Bioscience Methods 2025, Vol.16, No.2, 83-99 http://bioscipublisher.com/index.php/bm 89 transcriptome and chromatin openness can be measured simultaneously at the single-cell level, and then a certain open regulatory element can be linked to the gene expression of the cell in which it is located. This enables us to construct cell type-specific regulatory networks. For example, if the upstream enhancer of the MyoD promoter is specifically opened in satellite cells and closed in myoblasts, this suggests that the enhancer is essential for satellite cells to activate MyoD expression. Another important level is histone covalent modification, such as acetylation and methylation, which are involved in determining the active state of chromatin. Histone acetylation is generally associated with transcriptional activation because acetylation neutralizes the positive charge of histones, weakens DNA-histone interactions, and makes chromatin loose and easy to transcribe. The metabolism and differentiation of skeletal muscle cells are regulated by the level of histone acetylation: Studies have shown that in muscle cells, the dynamic balance of histone acetyltransferases (HATs) and deacetylases (HDACs) precisely regulates the expression of related genes. For example, HDAC inhibitors can promote myoblast differentiation because they increase histone acetylation, thereby activating the transcription of muscle differentiation genes (Xu et al., 2023). Specific to certain landmark modifications, such as histone H3 lysine 9 acetylation (H3K9ac) and H3 lysine 27 acetylation (H3K27ac), they are often enriched in active gene promoters and enhancer regions, and can be used as a marker to identify active enhancers in muscle cells. In the mouse C2C12 myoblast induced differentiation model, H3K27ac increased significantly near the promoters of key genes such as Myogenin, corresponding to the rapid upregulation of genes. In contrast, histone deacetylation and specific histone methylation (such as H3K27 trimethylation) are high when muscle stem cells are maintained in an undifferentiated state, thereby silencing the expression of differentiation genes. This has been observed in satellite cells: H3K27me3 mediated by the polycomb complex (PRC2) is enriched at the muscle differentiation loci of quiescent satellite cells to inhibit premature differentiation; when satellite cells are activated, this modification decreases and the transcription of related genes is unrepressed. A large amount of evidence shows that changes in histone modifications during muscle development are closely related to gene expression. For example, a review summarizes the effects of histone acetylation/deacetylation on skeletal muscle metabolism and phenotype, including regulation of myocyte cycle, fiber type conversion, muscle atrophy, and insulin sensitivity. For livestock and poultry such as goats, although there are not many studies on specific histone modification maps, similar mechanisms can be expected to exist. In addition, the role of histone methylation such as H3K4me3 (active mark) and H3K27me3 (repressive mark) in muscle development is also worthy of attention. For example, EZH2, as a methyltransferase of H3K27me3, plays a role in satellite cell self-renewal. A study detected high expression of EZH2 in some cells in single-cell sequencing of sheep spermatogonia (Xiong et al., 2022). It is speculated that EZH2 may also be involved in maintaining the stem cell state in muscle satellite cells. When EZH2 function is impaired, satellite cells may differentiate prematurely, deplete the stem cell pool, and lead to decreased muscle regeneration ability. On the contrary, if factors that regulate histone acetylation levels, such as HATs such as PCAF, are upregulated, they may promote the formation of muscle fibers. 4.2 DNA methylation patterns in muscle cells DNA methylation is another important epigenetic mechanism, that is, adding methyl groups to cytosine in DNA (mainly occurring at CpG sites), which is usually associated with transcriptional repression. In skeletal muscle development, DNA methylation has a regulatory effect on cell fate determination and gene temporal expression. In general, high methylation in gene promoter/enhancer regions often leads to gene silencing, while demethylation is associated with gene activation. When muscle stem cells transform into differentiated cells, the methylation status of many muscle-specific gene promoters will change. For example, low methylation in the promoter regions of key myogenic factors such as MYOD1 and MYF5 is usually a prerequisite for their high expression, and literature has reported that the methylation level of these sites is negatively correlated with myogenic differentiation ability. A study on maternal malnutrition in goats directly demonstrated the effect of DNA methylation on muscle gene expression: when ewes were malnourished in mid-pregnancy, the methylation level of specific sites on the MYF5 promoter in the skeletal muscle of their fetuses increased significantly, resulting in decreased MYF5 expression; correspondingly, some sites of the MYOD promoter in the muscle of newborn lambs
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