Min pigs, a Chinese indigenous pig breed, were obtained from the Institute of Animal Husbandry, Heilongjiang Academy of Agricultural Sciences, Harbin, China. For preadipocyte isolation and cDNA synthesis, three Min pigs at age of 30-day were used, and back fat tissues were sampled immediately after the pigs were slaughtered. Fresh tissues were used for preadipocyte isolation, and those for cDNA synthesis were stored at −80°C after snap frozen in liquid nitrogen. For polymorphism analysis, a total of 11 adult Min pigs were used and ear tissues were collected. Total RNA was extracted with TRIzol reagent (Invitrogen). Genomic DNA was obtained with normal phenol-chloroform method. cDNA was synthesized with HiScript III 1st Strand cDNA Synthesis Kit (+gDNA wiper) (Vazyme). All procedures of animal treatment were approved by the Laboratory Animal Welfare and Ethics Committee of Northeast Agricultural University.

Preadipocytes were obtained from backfat tissues of 30-day-old Min pigs as described previously [21]. Briefly, backfat tissues were first digested with 0.1% type I collagenase (Invitrogen), and then cultured in DMEM/F12 (Dulbecco’s Modified Eagle’s Medium/Nutrient Mixture F-12) medium supplemented with 10% FBS (fetal bovine serum: Gibco) and 1% penicillin-streptomycin (Invitrogen) at 37°C with 5% CO₂. When the cells reached contact inhibition, DMEM/F12 medium containing 10% FBS, 1 μmol/L dexamethasone, 0.5 mmol/L 3-isobytyl-1-methylxanthine and 5 μg/mL insulin was used to induce differentiation. At two days post induction, the cells were transferred to DMEM/F12 medium containing 10% FBS and 5 μg/mL insulin to maintain the differentiation for another six days. The medium was changed every 48 h. The mature adipocyte was stained with Oil Red O (Leagene), and the results were viewed and photographed with a light microscope (Analytik Jena). At the same time cellular Oil Red O was extracted with isopropanol, and the triglyceride content was quantified with optical absorbance (OD) at 510 nm.

Fragments spanning −1,576 to −90 bp of porcine PABPN1 promoter were amplified from genomic DNA. Polymerase chain reaction (PCR) was performed with Ex Taq (TaKaRa). The resultant products were inserted into pGL3-basic at Kpn I and Hind III sites to construct reporter genes, 15F/R. Here, the first nucleotide of start codon was designated as +1. The 5’ truncated fragments were then amplified using 15F/R as templates, and the reporter genes were constructed. Over-lap extension PCR was performed to mutate the fragments as described previously [21] and. Briefly, the mutation was first introduced into the end of two over-lap fragments, respectively, with primers, then the two fragments were spliced to introduce the mutation into the inner of the fragments. High-fidelity Pfu DNA polymerase (Trans Gene) was used in the over-lap extension PCR. The mutant fragment obtained were inserted into pGL3-basic at Kpn I and Hind III sites to construct mutant type reporter genes.

The complete coding sequences (CDS) of CCAAT-enhancer binding protein (C/EBP) α and β were amplified using cDNA template obtained from fat tissues, respectively, and inserted into pCMV-HA vector at EcoR I and Xho I sites. Overexpression vector of C/EBPα constructed with pcDNA3.1+ was obtained previously [21]. Plasmids pcDNA3.1-C/EBPα and pCMV-HA-C/EBPα were used for dual-luciferase reporter and electrophoretic mobility shift assay (EMSA), respectively. Plasmids overexpressing and siRNA against PABPN1 were obtained previously [18]. All primers here were synthesized by Genesoul Tech and listed in Supplements 1–3.

Transient transfection was carried out with lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. In PK-15 cells, at 48 h post-transfection the cells were collected for dual-luciferase reporter assay or real-time quantitative PCR (qPCR). In preadipocytes, at 24 h post-transfection the cells were induced to differentiate and the induction time was indicated in Results. The preadipocytes were then used for Oil Red O staining or qPCR.

PK-15 cells were cultured as described previously [21]. Each reporter gene was transfected into the cells individually or together with overexpression vector. Renilla luciferase reporter, pRL-TK, was used as internal reference. At 48 h post transfection, luciferase activities were measured with dual-luciferase reporter gene assay kit (Beyotime), and relative luciferase activity was calculated as a ratio of firefly to Renilla luciferase activities.

qPCR was performed with ChamQ Universal SYBR qPCR Master Mix (Vazyme) according to the protocol of manufacturer. The relative expression level of target gene was calculated with 2−ΔΔCt method with β-actin as a reference [22]. Primers for C/EBPα and C/EBPβ were synthesized by Genesoul Technology and those for PABPN1 and β-actin were obtained previously [18]. Primer sequences were listed in Supplement 4.

PK-15 cells, cultured in six-well plates, were transfected with plasmid pCMV-HA-C/EBPα and pCMV-HA-C/EBPβ, respectively, for 48 h. Total protein was isolated with RIPA buffer (Beyotime) containing a protease inhibitor (Invitrogen). BCA protein assay kit (Beyotime) was used to quantify the protein. 25–30 μg total protein was loaded on SDS-polyacrylamide gel and separated at 80 V. The protein was then transferred onto a polyvinyl difluoride membrane (Millipore) and incubated with anti-HA tag (1:5,000 dilution; Abmart) and anti-β-actin (1:3,000 dilution; Abmart) primary antibodies. Goat anti-mouse IgG (1:20,000 dilution; LI-COR) was used as secondary antibody. The results were visualized on UVP ChemStudioTM PLUS touch (Analytik Jena).

EMSA was performed as described previously [21]. Briefly, the biotin-labeled gene-specific binding probes, unlabeled specific and mutant competitors were synthesized by General Biol. Briefly, overexpression vector was transfected into HEK-293T cells using lipofectamine 2000 (Invitrogen). At 48 h post transfection, nuclear extracts were isolated with the kit purchased from Solarbio. Incubation was performed with Chemiluminescent kit (Beyotime) according to the manufacturer’s instructions. The biotin-labeled probe was incubated with 20 μg nuclear extracts singly or together with mutant competitor. For co-incubation of biotin-labeled probe and unlabeled specific competitor, the unlabeled probes were first incubated with nuclear extracts for 10 min, and then the labeled ones were added. Electrophoresis was performed on 6.5% polyacrylamide gel for 1.5 h at 90 V. After transferred to nylon membrane (Beyotime), the gels were viewed on Azure c300 Gel Imaging System (Zzure Biosystems). The probe sequences were given in Supplement 5.

PK-15 cells were cultured until 70%–80% confluence, and transfected for 48 h. The Chromatin immunoprecipitation (ChIP)-qPCR was performed with SimpleChIP Enzymatic Chromatin IP Kit (Cell Signaling) according to the manufacturer’s instructions. Briefly, the crosslinking reaction was performed in 1% formaldehyde for 10 min at room temperature, and terminated with 10× glycine solution. The cells were collected using 2 mL ice-cold PBS solution and 200× Protease Inhibitor Cocktail, and nuclear pellet was isolated at 2,000×g for 5 min at 4°C. Ultrasonic Homogenizer was used to shiver DNA to a length of about 150–900 bp, and the products were blotted with anti-HA tag (Abmart) overnight at 4°C with rotation. The immunoprecipitated DNA was quantified with qPCR, and the primers were listed in Supplement 6.

RNA sequencing (RNA-seq) was performed to identify genes regulated by PABPN1 in porcine preadipocytes. SiRNAs against PABPN1 and negative control (NC) sequences were transfected into cells, respectively, with lipofectamine 2000 (Invitrogen), each with triplicate. At 24 h post transfection, the cells were induced to differentiate for 48 h. RNA was isolated and detected with NanoDrop 2000 (IMPLEN), Qubit 2.0 (Thermo Fisher Scientific), and Agilent 2100 to examine the purity, concentration, and integrity of the sample. Qualified RNA was subjected to library construction and a total of six libraries (NC = 3, siPABPN1 = 3) were obtained. RNA-seq was performed on the NovaSeq6000 PE150 platform (Illumina) at Allwegene Technologies.

The clean reads were mapped to the pig reference genome (S. scrofa 11.1_release 109) using HISAT2 (v2.0.4) [23]. The mapped reads were assembled into transcripts with StringTie (v2.2.1) [24]. FPKM (Fragments Per Kilobase of transcript per Million fragments mapped) [25] was used to measure the gene expression level, and genes with FPKM≥0.5 in at least three biological repetitions were used for further analysis. Differentially expressed genes (DEGs) were identified using DESeq2 [26] in cells knocking down PABPN1 compared to that transfected with NC sequences, and the threshold was set as Fold Change (FC)≥1.2 and p<0.05. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG pathway) was performed as described previously [27].

Fragments spanning −1,094 to −90 were amplified with genomic DNA template. The products were sequenced directly by Genesoul Technology to analyze SNPs. The sequences obtained were aligned by using SnapGene (v6.0.2) to identify SNPs. At the same time manual inspection of the diagram was performed with the Chromas software to confirm the SNPs and the genotype. Linkage disequilibrium analysis was performed with the online program SHEsis ( http://analysis.bio-x.cn/myanalysis.php, accessed on 12 September 2024).

All experiments were performed in three independent experiments, each with three biological repetitions. Statistical analyses were carried out with GraphPad Prism (v9.5.1). Difference between two groups was analyzed with unpaired t-test, while that among multiple groups was analyzed with one-way ANNOVA. Multiple comparison was performed using default method in ANNOVA program of GraphPad Prism (v9.5.1).

To investigate the transcriptional regulation of porcine PABPN1 in detail, the 5’ flanking region were first subjected to bioinformatic analysis using online software. It is interesting that C/EBPα and β, two well-known TFs for fat accumulation, were predicted in the promoter of PABPN1. Each of the TFs has two putative binding sites (Figure 1A). To investigate the role of C/EBPα and β in PABPN1 transcription, reporter genes containing serial 5’ flanking region of PABPN1 were constructed to identify the promoter activity (Figure 1A). It was revealed that fragments spanning −1,094 to −90 bp have promoter activity with the highest level in region −534 to −90 bp, and the core promoter was situated in −209 to −90 bp; while the longest fragment (−1,576 to 90 bp) did not show activity to drive the expression of firefly luciferase gene, indicating there is motif inhibiting the transcription (Figures 1A, 1B).

Two plasmids overexpressing C/EBPα, pcDNA3.1-C/EBPα and pCMV-C/EBPα, were used here. The efficiencies of pcDNA3.1-C/EBPα were characterized previously [21]. Western blotting showed that the plasmids pCMV-C/EBPα were constructed successfully (Figure 1C). We first focused on the role of C/EBPα in PABPN1 transcription. The mRNA level of PABPN1 was significantly increased by ectopic C/EBPα in PK-15 cells (p<0.01; Figure 1D). C/EBPα overexpression significantly enhanced the activity of PABPN1 promoter as revealed by reporter gene analysis (p<0.01; Figure 1E). Absence of any of the two putative binding sites, mapped to −715 to −706 and −450 to −441 (named Site 1 and Site 2), respectively, resulted in significant decreases in the promoter activities compared to the wild type fragment. Compared to site 1, Site 2 has a more powerful impact on the promoter activity (Figure 1F). Additionally, deleting the motifs, either singly or simultaneously, abolished the promoting effects of ectopic C/EBPα on promoter activity (Figure 1G).

At the same time, EMSA was used to analyze the binding ability of C/EBPα to the motifs. It was revealed that the DNA-protein complexes could be formed clearly in groups containing biotin-labeled probe in both sites, addition of mutant competitor did not affect the formation of the complexes, while specific competitor weaken the complexes. The addition of antibody against HA tag also led to weakness of the complexes (Figure 1H). Furthermore, ChIP-qPCR showed that C/EBPα enriched significantly in the both motifs of PABPN1 promoter (Figure 1I). These results indicated C/EBPα regulated the expression of PABPN1 by directly binding to the promoter.

Two putative binding sites for C/EBPβ were mapped to −164 to −159 and −127 to −121 (named Site 1 and Site 2) in the promoter of porcine PABPN1, respectively. Western blotting showed that the plasmids C/EBPβ were constructed successfully (Figure 2A). We first showed that ectopic C/EBPβ increased the mRNA level of PABPN1 in PK-15 cells (p<0.01; Figure 2B). Dual-luciferase reporter gene analysis revealed that ectopic C/EBPβ led to significant increases in the promoter activity of PABPN1 (p<0.01; Figure 2C). Deletion of any one of the sites resulted in significant decreases in the promoter activity, but no synergistic effects were found in mutants absent of two sites (Figure 2D). As expected, deletion of the sites significantly decreased the promoting effects of ectopic C/EBPβ on PABPN1 promoter activity, and no differences were found between mutants deleting single sites (Figure 2E). EMSA assay showed that C/EBPβ could bind to Site 2 directly as complexes were formed clearly between nuclear extracts and biotin-labeled probes in both sites, and mutant competitor had no effects on the formation of complexes, while specific competitor weakened the complexes robustly. Additionally, antibody against HA tag significantly decreased the complex intensity (Figure 2F). Because the two sites are close to each other, ChIP-qPCR was performed on the two sites simultaneously, and the results showed that C/EBPβ were significantly enriched in the region (Figure 2G). However, the binding of C/EBPβ on Site 1 were not shown clearly by EMSA assay, and we did not perform ChIP-qPCR on the site individually. We cannot confirm whether it is a genuine site for C/EBPβ. Nevertheless, we made clear that there was at least one binding site for C/EBPβ in the promoter of PABPN1, and that C/EBPβ could regulate the transcription of PABPN1 by directly binding to the promoter.

It is well known that C/EBPα and β are key regulators of adipogenesis, suggesting a role of PABPN1 in the process. We then analyzed the effects of PABPN1 on adipogenesis through gain- and loss-of-function assays in primary preadipocytes isolated from subcutaneous fat tissues of 30-d-old pigs. Plasmids overexpressing and siRNA against PABPN1 were obtained successfully previously [18]. The ectopic PABPN1 promoted the differentiation of preadipocytes, while knockdown of PABPN1 inhibited it, as revealed by Oil Red O staining at 6-days post-induction (Figure 3A). Consistently, triglyceride contents were increased by overexpressing PABPN1, and decreased by interfering PABPN1, in porcine preadipocytes (Figure 3B). At the same time the expression of adipogenesis marker genes, C/EBPα and peroxisome proliferator-activated receptor (PPAR) γ, was significantly increased by ectopic PABPN1, and decreased by knockdown of PABPN1 (Figures 3C, 3D).

To further reveal the role of PABPN1 in adipogenesis, RNA-seq was performed on preadipocytes knocking down PABPN1 (Supplement 7). Principle component analysis showed that the RNAi and NC groups were separated well, and there were small differences between samples in the same group (Figure 4A). A total of 436 DEGs including 239 upregulated and 197 downregulated were identified in preadipocytes knocking down PABPN1 compared to NC groups (Figures 4B, 4C; Supplement 8). There are a lot of TFs among the DEGs including three members of nuclear receptor subfamily 4 group A, two members of homeobox family, three members of zinc finger protein, signal transducer and activator of transcription 3, etc. Additionally, known regulators for fat deposition such as lipoprotein lipase, apelin, secreted frizzled related protein 2, notch receptor 3, etc. were also differentially expressed. However, C/EBPα and PPAR γ were not found in DEGs, which might be caused by the fact that the induction time was limited. A series of KEGG pathways were significantly enriched by DEGs (q<0.05). Among the top 20 pathways enriched five have been involved in fat accumulation including RIG-I-like receptor signaling pathway, Wnt signaling pathway, IL-17 signaling pathway, mTOR signaling pathway and Hippo signaling pathway according to literature retrieval. Among them RIG-I-like receptor signaling pathway and IL-17 signaling pathway were the top 2 significantly enriched. Additionally, pathways involved in human diseases were also significantly enriched by DEGs (Figure 4D).

Three SNPs, −812A>G, −719C>A and −249C>T, were identified in the promoter of PABPN1 by directly sequencing of PCR products (Figure 5A). Genotyping analysis showed that these SNPs occurred frequently in Min pigs. Among 11 Min pigs analyzed, the allele frequencies of G, A and T in sites −812, −719 and −249 were 13.64%, 77.3% and 77.3%, respectively (Table 1). SNPs −719C>A and −249C>T are in complete linkage disequilibrium with the parameter D’ = 1.0 and r² = 1.0 as revealed by the online software SHEsis.

The effects of the SNPs on the expression of PABPN1 were analyzed with dual-luciferase reporter gene. We first double mutated CC to AT in the reporter gene 7F/R to analyze the effects of the two linkage SNPs (Figure 5B). Dual-luciferase reporter assay showed that the mutation significantly decreased the promoter activity (p<0.01; Figure 5C), indicating Haplotype AT had an inhibitory role on the expression of PABPN1 gene.

Next, the role of SNP −812A>G was analyzed. Mutagenesis was conducted on the reporter gene 10F/R containing A, C and C at sites −812, −719 and −249, respectively, and named Haplotype ACC. Haplotype GCC was obtained by changing nucleotide A in site −812 to G (Figure 5B). We found that this single mutation significantly increased luciferase activity (p<0.01; Figure 5D). Then haplotype GAT were created (Figure 5B), and no differences were found in luciferase activities between reporter genes containing Haplotype ACC and Haplotype GAT (p>0.05; Figure 5D). Compared to haplotype GCC, the activity of haplotype GAT was significantly decreased (p<0.01; Figures 5B, 5D). Thereafter we characterized a haplotype promoting the expression of PABPN1 gene, Haplotype GCC.