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FTO-mediated demethylation of GADD45B promotes myogenesis through the activation of p38 MAPK pathway

N6-methyladenosine (m6A) modification plays a critical role in mammalian development. However, the role of m6A in the skeletal muscle development remains largely unknown. Here, we report a global m6A modification pattern of goat skeletal muscle at two key development stages and identified that the m6A modification regulated the expression of the growth arrest and DNA damage-inducible 45B (GADD45B) gene, which is involved in myogenic differentiation. We showed that GADD45B expression increased during myoblast differentiation, whereas the downregulation of GADD45B inhibits myogenic differentiation and mitochondrial biogenesis. Moreover, the expression of GADD45B regulates the expression of myogenic regulatory factors and peroxisome proliferator-activated receptor gamma coactivator 1 alpha by activating the p38 mitogen-activated protein kinase (MAPK) pathway. Conversely, the inactivation of p38 MAPK abolished the GADD45B-mediated myogenic differentiation. Furthermore, we found that the knockdown of fat mass and obesity-associated protein (FTO) increases GADD45B m6A modification and decreases the stability of GADD45B mRNA, which impairs myogenic differentiation. Our results indicate that the FTO-mediated m6A modification in GADD45B mRNA drives skeletal muscle differentiation by activating the p38 MAPK pathway, which provides a molecular mechanism for the regulation of myogenesis via RNA methylation.


INTRODUCTION
Skeletal muscle is an indispensable component of the body, playing a vital role in locomotion, metabolism, and homeostasis. It originates from mesodermal progenitors, which undergo a complex, multistep process of myoblast proliferation and fusion, myotube formation, and myofiber maturation during embryogenesis. 1,2 Skeletal muscle development, also called myogenesis, occurs as a result of the coordination of complex transcriptional regulatory networks, consisting of myogenic regulatory factors (MRFs), the myocyte enhancer factor 2 family, and other related transcription factors, as well as several signaling molecules, such as mitogen-activated protein kinase (MAPK), Wnt, and transforming growth factor-b (TGF-b). 1,[3][4][5] Moreover, post-transcriptional regulation also contributes to myogenesis via RNA binding proteins and noncoding RNAs. [6][7][8] Although mRNA modifications play key roles in post-transcriptional gene regulation, 9 their roles in regulating myogenesis and the underlying mechanisms remain unclear.
More than 150 types of chemical modifications have been identified in cellular RNAs. 10 Among these, N6-methyladenosine (m 6 A) modification has attracted the most attention because of its prevalence and biological functions in mammals. [11][12][13] The m 6 A modification of mRNA is a reversible process that is dynamically regulated by m 6 A writers and erasers. The m 6 A writer complex consists of methyltransferase-like 3 (METTL3), methyltransferase-like 14 (METTL14), and Wilms tumor 1-associated protein (WTAP), which can catalyze the methylation of specific adenosines in mammalian mRNA. 14,15 Fat mass and obesity-associated protein (FTO) and alpha-ketoglutarate-dependent dioxygenase alkB homolog 5 (ALKBH5) have been identified as m 6 A erasers, in which their role is to remove m 6 A modifications. 16 m 6 A modifications have been shown to exert important functions in stem cell self-renewal and differentiation, cell fate determination, tissue development, carcinogenesis, and stress response regulation, as well as in RNA metabolism, including mRNA stability, splicing, transport, localization, and translation. [17][18][19][20] Recent in vivo studies have led to breakthroughs in elucidating the effects of m 6 A modifications on mammalian development. Aberrant m 6 A modifications were found to impair cell-cycle progression, stem cell differentiation, bone homeostasis balance, and brown adipogenesis and to retard neurogenesis. [21][22][23] It has also been reported that FTO knockout in mice reduces the volume of lean body mass; 24,25 therefore, it is crucial to understand the potential role of m 6 A modifications in skeletal muscle development in large animals, such as goats.
Here, we explored the RNA m 6 A modification profile of goat skeletal muscle at key developmental stages (75-day fetus, 1-dayold kid) and identified growth arrest and DNA damage-inducible 45B (GADD45B) as a potential m 6 A-mediated gene involved in myogenesis. Importantly, the molecular mechanism underling the role of GADD45B in myogenic differentiation and the effect of m 6 A modifications on GADD45B expression were also investigated. Taken together, our findings demonstrate that GADD45B expression is regulated by FTO-mediated m 6 A modification and promotes myogenic differentiation by activating the p38 MAPK pathway.

RESULTS m 6 A levels decreased during myogenic differentiation in vivo and in vitro
To determine the potential role of m 6 A modification in myogenesis, we evaluated the correlation between the m 6 A content and myogenic differentiation in vivo. First, the muscle collected from goats at different developmental stages was physiologically investigated. The size and number of myofibers in newly born kid skeletal muscle were markedly higher compared with that of myofibers in the fetal skeletal muscle (Figures 1A and 1B). Furthermore, based on the Im-ageJ analysis (National Institutes of Health, Bethesda, MD, USA) of the H&E staining of the longissimus muscle, it was found that the number of myofibers occupying a larger area (>80 mm 2 in area) was higher in the kid samples than in the fetus samples, whereas the number of smaller-sized myofibers (<80 mm 2 in area) was significantly lower in the kid samples than in the fetus samples ( Figure 1C). This corresponds to the muscular development phenotype of the two development stages. The myogenic differentiation marker actin, alpha skeletal muscle (ACTA1) was significantly upregulated in the kids compared to the fetuses; in contrast, the expression of paired box 7 (PAX7), the gene responsible for the maintenance of satellite cells, was reduced during development ( Figure 1D).
Next, colorimetry was used to quantify the global m 6 A modifications in skeletal muscle during the embryonic period and at the newborn stage. 26 As a result, fewer global m 6 A modifications were observed in the muscles of kids compared to those of fetuses ( Figure 1E). Consistently, m 6 A demethylase FTO was more highly expressed in kid muscle samples than in fetal muscle samples, whereas the expression of m 6 A methyltransferase METTL3 was lower in kid muscle samples than in fetal muscle samples ( Figure 1F). Furthermore, we sought to explore the correlation between m 6 A modifications and myogenic differentiation in vitro. Goat primary myoblasts (GPMs) were isolated, identified, and differentiated (Figures 1G and S1). Consistently, the level of global m 6 A modifications decreased markedly, along with the differentiation of GPMs (Figure 1H). In line with this finding, the expression of FTO increased, along with the myogenic differentiation of GPMs, whereas the level of METTL3 decreased after differentiation initiation ( Figure 1I). These results indicate that m 6 A modifications may negatively regulate myogenesis.
Identification of GADD45B as a potential m 6 A-modified gene related to myogenesis To identify the potential m 6 A-modified genes related to myogenesis, the m 6 A distribution in fetal (75 days) and kid (newborn stage) skeletal muscle was analyzed. First, the m 6 A methylomes of fetal and kid skeletal muscle were mapped using m 6 A sequencing (m 6 A-seq) and four biological replicates. As shown in Table S1, 64.0 to 111.1 million reads were obtained from each m 6 A-seq or RNA sequencing (RNAseq) library. Consistent with the published consensus motif RRACH (R = G or A; H = A, C or U), 11 the GGACU sequence was identified as the most common m 6 A motif in goat skeletal muscle (Figure 2A). Notably, a total of 4,632 and 2,789 m 6 A peaks from 3,270 to 2,227 m 6 A-modified transcripts in fetal and kid skeletal muscle were identified using m 6 A-seq, respectively ( Figures 2B and 2C). In kid skeletal muscle, 1,654 new peaks appeared, whereas 3,498 peaks disappeared. The other 1,134 peaks were observed in both fetal and kid skeletal muscle ( Figure 2B). Furthermore, the m 6 A peak distribution patterns within the whole transcripts were investigated. The m 6 A peaks mainly appeared in the coding sequence (CDS) and 3 0 UTR regions and were especially abundant close to the stop codon ( Figures 2D and S2A). A total of 937 common m 6 A-modified genes were identified in both fetal and kid muscle, and 2,333 and 1,290 unique m 6 A-modified genes were observed in fetal and kid skeletal muscle, respectively (Figure 2C). With the use of Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, the m 6 A-modified genes were found to be mainly involved in focal adhesion, actin binding, actin filament binding, and myotube differentiation, as well as in some signaling pathways related with skeletal muscle development, including the MAPK and Wnt signaling pathways ( Figures S2B and S2C).
Next, we compared the m 6 A peak abundance of common m 6 Amodified genes between the skeletal muscle of fetuses and kids. A total of 185 hyper-methylated m 6 A peaks and 372 hypo-methylated m 6 A peaks were discovered in the kid skeletal muscles relative to the fetal skeletal muscles (fold change >1.5, p < 0.05; Figure 2E; Table  S2). With the use of cross analysis of the m 6 A-seq and RNA-seq data, we identified 109 hyper-methylated m 6 A peaks in the mRNA transcripts, which were significantly (fold change >1.5, p < 0.05) upregulated (107; hyper up) or downregulated (2; hyper down) in the kid skeletal muscle relative to the fetal skeletal muscle ( Figure 2F; Table S3). Moreover, 222 hypo-methylated m 6 A peaks in the mRNA transcripts were found to be significantly (fold change >1.5, p < 0.05) upregulated (3; hypo up) or downregulated (209; hypo down) (Figure 2F; Table S3). Interestingly, based on the results of the associated analysis of m 6 A peak abundance and the mRNA transcripts levels, a positive correlation was found between the differentially methylated m 6 A peaks and the gene expression levels in these common m 6 Amodified genes ( Figure 2G). Therefore, given the potentially negative correlation between m 6 A modifications and myogenesis, hypo-methylated genes were considered as candidate genes in the regulation of muscle development. Notably, only three upregulated genes were found among these hypo-methylated transcripts, one of which, GADD45B, is involved in myogenesis 27 and could activate the MAPK pathway to regulate cell cycle and apoptosis ( Figure 2H). The m 6 A-seq data showed that GADD45B contained m 6 A sites in the 3 0 UTR, with the m 6 A levels at 3 0 UTR markedly lower in the kid skeletal muscle compared to the fetal skeletal muscle ( Figure 2I). In line with the m 6 A-seq data, the m 6 A quantitative real-time PCR results further confirmed that the m 6 A level in GADD45B 3 0 UTR was lower in the kid skeletal muscle than in the fetal skeletal muscle ( Figure 2J). Notably, GADD45B expression was found to be markedly upregulated during skeletal muscle development ( Figure 2K). These results suggest that GADD45B is a m 6 A-modified gene with the potential function of regulating skeletal muscle development. To demonstrate the fact that GADD45B is a m 6 A-modified gene and regulates myogenesis, the function of GADD45B in myogenesis was explored. First, we measured the levels of GADD45B mRNA and protein, both of which were found to increase during myogenic differentiation ( Figures 3A and 3B), further suggesting that GADD45B has a potential function in myogenesis. Interestingly, the depletion of GADD45B using small interfering RNA (siRNA) was found to dramatically suppress the formation of myotubes during differentiation ( Figure 3C). Consistently, the protein abundance and the mRNA level of myosin heavy chain (MyHC) and myogenin were markedly decreased upon GADD45B depletion (Figures 3DÀ3F). In contrast, the overexpression of GADD45B promoted the formation of myotubes during differentiation ( Figure 3G). Moreover, the forced expression of GADD45B significantly increased the protein and mRNA expression of MyHC and myogenin (Figures 3HÀ3J).
Given that mitochondrial remodeling is required for the differentiation of myoblasts into myotubes and that GADD45B may affect mitochondria biogenesis in brown adipocytes, [28][29][30] we sought to determine whether GADD45B plays a role in mitochondria biogenesis during myogenic differentiation. Transmission electron microscopy was used to detect the status of mitochondrial networks in differentiating cells. Electron micrographs showed that the mitochondrial populations were relatively dense and exhibited an elongated morphology in differentiated myoblasts compared to undifferentiated myoblasts ( Figure 4A). However, in differentiated myoblasts lacking GADD45B, mitochondria are sparse and mostly round ( Figure 4A). Moreover, the analysis of the mitochondrial mass (Figures 4B and 4C) and mitochondrial DNA content ( Figure 4D) indicated that mitochondrial biogenesis was repressed upon GADD45B depletion during myogenic differentiation. Interestingly, upon GADD45B deficiency, the protein level of mitochondrial fission protein dynamin 1 like (DNM1L) was elevated, whereas the protein levels of mitochondrial biogenesis-related protein peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PPARGC1A) and mitochondrial fusion protein OPA1 were decreased ( Figures 4E and 4F). In addition, GADD45B depletion resulted in the downregulation of mitochondrial-encoded genes (mtCytb, mtCo1, mtCo2, and mtNd1) in GPMs ( Figure 4G), as well as in a reduction in the ATP levels during differentiation ( Figure 4H). To further assess the influence of mitochondrial dysfunction caused by GADD45B deficiency on cellular homeostasis, we examined the levels of cell autophagy and apoptosis. Reactive oxygen species (ROS) production, autophagy related 7 (ATG7) and autophagy related 5 (ATG5) expression, and the microtubule-associated protein 1 light chain 3b-II/I (LC3B-II/I) ratio were elevated in GADD45B-depleted cells than in control cells ( Figures S3AÀS3D). Meanwhile, the expression of autophagic adaptor protein sequestosome 1 (SQSTM1) was reduced ( Figures  S3C and S3D), which indicated that GADD45B deficiency elevated the level of autophagy GPMs at day 3 of differentiation. Moreover,  (legend continued on next page) Molecular Therapy: Nucleic Acids apoptosis was found to be increased in GADD45B-depleted cells at day 3 of differentiation ( Figure S3E). Consistently, GADD45B deficiency significantly increased the Bcl-2-associated X protein (BAX) protein level and decreased the pro-survival protein B-cell Lymphoma-2 (BCL2) content in GPMs (Figures S3F and S3G). Taken together, these results suggest that GADD45B plays a vital role in mitochondrial biogenesis and myogenic differentiation in myoblasts.
GADD45B promotes myogenesis by activating the p38 MAPK pathway p38 MAPK is a major regulator of skeletal muscle development and regeneration. It regulates myogenesis and mitochondria biogenesis via MRFs and PPARCG1A. 27,31 Given that GADD45B could activate the p38 MAPK pathway, 32 we investigated whether the p38 MAPK pathway mediated the effect of GADD45B on myogenic differentiation and mitochondria biogenesis. First, we found that the expression of myogenic differentiation 1 (MyoD1), myogenic factor 5 (Myf5), and PPARC1A was downregulated upon depletion of GADD45B ( Figure 5A). Notably, by detecting the phosphorylation levels of p38 MAPK, we found that p38 MAPK was activated during myogenic differentiation ( Figure 5B). Furthermore, the formation of myotubes and the protein levels of myogenin and MyHC, which were found to be markedly repressed, were reduced in GPMs upon treatment with the p38 MAPK inhibitor SB203580 ( Figures S4AÀS4C). SB203580 treatment also suppressed the expression of Myf5 and PPARGC1A in cells at day 1 of differentiation ( Figures S4D and S4E). Taken together, these results indicate that p38 MAPK may play a crucial role in myogenic differentiation and mitochondria biogenesis.
Next, we explored whether GADD45B regulates MRFs and PPARGC1A expression through the p38 MAPK pathway during myogenic differentiation. Our results showed that the phosphor-p38 MAPK level was decreased in GADD45B-depleted cells at day 1 of differentiation ( Figures 5C and S5A). In contrast, GADD45B overexpression led to an increase in the level of phosphor-p38 MAPK ( Figures 5D and S5B). Furthermore, MRFs (MyoD1 and Myf5) and PPARGC1A were found to be dramatically downregulated in GADD45B-depleted cells (Figures 5C and S5A) but were upregulated in GADD45B-overexpressing cells (Figures 5D and S5B). These results indicate that GADD45B could affect the activity of p38 MAPK during myogenic differentiation.
To better understand the correlation among GADD45B, p38 MAPK, MRFs, and PPARGC1A, the p38 MAPK inhibitor SB203580 and p38 MAPK activator hesperetin were used to block and activate p38 MAPK, respectively. Interestingly, we found that hesperetin treatment restored the decrease in myotube formation in GADD45Bdepleted cells (Figures 5E and S5C), whereas SB203580 treatment dramatically antagonized the increase in myotube formation in GADD45B-overexpressing cells (Figures 5F and S5D). In addition, hesperetin treatment also restored the expression of MRFs and PPARGC1A in GADD45B-depleted cells (Figures 5G, 5I, and S5E). Similarly, SB203580 treatment also attenuated the upregulation effect of GADD45B overexpression on MRFs and PPARGC1A expression ( Figures 5H, 5J, and S5F). Taken together, these results suggest that GADD45B might regulate the expression of MyoD1, Myf5, and PPARGC1A, as well as mediate myogenic differentiation downstream of the p38 MAPK pathway.
FTO-mediated m 6 A demethylation of GADD45B mRNA affects its stability and expression level GADD45B was one of the upregulated genes both at the mRNA and the m 6 A modification level in kids compared to the fetuses and exhibited a positive correlation with FTO expression during myogenic differentiation ( Figure 6A). Moreover, the RNA-seq dataset of FTO-knockdown GPMs was analyzed, and the results showed that GADD45B was one of 869 downregulated genes (1.5-fold change), 33 suggesting that FTO could regulate the expression of GADD45B via m 6 A modification.
To further investigate the effect of m 6 A modification on GADD45B expression, quantitative real-time PCR and western blot assays were conducted. The results showed that FTO knockdown dramatically downregulated the RNA and protein levels of GADD45B ( Figures  6BÀ6D). The m 6 A levels were found to be upregulated in FTOknockdown cells ( Figure 6E), which could alter the mRNA instability and degradation, as well as protein translation. 12,13 We found that the m 6 A levels in GADD45B mRNA rose from 1.5-to 3-fold in the FTO-knockdown cells, according to gene-specific m 6 A quantitative real-time PCR assays ( Figure 6F). RNA stability assays 34 were used to measure the effect of m 6 A levels on the stability of GADD45B. A shortened half-life of GADD45B mRNA was observed in FTOknockdown cells treated with actinomycin D compared with control cells ( Figure 6G). Since GADD45B contains the RRACH m 6 A consensus motif in the 3 0 UTR, we constructed wild-type and mutant pmirGLO-GADD45B-3 0 UTR firefly luciferase reporters containing intact wild-type or mutant m 6 A sites ( Figure 6H), respectively. As expected, our results showed that the luciferase activity of wild-type pmirGLO-GADD45B-3 0 UTR reporter, but not of mutant pmir-GLO-GADD45B-3 0 UTR reporter, was markedly reduced upon FTO knockdown ( Figure 6H). Taken together, our data demonstrate that the FTO-mediated m 6 A demethylation of GADD45B mRNA regulates its stability, thereby affecting its expression level.

FTO knockdown attenuates GADD45B-induced p38 MAPK phosphorylation and inhibits myogenic differentiation
Given the important role of FTO-mediated m 6 A in regulating myogenesis and GADD45B expression, 25 we investigated whether the function of FTO in myogenesis depended on GADD45B-mediated p38 MAPK pathway activation. Consistent with the results of GADD45B deficiency, FTO knockdown was found to significantly decrease the phosphorylation level of p38 MAPK and subsequently suppressed the expression of MyoD1, Myf5, and PPARGC1A (Figures 7A and S6A). Moreover, the expression of MyHC and myogenin was reduced, and myotube formation was attenuated upon FTO knockdown ( Figures 7B, 7C, 7D, and S6B). Notably, in FTO-silenced cells, mitochondria biogenesis and the related gene expression were suppressed (Figures S7AÀS7G), along with a decrease in ATP production ( Figure S7H), similar to the results of GADD45B deficiency. Interestingly, the forced expression of GADD45B CDS alone rescued the inhibition of GADD45B expression caused by FTO knockdown ( Figures 7E, 7F, and S6C), subsequently improving myotube formation in FTO-depleted cells ( Figure 7G). Furthermore, GADD45B overexpression restored the reduced phosphorylation levels of p38 MAPK and the PPARGC1A, MyoD1, Myf5, myogenin, and MyHC expression levels in FTO-depleted cells (Figures 7H, 7I, and S6D). Consistently, hesperetin treatment achieved a similar restoration effect regarding myotube formation, phosphorylation levels of p38 MAPK, and MRFs and PPARGC1A expression levels (Figures S8AÀS8C), as observed upon GADD45B overexpression in FTOdepleted conditions. Taken together, our findings indicate that FTO regulates myogenic differentiation via the GADD45B-p38 MAPK pathway.

DISCUSSION
In the present study, we mapped the pattern of global m 6 A modifications in goat skeletal muscle during embryonic development and demonstrated that GADD45B, which is regulated by m 6 A modification, is an important regulator of myogenesis. GADD45B deficiency was found to decrease the phosphorylation levels of p38 MAPK, thereby downregulating the expression of MRFs and PPARGC1A, leading to the suppression of myogenic differentiation and mitochondria biogenesis. The knockdown of FTO was found to increase the level of m 6 A modification in GADD45B, which in turn accelerated the degradation of its mRNA and downregulated its transcription and protein levels. The knockdown of FTO significantly impaired myogenic differentiation, which was found to be restored by GADD45B overexpression. These results indicate that the FTO-mediated m 6 A modification of GADD45B mRNA plays a critical role in muscle differentiation.
The transcriptional regulatory networks that control the specification of muscle precursor cells and the expression of myogenic regulatory genes have been well defined in mammals. However, the role of post-transcriptional modifications in skeletal muscle development is only just being gradually discovered. m 6 A modifications represent a new type of post-transcriptional gene regulation and play a vital role in the development of a variety of tissues, including cerebellar development, 35 oocyte development, 36 and adipogenesis. 37 Interestingly, a reduction in the lean body mass was commonly observed in FTO knockout mice, 24,38 caused by the dysregulation of energy expenditure induced by disorders of FTO expression. Notably, the deficiency in the demethylase activity of FTO was a major factor leading to the impairment of muscle differentiation and skeletal muscle development. 25 In our study, the m 6 A level was decreased during myogenic differentiation in vivo and in vitro. Thus, it is likely that m 6 A modifications may be involved in the regulation of skeletal muscle development. However, the genes that are modified by m 6 A to regulate skeletal muscle development remain unknown.
GADD45B, also known was Myd118, is a member of the GADD45 gene family, which is involved in the process of response to cell injury caused by physiological or environmental stressors. 39 GADD45B is highly expressed in the chorion during embryonic development 40 and has been reported to play a vital role in brain development, including in neurogenesis 41 and in dendritic development. 42 Although there is still a lack of evidence showing that GADD45B directly controls muscle development, it has been demonstrated that GADD45B could regulate the downstream p38 MAPK signaling pathway, which is associated with myogenesis. 32,43 In our study, GADD45B was identified as a m 6 A-modified gene and was found to be upregulated during myogenic differentiation. Furthermore, GADD45B knockdown was found to impair myogenic differentiation and mitochondria biogenesis in myoblasts, whereas the forced expression of GADD45B promoted myogenic differentiation. Future studies will be needed to confirm the potential roles of GADD45B in skeletal muscle development in vivo using GADD45B knockout mice.
Next, we elucidated the mechanism underlying the role of GADD45B in myogenic differentiation and found that GADD45B expression influenced the activity of p38 MAPK during myogenic differentiation. p38 MAPK, a subgroup of the MAPK superfamily of intracellular serine/threonine protein kinases, is known to regulate the differentiation of various cell types, such as adipocytes and muscle stem cells. It also regulates the expression of MRFs, including Myf5, MyoD1, myogenin, and MRF4. 44,45 Interestingly, GADD45B can also regulate the expression of MyoD1, Myf5, and myogenin genes. However, when p38 MAPK activity was blocked by SB203580 treatment, the effect of GADD45B on the transcription of MRFs was attenuated, suggesting that the GADD45B-mediated regulation of MRFs depends on p38 MAPK activity. During the differentiation of skeletal myoblasts into myotubes, a cellular metabolic-style shift from glycolysis to oxidative phosphorylation occurs. 46 To adapt to this change, the complex mitochondrial network is remodeled through mitochondrial clearance and biogenesis during myogenic differentiation. 28 Therefore, maintaining the balance of mitochondrial clearance and biogenesis is indispensable for myogenesis. PPARGC1A, also known as peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1a), is a master regulator of mitochondrial biogenesis, modulating the transcription of nuclear genes encoding mitochondrial proteins. 47 It has been demonstrated that PPARGC1A sustains mitochondrial homeostasis during myogenesis by blocking excessive ROS production, which can result in the degradation of the mitochondrial network via mitophagy. 48 In line with these findings, GADD45B knockdown downregulates PPARGC1A, resulting in an increased ROS production, autophagy, and apoptosis, leading to a decrease in the mitochondrial content. Furthermore, our results show that the ability of GADD45B to regulate PPARGC1A expression relies on p38 MAPK activity, which is in agreement with the results of a previous study that found that the p38 MAPK pathway affects PPARGC1A expression. 49 Taken together, the results presented in this study indicate that GADD45B regulates myogenic differentiation via the p38 MAPK-MRFs and p38 MAPK-PPARGC1A-mitochondria axes, demonstrating that the p38 MAPK activation is essential for the GADD45B-mediated myogenic differentiation.
It is well known that the expression of the genes of the GADD45 family is induced by various endogenous and exogenous stress stimuli. To accurately respond to complex cellular stress stimuli, the stress sensors on GADD45 proteins need to be rapidly regulated at both formation in siCtrl, siFTO, and siFTO + GADD45B cells at day 3 of differentiation. Scale bars, 50 mm. (H) Western blot analysis of phosphorylation and non-phosphorylation of p38 MAPK, MRFs, and PPARGC1A in siCtrl, siFTO, and siFTO + GADD45B cells at day 3 of differentiation. (I) Quantitative real-time PCR analysis of MyHC and myogenin in siCtrl, siFTO, and siFTO + GADD45B cells at day 3 of differentiation. (J) Graphical representation of the role and underlying mechanism of the FTO-mediated demethylation of GADD45B in myogenesis. The data were obtained from at least three independent experiments. The values are expressed as the mean ± SEM; *p < 0.05, **p < 0.01. the transcriptional and post-transcriptional levels. 39 Under different stress induction signals, GADD45B is distinctly regulated at the mRNA transcription or mRNA stability level. 50 However, little is known about whether GADD45B is subject to m 6 A modifications. In the current study, we found that the knockdown of FTO leads to an increase in the m 6 A levels in the 3 0 UTR of GADD45B, which decreased the mRNA stability and reduced GADD45B expression. To our knowledge, this study is the first to demonstrate that posttranscriptional m 6 A modifications are involved in the regulation of the GADD45B mRNA levels during myogenic differentiation. Although FTO has been reported to regulate myogenic differentiation via the mechanistic target of rapamycin kinase (mTOR) pathway, 25 the specific genes subject to FTO-mediated m 6 A modifications in this process have not yet been identified. However, our results indicate that FTO demethylates GADD45B mRNA and regulates myogenic differentiation via the p38 MAPK pathway. It is worth noting that other genes carrying an m 6 A modification could be targeted by FTO and be involved in myogenic differentiation, in addition to GADD45B. More studies will be needed to investigate and identify additional genes in order to clarify the function of m 6 A modifications during skeletal muscle differentiation.
In conclusion, our results demonstrate that GADD45B promotes myogenic differentiation by activating the p38 MAPK pathway and is regulated by FTO-mediated m 6 A modifications ( Figure 7J). These findings highlight the importance of the m 6 A modification-mediated regulation of gene expression during myogenic differentiation and provide new insights into the molecular mechanisms underlying skeletal muscle development.

Animal samples
All animal studies were performed under the guidelines of the Ethics Committee of Nanjing Agricultural University, China. All goats used in this study were from Haimen goat breeding farm, in Nantong, China. Sixteen female goats in good body condition and suitable for pregnancy were selected. All selected goats underwent estrus synchronization treatment and were naturally mated. After 75 days of gestation, four male fetuses were removed from five pregnant goats during abortion operations, and their longissimus muscle samples were collected. After natural parturition of other pregnant goats, four male goats were selected from the newborn goats, and they were sacrificed via exsanguination for longissimus muscle sampling.

Histological analysis
The longissimus muscle samples from goats were collected and preserved in 4% paraformaldehyde overnight at room temperature before paraffin embedding. The H&E-stained cross-sections of the longissimus muscles from each animal were imaged using a microscope (Nikon, Tokyo, Japan) The fibers (2,500 mm 2 ) in five fields were counted from each animal (n = 4) using ImageJ software (National Institutes of Health, Bethesda, MD, USA). In addition, the cross-section area of each fiber (n > 1,000) in the five fields (n = 4) was determined using ImageJ (National Institutes of Health, Bethesda, MD, USA).

Cell culture
GPMs collected from goat fetuses at the embryonic stage (75 days) were cultured as previously described. 51 Briefly, the hindlimb muscle tissues were minced into pieces and digested with 1% collagenase I (Sigma-Aldrich) for 50 min at 37 C with slight agitation and then digested with 0.25% trypsin (Gibco) for 15 min at 37 C. The digested samples were filtered through a 70-mm filter and then centrifuged to collect the cells. Cells were plated in growth media (DMEM containing 20% fetal bovine serum (FBS), 10% horse serum, and 1% penicillin/streptomycin). After 2 h, the non-adherent cells were transferred to another culture dish. To obtain pure GPMs, the differential adhesion method was performed as previously described. 51 GPMs were cultured at 37 C and 5% CO 2 in growth media. To induce differentiation, GPMs were grown to about 80% confluence in growth media and then switched to differentiation medium (DMEM containing 2% horse serum and 1% penicillin/streptomycin).
Cell transfection siRNAs and plasmids were transfected into cells using Lipofectamine 3000 transfection reagent (Life Technologies) and LIPOFECTAMINE LTX transfection reagent (Life Technologies), respectively, according to the manufacturer's instructions. All siRNA sequences were designed and synthesized by Genepharma (Shanghai, China), as listed in Table S4. The GADD45B CDS was amplified from the Capra hircus GADD45B gene (XM_018050778.1) using PCR by using primers GADD45B-NotI-5.1 and GADD45B-EcoRI-3.1 and then cloned into a pEX3 vector (Genepharma) to generate the pEX3-GADD45B plasmid. The primers used for cloning GADD45B CDS are listed in Table S5.

RNA extraction and quantitative real-time PCR
Trizol reagent (Invitrogen) was used to extract the total RNA from the tissues or cells. The extracted RNA was then reversed transcribed into cDNA using reverse transcription reagent kits (Takara, Dalian, China), according to the manufacturer's instructions. Quantitative real-time PCR analysis was performed using the SYBR Green PCR Master Mix (Roche) and the QuantStudio 5 Real-Time PCR System (Applied Biosystems, Bedford, MA, USA). The relative gene expression was quantified using the 2 ÀDDCt method by using 18S rRNA as an endogenous control. The primers used are listed in Table S5.

Quantification of m 6 A modifications
The extracted RNA was purified using GenElute mRNA Miniprep (MRN10; Sigma-Aldrich). After analyzing the quality of the purified RNA using NanoDrop, the global m 6 A RNA methylation was measured using an EpiQuik m 6 A RNA Methylation Quantification Kit (P-9005-48; Epigentek), following the manufacturer's protocol. The global m 6 A levels were measured colorimetrically by reading the absorbance at 450 nm and calculated using the resulting standard curve.
www.moleculartherapy.org m 6 A-seq assays For m 6 A-seq, 200 mg of total RNA was subjected to poly(A) mRNA isolation using poly-T oligo-attached magnetic beads (Invitrogen). Following purification, the poly(A) mRNA fractions were fragmented using divalent cations at high temperatures. Then, random RNA fragments (~100 nucleotides) were incubated with an m 6 A-specific antibody (number [no.] 202003; Synaptic Systems) in immunoprecipitation (IP) buffer (10 mM Tris-HCl, 150 mM NaCl, and 0.1% Igepal CA-630) for 2 h at 4 C. The mixture was then incubated with protein-A beads and eluted using elution buffer (1 Â IP buffer and 6.7 mM m 6 A). The eluted RNA was precipitated using 75% ethanol. Eluted m 6 A-containing fragments (IP) and untreated input control fragments were used to obtain the final cDNA library in accordance with a strand-specific library preparation by using the deoxynucleotide triphosphate (dUTP) method. The resulting library was subjected to paired-end 2 Â 150 bp sequencing using an Illumina NovaSeq 6000 platform at LC-BIO Bio-Tech (Hangzhou, China).

Immunofluorescence and confocal imaging
Cells were fixed in 4% paraformaldehyde and washed with PBS. The samples were permeabilized in 0.25% Triton X-100 and incubated with 3% bovine serum albumin for 1 h at room temperature, followed by incubation with a diluted solution of the following antibodies: anti-PAX7, anti-Desmin, anti-Myf5, or anti-MyHC, at 4 C overnight. After washing with PBS, the cells were incubated with the secondary antibody for 1 h at room temperature. The cell nuclei were then colored with 4 0 ,6 0 -diamidino-2-phenylindole (DAPI), and the fluorescence images were analyzed using a fluorescence microscope (Jena).

Immunoblotting
Cells were lysed in radioimmunoprecipitation assay (RIPA) buffer (Thermo Fisher Scientific) containing a cocktail of protease inhibitors. Approximately 20 mg of extracted protein was loaded and separated on 8%~12% SDS-PAGE gels, transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, MA, USA), and blocked for 1 h at room temperature with 5% skim milk. After washing with Tris-buffered saline containing Tween-20 (TBST), the membranes were incubated overnight with the corresponding primary antibodies at 4 C. Then, the membranes were washed with TBST and incubated with the appropriate horseradish peroxidase secondary antibodies at room temperature for 1 h. The bands were visualized using ECL Plus Western Blotting Reagent Pack (Bio-Rad). The primary antibodies used are listed in Table S6.

Transmission electron microscopy
The cells were harvested from the cell culture dishes using trypsin and centrifuged for 7 min at 350 Â g. After washing with PBS, the cells were fixed in a glutaric dialdehyde solution (Solarbio) for 20 min. Then, the cells were post-fixed in 1% OsO 4 in 0.1 M phosphate buffer (PB; pH 7.4) for 2 h on a rotator. Subsequently, the cells were dehydrated using an ethanol-graded series and infiltrated using a Spurr's resin-graded series. After polymerization, the resin blocks were cut into 60-80 nm-thick sections using an ultramicrotome (Leica Micro-systems, Wetzlar, Germany). The sections were fished out onto 150 mesh cuprum grids using formvar film (G300-Cu; Electron Microscopy Sciences). The sections were stained with a 2% uranium acetate saturated alcohol solution and 2.6% lead citrate and examined using a transmission electron microscope (FEI, Hillsboro, OR, USA).

Mitochondrial content assay
Mitochondrial content assays were performed according to the protocol described by Iwabu et al. 52 The mitochondrial content was evaluated using mtDNA quantification and Mito-tracker staining. The mtDNA copy number was measured using quantitative real-time PCR by quantifying the ratio of the mitochondrial Cox2 gene to an endogenous gene of the nuclear 18S rRNA. The primer sequences used are listed in Table S5.
Cells were incubated with 100 nM Mito-tracker Green (Beyotime Biotechnology) for 30 min at 37 C. After washing with PBS, cells were collected via trypsin/EDTA digestion and resuspended in PBS. Fluorescence intensity was measured using excitation and emission wavelengths of 490 and 516 nm, respectively, and the values were corrected for total protein (micrograms per milliliter).

ATP measurement
The intracellular ATP content was measured using an ATP assay kit (Beyotime) following the manufacturer's protocol.

Intracellular ROS measurement
Cells were incubated with 50 mM dichlorofluorescin diacetate (DCF-DA; Beyotime Biotechnology) for 30 min at 37 C and then were washed with PBS. Immunofluorescence was observed using a fluorescence microscope (Leica, Germany). The fluorescence intensity of the images was analyzed using ImageJ software.

Flow cytometry
Cells were harvested and washed in PBS before being resuspended in annexin A5 (ANXA5) binding buffer and incubated with 5 mL of ANXA5-fluorescein isothiocyanate (FITC; Thermo Fisher Scientific) and 10 mL of propidium iodide (PI) (50 mg/mL; Sigma-Aldrich). The cells were then analyzed using a flow cytometer (BD FACSCalibur). The RNA IP (RIP) procedure was performed as previously described. 53 Briefly, the total RNA was obtained from cells using TRIzol and then purified using GenElute mRNA Miniprep (MRN10; Sigma-Aldrich). Poly(A) RNA was fragmented using an RNA fragmentation reagent and immunoprecipitated using m 6 A antibody for m 6 A IP. Finally, the m 6 A-bound fraction RNA was recovered via ethanol precipitation, and the m 6 A enrichment was determined using quantitative real-time PCR analysis.

RNA stability assays
The cells were transfected with either control siRNA (siCtrl) or FTO siRNA (siFTO) for 24 h and then treated with 5 mg/mL actinomycin D (HY-17559; MedChemExpress [MCE]) for 0, 1, or 3 h to inhibit global mRNA transcription, after which, the samples were collected to assess their degradation. The RNA was extracted using TRIzol reagent and then reverse transcribed, as previously described. The mRNA transcript levels were detected by using quantitative real-time PCR. The degradation rate of the RNA was estimated as previously described. 13,54 Dual-luciferase reporter and mutagenesis assays The DNA fragments of GADD45B-3 0 UTR containing the wild-type m 6 A and mutant motifs (m 6 A was replaced by T) were directly synthesized by Genepharma (Shanghai, China). The GADD45B-3 0 UTR sequences used are listed in Table S7. Wild-type and mutant GADD45B-3 0 UTR were then inserted into pmirGLO vector (Promega) between the SacI and XhoI enzyme sites. For the dual-luciferase reporter assay, 200 ng of wild-type or mutant pmirGLO-GADD45B-3 0 UTR and siFTO were co-transfected into GPMs in 24-well plates. After 48 h of transfection, the relative luciferase activity was measured using a dual-luciferase reporter assay system (Vazyme).

Statistical analysis
All experiments were carried out at least in triplicates. All data are expressed as the mean ± SEM. Statistical analysis was performed using SPSS software (version 24.0; SPSS, Chicago, IL, USA) using a twotailed Student's t test or one-way analysis of variance (ANOVA) followed by Tukey's post hoc analysis. For all the analyses, p < 0.05 was considered significant.