A novel transcript of MEF2D promotes myoblast differentiation and its variations associated with growth traits in chicken

Animals

The fertilized eggs of Xinghua chicken in this experiment were purchased from a livestock farm of South China Agricultural University (Guangzhou, China). They were hatched in a full-automatic incubator. During the period from the 10th embryo age (E10) to the 1st day post-hatching (P1), the breast muscle and leg muscle tissues of 20 chickens were collected each day and stored at −80 °C. Five 7-weeks-old Xinghua female chickens were purchased from a livestock farm of South China Agricultural University. A total of 15 tissues (cerebrum, cerebellum, hypothalamus, pituitary, heart, liver, spleen, lung, kidney, breast muscle, leg muscle, subcutaneous fat, abdominal fat, muscular stomach and glandular stomach) of each chicken were collected and stored at −80 °C.

DNA samples

The DNA samples were obtained from an F₂ resource population crossed from Xinghua and White Recessive Rock (XH & WRR) as described previously (Lei et al., 2005). The population consists of 17 full-sibling families, and 434 F₂ individuals (221 male and 213 female chickens) with a detailed record of growth traits, carcass traits and meat quality traits. Weight (body, semi-eviscerated, eviscerated, breast muscle, leg muscle and abdominal fat pad) was measured in grams using an electronic scale. The shank length, head width, breast width, breast depth and body length were measured with vernier caliper. The shank diameter was measured in the middle of the shank with string and straightedge.

RNA isolation, cDNA synthesis and quantitative real time PCR

Total RNA of all tissues were isolated using Trizol reagent (Invitrogen, Carlsbad, CA, USA), following the recommended manufacturer’s protocol. The quality and quantity of RNA samples were assessed by gel electrophoresis and a spectrophotometer (NanoDrop 2000c; Thermo, Waltham, MA, USA). The cDNA synthesis was performed with 1 μg of RNA for each sample using a RevertAid™ First Strand cDNA Synthesis Kit (Fementas, Waltham, MA, USA) in a total reaction volume of 20 μl.

The mRNA level of MEF2D and its four variant transcripts, RBFOX2, MHC and MYOD were measured by qPCR. The qPCR was performed using SsoFast Eva Green Supermix (BIO-RAD, Hercules, CA, USA) in CFX9600 (BIO-RAD, Hercules, CA, USA). Each sample was assayed in triplicate under the following conditions: 95 °C for 2 min, followed by 40 cycles of 10 s at 95 °C, 30 s at the annealing temperature (58–62 °C), 30 s at 72 °C, a melt curve by 65–95 °C, and increments 0.5 °C for 5 s. Chicken GAPDH was used as the reference gene for tissue-samples of 7-weeks-old chickens and myoblasts, whereas 18S rRNA was used as the reference gene for embryonic muscle samples. The relative mRNA level in each sample was calculated using the comparative 2^−ΔΔCt (CT is threshold cycle; ΔΔCt = ΔCt_{target sample} −ΔCt_{control sample}) method (Livak & Schmittgen, 2001).

Gene cloning and sequences analysis

Referring to the MEF2D gene sequence in chicken (NM_001031600.3) reported by National Center for Biotechnology Information (NCBI), primers were designed to amplify MEF2D gene by PCR. Products of PCR were purified using an Agarose Gel DNA Extraction Kit (Takara, Osaka, Japan) and then cloned into the pMD-18T vector (Takara, Osaka, Japan) according to the manufacturer’s protocol. Positive clones were identified by PCR and then sequenced by Invitrogen Co. Ltd (Guangzhou, China).

The sequencing results were analyzed and compared with the chicken genome (Gallus_Gallus-5.0/Galgalgal5; http://genome.ucsc.edu/cgi-bin/hgBlat) and MEF2D sequence (NM_001031600.3). DNAStar software (DNASTAR, Madison, WI, USA) was used to analyze the homology of the amino acid (AA) sequence of MEF2D between different species and the conserved regions of the sequence. The AA sequences of MEF2D from the other species were obtained from GenBank (Table S1).

Plasmid construction, cell culture and transfection

The coding sequences of chicken RBFOX2 and MEF2D-V4 were amplified from cDNA of chicken leg muscle using PCR, and then cloned into the pEGFP-C1 vector (Invitrogen, Guangzhou, China) using the EcoRI and BamHI restriction sites.

Chicken primary myoblasts were isolated from the leg muscle of chickens at 10–11 embryo age as described previously (Luo et al., 2014). Cells were maintained in RPMI-1640 medium (Gibco, Grand Island, NY, USA) supplemented with 20% (v/v) fetal bovine serum (Gibco, Grand Island, NY, USA), and 100 μg/ml penicillin/streptomycin (Invitrogen, Guangzhou, China) at 37 °C with 5% CO₂, humidified atmosphere. Cells were seeded in 12-well plates with one ml per well at 10⁵ cells/ml. When the cells had grown to 70–80% confluence, they were transfected with plasmids (one μg/ml) of MEF2D or RBFOX2 or pEGFP-C1 vector control using lipofectamine 3,000 reagent (Invitrogen, Guangzhou, China) according to the manufacturer’s instructions.

Cell proliferation assay

After overexpressing RBFOX2 and MEF2D genes in myoblasts for 48 h, respectively, cells were collected and fixed with 70% ethanol overnight at −20 °C. The fixed cells were collected by centrifugation at 1,000×g, washed once with PBS, and stained with 0.5 ml propidium iodide (PI) dye solution (5 mg PI + 0.1 ml Triton X-100 + 3.7 mg EDTA +10 ml PBS), and then incubate for 30 min at 4 °C in the dark. After staining, cells were detected by BD FACSAriaII flow cytometer (BD, Franklin Lakes, NJ, USA). The results were analyzed by software ModFit Lt 4.1.

Western blotting

Proteins of transfected myoblasts were extracted using RIPA lysis buffer (Beyotime, Shanghai, China) and the concentration was determined by a bicinchoninic acid protein assay kit (Beyotime, Shanghai, China). The primary antibodies MYOG (1:500 dilution; Biorbyt, Cambridge, UK) and MHC (1:1,000 dilution; DSHB, Iowa, IA, USA) were using to measure the protein levels of MYOG and MHC respectively by Western blotting as described previously (Ouyang et al., 2018). GAPDH (1:1,000 dilution; Bioworld, St Louis Park, MN, USA) was used as the reference gene.

Primers

Primers were designed using primer premier 5 software (PREMIER Biosoft, Palo Alto, CA, USA) and synthesized by Bioengineering Co., Ltd. (Shanghai, China). Specific primer sequences are shown in Tables S2 and S3.

Identification and genotyping of SNPs

Variations in the coding sequences of chicken MEF2D were identified using PCR with primers PM1–PM9 in our F₂ resource population (XH & WRR). The locations of primers were shown in Fig. S1. PCR was performed in 50 μl of a mixture containing 50 ng of chicken genomic DNA, 25 pmol of primers and 25 μl PCR Master Mix (Transgen, Beijing, China), and using the following protocol: 94 °C for 3 min, followed by 32 cycles of 30 s at 94 °C, 30 s at the annealing temperature (58–63 °C), 30 s at 72 °C and 72 °C for 5 min at last. Twenty DNA samples were selected randomly from the F₂ resource population (XH & WRR) for PCR using primers PM1–PM9. PCR products were sequenced by Bioengineering Co., Ltd. (Shanghai, China) and the results were then blasted with each other to identify variations. The special Single-nucleotide polymorphisms (SNPs) we were interested were genotyped by PCR and sequencing in all DNA samples of the F₂ resource population (XH & WRR).

Statistical analysis

Single-nucleotide polymorphism frequencies were calculated using the observed numbers of alleles for each SNP. SNP genotypes were tested for Hardy–Weinberg equilibrium with the Chi-square test. Association analysis of SNPs and fatness traits were performed using the General Linear Models Procedures of SAS 9.0 (SAS Institute Inc., Cary, NC, USA) using the following model: $$Y_{\mathrm{ijkl}}=\mathrm{\mu }+S_{\mathrm{i}}+G_{\mathrm{j}}+H_{\mathrm{k}}+F_{\mathrm{l}}+e_{\mathrm{ijkl}}$$Where Y = the traits phenotypic values; µ = the overall population mean; S = the effect of gender; G = the effect of genotype; H = the effect of incubation batch; F = the effect of family; e = the random residuals.

Data on gene expression were analyzed using SPSS 21.0 (IBM, Armonk, NY, USA). The ANOVA was used to compare expression levels among different groups. All values are presented as means ± standard error of mean (SEM). The threshold for significance was set at P < 0.05 and for high significance at P < 0.01.

Animal ethics

Animal experiments were handled in compliance and all efforts were made to minimize suffering. It was approved by the Animal Care Committee of South China Agricultural University (Guangzhou, People’s Republic of China) with approval number SCAU#0014.

Sequence alignment and phylogeny analysis of MEF2D

According to the information from NCBI database, the chicken MEF2D gene cDNA sequence (NM_001031600.3) is 4,111 bp in length, the coding region is 715–2,271 nt, and it encodes 518 AAs (NP_001026771.3). Blast with the chicken genome (GRCg6a/galGal6), this gene is located on chicken chromosome 25 (2,742,900–2,782,225), the full length of the gene is 39,326 bp, and it contains 10 exons and nine introns.

The protein sequences of MEF2D in 10 species (Gallus gallus, Meleagris gallopavo, Coturnix japonica, Homo sapiens, Mus musculus, Rattus norvegicus, Sus scrofa, Bos taurus, Danio rerio and Xenopus laevis) were compared and analyzed by homologous clustering. The results showed that the protein sequences of MEF2D were highly conserved, and had conserved domain of MADS-Box (2–57 AA) and MEF2-Domain (58–86 AA) in chicken and the other nine species tested (Fig. 1A). Phylogenetic tree clustering showed that 10 species were divided into four distinct groups: birds (Gallus gallus, Meleagris gallopavo and Coturnix japonica), mammals (Homo sapiens, Mus musculus, Rattus norvegicus, Sus scrofa and Bos taurus), Danio rerio and Xenopus laevis (Fig. 1B). Homology between chicken, turkey and quail was more than 96%. Homology among mammals (human, mouse, rat, pig and cow) was also very high, while the homology between zebrafish and frogs and other species was relatively low (Fig. 1C).

Variant transcripts of chicken MEF2D

In this experiment, cDNA samples from liver, hypothalamus and muscle tissue at different stages were used as PCR templates to clone chicken MEF2D gene, and positively clone PCR products were detected by agarose gel electrophoresis (Fig. 2A). Sequencing analysis of PCR products identified four novel variant transcripts (V1–V4) of MEF2D (Fig. 2B). Compared with the transcript of MEF2D gene on NCBI, the transcript V1 (NCBI Accession Number: KY680649) was 3,222 bp in length, had a deletion of 889 bp (1,446–2,334 nt), and was predicted to encode 251 AA. The transcript V2 (KY680650) was 3,616 bp in length, had a deletion of 498 bp (1,139–1,636 nt) and was predicted to encode 353 AA. The transcript V3 (KY680651) was 4,135 bp in length, had an insertion of 21 bp after exon 8 (1,570 nt) and an AAC insertion at 1,813 nt, and was predicted to encode 526 AA. The full length of the transcript V4 (KY680652) was 4,132 bp, and a 21 bp is inserted after exon 8 (1,570 nt), and was predicted to encode 526 AA. The complete DNA and protein sequences of these four variants are shown in File S2.

Comparative analysis of the AA sequences of the four novel MEF2D transcripts V1–V4 revealed that they contained conserved functional MADS-Box and MEF2-Domain. The position and sequence of exon 4 (87–132 AA) of the transcript V4 was different from that of the other transcripts. This was the same as the variant transcripts found in humans and mice, and they also mutated in the AA sequence 87–132 (Fig. S2).

Tissue specific expression of MEF2D

The expression of MEF2D transcripts in the different tissues of chickens was measured. Two deletion transcripts, MEF2D-V1 and MEF2D-V2 were barely expressed. The main transcripts expressed were MEF2D-1 (the same transcription as reported by NCBI), MEF2D-V3 and MEF2D-V4. MEF2D-1 and MEF2D-V3 were expressed widely in various tissues, and the relative expression levels in adipose tissue and brain tissue were higher, and in the liver and kidney were lower (Figs. 3A and 3B). The transcript MEF2D-V4 exhibited muscle-specific expression and was highly expressed in the heart, chest muscles and leg muscles, but its expression in other tissues was extremely low (Fig. 3C). We measured expression level of MEF2D-V4 in embryonic leg muscles, and found that the expression level of MEF2D-V4 increased from E11 to E19. The expression level of MEF2D-V4 increased significantly at E15 and E17, and it was stably expressed at E17 to E19 (Fig. 4).

Novel transcript MEF2D-V4 promotes myoblast differentiation in chicken

The sequence of MEF2D-V4 was similar to that of the human variant transcript Mef2Dα2, expression of which was regulated by RBFOX2 and was required for muscle differentiation (Singh et al., 2014; Runfola et al., 2015). Thus, to explore the effects of MEF2D-V4 and RBFOX2 on muscle differentiation, the eukaryotic overexpression vector of RBFOX2 and MEF2D-V4 were constructed, and transfected into chicken myoblasts respectively. After 48 h, the expression levels of RBFOX2 and MEF2D-V4 were measured by qPCR: both of these two vectors could induce overexpression of the corresponding genes effectively. Furthermore, overexpression of RBFOX2 gene could also increased the expression level of the MEF2D-V4 significantly (Fig. 5).

After overexpressing MEF2D-V4 and RBFOX2 in chicken primary myoblasts, the cell cycle was detected by flow cytometry. Compared with the control group, the number of S phase cells was increased in overexpressed MEF2D-V4 or RBFOX2 group, but did not reach significant levels (P > 0.05; Fig. S3). After overexpressing MEF2D-V4 and RBFOX2 for 48 h in myoblast differentiation, the mRNA level of MYOG and MHC was both increased (P < 0.05) in cells overexpressing MEF2D-V4 or RBFOX2 (Fig. 6A). The expression of MYOG and MHC was also detected by Western blot. Protein levels of MYOG and MHC were also increased, in accordance with mRNA levels (Fig. 6B).

SNPs identification and its association analysis with production traits

In the F₂ resource population (XH & WRR), 31 SNPs were identified in the full length chicken MEF2D DNA through PCR sequencing (Table 1), including one insertion/deletion, 14 synonymous mutations and 16 missense mutations. Interestingly, there was a T–C mutation at exon 9, g.36186C > T, generate a TAG stop codon, resulting in a change in the coding sequence and termination of translation in both MEF2D-1 and MEF2D-V4. Therefore, we genotyped this SNP by PCR amplification and sequencing on exon 9, and carried out association analysis in the F₂ resource population (XH & WRR). Several growth traits were associated significantly with this SNP g.36186C > T, including first days, 7, 14, 21, 28 and 63 days of body weight, 42, 77 and 88 days of shank length, 42 and 56 days of shank diameter, and 0–4 weeks of average weight gain (Table 2). The dominant genotype of SNP g.36195C > T was the CC type, and the average early body weight of TT type individuals was lower than that of CC type individuals.

In addition, g.36094CAGIns/Del (another SNP site of exon 9) was associated with carcass traits in chickens (Table 3), including eviscerated weight (EW), leg muscle weight (LMW), abdominal fat pad weight (AFW) and small intestine length (SIL). The dominant genotype of g.36094CAGIns/Del was the Del/del type. EW and LMW of the Del/del type were lower than that of the Ins/ins type.