Identification of key differentially methylated genes regulating muscle development in chickens: insights from Jingyuan breed

Abstract

Skeletal muscle development is a complex, regulated physiological process that involves myoblast proliferation and differentiation and the fusion of myotubes. In this study, phenotypic differences in the breast and leg muscles of 180-day-old Jingyuan chickens were investigated. Differentially methylated genes (DMG) that regulate muscle development were identified through differential expression analysis and weighted gene co-expression network analysis. Moreover, myoblasts were used as test material and treated with cycloleucine to investigate the effect of N⁶-methyladenosine (m⁶A) modification on their proliferation and differentiation. The results revealed that the myofiber diameter and cross-sectional area in the breast muscle of Jingyuan chickens were significantly smaller than those in the leg muscle, while myofiber density in the breast muscle was significantly higher. A total of 484 DMG were identified in both muscle types. Module gene association analysis with DMGs revealed multiple DMG associated with muscle development. In vitro cell model analysis revealed that cycloleucine treatment significantly downregulated the m⁶A modification level of myoblasts and inhibited their proliferation and differentiation. Additionally, stage-specific differences in LDHA, LDHB, and GAPDH expressions were observed during myoblast differentiation. Cycloleucine treatment significantly inhibited LDHA, LDHB, and GAPDH expression. These findings indicate that m⁶A methylation modifications play significant regulatory roles in muscle development, with LDHA, LDHB, and GAPDH being potential candidate genes for regulating muscle development. This study provides an essential theoretical basis for further study on the functional mechanisms of m⁶A modifications involved in muscle development.

Keywords: Jingyuan chicken; N(6)-methyladenosine; WGCNA; muscle development.

Figure 1

Muscle phenotypes of breast as well as leg muscles of Jingyuan chickens (B: breast; L: leg). (A) Differences in muscle rate between breast and leg muscles were studied (n = 5). (B) HE staining results of the breast and leg muscles. (C) Myofiber diameter, cross-sectional area, and density analysis of the breast and leg muscles (n = 5).

Figure 2

Identification of differential peak in the breast leg muscles and functional enrichment analysis (B: breast; L: leg) (A) Differential peak volcano plots in the L vs. B group. (B–C) Analysis of differential peak GO and KEGG enrichment in L vs. B.

Figure 3

Expression pattern and analysis of L vs. B DEGs (B: breast; L: leg) (A) Volcanic map of L vs. B DEGs. (B–C) GO and KEGG enrichment analysis of L vs. B DEGs.

Figure 4

Different DMGs identification and functional enrichment analysis of L vs. B (B: breast; L: leg) (A) Distribution of genes with changes in m⁶A methylation level and mRNA level in group L vs. B (Hyper-up: m⁶A methylation level and mRNA expression level are up-regulated, Hyper-down: m⁶A methylation level is up-regulated while mRNA expression level is down-regulated, Hypo-up: m⁶A methylation level is down-regulated while mRNA expression level is up-regulated. Hypo-down: m⁶A methylation level and mRNA expression level are down-regulated). (B–C) GO enrichment analysis of Leg vs. Breast differential DMGs. (D) KEGG enrichment analysis of L vs. B differential DMGs.

Figure 5

Analysis of coexpression network of genes in group L vs. B. (A) The power value curve. (B) Gene cluster analysis (each color represents 1 module). (C) Heat map of correlation between modules and phenotypes.

Figure 6

Functional enrichment analysis of blue module, magenta module and black module genes Scatter plot of gene significance and module membership for (A)-1 (blue),(B)-1 (Magenta),(C)-1 (black). KEGG pathway enrichment analysis of hub genes in (A)-2 (blue),(B)-2 (Magenta),(C)-2 (black). GO functional enrichment analysis in (A)-3 (blue),(B)-3 (Magenta),(C)-3 (black).

Figure 7

Identification of Hub genes in key modules and validation at the m⁶A and mRNA levels (A) Identification of DMGs in key modules. (B–C) GO and KEGG enrichment analysis of DMGs in key modules. (D) Network diagram of protein interactions of LDHA, LDHB, and GAPDH (E) Validation of LDHA, LDHB, and GAPDH at the m⁶A and mRNA levels.

Figure 8

10mM cycloleucine inhibits myoblast proliferation and differentiation. (A–B) After myoblasts were treated with 10mM cycloleucine for 24 h, m⁶A Dot Blot and m⁶A colorimetry were used to detect m⁶A modification levels (C) After cycloleucine treatment for 24h, the expression levels of genes related to proliferation and differentiation in myoblasts were detected by qPCR (n = 3). (D) Proliferation of cycloleucine -treated myoblasts was detected by EDU after 24 h (n = 3). (E) Proliferation of cycloleucine -treated myoblasts after 24 h of treatment by CCK8 (n = 8).

Figure 9

Functional analysis of LDHA, LDHB and GAPDH in myoblasts. (A) Expression trends of LDHA, LDHB, and GAPDH during myoblast differentiation. (n = 3) Different letters indicate significant differences (P<0.01), and the same letters indicate nonsignificant differences (P>0.05). (B) Changes in the expression of LDHA, LDHB, and GAPDH after 10 mM cycloleucine treatment of myoblasts for 24 h (n = 3).