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Abstract Background Chinese indigenous pigs are popular with consumers for their juiciness, flavour and meat quality, but they have lower meat production. Insulin-like growth factor 2 (IGF2) is a maternally imprinted growth factor that promotes skeletal muscle growth by regulating cell proliferation and differentiation. A single nucleotide polymorphism (SNP) within intron 3 of porcine IGF2 disrupts a binding site for the repressor, zinc finger BED-type containing 6 (ZBED6), leading to up-regulation of IGF2 and causing major effects on muscle growth, heart size, and backfat thickness. This favorable mutation is common in Western commercial pig populations, but absent in most Chinese indigenous pig breeds. To improve meat production of Chinese indigenous pigs, we used cytosine base editor 3 (CBE3) to introduce IGF2-intron3-C3071T mutation into porcine embryonic fibroblasts (PEFs) isolated from a male Liang Guang Small Spotted pig (LGSS), and single-cell clones harboring the desired mutation were selected for somatic cell nuclear transfer (SCNT) to generate the founder line of IGF2 T/T pigs. Results We found the heterozygous progeny IGF2 C/T pigs exhibited enhanced expression of IGF2, increased lean meat by 18%–36%, enlarged loin muscle area by 3%–17%, improved intramuscular fat (IMF) content by 18%–39%, marbling score by 0.75–1, meat color score by 0.53–1.25, and reduced backfat thickness by 5%–16%. The enhanced accumulation of intramuscular fat in IGF2 C/T pigs was identified to be regulated by the PI3K-AKT/AMPK pathway, which activated SREBP1 to promote adipogenesis. Conclusions We demonstrated the introduction of IGF2-intron3-C3071T in Chinese LGSS can improve both meat production and quality, and first identified the regulation of IMF deposition by IGF2 through SREBP1 via the PI3K-AKT/AMPK signaling pathways. Our study provides a further understanding of the biological functions of IGF2 and an example for improving porcine economic traits through precise base editing.

Abstract Krüppel-like factor 4 (KLF4) is an evolutionarily conserved zinc finger-containing transcription factor that regulates diverse cellular processes such as cell proliferation, apoptosis, and differentiation. Our previous study showed that KLF4 expression is upregulated in skeletal muscle ontogeny during embryonic development in pigs, suggesting its importance for skeletal muscle development and muscle function. We revealed here that KLF4 plays a critical role in skeletal muscle development and regeneration. Specific knockout of KLF4 in skeletal muscle impaired muscle formation further affecting physical activity and also defected skeletal muscle regeneration. In vitro, KLF4 was highly expressed in proliferating myoblasts and early differentiated cells. KLF4 knockdown promoted myoblast proliferation and inhibited myoblast fusion, while its overexpression showed opposite results. Mechanically, in proliferating myoblasts, KLF4 inhibits myoblast proliferation through regulating cell cycle arrest protein P57 by directly targeting its promoter; while in differentiated myoblasts, KLF4 promotes myoblast fusion by transcriptionally activating Myomixer. Our study provides mechanistic information for skeletal muscle development, reduced muscle strength and impaired regeneration after injury and unveiling the mechanism of KLF4 in myogenic regulation.

Abstract Background Skeletal muscle development is a multistep process whose understanding is central in a broad range of fields and applications, from the potential medical value to human society, to its economic value associated with improvement of agricultural animals. Skeletal muscle initiates in the somites, with muscle precursor cells generated in the dermomyotome and dermomyotome-derived myotome before muscle differentiation ensues, a developmentally regulated process that is well characterized in model organisms. However, the regulation of skeletal muscle ontogeny during embryonic development remains poorly defined in farm animals, for instance in pig. Here, we profiled gene expression and chromatin accessibility in developing pig somites and myotomes at single-cell resolution. Results We identified myogenic cells and other cell types and constructed a differentiation trajectory of pig skeletal muscle ontogeny. Along this trajectory, the dynamic changes in gene expression and chromatin accessibility coincided with the activities of distinct cell type-specific transcription factors. Some novel genes upregulated along the differentiation trajectory showed higher expression levels in muscular dystrophy mice than that in healthy mice, suggesting their involvement in myogenesis. Integrative analysis of chromatin accessibility, gene expression data, and in vitro experiments identified EGR1 and RHOB as critical regulators of pig embryonic myogenesis. Conclusions Collectively, our results enhance our understanding of the molecular and cellular dynamics in pig embryonic myogenesis and offer a high-quality resource for the further study of pig skeletal muscle development and human muscle disease.

Abstract Histone lysine demethylase 4A (KDM4A) plays a crucial role in regulating cell proliferation, cell differentiation, development and tumorigenesis. However, little is known about the function of KDM4A in muscle development and regeneration. Here, we found that the conditional ablation of KDM4A in skeletal muscle caused impairment of embryonic and postnatal muscle formation. The loss of KDM4A in satellite cells led to defective muscle regeneration and blocked the proliferation and differentiation of satellite cells. Myogenic differentiation and myotube formation in KDM4A-deficient myoblasts were inhibited. Chromatin immunoprecipitation assay revealed that KDM4A promoted myogenesis by removing the histone methylation mark H3K9me3 at MyoD, MyoG and Myf5 locus. Furthermore, inactivation of KDM4A in myoblasts suppressed myoblast differentiation and accelerated H3K9me3 level. Knockdown of KDM4A in vitro reduced myoblast proliferation through enhancing the expression of the cyclin-dependent kinase inhibitor P21 and decreasing the expression of cell cycle regulator Cyclin D1. Together, our findings identify KDM4A as an important regulator for skeletal muscle development and regeneration, orchestrating myogenic cell proliferation and differentiation.