Background: The genetic mechanisms that underlie phenotypic differentiation in breeding animals have important implications in evolutionary biology and agriculture. However, the contribution of cis-regulatory variants to pig phenotypes is poorly understood. Therefore, our aim was to elucidate the molecular mechanisms by which non-coding variants cause phenotypic differences in pigs by combining evolutionary biology analyses and functional genomics. Results: We obtained a high-resolution phased chromosome-scale reference genome with a contig N50 of 18.03 Mb for the Luchuan pig breed (a representative eastern breed) and profiled potential selective sweeps in eastern and western pigs by resequencing the genomes of 234 pigs. Multi-tissue transcriptome and chromatin accessibility analyses of these regions suggest that tissue-specific selection pressure is mediated by promoters and distal cis-regulatory elements. Promoter variants that are associated with increased expression of the lysozyme ( LYZ ) gene in the small intestine might enhance the immunity of the gastrointestinal tract and roughage tolerance in pigs. In skeletal muscle, an enhancer-modulating single-nucleotide polymorphism that is associated with up-regulation of the expression of the troponin C1, slow skeletal and cardiac type ( TNNC1 ) gene might increase the proportion of slow muscle fibers and affect meat quality. Conclusions: Our work sheds light on the molecular mechanisms by which non-coding variants shape phenotypic differences in pigs and provides valuable resources and novel perspectives to dissect the role of gene regulatory evolution in animal domestication and breeding.

Adipose tissue is the most important energy metabolism and secretion organ, and these functions are conferred during the adipogenesis process. However, the cause and the molecular events underlying adipogenesis are still unclear. In this study, we performed integrated bioinformatics analyses to identify vital genes involved in adipogenesis and reveal potential molecular mechanisms. Five mouse high-throughput expression profile datasets were downloaded from the Gene Expression Omnibus (GEO) database; these datasets contained 24 samples of 3T3-L1 cells during adipogenesis, including 12 undifferentiated samples and 12 differentiated samples. The five datasets were reanalyzed and integrated to select differentially expressed genes (DEGs) during adipogenesis via the robust rank aggregation (RRA) method. Functional annotation of these DEGs and mining of key genes were then performed. We also verified the expression levels of some potential key genes during adipogenesis. A total of 386 consistent DEGs were identified, with 230 upregulated genes and 156 downregulated genes. Gene Ontology (GO) analysis showed that the biological functions of the DEGs primarily included fat cell differentiation, lipid metabolic processes, and cell adhesion. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that these DEGs were mainly associated with metabolic pathways, the peroxisome proliferator-activated receptor (PPAR) signaling pathway, regulation of lipolysis in adipocytes, the tumor necrosis factor (TNF) signaling pathway, and the FoxO signaling pathway. The 30 most closely related genes among the DEGs were identified from the protein–protein interaction (PPI) network and verified by real-time quantification during 3T3-L1 preadipocyte differentiation. In conclusion, we obtained a list of consistent DEGs during adipogenesis through integrated analysis, which may offer potential targets for the regulation of adipogenesis and treatment of adipose dysfunction.

Myogenesis is a central step in prenatal myofiber formation, postnatal myofiber hypertrophy, and muscle damage repair in adulthood. RNA-Seq technology has greatly helped reveal the molecular mechanism of myogenesis, but batch effects in different experiments inevitably lead to misinterpretation of differentially expressed genes (DEGs). We previously applied the robust rank aggregation (RRA) method to effectively circumvent batch effects across multiple RNA-Seq datasets from 3T3-L1 cells. Here, we also used the RRA method to integrate nine RNA-Seq datasets from C2C12 cells and obtained 3140 robust DEGs between myoblasts and myotubes, which were then validated with array expression profiles and H3K27ac signals. The upregulated robust DEGs were highly enriched in gene ontology (GO) terms related to muscle cell differentiation and development. Considering that the cooperative binding of transcription factors (TFs) to enhancers to regulate downstream gene expression is a classical epigenetic mechanism, differentially expressed TFs (DETFs) were screened, and potential novel myogenic factors (MAF, BCL6, and ESR1) with high connection degree in protein–protein interaction (PPI) network were presented. Moreover, KLF5 cooperatively binds with the three key myogenic factors (MYOD, MYOG, and MEF2D) in C2C12 cells. Motif analysis speculates that the binding of MYOD and MYOG is KLF5-independent, while MEF2D is KLF5-dependent. It was revealed that KLF5-binding sites could be exploited to filter redundant MYOD-, MYOG-, and MEF2D-binding sites to focus on key enhancers for myogenesis. Further functional annotation of KLF5-binding sites suggested that KLF5 may regulate myogenesis through the PI3K-AKt signaling pathway, Rap1 signaling pathway, and the Hippo signaling pathway. In general, our study provides a wealth of untapped candidate targets for myogenesis and contributes new insights into the core regulatory mechanisms of myogenesis relying on KLF5-binding signal.

Background: Enhancer RNAs (eRNAs) play a crucial role in transcriptional regulation. While significant progress has been made in understanding epigenetic regulation mediated by eRNAs, research on the construction of eRNA-mediated gene regulatory networks (eGRN) and the identification of critical network components that influence complex traits is lacking. Results: Here, employing the pig as a model, we conducted a comprehensive study using H3K27ac histone ChIP-seq and RNA-seq data to construct eRNA expression profiles from multiple tissues of two distinct pig breeds, namely Enshi Black (ES) and Duroc. In addition to revealing the regulatory landscape of eRNAs at the tissue level, we developed an innovative network construction and refinement method by integrating RNA-seq, ChIP-seq, genome-wide association study (GWAS) signals and enhancer-modulating effects of single nucleotide polymorphisms (SNPs) measured by self-transcribing active regulatory region sequencing (STARR-seq) experiments. Using this approach, we unraveled eGRN that significantly influence the growth and development of muscle and fat tissues, and identified several novel genes that affect adipocyte differentiation in a cell line model. Conclusions: Our work not only provides novel insights into the genetic basis of economic pig traits, but also offers a generalizable approach to elucidate the eRNA-mediated transcriptional regulation underlying a wide spectrum of complex traits for diverse organisms.

Exercise is increasingly recognized as an effective strategy to counteract skeletal muscle aging and conditions such as sarcopenia. However, the specific exercise-induced genes responsible for these protective effects remain unclear. To address this, we conducted an eight-week aerobic exercise regimen on late-middle-aged mice and developed an integrated approach that combines mouse exercise-induced genes with human GWAS datasets to identify causal genes for sarcopenia. This approach led to significant improvements in the skeletal muscle phenotype of the mice and the identification of exercise-induced genes and miRNAs. By constructing a miRNA regulatory network enriched with transcription factors and GWAS signals related to muscle function and traits, we focused on 896 exercise-induced genes. Using human skeletal muscle cis-eQTLs as instrumental variables, 250 of these exercise-induced genes underwent two-sample Mendelian randomization analysis, identifying 40, 68, and 62 causal genes associated with sarcopenia and its clinical indicators—appendicular lean mass (ALM) and hand grip strength (HGS), respectively. Sensitivity analyses and cross-phenotype validation confirmed the robustness of our findings. Consistently across the three outcomes, RXRA, MDM1, RBL2, KCNJ2, and ADHFE1 were identified as risk factors, while NMB, TECPR2, MGAT3, ECHDC2, and GINM1 were identified as protective factors, all with potential as biomarkers for sarcopenia progression. Biological activity and disease association analyses suggested that exercise exerts its anti-sarcopenia effects primarily through the regulation of fatty acid oxidation. Based on available drug–gene interaction data, 21 of the causal genes are druggable, offering potential therapeutic targets. Our findings highlight key genes and molecular pathways potentially responsible for the anti-sarcopenia benefits of exercise, offering insights into future therapeutic strategies that could mimic the safe and mild protective effects of exercise on age-related skeletal muscle degeneration.

Introduction: We investigated epigenetic mechanisms underlying muscle development in black Tibetan sheep fed different dietary ratios of non-fiber carbohydrate to neutral detergent fiber (NFC/NDF), integrating phenotypic analyses (growth performance, meat quality, muscle histomorphology) with multi-omics approaches (ATAC-seq, H3K27ac CUT&Tag, RNA-seq).Methods: The subjects were 2-month-old weaned male Tibetan sheep with an initial body weight of 10.45 ± 0.96 kg. Using a completely randomized study design, 90 sheep were randomly assigned to three treatment groups with 30 sheep per group. Each treatment group consisted of 5 pens with 6 sheep per pen. The dietary NFC/NDF ratios of these three treatment groups were 0.71 (Group L), 1.16 (Group M) and 1.82 (Group H).Results: An NFC/NDF ratio of 1.82 yielded optimal growth performance with increased live weight, carcass weight, and dressing percentage. This ratio significantly improved meat tenderness, water-holding capacity, and pH values while reducing hardness and chewiness, accompanied by compact muscle fiber architecture. Epigenomic analyses revealed epigenetic signals predominantly localized within distal intergenic regions (10–100 kb). Enhancers showed dynamic responsiveness to dietary NFC/NDF changes, with MEF2 and SIX transcription factor binding sites enriched in upregulated enhancers. RNA-seq confirmed functional associations between epigenetic modifications and gene expression, revealing enrichment of fatty acid metabolism, epithelial cell proliferation, and macrophage chemotaxis pathways. Differential expression of key genes (EGR3, FGF7, HPGD, C5, EDNRB) corroborated MEF2 and SIX families’ central role in nutrient-mediated muscle remodeling.Discussion: These findings elucidate how dietary NFC/NDF ratios optimize muscle development through enhancer activation, providing insights for precision nutrition strategies in livestock production.