Genome-wide DNA methylation profiles provide insight into epigenetic regulation of red and white muscle development in Chinese perch Siniperca chuatsi

https://doi.org/10.1016/j.cbpb.2021.110647Get rights and content

Highlights

  • The overall DNA methylation levels and dnmts expression in the red muscles are higher than that in the white muscle of Chinese perch.

  • WGBS identifies 4192 differential methylated genes (DMGs) between red and white muscle of Chinese perch.

  • DMGs are linked to cell signaling pathway related to skeletal muscle differentiation and growth.

Abstract

Fish skeletal muscles are composed of spatially well-separated fiber types, namely, red and white muscles with different physiological functions and metabolism. To compare the DNA methylation profiles of the two types of muscle tissues and identify potential candidate genes for the muscle growth and development under epigenetic regulation, genome-wide DNA methylation of the red and white muscle in Chinese perch Siniperca chuatsi were comparatively analyzed using bisulfate sequencing methods. An average of 0.9 billion 150-bp paired-end reads were obtained, of which 86% were uniquely mapped to the genome. Methylation mostly occurred at CG sites at a ratio of 94.43% in the red muscle and 93.16% in the white muscle. The mean methylation levels at C-sites were 5.95% in red muscle and 5.83% in white muscle, whereas the mean methylation levels of CG, CHG, and CHH were 73.23%, 0.62%, and 0.67% in red muscle, and 71.01%, 0.62%, and 0.67% in white muscle, respectively. A total of 4192 differentially methylated genes (DMGs) were identified significantly enriched in cell signaling pathways related to skeletal muscle differentiation and growth. Various muscle-related genes, including myosin gene isoforms and regulatory factors, are differentially methylated in the promoter region between the red and white muscles. Further analysis of the transcriptional expression of these genes showed that the muscle regulatory factors (myf5, myog, pax3, pax7, and twitst2) and myosin genes (myh10, myh16, myo18a, myo7a, myo9a, and myl3) were differentially expressed between the two kinds of muscles, consistent with the DNA methylation analysis results. ELISA assays confirmed that the level of 5mC in red muscle was significantly higher than in white muscle (P < 0.05). The RT-qPCR assays revealed that the expression levels of the three DNA methylation transferase (dnmt) subtypes, dnmt1, dnmt3ab, and dnmt3bb1, were significantly higher in red muscle than in white muscle. The higher DNA methylation levels in the red muscle may result from higher DNA methylation transferase expression in the red muscles. Thus, this study might provide a theoretical foundation to better understand epigenetic regulation in the growth and development of red and white muscles in animals, at least in Chinese perch fish.

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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