Gene expression and promoter methylation of porcine uncoupling protein 3 gene

Objective Uncoupling protein 3 gene (UCP3) is a candidate gene associated with the meat quality of pigs. The aim of this study was to explore the regulation mechanism of UCP3 expression and provide a theoretical basis for the research of the function of porcine UCP3 gene in meat quality. Methods Bisulfite sequencing polymerase chain reaction (PCR) and quantitative real-time PCR (Q-PCR) were used to analyze the methylation of UCP3 5′-flanking region and UCP3 mRNA expression in the adipose tissue or skeletal muscle of three pig breeds at different ages (1, 90, 210-day-old Putian Black pig; 90-day-old Duroc; and 90-day-old Dupu). Results Results showed that two cytosine-guanine dinucleotide (CpG) islands are present in the promoter region of porcine UCP3 gene. The second CpG island located in the core promoter region contained 9 CpG sites. The methylation level of CpG island 2 was lower in the adipose tissue and skeletal muscle of 90-day-old Putian Black pigs compared with 1-day-old and 210-day-old Putian Black pigs, and the difference also existed in the skeletal muscle among the three 90-day-old pig breeds. Furthermore, the obvious changing difference of UCP3 mRNA expression was observed in the skeletal muscle of different groups. However, the difference of methylation status and expression level of UCP3 gene was not significant in the adipose tissue. Conclusion Our data indicate that UCP3 mRNA expression level was associated with the methylation status of UCP3 promoter in the skeletal muscle of pigs.


INTRODUCTION
With the improvements in living standards, people tend to be interested in eating highquality pork. Improvement of porcine meat quality has become a priority when it comes to pig breed ing and is also one of the ways in which breeding enterprises can earn more [1]. Uncoupling protein 3 gene (UCP3) was reported to wield a significant influence on fat deposition in pigs, which is an important factor affecting meat quality, especially influencing the juiciness, tenderness, and color of pork [24]. UCP3 gene belongs to the mitochondrial anion carrier gene family, mainly involved in the uncoupling effect of mitochondrial respiratory chain and has an important impact on separating oxidative phosphorylation from the synthesis of ATP as energy which is anticipated as heat [5,6].
Porcine UCP3 gene is located on chromosome 9p21p24 with the fulllength open reading frame of 927 bp, coding 308 amino acids [7,8]. The chromosome location of UCP3 gene is consistent with the notion that numerous quantitative trait loci (QTLs) related to carcass and meat quality traits were located on SSC9 in swine (https://www.animalgenome.org/cgi bin/QTLdb/SS/viewmap). In a study on pigs at high ambient temperature, it was reported that the UCP3 expression was upregulated in the muscle [9]. UCP3 mRNA expression in adipose tissue was found to be differentially associated with leptin between groups according to the birth weight of piglets [10]. In mice, the overexpression of UCP3 protected against the accumulation of triglyceride in both muscle and adipose tissue [11]. Lin et al [12] discovered that UCP3 could mediate heat production in coldresistant pigs to adapt to cold environments; furthermore, eight domi nant pig breeds from China could be classified into cold sensitive and coldresistant breeds based on the sequence of UCP3.
The polymorphisms of UCP3 gene have been reported to be associated with meat quality traits in pigs. Cieslak et al [13] found a novel missense substitution in the UCP3 and the poly morphism of UCP3 (g.946C>T) was potentially associated with porcine abdominal fat weight. One 9base continuous mutated site in 3′UTR of pig UCP3 gene was screened, and the site was identified in significantly close association with the backfat thickness at the sixth and seventh rib [14]. Tu et al [15] detected three codingregion single nucleotide polymor phisms in UCP3 gene and found that one of the mutations may have a significant effect on several carcass and meat quality traits.
However, few studies have focused on the UCP3 5′flanking or promoter region, the correlation between promoter cytosine guanine dinucleotide (CpG) island methylation status, and UCP3 mRNA expression. In this study, bisulfite sequencing polymerase chain reaction (PCR) was used to investigate the methylation status of the UCP3 gene promoter and quantitative realtime PCR (QPCR) was used to measure UCP3 mRNA expression in the adipose tissue or skeletal muscle of three pig breeds at different ages.

Animals and tissues
All animal procedures were conducted according to the Regulations for the Administration of Affairs Concerning Experimental Animals (Ministry of Science and Technology, China, revised in July 2013) and approved by the Experimen tal Animal Care and Use Committee of Fujian Agriculture and Forestry University (Fuzhou, Fujian, China).
Putian Black pig is a quality native breed in Fujian province and included in the list of National Livestock and Poultry Genetic Resources Protection of China. Dupu pig is a hybrid of Duroc×Putian Black pig. All pigs (1, 90, and 210dayold Putian Black pigs; 90dayold Duroc; and 90dayold Dupu, n = 4 per group) included in the study were euthanized at a commercial slaughterhouse. The adipose and skeletal muscle were removed immediately, washed briefly with phosphate buffered saline, rapidly frozen in liquid nitrogen, and stored at -80°C.

Methylation detection of CpG island
Genomic DNA was extracted from the samples by standard phenol/chloroform procedure and modified using the EZ DNA MethylationGold Kit (ZYMO Research, Irvine, CA, USA) according to the manufacturer's instructions.
The reaction mixture for the analysis included 20 μL of PCR product containing 100 ng genomic DNA, 2 μL of 10×PCR buffer, 150 μM dNTPs, 1 μM of each PCR primer (Table 1), 1 U Taq DNA polymerase (TaKaRa, Tokyo, Japan), and ster ilized distilled water up to 20 μL. The amplifying conditions of PCR for normal DNA were 94°C for 5 min, followed by 40 cycles of 94°C for 30 s, 51°C for 30 s, 72°C for 30 s, and 72°C for 10 min. The annealing temperature used to amplify the bisulfitetreated DNA was 58°C. The PCR products were visu alized by electrophoresis in 1.5% agarose gel electrophoresis The expected PCR fragment was cloned and inserted into the pMD18T vector. The recombinant clones were used to transform Trans5α Chemically Competent Cell (TransGen Biotech, Beijing, China). The positive recombinant clones were selected on LuriaBertani medium containing 60 μg/mL am picillin and confirmed by PCR. Five randomly selected positive recombinant clones from each individual were sequenced through a commercial sequencing facility (Sangon, Shanghai, China). The sequencing results were analyzed using QUMA software [20].

RNA isolation and reverse transcription
Total RNA was extracted from frozen tissues using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol. The concentration and quality of the isolated RNA were determined using Nanodrop 2000 (Thermo Fisher Scientific, Leicester, UK) and denatured gel electro phoresis. Reverse transcription polymerase chain reaction (RTPCR) was conducted using PrimeScript RT reagent Kit with gDNA Eraser (TaKaRa, Japan), containing oligo (dT), random primers and gDNA Eraser. After treating 1 μg RNA with gDNA Eraser, the reverse transcription reaction was carried out at 37°C for 15 min, 85°C for 5 s, and 4°C for 2 min.

Quantitative real-time polymerase chain reaction analysis
Quantitative realtime PCR (QPCR) was performed using an ABI Prism 7500 sequencedetection system (Applied Bio systems, Foster City, CA, USA) with SYBR Green Realtime PCR Master Mix (Toyobo, Osaka, Japan), under the manu facturer's instructions.
The UCP3 fragment was amplified using the primers listed in Table 1 and the β-actin primers were used as internal con trol. The primers for QPCR were designed by Lin et al [12] and Erkens et al [21]. All PCRs were performed in triplicate and the expression level of UCP3 was quantified relative to the expression of β-actin by employing the 2 -ΔΔCt value method [22].

Data analysis
Differences between groups were performed with oneway analysis of variance and least significant difference test (V19.0, SPSS Inc., Chicago, IL, USA). Methylation levels and mRNA expression were analyzed by Pearson's correlation.

Bioinformatics analysis of the porcine UCP3 promoter
Analysis of the porcine UCP3 gene 5′flanking region (2 kb upstream of the initiation codon) revealed that the core pro moter region is located between nucleotides -870 and -550 and the 2 kbfragment contains two CpG islands: -1,603 to -1,501 and -910 to -777 ( Figure 1). Here, the second CpG island located within the putative promoter region was specu lated to play a role in regulating gene expression. Several putative transcription factor binding sites were also identi fied in this CpG island, including Sp1 transcription factor, neurofibromin 1, CCAAT enhancer binding protein beta, transcription factor AP2 alpha, CCAAT enhancer binding protein alpha, and organic cation/carnitine transporter 1. Therefore, primers were designed to amplify the fragment containing 9 CpG sites of CpG island 2.

Methylation status of the porcine UCP3 promoter
The PCR products of normal and bisulfitetreated DNA were analyzed by using 1.5% agarose gel electrophoresis, and the positive recombinant clones were sequenced by Sangon Biotech. By comparative sequencing of the putative UCP3 promoter region, we identified two novel single nucleotide polymor phisms (-882 A/T, -852 G/A) in the 5′flanking region of The methylation status of the putative UCP3 promoter re gion was examined in the adipose tissue and skeletal muscle of Putian Black pig at three different ages (Figure 2). Data in Table 2 showed that the degree of methylation in 90dayold Putian Black pig was lower compared with the other two ages in skeletal muscle and adipose tissue. However, the changing of methylation status did not reach the statistical significance level in adipose tissue. Furthermore, we measured the meth ylation level of skeletal muscle in other two 90dayold pig breeds (Duroc, 19.44%±5.56%, Dupu, 11.11%, Supplementary Figure S1). The methylation level in Dupu was lower than that in Duroc (p<0.05), and there was no significant differ ence between that of Dupu and Putian Black pig.

mRNA expression level of UCP3 gene
The relative expression level of UCP3 in the skeletal muscle of the Putian Black pig increased to the highest at 90 days (p< 0.01) and then decreased at 210 days ( Figure 3). However, the UCP3 mRNA expression level was not different in the adipose tissue of different ages. Moreover, the expression level of UCP3 in skeletal muscle was the highest in Dupu pig and the low est in Duroc at 90dayold (Figure 4). Pearson's correlation analysis showed that the methylation status of the CpG island correlated negatively with UCP3 expression in skeletal muscle (r = -0.82 or -0.72; p<0.01); with significant correlation co efficients for CpG_9 in Putian Black pig at three stages (r = -0.632; p<0.05).

DISCUSSION
It is well known that UCP3 is an important candidate gene for regulating intramuscular fat to affect meat quality traits [13,14]. Putian Black pig, which is one of the native breeds in Fujian province, is famous for its meat fragrance and taste. We measured the UCP3 promoter methylation and mRNA expression in the adipose tissue and skeletal muscle of Putian Black pig at three ages. The results showed that the methyla tion level in the skeletal muscle of the 90dayold Putian Black pig was lower than that of the other two ages. Even though the changing of methylation status existed in the adipose tissue, it did not reach the statistical significance level. Similarly, the UCP3 mRNA expression level only differed in skeletal mus cle at various stages. These findings were consistent with the research that UCP3 mRNA in adipose tissue was similar be tween Meishan and commercial sows at different postnatal ages, and the expression in skeletal muscle was greater on day 7 in commercial sows (p<0.05) [2]. In contrast, it has been reported that ucp3 mRNA was highly expressed in the brown adipose tissue (BAT) of rats soon after birth, decreased until three months, and recovered thereafter [23]. Pigs however, are one of the few species so far found to lack functional BAT [24], because the functional uncoupling protein 1 was lost in pigs in a genetic event [25]. We suspected that the lack of functional BAT in pigs is responsible for the undifferentiated expression of UCP3 gene in their adipose tissue.
Furthermore, we compared the gene expression level and promoter methylation status of UCP3 in the skeletal muscle of 90dayold Putian Black pig, Duroc and their crossing pro genies (Dupu). The methylation level in Dupu was lower than that in Duroc (p<0.05), and there was no significant differ ence in Dupu and Putian Black pig. The expression level of UCP3 gene was higher in Dupu and Putian Black pig than that in Duroc. Another study also suggested that breed can affect the methylation of UCP3 in chicken breast muscle [26].
In the previous work, we found that the pork flavor of Dupu and Putian Black was better than that of Duroc, as Dupu and Putian Black had higher concentrations of saturated and mono unsaturated fatty acids and lower concentrations of poly unsaturated fatty acids than Duroc [27]. Therefore, the different expression of UCP3 gene in different pig breeds may be re lated to meat quality. Interestingly, two novel SNPs (-882 A/T, -852 G/A) were detected in the various pig species. Further research is needed to determine whether the allelic variations of UCP3 gene will affect meat quality.
Studies have shown that the methylation of CpG islands in the promoter region played a vital role in gene regulation, cell differentiation, embryonic development, and so on [2830]. In the skeletal muscle, the methylation of UCP3 promoter experienced a dynamic process, with the development of Putian Black pig, which agreed with the fluctuating trend of UCP3 expression. Meanwhile, breed has a significant influence on the expression of UCP3 via the distinctive methylation status of CpG island. This indicates that methylation of the CpG is land suppresses UCP3 gene expression.
In summary, the major findings of the present study were the differential influences of porcine breed and postnatal age on CpG methylation and expression of UCP3 gene in skeletal muscle. These results might contribute to studies on the func tion of UCP3 gene in pig breeding for meat quality.

CONFLICT OF INTEREST
We certify that there is no conflict of interest with any financial organization regarding the material discussed in the manu script.