We used a combination of genome-wide and promoter-specific DNA binding and expression analyses to assess the functional roles of Myod and Myog in regulating the program of skeletal muscle gene expression. Our findings indicate that Myod and Myog have distinct regulatory roles at a similar set of target genes. At genes expressed throughout the program of myogenic differentiation, Myod can bind and recruit histone acetyltransferases. At early targets, Myod is sufficient for near full expression, whereas, at late expressed genes, Myod initiates regional histone modification but is not sufficient for gene expression. At these late genes, Myog does not bind efficiently without Myod; however, transcriptional activation requires the combined activity of Myod and Myog. Therefore, the role of Myog in mediating terminal differentiation is, in part, to enhance expression of a subset of genes previously initiated by Myod.

BACKGROUND

Dramatic changes in gene expression occur in response to extracellular stimuli and during differentiation. Although transcriptional effects are important, alterations in mRNA decay also play a major role in achieving rapid and massive changes in mRNA abundance. Moreover, just as transcription factor activity varies between different cell types, the factors influencing mRNA decay are also cell-type specific.

RESULTS

We have established the rates of decay for over 7000 transcripts expressed in mouse C2C12 myoblasts. We found that GU-rich (GRE) and AU-rich (ARE) elements are over-represented in the 3'UTRs of short-lived mRNAs and that these mRNAs tend to encode factors involved in cell cycle and transcription regulation. Stabilizing elements were also identified. By comparing mRNA decay rates in C2C12 cells with those previously measured for pluripotent and differentiating embryonic stem (ES) cells, we identified several groups of transcripts that exhibit cell-type specific decay rates. Further, whereas in C2C12 cells the impact of GREs on mRNA decay appears to be greater than that of AREs, AREs are more significant in ES cells, supporting the idea that cis elements make a cell-specific contribution to mRNA stability. GREs are recognized by CUGBP1, an RNA-binding protein and instability factor whose function is affected in several neuromuscular diseases. We therefore utilized RNA immunoprecipitation followed by microarray (RIP-Chip) to identify CUGBP1-associated transcripts. These mRNAs also showed dramatic enrichment of GREs in their 3'UTRs and encode proteins linked with cell cycle, and intracellular transport. Interestingly several CUGBP1 substrate mRNAs, including those encoding the myogenic transcription factors Myod1 and Myog, are also bound by the stabilizing factor HuR in C2C12 cells. Finally, we show that several CUGBP1-associated mRNAs containing 3'UTR GREs, including Myod1, are stabilized in cells depleted of CUGBP1, consistent with the role of CUGBP1 as a destabilizing factor.

CONCLUSIONS

Taken together, our results systematically establish cis-acting determinants of mRNA decay rates in C2C12 myoblast cells and demonstrate that CUGBP1 associates with GREs to regulate decay of a wide range of mRNAs including several that are critical for muscle development.

Recent studies have demonstrated the effect of matrix stiffness on the phenotype and differentiation pathway of mesenchymal stem cells (MSCs). MSCs differentiated into neural, myogenic or osteogenic phenotypes depending on whether they were cultured on two-dimensional (2D) substrates of elastic moduli in the lower (0.1-1 kPa), intermediate (8-17 kPa) or higher ranges (34 kPa). In this study, MSCs were cultured in thixotropic gels of varying rheological properties, and similar results were found for the three-dimensional (3D) culture as for the previous findings in 2D culture. For the 3D cell cultures in thixotropic gels, the liquefaction stress (tau(y)), the minimum shear stress required to liquefy the gel, was used to characterize the matrix stiffness. The highest expressions of neural (ENO2), myogenic (MYOG) and osteogenic (Runx2, OC) transcription factors were obtained for gels with tau(y) of 7, 25 and 75 Pa, respectively. Immobilization of the cell-adhesion peptide, RGD, promoted both proliferation and differentiation of MSCs, especially for the case of the stiffer gels (>75 Pa). This study demonstrated the usefulness of thixotropic gels for 3D cell culture studies, as well as the use of tau(y) as an effective measure of matrix stiffness that could be correlated to MSC differentiation.

The basic helix-loop-helix (bHLH) transcription factor Myod directly regulates gene expression throughout the program of skeletal muscle differentiation. It is not known how a Myod-driven myogenic program is modulated to achieve muscle fiber-type-specific gene expression. Pbx homeodomain proteins mark promoters of a subset of Myod target genes, including myogenin (Myog); thus, Pbx proteins might modulate the program of myogenesis driven by Myod. By inhibiting Pbx function in zebrafish embryos, we show that Pbx proteins are required in order for Myod to induce the expression of a subset of muscle genes in the somites. In the absence of Pbx function, expression of myog and of fast-muscle genes is inhibited, whereas slow-muscle gene expression appears normal. By knocking down Pbx or Myod function in combination with another bHLH myogenic factor, Myf5, we show that Pbx is required for Myod to regulate fast-muscle, but not slow-muscle, development. Furthermore, we show that Sonic hedgehog requires Myod in order to induce both fast- and slow-muscle markers but requires Pbx only to induce fast-muscle markers. Our results reveal that Pbx proteins modulate Myod activity to drive fast-muscle gene expression, thus showing that homeodomain proteins can direct bHLH proteins to establish a specific cell-type identity.

In addition to generating movement, skeletal muscle may have a function as a secretory organ. The aim of the present study was to identify novel proteins with signaling capabilities secreted from skeletal muscle cells. IL-7 was detected in media conditioned by primary cultures of human myotubes differentiated from satellite cells, and concentrations increased with incubation time. By immunoblotting and real-time RT-PCR IL-7 expression was confirmed at both protein and mRNA levels. Furthermore, with immunofluorescence and specific antisera, multinucleated myotubes were found to coexpress IL-7 and myosin heavy chain. During differentiation of human myotubes from satellite cells, IL-7 expression increased at mRNA and protein levels. In contrast, mRNA expression of the IL-7 receptor was 80% lower in myotubes compared with satellite cells. Incubations with recombinant IL-7 under differentiation conditions caused approximately 35% reduction in mRNA for the terminal myogenic markers myosin heavy chain 2 (MYH2) and myogenin (MYOG), suggesting that IL-7 may act on satellite cells to inhibit development of the muscle fiber phenotype. Alternative routes of cell development were investigated, and IL-7 increased migration of satellite cells by 40% after 48 h in a Transwell system, whereas cell proliferation remained unchanged. In vivo, real-time RT-PCR analysis of musculus vastus lateralis (n = 10) and musculus trapezius (n = 7) biopsies taken from male individuals undergoing a strength training program demonstrated that after 11 wk mean IL-7 mRNA increased by threefold (P = 0.01) and fourfold (P = 0.04), respectively. In conclusion, we have demonstrated that IL-7 is a novel myokine regulated both in vitro and in vivo, and it may play a role in the regulation of muscle cell development.

BACKGROUND

Polycomb group (PcG) genes code for chromatin multiprotein complexes that are responsible for maintaining gene silencing of transcriptional programs during differentiation and in adult tissues. Despite the large amount of information on PcG function during development and cell identity homeostasis, little is known regarding the dynamics of PcG complexes and their role during terminal differentiation.

RESULTS

We show that two distinct polycomb repressive complex (PRC)2 complexes contribute to skeletal muscle cell differentiation: the PRC2-Ezh2 complex, which is bound to the myogenin (MyoG) promoter and muscle creatine kinase (mCK) enhancer in proliferating myoblasts, and the PRC2-Ezh1 complex, which replaces PRC2-Ezh2 on MyoG promoter in post-mitotic myotubes. Interestingly, the opposing dynamics of PRC2-Ezh2 and PRC2-Ezh1 at these muscle regulatory regions is differentially regulated at the chromatin level by Msk1 dependent methyl/phospho switch mechanism involving phosphorylation of serine 28 of the H3 histone (H3S28ph). While Msk1/H3S28ph is critical for the displacement of the PRC2-Ezh2 complex, this pathway does not influence the binding of PRC2-Ezh1 on the chromatin. Importantly, depletion of Ezh1 impairs muscle differentiation and the chromatin recruitment of MyoD to the MyoG promoter in differentiating myotubes. We propose that PRC2-Ezh1 is necessary for controlling the proper timing of MyoG transcriptional activation and thus, in contrast to PRC2-Ezh2, is required for myogenic differentiation.

CONCLUSIONS

Our data reveal another important layer of epigenetic control orchestrating skeletal muscle cell terminal differentiation, and introduce a novel function of the PRC2-Ezh1 complex in promoter setting.

Although the mechanisms regulating the formation of embryonic skeletal muscle in vertebrates are well characterized, less is known about postnatal muscle formation even though the largest increases in skeletal muscle mass occur after birth. Adult muscle stem cells (satellite cells) appear to recapitulate the events that occur in embryonic myoblasts. In particular, the myogenic basic helix-loop-helix factors, which have crucial functions in embryonic muscle development, are assumed to have similar roles in postnatal muscle formation. Here, we test this assumption by determining the role of the myogenic regulator myogenin in postnatal life. Because Myog-null mice die at birth, we generated mice with floxed alleles of Myog and mated them to transgenic mice expressing Cre recombinase to delete Myog before and after embryonic muscle development. Removing myogenin before embryonic muscle development resulted in myofiber deficiencies identical to those observed in Myog-null mice. However, mice in which Myog was deleted following embryonic muscle development had normal skeletal muscle, except for modest alterations in the levels of transcripts encoding Mrf4 (Myf6) and Myod1 (MyoD). Notably, Myog-deleted mice were 30% smaller than control mice, suggesting that the absence of myogenin disrupted general body growth. Our results suggest that postnatal skeletal muscle growth is controlled by mechanisms distinct from those occurring in embryonic muscle development and uncover an unsuspected non-cell autonomous role for myogenin in the regulation of tissue growth.

We have devised a PCR-based sexing method that is quick, simple, and highly reproducible. DNA is first extracted from embryonic mouse yolk sac via a 15 min, two-step incubation procedure utilizing PCR-compatible proteinase K buffer. Without any further manipulation the lysate is subjected to 30 cycles of PCR, optimized to run in less than 1 hr. The reaction includes multiplexed primer pairs for Sry and Myog (myogenin) that generate a male specific band of 441 bp and an internal control band of 245 bp, respectively. This robust method is used routinely in our laboratory and gives rapid genotyping results with 98% reliability and 100% accuracy.

Mesenchymal stem cells from adipose tissue can differentiate into mesodermal lineages. Differentiation potential, however, varies between clones of adipose stem cells (ASCs), raising the hypothesis that epigenetic differences account for this variability. We report here a bisulfite sequencing analysis of CpG methylation of adipogenic (leptin [LEP], peroxisome proliferator-activated receptor gamma 2 [PPARG2], fatty acid-binding protein 4 [FABP4], and lipoprotein lipase [LPL]) promoters and of nonadipogenic (myogenin [MYOG], CD31, and GAPDH) loci in freshly isolated human ASCs and in cultured ASCs, in relation to gene expression and differentiation potential. Uncultured ASCs display hypomethylated adipogenic promoters, in contrast to myogenic and endothelial loci, which are methylated. Adipogenic promoters exhibit mosaic CpG methylation, on the basis of heterogeneous methylation between cells and of variation in the extent of methylation of a given CpG between donors, and both between and within clonal cell lines. DNA methylation reflects neither transcriptional status nor potential for gene expression upon differentiation. ASC culture preserves hypomethylation of adipogenic promoters; however, between- and within-clone mosaic methylation is detected. Adipogenic differentiation also maintains the overall CpG hypomethylation of LEP, PPARG2, FABP4, and LPL despite demethylation of specific CpGs and transcriptional induction. Furthermore, enhanced methylation at adipogenic loci in primary differentiated cells unrelated to adipogenesis argues for ASC specificity of the hypomethylated state of these loci. Therefore, mosaic hypomethylation of adipogenic promoters may constitute a molecular signature of ASCs, and DNA methylation does not seem to be a determinant of differentiation potential of these cells.

Mitochondrial biogenesis and mitophagy are recognized as critical processes underlying mitochondrial homeostasis. However, the molecular pathway(s) coordinating the balance between these cellular programs is still poorly investigated. Here, we show an induction of the nuclear and mitochondrial peroxisome proliferator-activated receptor gamma, coactivator 1 alpha (PGC-1α) during myogenesis, which in turn co-activates the transcription of nuclear and mtDNA-encoded mitochondrial genes. We demonstrate that PGC-1α also buffers oxidative stress occurring during differentiation by promoting the expression of antioxidant enzymes. Indeed, by downregulating PGC-1α, we observed an impairment of antioxidants expression, which was accompanied by a significant reactive oxygen species (ROS) burst and increase of oxidative damage to proteins. In parallel, we detected a decrease of mitochondrial mass and function as well as increased mitophagy through the ROS/FOXO1 pathway. Upon PGC-1α downregulation, we found ROS-dependent nuclear translocation of FOXO1 and transcription of its downstream targets including mitophagic genes such as LC3 and PINK1. Such events were significantly reverted after treatment with the antioxidant Trolox, suggesting that PGC-1α assures mitochondrial integrity by indirectly buffering ROS. Finally, the lack of PGC-1α gave rise to a decrease in MYOG and a strong induction of atrophy-related ubiquitin ligases FBXO32 (FBXO32), indicative of a degenerative process. Overall, our results reveal that in myotubes, PGC-1α takes center place in mitochondrial homeostasis during differentiation because of its ability to avoid ROS-mediated removal of mitochondria.

MyoD is a master regulator of the skeletal muscle gene expression program. ChIP-Seq analysis has recently revealed that MyoD binds to a large number of genomic loci in differentiating myoblasts, yet only activates transcription at a subset of these genes. Here we discuss recent data suggesting that the ability of MyoD to mediate gene expression is regulated through the function of Polycomb and Trithorax Group proteins. Based on studies of the muscle-specific myog gene, we propose a model where the transcriptional activators Mef2d and Six4 mediate recruitment of Trithorax Group proteins Ash2L/MLL2 and UTX to MyoD-bound promoters to overcome the Polycomb-mediated repression of muscle genes. Modulation of the interaction between Ash2L/MLL2 and Mef2d by the p38α MAPK signaling pathway in turns provides fine-tuning of the muscle-specific gene expression program. Thus Mef2d, Six4, and p38α MAPK function coordinately as regulators of a master regulator to mediate expression of MyoD target genes.

OBJECTIVE

PITX2 gene dose plays a central role in Axenfeld-Rieger syndrome. The purpose of this study was to test the hypothesis that the effects of Pitx2 gene dose on eye development can be molecularly dissected in available Pitx2 mutant mice.

METHODS

A panel of mice with Pitx2 gene dose ranging from wild-type (+/+) to none (-/-) was generated. Eye morphogenesis was assessed in animals with each Pitx2 gene dose. We also compared global gene expression in eye primordia taken from e12.5 Pitx2+/+, Pitx2+/-, Pitx2-/- embryos using gene microarrays. The validity of microarray results was confirmed by qRT-PCR.

RESULTS

Morphogenesis of all extraocular muscle bundles correlated highly with Pitx2 gene dose, but there were some differences in sensitivity among muscle groups. Superior and inferior oblique muscles were most sensitive and disappeared before the four rectus muscles. Expression of muscle-specific genes was globally sensitive to Pitx2 gene dose, including the muscle-specific transcription factor genes Myf5, Myog, Myod1, Smyd1, Msc, and Csrp3.

CONCLUSIONS

Pitx2 gene dose regulates both morphogenesis and gene expression in developing extraocular muscles. The expression of key muscle-specific transcription factor genes is regulated by Pitx2 gene dose, suggesting that sufficient levels of PITX2 protein are essential for early initiation of the myogenic regulatory cascade in extraocular muscles. These results document the first ocular tissue affected by Pitx2 gene dose in a model organism, where the underlying mechanisms can be analyzed, and provide a paradigm for future experiments designed to elucidate additional effects of Pitx2 gene dose during eye development.

In contrast to the detailed understanding we have for the regulation of skeletal muscle gene expression in embryos, similar insights into postnatal muscle growth and regeneration are largely inferential or do not directly address gene regulatory mechanisms. Muscle stem cells (satellite cells) are chiefly responsible for providing new muscle during postnatal and adult life. The purpose of this study was to determine the role that the myogenic basic helix-loop-helix regulatory factor myogenin has in postnatal muscle growth and adult muscle stem cell gene expression. We found that myogenin is absolutely required for skeletal muscle development and survival until birth, but it is dispensable for postnatal life. However, Myog deletion after birth led to reduced body size implying a role for myogenin in regulating body homeostasis. Despite a lack of skeletal muscle defects in Myog-deleted mice during postnatal life and the efficient differentiation of cultured Myog-deleted adult muscle stem cells, the loss of myogenin profoundly altered the pattern of gene expression in cultured muscle stem cells and adult skeletal muscle. Remarkably, these changes in gene expression were distinct from those found in Myog-null embryonic skeletal muscle, indicating that myogenin has separate functions during postnatal life.

The muscle-specific basic helix-loop-helix proteins MyoD, Myf5, myogenin (Myog) and MRF4 constitute the myogenic regulatory factor (MRF) family of transcription factors that drive muscle gene expression during myogenesis. Having evolved from a single ancestral gene, the spatial and temporal specificity of expression for each family member has been used to define a hierarchical relationship between the four MRFs. Molecular characterization of two of the MRFs (MyoD and Myog) suggests an important distinction between these factors, whereby MyoD establishes an open chromatin structure at muscle-specific genes, whereas Myog drives high levels of transcription of genes within this open chromatin state. Furthermore, recent data have provided an additional distinction between MRF function with respect to cell cycle regulation. Indeed, MyoD has been shown to directly activate genes involved in cell cycle progression, leading to myoblast proliferation. In contrast, Myog has antiproliferative activity through the activation of genes that shut down the cell proliferation machinery, leading to cell cycle exit and myoblast differentiation. Although the transcriptional activities of MyoD and Myog synergize to drive muscle differentiation, it is the expression of Myog that sets in motion a gene expression program that constitutes a 'point of no return', leading to cell cycle exit. In this review, we compare and contrast the current literature with respect to MRF function, with a particular emphasis on the differential role of MRFs in modulating the cell cycle.

Skeletal and smooth muscle can mutually transdifferentiate, but little molecular insight exists as to how each muscle program may be subverted to the other. The myogenic basic helix-loop-helix transcription factors MyoD and myogenin (Myog) direct the development of skeletal muscle and are thought to be dominant over the program of smooth muscle cell (SMC) differentiation. Myocardin (Myocd) is a serum response factor (SRF) coactivator that promotes SMC differentiation through transcriptional stimulation of SRF-dependent smooth muscle genes. Here we show by lineage-tracing studies that Myocd is expressed transiently in skeletal muscle progenitor cells of the somite, and a majority of skeletal muscle is derived from Myocd-expressing cell lineages. However, rather than activating skeletal muscle-specific gene expression, Myocd functions as a transcriptional repressor of Myog, inhibiting skeletal muscle differentiation while activating SMC-specific genes. This repressor function of Myocd is complex, involving histone deacetylase 5 silencing of the Myog promoter and Myocd's physical contact with MyoD, which undermines MyoD DNA binding and transcriptional synergy with MEF2. These results reveal a previously unrecognized role for Myocd in repressing the skeletal muscle differentiation program and suggest that this transcriptional coregulator acts as a bifunctional molecular switch for the smooth versus skeletal muscle phenotypes.

BACKGROUND

Birt-Hogg-Dubé (BHD) syndrome is a hereditary hamartoma syndrome that predisposes patients to develop hair follicle tumors, lung cysts, and kidney cancer. Genetic studies of BHD patients have uncovered the causative gene, FLCN, but its function is incompletely understood.

METHODS

Mice with conditional alleles of FLCN and/or peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PPARGC1A), a transcriptional coactivator that regulates mitochondrial biogenesis, were crossbred with mice harboring either muscle creatine kinase (CKM) -Cre or myogenin (MYOG) -Cre transgenes to knock out FLCN and/or PPARGC1A in muscle, or cadherin 16 (CDH16)- Cre transgenes to knock out FLCN and/or PPARGC1A in kidney. Real-time polymerase chain reaction, immunoblotting, electron microscopy, and metabolic profiling assay were performed to evaluate mitochondrial biogenesis and function in muscle. Immunoblotting, electron microscopy, and histological analysis were used to investigate expression and the pathological role of PPARGC1A in FLCN-deficient kidney. Real-time polymerase chain reaction, oxygen consumption measurement, and flow cytometry were carried out using a FLCN-null kidney cancer cell line. All statistical analyses were two-sided.

RESULTS

Muscle-targeted FLCN knockout mice underwent a pronounced metabolic shift toward oxidative phosphorylation, including increased mitochondrial biogenesis (FLCN ( f/f ) vs FLCN ( f/f ) /CKM-Cre: % mitochondrial area mean = 7.8% vs 17.8%; difference = 10.0%; 95% confidence interval = 5.7% to 14.3%; P < .001), and the observed increase in mitochondrial biogenesis was PPARGC1A dependent. Reconstitution of FLCN-null kidney cancer cells with wild-type FLCN suppressed mitochondrial metabolism and PPARGC1A expression. Kidney-targeted PPARGC1A inactivation partially rescued the enlarged kidney phenotype and abrogated the hyperplastic cells observed in the FLCN-deficient kidney.

CONCLUSIONS

FLCN deficiency and subsequent increased PPARGC1A expression result in increased mitochondrial function and oxidative metabolism as the source of cellular energy, which may give FLCN-null kidney cells a growth advantage and drive hyperplastic transformation.

Rhabdomyosarcomas (RMS) are the most frequent soft-tissue sarcoma in children and characteristically show features of developing skeletal muscle. The alveolar subtype is frequently associated with a PAX3-FOXO1 fusion protein that is known to contribute to the undifferentiated myogenic phenotype of RMS cells. Histone methylation of lysine residues controls developmental processes in both normal and malignant cell contexts. Here we show that JARID2, which encodes a protein known to recruit various complexes with histone-methylating activity to their target genes, is significantly overexpressed in RMS with PAX3-FOXO1 compared with the fusion gene-negative RMS (t-test; P < 0.0001). Multivariate analyses showed that higher JARID2 levels are also associated with metastases at diagnosis, independent of fusion gene status and RMS subtype (n = 120; P = 0.039). JARID2 levels were altered by silencing or overexpressing PAX3-FOXO1 in RMS cell lines with and without the fusion gene, respectively. Consistent with this, we demonstrated that JARID2 is a direct transcriptional target of the PAX3-FOXO1 fusion protein. Silencing JARID2 resulted in reduced cell proliferation coupled with myogenic differentiation, including increased expression of Myogenin (MYOG) and Myosin Light Chain (MYL1) in RMS cell lines representative of both the alveolar and embryonal subtypes. Induced myogenic differentiation was associated with a decrease in JARID2 levels and this phenotype could be rescued by overexpressing JARID2. Furthermore, we that showed JARID2 binds to and alters the methylation status of histone H3 lysine 27 in the promoter regions of MYOG and MYL1 and that the interaction of JARID2 at these promoters is dependent on EED, a core component of the polycomb repressive complex 2 (PRC2). Therefore, JARID2 is a downstream effector of PAX3-FOXO1 that maintains an undifferentiated myogenic phenotype that is characteristic of RMS. JARID2 and other components of PRC2 may represent novel therapeutic targets for treating RMS patients.

Alveolar rhabdomyosarcoma is a life-threatening myogenic cancer of children and adolescent young adults, driven primarily by the chimeric transcription factor PAX3-FOXO1. The mechanisms by which PAX3-FOXO1 dysregulates chromatin are unknown. We find PAX3-FOXO1 reprograms the cis-regulatory landscape by inducing de novo super enhancers. PAX3-FOXO1 uses super enhancers to set up autoregulatory loops in collaboration with the master transcription factors MYOG, MYOD, and MYCN. This myogenic super enhancer circuitry is consistent across cell lines and primary tumors. Cells harboring the fusion gene are selectively sensitive to small-molecule inhibition of protein targets induced by, or bound to, PAX3-FOXO1-occupied super enhancers. Furthermore, PAX3-FOXO1 recruits and requires the BET bromodomain protein BRD4 to function at super enhancers, resulting in a complete dependence on BRD4 and a significant susceptibility to BRD inhibition. These results yield insights into the epigenetic functions of PAX3-FOXO1 and reveal a specific vulnerability that can be exploited for precision therapy.Significance: PAX3-FOXO1 drives pediatric fusion-positive rhabdomyosarcoma, and its chromatin-level functions are critical to understanding its oncogenic activity. We find that PAX3-FOXO1 establishes a myoblastic super enhancer landscape and creates a profound subtype-unique dependence on BET bromodomains, the inhibition of which ablates PAX3-FOXO1 function, providing a mechanistic rationale for exploring BET inhibitors for patients bearing PAX-fusion rhabdomyosarcoma. Cancer Discov; 7(8); 884-99. ©2017 AACR.This article is highlighted in the In This Issue feature, p. 783.

Myogenesis has been a leading model for elucidating the molecular mechanisms that underlie tissue differentiation and development since the discovery of MyoD. During myogenesis, the fate of myogenic precursor cells is first determined by Pax3/Pax7. This is followed by regulation of the myogenic differentiation program by muscle regulatory factors (Myf5, MyoD, Myog, and Mrf4) to form muscle tissues. Recent studies have uncovered a detailed myogenic program that involves the RP58 (Zfp238)-dependent regulatory network, which is critical for repressing the expression of inhibitor of DNA binding (Id) proteins. These novel findings contribute to a comprehensive understanding of the muscle differentiation transcriptional program.

Muscle inactivity due to injury or disease results in muscle atrophy. The molecular mechanisms contributing to muscle atrophy are poorly understood. However, it is clear that expression of atrophy-related genes, like Atrogin-1 and MuRF-1, are intimately tied to loss of muscle mass. When these atrophy-related genes are knocked out, inactive muscles retain mass. Muscle denervation stimulates muscle atrophy and Myogenin (Myog) is a muscle-specific transcription factor that is highly induced following muscle denervation. To investigate if Myog contributes to muscle atrophy, we have taken advantage of conditional Myog null mice. We show that in the denervated soleus muscle Myog expression contributes to reduced muscle force, mass, and cross-sectional area. We found that Myog mediates these effects, at least in part, by regulating expression of the Atrogin-1 and MuRF-1 genes. Indeed Myog over-expression in innervated muscle stimulates Atrogin-1 gene expression and Myog over-expression stimulates Atrogin-1 promoter activity. Thus, Myog and the signaling cascades regulating its induction following muscle denervation may represent novel targets for therapies aimed at reducing denervation-induced muscle atrophy.

BACKGROUND

Analysis of RNA expression using real-time PCR (qRT-PCR) traditionally includes reference genes (RG) as an internal control. This practice is being questioned as it becomes increasingly clear that RG may vary considerably under certain experimental conditions. Thus, the validity of a particular RG must be determined for each experimental setting. We used qRT-PCR to measure the levels of six RG, which have been reported in the literature to be invariant. The RG were analyzed in human myoblast cultures under differentiation conditions. We examined the expression by qRT-PCR of mRNA encoding Beta-actin (ACTB), Beta-2-microglobulin (B2M), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), peptidylprolyl isomerase A (PPIA), TATA box binding protein (TBP) and ribosomal protein, large, P0 (RPLPO). The mRNA expression of the following genes of interest (GOI) were analyzed: skeletal muscle alpha 1 actin (ACTA1), myogenin/myogenic factor 4 (MYOG), embryonic skeletal muscle myosin heavy chain 3 (MYH3) and the activity of creatine phosphokinase (CK). The geNorm, NormFinder and BestKeeper software programs were used to ascertain the most suitable RG to normalize the RNA input.

RESULTS

Using the geNorm program, RPLPO and TBP were found to be the most stable genes, additionally a suitable normalization factor (NF) was calculated. The NormFinder software showed that RPLPO was the most stable, whereas TBP ranked second. BestKeeper program also revealed that RPLPO and TBP as stable genes, but PPIA was the most stable reference gene, whereas GAPDH and ACTB were the worst ranked.

CONCLUSIONS

RNA expression analyses including three independent softwares revealed that RPLPO, TBP as reference genes or NF calculated by geNorm software, are suitable to normalize the mRNA expression in myoblast after culture under differentiation conditions. Significant correlations can be identified between the differentiations markers ACTA1, MYOG, MYH3 and creatine phosphokinase (CK) activity, when the expression is normalized with the NF calculated with RPLPO and TBP.

BACKGROUND

Despite modern technologies and novel computational approaches, decoding causal transcriptional regulation remains challenging. This is particularly true for less well studied organisms and when only gene expression data is available. In muscle a small number of well characterised transcription factors are proposed to regulate development. Therefore, muscle appears to be a tractable system for proposing new computational approaches.

RESULTS

Here we report a simple algorithm that asks "which transcriptional regulator has the highest average absolute co-expression correlation to the genes in a co-expression module?" It correctly infers a number of known causal regulators of fundamental biological processes, including cell cycle activity (E2F1), glycolysis (HLF), mitochondrial transcription (TFB2M), adipogenesis (PIAS1), neuronal development (TLX3), immune function (IRF1) and vasculogenesis (SOX17), within a skeletal muscle context. However, none of the canonical pro-myogenic transcription factors (MYOD1, MYOG, MYF5, MYF6 and MEF2C) were linked to muscle structural gene expression modules. Co-expression values were computed using developing bovine muscle from 60 days post conception (early foetal) to 30 months post natal (adulthood) for two breeds of cattle, in addition to a nutritional comparison with a third breed. A number of transcriptional landscapes were constructed and integrated into an always correlated landscape. One notable feature was a 'metabolic axis' formed from glycolysis genes at one end, nuclear-encoded mitochondrial protein genes at the other, and centrally tethered by mitochondrially-encoded mitochondrial protein genes.

CONCLUSIONS

The new module-to-regulator algorithm complements our recently described Regulatory Impact Factor analysis. Together with a simple examination of a co-expression module's contents, these three gene expression approaches are starting to illuminate the in vivo transcriptional regulation of skeletal muscle development.

OBJECTIVE

Exosomes released from cells can transfer both functional proteins and RNAs between cells. In this study we tested the hypothesis that muscle cells might transmit specific signals during lipid-induced insulin resistance through the exosomal route.

METHODS

Exosomes were collected from quadriceps muscles of C57Bl/6 mice fed for 16 weeks with either a standard chow diet (SD) or an SD enriched with 20% palm oil (HP) and from C2C12 cells exposed to 0.5 mmol/l palmitate (EXO-Post Palm), oleate (EXO-Post Oleate) or BSA (EXO-Post BSA).

RESULTS

HP-fed mice were obese and insulin resistant and had altered insulin-induced Akt phosphorylation in skeletal muscle (SkM). They also had reduced expression of Myod1 and Myog and increased levels of Ccnd1 mRNA, indicating that palm oil had a deep impact on SkM homeostasis in addition to insulin resistance. HP-fed mouse SkM secreted more exosomes than SD-fed mouse SkM. This was reproduced in-vitro using C2C12 cells pre-treated with palmitate, the most abundant saturated fatty acid of palm oil. Exosomes from HP-fed mice, EXO-Post Palm and EXO-Post Oleate induced myoblast proliferation and modified the expressions of genes involved in the cell cycle and muscle differentiation but did not alter insulin-induced Akt phosphorylation. Lipidomic analyses showed that exosomes from palmitate-treated cells were enriched in palmitate, indicating that exosomes likely transfer the deleterious effect of palm oil between muscle cells by transferring lipids. Muscle exosomes were incorporated into various tissues in vivo, including the pancreas and liver, suggesting that SkM could transfer specific signals through the exosomal route to key metabolic tissues.

CONCLUSIONS

Exosomes act as 'paracrine-like' signals and modify muscle homeostasis during high-fat diets.

Elucidation of new biomarkers and potential drug targets from high-throughput profiling data is a challenging task due to a limited number of available biological samples and questionable reproducibility of differential changes in cross-dataset comparisons. In this paper we propose a novel computational approach for drug and biomarkers discovery using comprehensive analysis of multiple expression profiling datasets.The new method relies on aggregation of individual profiling experiments combined with leave-one-dataset-out validation approach. Aggregated datasets were studied using Sub-Network Enrichment Analysis algorithm (SNEA) to find consistent statistically significant key regulators within the global literature-extracted expression regulation network. These regulators were linked to the consistent differentially expressed genes.We have applied our approach to several publicly available human muscle gene expression profiling datasets related to Duchenne muscular dystrophy (DMD). In order to detect both enhanced and repressed processes we considered up- and down-regulated genes separately. Applying the proposed approach to the regulators search we discovered the disturbance in the activity of several muscle-related transcription factors (e.g. MYOG and MYOD1), regulators of inflammation, regeneration, and fibrosis. Almost all SNEA-derived regulators of down-regulated genes (e.g. AMPK, TORC2, PPARGC1A) correspond to a single common pathway important for fast-to-slow twitch fiber type transition. We hypothesize that this process can affect the severity of DMD symptoms, making corresponding regulators and downstream genes valuable candidates for being potential drug targets and exploratory biomarkers.