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Journal of Animal Science - Animal Genetics

Study of bovine Mef2B gene: the temporal-spatial expression patterns, polymorphism and association analysis with meat production traits12

 

This article in JAS

  1. Vol. 94 No. 11, p. 4536-4548
     
    Received: June 22, 2016
    Accepted: Aug 31, 2016
    Published: October 27, 2016


    3 Corresponding author(s): e.kubiak@ighz.pl
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doi:10.2527/jas.2016-0741
  1. E. Juszczuk-Kubiak 3*,
  2. K. Bujko*,
  3. M. Grześ*,
  4. M. Cymer*,
  5. K. Wicińska*,
  6. A. Szostak and
  7. M. Pierzchała
  1. * Department of Molecular Biology, Laboratory of Genome and Transcriptome Sequencing, Institute of Genetics and Animal Breeding of the Polish Academy of Science, Jastrzębiec, Poland
     Department of Genomics, Institute of Genetics and Animal Breeding of the Polish Academy of Science, Jastrzębiec, Poland

Abstract

The Mef2B gene (myocyte enhancer factor 2B) encodes a transcription factor belonging to the MEF2 family that plays an important role in myogenesis by transcriptional regulation of genes involved in skeletal muscle growth and development. Despite the established importance of the Mef2 factors in the muscular growth and development, the temporal-spatial expression and biological function of Mef2B have not been reported in cattle. The aim of this study was to analyze the level of Mef2B expression in the developing longissimus dorsi muscle (LM) of 4 cattle breeds (Polish Holstein-Friesian [HF], Limousine [LIM], Hereford [HER], Polish Red [PR]), differing in terms of meat production and utility type, at 6, 9, and 12 mo of age. The Mef2B genetic polymorphism and expression patterns in 6 tissues (heart, spleen, liver, semitendinosus muscle [ST], gluteus medius muscle [GM], and LM) were also investigated. The results showed that Mef2B mRNA was expressed at a high level in adult skeletal and cardiac muscles. Moreover, Mef2B expression was markedly greater in the GM than in the LM (P < 0.05) and ST (P < 0.01). An age-dependent and breed-specific comparison of Mef2B mRNA level in skeletal muscle of HF, LIM, HER, and PR bulls showed that age was significant differentiating factor of Mef2B transcript/protein abundance in the LM of HER and LIM (P < 0.001) compared to HF and PR, for which the differences in Mef2B mRNA level were not significant (P > 0.05). Regarding the breed effect on the Mef2B expression, significantly greater Mef2B mRNA/protein level was noticed in the LM of 9 and 12 mo-old HER than of LIM (P < 0.01), HF (P < 0.001), and PR (P < 0.001). Four novel SNP, namely, HQ591462:g.909A > G (promoter), JX065116:g.3867T > C (exon 7), JX065116:g.4359G > C (exon 8), and JX065116:g.4546G > A (3’UTR), were identified. We found that Mef2B 3’UTR variant, situated within the seed region of the miR-5187–3p and miR-6931–5p binding sites, was associated with the level of Mef2B mRNA/protein in LM of 12-mo-old HF bulls. In addition, we observed a significant association between some carcass quality traits, including meat and carcass fatness quality traits, and various Mef2B 3’UTR genotypes in the investigated population of HF cattle. Our finding provides new evidence of the Mef2B significant role in the postnatal muscle growth and development in cattle, and indicates that Mef2B can be a promising molecular marker for carcass quality-related traits in adult cattle.



INTRODUCTION

The myocyte enhancer factor 2 (MEF2) family of transcription factors has been shown to play a crucial role in the activation of muscle-specific gene transcription in skeletal, cardiac, and smooth muscle (Naya and Olson, 1999). The products of 4 Mef2 genes, namely, Mef2A, Mef2B, Mef2C, and Mef2D, bind to A/T-rich DNA sequences that are present in many promoters and enhancers of skeletal muscle-specific genes (Black and Olson, 1998). The well-established role of Mef2 factors in muscle development is cooperation with the myogenic (bHLH) transcription factors (MyoD, Myf5, MyoG, and Myf6), the homeobox proteins (e.g., tinman, GAX) or GATA factors (e.g., GATA4; Onteru et al., 2012) as well as in regulation of myogenesis by controlling the expression of miRNAs, such as muscle-specific miR-1 and miR-133 (Zhao et al., 2005). Mef2 genes show distinct, but overlapping, temporal and spatial expression patterns in embryonic and adult tissues (Chen et al., 2015).

The bovine Mef2B gene was mapped on chromosome 7, within QTL for average daily gain, body and carcass weight (Casas et al., 2003), as well as for fat thickness (Ferraz et al., 2009). Thus, it can be considered as a positional and functional candidate for carcass and meat quality traits in cattle. To our knowledge, this is the first investigation of the Mef2B expression profile, polymorphism distribution, and biological function in the postnatal skeletal muscle growth and development in cattle.

Hence, the aim of this study was to: (1) determine the Mef2B expression patterns in different tissues, (2) assess the postnatal Mef2B mRNA level in the developing longissimus muscle (LM) of 4 cattle breeds (Polish Holstein-Friesian, Limousine, Hereford, and Polish Red), (3) investigate the Mef2B gene for functional polymorphisms and evaluate their effect on Mef2B mRNA and protein levels in the LM of 12-mo-old HF bulls, and, finally, (4) test the associations with production traits.

MATERIALS AND METODS

Animals, Blood, and Tissue Collection

All bulls were maintained at the Experimental Farm of IGAB PAS, Jastrzębiec and kept under identical environmental conditions, fed ad libitum with the same commercial mix fodder, slaughtered, and dissected at a IGAB PAS abattoir. All tissues for gene expression were harvested from carcasses within 20 min after slaughtered, immediately frozen in liquid nitrogen and stored at ‒80°C until further analyses. Blood was collected at slaughter and stored at ‒20°C for the purpose of genomic DNA extraction. All animal procedures performed for the purpose of this study were approved by the Local Ethical Commission on Experiments on Animals at the Warsaw Agricultural University, permission No. 29/2010. Three panels of bulls were included in the study:

Panel 1.

Panel 1 included a total of 72 bulls representing 4 breeds: Polish Holstein-Friesian (HF, n = 18), Polish Red (Polish native breed; PR, n = 18), Limousine (LIM, n = 18), and Hereford (HER, n = 18), which were used for the temporal and spatial Mef2B expression analysis. Level of Mef2B transcript/protein at different developmental stages was determined in the LM samples of HF, PR, LIM, and HER bulls, assigned to 3 groups (6 bulls of each breed per group), according to the age at which they were slaughtered, i.e., of 6, 9, and 12 mo. In addition, for spatial expression analysis of Mef2B, 6 different tissues: heart, spleen, liver, semitendinosus muscle (ST), gluteus medius muscle (GM) and LM were collected from ten 12-mo-old HF bulls (in total, 10 samples for each tissue).

Panel 2.

A total of 375 DNA samples derived from unrelated bulls representing 4 breeds, HF (n = 278), HER (n = 35), LIM (n = 27), and PR (n = 35), were used for Mef2B polymorphism screening and genotyping to determine the genotype and allelic frequency.

Panel 3.

A total of 278 HF bulls, progeny from 24 AI Holstein sires, slaughtered at the age of 12 mo, with body weight of about 380 kg, were used to determine the association of SNP with carcass traits. The following carcass traits were measured postmortem: carcass dressing percentage (CDP; %), weight of valuable cuts (WVC; kg), weight of lean in valuable cuts (WLVC; kg), weight of fat in valuable cuts (WFVC; kg), percent of lean in valuable cuts (PLVC; %), and percent of fat in valuable cuts (PFVC; %). Blood collected from HF bulls was used for screening for Mef2B polymorphism and subsequently for genotyping analysis. Additionally, LM samples from 30 bulls (10 animals for each genotype) were analyzed to establish the effect of the HQ591462:g.909A > G and JX065116:g.4546G > A SNP on the Mef2B transcript level and MEF2B protein abundance.

RNA Isolation, cDNA Synthesis, and Real-Time Quantitative PCR Assays (qRT-PCR)

Total RNA was isolated from tissue samples using the High Pure RNA Tissue Kit (Roche, Switzerland), according to the manufacturer’s protocol. A qualitative and quantitative assessment of the isolated RNA was conducted in ND-1000 NanoDrop spectrophotometer (NanoDrop Technologies, Wilmington, DE) and by 2% gel electrophoresis. Only samples containing more than 100 ng RNA and with absorbance ratios A260/280 and A260/230 of around 2.0 were used for further analyses. 1µg of total RNA was reverse transcribed with oligo(dT) primers using Transcription First Strand cDNA Synthesis Kit (Roche, Switzerland), according to the manufacturer’s instructions. Obtained cDNA was stored at ‒20°C. The expression level of bovine Mef2B was measured in triplicate by qRT-PCR using LightCycler96 System (Roche, Switzerland) in 96-well optical reaction plates with SYBR Green detection. PCR reactions were performed in a total volume of 10 mL, according to the LightCycler SYBR Green I Master kit protocol (Roche, Switzerland). The following amplification program was used: 10 min of initial denaturation at 95°C, 40 cycles of denaturation (10 s, 95°C), annealing (9 s, 60–61°C), and elongation (20 s, 72°C). qPCR amplifications were conducted in triplicates for both target and reference (splicing factor 3 subunit 1 [SF3A1]), eukaryotic translation elongation factor 1 a 2 (EEFIA2), and TATA-binding protein (TBP) genes. Negative controls containing the template RNA and all PCR reagents, but not reverse transcriptase, were included to determine the RNA purity from DNA contamination. Primer pairs were designed at the junction of exons to avoid amplification of any genomic DNA (Supplementary Table S1; see online version of the journal). Raw results were normalized relatively to the geometric mean of mRNA of 3 reference genes. The Mef2B expression level was calculated from Ct value by using the 2-ΔΔCt method (Livak and Schmittgen, 2001).

Western Blot Analysis

For detection of the MEF2B protein, nuclear extracts were prepared from frozen LM of the same animals used for Mef2B mRNA expression analysis, in accordance with Andrews and Faller (1991). Protein concentration was determined spectrophotometrically using the Bio-Rad protein assay. Nuclear extracts (25 µg) were subsequently resolved on 10% SDS-polyacrylamide gel and transferred to PVDF membrane (Bio-Rad). The membranes were initially blocked by gentle agitation in TBST (0.15% Tween 20 in Tris-buffered saline) containing 5% fat-free dried milk for 1 h at room temperature followed by overnight incubation at 4°C with the goat polyclonal anti-MEF2B (sc-30244; Santa Cruz Biotechnology, Santa Cruz, CA), or mouse monoclonal anti-GAPDH (Santa Cruz Biotechnology). Membranes were then washed and incubated with peroxidase-conjugated antimouse antibody (Santa Cruz Biotechnology) for 1 h at room temperature. Immunoreactive bands were detected using ClarityTM Western ECL Substrate (Bio-Rad), according to the manufacturer’s instruction. For densitometry, digital images were captured using Chemi Doc (Bio-Rad), and the intensity of the MEF2B bands (relative to the intensity of GAPDH bands) was quantified with a Molecular Imager using Quantity One software (Bio-Rad). Reactions were performed in triplicate for each sample.

SNP Identification and Genotyping

Genomic DNA was extracted from the blood samples using a Genomic DNA Purification Kit (Promega, Madison, WI) and stored at– 20°C. Promoter region, exons 4, 5, 6, 7, and exon 8 including 3’untranslated region (3’UTR) of Mef2B gene were targeted for selective amplification by PCR. Six pairs of nucleotide primers (Table S1) were designed using Primer 3.0 software (http://www.frodo.wi.mit.edu/) based on reference sequence AC_000164.1 and synthesized by Genomed Co. (Warsaw, Poland). Polymorphism screening was performed using the comparative resequencing approach in 20 bulls representing 4 cattle breeds. PCR reactions were performed in a total volume of 10 mL containing 0.5 mL of 10µM forward and reverse primers, 5 mL of HotStar Taq Master Mix Kit (Qiagen, Hilden, Germany), and approximately 100 ng of genomic DNA. The PCR amplification reactions were optimized and performed in a C1000 Thermal Cycler (BioRad Laboratories, Inc., Hercules, CA) using the following cycling parameters: 95°C for 15 min, followed by 35 cycles of 94°C for 1 min, 59.5°C to 61.0°C for 40 s, and final extension at 72°C for 5 min (Table S1). The purified PCR products were directly sequenced using a 3130xl Genetic Analyzer (Applied Biosystems, Carlsbad, CA). Two SNP, HQ591462:g.909A > G, and JX065116:g.4359G > C, were genotyped using AflIII and BfaI restriction enzymes (New England Biolabs, Inc., Ipswich, MA), respectively. For genotyping of 2 other SNP (JX065116:g.3867T > C and JX065116:g.4546G > A) the multitemperature Single Strand Chain Polymorphism (MSSCP) technique was applied. MSSCP electrophoresis was performed in the Pointer System (Kucharczyk Co., Warsaw, Poland) at constant power (40W) for 70 min. The electrophoresis temperatures were as follows: 35°C, 15°C, and 5°C for 350Vh. The gels were subsequently silver stained for 30 min using the Silver Stain Kit (Kucharczyk Co., Warsaw, Poland) and scanned with the Molecular Imager System FX (BioRad Laboratories, Inc., Hercules, CA). Sequence alignments and identification of variations were performed using the Clustal W (http://www.ebi.ac.uk/tools/msa/clustalW2) and Chromas Lite v2.01 (http://www.technelysium,com.au/chromas) programs. POPGENE V3.1 software (http://www.ualberta.ca/∼fyech) was applied to calculate the genotype and haplotype frequencies and deviation from the Hardy-Weinberg equilibrium.

In Silico Analysis

The search for putative transcription factor binding sites (TFBS) for HQ591462:g.909A > G SNP in promoter region was performed using TFBIND software (http://tfbind.hgc.jp/) and the TRANSFAC database (http://www.gene-regulation.com/). Effect of 3’UTR JX065116:g.4546G > A variant on miRNA binding sites was tested with miRBase database (http://www.mirbase.org/) and TargetScan (www.targetscan.org/). For analysis of binding stability of miRNAs to Mef2B polymorphic variants the RNAhybrid tool was applied (http://bibiserv.techfak.uni-bielefeld.de/rnahybrid/submission.html)

Statistical Analyses

Statistical significance of the differences in expression/protein level of Mef2B in relation to age, breed, tissue, and its genotype was determined by multi-factors ANOVA and Tukey’s adjustment. Differences were considered significant when P values were less than 0.05 (indicated by * for P < 0.05, by ** for P < 0.01, and by *** for P < 0.001). The association between JX065116:g.4546G > A variants and carcass traits of the HF bulls was estimated by least squares method as applied in the general linear model (GLM) procedure of SAS (9.2 version, SAS Inst. Inc., Cary, NC), according to the model: Yijkl = µ + Fi + Gj + SYk + b(xijkl ‒ x) + eijkl, where: Yijkl studied traits; µ ‒ overall mean; Fi ‒ the random effect of sire (i = 1,…, 24); Gj– the fixed effect of the Mef2B genotype (j = 1,…, 3); SYk– the fixed effect of year and season at start of fattening (k = 1, 2); b(xijkl ‒ x)– the regression on cold carcass weight; eijkl ‒ the random residual effect. Significant differences in carcass traits in respect to Mef2B genotypes were tested by Duncan’s test.


RESULTS

Expression Patterns of Mef2B in Cattle Tissues

Mef2B expression patterns were determined by qRT-PCR in 6 different tissues. The results revealed that the bovine Mef2B gene was expressed in all of the examined tissues, but at different levels. A high level of Mef2B transcript was detected in the GM, LM, and ST and heart, lower in the spleen, and the lowest level of Mef2B was observed in the liver (Fig. 1). Moreover, a significantly greater Mef2B mRNA abundance was noticed in the GM than in the LM (P < 0.05) and ST (P < 0.05).

Figure 1.
Figure 1.

Analysis of Mef2B expression patterns in different tissues of 12-mo-old HF bulls. Bars represent the mean ± SE (n = 10). *P < 0.05; **P < 0.01. LM, longissimus dorsi; GM, gluteus medium; ST, semitendinosus.

 

Developmental Expression of Mef2B in the LM of 4 Cattle Breeds

The qRT-PCR results showed that within-breed, age was significant differentiating factor of Mef2B expression in HER and LIM breeds. In both HER and LIM, a markedly greater level of the Mef2B mRNA was noticed in the muscle of bulls at the age of 9 mo as compared to relatively lower Mef2B expression level in bulls at age of 6 and 12 mo (P < 0.001 and P < 0.05, respectively). In HF and PR bulls age-dependent differences in Mef2B transcript level were not significant (Fig. 2A), although in PR a decrease of the Mef2B mRNA abundance was observed among subsequent age groups.

Figure 2.
Figure 2.

Breed-specific (A) and age-dependent (B) expression level of the Mef2B in LM of four cattle breeds. Data are means ± SE (n = 10). *P < 0.05; **P < 0.01; ***P < 0.001. HF, Polish Holstein-Friesian; LIM, Limousine; HER, Hereford; PR, Polish Red.

 

Comparison of the different breeds at the same age revealed a significantly greater expression level of Mef2B in the muscle of HER at the age of 9 and 12 mo as compared to LIM (P < 0.01), HF (P < 0.001), and PR (P < 0.001). Moreover, a relatively greater level of Mef2B transcript was observed in LM of HER and LIM at the age of 6 mo than in HF (P < 0.01) and PR (P < 0.05; Fig. 2B). Furthermore, by using Western blot analysis, we confirmed that the differences in Mef2B mRNA expression between breeds and age reflect variations in MEF2B protein abundance (Fig. 3). The relatively greater level of MEF2B protein was detected in the LM of HER bulls as compared to LIM, HF, and PR at the 3 postnatal stages (Fig. 3A, 3B). In addition, for LIM and HER bulls a significant increase of MEF2B protein abundance in the LM was estimated at the age of 12 mo, although this change was not observed in case of the Mef2B expression. No significant age-dependent differences in the MEF2B protein level were found in tested groups of HF and PR bulls (P > 0.05).

Figure 3.
Figure 3.

Western blot analysis of MEF2B protein level in LM of 4 cattle breeds at 3 stages of postnatal development. (A) Representative blots of MEF2B and GAPDH protein; (B) ratio of relative protein levels of MEF2B expressed to GAPDH. GAPDH was used as a loading control, as indicated. Data are representative for three independent Western blot analyses performed using extracts from 3 bulls of each experimental group. Results are expressed as mean ± SE. Statistically significant differences (**P < 0.01; ***P < 0.001) in MEF2B protein levels were calculated within breed in a given age group. LIM, Limousine; HER, Hereford; HF, Polish Holstein-Friesian; PR, Polish Red.

 

Sequencing Results of the Mef2B Gene and Polymorphism Analysis

Six selected fragments of Mef2B gene, encompassing promoter region, coding region, and 3’UTR, were resequenced to identify genetic variations in cattle. Overall, 4 novel SNP were identified, namely, HQ591462:g.909A > G in the promoter region, JX065116:g.3867C > T in exon 7, JX065116:g.4359G > C in exon 8 as well as JX065116:g.4546G > A transition in 3’UTR (exon 8) and all of them have been deposited in the GenBank. No polymorphism was detected in exon 4, 5, and 6. The genotype distributions and allele frequencies of these SNP have been shown in Table 1. The substitution JX065116:g.4359G > C in exon 8 was a nonsynonymous switch resulting in an amino acid substitution (Ala/Pro) at residue 348 (AFN86200). Of the 4 SNP identified, 2 (HQ591462:g.909A > G and JX065116:g.4359G > C) were genotyped by digestion with AflIII and BfaI, respectively, while 2 other substitutions (JX065116:g.3867C > T and JX065116:g.4546C > T) were analyzed by MSSCP technique. The MSSCP results showed that genotype frequencies of JX065116:g.3867T > C in HER, PR, and LIM breeds did not follow Hardy-Weinberg proportions (P < 0.01; P < 0.05; P < 0.05). In addition, the TT genotype occurred with the low frequency in HF (0.042), HER (0.029), and PR (0.057) breeds, and it was not detected in LIM breed. The most widely distributed SNP were HQ591462:g.909A > G, JX065116:g.4359G > C, JX065116:g.4546G > A, but the deviations from the Hardy-Weinberg proportion were observed in HER (P < 0.05) and PR (P < 0.05) for HQ591462:g.909A > G and JX065116:g.4546G > A, respectively. Concerning HQ591462:g.909A > G SNP, genotyping revealed the highest prevalence of the AG genotype in all studied breeds. In turn, at the JX065116:g.4546G > A locus, the allele G was dominant in HF, PR, and LIM breeds with a frequency ranged from 0.611 (LIM) to 0.686 (PR).


View Full Table | Close Full ViewTable 1.

Genotypic distribution and allelic frequencies of 4 SNP of the Mef2B gene in 4 cattle breeds

 
Breed
SNP Detection method Genotype/allele HF (278)a HER (35) PR (35) LIM (27)
g.909A>G (promoter) RFLP/AflIII GG
AG
AA
G
A
Χ2 (P-value)
0.202 (58)
0.526 (151)
0.276 (78)
0.465
0.535
0.95 (0.330)
0.314 (11)
0.629 (21)
0.057 (3)
0.629
0.371
4.19 (0.040)*
0.286 (10)
0.543 (19)
0.171 (6)
0.557
0.443
0.35 (0.553)
0.259 (7)
0.407 (11)
0.333 (9)
0.463
0.537
0.88 (0.347)
g.3867C>T (exon 7) MSSCP/sequencing CC
CT
TT
C
T
Χ2 (P-value)
0.693 (199)
0.265 (76)
0.042 (12)
0.826
0.174
1.80 (0.177)
0.629 (12)
0.343 (22)
0.029 (1)
0.657
0.343
5.46 (0.019)**
0.286 (10)
0.657 (21)
0.057 (4)
0.614
0.386
5.23 (0.022)*
0.444 (12)
0.556 (15)
0.000 (0)
0.722
0.278
3.99 (0.045)*
g.4359G>C (exon 8) RFLP/BfaI CC
GC
GG
C
T
Χ2 (P-value)
0.329 (132)
0.461 (98)
0.209 (65)
0.560
0.440
1.64 (0.200)
0.556 (20)
0.333 (12)
0.111 (4)
0.722
0.278
1.03 (0.309)
0.368 (7)
0.526 (10)
0.105 (2)
0.632
0.368
0.33 (0.568)
0.375 (6)
0.563 (9)
0.063 (1)
0.656
0.344
1.03 (0.309)
g.4546G>A (3’UTR) MSSCP/sequencing GG
GA
AA
G
A
Χ2 (P-value)
0.353 (98)
0.514 (143)
0.133 (37)
0.610
0.390
1.82 (0.177)
0.314 (11)
0.600 (21)
0.086 (3)
0.386
0.614
2.48 (0.115)
0.543 (19)
0.286 (10)
0.171 (6)
0.686
0.314
3.98 (0.046)*
0.259 (9)
0.407 (15)
0.333 (3)
0.611
0.389
0.77 (0.380)
aIn brackets, number of animals; HF, Polish Holstein Friesian; HER, Hereford; CH, Charolaise; PR, Polish Red; LIM, Limousine; *P < 0.05, **P < 0.01.

Effect of HQ591462:g.909A > G and JX065116:g.4546G > A Genotypes on Mef2B mRNA and Protein Level in LM

Using TFBIND and TRANSFAC we examined the impact of promoter variant HQ591462:g.909A > G on the transcription factors binding probability. Results showed that allele A creates putative binding site for GATA-2 transcription factor, which is disrupted by the G allele. Therefore, we investigated the influence of the g.909A > G variant on the Mef2B mRNA level in LM of 12-mo-old HF bulls, but no significant differences between GG, AA, and GA genotypes were found (data not shown). We also performed a search for miRNA target sequences within 3’UTR of Mef2B gene. The miRBase analysis revealed that JX065116:g.4546G > A is situated within the potential target sequence for miR-5187–3p and miR-6931–5p, which is abrogated by the presence of the A allele. To assess the effect of g.4546G > A substitution on the Mef2B mRNA level, we quantified the relative transcript level in the LM of 12-mo-old HF bulls with the AA, AG and GG genotypes. The relative Mef2B transcript level was significantly lower (P < 0.05) in the GG in comparison to the AA bulls (Fig. 4A). Moreover, by using Western blot analysis, we confirmed that the Mef2B g.4546G > A variants were also highly correlated with MEF2B protein abundance in LM. The significantly lower MEF2B protein level was estimated in muscle of bulls with the GG genotype as compared to these with the AA (P < 0.05) and GA (P < 0.05; Fig. 4B, 4C). For each allele of g.4546G > A SNP we evaluated minimum free energy (mfe), which indicated the probability of stable duplex formation between miRNA and mRNA, and our results showed that G > A substitution reduced the predicted mfe from -19.5 kcal/mol (G allele) to -18.3 kcal/mol (A allele) in the case of miR-5187–3p (Supplementary Fig. S1; see online version of the journal). This may potentially increase the probability of miR-5187–3p binding to the Mef2B target region and reduce the Mef2B transcript level in case of the G allele.

Figure 4.
Figure 4.

The effect of JX065116:g.4546G > A in the 3’UTR on Mef2B mRNA and MEF2B protein abundance in LM of 12-mo-old HF bulls. (A) Relative Mef2B mRNA level in LM of HF bulls representing GG (n = 10), AA (n = 10), and AG (n = 10) genotypes for SNP JX065116:g.4546G > A. Data are shown as the mean ± SE. A statistically significant difference at P < 0.05 was calculated between the GG and AA genotypes. (B) Western blot analysis of the MEF2B protein level in LM of HF bulls with different JX065116:g.4546G > A variants. Representative blots of MEF2B and GAPDH protein. GAPDH is shown as a loading control. (C) Bar graphs shows densitometry quantification of blots from MEF2B normalized to GAPDH. Relative protein abundance differences are shown as densitometry mean ± SE (n = 10). *P < 0.0.

 

Association Analysis between g.4546G > A SNP in 3’UTR of Mef2B Gene and Production Traits

Preliminary association analysis between g.4546G > A SNP and the carcass traits in 278 HF bulls showed that transition in Mef2B 3’UTR was significantly associated with WLVC and WFVC (Table 2). Animals with the GG genotype had a lower WLVC (P < 0.05) than those with the AA and GA genotype. In addition, greater value of WFVC was observed for animals with GG genotype than for those with the AA (P < 0.05) and GA (P < 0.05). No associations of g.4546G > A SNP with CDP, WVC, PLVC, and PFVC were found in this study.


View Full Table | Close Full ViewTable 2.

Association between SNP JX065116:g.4546G>A in the 3’UTR of Mef2B gene and carcass quality traits of 12-month-old Polish Holstein-Friesian (HF) bulls

 
Genotype
Traits AA (n = 37) GA (n = 143) GG (n = 98) P-value
CDP (%) 52.4 ± 0.91 52.2 ± 0.3 51.8 ± 0.6 ns
WVC (kg) 59.4 ± 0.2 59.2 ± 0.6 59.0 ± 0.4 ns
WLVC (kg) 41.6a ± 0.3 41.5 ± 0.7 40.6a ± 0.3 P = 0.040
WFVC (kg) 6.0a ± 0.1 6.2b ± 0.1 6.7ab ± 0.2 aP = 0.049; bP = 0.044
PLVC (%) 68.7 ± 1.3 68.4 ± 0.5 67.2 ± 0.9 ns
PFVC (%) 11.0 ± 1.3 11.7 ± 0.7 12.5 ± 1.3 ns
a,bSignificant differences between the genotypes are indicates with superscript lowercase letters.
1Least squares means ± SE; n, number of animals; ns, non-significant; CDP (%), carcass dressing percentage cold; WVC (kg), weight of valuable cut; WLVC (kg), weight of lean in valuable cuts; WFVC (kg), weight of fat in valuable cuts; PLVC (%), percentage of lean in valuable cuts; PFVC (%), percentage of fat in valuable cuts.


DISCUSSION

General Remarks

It is known that the expression level of the genes in different tissues determines the quantitative and qualitative traits and affects the variation between breeds of livestock including cattle (Bartz et al., 2014; Stachowiak et al., 2014). Recently, a number of published studies have clearly shown that family of Mef2 transcription factors is involved in the postnatal skeletal muscle growth and development (Knapp et al., 2006; Potthoff et al., 2007; Juszczuk-Kubiak et al., 2014) as well as homeostasis (Jung and Ko, 2010; Zhao et al., 2011) by adjusting the expression levels of muscle-specific genes. It has been reported that the expression level of Mef2 factors was downregulated after birth (Sun et al., 2013), while the substantial increase of their transcript abundance was observed during postnatal skeletal muscle and cardiac hypertrophy (Black, 2007), regeneration process (Liu et al., 2014), and endurance exercise (Vissing et al., 2008) as well as with age (Musarò et al., 1995).

Thus, study of the muscle expression of particular genes, such as Mef2 factors, might be useful to unravel the molecular background underlying breed-specific differences in phenotype of muscle traits.

Mef2B Transcript and Protein Level in Relation to Tissue, Age, and Breed

The tissue-specific expression patterns of the bovine Mef2B gene have not been investigated in previous studies, but the presence of 3 other Mef2 transcripts (Mef2A, Mef2C, and Mef2D) in adult skeletal muscles of humans (Bachinski et al., 2010), mice (Hakim et al., 2010), ducks (Sun et al., 2013), cattle (Juszczuk-Kubiak et al., 2012a; 2012b; 2014), goats (Chen et al., 2015), and in human cardiac muscle (Alonso-Montes et al., 2012), as well as in the brain and spleen of mice (Hakim et al., 2010; Sekiyama et al., 2012) has been reported. According to earlier studies, it has been stated that the Mef2B gene is not expressed in the adult tissues and organs. For instant, Morisaki et al. (1997) reported, that Mef2B expression was not detected in adult mouse heart, skeletal muscle, and brain, but an elevated level of Mef2B transcript was observed in the developing cardiac and skeletal muscle lineages during mouse embryogenesis (Molkentin et al., 1996). In the current study, we clearly demonstrated that bovine Mef2B was expressed ubiquitously in all studied tissues, and was preferentially accumulated in adult skeletal and cardiac muscles, which is consistent with previous outcomes that have shown an essential role of Mef2 factors in postnatal skeletal and cardiac muscle hypertrophy (Knapp et al., 2006; Potthoff and Olson 2007; Hakim et al., 2010; Juszczuk-Kubiak et al., 2014). Furthermore, the presence of the Mef2B transcripts in the adult bovine liver and spleen suggests the possible role of Mef2B in transcriptional pathways regulating postnatal development and physiological processes in these vital organs. Recently, the tissue-specific expression patterns of goat Mef2B in 6 adult tissues, including liver, kidney, myocardium, and 3 skeletal muscles (LM, triceps brachii, and semimembranosus) were also investigated by Chen et al. (2015), who observed a higher level of Mef2B expression in the skeletal muscles as compared to barely detectable Mef2B transcripts in other tissues.

In our study, the Mef2B mRNA spatial expression pattern showed that Mef2B was differentially expressed in the tested skeletal muscles. The significantly greater Mef2B mRNA level noticed in the GM as compared to the LM and ST indicates that Mef2B expression depends on the type of muscle. These data suggest that observed differences in the Mef2B mRNA levels between various skeletal muscles may reflect differences in fiber-type composition and/or the rate of their maturation. A muscle-dependent expression of Mef2B was also observed in the goat (Chen et al., 2015), but relatively greater level of Mef2B transcript was noticed in the triceps brachii muscle than in the LM and semimembranosus. Moreover, in our previous study, we have investigated tissue-specific differences in expression profile of the bovine Mef2C (Juszczuk-Kubiak et al., 2014), and we found that Mef2C was expressed ubiquitously in all studied tissues, but preferentially was accumulated in the cardiac and skeletal muscles. In addition, a significantly lower mRNA level was observed in the ST than in the LM and GM. Thus, our findings indicate a possible essential role of Mef2B in the postnatal muscle development and also suggest involvement of tissue-specific regulatory mechanisms controlling its mRNA expression.

To understand the molecular mechanism underlying breed-specific differences, the Mef2B mRNA expression patterns were compared at 3 different developmental stages in the LM of 4 cattle breeds differing with respect to meat and fat share in carcass, quality of meat, intramuscular fat content, as well as growth rate and the age of achieving maturity. Moreover, the carcasses of included breeds vary in their muscle phenotype traits such as myofiber numbers, size, and type (Iwanowska and Pospiech, 2010).

In the current study, we have found highly significant differences in the relative Mef2B expression among 4 cattle breeds at various developmental stages. The significant breed- and age-dependent differences in both Mef2B mRNA and MEF2B protein abundance were observed between beef (HER and LIM) versus dairy HF and dual-purpose PR bulls. Our study revealed substantially greater level of Mef2B mRNA in LM of HER bulls than LIM, HF, and PR bulls at the 3 postnatal stages. Moreover, in both HER and LIM, the level of Mef2B mRNA and MEF2B protein fluctuated with age, being greater at 9 mo of age for mRNA, and at 9 (HER) and 12 (LIM, HER) months of age with regard to protein. The observed mismatch between Mef2B mRNA and protein expression in LIM muscle at the age of 12 mo may be due to a posttranscriptional modification of Mef2B mRNA. Thus, it is possible that observed differences in Mef2B expression might contribute to the distinct postnatal myogenesis process between the tested breeds. According to the study of Chen et al. (2015), concerning age-dependent expression profiles of all 4 Mef2 genes, including Mef2B, in the adult skeletal muscles of Nanjiang Yellow goats, the overall expression levels of Mef2A and Mef2D were greater than those of Mef2B and Mef2C, and their expression peaks varied depending on the muscle type and stage of development; the relatively greater levels of Mef2B and Mef2C were detected between 30 and 120 d of age. The breed and age relationship in terms of the Mef2C expression in the bovine skeletal muscle was also reported in our previous study (Juszczuk-Kubiak et al., 2014), in which a comparative analysis of the Mef2C mRNA level between dairy-HF and meat-LIM bulls revealed the opposite trend at 2 developmental stages. In LIM, a significantly greater level of Mef2C mRNA was estimated at the age of 6 mo followed by a subsequent decrease with age. In contrast, a substantial increase of Mef2C mRNA level was noticed in the muscle of 12-mo-old HF bulls. A similar complex crosstalk between age and postnatal muscle development was also observed in the case of chicken Mef2A gene (Liu et al., 2012).

Taken together, our outcomes may suggest that elevated Mef2B gene and protein expression could be related to greater muscularity of carcasses, as it is in HER and LIM breeds. Moreover, the significant increase in both Mef2B mRNA and protein abundance, observed in HER at the age of 9 mo, may have a connection with early maturing of this breed (Iwanowska and Pospiech, 2010). It has been proven that in the skeletal muscle, Mef2 factors play a cooperative role with the MyoD family of bHLH transcription factors in inducing muscle hypertrophy (Black, 2007), and high levels of Mef2 expression were significantly correlated with an increase of myofiber diameter in goat LM during the 5 postnatal stages (Chen et al., 2015). Several studies have shown that expression of the Mef2 transcription factors is required for increase and maintenance of Myod1 expression during postnatal activation and differentiation of satellite cells in mice (L’honore et al., 2007; Bachiński et al., 2010). Moreover, Naidu et al. (1995) reported that Mef2 plays a key role in the activation of the Myf6 gene transcription, since a proximal MEF2-site is required for constitutive Myf6 expression during muscle fiber maturation. Therefore, it is very likely that elevated level of Mef2B mRNA may be required for the Myod1 and Myf6 expression and in maintaining the greater muscle growth of HER and LIM bulls. On the other hand, recent studies demonstrated that Mef2 along with MyoD factors play a crucial role in maintaining the balance between intramuscular adipogenesis and myogenesis by regulating the expression of C/EBPa (Zhao et al., 2011) and PPAR-b in the skeletal muscle, heart, and adipose tissue (Gan et al., 2011), thus the effect of Mef2B on fat deposition, especially in HER, is also possible. In addition, the elevations in the Mef2B expression in HER and LIM could have also facilitated and enhanced transcription of type I, IIa, and IIx myosin heavy chain (MHC) mRNA molecules. It is well established that postnatally MEF2 proteins induce a remodeling of skeletal muscle fiber-type composition (Knapp et al., 2006; Potthoff et al., 2007; Hennebry et al., 2009), and the fiber composition displayed differences among different cattle breeds (McPherron et al., 1997; Grześ et al., 2007). MEF2 factors have been shown to induce a shift in enzyme activity from glycolytic to oxidative metabolism when overexpressed in adult mice (Czubryt et al., 2003). Therefore, it is very likely that observed differences in the Mef2B mRNA level and MEF2B protein abundance might result from changes in the muscle’s glycolytic/oxidative profile between tested breeds. We speculated, that in the case of HER and LIM breeds, the period between 9 and 12 mo of age could be critical for fiber transition and maturation. On the other hand, the lack of age-dependent differences in the Mef2B expression in HF and PR, and its lower level of mRNA and protein expression compared with HER and LIM, might contribute to slower muscle development and lead to the less muscle mass in these breeds. Thus, our data indicate that the expression profile of the Mef2B could be related to muscle fiber hypertrophy and meat quality which is affected by muscle fiber type.

Mef2B Polymorphism Analysis

Another possibility is that age-dependent differences in the transcript level might be breed-specific and caused by a varied genetic background resulting from a different genetic origin. This discrepancy could be explained by breed-specific polymorphisms, such as the presence of the genetic variations in the promoter and 3’UTR sequences of the Mef2B gene. So far, the polymorphism of the bovine Mef2B gene was not reported in any available study, and its effect on gene expression level and muscle development is not known. Also, there are few reports addressing this issue in livestock. Several SNP in the Mef2A and Mef2C have been found in cattle (Chen et al., 2010; Juszczuk-Kubiak et al., 2011; Juszczuk-Kubiak et al., 2012a) and chickens (Zhou et al., 2010), as well as in the Mef2D gene of ducks (Wang et al., 2016) and cattle (Juszczuk-Kubiak et al., 2012b, Juszczuk-Kubiak et al., 2013).

In the current study, 4 novel SNP in the Mef2B promoter, exon 7 and exon 8 as well as in 3’UTR were identified; no polymorphism was found in exon 4, 5, and 6. In the coding region, only one nonsynonymous SNP (Ala348Pro) in the exon 8 was detected, suggesting that the coding sequence of the Mef2B is lowly polymorphic and evolutionary conserved. The 4 SNP were widely distributed in the all tested breeds and revealed the presence of the 3 genotypes, except of JX065116:g.3867C > T SNP for LIM. In addition, the genotype frequencies for JX065116:g.3867C > T in HER, PR, as well as LIM did not follow the Hardy-Weinberg proportion. This may be due to variations in breed productivity and breeding purpose or a small number of the investigated bulls.

It is known that SNP in the promoter and 3’UTR sequences may be situated within transcriptional factor and microRNA binding sites and potentially affect the gene expression and mRNA stability. The results of the current study indicated that the Mef2B promoter variant does not affect the level of mRNA in the LM of HF bulls, which implies that HQ591462:g.909A > G substitution is presumably not involved in the cis-regulation of Mef2B expression in the muscles of tested bulls. Similarly, in our previous study, we showed that the Mef2C promoter variant did not affect the level of its mRNA in the LM and ST of HF bulls (Juszczuk-Kubiak et al., 2014), whereas the allele-dependent impact of SNP in the promoter of the Mef2A (Juszczuk-Kubiak et al., 2012a) and Mef2D (Juszczuk-Kubiak et al., 2012b) on mRNA levels, as well as on MEF2D protein abundance in the LM of HF bulls, have been noticed. On the other hand, the JX065116:g.4546G > A transition in the 3’UTR, which was perfectly situated within the seed region of the miR-5187–3p and miR-6931–5p binding sites, was associated with the significant reduction of Mef2B mRNA and protein levels in LM of bulls with GG genotype. This finding supported our in silico-based hypothesis, that the predicted miRNAs binding to 3’UTR in the variant with G allele could be stronger and thus potentially decrease the Mef2B transcript stability. Moreover, the polymorphisms in 3’UTR may influence the secondary structure of mRNA, which is important to ensure properly high accessibility of miRNA targets to interacting miRNA. Thus, we also hypothesize, that these 2 genotypes (AA and GG) present a different architecture of its 3’UTR and this could, in turn, affect the level of the Mef2B expression. Although the specific function of miR-5187–3p in skeletal muscle development has not been reported to date, in this study we suggest that miR-5187–3p may act as a negative regulator of the Mef2B expression during skeletal muscle development and growth in cattle. Several studies have reported that Mef2 factors are regulated by miRNAs. For instance, Mef2C was identified as a target for regulation by miR-27b, and overexpression of miR-27b in mouse cardiomyocytes, similarly as in skeletal muscle myoblast, led to a decrease of Mef2C transcript level (Chinchilla et al., 2011). Recently, Shen et al. (2016) reported that overexpression of the miR-23a significantly suppressed the expression of porcine Mef2C at both mRNA and protein levels, thus causing downregulation of the expression of some key downstream genes of Mef2C (PGC1-a, NRF1, and mtTFA). On the other hand, it has been reported that Mef2-dependent intronic enhancers regulate the skeletal muscle expression of miR-1 and miR-133a (Liu et al., 2007).

The evaluation of the potential impact of polymorphisms in 3’UTR on gene expression via miRNA-target interactions is still limited, especially in livestock. Nevertheless, recent reports have confirmed that the existence of these interactions may contribute to phenotypic variability by altering the expression of the target genes that directly impact economic traits in livestock. Examples of such functional SNP were reported in 3’UTR of the Texel sheep (Clop et al., 2006) and Piemontese cattle MSTN gene (Miretti et al., 2013), as well as in the 3’UTR of the porcine PGC-1a and PPARA genes (Lee et al., 2013; Stachowiak et al., 2014). We showed in this study that the JX065116:g.4546G > A SNP in the Mef2B gene 3’UTR also contributes to the variability of carcass traits in the investigated population of the HF cattle, and the observed associations appeared to be the most promising. The tested SNP showed a significant correlation with WLVC and WFVC, and favorable values for these traits (higher WLVC and lower WFVC) have been recorded for the AA genotype, which suggests that allele A presented a positive and desirable effect on carcass composition and gain in muscle mass. This correlation might be due to the fact that allele A at the g.4546G > A locus abolishes a target binding sequence for 2 miRNA (miR-5187–3p and miR-6931–5p) and reduces the probability of these miRNA binding to the Mef2B target region. Therefore, one can speculate that the allele A is responsible for an elevated expression and translation efficiency of the Mef2B due to its ineffective posttranscriptional downregulation by miRNA. So far, there are few studies on the Mef2 genes polymorphisms focusing on their association with economic traits in livestock. Zhou et al. (2010) found that SNP in 5’UTR, exon 4, and intron 7 of the chicken Mef2A gene have been positively correlated with body and muscle weight. Furthermore, Chen et al. (2010) reported that 3 linked SNP in exon 11 of the Mef2A affect the average daily gain and body weight in Chinese cattle breeds. Moreover, an association of the Mef2D polymorphisms with production traits in cattle was demonstrated in our previous studies (Juszczuk-Kubiak et al., 2012b; Juszczuk-Kubiak et al., 2013). We found that silent SNP (g.93C > T) in the exon 8 was strongly associated with WLVC and WFVC in the HF bulls. No relationship with carcass traits was observed for the SNP (g.47C > T) in intron 9, but a significant effect of combined genotypes of these 2 SNP on WFVC, percent of lean (PLVC), and fat (PFVC) in valuable cuts has been found (Juszczuk-Kubiak et al., 2013). Recently, positive correlation between the expansion of the CAG repeat in the exon 9 of the duck Mef2D and 5 muscle-related traits, including breast muscle weight, has also been documented (Wang et al., 2016). Thus, the above mentioned data indicate that polymorphism (JX065116:g.4546G > A) in the 3’UTR of the Mef2B gene appears to be a promising genetic marker linking quantitative trait loci with effects on carcass traits in cattle.

Conclusions

In summary, our study showed significant age-and breed-dependent differences in both Mef2B mRNA and protein expression, observed between bulls of various breeds. The trend of elevated abundance of Mef2B transcript/protein was found in bulls with higher muscularity of carcasses (HER and LIM). It is possible that these differences contribute to a distinct postnatal myogenesis process between these breeds, and may be one of the genetic factors determining the phenotype of the animals toward different meat production and utility type. We also observed that the Mef2B mRNA level depended on the type of muscle. Moreover, the association between the SNP (JX065116:g.4546G > A) in 3’UTR and phenotype traits showed that the Mef2B can be considered as a potential candidate gene and molecular marker for improvement of meat production in livestock. In line with available information, we are presenting one of the first reports considering breed- and age-dependent, specific expression of Mef2B in skeletal muscle during postnatal development of cattle. Therefore, we believe that the results presented in our paper provide useful molecular information for the further investigation of the function of bovine Mef2B gene in postnatal muscle growth and development.

 

References

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