The SNPs in myoD gene from normal muscle developing individuals have no effect on muscle mass

Background Myogenic Differentiation 1 (MyoD) is a crucial master switch in regulating muscle-specific gene transcription. Forced expression of myoD is equipped to induce several cell lineages into myoblast, which then differentiate and fuse into myotube. Pig is one of the most significant livestock supplying meat, and has been classified into lean, fat and miniature pig breeds. However, the mechanisms underlying muscle mass variation among different pig breeds have remained unclear. Considering the important effect of MyoD on muscle development, it remains to be investigated whether the difference in muscle mass is caused by its single nucleotide polymorphisms (SNPs) which are the major differences among pig breeds at DNA level. Results In this study, we identified the locations of porcine myoD regulatory regions including proximal regulatory region (PRR), distal regulatory region (DRR), and core enhancer (CE) region. There are 8 SNPs in the regulatory regions and 6 SNPs in gene body region, which were identified from lean, fat and miniature pig populations. However, these SNPs have no effects on its temporal expression and transcriptional activity which might lead to the distinction in postnatal muscle mass. In addition, overexpression of myoD clones across from amphibious to mammals including xenopus tropicalis, chicken, mouse and pig whose gene identities vary from 68 to 84%, could promote myogenesis in NIH3T3 fibroblasts cells. Conclusions These results proved that myoD nucleotide variations from different pig populations have no effect on muscle mass, suggesting that the function of myoD is highly conserved not only among different pig breeds, but also across different species. Thus, it would be futile to discover SNPs affecting muscle mass in pig populations with normal muscle development. Electronic supplementary material The online version of this article (10.1186/s12863-019-0772-6) contains supplementary material, which is available to authorized users.


Background
Different pig breeds vary in muscle mass because of genetic differences and intensive selection. Development of skeletal muscle determines muscle mass. Numerous researches on mechanisms of skeletal muscle development have been reported, and many genes regulating myogenesis have been found [1][2][3]. Among them, MyoD, containing basic helixloop-helix (bHLH) domain, can bind to muscle-specific genes and promote their expression robustly [4,5]. Previous study revealed that earlier myoD expression exhibits precocious myogenic differentiation and causes severe muscle hypotrophy [6]. In addition, our earlier research found that myoD showed differential expression at 35 dayspost-coitus in Landrace and Lantang pigs [7]. These suggest the expression time and level of MyoD have an important effect on muscle mass. However, there are many factors affecting myoD expression, the primary one is its regulatory elements. So far, three regulatory regions have been identified to regulate myoD expression: proximal regulatory region (PRR), distal regulatory region (DRR), and core enhancer (CE) region. The CE region and DRR are essential to regulate myoD expression [8,9]. Recent findings have found that myoD transcripts corresponding to CE and DRR enhancers can promote chromatin accessibility and RNA polymerase II recruitment at myoD and Myogenin loci, respectively [10][11][12]. In this case, is the difference in muscle mass among different pig breeds related to the SNPs in myoD gene? Therefore, we obtained myoD SNPs in its regulatory regions and gene body by resequencing, and analyzed them to explore whether these SNPs caused alteration in muscle mass in pigs. This study not only helps us to better understand the mechanisms of different muscle mass among pigs, but also provides new clues to study these mechanisms in the future.

Determination of myoD regulatory regions in pigs
The location of myoD regulatory regions in pigs have not been identified before. Depending on the CE, DRR and PRR of human and mouse myoD reported in documents [13][14][15], we finally identified the locations of porcine myoD CE regions (Fig. 1a), DRR (Fig. 1b) and PRR (Fig. 1c) by comparing nucleotide sequence similarities using BLAST. The CE region of porcine myoD was localized to a 258 bp fragment approximately 21,924 to 22,182 bp upstream of myoD transcriptional start site (TSS), which shows 94% sequence homology with human (Additional file 3: Table  S3, Additional file 4: Table S4.). The DRR was located at 5 kb upstream relative to the TSS and the PRR was located between -197 bp and -485 bp (Additional file 4: Table S4).
In these regions, it was found that the CE region contains three E-boxes (Fig. 1a, d). In addition, the DRR contains consensus sequences for three E-boxes, two MEF2 binding sites and so on (Fig. 1b, d), which are nearly identical to human and mice [13,15,16]. The PRR contains consensus sequences for an E-box, two SP1 sites, an AP2 binding sites, etc. (Fig. 1c, d), which are necessary elements for mouse PRR to regulate muscle-specific transcription [15,16].
The SNPs in regulatory regions of myoD gene have no effect on muscle mass Considering the significant role of myoD regulatory region in itself expression, we hypothesized that the polymorphisms of myoD are associated with its expression time and expression level resulting in the divergence of pig muscle mass. To find out whether the SNPs in myoD gene affect muscle mass, whole myoD genome sequence including regulatory regions and gene body were obtained from resequencing results. One SNP existed in all breeds in the CE region (C41446175T) listed in Table 1. However, this SNP also existed within the pig breed. Therefore, this SNP was unlikely to be the major cause that leads to the different muscle mass. Another SNP at locus 41,446,192 in the CE region was found only in individuals, but no breed specificity. The same results were also observed in DRR and PRR (Table 1). Moreover, these SNPs in the three regulatory regions did not appear in their binding motifs which were marked in black boxes (Fig. 1a, b and c). Therefore, these results indicate that SNPs in myoD regulatory region obtained from normally developed pig populations do not contribute to muscle mass.
The mutation (Arg76Pro) has no effect on muscle mass Six SNPs in myoD coding region (CDS) were discovered through whole-genome resequencing ( Table 2). Among them, four SNPs do not result in amino acids alterations, whereas the other two SNPs at locus 41,424,009 (A → G) and 41,424,010 (C → G) synchronously encoded an amino acid, which resulted in the substitution of amino acid from arginine (Arg) to proline (Pro) at 76th amino acid of MyoD protein sequence (Fig. 2a). Of note, the substitution is mainly led by the alteration at locus 41, 424,010 from C to G, which was appeared only in the lean pigs.
To determine whether this mutation can affect MyoD expression and then result in distinction in muscle mass, we constructed a myoD expressing plasmid with a C-terminal FLAG tag (PCDNA3.1-flag-MyoD WT ) and a mutagenized plasmid to substitute Arg76 with Pro (PCDNA3.1-flag -MyoD R76P ). These plasmids were separately transfected into NIH3T3 cells which cannot differentiate into myotubes because of lacking MyoD expression. When MyoD is forced to be expressed, NIH3T3 cell can differentiate into myotubes. So, it is considered as an ideal cell model for testing exogenous MyoD function. As a result, MyoD expression was upregulated significantly both at mRNA level and protein level (Fig. 2b, c), which remarkably increased the mRNA levels of Ckm, Cdh15 and Myh3 (Fig. 2d). Actually, Ckm, Cdh15 and Myog are the targets of MyoD and promote myogenic differentiation. However, there was no significant difference between MyoD WT and MyoD R76P plasmid in the expression levels of Ckm, Cdh15 and Myh3 (Fig. 2d). MyoD WT and MyoD R76P plasmids were respectively co-transfected into 293 T cells with a 4Rtk-luc reporter plasmid which was used as MyoD-responsive reporter [17] (Fig. 2e). The transfection of MyoD WT and MyoD R76P both could activate 4Rtk-luc transcription whereas there was no difference between them (Fig. 2e). Immunofluorescence assay showed that overexpression of MyoD WT or MyoD R76P in NIH3T3 cells equally promoted MyHC expression and generated myotubes ( Fig. 2f, g). Collectively, these data made it clear that the mutant protein has neither effect on MyoD transcriptional activity in vitro, nor effect on myogenesis.

MyoD clones across from amphibious to mammals can promote myogenesis
Based on the above-mentioned studies, we demonstrated that these SNPs in CDS and regulatory regions of myoD do not result in alteration of its expression and function. It should be noted that none of these mutations occurred in bHLH domain which presented high conservation among these pig populations. This phenomenon makes us have to believe that the bHLH domain of MyoD is conserved in function among normally growing animals, even different species.
To validate the hypothesis, FLAG-tagged myoD plasmids were constructed using pig, mouse, chicken and xenopus tropicalis, respectively. The sequence homologies of MyoD protein among these four species vary from 66 to 88% (Table 3, Additional file 5: Figure S1.), whereas their bHLH regions are highly-conserved (98.08% identity) (Fig. 3a). When these myoD plasmids were transfected into NIH3T3 cells, the mRNA levels of Ckm, Cdh15 and Myh3 were all significantly increased (Fig. 3d, e). However, from the result, it was indicated that there was no significant disparity among the four species at the expression levels of Ckm, and Myh3 regulated by MyoD (Fig. 3f, g). However, the expression level of Cdh15 in mice was significantly higher than that in other three species (Fig. 3f), Which may be related to the fact that NIH3T3 is a mouse-derived cells. In order to assess the transcriptional activity of MyoD from different species, myoD plasmids was co-transfected into 293 T cells with 4Rtk-luc reporter  Table 1 The SNPs in myoD regulatory region (1) The SNPs in regulatory regions of myoD are shown and variations in each region are represented by the colors indicated in the table (2) "./." represents that the site has not been detected Table 2 The SNPs in myoD CDS (1) The SNPs in myoD CDS are shown and variations in each locus are represented by the colors indicated in the table (2) "./." represents that the site has not been detected plasmid. As expected, there is no difference of luciferase activity among them (Fig. 3h). Immunofluorescence assay also showed that overexpression of myoD clones promoted MyHC expression and resulted in the similar phenotype in NIH3T3 cells (Fig. 3i).
Together, these results indicated that all myoD clones across from amphibious to mammals can promote myogenesis of NIH3T3 fibroblasts cells owing to the highlyconserved bHLH domain, even though the rest amino acid residues are different. Firefly and Renilla luciferase activities were measured using the Dual-Luciferase Reporter Assay System Kit (Promega). The data was presented as the normalized ratio of Firefly luciferase activity to the Renilla luciferase activity. f The myotube formation was observed under white light after differentiation for 5 days. Scale bar =50 μm. g After transfection and differentiation for 6 days, MyHC was detected by immunofluorescence staining. Scale bar = 100 μm. Data are presented as mean ± S.E.M.; n = 3; *p < 0.05, ***p < 0.001 (Student's t-test)

Discussion
The ongoing studies about swine have received extensive attention owning to its association to agricultural, clinical and dietary needs. Also, as one of the most significant livestock worldwide, pigs have been classified into lean, fat and miniature categories according to the differences in muscle and fat proportion, and it is well known that there are genetic differences among these three types of pig breeds. Lean pigs, such as Landrace, Duroc and Pietrain, are characterized by high lean meat percentage and fastgrowing muscle [18][19][20]. In contrast, fat pigs including Lantang, Luchuan and Laiwu Blacks, as China indigenous pig breeds, are recognized by high subcutaneous fat content, slow-growing muscle as well as low lean percentage [18,21]. As miniature pigs, Bamaxiang and Wuzhishan are not only relative slow in growth rate and feed conversion rate, but also less in muscle mass [22]. However, the genetic mechanism underlying the difference of muscle mass has remained unclear.

The distinct muscle mass is not relevant to the SNPs in myoD gene
All SNPs in regulatory regions of myoD gene from 10 pig populations were listed in Tables 1 and 2. After analysis, it was inferred that the initiate up-regulated time and level of myoD expression should be not affected by the SNPs of myoD gene. The initiate expression of myoD occurred in myotome in mice and had crucial role in the specification of progenitor cells [3,23]. Myotube formation was delayed in MyoD-deficient skeletal muscle [1,24]. In particular, loss of MyoD facilitated adipogenic trans-differentiation of myoblasts [25]. So importantly myoD acts, it is not workable to expect myoD SNPs cause the alteration of myoD expression characteristics in normal muscle developing individuals. The mutations that affect muscle mass should generally exist in diseased individuals or individuals with phenotypic abnormalities. Although a novel variation of amino acid (Arg76Pro) was found in MyoD CDS, and only appeared in lean pigs (Table 2), this mutation did not alter the characteristics of myoD expression (Fig. 2a). Besides, this mutation also occurred in different species including pig, mouse, chicken and xenopus tropicalis (Additional file 5: Figure S1) whose MyoD transcriptional activity were similar in our experiment (Fig. 3). Therefore, this mutation (Arg76Pro) does not affect the transcriptional activity of MyoD, which was verified in NIH3T3 cells (Fig. 2). These data explained detailly that the distinct muscle mass among pig breeds is not relevant to the myoD SNPs which present in different pig populations with normal muscle development.

No mutation occurs in the bHLH domain of MyoD in animals with normal muscle development
MyoD protein contains a bHLH motif, which can form dimers with the E-protein to promote MyoD transcriptional activity [26]. Previous studies have revealed that the bHLH domain of MyoD is sufficient to convert C3H10T1/ 2 cells to myoblasts [27]. What's more, when the bHLH domain of MyoD is replaced by the bHLH domain of Neu-roD2, MyoD will become a vital monitor of neurogenesis, other than a master regulator of myogenesis [28]. These results suggest that the bHLH domain of MyoD confers its own lineage determination potential. In addition, Myf5, a bHLH protein highly related with MyoD, is also expressed in skeletal muscle and has a crucial role in muscle cell specification. The mutation (p.Arg95Cys) located at the bHLH domain of Myf5 can impair Myf5 nuclear localization and transcriptional activity, which leads to external ophthalmoplegia, rib, and vertebral anomalies in humans [29]. Notably, the bHLH domain of MyoD is highly-conserved among pig breeds, even species in our study, indicating its importance in transcriptional activation. Since this domain is very important in myogenesis, it is unrealistic to expect functional mutations in this domain for normally developing individuals. In addition, it also indicates that difference in muscle mass among breeds is not triggered by the SNPs in bHLH domain, which was confirmed by our sequencing results.
The prospect for exploring the mechanisms of different muscle mass The difference in muscle mass among pig breeds is not due to the SNPs in myoD gene, which makes us more strongly believe that it is futile to explore the causes of the differences in muscle mass only based on SNPs in populations with normal muscle development. In general, if there is such a mutation that affects muscle mass, there will be abnormal muscle development, such as double muscle rump. After exclusion of SNPs at DNA level, the probable reason resulting in the difference of muscle mass could be attributed to epigenetic modifications that can also be inherited [9,30,31]. These modifications may affect the expression of MyoD by affecting its upstream regulatory factors and then influence the differentiation time of myoblasts, eventually leading to differences in muscle mass. Overall, our study excludes a SNP-based strategy for exploring the mechanisms of different muscle mass among pigs in myoD or other important myogenic genes. At the same time, it also opens another door for researchers.

Conclusion
In summary, our study has showed that the distinction of muscle mass among pig breeds is not caused by the i Immunofluorescence detection of MyHC (green) in myoD plasmid of mouse, pig, chicken and xenopus or control transfected NIH3T3 cells after 7 days of differentiation. Data are presented as mean ± S.E.M.; n = 3; *p < 0.05, **p < 0.01, ***p < 0.001 (Student's t-test); Scale bar =100 μm SNPs in myoD gene, but might be attributed by epigenetic modifications, which conduce to understand the mechanisms underlying muscle mass among different pig breeds.

DNA library construction and whole-genome resequencing
Porcine genome DNA was obtained using standard phenol chloroform method. One DNA library was constructed for each sample (Guangdong Small-ear Spotted and Lantang pigs) and then sequenced on an Illumina HiSeq X Ten system by Beijing Novogene (commercial service). We also downloaded other whole genome resequencing data of Bamaxiang, Wuzhishan, Duroc, Landrace, Large white, Pietrain, Luchuan and Laiwu Black pigs from GenBank. All the information of these samples was listed in Additional file 1: Table S1. Sequencing reads were mapped with porcine myoD in Sscrofa11.1 version by BWA-MEM [32] with default parameters and the polymorphisms of myoD were analyzed by GATK3.7 [33]. The sequencing depth of whole genome resequencing for each pig breed was listed in Table 4.

Cell culture and differentiation
Mouse NIH3T3 cell line was purchased from ATCC, and were cultured in high-glucose DMEM with 10% fetal bovine serum (growth medium, GM) until confluence. NIH3T3 cells were switched into DMEM with 2% horse serum (differentiation medium, DM) when cells reached confluence. All cells were cultured at 37°C in 5% CO2 incubator.

Plasmids and transfection
The cDNA of myoD from mice, pigs, chicken and xenopus tropicalis were separately cloned into pcDNA3.1flag vector (Invitrogen, Shanghai, China). NIH3T3 cells were transfected with myoD plasmid or control vector using Lipofectamine 3000 (Invitrogen) according to the manufacturer's instruction. NIH3T3 cells were passaged to 6-well or 12-well plates 12 h before transfection. All transfections were performed in triplicate for each experiment.

RNA isolation and quantitative real-time PCR
Total RNA was isolated from NIH3T3 cells using TRIzol® Reagent (Invitrogen, Shanghai, China) and cDNA was synthesized from total RNA by Reverse Transcription Kit (Promega, Shanghai, China). Quantitative real-time PCR (qPCR) assays was performed on LightCycler 480 II system (Roche, Basel, Switzerland) by using a SYBR Green qPCR Kit (Genestar, Beijing, China). GAPDH is used as an internal control for normalization. The sequences of qPCR primers are shown in Additional file 2: Table S2. The experimental data were analyzed using 2 -ΔΔCT method.

Western blotting
NIH3T3 cells were treated with cell Lysis Buffer to completely release total protein. Cell extracts were separated by SDS-PAGE and the proteins were transferred to 0.45 μm PVDF membrane (Bio-Rad, Shanghai, China). Then the PVDF membranes were blocked with 3% BSA for 1 h at room temperature and then incubated at 4°C overnight with primary antibodies. After washing, the membranes were labeled with proper secondary antibodies. Blots were visualized using a commercial enhanced chemiluminescene detection kit (Thermo Scientific, Beijing, China). GAPDH was used as the internal control. Primary antibodies used in this study included Flag (#8146S, CST) and GAPDH (#AP0063, Bioworld). Secondary antibodies included either anti-rabbit HRP-linked (#7074 S, CST) or anti-mouse HRPlinked (#7076 S, CST) antibodies.
Luciferase reporter assays 293 T Cells were seeded into a 24-well plate well and each plate of cells was transfected with 100 ng of 4R-TK-Luc and 100 ng of MyoD expression vectors or control vectors. Reporter activity was measured at 48 h later with a dual luciferase assay kit (Promega) and BioTek Synergy2. The luciferase activity was normalized by Renilla activity and the total transfection amount was normalized by an empty expression vector.