Y, beta (NF-YB) overnight (sc-13045; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), followed by incubation with peroxidase-conjugated anti-rabbit IgG (no. 7074; Cell Signaling Technology, Inc., Danvers, MA, USA) for 1 h. Visualization was accomplished using enhanced chemiluminescense reagents (Millipore) and analyzed using the ChemiDoc XRS+ imaging system (Bio-Rad, Hercules, CA, USA).

Complementary DNA (cDNA) for nuclear transcription factor Y, alpha (NF-YA), NF-YB, nuclear transcription factor Y, gamma (NF-YC) and cut-like homeobox 1 (CUX1) were amplified by RT-PCR from the total RNA prepared from HeLa cells, inserted into the pUC119 vector, and sequenced using an ABI PRISM 3700 sequence analyzer (Applied Biosystems, Foster City, CA, USA). The preparation of radiolabeled recombinant proteins was then performed as previously described (28). T7 promoter-tagged DNA fragments of these cDNAs were amplified using PCR with two primers: T7 promoter fused to a 5′ sequence of each cDNA and polyT fused to a 3′ complementary sequence of each cDNA. These amplified DNA fragments were used to synthesize 35S-radiolabeled recombinant proteins in a coupled transcription/translation system according to the protocol of the manufacturer (Promega Corp.).

DNA-immobilized nanobeads or control nanobeads (200 µg) were equilibrated with binding buffer as described above and incubated with 200 µl of the radiolabeled proteins (NF-YA, NF-YB, NF-YC and CUX1) at 4°C for 4 h using a RT-50 rotator. After washing with binding buffer, the bound proteins were eluted by boiling for 5 min with SDS-PAGE sample buffer. The eluates and inputs were subjected to SDS-PAGE. The gels were dried, and autoradiography was then performed to visualize the radiolabeled proteins.

Reporter plasmids that contain the luciferase gene under the control of the SNP rs2074196 region were constructed as follows: complementary oligonucleotides containing 3 copies of the SNP region were synthesized. These oligonucleotides were annealed and cloned into the pGL4.10 vector (Promega Corp.) along with the thymidine kinase promoter derived from pGL4.74 (Promega Corp.).

To construct mammalian expression plasmids, cDNA of NF-YA, NF-YB, NF-YC and CUX1 were amplified by PCR and subcloned into the pcDNA6 vector (Invitrogen Life Technologies, Carlsbad, CA, USA) to allow the expression of these cDNAs under the control of a CMV promoter. During PCR amplification, FLAG-tag was fused at the C-terminus of NF-YA; HA-tag was fused at the C-terminus of NF-YB; and Myc-tag was fused at the C-terminus of NF-YC. All the sequences were confirmed using an ABI PRISM 3700 sequence analyzer.

One of the constructed luciferase reporter plasmids (100 ng) and the control Renilla luciferase reporter vector pGL4.74 (10 ng) were co-transfected into the 293T cells (2x10⁵ cells) along with mammalian expression vectors: NF-Ys (NF-YA-FLAG, NF-YB-HA, and NF-YC-Myc, 300 ng each), CUX1 (900 ng), or empty pcDNA6 (900 ng) using Lipofectamine 2000 (Invitrogen Life Technologies) and seeded into a 24-well plate. Twelve hours after transfection, the medium was changed. Forty-eight hours after transfection, cell extracts were prepared, and Firefly luciferase activities and Renilla luciferase activities were determined using a Dual-Glo Luciferase assay system (Promega Corp.) and a CentroPRO LB 962 Microplate Luminometer (Berthold Technologies, Bad Wildbad, Germany).

The preparation of the samples was performed using a Magna ChIP G kit according to the manufacturer’s instructions (Millipore, Temecula, CA, USA). The samples were sonicated for 10 rounds of 30 pulses each (0.5 sec on and 0.5 sec off) using a Branson Sonifier S-450 digital ultrasonic cell disruptor/homogenizer (Branson Ultrasonics Corp., Danbury, CT, USA) at 15% amplitude. The efficiency of sonication was assessed to ensure that the majority of chromatin fragments were in the range of 200–1,000 bp. The sonicated samples were immunoprecipitated overnight at 4°C with 2.5 µg of anti-Myc-tag antibody (no. 2276; Cell Signaling Technology, Inc.) or normal mouse IgG (Millipore). The immunoprecipitated DNA was quantified by qPCR performed on the input and bound fractions, using the Fast SYBR-Green Master Mix and a StepOne Real-Time PCR system (both from Applied Biosystems). The primers used for qPCR were as follows: 5′-GAGCCAGTTGTTCCCAAACC-3′ and 5′-TAGGCTTGTGTCCCCAGTCC-3′ for SNP rs2074196, and 5′-CGCAAAAGGAGGGGAGAG-3′ and 5′-GCCGCTGGGTTTTATAGGG-3′ for the ACTB promoter.

All data are expressed as the means ± SD. Statistical analysis was performed using an unpaired two-tailed Student’s t-test. A value of P<0.01 was considered to indicate a statistically significant difference.

In this study, we aimed to identify proteins that bind the genomic region encompassing the type 2 diabetes-associated SNPs: rs151290, rs2074196, rs2237892, rs2283228, rs2237895 and rs2237897 (Fig. 1A). In order to identify the allele-specific binding proteins, we first prepared 2 types of beads, risk and non-risk allele beads, by immobilizing each allelic DNA onto magnetic nanobeads (Table I and Fig. 1B). The lengths of the immobilized DNA fragments were almost the same (100–3,000 bp) for the risk and non-risk alleles (Fig. 2A). Using these nanobeads, we purified DNA-binding proteins from the nuclear extract of INS-1 pancreatic β cells and analyzed the bound proteins using SDS-PAGE (Fig. 2B). We found that the affinities of several proteins for each examined SNP differed between alleles. There were obvious allele-specific bands, particularly in the SNP rs151290 and SNP rs2074196 regions. We decided to perform the following analysis with SNP rs2074196, as this was among the 3 SNPs (rs2074196, rs2237892 and rs2237895) which showed the best association P-values with type 2 diabetes in our previous study (2).

We compared the bound proteins of the risk and non-risk alleles and selected 4 differentially bound protein bands (Fig. 3A). Following mass spectrometry, we identified 3 proteins, CUX1, NF-YC and NF-YA. One protein band was not determined using mass spectrometry. However, we speculated that the band was NF-YB, due to the size of the protein band and the lack of a partner protein for the NF-Y ternary complex. Immunoblot analysis using a specific antibody against NF-YB confirmed that NF-YB had been purified using DNA-immobilized nano-beads in an allele-specific manner (Fig. 3B).

We then confirmed the DNA-binding activities of NF-Y and CUX1 in vitro using recombinant proteins. NF-YA, NF-YB or NF-YC alone exhibited no binding activity or only non-specific binding activity against the genomic region encompassing SNP rs2074196 (Fig. 4A). The ternary complex of NF-YA, NF-YB and NF-YC exhibited specific binding activity against the non-risk allele of the genomic region encompassing SNP rs2074196 (Fig. 4B). CUX1 also exhibited specific binding activity against the non-risk allele of the genomic region encompassing SNP rs2074196 (Fig. 4B). These DNA-binding specificities against the non-risk allele of recombinant NF-Y and CUX1 were consistent with those observed in the silver staining of purified proteins from the INS-1 cell nuclear extracts (Figs. 2B and 3A).

We then examined the transcriptional activities of NF-Y and CUX1 using luciferase reporter assay. As illustrated in Fig. 5A, we prepared 4 reporter constructs: risk allele and forward orientation, non-risk allele and forward orientation, risk allele and reverse orientation, and non-risk allele and reverse orientation. The basal luciferase activities, which are mediated by endogenous transcription factors possibly including NF-Y and CUX1, differed among the constructs (Fig. 5B): non-risk-forward, 1.60-fold; risk-reverse, 0.57-fold; and non-risk-reverse, 0.84-fold higher compared to risk-forward. Hence, the basal luciferase activities of non-risk-forward were the highest for these 4 constructions in 293T cells.

When the expression constructs of NF-YA, NF-YB and NF-YC were co-transfected, the luciferase activities of each reporter construct were upregulated (Fig. 5B): risk-forward, 1.16-fold; non-risk-forward, 3.45-fold, risk-reverse, 1.07-fold; and non-risk-reverse, 1.66-fold compared to basal risk-rorward; in addition non-risk-forward, 2.16-fold, risk-reverse, 1.87-fold, and non-risk-reverse, 1.98-fold compared to the basal value of each construction. These results confirmed that the NF-Y complex upregulated the transcription under the control of the genomic region encompassing SNP rs2074196, particularly for the non-risk allele and forward orientation.

When the expression construct of CUX1 was co-trans-fected, the luciferase activities of each reporter construct were downregulated (Fig. 5B). These results indicated that CUX1 downregulated the transcription under the control of the genomic region encompassing SNP rs2074196; however, no allele specificity was observed.

We further examined the DNA-binding activities of NF-Y in cell lines using chromatin immunoprecipitation assay using 6 cell lines: QGP-1, SUIT-2, KP-4, KP-3, AsPC-1 and Capan-1. The QGP-1 and SUIT-2 cells were found to be homozygous for the non-risk allele, the KP-4 and KP-3 were heterozygous for the risk and non-risk allele, and the AsPC-1 and Capan-1 cells were homozygous for the risk allele. We transfected these cells with the expression vectors, NF-YA-FLAG, NF-YB-HA, and NF-YC-Myc. As illustrated in Fig. 6A, the genomic region encompassing SNP rs2074196 was enriched in the QGP-1, SUIT-2, KP-4 and KP-3 cells, but not in the AsPC-1 and Capan-1 cells. As a positive control, the genomic region of the ACTB promoter containing NF-Y-binding CCAAT-box elements was enriched in all the examined cell lines (Fig. 6B). These results confirmed that NF-Y specifically bound the non-risk allele of the endogenous KCNQ1 intronic region encompassing SNP rs2074196.