Porcine skeletal muscle cells (BIOSPECIES-0017a) were purchased from Guangzhou Suyan Biotechnology Co., Ltd., Guangdong, China. C2C12 myoblasts and 293 T cells were purchased from the American Type Culture Collection (ATCC). The growth medium was Dulbecco’s Modified Eagle’s Medium (DMEM, Corning, China). contained 10% fetal bovine serum (FBS, Gibco, California, USA) and 1% penicillin–streptomycin (PS, Thermo Scientific, Massachusetts, USA). Cells were then placed in a 37°C cell incubator containing 5% oxygen and 95% carbon dioxide.

Longissimus dorsi muscle samples were meticulously collected from Landrace and Tongcheng pigs across a comprehensive spectrum of 27 developmental time points. These animals were granted unrestricted access to food and water, and they were uniformly housed under controlled conditions to minimize environmental variations. Notably, at each time point, tissue samples were meticulously harvested from three distinct pigs to ensure robust biological replication. Following collection, all specimens were promptly flash-frozen in liquid nitrogen and preserved until subsequent RNA-seq analyses (GSE157045 and PRJNA754250) (18, 30).

For RNA interference, negative control siRNA (siRNA-NC) and mouse siRNA-TP63 were purchased from Gemma Pharmaceutical Technology (Shanghai, China). The sequences of the TP63-targeted siRNAs are listed in Supplementary Table S1. The TP63 expression vector (pcDNA3.1-TP63) was synthesized by Gene Create (Wuhan, China). The control plasmid pcDNA3.1 was obtained from our laboratory. The coding sequences (CDSs) of the porcine TP63 gene were inserted into the PLV3 vector (Invitrogen). Porcine skeletal muscle cells and C2C12 cells were inoculated into 6 or 12 well plates 12 h before treatment and then transfected with siRNA or plasmid using Attractene transfection reagent (Qiagen) according to its instructions. The small interfering RNA (siRNA) and plasmid were transfected at a final concentration of 50 nM. This precise concentration was selected following thorough optimization experiments, aligning with the widely reported effective range documented in the existing literature for proficient gene knockdown.

Cells or tissues were lysed with Triol (Invitrogen, Shanghai, China), chloroform, and denatured with isopropanol to precipitate RNA, then washed with 75 and 100% ethanol, respectively, and finally solubilized in DEPC water. RNA quality was determined by NanoDrop 2000 (Thermo Fisher Scientific, Massachusetts, USA). The identified RNAs were available for further studies. HiScript III First Strand cDNA Synthesis Kit (+gDNA wiper) (R312-01, Vazyme, Nanjing, China) was used for cDNA reverse transcription synthesis of mRNA, respectively, according to the instructions. In addition, Taq Pro Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China) was used in the total reaction for messenger RNA qPCR in a volume of 20 μL, including 10 μL 2× SYBR Master Mix, 0.4 μL PCR forward primer, 0.4 μL polymerase Chain Reaction Reverse Primer, 2 μL cDNA, and 7.2 μL sterile enzyme-free water. Reaction conditions were 95°C for 30 s, followed by 95°C for 10 s and 65°C for 30 s for 40 cycles; this reference gene was Gapdh. Relative expression levels of messenger ribonucleic acid (RNA) were analyzed by the 2-∆∆CT method. Sequence information of the primers used for reverse transcription and quantification (Sangon Biotech, Shanghai, China) is shown in Supplementary Tables S2, S3.

EdU assay was performed using EdU assay kit (Beyotime, China) and cell proliferation ability was determined using EdU-488 cell proliferation assay kit (Beyotime, China). Cells were spread onto 6-well plates, which were disrupted and overexpressed, and the final concentration of EdU was adjusted to 10% μm was added to each well, and incubation was continued for 1–2 h in the cell incubator. Cells were washed three times with PBS (Thermo Fisher) and then fixed with 4% formaldehyde fixative for 15 min at room temperature. The fixative was removed and the cells were washed three times with detergent. 1 mL of 0.3% Triton X-100 diluted in PBS was added and incubated at room temperature for 15 min to increase cell membrane permeability. In addition, 500 μL of the prepared click reaction solution was added to each well, and the cells were incubated for 30 min at room temperature and protected from light to observe and quantify the number of EdU-stained cells, and the nuclei were stained with DAPI (1:1000 PBS). Three fields were randomly selected for statistical analysis.

C2C12 adult myoblasts and skeletal myoblasts were inoculated into 96-well plates and harvested at 0 h, 24 h, 36 h, 48 h, and 72 h post-transfection, respectively. The proliferation of adult myocytes was measured using Cell Counting Kit-8 (CCK-8) (Beyotime C0038, Beijing, China). A 1:9 mixture of CCK-8 reagent and complete culture medium was added to 96-well plates and continued at 37°C for 1 h. The cells were counted using a microplate reader. The optical density (OD) at 450 nm of each sample was measured with a microplate reader and growth curves were plotted.

Skeletal muscle cells were spread into 6-well plates, disrupted and overexpressed, and collected after 48 h. The cells were digested with trypsin, centrifuged at 1000 g for 3–5 min, precipitated, and single-cell suspensions were prepared. The cells were washed with pre-cooled PBS and centrifuged again. Fix the cells, resuspend the cells with 1 mL of pre-cooled 70% ethanol, and resuspend at 4°C for 2 h or overnight. An appropriate amount of propidium iodide (PI) staining solution was prepared according to the Cell Cycle Assay Kit (#C1052, Beyotime Biotechnology, Shanghai, China). Cells were resuspended with 500 μL of PI staining solution per tube, incubated at 37°C for 30 min, protected from light, and subjected to flow cytometry (CytoFLEX, BD Biosciences, NY, United States) upon excitation.

PCR amplification was employed to isolate a 400 bp DNA fragment located upstream and downstream of the SNP rs327571319 locus. Subsequently, this fragment was cloned into a PGK vector for the validation of enhancer activity. The constructed plasmid was then transfected into 293 T cells using a transfection reagent (Qiagen). Concurrently, co-transfection involved a plasmid harboring Renilla luciferase (PRL-TK) to serve as an internal control. After 24 h of transfection, luciferase signal transduction was quantified using the dual luciferase reporter assay kit (Vazyme).

To find the bound transcription factors, transcription factor motifs (TFs) bound to rs327571319 were looked at using TFs from the JASPAR public database.

The mRNA expression levels were calculated with the 2-ΔΔCt method and displayed as mean ± standard deviation. Unpaired two-tailed Student’s t-test was used to calculate the p-value. The t-test was adopted to detect differences between groups for statistical significance. All analytical data were obtained from three independent experiments and each experiment was performed in triplicate.

The intricate molecular mechanisms governing skeletal muscle development have garnered significant attention due to their impact on muscle development and therapeutic strategies for muscular disorders. Selective sweep analyses demonstrated TP63 as a highly selected gene in Western lean pig breeds (WED) compared to Eastern pigs (EAD) (Figure 1A), indicating its potential involvement in skeletal muscle development. To validate its tissue-specific expression, we leveraged the Pig GTEx Atlas database, revealing robust TP63 expression in muscle tissue relative to other tissues (Figure 1B). Additionally, based on our previous RNA-seq data from skeletal muscle at different developmental stages, we examined the expression profiles of TP63 in Tongcheng and Landrace pigs during various developmental stages of skeletal muscle. We observed a significant disparity in TP63 expression levels between these two pig breeds, with Tongcheng pigs exhibiting notably higher expression of TP63 compared to Landrace pigs across fetal, neonatal, and adult stages of skeletal muscle development (Figure 1C). The above findings highlight TP63 as a promising candidate gene implicated in the regulation of skeletal muscle development.

To investigate the effect of TP63 on myoblast proliferation, we first utilized mouse C2C12 adult myoblasts and performed functional experiments involving transfection with TP63 overexpression vectors and siRNA-TP63 designed to target TP63. Our results demonstrated that the knockdown of TP63 effectively attenuated the mRNA level of TP63 in mouse C2C12 myoblasts, this knockdown rate is about 60% compared to the control group (Figure 2A), as well as several proliferation marker genes, including CYCLINE, CYCLIND, and KI67 (Figure 2B). EdU assay results further revealed a decrease in cell proliferation activity upon TP63 knockdown (Figure 2C). The CCK8 assay revealed a decline in cell proliferation viability upon TP63 knockdown, but not significant change. (Figure 2D). Furthermore, the RT-qPCR experiments showed that overexpression of TP63, this overexpression efficiency increased about 100-fold compared to the control (Figure 2E) significantly up-regulated the expression of proliferation marker genes (Figure 2F). EdU assays showed an increased number of EdU-positive cells following TP63 overexpression (Figure 2G), suggestive of enhanced proliferation. Moreover, cell viability assays demonstrated a significant increase in cell viability upon TP63 overexpression (Figure 2H). The findings indicate that TP63 enhances the proliferation of murine myoblasts and potentially contributes to the process of myogenesis.

To further validate the function of TP63 in cell proliferation, we performed multiple experiments in the porcine skeletal muscle cells. Our findings demonstrated that siRNA-mediated TP63 knockdown, The knockdown efficiency reached approximately 80% when compared to the control group, underscoring a significant reduction in target gene expression consequent to the experimental intervention (p < 0.01; Figure 3A) and efficiently reduced the mRNA expression levels of critical proliferation marker genes, namely KI67, PCNA, and CYCLINA (Figure 3B). Consistently, TP63 depletion resulted in a decrease in the number of EdU-positive myoblasts, reflecting diminished proliferative capacity (p < 0.05; Figure 3C). Furthermore, the CCK8 assay revealed a significant decline in cell proliferation viability upon TP63 knockdown (Figure 3D). Remarkably, cell cycle analysis unveiled an altered distribution of cells progressing through the cell cycle, with an increased proportion of cells arrested at the G0/G1 phase and a reduced number of cells transitioning to the S phase (Figures 3E,F). Conversely, TP63 overexpression in porcine skeletal basal cells. The overexpression efficiency surged approximately 130-fold relative to the control (p < 0.001; Figure 3G), leading to a remarkable increase in the mRNA expression levels of TP63 and key proliferation marker genes (Figure 3H). Intriguingly, both EdU and CCK-8 assays confirmed that TP63 overexpression significantly enhanced cell proliferative activity (Figures 3I,J). Moreover, cell cycle analysis demonstrated an augmented number of cells progressing to the S phase upon up-regulation of TP63 (Figures 3K,L). These findings shed light on the essential function of TP63 in orchestrating the proliferation of porcine skeletal muscle cells, potentially contributing to our understanding of muscle development and regeneration mechanisms.

To explore the functional genetic variants regulating TP63 in myogenesis, we conducted systematic analyses based on public datasets. We utilized genotype files comprising 570 samples from eastern (EAD) population and 296 samples from western pigs (WED) to identify SNPs in the promoter or intron region of TP63 that exhibited a gene frequency difference greater than 0.9 between the two community from our previous study (18). One candidate SNP, chr13: 127266445 (rs327571319 T/C) located in the fifth intron of TP63, stood out as it displayed a T allele frequency of 0.972 in Western pigs and only 0.051 in Eastern pigs (Figure 4A). Chromatin state data showed that the mutation was located in the TP63 enhancer region (Figure 4B). Further investigation revealed that this SNP was associated with loin muscle area at 100 kg (LMA) (Figure 4C) (31). To determine its functional impact, we performed a dual luciferase assay using vectors containing the wild-type sequence (PGL4.23-rs327571319-T) or the mutated sequence (PGL4.23-rs327571319-C). The results demonstrated that the T-to-C mutation significantly altered enhancer activity (p < 0.001; Figure 4D). Moreover, in 293 T cells co-transfected with TP63 overexpression (TP63-OE) along with rs327571319T/C and PRL-TK plasmid, luciferase activity was significantly higher in the presence of OE-TP63-rs327571319-C + TK compared to OE-TP63-rs327571319-T + TK, confirming the regulatory relationship between TP63 and rs327571319 (p < 0.001; Figure 4E). These findings suggest that rs327571319 could potentially be a functional candidate SNP involved in the regulation of TP63 and myogenesis.

Skeletal muscle development is a highly intricate process governed by the coordinated actions of numerous transcription factors (TFs) and genetic variants. Here, we endeavored to investigate the impact of a putative functional single nucleotide polymorphism (SNP), rs327571319, located within the intronic region of TP63, on skeletal muscle development. Through motif analysis using JASPAR, MEF2A caught our attention due to its reported promotion of myoblast differentiation (Figure 5A) (32). To elucidate the specific regulatory role of MEF2A in relation to the rs327571319 (T/C) allele, we conducted co-transfection experiments by overexpressing MEF2A along with plasmids containing the rs327571319-C or rs327571319-T allele in 293 T cells. Co-transfection experiments revealed a significant increase in enhancer activity associated with the rs327571319-C allele, suggesting the greater binding capacity of MEF2A on the C allele (p < 0.01; Figure 5B). Moreover, overexpression of MEF2A in porcine skeletal muscle cells (p < 0.001; Figure 5C) resulted in a substantial upregulation of TP63 expression (p < 0.001; Figure 5D). Consistent with these findings, the EdU assay demonstrated an augmented cell proliferative activity (p < 0.01; Figures 5E,F). Our study provides evidence that the rs327571319-C allele within the TP63 intronic region interacts with MEF2A, leading to increased TP63 expression and enhancer activity (Figure 5G). These regulatory changes promote cell proliferation and contribute to the regulation of skeletal muscle development.