Myostatin Promoter Research Articles

Myocyte enhancer factor 2 upregulates expression of myostatin promoter in Yesso scallop, Patinopecten yessoensis

Myostatin (MSTN) plays an important role in muscle development in animals, especially for mammals and fishes. However, little information has been reported on the regulation of MSTN in marine invertebrates, such as bivalves. In the present study, we cloned the MSTN promoter sequence of Yesso scallop Patinopecten yessoensis, identifying 4 transcription start sites, eleven TATA boxes and one E-box. Additionally, transcription factor binding sites, including myocyte enhancer factor 2 (MEF2) and POU homeodomain protein, were identified. The interaction between the MSTN promoter and MEF2 was analyzed to reveal the transcriptional activity of different fragment sizes of promoters through the dual-luciferase reporter assays. The highest transcriptional activity was found in recombinant plasmids with the most MEF2 binding sites, indicating that this transcription factor upregulates MSTN in Yesso scallop. This study provides new insight into the regulation of muscle growth and development in this species.

Systematic Myostatin Expression Screening Platform for Identification and Evaluation of Myogenesis-Related Phytogenic in Pigs.

Skeletal muscle growth in livestock impacts meat quantity and quality. Concerns arise because certain feed additives, like beta-agonists, may affect food safety. Skeletal muscle is a specialized tissue consisting of nondividing and multinucleated muscle fibers. Myostatin (MSTN), a protein specific to skeletal muscle, is secreted and functions as a negative regulator of muscle mass by inhibiting the proliferation and differentiation of myoblasts. To enhance livestock muscle growth, phytogenic feed additives could be an alternative as they inhibit MSTN activity. The objective of this study was to establish a systematic screening platform using MSTN activity to evaluate phytogenics, providing scientific evidence of their assessment and potency. In this study, we established a screening platform to monitor myostatin promoter activity in rat L8 myoblasts. Extract of Glycyrrhiza uralensis (GUE), an oriental herbal medicine, was identified through this screening platform, and the active fractions of GUE were identified using a process-scale liquid column chromatography system. For in vivo study, GUE as a feed additive was investigated in growth-finishing pigs. The results showed that GUE significantly increased body weight, carcass weight, and lean content in pigs. Microbiota analysis indicated that GUE did not affect the composition of gut microbiota in pigs. In summary, this established rodent myoblast screening platform was used to identify a myogenesis-related phytogenic, GUE, and further demonstrated that the active fractions and compounds inhibited MSTN expression. These findings suggest a novel application for GUE in growth performance enhancement through modulation of MSTN expression. Moreover, this well-established screening platform holds significant potential for identifying and assessing a diverse range of phytogenics that contribute to the process of myogenesis.

Metformin induces muscle atrophy by transcriptional regulation of myostatin via HDAC6 and FoxO3a

BackgroundSkeletal muscle atrophy is a severe condition that involves loss of muscle mass and quality. Drug intake can also cause muscle atrophy. Biguanide metformin is the first‐line and most widely prescribed anti‐diabetic drug for patients with type 2 diabetes. The molecular mechanism of metformin in muscle is unclear.MethodsMyostatin expression was investigated at the protein and transcript levels after metformin administration. To investigate the pathways associated with myostatin signalling, we used real‐time polymerase chain reaction, immunoblotting, luciferase assay, chromatin immunoprecipitation assay, co‐immunoprecipitation, immunofluorescence, primary culture, and confocal microscopy. Serum analysis, physical performance, and immunohistochemistry were performed using our in vivo model.ResultsMetformin induced the expression of myostatin, a key molecule that regulates muscle volume and triggers the phosphorylation of AMPK. AMPK alpha2 knockdown in the background of metformin treatment reduced the myostatin expression of C2C12 myotubes (−49.86 ± 12.03%, P < 0.01) and resulted in increased myotube diameter compared with metformin (+46.62 ± 0.88%, P < 0.001). Metformin induced the interaction between AMPK and FoxO3a, a key transcription factor of myostatin. Metformin also altered the histone deacetylase activity in muscle cells (>3.12‐fold ± 0.13, P < 0.001). The interaction between HDAC6 and FoxO3a induced after metformin treatment. Confocal microscopy revealed that metformin increased the nuclear localization of FoxO3a (>3.3‐fold, P < 0.001). Chromatin immunoprecipitation revealed that metformin induced the binding of FoxO3a to the myostatin promoter. The transcript‐level expression of myostatin was higher in the gastrocnemius (GC) muscles of metformin‐treated wild‐type (WT) (+68.9 ± 10.01%, P < 0.001) and db/db mice (+55.84 ± 6.62%, P < 0.001) than that in the GC of controls (n = 4 per group). Average fibre cross‐sectional area data also showed that the metformin‐treated C57BL/6J (WT) (−31.74 ± 0.75%, P < 0.001) and C57BLKS/J‐db/db (−18.11 ± 0.94%, P < 0.001) mice had decreased fibre size of GC compared to the controls. The serum myoglobin level was significantly decreased in metformin‐treated WT mice (−66.6 ± 9.03%, P < 0.01).ConclusionsOur results demonstrate that metformin treatment impairs muscle function through the regulation of myostatin in skeletal muscle cells via AMPK‐FoxO3a‐HDAC6 axis. The muscle‐wasting effect of metformin is more evident in WT than in db/db mice, indicating that more complicated mechanisms may be involved in metformin‐mediated muscular dysfunction.

Effect on Myogenesis and Anti-Inflammation of Mealworm (Tenebrio Molitor) Larvae Protein Hydrolysate

Abstract Objectives Sarcopenia, a loss of skeletal muscle mass and functions, becomes prevalent in older populations. Adequate dietary protein intake is one of the most effective ways to improve sarcopenia. The mealworm is an edible insect that comprises high levels of proteins and micro-nutrients. It could be used a potential protein source for enhancing muscle growth. Also, protein hydrolysate is easily digested and absorbed by the human body with greater functional properties. In investigating the prevention of sarcopenia with mealworm larvae, we optimize the hydrolysis condition of mealworm larvae protein isolate (MPI) and the effect of myogenesis and anti-inflammation of mealworm larvae protein hydrolysate (MPH). Methods The optimization of MPH by alcalase and flavorzyme was investigated based on the amount of available α-amino acids (L-Leucine) by TNBS assay and the distribution patterns of molecular weight by SDS-PAGE. ABTS radical scavenging capacity, expression of myostatin (MSTN), MSTN promoter activity, and anti-inflammatory induced by palmitate and LPS were examined. Results The hydrolysate prepared for 12 hours by adding flavourzyme after hydrolysis of the MPI with alcalase for 12 hrs under 1% substrate and 1% enzyme/substrate at 55˚C, pH 8 showed the highest available α-amino acid (22.09 mg/g) and had the largest amount of low-molecular peptides at 10 kDa or less. Antioxidant capacity was also the highest (97.8%) among the hydrolysates prepared under different conditions. In the MSTN level which negatively regulate muscle growth, MPH (0.28 MSTN/GAPDH) was lower than MPI (0.38 MSTN/GAPDH). Also, in the MSTN promoter activity, MPI (16.94 Luc/β-gal) was higher than the control while MPH (9.99 Luc/β-gal) was lower activity than the control (12.47 Luc/β-gal). MPI and MPH induced lower expression levels of IL-6, TNF-a, and IL-1b cytokines. Conclusions MPH had better capacity in myogenesis compared to MPI, particularly hydrolyzed by flavourzyme after hydrolysis of the MPI with alcalase. Also, the MPH decreased proinflammatory cytokines which increase proteolysis and muscle atrophy. These results demonstrate that MPH could be used as a source for preventing sarcopenia. Funding Sources This study was supported in part by the National Research Foundation of Korea.

IMB0901 inhibits muscle atrophy induced by cancer cachexia through MSTN signaling pathway

BackgroundCancer cachexia as a metabolic syndrome can lead to at least 25% of cancer deaths. The inhibition of muscle atrophy is a main strategy to treat cancer cachexia. In this process, myostatin (MSTN) can exert a dual effect on protein metabolism, including inhibition of protein biosynthesis and enhancement of protein degradation. In this study, we will test the effect on muscle atrophy induced by cancer cachexia of IMB0901, a MSTN inhibitor.MethodsTwo high-throughput screening models against MSTN were developed. By screening, IMB0901, 2-((1-(3,4-dichlorophenyl)-1H-pyrazolo [3,4-d] pyrimidin-4-yl) amino) butan-1-ol, was picked out from the compound library. The in vitro cell model and the C26 animal model of muscle atrophy induced by cancer cachexia were used to determine the pharmacological activity of IMB0901. Whether IMB0901 could inhibit the aggravating effect of doxorubicin on muscle wasting was examined in vitro and in vivo.ResultsIMB0901 inhibited the MSTN promoter activity, the MSTN signaling pathway, and the MSTN positive feedback regulation. In atrophied C2C12 myotubes, IMB0901 had a potent efficiency of decreasing MSTN expression and modulating MSTN signaling pathway which was activated by C26-conditioned medium (CM). In C2C12 myotubes, the expressions of three common myotube markers, myosin heavy chain (MyHC), myogenic differentiation 1 (MyoD), and myogenin (MyoG), were downregulated by CM, which could be efficiently reversed by IMB0901 via reduction of ubiquitin-mediated proteolysis and enhancement of AKT/mTOR-mediated protein synthesis. In the C26 animal model, IMB0901 mitigated the weight loss of body, quadricep and liver, and protected the quadriceps cell morphology. Furthermore, IMB0901 decreased the expression of two E3 ligases Atrogin-1 and MuRF-1 in the quadriceps in vivo. At the cellular level, IMB0901 had no influence on anti-tumor effect of three chemotherapeutic agents (cisplatin, doxorubicin, and gemcitabine) and lowered doxorubicin-induced upregulation of MSTN in C2C12 myotubes. IMB0901 did not affect the inhibitory effect of doxorubicin on C26 tumor and delayed the weight loss of muscle and adipose tissue caused by C26 tumor and doxorubicin.ConclusionsIMB0901 inhibits muscle atrophy induced by cancer cachexia by suppressing ubiquitin-mediated proteolysis and promoting protein synthesis. These findings collectively suggest that IMB0901 is a promising leading compound for the management of muscle atrophy induced by cancer cachexia.

291 Segregation of a triple nucleotide insertion/deletion polymorphism in Activin receptor II B-promoter and a transversion in the Myostatin promoter in Senepol cows.

291 Segregation of a triple nucleotide insertion/deletion polymorphism in Activin receptor II B-promoter and a transversion in the Myostatin promoter in Senepol cows.

PO-273 Exercise, myocardial insulin resistance and myostatin

Objective Myostatin (MSTN) is a member of the TGF-β superfamily and is a negative regulator of skeletal muscle growth. MSTN is also expressed in other tissues such as heart and fat. The purpose of this study was to elucidate the occurrence of myocardial IR caused by myocardial MSTN expression, and to elucidate the regulation of MSTN expression by exercise and improve myocardial IR status and myocardial interstitial fibrosis.&#x0D; Methods The literatures on exercise, myocardial IR, and myocardial MSTN published in recent years were reviewed and summarized.&#x0D; Results 1.Myostatin, which is rarely expressed in myocardium under normal conditions, the appropriate amount of MSTN expression has anti-cardiac hypertrophy, and excessive MSTN expression can cause various cardiovascular diseases. In the IR state, the expression of myocardial inflammatory factors is increased, and MSTN is highly expressed. MSTN acts on the myocardium through various pathways, leading to the occurrence of myocardial fibrosis. In the IR state, possible pathways for inducing high expression of MSTN include: (1) inflammatory factor TNF-α is elevated, and MSTN expression is induced by P38MAPK and NF-KB; (2) The transcription factor Smad-2/3/4 up-regulates the MSTN promoter activity; (3) Glucocorticoid up-regulates MSTN expression; (4) IGF-1 up-regulates MSTN expression via p38MAPK and transcription factor MEF-2; (5) AngII up-regulates MSTN via Ang1 receptor, p38MAPK and MEF-2;(6) urotensin-II and urocortin can stimulate MSTN expression;(7) The ERK-1/2-MEF-2 pathway up-regulates MSTN expression. 3. Possible pathways by which MSTN acts on the myocardium include:(1) MSTN induces Smad2/3 overexpression; (2) MSTN activates the NF-KB pathway; (3) MSTN activates miR-1 expression, inhibits HSP70, and decreases pAkt levels; (4) MSTN inhibits the PI3K-Akt pathway, leading to induction or damage of the downstream pathway. Regular endurance exercise and resistance exercise improve myocardial IR and myocardial interstitial fibrosis involved in MSTN. This effect is mainly dependent on exercise can effectively improve the body's chronic inflammatory state, down-regulate the MSTN superior inducer, resulting in a series of cascade reactions.&#x0D; Conclusions 1.MSTN expression in myocardial tissue is dose-dependent,proper expression of MSTN in the myocardium can prevent cardiac hypertrophy, and overexpression of MSTN can lead to various cardiovascular diseases. 2.Under the hyperglycemia and inflammation state, it can induce high expression of MSTN. After high expression of myocardial MSTN, it can participate in myocardial IR and can lead to myocardial interstitial fibrosis. 3. Exercise can effectively improve the chronic inflammatory state of the body, down-regulate the level of MSTN induction, improve myocardial IR status and IR-induced myocardial interstitial fibrosis.&#x0D;

Viral infection upregulates myostatin promoter activity in orange-spotted grouper (Epinephelus coioides).

Myostatin is a negative regulator of myogenesis and has been suggested to be an important factor in the development of muscle wasting during viral infection. The objective of this study was to characterize the main regulatory element of the grouper myostatin promoter and to study changes in promoter activity due to viral stimulation. In vitro and in vivo experiments indicated that the E-box E6 is a positive cis-and trans-regulation motif, and an essential binding site for MyoD. In contrast, the E-box E5 is a dominant negative cis-regulatory. The characteristics of grouper myostatin promoter are similar in regulation of muscle growth to that of other species, but mainly through specific regulatory elements. According to these results, we conducted a study to investigate the effect of viral infection on myostatin promoter activity and its regulation. The nervous necrosis virus (NNV) treatment significantly induced myostatin promoter activity. The present study is the first report describing that specific myostatin motifs regulate promoter activity and response to viral infection.

Glucocorticoid‐induced inhibition of AKT leads to CREB phosphorylation and increased myostatin expression via a PDE/cAMP/PKA pathway in skeletal muscle

Muscle atrophy in diseases like chronic kidney disease and diabetes is associated with dysfunctional regulation of insulin/Akt/FoxO3 and myostatin‐SMAD3 signaling pathways. We recently found CREB, a transcription factor that regulates maintenance and regeneration of skeletal muscle, is phosphorylated in wasting conditions. In silico analysis identifies a CREB binding site in the myostatin promoter. This study investigated the mechanism by which atrophy‐inducing conditions regulate CREB phosphorylation and its impact on myostatin expression. Differentiated C2C12 myotubes were incubated with combinations of dexamethasone (Dex), LY294002 (PI‐3 kinase inhibitor), H89 (protein kinase A inhibitor), isobutylmethyl xanthine (IBMX, a general phosphodiesterase (PDE) inhibitor), milrinone (PDE3 inhibitor), or rolipram (PDE4 inhibitor) for up to 24 h. Cells were harvested for analysis of proteins or mRNAs. Treatment of myotubes with Dex for 6–24h increased CREB phosphorylation (pCREB) within 3 h and sustained the increase over 24h. Dex also inhibited Akt phosphorylation (pAkt) over a similar time frame. Inhibition of pAkt by LY294002 mimicked Dex by increasing pCREB; the actions of LY294002 and Dex on pCREB were not additive. Since Akt can activate PDE3/PDE4 and reduce cAMP/PKA signaling in non‐muscle cells, we tested whether Dex‐induced inhibition of PDE3/4 increases pCREB. Without Dex, IBMX, milrinone, and rolipram increased pCREB. The Dex‐induced increase in pCREB was attenuated by the PKA inhibitor H89. When added individually, Dex, milrinone, rolipram and IBMX increase myostatin mRNA and protein significantly. Our study identifies a new mechanism by which atrophy‐inducing conditions activate CREB and contribute to myostatin expression. Signals that attenuate insulin/Akt signaling reduce the PDE3/4 activity. This leads to an increase in cAMP, PKA activity, CREB phosphorylation and myostatin expression.Support or Funding InformationFunded by NIH RO1 DK95610 and VA Merit X01BX001456

207 EFFICIENT GENERATION OF MYOSTATIN PROMOTER MUTATIONS IN BOVINE EMBRYOS USING THE CRISPR/Cas9 SYSTEM

The myostatin gene or growth differentiation factor 8 is a member of the transforming growth factor-β superfamily that acts as a negative regulator of muscle growth. Mutations inactivating this gene occur naturally in Piedmontese and Belgian Blue cattle breeds, resulting in a dramatic increase in muscle mass, albeit with unwanted consequences of increased dystocia and decreased fertility. Modulation of muscle mass increase without the unwanted effects would be of great value for improving livestock growth and economic value of livestock. The objective of our work was to use the CRISPR-Cas9 genetic engineering tool to generate deletions of different elements in the myostatin promoter in order to decrease the level of expression and obtain an attenuated phenotype without the detrimental consequences of an inactivating mutation. To achieve this objective 4 different small guide RNA (sgRNA) targeting the promoter near the mutation were designed with PAM positions from transcription starting site of −1577, −689, −555, and −116. These sgRNA were cloned individually into the Cas9 plasmids (px461, and px462; Addgene®). These plasmids allow for a dual puromycin resistance (px462) and green fluorescent protein (px461) selection. We first tested the functionality of these sgRNA in vitro by co-transfecting bovine fetal fibroblasts with a combination of both plasmids (Set 1 = sgRNA 1–4; Set 2 = sgRNA 2–3). Cells were exposed to puromycin (0.2 µg mL−1) for 72 h, then single and mixed colonies positive for green fluorescent protein expression were separated for propagation. The DNA was extracted for PCR amplification of the targeted region. Multiple deletions and a few insertion events were observed after PCR, bands were cloned into TOPO® vector (Thermo Fisher Scientific, Waltham, MA, USA) and sequenced. Sequencing results confirmed the PCR products as insertions or deletions in the myostatin promoter region. We proceeded to modify the myostatin promoter directly in bovine zygotes. For this, IVF-derived zygotes were randomly assigned to 3 different treatment groups Set 1, Set 2, or Null (no sgRNA) for microinjections. Each zygote was injected with ~100 pL of trophectoderm buffer containing 50 ng µL−1 of total sgRNA, 10 ng µL−1 of Cas9 mRNA, and 30 ng µL−1 of Cas9 protein with 1 mg mL−1 of fluorescent dextran. Day 7 post-IVF blastocysts were lysed and DNA was extracted for PCR amplification of the target region. In Set 1, 16 of 19 embryos (94.12%) were successfully edited, whereas in Set 2 there were 11 of 17 embryos (64.7%) edited. In both sets of sgRNA there was a high degree of mosaicism, with only 1 embryo demonstrating a homozygous deletion. In conclusion, CRISPR/Cas9 acts over the course of the first few cleavage divisions Further research is necessary to refine the CRISPR/Cas9 system for inducing genetic mutations in bovine embryos.

Suppression of muscle wasting by the plant-derived compound ursolic acid in a model of chronic kidney disease.

BackgroundMuscle wasting in chronic kidney disease (CKD) and other catabolic disorders contributes to morbidity and mortality, and there are no therapeutic interventions that regularly and safely block losses of muscle mass. We have obtained evidence that impaired IGF‐1/insulin signalling and increases in glucocorticoids, myostatin and/or inflammatory cytokines that contribute to the development of muscle wasting in catabolic disorders by activating protein degradation.MethodsUsing in vitro and in vivo models of muscle wasting associated with CKD or dexamethasone administration, we measured protein synthesis and degradation and examined mechanisms by which ursolic acid, derived from plants, could block the loss of muscle mass stimulated by CKD or excessive levels of dexamethasone.ResultsUsing cultured C2C12 myotubes to study muscle wasting, we found that exposure to glucocorticoids cause loss of cell proteins plus an increase in myostatin; both responses are significantly suppressed by ursolic acid. Results from promoter and ChIP assays demonstrated a mechanism involving ursolic acid blockade of myostatin promoter activity that is related to CEBP/δ expression. In mouse models of CKD‐induced or dexamethasone‐induced muscle wasting, we found that ursolic acid blocked the loss of muscle mass by stimulating protein synthesis and decreasing protein degradation. These beneficial responses included decreased expression of myostatin and inflammatory cytokines (e.g. TGF‐β, IL‐6 and TNFα), which are initiators of muscle‐specific ubiquitin‐E3 ligases (e.g. Atrogin‐1, MuRF‐1 and MUSA1).ConclusionsUrsolic acid improves CKD‐induced muscle mass by suppressing the expression of myostatin and inflammatory cytokines via increasing protein synthesis and reducing proteolysis.

Polymorphism of Myostatin (MSTN) Promoter Gene and its Association with Growth and Muscling Traits in Bali Cattle

Myostatin (MSTN) gene plays a key role in skletal muscle homeostasis such as inducing muscle athrophy, poliferation of myoblast, increasing ubiquitin-proteasomal, downregulating IGF pathway, and glucolysis. Myostatin gene expression is controled by CpG island located in promoter region. The objectives of this research were to identify polymorphism of MSTN promoter gene and to associate the polymorphism of SNP with growth and muscling traits in Bali cattle. A total of 48 Bali cattle from BPTU-HMT Bali island was screened to identify genetic polymorphisms in MSTN promoter region using sequencing method. The growth and muscling traits were measured at 12 months of age. The muscling traits were evaluated using ultrasound console with linear transducer having frequency 6.5 Hz and scaning we conducted at 130 mm in deep. Analysis of polymorphism was conducted by using PopGen 1.32 software. The association of MSTN with growth and muscling traits were analyzed by using General Linear Model (GLM) procedure. This result showed that a total of 20 polymorphic SNPs (seven SNPs in CpG island) were detected in this region. Although, only 3 SNPs (g.-8078C>T, g.-7996G>C, and g.-7930A>G) had equilibrium condition in Hardy-Weinberg analysis. The association result showed that 2 SNPs (g.-7799T>C and g.-7941C>T) were significantly associated with intramuscular fat percentage (P≤0.05) in Bali cattle. Although the 2 SNPs were nominally significant at nominal P≤0.05 threshold, they were not significant after Bonferroni correction for multiple testing. It could be concluded that MSTN promoter gene was polymorphic in Bali cattle and there were 2 SNPs associated with carcass quality.

Nfix Regulates Temporal Progression of Muscle Regeneration through Modulation of Myostatin Expression.

SummaryNfix belongs to a family of four highly conserved proteins that act as transcriptional activators and/or repressors of cellular and viral genes. We previously showed a pivotal role for Nfix in regulating the transcriptional switch from embryonic to fetal myogenesis. Here, we show that Nfix directly represses the Myostatin promoter, thus controlling the proper timing of satellite cell differentiation and muscle regeneration. Nfix-null mice display delayed regeneration after injury, and this deficit is reversed upon in vivo Myostatin silencing. Conditional deletion of Nfix in satellite cells results in a similar delay in regeneration, confirming the functional requirement for Nfix in satellite cells. Moreover, mice lacking Nfix show reduced myofiber cross sectional area and a predominant slow twitching phenotype. These data define a role for Nfix in postnatal skeletal muscle and unveil a mechanism for Myostatin regulation, thus providing insights into the modulation of its complex signaling pathway.

Construction of a new T-vector: Nickase (Nt.BspQI)-generated T-vector bearing a reddish-orange indicator gene.

T-vectors are widely used for cloning the polymerase chain reaction (PCR) products. However, the low conversion efficiency of a plasmid into the linear T-vector usually results in non-recombinants. Here, we designed a new plasmid pNBQ-T to easily select the recombinant colonies harboring PCR products. pNBQ-T plasmid, which contains a DsRed indicator gene between two Nt.BspQI restriction cassettes, each of which contains palindromic sequences susceptible to Nt.BspQI nickase (5'-GCTCTTCT^GAAGAGC-3') at each end. Thus, this plasmid can be easily converted into T-vectors by a nickase (quadruple nicking), which results in two double strand breaks with 3'-thymidine overhangs. DsRed indicator gene, which is inserted between the restriction sites, helps identifying the PCR recombinants. Using this pNBQ-T plasmid the insertion efficiency of a PCR product was examined. We successfully identified white colony of the recombinants with the inserted myostatin promoter gene: the cloning efficiency was 93%. Therefore, this simple method utilizing pNBQ-T plasmid will serve as a useful and efficient technique for preparation of home-made T-vectors.

Genome-wide evolutionary and functional analysis of the Equine Repetitive Element 1: an insertion in the myostatin promoter affects gene expression.

BackgroundIn mammals, an important source of genomic variation is insertion polymorphism of retrotransposons. These may acquire a functional role when inserted inside genes or in their proximity. The aim of this work was to carry out a genome wide analysis of ERE1 retrotransposons in the horse and to analyze insertion polymorphism in relation to evolution and function. The effect of an ERE1 insertion in the promoter of the myostatin gene, which is involved in muscle development, was also investigated.ResultsIn the horse population, the fraction of ERE1 polymorphic loci is related to the degree of similarity to their consensus sequence. Through the analysis of ERE1 conservation in seven equid species, we established that the level of identity to their consensus is indicative of evolutionary age of insertion. The position of ERE1s relative to genes suggests that some elements have acquired a functional role. Reporter gene assays showed that the ERE1 insertion within the horse myostatin promoter affects gene expression. The frequency of this variant promoter correlates with sport aptitude and racing performance.ConclusionsSequence conservation and insertion polymorphism of ERE1 elements are related to the time of their appearance in the horse lineage, therefore, ERE1s are a useful tool for evolutionary and population studies. Our results suggest that the ERE1 insertion at the myostatin locus has been unwittingly selected by breeders to obtain horses with specific racing abilities. Although a complex combination of environmental and genetic factors contributes to athletic performance, breeding schemes may take into account ERE1 insertion polymorphism at the myostatin promoter.Electronic supplementary materialThe online version of this article (doi:10.1186/s12863-015-0281-1) contains supplementary material, which is available to authorized users.

Rosa26 locus supports tissue-specific promoter driving transgene expression specifically in pig.

Genetically modified pigs have become a popular model system in fundamental research, agricultural and biomedical applications. However, random integration often result in unstable expression of transgene and unpredictable phenotypes. The Rosa26 locus has been widely used to produce genetic modified animals with high and consistent expressing of transgene in mouse, human and rat, as it can be targeted efficiently and is not subject to gene-silencing effects. Recently, the first case of reporter gene targeting pigs in porcine Rosa26 (pRosa26) locus was reported. In the study, full sequence of pRosa26 locus was further characterized, and the pRosa26 promoter (pR26) was cloned and we evidenced that the new porcine endogenous promoter is suitable for driving transgene expression in a high and stable manner by avoiding DNA methylation. Furthermore, elongation factor 1a promoter (EF1a) -driven GFP reporter and Myostatin promoter (MyoP)-driven Follistatin (Fst) were successfully targeted into the pRosa26 locusby traditional homologous recombination (HR) strategy. EF1a showed high activity and hypomethylation at the locus. And, muscle-specific promoter MyoP was activated strictly in muscle of the pRosa26 targeted pigs, indicating Rosa26 locus supports tissue-specific promoter driving transgene expression in its own manner. The study provided further demonstration on biomedical and agricultural applications of porcine Rosa26 promoter and locus.

The cAMP Response Element Binding protein (CREB) is activated by Insulin-like Growth Factor-1 (IGF-1) and regulates myostatin gene expression in skeletal myoblast

Myostatin, a member of the Transforming Growth Factor beta (TGF-β) superfamily, plays an important role as a negative regulator of skeletal muscle growth and differentiation. We have previously reported that IGF-1 induces a transient myostatin mRNA expression, through the activation of the Nuclear Factor of Activated T cells (NFAT) in an IP3/calcium-dependent manner. Here we examined the activation of CREB transcription factor as downstream targets of IGF-1 during myoblast differentiation and its role as a regulator of myostatin gene expression.In cultured skeletal myoblast, IGF-1 induced the phosphorylation and transcriptional activation of CREB via IGF-1 Receptor/Phosphatidylinositol 3-Kinase (PI3K)/Phospholipase C gamma (PLC γ), signaling pathways. Also, IGF-1 induced calcium-dependent molecules such as Calmodulin Kinase II (CaMK II), Extracellular signal-regulated Kinases (ERK), Protein Kinase C (PKC). Additionally, we examined myostatin mRNA levels and myostatin promoter activity in differentiated myoblasts stimulated with IGF-1. We found a significant increase in mRNA contents of myostatin and its reporter activity after treatment with IGF-1. The expression of myostatin in differentiated myoblast was downregulated by the transfection of siRNA–CREB and by pharmacological inhibitors of the signaling pathways involved in CREB activation. By using pharmacological and genetic approaches together these data demonstrate that IGF-1 regulates the myostatin gene expression via CREB transcription factor during muscle cell differentiation.

Single nucleotide polymorphisms in the upstream regulatory region alter the expression of myostatin

The expression of the gene encoding myostatin (MSTN), the product of which is a negative regulator of skeletal muscle growth and development in mammals, is regulated by many cis-regulatory elements, including enhancer box (E-box) motifs. While E-box motif mutants of MSTN exhibit altered expression of myostatin in many animal models, the phenotypes of these mutations in chicken are not investigated. In this study, we cloned and sequenced the full encoded DNA sequence of MSTN gene and its upstream promoter region in Wenshang Luhua chicken breed. After analysis of the sequence, 13 E-box motifs were identified in the MSTN promoter region, which were denoted by E1 to E13 according to their positions in the region. Although many single nucleotide polymorphisms (SNPs) were revealed in the MSTN promoter region, only two SNPs were in the E-boxes, i.e., the first nucleotide of the E3 and the fifth nucleotide of E4. The effects of these two polymorphisms on the expression of MSTN gene were explored both with MSTN-GFP reporter constructs in vitro and real-time PCR in vivo. The results suggested that the E-boxes in the chicken MSTN promoter region are involved in the regulation of myostatin expression and the polymorphisms in E3 and E4 altered the expression of myostatin.

Comparative analysis of myostatin gene and promoter sequences of Qinchuan and Red Angus cattle

To better understand the function of the myostatin gene and its promoter region in bovine, we amplified and sequenced the myostatin gene and promoter from the blood of Qinchuan and Red Angus cattle by using polymerase chain reaction. The sequences of Qinchuan and Red Angus cattle were compared with those of other cattle breeds available in GenBank. Exon splice sites were confirmed by mRNA sequencing. Compared to the published sequence (GenBank accession No. AF320998), 69 single nucleotide polymorphisms (SNPs) were identified in the Qinchuan myostatin gene, only one of which was an insertion mutation in Qinchuan cattle. There was a 16-bp insertion in the first 705-bp intron in 3 Qinchuan cattle. A total of 7 SNPs were identified in exon 3, in which the mutation occurred in the third base of the codon and was synonymous. On comparing the Qinchuan myostatin gene sequence to that of Red Angus cattle, a total of 50 SNPs were identified in the first and third exons. In addition, there were 18 SNPs identified in the Qinchuan cattle promoter region compared with those of other cattle compared to the Red Angus cattle myostatin promoter region. breeds (GenBank accession No. AF348479), but only 14 SNPs when compared to the Red Angus cattle myostatin promoter region.

Sulforaphane causes a major epigenetic repression of myostatin in porcine satellite cells

Satellite cells function as skeletal muscle stem cells to support postnatal muscle growth and regeneration following injury or disease. There is great promise for the improvement of muscle performance in livestock and for the therapy of muscle pathologies in humans by the targeting of myostatin (MSTN) in this cell population. Human diet contains many histone deacetylase (HDAC) inhibitors, such as the bioactive component sulforaphane (SFN), whose epigenetic effects on MSTN gene in satellite cells are unknown. Therefore, we aimed to investigate the epigenetic influences of SFN on the MSTN gene in satellite cells. The present work provides the first evidence, which is distinct from the effects of trichostatin A (TSA), that SFN supplementation in vitro not only acts as a HDAC inhibitor but also as a DNA methyltransferase (DNMT) inhibitor in porcine satellite cells. Compared with TSA and 5-aza-2′-deoxycytidine (5-aza-dC), SFN treatment significantly represses MSTN expression, accompanied by strongly attenuated expression of negative feedback inhibitors of the MSTN signaling pathway. miRNAs targeting MSTN are not implicated in posttranscriptional regulation of MSTN. Nevertheless, a weakly enriched myoblast determination (MyoD) protein associated with diminished histone acetylation in the MyoD binding site located in the MSTN promoter region may contribute to the transcriptional repression of MSTN by SFN. These findings reveal a new mode of epigenetic repression of MSTN by the bioactive compound SFN. This novel pharmacological, biological activity of SFN in satellite cells may thus allow for the development of novel approaches to weaken the MSTN signaling pathway, both for therapies of human skeletal muscle disorders and for livestock production improvement.