Transcription factor organic cation transporter 1 (OCT-1) affects the expression of porcine Klotho (KL) gene

Ethics statements

All animal procedures were performed according to the protocols approved by the Biological Studies Animal Care and Use Committee of Hubei Province, PR China. Sample collection was approved by the ethics committee of Huazhong Agricultural University (No. 30700571 for this study).

MARC0022311 polymorphism in pigs

Nineteen Landrace × DIV crossbred pigs were genotyped with the Porcine SNP60 BeadChip (Illumina) using the Infinium HD Assay Ultra protocol, which was conducted under the technical assistance by Compass Biotechnology Corporation. DIV was a synthetic dam line derived by crossing Landrace, Large White, Tongcheng or Meishan pigs. Raw data had high genotyping quality (call rate >0.99) and were analyzed with the GenomeStudio software.

In silico sequence analysis

KL gene sequence ENSSSCG00000009347 was available on the ENSEMBL online website (http://asia.ensembl.org/index.html). We obtained the up-stream sequence of porcine KL gene for promoter prediction. The potential promoter was analyzed using the online neural network promoter prediction (NNPP) (http://www.fruitfly.org/seq_tools/promoter.html) and Promoter 2.0 prediction server (http://www.cbs.dtu.dk/services/Promoter/). Transcription factor binding sites were predicted using biological databases (BIOBASE) (http://www.gene-regulation.com/pub/programs.html) with a threshold of 0.90 and TFsearch with a threshold of 85 (Akiyama, 1995). Threshold represents minimum probability of predicted transcription factor.

Cell culture, transient transfection and luciferase assay

The porcine kidney (PK) cells and swine testis (ST) cells obtained from China Center for Type Culture Collection (CCTCC) were cultured at 37 °C in a humidified atmosphere of 5% CO2 using DMEM supplemented with 10% FBS (Gibco).

Four KL promoter deletion fragments (KL-D1: −178 bp to −3 bp, KL-D2: −418 bp to −3 bp, KL-D3: −599 bp to −3 bp and KL-D4: −835 bp to −3 bp) were cloned into pGL3-Basic vector to determine the core promoter region. The plasmids contained pig KL intron 1 fragments (g.1474 A and g.1474 G) followed by KL-D2 promoter (−418 bp to −3 bp) were reconstructed, then transfected using lipofectamine 2000 (Invitrogen) into PK cells and ST cells. Plasmid DNA of each well used in the transfection containing 0.8 µg of KL promoter constructs and 0.04 µg of the internal control vector pRL-TK Renilla/luciferase plasmid. The enzymatic activity of luciferase was then measured with PerkinElmer 2030 Multilabel Reader (PerkinElmer).

RNA interference

Double-stranded small interfering RNAs (siRNAs) targeting OCT-1 were obtained from GenePharma. Cells were co-transfected with 2 µl of siRNA, 0.2 µg of reconstructed plasmids using Lipofetamine 2000™ reagent for 24 h. Transfection mixtures were removed, and fresh complete DMEM medium was added to each well. Finally, the enzymatic activity of luciferase was then measured with PerkinElmer 2030 Multilabel Reader (PerkinElmer).

Quantitative real time PCR (qPCR)

qPCR was performed on the LightCycler^® 480 (Roche) using SYBR^® Green Real-time PCR Master Mix (Toyobo). Primers used in the qPCR were shown in Table 1. qPCR conditions consisted of 1 cycle at 94 °C for 3 min, followed by 40 cycles at 94 °C for 40 s, 61 °C for 40 s, and 72 °C for 20 s, with fluorescence acquisition at 74 °C. All PCRs were performed in triplicate and gene expression levels were quantified relatively to the expression of β-actin. Analysis of expression level was performed using the 2^−ΔΔCt method (Livak & Schmittgen, 2001). Student’s t-test was used for statistical comparisons.

Western blotting

Western blotting was performed as described previously (Tao et al., 2014). Five µg proteins were boiled in 5 × SDS buffer for 5 min, separated by SDS-PAGE, and transferred to PVDF membranes (Millipore). Then, the membranes were blocked with skim milk and probed with anti-KL (ABclonal). β-actin (Santa Cruz) was used as a loading control. The results were visualized with horseradish peroxidase-conjugated secondary antibodies (Santa Cruz) and enhanced chemiluminescence.

Electrophoretic mobility shift assays (EMSA)

Nuclear protein of PK and ST cells was extracted with Nucleoprotein Extraction Kit (Beyotime). The oligonucleotides (Sangon) corresponding to the OCT-1 binding sites of KL intron 1 (Table 1) were synthesized and annealed into double strands. The DNA binding activity of OCT-1 protein was detected by LightShift^® Chemiluminescent EMSA Kit (Pierce). Ten µg nuclear extract was added to 20 fmol biotin-labeled double stranded oligonucleotides, 0.1 mM EDTA, 2.5% Glycerol, 1× binding buffer, 5 mM MgCl₂, 50 ng Poly (dI⋅dC) and 0.05% NP-40. In addition, control group added 2 pmol unlabeled competitor oligonucleotides, while the super-shift group added 10 µg OCT-1 antibodies (Santa Cruz). The mixtures were then incubated at 24 °C for 20 min. The reactions were analyzed by electrophoresis in 5.5% polyacrylamide gels at 180 V for 1 h, and then transferred to a nylon membrane. The dried nylon was scanned with GE ImageQuant LAS4000 mini (GE-Healthcare).

Chromatin immunoprecipitation (ChIP) assay

ChIP assays were performed using a commercially available ChIP Assay Kit (Beyotime) as previously described (Tao et al., 2015). Briefly, after crosslinking the chromatin with 1% formaldehyde at 37 °C for 10 min and neutralizing with glycine for 5 min at room temperature, PK and ST cells were washed with cold PBS, scraped and collected on ice. Then, cells were harvested, lysed and treated by sonication. Nuclear lysates were processed 20 times for 10 s with 20 min intervals on ice water using a Scientz-IID (Scientz). An equal amount of chromatin was immune-precipitated at 4 °C overnight with at least 1.5 µg of OCT-1 antibody (Santa Cruz) and normal mouse IgG antibody (Millipore). Immune-precipitated products were collected after incubation with Protein A + G coated magnetic beads. The beads were washed, and the bound chromatin was eluted in ChIP elution buffer. Then the proteins were digested with Proteinase K for 4 h at 45 °C. The DNA was purified using the AxyPrep PCR Cleanup Kit (Axygen). The DNA fragment of OCT-1 binding sites in KL intron 1 was amplified with the specific primers (Table 1). The PCR procedure was executed with 36 rounds and in the linear range, ChIP assay had 3 biological replicates.

Statistical analysis

Statistical analyses based on two-tailed Student’s t-tests were performed using the Statistical Package for the Social Sciences software. Significance was determined at a 95% confidence interval. All data were expressed as the mean ± standard deviation (S.D.).

MARC0022311 status in pigs

MARC0022311 in KL intron 1 appeared a polymorphism (A or G) in 19 Landrace × DIV pigs, with 12 AA pigs and AG pigs genotyped using the Illumina PorcineSNP60 chip (Data S1) . The SNP (MARC0022311) in pig KL intron 1 was renamed as KL g.1474 A > G according to the standard mutation nomenclature (Den Dunnen & Antonarakis, 2000).

Identification of promoter region of the porcine KL gene

An 833 bp contig in 5′ flanking region of pig KL gene was obtained by PCR. To determine the promoter region, four promoter deletions (KL-D1, KL-D2, KL-D3 and KL-D4) were cloned into fluorescent vector based on the prediction of NNPP online software and Promoter 2.0 (Fig. 1A). Luciferase activity analysis in both PK and ST cells revealed that KL-D2 (−418 bp to −3 bp) was essential for its transcriptional activity and was defined as the KL promoter region (Fig. 1B).

MARC0022311 SNP affects the KL expression

Intron SNPs could not change the amino acid sequence, but might alter gene promoter activity by affecting the binding ability of transcription factors (Van Laere et al., 2003). The plasmids contained the wild-type A (g.1,474 A) or mutant G (g.1474 G) sequence followed by KL-D2 were named as pGL3-D2-A and pGL3-D2-G, respectively. Then recombinant DNA fragments were inserted in the downstream of the luc+ gene between the KpnI and HindIII sites. Results showed that luciferase activity of pGL3-D2-G was significantly higher than pGL3-D2-A in both PK cells (P < 0.05) and ST cells (P < 0.01) (Fig. 2A), and indicated that MARC0022311 SNP changed the binding ability of certain regulatory elements affected KL promoter activity.

The SNP (MARC0022311) located in the first intron of KL gene (+1, 474 bp) was predicted to change the binding ability of OCT-1 by BIOBASE and TFsearch (Fig. S1). After silencing OCT-1 using siRNAs in PK and ST cells, luciferase activity of pGL3-D2-G was significantly lower than pGL3-D2-A (P < 0.05) (Figs. 2B and 2C). Furthermore, compared with the negative control, the luciferase activity of pGL3-D2-A was significantly decreased (P < 0.05) (Figs. 2B and 2C). Thus, MARC0022311 regulated the promoter activity via OCT-1.

However, inhibition of OCT-1 expression significantly suppressed KL expression in PK and ST cells (P < 0.05) (Fig. 3), possibly because OCT-1 could stimulate KL expression by binding KL gene at other sites.

Transcription factor OCT-1 binds to the KL intron 1 both in vitro and in vivo

To address whether KL intron 1 contained OCT-1 binding sites in vitro, we used two oligonucleotides (A allele and G allele oligonucleotides) as porcine OCT-1 probes in EMSA. EMSA revealed a highly specific interaction with allele A oligonucleotide, and a 100 fold excess of mutant allele G oligonucleotide could not outcompete the interaction (Fig. 4A). A super-shift was obtained when nuclear extracts from PK and ST cells were incubated with OCT-1 antibodies, providing further biochemical evidence for the presence of OCT-1 in vitro (Fig. 4A). We found the KL genotype at g.1474 A > G locus was AA in PK and ST cells by PCR-sequencing, indicating the endogenous binding of OCT-1 to KL in above two cell lines (Fig. S2). The chromatin was immune-precipitated using an OCT-1 antibody and DNA fragments of the expected size were used as a template to perform PCR amplification. ChIP analysis showed that OCT-1 interacted with KL intron 1 (Fig. 4B). These results showed that transcription factor OCT-1 bound to KL intron 1 both in vitro and in vivo.