DNA methylation is important for the epigenetic regulation of gene expression and plays a critical role in mammalian development. However, the dynamic regulation of genome-wide DNA methylation in skeletal muscle development remains largely unknown. Here, we generated the first single-base resolution DNA methylome and transcriptome maps of porcine skeletal muscle across 27 developmental stages. The overall methylation level decreased from the embryo to the adult, which was highly correlated with the downregulated expression of DNMT1 and an increase in partially methylated domains. Notably, we identified over 40 000 developmentally differentially methylated CpGs (dDMCs) that reconstitute the developmental trajectory of skeletal muscle and associate with muscle developmental genes and transcription factors (TFs). The dDMCs were significantly under-represented in promoter regulatory regions but strongly enriched as enhancer histone markers and in chromatin-accessible regions. Integrative analysis revealed the negative regulation of both promoter and gene body methylation in genes associated with muscle contraction and insulin signaling during skeletal muscle development. Mechanistically, DNA methylation affected the expression of muscle-related genes by modulating the accessibly of upstream myogenesis TF binding, indicating the involvement of the DNA methylation/SP1/IGF2BP3 axis in skeletal myogenesis. Our results highlight the function and regulation of dynamic DNA methylation in skeletal muscle development.

Adenosine-to-inosine (A-to-I) RNA editing meditated by adenosine deaminases acting on RNA (ADARs) enzymes is a widespread post-transcriptional event in mammals. However, A-to-I editing in skeletal muscle remains poorly understood. By integrating strand-specific RNA-seq, whole genome bisulphite sequencing, and genome sequencing data, we comprehensively profiled the A-to-I editome in developing skeletal muscles across 27 prenatal and postnatal stages in pig, an important farm animal and biomedical model. We detected 198,892 A-to-I editing sites and found that they occurred more frequently at prenatal stages and showed low conservation among pig, human, and mouse. Both the editing level and frequency decreased during development and were positively correlated with ADAR enzymes expression. The hyper-edited genes were functionally related to the cell cycle and cell division. A co-editing module associated with myogenesis was identified. The developmentally differential editing sites were functionally enriched in genes associated with muscle development, their editing levels were highly correlated with expression of their host mRNAs, and they potentially influenced the gain/loss of miRNA binding sites. Finally, we developed a database to visualize the Sus scrofa RNA editome. Our study presents the first profile of the dynamic A-to-I editome in developing animal skeletal muscle and provides evidences that RNA editing is a vital regulator of myogenesis.

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Background Circular RNAs (circRNAs) represent a novel class of non‐coding RNAs formed by a covalently closed loop and play crucial roles in many biological processes. Several circRNAs associated with myogenesis have been reported. However, the dynamic expression, function, and mechanism of circRNAs during myogenesis and skeletal muscle development are largely unknown. Methods Strand‐specific RNA‐sequencing (RNA‐seq) and microarray datasets were used to profile the dynamic circRNAome landscape during skeletal muscle development and myogenic differentiation. Bioinformatics analyses were used to characterize the circRNAome and identify candidate circRNAs associated with myogenesis. Bulk and single‐cell RNA‐seq were performed to identify the downstream genes and pathways of circFgfr2. The primary myoblast cells, C2C12 cells, and animal model were used to assess the function and mechanism of circFgfr2 in myogenesis and muscle regeneration in vitro or in vivo by RT‐qPCR, western blotting, dual‐luciferase activity assay, RNA immunoprecipitation, RNA fluorescence in situ hybridization, and chromatin immunoprecipitation. Results We profiled the dynamic circRNAome in pig skeletal muscle across 27 developmental stages and detected 52 918 high‐confidence circRNAs. A total of 2916 of these circRNAs are conserved across human, mouse, and pig, including four circRNAs (circFgfr2, circQrich1, circMettl9, and circCamta1) that were differentially expressed (|log2 fold change| > 1 and adjusted P value < 0.05) in various myogenesis systems. We further focused on a conserved circRNA produced from the fibroblast growth factor receptor 2 (Fgfr2) gene, termed circFgfr2, which was found to inhibit myoblast proliferation and promote differentiation and skeletal muscle regeneration. Mechanistically, circFgfr2 acted as a sponge for miR‐133 to regulate the mitogen‐activated protein kinase kinase kinase 20 (Map3k20) gene and JNK/MAPK pathway. Importantly, transcription factor Kruppel like factor 4 (Klf4), the downstream target of the JNK/MAPK pathway, directly bound to the promoter of circFgfr2 and affected its expression via an miR‐133/Map3k20/JNK/Klf4 auto‐regulatory feedback loop. RNA binding protein G3BP stress granule assembly factor 1 (G3bp1) inhibited the biogenesis of circFgfr2. Conclusions The present study provides a comprehensive circRNA resource for skeletal muscle study. The functional and mechanistic analysis of circFgfr2 uncovered a circRNA‐mediated auto‐regulatory feedback loop regulating myogenesis and muscle regeneration, which provides new insight to further understand the regulatory mechanism of circRNAs.

Forkhead Box F1 (FOXF1) has been recently implicated in cancer progression and metastasis of lung cancer and breast cancer. However, the biological functions and underlying mechanisms of FOXF1 in the regulation of the progression of colorectal cancer (CRC) are largely unknown. We showed that FOXF1 was up-regulated in 93 paraffin-embedded archived human CRC tissue, and both high expression and nuclear location of FOXF1 were significantly associated with the aggressive characteristics and poorer survival of CRC patients. The GSEA analysis showed that the higher level of FOXF1 was positively associated with an enrichment of EMT gene signatures, and exogenous overexpression of FOXF1 induced EMT by transcriptionally activating SNAI1. Exogenous overexpression FOXF1 functionally promoted invasion and metastasis features of CRC cells, and inhibition of SNAI1 attenuates the invasive phenotype and metastatic potential of FOXF1-overexpressing CRC cells. Furthermore, the results of the tissue chip showed that the expression of FOXF1 was positively correlated with SNAI1 in CRC tissues chip. These results suggested that FOXF1 plays a critical role in CRC metastasis by inducing EMT via transcriptional activation of SNAI1, highlighting a potential new therapeutic strategy for CRC.

MeiShan and Large White pigs differ in their female fecundity. However, the mechanisms behind the gene expression and regulation that cause these differences remain unclear. In this study, we profiled circRNAs and identified 5,879 circRNAs from the ovaries of MeiShan and Large White pigs. Eighty-five circRNAs were differentially expressed between the two pig breeds. Of these, 37 were up-regulated and 48 were down-regulated in MeiShan pigs. Gene ontology enrichment analysis suggested that the differentially expressed circRNA were involved in the hormone-mediated signaling pathway. We verified that circSCIN and its parent gene, scinderin (SCIN), were differentially expressed by reverse transcription and quantitative PCR (RT-qPCR). Luciferase assays demonstrated that circSCIN can target and sponge miR-133 and miR-148a/b. The identification of differentially expressed circRNAs (DECs) and their regulatory functions increased our understanding of the differences in reproductive efficiency between MeiShan and Large White pigs.

Circular RNAs (circRNAs) represent a class of covalently closed single-stranded RNA molecules that are emerging as essential regulators of various biological processes. The circRNA circHipk2 originates from exon 2 of the Hipk2 gene in mice and was reported to be involved in acute promyelocytic leukemia and myocardial injury. However, the functions and mechanisms of circHipk2 in myogenesis are largely unknown. Here, to deepen our knowledge about the role of circHipk2, we studied the expression and function of circHipk2 during skeletal myogenesis. We found that circHipk2 was mostly distributed in the cytoplasm, and dynamically and differentially expressed in various myogenesis systems in vitro and in vivo. Functionally, overexpression of circHipk2 inhibited myoblast proliferation and promoted myotube formation in C2C12 cells, whereas the opposite effects were observed after circHipk2 knockdown. Mechanistically, circHipk2 could directly bind to ribosomal protein Rpl7, an essential 60S preribosomal assembly factor, to inhibit ribosome translation. In addition, we verified that transcription factor Sp1 directly bound to the promoter of circHipk2 and affected the expression of Hipk2 and circHipk2 in C2C12 myoblasts. Collectively, these findings identify circHipk2 as a candidate circRNA regulating ribosome biogenesis and myogenesis proliferation and differentiation.

Natural and artificial directional selections have resulted in significantly genetic and phenotypic differences across breeds in domestic animals. However, the molecular regulation of skeletal muscle diversity remains largely unknown. Here, we conducted transcriptome profiling of skeletal muscle across 27 time points, and performed whole-genome re-sequencing in Landrace (lean-type) and Tongcheng (obese-type) pigs. The transcription activity decreased with development, and the high-resolution transcriptome precisely captured the characterizations of skeletal muscle with distinct biological events in four developmental phases: Embryonic, Fetal, Neonatal, and Adult. A divergence in the developmental timing and asynchronous development between the two breeds was observed; Landrace showed a developmental lag and stronger abilities of myoblast proliferation and cell migration, whereas Tongcheng had higher ATP synthase activity in postnatal periods. The miR-24-3p driven network targeting insulin signaling pathway regulated glucose metabolism. Notably, integrated analysis suggested SATB2 and XLOC_036765 contributed to skeletal muscle diversity via regulating the myoblast migration and proliferation, respectively. Overall, our results provide insights into the molecular regulation of skeletal muscle development and diversity in mammals.