Increased chromatin accessibility facilitates intron retention in specific cell differentiation states

Dynamic intron retention (IR) in vertebrate cells is of widespread biological importance. Aberrant IR is associated with numerous human diseases including several cancers. Despite consistent reports demonstrating that intrinsic sequence features can help introns evade splicing, conflicting findings about cell type- or condition-specific IR regulation by trans-regulatory and epigenetic mechanisms demand an unbiased and systematic analysis of IR in a controlled experimental setting. We integrated matched mRNA sequencing (mRNA-Seq), whole-genome bisulfite sequencing (WGBS), nucleosome occupancy methylome sequencing (NOMe-Seq) and chromatin immunoprecipitation sequencing (ChIP-Seq) data from primary human myeloid and lymphoid cells. Using these multi-omics data and machine learning, we trained two complementary models to determine the role of epigenetic factors in the regulation of IR in cells of the innate immune system. We show that increased chromatin accessibility, as revealed by nucleosome-free regions, contributes substantially to the retention of introns in a cell-specific manner. We also confirm that intrinsic characteristics of introns are key for them to evade splicing. This study suggests an important role for chromatin architecture in IR regulation. With an increasing appreciation that pathogenic alterations are linked to RNA processing, our findings may provide useful insights for the development of novel therapeutic approaches that target aberrant splicing.     

Expression of Transthyretin during bovine myogenic satellite cell differentiation

Adult myogenesis responsible for the maintenance and repair of muscle tissue is mainly under the control of myogenic regulatory factors (MRFs) and a few other genes. Transthyretin gene (TTR), codes for a carrier protein for thyroxin (T₄) and retinol binding protein bound with retinol in blood plasma, plays a critical role during the early stages of myogenesis. Herein, we investigated the relationship of TTR with other muscle-specific genes and report their expression in muscle satellite cells (MSCs), and increased messenger RNA (mRNA) and protein expression of TTR during MSCs differentiation. Silencing of TTR resulted in decreased myotube formation and decreased expression of myosin light chain (MYL2), myosin heavy chain 3 (MYH3), matrix gla protein (MGP), and voltage-dependent L type calcium channel (Cav1.1) genes. Increased mRNA expression observed in TTR and other myogenic genes with the addition of T₄ decreased significantly following TTR knockdown, indicating the critical role of TTR in T₄ transportation. Similarly, decreased expression of MGP and Cav1.1 following TTR knockdown signifies the dual role of TTR in controlling muscle myogenesis via regulation of T₄ and calcium channel. Our computational and experimental evidences indicate that TTR has a relationship with MRFs and may act on calcium channel and related genes.     

Comparison of the proliferation and differentiation of myogenic satellite cells and embryonic myoblasts derived from the turkey

• 1.1. Embryonic and posthatch turkey skeletal muscle development was compared in in vitro studies using clonal-derived embryonic myoblasts and satellite cells.
• 2.2. Although population doubling times were similar between the two lines (25.4 hr for satellite cells and 26.4 hr for embryonic myoblasts), embryonic myoblasts consistently began log phase growth 24 hr earlier than satellite cells.
• 3.3. Differentiation (fusion) of embryonic myoblasts was maximized by 36 hr in Dulbecco's Modified Eagle's Medium containing 1% horse serum compared with 72 hr for satellite cells.
• 4.4. When administered a serum-free medium which supports proliferation of turkey satellite cells, embryonic myoblasts differentiated to form myotubes.

     

Gene expression profiles during differentiation and transdifferentiation of bovine myogenic satellite cells

The molecular mechanisms underlying myogenic satellite cells (MSCs) differentiation into myotube-formed cells (MFCs) and transdifferentiation into adipocyte-like cells (ALCs) are unclear. As a step towards understanding the molecular mechanisms underlying MSC differentiation and transdifferentiation, we attempted to identify the genes differentially expressed during differentiation and transdifferentiation using gene microarray analysis (GMA). Thirty oligonucleotide arrays were used with two technical replicates and nine and six biological replicates for MFCs vs. MSCs and ALCs vs. MSCs, respectively, to contrast expression profile differences. GMA identified 1,224 differentially expressed genes by at least 2-fold during differentiation and transdifferentiation of MSCs. To select the highly expressed genes for future functional study, genes with a 4-fold expression difference were selected for validation by real time RT-PCR and approximately 96.9% of the genes were validated. The up-regulation of marker genes for myogenesis (MYL2, MYH3) and adipogenesis (PPAR??, and FABP4) was observed during the differentiation and transdifferentiation of MSCs into MFCs and ALCs, respectively. KOG analysis revealed that the most of the genes up-regulated during differentiation and transdifferentiation of MSCs were related to signal transduction. Again the exact location of 109 differentially expressed genes by 4-fold were analyzed by chromosome mapping. Among those, co-localization of 29 genes up-regulated during transdifferentiation with QTL for marbling score and intramuscular fat percentage supports the involvement of these genes in cellular transdifferentiation. Interestingly, some genes with unknown function were also identified during the process. Functional studies on these genes may unfold the molecular mechanisms controlling MSC differentiation and transdifferentiation.     

Effects of gender-specific adult bovine serum on myogenic satellite cell proliferation, differentiation and lipid accumulation

The study was performed to explore the effects of adult bovine male serum (MS), female serum (FS), and castrated male serum (C-MS) on myogenic satellite cells (MSCs) proliferation and differentiation into myotubes or into adipocyte-like cells (ALCs). MSC proliferation and differentiation was highest in the medium supplemented with MS, implying the important role of male steroid hormones. Myogenin and desmin were highly upregulated in cells cultured in MS-supplemented medium. In contrast, lipid accumulation in ALCs was highest in the medium supplemented with FS. Fatty acid transporter (FAT/CD36) was upregulated in FS-supplemented cultures. Detection of higher FAT/CD36 inducing fatty acids (arachidic acid and eicosapentaenoic acid) in FS compared with MS and C-MS suggests that these fatty acids may have influenced the enhanced formation of lipid droplets in ALCs. Effect of sex steroids on cell proliferation and cell growth of bovine MSCs and C2C12 cell in C-MS was greater than charcoal-dextran-treated fetal bovine serum (CDFBS). Concluding the above facts, the results indicate that each gender-specific bovine serum constitutes of different component, which leads to unique effects on cell behavior.     

TRAF6 regulates proliferation and differentiation of skeletal myoblasts

We could recently demonstrate an important role of receptor interacting protein-2 (RIP2), an activator of nuclear factor kappa B (NF-κB) and a target of activated receptors of the tumor necrosis factor receptor (TNFR) type, in myogenic differentiation and regeneration. Here, we analyze a potential role of TNFR associated factor 6 (TRAF6), which also associates with the cytoplasmic domain of TNFR type, but also IL-1-R and TLR type receptors, and activates NF-κB, in these processes. Specifically, we show that during myogenic differentiation in vitro, traf6 gene expression is downregulated in normal myoblasts, but not in rhabdomyosarcoma cells, suggesting a role of the TRAF6 protein in this process. Inhibition of traf6 expression using specific siRNAs led to an inhibition of both myoblast proliferation and differentiation, whereas inhibition of the TRAF6 effector NF-κB alone in our system only blocked proliferation. Finally, we demonstrate that the traf6 gene is downregulated in skeletal muscle tissue of the dystrophic mdx mouse. Taken together, these data argue for a role of TRAF6 in the regulation of skeletal muscle differentiation and regeneration.     

Cytoskeleton/stretch-activated ion channel interaction regulates myogenic differentiation of skeletal myoblasts

In the present study, we investigated the functional interaction between stress fibers (SFs) and stretch-activated channels (SACs) and its possible role in the regulation of myoblast differentiation induced by switch to differentiation culture in the presence or absence of sphingosine 1-phosphate. It was found that there was a clear temporal correlation between SF formation and SAC activation in differentiating C2C12 myoblasts. Inhibition of actin polymerization with the specific Rho kinase inhibitor Y-27632, significantly decreased SAC sensitivity in these cells, suggesting a role for Rho-dependent actin remodeling in the regulation of the channel opening. The alteration of cytoskeletal/SAC functional correlation had also deleterious effects on myogenic differentiation of C2C12 cells as judged by combined confocal immunofluorescence, biochemical and electrophysiological analyses. Indeed, the treatment with Y-27632 or with DHCB, an actin disrupting agent, inhibited the expression of the myogenic markers (myogenin and sarcomeric proteins) and myoblast-myotube transition. The treatment with the channel blocker, GdCl(3), also affected myogenesis in these cells. It impaired, in fact, myoblast phenotypic maturation (i.e., reduced the expression of alpha-sarcomeric actin and skeletal myosin and the activity of creatine kinase) but did not modify promoter activity and protein expression levels of myogenin. The results of this study, together with being in agreement with the general idea that cytoskeletal remodeling is essential for muscle differentiation, describe a novel pathway whereby the formation of SFs and their contraction, generate a mechanical tension to the plasma membrane, activate SACs and trigger Ca(2+)-dependent signals, thus influencing the phenotypic maturation of myoblasts.     

Hypoxia affects positively the proliferation of bovine satellite cells and their myogenic differentiation through up-regulation of MyoD

Hypoxia alters the biological functions of skeletal muscle cells to proliferate and differentiate into myotubes. However, the cellular responses of myoblasts to hypoxia differ according to the levels of oxygen and the types of cells studied. This study examined the effect of hypoxia (1% oxygen) on bovine satellite cells. Hypoxia significantly increased the proliferation of satellite cells cultured in a growth medium. In addition, the levels of PCNA, cyclin D1, cyclin-dependent kinase-1 (CDK1) and CDK2 expression were increased. Hypoxia facilitated the formation of myotubes as well as the stimulation of MyoD, myogenin, and myosin heavy chain (MHC) expression in differentiating medium (DM) cultures. In particular, satellite cells cultured under hypoxic/DM conditions showed increased p21 expression but not p27. The transfection of satellite cells with antisense MyoD oligonucleotides resulted in a decrease in the MHC, myogenin, MRF4 RNA and protein levels with the concomitant decrease in fused cells to levels similar to those observed under normoxia/DM conditions. This indicates that MyoD up-regulation is closely associated with hypoxia-stimulated myogenic differentiation. In conclusion, hypoxia stimulates the proliferation of satellite cells and promotes their myogenic differentiation with MyoD playing an important role.     

Leptin promotes proliferation and inhibits differentiation in porcine skeletal myoblasts

Leptin, a major regulator of body weight, was recently suggested to play a role in myoblasts. We conducted an experiment to determine whether leptin can influence the proliferation and differentiation of porcine skeletal myoblasts. Myoblasts occurred in non-leptin and leptin forms in various concentrations for various periods of cell states. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and flow cytometry assays demonstrated that leptin significantly promoted myoblast proliferation and increased cell accumulation in the S + G2/M phase, in a dose-dependent manner. Furthermore, in morphologic experiments, the formation of myotubes and the myogenic index was markedly reduced by leptin. In addition, biochemical analysis showed that leptin decreased creatine kinase (CK) activity and the amount of myogenin and myosin heavy chain (MyHC) protein. Taking all this together, our study indicated that exogenous leptin promoted proliferation but inhibited differentiation in porcine skeletal myoblasts, suggesting that leptin might be an important mediator in the regulation of the growth and development of muscle cells.     

Angiopoietin 1 enhances the proliferation and differentiation of skeletal myoblasts

Angiopoietin 1 (Ang1) plays an important role in various endothelial functions, such as vascular integrity and angiogenesis; however, less is known about its function outside of the endothelium. In this study, we examined whether Ang1 has direct effects on skeletal muscle cells. We found that Ang1 exhibited myogenic potential, as it promoted the proliferation, migration, and differentiation of mouse primary skeletal myoblasts. The positive effect of Ang1 on myoblast proliferation could have been mediated by the α7 and β1 integrins. We also found that Ang1 potentiated cellular Ca²⁺ movements in differentiated myotubes in response to stimuli, possibly through the increased expression of two Ca²⁺‐related proteins, namely, Orai1 and calmodulin. Ang1 also increased Orai1 and calmodulin expression in mouse hearts in vivo. These results provide an insight into the molecular mechanisms by which Ang1 directly affects the myogenesis of striated muscle. J. Cell. Physiol. © 2012 Wiley Periodicals, Inc.     

BTB-Kelch protein Krp1 regulates proliferation and differentiation of myoblasts

The BTB-Kelch protein Krp1 is highly and specifically expressed in skeletal muscle, where it is proposed to have a role in myofibril formation. We observed significant upregulation of Krp1 in C2 cells early in myoblast differentiation, well before myofibrillogenesis. Krp1 has a role in cytoskeletal organization and cell motility; since myoblast migration and elongation/alignment are important events in early myogenesis, we hypothesized that Krp1 is involved with earlier regulation of differentiation. Krp1 protein levels were detectable by 24 h after induction of differentiation in C2 cells and were significantly upregulated by 48 h, i.e., following the onset myogenin expression and preceding myosin heavy chain (MHC) upregulation. Upregulation of Krp1 required a myogenic stimulus as signaling derived from increased myoblast cell density was insufficient to activate Krp1 expression. Examination of putative Krp1 proximal promoter regions revealed consensus E box elements associated with myogenic basic helix-loop-helix binding. The activity of a luciferase promoter-reporter construct encompassing this 2,000-bp region increased in differentiating C2 myoblasts and in C2 cells transfected with myogenin and/or MyoD. Knockdown of Krp1 via short hairpin RNA resulted in increased C2 cell number and proliferation rate as assessed by bromodeoxyuridine incorporation, whereas overexpression of Krp1-myc had the opposite effect; apoptosis was unchanged. No effects of changed Krp1 protein levels on cell migration were observed, either by scratch wound assay or live cell imaging. Paradoxically, both knockdown and overexpression of Krp1 inhibited myoblast differentiation assessed by expression of myogenin, MEF2C, MHC, and cell fusion.     

Smad3 signaling is required for satellite cell function and myogenic differentiation of myoblasts

TGF-β and myostatin are the two most important regulators of muscle growth. Both growth factors have been shown to signal through a Smad3-dependent pathway. However to date, the role of Smad3 in muscle growth and differentiation is not investigated. Here, we demonstrate that Smad3-null mice have decreased muscle mass and pronounced skeletal muscle atrophy. Consistent with this, we also find increased protein ubiquitination and elevated levels of the ubiquitin E3 ligase MuRF1 in muscle tissue isolated from Smad3-null mice. Loss of Smad3 also led to defective satellite cell (SC) functionality. Smad3-null SCs showed reduced propensity for self-renewal, which may lead to a progressive loss of SC number. Indeed, decreased SC number was observed in skeletal muscle from Smad3-null mice showing signs of severe muscle wasting. Further in vitro analysis of primary myoblast cultures identified that Smad3-null myoblasts exhibit impaired proliferation, differentiation and fusion, resulting in the formation of atrophied myotubes. A search for the molecular mechanism revealed that loss of Smad3 results in increased myostatin expression in Smad3-null muscle and myoblasts. Given that myostatin is a negative regulator, we hypothesize that increased myostatin levels are responsible for the atrophic phenotype in Smad3-null mice. Consistent with this theory, inactivation of myostatin in Smad3-null mice rescues the muscle atrophy phenotype.     

Interplay between proliferation and differentiation within the myogenic lineage.

In muscle cells, as in a variety of cell types, proliferation and differentiation are mutually exclusive events controlled by a balance of opposing cellular signals. Members of the MyoD family of muscle-specific helix-loop-helix proteins which, in collaboration with ubiquitous factors, activate muscle differentiation and inhibit cell proliferation function at the nexus of the cellular circuits that control proliferation and differentiation of muscle cells. The activities of these myogenic regulators are negatively regulated by peptide growth factors and activated oncogenes whose products transmit growth signals from the membrane to the nucleus. Recent studies have revealed multiple mechanisms through which intracellular growth factor signals may interfere with the functions of the myogenic regulators. When expressed at high levels, members of the MyoD family can override mitogenic signals and can cause growth arrest independent of their effects on differentiation. The ability of these myogenic regulators to inhibit proliferation of normal as well as transformed cells from multiple lineages suggests that they interact with conserved components of the cellular machinery involved in cell cycle progression and that similar types of regulatory factors participate in differentiation and cell cycle control in diverse cell types.     

Serum-free culture conditions for analysis of secretory proteinases during myogenic differentiation of mouse C2C12 myoblasts.

We have been studying extracellular proteins such as proteinases and attachment factors under serum-free culture conditions. A number of studies on myogenesis using an in vitro culture system have reported that proteinases and ECM components play significant roles in muscle differentiation. However, most of the studies were performed in the presence of serum. Serum is abundant in the aforementioned proteins and its use in serum-free culture affects many cellular functions significantly. In this study, we tried to establish serum-free culture conditions for analyzing extracellular proteins involved in mouse myogenic differentiation. By evaluating media, supplements, and procedure of cell inoculation under serum-free conditions and by comparing the resultant conditions with conventional conditions on differentiated characteristics of the cells, it was revealed that serum-free Dulbecco's modified Eagle's medium/Ham's F-12 plus insulin more efficiently supported myogenesis morphologically and biochemically than conventional 2% horse serum-containing culture and that secretory proteinases obtained from our serum-free culture were different from those obtained utilizing conventional serum-free cultures in their activities and patterns. Since our serum-free medium consists of simple components, the medium is low cost and easy to prepare. Furthermore, the results suggest that our culture conditions are superior to conventional conditions biochemically and morphologically and will provide more precise and accurate information on extracellular proteins involved in myogenesis.