Regulating Muscle Gene Expression Research Articles

Eutherian-Specific Functions of BetaM Acquired through Atp1b4 Gene Co-Option in the Regulation of MyoD Expression.

Vertebrate ATP1B4 genes represent a rare instance of orthologous gene co-option, resulting in radically different functions of the encoded BetaM proteins. In lower vertebrates, BetaM is a Na, K-ATPase β-subunit that is a component of ion pumps in the plasma membrane. In placental mammals, BetaM lost its ancestral role and, through structural alterations of the N-terminal domain, became a skeletal and cardiac muscle-specific protein of the inner nuclear membrane, highly expressed during late fetal and early postnatal development. We previously determined that BetaM directly interacts with the transcriptional co-regulator SKI-interacting protein (SKIP) and is implicated in the regulation of gene expression. This prompted us to investigate a potential role for BetaM in the regulation of muscle-specific gene expression in neonatal skeletal muscle and cultured C2C12 myoblasts. We found that BetaM can stimulate expression of the muscle regulatory factor (MRF), MyoD, independently of SKIP. BetaM binds to the distal regulatory region (DRR) of MyoD, promotes epigenetic changes associated with activation of transcription, and recruits the SWI/SNF chromatin remodeling subunit, BRG1. These results indicate that eutherian BetaM regulates muscle gene expression by promoting changes in chromatin structure. These evolutionarily acquired new functions of BetaM might be very essential and provide evolutionary advantages to placental mammals.

Med12 is Essential for Skeletal Muscle Development

Tight regulation of tissue‐ and cell‐specific gene expression is essential for healthy physiological development. Mediator is a multisubunit protein complex that links basal transcriptional machinery, transcription factors, and epigenetic modifiers to gene targets to coordinate transcriptional regulation during development. One subunit, MED12, controls tissue‐ and cell‐specific gene expression. Some mutations in MED12 cause neurological developmental disorders, while others are associated with uterine leiomyomas. In addition to these diseases, human MED12 mutations have been linked to muscle disorders such as skeletal muscle hypotonia and congenital heart defects, but the function of MED12 in muscle and muscle cell development has not been elucidated. The objective in this study was to discover a molecular role of MED12 in transcriptional control of muscle development. We hypothesized that MED12 is required for healthy muscle development, and that MED12 helps recruit myogenic transcription factors (TFs) to Mediator at the Pre‐Initiation Complex (PIC) to achieve transcriptional control. Our lab generated a conditional skeletal muscle‐specific Med12 knockout mouse model (Med12mKO) driven by a Myogenin‐Cre transgene that is expressed during embryonic muscle development. Med12mKOmice have a runted phenotype, smaller muscles, disorganized muscle architecture, and suffer premature death, compared to littermate controls. Gene expression analysis by RNA‐sequencing (RNA‐seq) of muscle from 1‐day old Med12mKO and control mice revealed that Med12 deletion dysregulates metabolic and structural genes, including several myosin genes. MED12 is a transcriptional regulator, but does not directly bind DNA. To identify putative candidate TFs that regulate muscle gene expression in a MED12‐dependent manner we performed Upstream Regulator Analysis (URA) of dysregulated genes in Med12mKO muscle. Multiple TFs that regulate muscle gene expression were predicted to do so via MED12, including SRF, which is known to regulate expression of many myosin genes. Using differentiating C2C12 mouse myoblasts as a cell model of myogenesis, knockdown of Med12by shRNA resulted in diminished myotube size and reduced expression of myosin genes, recapitulating the gene expression and muscle phenotype observed in the Med12mKOmodel. However, no interaction was detected between SRF and MED12 in C2C12 myoblasts by Co‐Immunoprecipitation (Co‐IP), which did not support our hypothesis of muscle gene expression regulation through a MED12‐SRF mechanism. To further investigate how loss of MED12 dysregulates muscle gene expression, we used an unbiased approach to identify TFs that interact with MED12. We immunoprecipitated MED12 from C2C12 myoblasts and identified interacting TFs using mass spectrometry. We are currently investigating other TFs through which MED12 could be regulating gene expression during muscle development. We conclude that Med12 is required for normal skeletal muscle development, including normal gene expression of structural and metabolic genes during myogenesis.

Lack of skeletal muscle liver kinase B1 alters gene expression, mitochondrial content, inflammation and oxidative stress without affecting high-fat diet-induced obesity or insulin resistance

Ad libitum high-fat diet (HFD) induces obesity and skeletal muscle metabolic dysfunction. Liver kinase B1 (LKB1) regulates skeletal muscle metabolism by controlling the AMP-activated protein kinase family, but its importance in regulating muscle gene expression and glucose tolerance in obese mice has not been established. The purpose of this study was to determine how the lack of LKB1 in skeletal muscle (KO) affects gene expression and glucose tolerance in HFD-fed, obese mice.KO and littermate control wild-type (WT) mice were fed a standard diet or HFD for 14 weeks. RNA sequencing, and subsequent analysis were performed to assess mitochondrial content and respiration, inflammatory status, glucose and insulin tolerance, and muscle anabolic signaling.KO did not affect body weight gain on HFD, but heavily impacted mitochondria-, oxidative stress-, and inflammation-related gene expression. Accordingly, mitochondrial protein content and respiration were suppressed while inflammatory signaling and markers of oxidative stress were elevated in obese KO muscles. KO did not affect glucose or insulin tolerance. However, fasting serum insulin and skeletal muscle insulin signaling were higher in the KO mice. Furthermore, decreased muscle fiber size in skmLKB1-KO mice was associated with increased general protein ubiquitination and increased expression of several ubiquitin ligases, but not muscle ring finger 1 or atrogin-1. Taken together, these data suggest that the lack of LKB1 in skeletal muscle does not exacerbate obesity or insulin resistance in mice on a HFD, despite impaired mitochondrial content and function and elevated inflammatory signaling and oxidative stress.

Profiling of the muscle-specific dystroglycan interactome reveals the role of Hippo signaling in muscular dystrophy and age-dependent muscle atrophy

BackgroundDystroglycanopathies are a group of inherited disorders characterized by vast clinical and genetic heterogeneity and caused by abnormal functioning of the ECM receptor dystroglycan (Dg). Remarkably, among many cases of diagnosed dystroglycanopathies, only a small fraction can be linked directly to mutations in Dg or its regulatory enzymes, implying the involvement of other, not-yet-characterized, Dg-regulating factors. To advance disease diagnostics and develop new treatment strategies, new approaches to find dystroglycanopathy-related factors should be considered. The Dg complex is highly evolutionarily conserved; therefore, model genetic organisms provide excellent systems to address this challenge. In particular, Drosophila is amenable to experiments not feasible in any other system, allowing original insights about the functional interactors of the Dg complex.MethodsTo identify new players contributing to dystroglycanopathies, we used Drosophila as a genetic muscular dystrophy model. Using mass spectrometry, we searched for muscle-specific Dg interactors. Next, in silico analyses allowed us to determine their association with diseases and pathological conditions in humans. Using immunohistochemical, biochemical, and genetic interaction approaches followed by the detailed analysis of the muscle tissue architecture, we verified Dg interaction with some of the discovered factors. Analyses of mouse muscles and myocytes were used to test if interactions are conserved in vertebrates.ResultsThe muscle-specific Dg complexome revealed novel components that influence the efficiency of Dg function in the muscles. We identified the closest human homologs for Dg-interacting partners, determined their significant enrichment in disease-associations, and verified some of the newly identified Dg interactions. We found that Dg associates with two components of the mechanosignaling Hippo pathway: the WW domain-containing proteins Kibra and Yorkie. Importantly, this conserved interaction manages adult muscle size and integrity.ConclusionsThe results presented in this study provide a new list of muscle-specific Dg interactors, further analysis of which could aid not only in the diagnosis of muscular dystrophies, but also in the development of new therapeutics. To regulate muscle fitness during aging and disease, Dg associates with Kibra and Yorkie and acts as a transmembrane Hippo signaling receptor that transmits extracellular information to intracellular signaling cascades, regulating muscle gene expression.

The Circular RNA circHUWE1 Sponges the miR-29b-AKT3 Axis to Regulate Myoblast Development

Myogenesis is controlled by a well-established transcriptional hierarchy that coordinates the activities of a set of muscle genes. Recently, roles in myogenesis have been described for non-coding RNAs, including a role of circular RNA (circRNA) to regulate muscle gene expression. However, the functions of circRNA and the underlying mechanism by which circRNAs affect myogenesis remain poorly understood. In this study, we analyzed circRNA high-throughput sequencing results of bovine skeletal muscle samples and constructed a circRNA-miRNA-mRNA network according to the competitive endogenous RNA (ceRNA) theory. The putative circHUWE1-miR-29b-AKT3 network was analyzed and its involvement in myogenesis was confirmed through a series of assays. To assess the potential function of this regulation, bovine myoblasts were infected with overexpression plasmids and small interfering RNAs (siRNAs) that target circHUWE1. Next, cell proliferation, apoptosis, and differentiation were analyzed using Cell Counting Kit-8 (CCK-8), 5-ethynyl-2′-deoxyuridine (EdU), flow cytometry, western blotting, and qRT-PCR assays. The results suggest that circHUWE1 facilitates bovine myoblast proliferation and inhibits cell apoptosis and differentiation. Next, bioinformatics, dual-luciferase reporter assay, and AGO2 RNA immunoprecipitation (RIP) approaches were used to verify the interaction between circHUWE1, miR-29b, and AKT3. Subsequently, we identified that circHUWE1 could directly interfere with the ability of miR-29b to relieve AKT3 suppression, which ultimately activates the AKT signaling pathway. These findings suggested a new regulatory pathway for bovine skeletal muscle development, and they also expand our understanding of circRNA functions in mammals.

Genome-Wide Analysis of Circular RNAs Mediated ceRNA Regulation in Porcine Embryonic Muscle Development.

Many circular RNAs (circRNAs) have been discovered in various tissues and cell types in pig. However, the temporal expression pattern of circRNAs during porcine embryonic muscle development remains unclear. Here, we present a panorama view of circRNA expression in embryonic muscle development at 33-, 65-, and 90-days post-coitus (dpc) from Duroc pigs. An unbiased analysis reveals that more than 5,000 circRNAs specifically express in embryonic muscle development. The amount and complexity of circRNA expression is most pronounced in skeletal muscle at day 33 of gestation. Our circRNAs annotation analyses show that “hot-spot” genes produce multiple circRNA isoforms and RNA binding protein (RBPs) may regulate the biogenesis of circRNAs. Furthermore, we observed that host genes of differentially expressed circRNA across porcine muscle development are enriched in skeletal muscle function. A competing endogenous RNA (ceRNA) network analysis of circRNAs reveals that circRNAs regulate muscle gene expression by functioning as miRNA sponges. Finally, our experimental validation demonstrated that circTUT7 regulate the expression of HMG20B in a ceRNA mechanism. Our analyses show that circRNAs are dynamically expressed and interacting with muscle genes through ceRNA manner, suggesting their critical functions in embryonic skeletal muscle development.

Profiling of the Muscle-Specific Dystroglycan Complexome Identifies Novel Muscular Dystrophy Factors

Dystroglycanopathies are a group of inherited disorders characterized by vast clinical and genetic heterogeneity caused by abnormal functioning of the ECM receptor Dystroglycan (Dg). Remarkably, among many cases of diagnosed dystroglycanopathies, only a small fraction can be directly linked to mutations in Dg or its regulatory enzymes, implying the involvement of other, not yet characterized Dg-regulating factors. To identify new players contributing to dystroglycanopathies, we used Drosophila as a genetic muscular dystrophy model. We analyzed the muscle-specific Dg complexome and identified novel Dg interactors, the majority of which are conserved and have human disease-associated homologs. We discovered that components of the Hippo pathway interact physically and genetically with Dg in adult muscles. Dg associates with the WW domain-containing proteins, Kibra and Yorkie, controlling muscle size and integrity. We propose that Dg acts as a transmembrane Hippo signaling receptor that transmits extracellular information to intracellular signaling cascades, regulating muscle gene expression.

YY1 Positively Regulates Transcription by Targeting Promoters and Super-Enhancers through the BAF Complex in Embryonic Stem Cells.

SummaryYin Yang 1 (YY1) regulates early embryogenesis and adult tissue formation. However, the role of YY1 in stem cell regulation remains unclear. YY1 has a Polycomb group (PcG) protein-dependent role in mammalian cells. The PcG-independent functions of YY1 are also reported, although their underlying mechanism is still undefined. This paper reports the role of YY1 and BAF complex in the OCT4-mediated pluripotency network in mouse embryonic stem cells (mESCs). The interaction between YY1 and BAF complex promotes mESC proliferation and pluripotency. Knockdown of Yy1 or Smarca4, the core component of the BAF complex, downregulates pluripotency markers and upregulates several differentiation markers. Moreover, YY1 enriches at both promoter and super-enhancer regions to stimulate transcription. Thus, this study elucidates the role of YY1 in regulating pluripotency through its interaction with OCT4 and the BAF complex and the role of BAF complex in integrating YY1 into the core pluripotency network.

Myotube-derived exosomal miRNAs downregulate Sirtuin1 in myoblasts during muscle cell differentiation

It has recently been established that exosomes can mediate intercellular cross-talk under normal and pathological conditions through the transfer of specific miRNAs. As muscle cells secrete exosomes, we addressed the question of whether skeletal muscle (SkM) exosomes contained specific miRNAs, and whether they could act as “endocrine signals” during myogenesis. We compared the miRNA repertoires found in exosomes released from C2C12 myoblasts and myotubes and found that 171 and 182 miRNAs were exported into exosomes from myoblasts and myotubes, respectively. Interestingly, some miRNAs were expressed at higher levels in exosomes than in their donor cells and vice versa, indicating a selectivity in the incorporation of miRNAs into exosomes. Moreover miRNAs from C2C12 exosomes were regulated during myogenesis. The predicted target genes of regulated exosomal miRNAs are mainly involved in the control of important signaling pathways for muscle cell differentiation (e.g., Wnt signaling pathway). We demonstrated that exosomes from myotubes can transfer small RNAs (C. elegans miRNAs and siRNA) into myoblasts. Moreover, we present evidence that exosome miRNAs secreted by myotubes are functionally able to silence Sirt1 in myoblasts. As Sirt1 regulates muscle gene expression and differentiation, our results show that myotube–exosome miRNAs could contribute to the commitment of myoblasts in the process of differentiation. Until now, myokines in muscle cell secretome provided a conceptual basis for communication between muscles. Here, we show that miRNA exosomal transfer would be a powerful means by which gene expression is orchestrated to regulate SkM metabolic homeostasis.

SEEING AND COMMUNICATING THROUGH WEAK ELECTRIC FIELDS

![][1] Weakly electric fish spend their lives bathed in their own internally generated mild electric field, interpreting perturbations in the field as objects pass through and when communicating with members of their own species through high frequency electric ‘chirps’. Rudiger

Integrative analysis of the zinc finger transcription factor Lame duck in the Drosophila myogenic gene regulatory network

Contemporary high-throughput technologies permit the rapid identification of transcription factor (TF) target genes on a genome-wide scale, yet the functional significance of TFs requires knowledge of target gene expression patterns, cooperating TFs, and cis-regulatory element (CRE) structures. Here we investigated the myogenic regulatory network downstream of the Drosophila zinc finger TF Lame duck (Lmd) by combining both previously published and newly performed genomic data sets, including ChIP sequencing (ChIP-seq), genome-wide mRNA profiling, cell-specific expression patterns of putative transcriptional targets, analysis of histone mark signatures, studies of TF cooccupancy by additional mesodermal regulators, TF binding site determination using protein binding microarrays (PBMs), and machine learning of candidate CRE motif compositions. Our findings suggest that Lmd orchestrates an extensive myogenic regulatory network, a conclusion supported by the identification of Lmd-dependent genes, histone signatures of Lmd-bound genomic regions, and the relationship of these features to cell-specific gene expression patterns. The heterogeneous cooccupancy of Lmd-bound regions with additional mesodermal regulators revealed that different transcriptional inputs are used to mediate similar myogenic gene expression patterns. Machine learning further demonstrated diverse combinatorial motif patterns within tissue-specific Lmd-bound regions. PBM analysis established the complete spectrum of Lmd DNA binding specificities, and site-directed mutagenesis of Lmd and additional newly discovered motifs in known enhancers demonstrated the critical role of these TF binding sites in supporting full enhancer activity. Collectively, these findings provide insights into the transcriptional codes regulating muscle gene expression and offer a generalizable approach for similar studies in other systems.

Downregulation of the serum response factor/miR-1 axis in the quadriceps of patients with COPD

RationaleMuscle atrophy confers a poor prognosis in patients with chronic obstructive pulmonary disease (COPD), yet the molecular pathways responsible are poorly characterised. Muscle-specific microRNAs and serum response factor (SRF) are...

Long-term Training with Electrical Stimulation Regulates Muscle Gene Expression and Bone Architecture after Spinal Cord Injury

Long-term training with electrical stimulation regulates muscle gene expression and bone architecture after SCI: Adams CM, Suneja M, McHenry CM, Littmann AL, Dudley-Javoroski S, Shields RK, Physical Therapy and Rehabilitation Science, Carver College of Medicine, University of Iowa Background: Musculoskeletal deterioration is a serious secondary complication associated with paralysis from spinal cord injury (SCI). PURPOSE: We present the long-term effects of regular electrical stimulation training on muscle gene regulation and bone architecture after SCI. METHODS: Individuals with complete paralysis from SCI trained the calf muscle (soleus) or the thigh muscle (quadricep) during novel resistive tasks. The electrical stimulation protocol induced 2-4 bouts of 60-120 isometric contractions at a 15-20 Hz stimulation. After at least 2 years of unilateral training, we obtained muscle biopsies from the calf (soleus) and thigh muscles (vastus lateralis) of (trained and untrained) subjects with SCI. We then used Affymetrix human exon 1.0 arrays to identify mRNAs that were ≥ 1.5 fold increased or decreased in the atrophied untrained muscles. We next examined the trabecular bone using mDCT to quantify the sites and quality of the trabecular lattice. RESULTS: Out of >17,000 transcripts examined, in the untrained limb 51 mRNAs were increased including mRNAs encoding fast-twitch isoforms of myosin and troponin, as well as the mRNA encoding myostatin. Fifty-four mRNAs were decreased, including mRNAs encoding slow-twitch isoforms of myosin and troponin, 11 enzymes for oxidative phosphorylation, as well as PGC1α, a transcriptional co-activator that promotes the slow twitch, oxidative phenotype and inhibits FoxO3-mediated muscle atrophy. Training increased the mRNAs encoding slow-twitch isoforms and isoforms associated with glucose metabolism. The bone trabecular lattice was denser with distinct rods and plates within the trained femur and tibia. CONCLUSIONS: These findings support that thirty minutes/day of electrical stimulation stress induced significant muscle molecular adaptations and prevented the deterioration of bone after SCI. Funded by Nielsen Foundation and National Institutes of Health (R01-NR-01028505; R01-HD-04583233; R01HD062507).

Genome-wide MyoD Binding in Skeletal Muscle Cells: A Potential for Broad Cellular Reprogramming

Recent studies have demonstrated that MyoD initiates a feed-forward regulation of skeletal muscle gene expression, predicting that MyoD binds directly to many genes expressed during differentiation. We have used chromatin immunoprecipitation and high-throughput sequencing to identify genome-wide binding of MyoD in several skeletal muscle cell types. As anticipated, MyoD preferentially binds to a VCASCTG sequence that resembles the in vitro-selected site for a MyoD:E-protein heterodimer, and MyoD binding increases during differentiation at many of the regulatory regions of genes expressed in skeletal muscle. Unanticipated findings were that MyoD was constitutively bound to thousands of additional sites in both myoblasts and myotubes, and that the genome-wide binding of MyoD was associated with regional histone acetylation. Therefore, in addition to regulating muscle gene expression, MyoD binds genome wide and has the ability to broadly alter the epigenome in myoblasts and myotubes.

Exploring the Function of Titin Kinase by Mechanical Single-Molecule Pump-And-Probe

Protein function like catalytic activity or molecular recognition is tightly coupled to conformation and its dynamics. Since protein conformation can be controlled by forces, diverse mechanisms evolved allowing biological systems to respond to mechanical strain. Recently, it was shown in combination with single-molecule force spectroscopy, MD-simulations and enzymatics that titin kinase acts as a force sensor regulating muscle gene expression and protein turnover [1]. However, there is no experimental access to investigate which force-induced conformation during the activation pathway is competent for ATP-binding. Here we develop a new AFM-based single-molecule pump-and-probe protocol to mechanically prepare a predefined conformation and to read out afterwards whether ATP bound. We show that ATP only binds to the conformation prepared after barrier two. Therefore, titin kinase exhibits a dual mechanical autoinhibition, which cannot be overcome by thermal fluctuations but by physiological forces acting on the M-band structure of the muscle sarcomere. This single-molecule approach might also become useful for the investigation of other conformation controlled processes such as hidden binding pockets, catch bonds or motor proteins. [1] Puchner, E.M. et al. Mechanoenzymatics of titin kinase.Proc Natl Acad Sci U S A, 2008. 105(36): p. 13385-90.

The Histone Deacetylase HDAC4 Connects Neural Activity to Muscle Transcriptional Reprogramming

Neural activity actively regulates muscle gene expression. This regulation is crucial for specifying muscle functionality and synaptic protein expression. How neural activity is relayed into nuclei and connected to the muscle transcriptional machinery, however, is not known. Here we identify the histone deacetylase HDAC4 as the critical linker connecting neural activity to muscle transcription. We found that HDAC4 is normally concentrated at the neuromuscular junction (NMJ), where nerve innervates muscle. Remarkably, reduced neural input by surgical denervation or neuromuscular diseases dissociates HDAC4 from the NMJ and dramatically induces its expression, leading to robust HDAC4 nuclear accumulation. We present evidence that nuclear accumulated HDAC4 is responsible for the coordinated induction of synaptic genes upon denervation. Inactivation of HDAC4 prevents denervation-induced synaptic acetyl-choline receptor (nAChR) and MUSK transcription whereas forced expression of HDAC4 mimics denervation and activates ectopic nAChR transcription throughout myofibers. We determined that HDAC4 executes activity-dependent transcription by regulating the Dach2-myogenin transcriptional cascade where inhibition of the repressor Dach2 by HDAC4 permits the induction of the transcription factor myogenin, which in turn activates synaptic gene expression. Our findings establish HDAC4 as a neural activity-regulated deacetylase and a key signaling component that relays neural activity to the muscle transcriptional machinery.

Chromatin modification and muscle differentiation

Skeletal muscle differentiation is a multistep process, which begins with the commitment of multi-potent mesodermal precursor cells to the muscle fate. These committed cells, the myoblasts, then differentiate and fuse into multinucleated myotubes. The final step of muscle differentiation is the maturation of differentiated myotubes into myofibres. Skeletal muscle development requires the coordinated expression of various transcription factors like the members of the myocyte enhancer binding-factor 2 family and the muscle regulatory factors. These transcription factors, in collaboration with chromatin-remodelling complexes, act in specific combinations and within complex transcriptional regulatory networks to achieve skeletal myogenesis. Additional factors involved in the epigenetic regulation of this process continue to be discovered. In this review, the authors discuss the recent discoveries in the epigenetic regulation of myogenesis. They also summarise the role of chromatin-modifying enzymes regulating muscle gene expression. These different factors are often involved in multiple steps of muscle differentiation and have redundant activities. Altogether, the recent findings have allowed a better understanding of myogenesis and have raised new hopes for the pharmacological development of new therapies aimed at muscle degeneration diseases, such as myotonic dystrophy or Duchenne muscular dystrophy.

The Polycomb Ezh2 methyltransferase regulates muscle gene expression and skeletal muscle differentiation

The Ezh2 protein endows the Polycomb PRC2 and PRC3 complexes with histone lysine methyltransferase (HKMT) activity that is associated with transcriptional repression. We report that Ezh2 expression was developmentally regulated in the myotome compartment of mouse somites and that its down-regulation coincided with activation of muscle gene expression and differentiation of satellite-cell-derived myoblasts. Increased Ezh2 expression inhibited muscle differentiation, and this property was conferred by its SET domain, required for the HKMT activity. In undifferentiated myoblasts, endogenous Ezh2 was associated with the transcriptional regulator YY1. Both Ezh2 and YY1 were detected, with the deacetylase HDAC1, at genomic regions of silent muscle-specific genes. Their presence correlated with methylation of K27 of histone H3. YY1 was required for Ezh2 binding because RNA interference of YY1 abrogated chromatin recruitment of Ezh2 and prevented H3-K27 methylation. Upon gene activation, Ezh2, HDAC1, and YY1 dissociated from muscle loci, H3-K27 became hypomethylated and MyoD and SRF were recruited to the chromatin. These findings suggest the existence of a two-step activation mechanism whereby removal of H3-K27 methylation, conferred by an active Ezh2-containing protein complex, followed by recruitment of positive transcriptional regulators at discrete genomic loci are required to promote muscle gene expression and cell differentiation.

Sir2 Regulates Skeletal Muscle Differentiation as a Potential Sensor of the Redox State

Sir2 is a NAD +-dependent histone deacetylase that controls gene silencing, cell cycle, DNA damage repair, and life span. Prompted by the observation that the [NAD +]/[NADH] ratio is subjected to dynamic fluctuations in skeletal muscle, we have tested whether Sir2 regulates muscle gene expression and differentiation. Sir2 forms a complex with the acetyltransferase PCAF and MyoD and, when overexpressed, retards muscle differentiation. Conversely, cells with decreased Sir2 differentiate prematurely. To inhibit myogenesis, Sir2 requires its NAD +-dependent deacetylase activity. The [NAD +]/[NADH] ratio decreases as muscle cells differentiate, while an increased [NAD +]/[NADH] ratio inhibits muscle gene expression. Cells with reduced Sir2 levels are less sensitive to the inhibition imposed by an elevated [NAD +]/[NADH] ratio. These results indicate that Sir2 regulates muscle gene expression and differentiation by possibly functioning as a redox sensor. In response to exercise, food intake, and starvation, Sir2 may sense modifications of the redox state and promptly modulate gene expression.

Beta(1)-integrin and PI 3-kinase regulate RhoA-dependent activation of skeletal alpha-actin promoter in myoblasts.

RhoA GTPase, a regulator of actin cytoskeleton, is also involved in regulating c-fos gene expression through its effect on serum response factor (SRF) transcriptional activity. We have also shown that RhoA plays a critical role in myogenesis and regulates expression of SRF-dependent muscle genes, including skeletal alpha-actin. In the present study, we examined whether the RhoA signaling pathway cross talks with other myogenic signaling pathways to modulate skeletal alpha-actin promoter activity in myoblasts. We found that extracellular matrix proteins and the beta(1)-integrin stimulated RhoA-dependent activation of the alpha-actin promoter. The muscle-specific isoform beta(1D) selectively activated the alpha-actin promoter in concert with RhoA but inhibited the c-fos promoter. In addition, focal adhesion kinase (FAK) and phosphatidylinositol (PI) 3-kinase were required for full activation of the alpha-actin promoter by RhoA. Expression of a dominant negative mutant of FAK, application of wortmannin to cultured myoblasts, or expression of a dominant negative mutant of PI 3-kinase inhibited alpha-actin promoter activity induced by RhoA. These results suggest that RhoA, beta(1)-integrin, FAK, and PI 3-kinase serve together as an important signaling network in regulating muscle gene expression.