Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology
Genome-wide DNA methylation profiles provide insight into epigenetic regulation of red and white muscle development in Chinese perch Siniperca chuatsi
Graphical abstract
Introduction
Epigenetics refers to DNA or associated protein modification without DNA sequence changes, and it mainly includes DNA methylation, histone modification, and related non-coring microRNAs (Zuo et al., 2009). Among these, DNA methylation is one of the most important epigenetic modifications. It is catalyzed by DNA methyltransferases by the addition of a methyl group to the 5‑carbon of the cytosine ring, resulting in 5-methylcytosine (5-mC), and it mostly occurs in the CpG enriched promoter region of a gene (Lister and Mukamel, 2015). Under normal conditions, CpG islands remain unmethylated in the promoter region; however, once they are methylated, they cause gene silencing (Zhang et al., 2017). In addition, hypermethylation of the first exon or first intron has also been reported to negatively regulate gene expression (Anastasiadi et al., 2018; Brenet et al., 2011). Although hypermethylation of gene regions is involved in the negative regulation of gene expression, some hypermethylation has been reported to be linked with gene expression upregulation (McGuire et al., 2019; Rauluseviciute et al., 2020). The mechanism of DNA methylation associated with gene regulation and expression is conserved in all invertebrates and vertebrates. It is involved in many cellular and molecular processes, such as ensuring chromosomal stability and genomic chromosome inactivation through transcription etc. (Yamazaki et al., 2020). In addition, DNA methylation also plays an important role in gene regulation in different DNA sequence context, such as promoter methylation may cause gene silencing (Shen et al., 2016), gene body methylation could affect gene transcription (Liang and Peng, 2021; Teissandier and Bourc'his, 2017) and intergenic methylation may suppress gene expression (Earley et al., 2010; Yan et al., 2016).
Fish skeletal muscles are highly differential multicellular tissues composed of two types of muscle fibers, such as red muscle (slow-twitch muscle) and white muscle (fast-twitch muscle), and they perform different physiological functions and metabolic processes (Wakeling and Johnston, 1999; Wu et al., 2018). Skeletal muscle differentiation and growth are regulated by many extracellular signaling molecules combined with intracellular transcription factors such as MyoD, Myf5, myogenin, and MRF4. They play crucial roles in muscle cell specification, proliferation, and differentiation during muscle development (Johnston et al., 2011). Studies have revealed that skeletal muscle development is regulated by the methylation and demethylation of DNA at specific structural or regulatory genes (Simo-Mirabet et al., 2020). Campos et al. (2013) reported that increased muscle development in Senegalese sole (Solea senegalensis) was associated with upregulated myogenin expression, decreased methylation of the myogenin promoter, and decreased DNA methyltransferase (dnmt1 and dnmt3b) expression. Burgerhout et al. (2017) also demonstrated that in Atlantic salmon (Salmo salar), higher larval myogenin expression was related to relatively low DNA methylation levels. Differential cytosine methylation was also reported to affect the sex-specific growth phenotypes in hybrid tilapia (Wan et al., 2016). Several studies also revealed that the epigenetic regulation of muscle growth involved in different gene networks between males and females in tilapia fish (Podgorniak et al., 2019). Therefore, further studies on the complex epigenic network regulating skeletal muscle differentiation and growth would improve our understanding of the potential mechanism of skeletal muscle growth and development.
The combination of DNA bisulfite treatment and high-throughput sequencing technologies has led us to investigate DNA methylation at the genome-wide level in a near-base-pair-level resolution (Adusumalli et al., 2015). With advent of these technologies, many studies have focused on the DNA methylation profiles of mammalian skeletal muscles. Kim et al. (2018) examined the genome-wide DNA methylome in the loin (longissimus dorsi) muscle (LDM) of swine and revealed the functional role of DNA methylation in gene expression of LDM. Studies on pigs verified genome-wide DNA methylation profile changes in skeletal muscle with distinct metabolic types and ryanodine receptor variation (Ponsuksili et al., 2019) or constant heat stress(Hao et al., 2016). Furthermore, Cao et al. (2017) analyzed the DNA methylation profile in the longissimus dorsi muscle between Small Tailed Han and Dorperx Small Tailed Han crossbred sheep and found that the DNA methylation levels in DMRs of the function genes may influence the expression. However, there has been limited progress in the genome-wide mapping of the DNA methylation profile of skeletal muscles in commercially important fish species.
Chinese perch (Siniperca chuatsi) is one of the most commercially important freshwater fish in China, with good meat quality, high content of essential amino acids, and an abundance of unsaturated fatty acids (Chu et al., 2011; Zhang et al., 2011). Similar to other teleost fish, skeletal muscles in fish species are mainly composed of red and white muscle fibers, and they are separated to a much greater degree than in mammals (Johnston and Moon, 1981). The two types of muscle fibers of Chinese perch are structurally and functionally different with specific genetic and metabolic characteristics (Chu et al., 2017; Wen et al., 2015; Zhu et al., 2015). Consequently, studies focusing on genome-wide DNA methylation status between the red and white muscle tissues, particularly specific candidate gene methylation related to two kinds of muscle fibers, have not been conducted. Therefore, the genome-wide DNA methylation profiles of the white and red muscle tissues of Chinese perch were analyzed by bisulfite sequencing in this study. This is the first systematic comparative analysis of genome-wide DNA methylation profiles between the two types of muscle fibers in fish. The results revealed that the overall DNA methylation levels in the red muscles were hypermethylated than those in the white muscle. Furthermore, the methylation levels of 20 different signaling pathways, several muscle-related structural gene myosin isoforms, and regulatory factors were identified to be differentially methylated between the two muscle types, whereas the expression levels of DNA methyltransferases in the red muscle were much higher than those in the white muscle. The obtained data suggest a potential mechanism of the differential degree of growth and development between the red and white muscle in fish, at least in Chinese perch.
Section snippets
Ethics approval and consent for participation
This study was conducted in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health of Changsha University. All fish handling procedures were performed after the fish were anesthetized in MS-222.
Experimental muscle tissue sampling
All the test fish were reared at the Hunan Fisheries Science Institute (Changsha, Hunan, China). Six fish were taken by immersing them in water containing 0.2 g/L MS-222. The dorsal epaxial fast and slow muscles were collected from the fish of
Genome-wide mapping of DNA methylation and genome coverage statistics in both red and white muscle tissues
To reveal the difference in DNA methylation in red and white muscles of Chinese perch at the whole genome level, we performed WGBS on three of both red and white muscle tissues from Chinese perch with a mean yield of 0.9 billion 150-bp. After quality control, 85.9–87.4% of the reads were uniquely mapped to the genome, and an average of 90% base covered ≥10×. The coverage depths in the red and white muscle tissues were at least 26% above (Table 1). All the cleaning reads have been deposited in
Discussion
Fish skeletal muscles are composed of two types of muscle fiber types: slow (or red) and fast (white) muscles. They perform different metabolic activities and functions. Skeletal muscle differentiation and growth are regulated at the cellular and genomic levels (Zanou and Gailly, 2013) and influenced by epigenetic regulation via DNA methylation and demethylation (Carrió and Suelves, 2015). To reveal the differences in DNA methylation between red and white muscles at the genome-wide level, we
Declaration of Competing Interest
We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, there is no professional or other personal interest of any nature or kind in any product, service and company that could be construed as influencing the position presented in the manuscript entitled “Genome-wide DNA methylation profiles provide insight into epigenetic regulation of red and white muscle development in Chinese perch Siniperca chuatsi”.
Acknowledgements
This study was supported by the National Natural Science Foundation of China (Nos. 31820103016, 32002364, 31972766), and Scientific Research Fund of Education Department of Hunan Province of China (19B065, 20K014, 18A375).
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The authors made equal contribution to the work.