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Reporting in this issue of Developmental Cell , L'honore et al. (2014) show that a network composed of Pitx2/Pitx3 and downstream antioxidant enzymes protects differentiating skeletal muscle from excessive reactive oxygen species production during fetal myogenesis. Genetic deficiency of Pitx2/Pitx3 results in irreversible oxidative DNA damage and apoptosis, impairing skeletal muscle development.

Skeletal muscle differentiation is influenced by multiple pathways, which regulate the activity of myogenic regulatory factors (MRFs)—the myogenic basic helix-loop-helix proteins and the MEF2-family members—in positive or negative ways. Here we will review and discuss the network of signals that regulate MRF function during myocyte proliferation, differentiation, and post-mitotic growth. Elucidating the mechanisms governing muscle-specific transcription will provide important insight in better understanding the embryonic development of muscle at the molecular level and will have important implications in setting out strategies aimed at muscle regeneration. Since the activity of MRFs are compromised in tumors of myogenic derivation—the rhabdomyosarcomas—the studies summarized in this review can provide a useful tool to uncover the molecular basis underlying the formation of these tumors. J. Cell. Physiol. 185:155–173, 2000. © 2000 Wiley-Liss, Inc.

Histone acetyltransferases (HAT) play a critical role in transcriptional control by relieving repressive effects of chromatin, and yet how HATs themselves are regulated remains largely unknown. Here, it is shown that Twist directly binds two independent HAT domains of acetyltransferases, p300 and p300/CBP–associated factor (PCAF), and directly regulates their HAT activities. The N terminus of Twist is a primary domain interacting with both acetyltransferases, and the same domain is required for inhibition of p300-dependent transcription by Twist. Adenovirus E1A protein mimics the effects of Twist by inhibiting the HAT activities of p300 and PCAF. These findings establish a cogent argument for considering the HAT domains as a direct target for acetyltransferase regulation by both a cellular transcription factor and a viral oncoprotein.

PCAF is a histone acetyltransferase that associates with p300/CBP and competes with E1A for access to them. While exogenous expression of PCAF potentiates both MyoD-directed transcription and myogenic differentiation, PCAF inactivation by anti-PCAF antibody microinjection prevents differentiation. MyoD interacts directly with both p300/CBP and PCAF, forming a multimeric protein complex on the promoter elements. Viral transforming factors that interfere with muscle differentiation disrupt this complex without affecting the MyoD–DNA interaction, indicating functional significance of the complex formation. Exogenous expression of PCAF or p300 promotes p21 expression and terminal cell-cycle arrest. Both of these activities are dependent on the histone acetyltransferase activity of PCAF, but not on that of p300. These results indicate that recruitment of histone acetyltransferase activity of PCAF by MyoD, through p300/CBP, is crucial for activation of the myogenic program.

The human hepatitis B virus (HBV) protein pX is a multifunctional regulatory protein that is known to affect both transcription and cell growth. Here we describe induction of apoptosis in NIH 3T3 polyclonal cell lines upon stimulation of pX expression from a dexamethasone inducible mouse mammary tumor virus (MMTV)-X expression vector. The effect of long-term pX expression on the cell survival of mouse fibroblasts was confirmed in colony generation assays. This effect is not shared either by the other HBV products and it is c- myc mediated, as shown by the use of a dominant negative deletion mutant of c- myc . pX also sensitize cells to programmed cell death after exposure to DNA damaging agents. Taking advantage of stable transfectants carrying the p53val135 temperature-sensitive allele, we directly demonstrate that induction of apoptosis by pX requires p53. In p53 null mouse embryo fibroblasts pX activates transcription and confers an evident growth advantage without loss of cell viability. Although pX protein was not detectable in the experimental conditions we used, our results indicate that its expression affects both cell growth and cell death control.

International audience; HDAC inhibitors (HDACi) exert beneficial effects in mdx mice, by promoting endogenous regeneration; however, the cellular determinants of HDACi activity on dystrophic muscles have not been determined. We show that fibroadipogenic progenitors (FAP) influence the regeneration potential of satellite cells during disease progression in mdx mice and mediate HDACi ability to selectively promote regeneration at early stages of disease. FAPs from young mdx mice promote, while FAPs from old mdx mice repress, satellite cell-mediated formation of myotubes. In young mdx mice HDACi inhibited FAP adipogenic potential, while enhancing their ability to promote differentiation of adjacent satellite cells, through upregulation of the soluble factor follistatin. By contrast, FAPs from old mdx mice were resistant to HDACi-mediated inhibition of adipogenesis and constitutively repressed satellite cell-mediated formation of myotubes. We show that transplantation of FAPs from regenerating young muscles restored HDACi ability to increase myofibre size in old mdx mice. These results reveal that FAPs are key cellular determinants of disease progression in mdx mice and mediate a previously unappreciated stage-specific beneficial effect of HDACi in dystrophic muscles.

Previous works have established a unique function of MyoD in the control of muscle gene expression during DNA damage response in myoblasts. Phosphorylation by DNA damage-activated ABL tyrosine kinase transiently inhibits MyoD-dependent activation of transcription in response to genotoxic stress. We show here that ABL-MyoD signaling is also an essential component of the DNA repair machinery in myoblasts exposed to genotoxic stress. DNA damage promoted the recruitment of MyoD to phosphorylated Nbs1 (pNbs1)-containing repair foci, and this effect was abrogated by either ABL knockdown or the ABL kinase inhibitor imatinib. Upon DNA damage, MyoD and pNbs1 were detected on the chromatin to MyoD target genes without activating transcription. DNA damage-mediated tyrosine phosphorylation was required for MyoD recruitment to target genes, as the ABL phosphorylation-resistant MyoD mutant (MyoD Y30F) failed to bind the chromatin following DNA damage, while retaining the ability to activate transcription in response to differentiation signals. Moreover, MyoD Y30F exhibited an impaired ability to promote repair in a heterologous system, as compared with MyoD wild type (WT). Consistently, MyoD-null satellite cells (SCs) displayed impaired DNA repair that was rescued by reintroduction of MyoD WT but not by MyoD Y30F. In addition, inhibition of ABL kinase prevented MyoD WT-mediated rescue of DNA repair in MyoD-null SCs. These results identify an unprecedented contribution of MyoD to DNA repair and suggest that ABL-MyoD signaling coordinates DNA repair and transcription in myoblasts.

In this study, we characterized the molecular events involved in the activation of the ubiquitous transcription factor NF-kappa B by the viral transactivator pX. pX expression in HeLa cells determines a manyfold increase in NF-kappa B-dependent transcription, which is associated with an increase in p50/p65 heterodimer DNA-binding activity. Since the I kappa B-alpha inhibitory subunit proteolytic degradation, which follows its phosphorylation/modification, is a key event in NF-kappa B activation by different stimuli (such as growth factors, phorbol esters, tumor necrosis factor, UV irradiation, and oxygen radicals), we investigated pX effects on I kappa B-alpha, as well as the possible involvement of known signalling pathways in pX-induced NF-kappa B-dependent transcription. We observed that although pX had no direct effect on p50 or p65, it was able to restore the I kappa B-alpha-suppressed p50/p65 activity. More directly, the stable expression of pX in HeLa cells resulted in reduced levels of I kappa B-alpha in the cytoplasm. Pretreatment of the cells with H7, calphostin C, tyrphostin 25, or N-acetylcysteine did not impair the effects of pX on NF-kappa B, thus ruling out the involvement of protein kinase C, tyrosine kinases, and oxygen radicals. Finally, while most of the known NF-kappa B-activating agents converge on Raf-1 protein kinase, when Raf-1 activity is blocked by overexpression of a dominant negative mutant, the effects of pX on NF-kappa B are not impaired. Thus, we suggest that although pX is able to activate the Ras/Raf-1-signalling pathway, it triggers NF-kappa B activation by an as yet unidentified Raf-1-independent pathway.

Developmental biologists have defined many of the diffusible and transcription factors that control muscle differentiation, yet we still have only rudimentary knowledge of the mechanisms that dictate whether a myogenic progenitor cell forms muscle versus alternate lineages, including those that can be pathological in a state of disease or degeneration. Clues about the molecular basis for lineage determination in muscle progenitors are only now emerging from studies of chromatin modifications that avail myogenic genes for transcription, together with analysis of the composition and activities of the chromatin-modifying complexes themselves. Here we review recent progress on muscle determination and explore a unifying theme that environmental cues from the stem or progenitor niche control the selection of specific subunit variants of the switch/sucrose nonfermentable (SWI/SNF) chromatin-modifying complex, creating a combinatorial code that dictates whether cells adopt myogenic versus nonmyogenic cell fates. A key component of the code appears to be the mutually exclusive usage of the a, b, and c variants of the 60-kD structural subunit BAF60 (BRG1/BRM-associated factor 60), of which BAF60c is essential to activate both skeletal and cardiac muscle programs. Since chromatin remodeling governs myogenic fate, the combinatorial assembly of the SWI/SNF complex might be targeted to develop drugs aimed at the therapeutic reduction of compensatory fibrosis and fatty deposition in chronic muscular disorders.

Recently, numerous interdisciplinary approaches that include integration of bioinformatics, epigenetics, and molecular and cellular biology have started to shed light on the complex dynamics that regulate cellular processes such as cell “stemness,” reprogramming, and differentiation. From the sequencing of the first complete genomes to the recent high-throughput genomic and proteomic approaches, a huge amount of information has been generated to improve our understanding of how the eukaryotic genome works to activate and coordinate specific programs of gene expression. The focus of this review is to provide an overview of the epigenetic regulatory mechanisms controlling crucial cellular fate decisions, in particular during differentiation and reprogramming. A special emphasis has been given to skeletal myogenesis, as it provides an amenable model for in vitro and in vivo studies. Y. Ito et al. in “A system approach and skeletal myogenesis” provide an overview of the most common approaches currently used to address transcriptional regulation. They discuss deep sequencing and microarray-based methods, cell-based high throughput assays, and the generation of in situ gene expression databases as potent tools to dissect complex networks of gene expression. Using skeletal muscle as a paradigm, they highlight the main discoveries obtained through the different methods and underline the advantages of an integrated approach to obtain a full picture of cellular differentiation. An increasing number of studies suggest an epigenetic component in the development of many human diseases. In the review article “Epigenetic alterations in muscular disorders,” Dr. C. Lanzuolo discusses how altered epigenetic mechanisms contribute to human pathology and in particular to the development and progression of neuromuscular disorders. Muscular dystrophies arise from either mutations of proteins that play a role in the integrity of the sarcolemma—the plasma membrane that wraps the myofibers in skeletal muscles—or are due to a dysfunction within the cell nucleus. Dr. C. Lanzuolo focuses on the so-called nuclear muscular dystrophies and she analyzes the possible contribution of an important group of epigenetic modifiers, the polycomb-group (PcG) complexes, to the pathogenesis of these diseases. The dissection of the aberrant epigenetic profiles associated to several human pathologies has contributed to the design of experimental therapies with epigenetic drugs. The review “Tackling skeletal muscle cells epigenome in the next-generation sequencing era” by R. Fittipaldi and G. Caretti points to the significance of genome-wide study of the epigenetic modifications and the transcription factors that are activated during skeletal muscle differentiation. The authors discuss on the new genome-wide technologies to monitor the epigenetic landscape that characterizes myoblast-to-myotube transition in order to obtain innovative information on molecular mechanisms orchestrating biological processes, such myogenic differentiation. The fine regulation of a myogenic gene such as myogenin is discussed in “Turning on myogenin in muscle: A paradigm for understanding mechanisms of tissue-specific gene expression” by H. Faralli and F. J. Dilworth. Transcription factors, cofactors, and epigenetic modifications contribute to regulate muscle-specific gene expression during development. In particular, the authors highlight the fact that a tissue-specific factors such as MyoD cooperate with more ubiquitously expressed proteins (Six4, Mef2, and Pbx1) to establish a transcriptionally permissive environment to ensure proper spatiotemporal myogenin expression. The current challenge of epigenetics is how to analyse and extract biological knowledge from the large volumes of data produced with new high-throughput technologies. In this regard, the paper of S. Althammer et al., “Predictive models of gene regulation from high-throughput epigenomics data” describes a computational framework using integrative tools and machine learning algorithms that facilitate the systematic analysis of high-throughput sequencing epigenetic data for integrative studies. Their analysis shows a different epigenetic code of expression for intron-less and intron-containing genes, with more prominent differences for genes with low GC content (LGC) in their promoter, showing for instance that at the promoter regions of LGC intron-less genes, H3K36me3 and H3K4me1 are the most informative marks while in polyadenylation sites of expressed genes, the H3K36me3 signal is much weaker than in intron-containing genes. Based on epigenetic data, their model predicts very strongly gene expression between two conditions; therefore, this approach can be applied to study gene expression in different developmental stages, disease states, or treatments. Of tremendous relevance for its therapeutic potential, is the possibility to manipulate cell identity. In the review titled “The stability of the induced epigenetic programs”, M. J. Barrero discusses the epigenetic changes that occur during differentiation and the way back upon cellular reprogramming emphasizing on the thin regulation of pluripotent and lineage-specific genes expression to ensure a correct differentiation during development and in adult life. “The epigenetic regulation of B lymphocyte differentiation, transdifferentiation, and reprogramming” by B. Barneda-Zahonero et al. reviews the hierarchical network of transcription factors that mediate the epigenetic signature regulating the transcriptome during B cell development. B lymphocytes are highly specialized terminally differentiated cells responsible for generating high-affinity antibodies, providing the humoral immunity against pathogens. Every day the human body generates millions of B lymphocytes from precursor hematopoietic stem cells by a differentiation process that is a tightly regulated. This multistep differentiation process, vital to immunity, combines the successive expression of specific transcriptional factors and associated epigenetic changes to restrict the developmental potential of lymphoid progenitors to the B cell pathway by repressing B-lineage-inappropriate genes, while simultaneously promoting B cell development by activating B-lymphoid-specific genes. Lucia Latella Daniela Palacios Sonia Forcales Pier Lorenzo Puri

Reduction of nutrient intake without malnutrition positively influences lifespan and healthspan from yeast to mice and exerts some beneficial effects also in humans. The AMPK-FoxO axis is one of the evolutionarily conserved nutrient-sensing pathways, and the FOXO3A locus is associated with human longevity. Interestingly, FoxO3A has been reported to be also a mitochondrial protein in mammalian cells and tissues. Here we report that glucose restriction triggers FoxO3A accumulation into mitochondria of fibroblasts and skeletal myotubes in an AMPK-dependent manner. A low-glucose regimen induces the formation of a protein complex containing FoxO3A, SIRT3, and mitochondrial RNA polymerase (mtRNAPol) at mitochondrial DNA-regulatory regions causing activation of the mitochondrial genome and a subsequent increase in mitochondrial respiration. Consistently, mitochondrial transcription increases in skeletal muscle of fasted mice, with a mitochondrial DNA-bound FoxO3A/SIRT3/mtRNAPol complex detectable also in vivo. Our results unveil a mitochondrial arm of the AMPK-FoxO3A axis acting as a recovery mechanism to sustain energy metabolism upon nutrient restriction.

The capacity of adult skeletal muscle for regeneration appears to be limited, with progressive impairment in repair efficiency of injured muscles observed in chronic muscular disorders and during aging. While satellite cells, the committed adult muscle stem cells, are the main direct cell source supporting the regenerative potential of adult skeletal muscles, the characterization of the cell types and signals that constitute the functional "niche" of satellite cells is currently the object of intense investigation. Recent studies have identified a functional relationship between satellite cells and various cell types located in key anatomical position, such as the interstitium of skeletal muscles. This heterogeneous population of muscle interstitial cells (MICs) appears to retain an intrinsic multipotency within the mesodermal lineage, and their direct or indirect contribution to myofiber turnover, repair and degeneration has been suggested by many studies that will be reviewed here. Given the existing gap of knowledge on lineage identity and functional properties of MICs, their detailed characterization at the single cell level holds the promise to provide key insight into the composition of this heterogeneous population and the dynamic transition through distinct sub-populations in healthy, diseased and aging muscles. This review provides an overview of the results of various studies describing the phenotype and the function of cells isolated from skeletal muscle interstitium, and discusses the importance of single cell transcription profiling in order to decipher the functional and phenotypical heterogeneity of muscle interstitial cells (MICs).

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.

Upon exposure to genotoxic stress, skeletal muscle progenitors coordinate DNA repair and the activation of the differentiation program through the DNA damage-activated differentiation checkpoint, which holds the transcription of differentiation genes while the DNA is repaired. A conceptual hurdle intrinsic to this process relates to the coordination of DNA repair and muscle-specific gene transcription within specific cell cycle boundaries (cell cycle checkpoints) activated by different types of genotoxins. Here, we show that, in proliferating myoblasts, the inhibition of muscle gene transcription occurs by either a G 1- or G 2-specific differentiation checkpoint. In response to genotoxins that induce G 1 arrest, MyoD binds target genes but is functionally inactivated by a c-Abl-dependent phosphorylation. In contrast, DNA damage-activated G 2 checkpoint relies on the inability of MyoD to bind the chromatin at the G 2 phase of the cell cycle. These results indicate an intimate relationship between DNA damage-activated cell cycle checkpoints and the control of tissue-specific gene expression to allow DNA repair in myoblasts prior to the activation of the differentiation program.

Histone deacetylases inhibitors (HDACi) include a growing number of drugs that share the ability to inhibit the enzymatic activity of some or all the HDACs. Experimental and preclinical evidence indicates that these epigenetic drugs not only can be effective in the treatment of malignancies, inflammatory diseases and degenerative disorders, but also in the treatment of genetic diseases, such as muscular dystrophies. The ability of HDACi to counter the progression of muscular dystrophies points to HDACs as a crucial link between specific genetic mutations and downstream determinants of disease progression. It also suggests the contribution of epigenetic events to the pathogenesis of muscular dystrophies. Here we describe the experimental evidence supporting the key role of HDACs in the control of the transcriptional networks underlying the potential of dystrophic muscles either to activate compensatory regeneration or to undergo fibroadipogenic degeneration. Studies performed in mouse models of Duchenne muscular dystrophy (DMD) indicate that dystrophin deficiency leads to deregulated HDAC activity, which perturbs downstream networks and can be restored directly, by HDAC blockade, or indirectly, by reexpression of dystrophin. This evidence supports the current view that HDACi are emerging candidate drugs for pharmacological interventions in muscular dystrophies, and reveals unexpected common beneficial outcomes of pharmacological treatment or gene therapy.

Muscle regeneration relies on adult muscle stem (satellite) cells. Inflammatory cues released within the regenerative microenvironment, such as TNFα, instruct different components of the satellite cell niche toward specialized tasks by regulating specific subsets of genes in each individual cell type. However, how regeneration cues are deciphered and interpreted by the multitude of cell types within the regenerative environment is unknown. We have recently identified an inflammation-activated signaling, consisting of p38α-mediated recruitment of polycomb repressive complex 2 (PRC2) to the Pax7 promoter, in satellite cells. Here we show that p38α-PRC2 regulation of Pax7 expression is restricted to a discrete stage of satellite cell-mediated regeneration. In activated satellite cells, Pax7 locus shows a "bivalent" chromatin signature, with co-existence of H3-K27(3me) and H3-K4(3me), that appears to confer responsiveness to p38α-PRC2 signaling. p38α activation resolves bivalence to H3-K27(3me) which results in Pax7 repression, while p38α blockade promotes Pax7 expression by preventing PRC2-mediated H3-K27(3me) and leading to relative increase in H3-K4(3me). Interestingly, in satellite cell-derived myotubes Pax7 expression cannot be re-induced by p38α blockade, revealing a post-mitotic resistance of Pax7 gene to inflammatory cues. Likewise, in other cell types, such as muscle-derived fibroblasts, Pax7 locus is constitutively repressed by PRC2 and is unresponsive to p38α signaling. Finally, we show that Pax7 repression in embryonic stem cells is not directed by p38α signaling, although it is mediated by PRC2. This evidence indicates a cell type- and differentiation-stage specific control of Pax7 transcription by the p38α-PRC2.

Publisher Summary This chapter focuses on the epigenetic regulation of muscle regeneration, as a paradigm for other tissues. Tissue and organ precursors are often referred to as adult “somatic stem cells” (SSCs) because of their functional analogies with the embryonic stem cells (ESCs), including the ability for long-term self-renewal and the potential to commit into multiple lineages. However, while ESCs are totipotent and can adopt virtually all lineages, SSCs are located within differentiated tissues and organs, have restricted “potency,” and provide an immediate reservoir for repair upon injury or disease associated events. Lineage commitment, migration, proliferation, and differentiation of SSCs are regulated by the coordinated activation and repression of distinct subsets of genes in response to cues released within the regenerative environment. The extensive knowledge gained on muscle stem cells (MSCs) makes muscle regeneration an interesting paradigm to unveil general principles of epigenetic regulation of tissue regeneration and to investigate strategies for regenerative medicine using SSCs.

Previous work linked nitric oxide (NO) signaling to histone deacetelyases (HDACs) in the control of tissue homeostasis and suggested that deregulation of this signaling contributes to human diseases. In the previous issue of Chemistry & Biology, Kong and colleagues showed that coordinated NO signaling and histone acetylation are required for proper cranial neural crest development and craniofacial morphogenesis and suggested that alterations of NO/acetylation network can contribute to the pathogenesis of craniofacial malformations.

During embryogenesis a timely and coordinated expression of different subsets of genes drives the formation of skeletal muscles in response to developmental cues. In this review, we will summarize the most recent advances on the "epigenetic network" that promotes the transcription of selective groups of genes in muscle progenitors, through the concerted action of chromatin-associated complexes that modify histone tails and microRNAs (miRNAs). These epigenetic players cooperate to establish focal domains of euchromatin, which facilitates gene transcription, and large portions of heterochromatin, which precludes inappropriate gene expression. We also discuss the analogies and differences in the transcriptional and the epigenetic networks driving developmental and adult myogenesis. The elucidation of the epigenetic basis controlling skeletal myogenesis during development and adult life will facilitate experimental strategies toward generating muscle stem cells, either by reprogramming embryonic stem cells or by inducing pluripotency in adult skeletal muscles. During embryogenesis a timely and coordinated expression of different subsets of genes drives the formation of skeletal muscles in response to developmental cues. In this review, we will summarize the most recent advances on the "epigenetic network" that promotes the transcription of selective groups of genes in muscle progenitors, through the concerted action of chromatin-associated complexes that modify histone tails and microRNAs (miRNAs). These epigenetic players cooperate to establish focal domains of euchromatin, which facilitates gene transcription, and large portions of heterochromatin, which precludes inappropriate gene expression. We also discuss the analogies and differences in the transcriptional and the epigenetic networks driving developmental and adult myogenesis. The elucidation of the epigenetic basis controlling skeletal myogenesis during development and adult life will facilitate experimental strategies toward generating muscle stem cells, either by reprogramming embryonic stem cells or by inducing pluripotency in adult skeletal muscles.

SummaryWe created a whole-mount in situ hybridization (WISH) database, termed EMBRYS, containing expression data of 1520 transcription factors and cofactors expressed in E9.5, E10.5, and E11.5 mouse embryos—a highly dynamic stage of skeletal myogenesis. This approach implicated 43 genes in regulation of embryonic myogenesis, including a transcriptional repressor, the zinc-finger protein RP58 (also known as Zfp238). Knockout and knockdown approaches confirmed an essential role for RP58 in skeletal myogenesis. Cell-based high-throughput transfection screening revealed that RP58 is a direct MyoD target. Microarray analysis identified two inhibitors of skeletal myogenesis, Id2 and Id3, as targets for RP58-mediated repression. Consistently, MyoD-dependent activation of the myogenic program is impaired in RP58 null fibroblasts and downregulation of Id2 and Id3 rescues MyoD's ability to promote myogenesis in these cells. Our combined, multi-system approach reveals a MyoD-activated regulatory loop relying on RP58-mediated repression of muscle regulatory factor (MRF) inhibitors.

Current evidence supports the therapeutic potential of pharmacological interventions that counter the progression of genetic disorders by promoting regeneration of the affected organs or tissues. The rationale behind this concept lies on the evidence that targeting key events downstream of the genetic defect can compensate, at least partially, the pathological consequence of the related disease. In this regard, the beneficial effect exerted on animal models of muscular dystrophy by pharmacological strategies that enhance muscle regeneration provides an interesting paradigm. In this review, we describe and discuss the potential targets of pharmacological strategies that promote regeneration of dystrophic muscles and alleviate the consequence of the primary genetic defect. Regenerative pharmacology provides an immediate and suitable therapeutic opportunity to slow down the decline of muscles in the present generation of dystrophic patients, with the perspective to hold them in conditions such that they could benefit of future, more definitive, therapies.

The overlapping histological and biochemical features underlying the beneficial effect of deacetylase inhibitors and NO donors in dystrophic muscles suggest an unanticipated molecular link among dystrophin, NO signaling, and the histone deacetylases (HDACs). Higher global deacetylase activity and selective increased expression of the class I histone deacetylase HDAC2 were detected in muscles of dystrophin-deficient MDX mice. In vitro and in vivo siRNA-mediated down-regulation of HDAC2 in dystrophic muscles was sufficient to replicate the morphological and functional benefits observed with deacetylase inhibitors and NO donors. We found that restoration of NO signaling in vivo, by adenoviral-mediated expression of a constitutively active endothelial NOS mutant in MDX muscles, and in vitro, by exposing MDX-derived satellite cells to NO donors, resulted in HDAC2 blockade by cysteine S-nitrosylation. These data reveal a special contribution of HDAC2 in the pathogenesis of Duchenne muscular dystrophy and indicate that HDAC2 inhibition by NO-dependent S-nitrosylation is important for the therapeutic response to NO donors in MDX mice. They also define a common target for independent pharmacological interventions in the treatment of Duchenne muscular dystrophy.

Studies on DNA damage responses in proliferating cells have revealed the relationship between sensing and repair of the DNA lesions and the regulation of the cell cycle, leading to the discovery and molecular characterization of the DNA damage-activated cell cycle checkpoints. Much less is known about the DNA damage response in progenitors of differentiated cells, in which cell cycle arrest is a critical signal to trigger the differentiation program, and in terminally differentiated cells, which are typically post-mitotic. How DNA lesions are detected, processed and repaired in these cells, the functional impact of DNA damage on transcription of differentiation-specific genes, how these events are coordinated at the molecular level, the consequence of defective DNA damage response on tissue-specific functions and its potential relationship with age-related diseases are currently open questions. In particular the biological complexity inherent to the global genome reprogramming of tissue progenitors, such as embryonic or adult stem cells, suggests the importance of an accurate DNA damage response at the transcription level in these cells to ensure the genomic integrity of regenerating tissues.

The pivotal role of the Retinoblastoma gene product pllO (pRb) in cellular differentiation has been postulated since the identification of pRb as a target of oncogenic events. The demonstration of the essential role of pRb during terminal differentiation of many tissues appeared evident along with the identification of the critical properties of pRb in regu-lating cell cycle progression and apoptosis. Permanent cell cycle arrest and resistance to both tumor formation and apoptosis are three cardinal features of terminal differentiation. Upon functional or genetic inactivation of pRb, cells from skeletal muscle, neuronal and hematopoi-etic lineages exhibit higher extent of apoptosis, fail to permanently exit the cell cycle and showincomplete differentiation.

During muscle regeneration, the mechanism integrating environmental cues at the chromatin of muscle progenitors is unknown. We show that inflammation-activated MKK6-p38 and insulin growth factor 1 (IGF1)-induced PI3K/AKT pathways converge on the chromatin of muscle genes to target distinct components of the muscle transcriptosome. p38 alpha/beta kinases recruit the SWI/SNF chromatin-remodeling complex; AKT1 and 2 promote the association of MyoD with p300 and PCAF acetyltransferases, via direct phosphorylation of p300. Pharmacological or genetic interference with either pathway led to partial assembly of discrete chromatin-bound complexes, which reflected two reversible and distinct cellular phenotypes. Remarkably, PI3K/AKT blockade was permissive for chromatin recruitment of MEF2-SWI/SNF complex, whose remodeling activity was compromised in the absence of MyoD and acetyltransferases. The functional interdependence between p38 and IGF1/PI3K/AKT pathways was further established by the evidence that blockade of AKT chromatin targets was sufficient to prevent the activation of the myogenic program triggered by deliberate activation of p38 signaling.

Recent evidence has revealed the importance of reciprocal functional interactions between different types of mononuclear cells in coordinating the repair of injured muscles. In particular, signals released from the inflammatory infiltrate and from mesenchymal interstitial cells (also known as fibro-adipogenic progenitors (FAPs)) appear to instruct muscle stem cells (satellite cells) to break quiescence, proliferate and differentiate. Interestingly, conditions that compromise the functional integrity of this network can bias muscle repair toward pathological outcomes that are typically observed in chronic muscular disorders, that is, fibrotic and fatty muscle degeneration as well as myofiber atrophy. In this review, we will summarize the current knowledge on the regulation of this network in physiological and pathological conditions, and anticipate the potential contribution of its cellular components to relatively unexplored conditions, such as aging and physical exercise.

A polyamide-chlorambucil conjugate (1R-Chl) arrests a wide range of human cancer cell lines at the G2/M phase of the cell cycle and downregulates histone H4c gene expression. However, an siRNA against H4c mRNA causes G1/S arrest. Here, we report that 1R-Chl downregulates H4c prior to G2/M arrest. G2/M arrest is the result of extensive DNA damage by 1R-Chl, which leads to phosphorylation of H2A.X at serine 139, recruitment of the Nbs1 repair protein, and a cascade of unknown events culminating with cdc2 phosphorylation at tyrosine 15 and abolishment of cdc2 kinase activity. A control polyamide-Chl conjugate, which neither binds to the H4c gene nor has an anti-proliferative effect by itself, causes G2/M arrest when cells are treated with siRNAs specific for H3 or H4c.

Pharmacological interventions that increase myofiber size counter the functional decline of dystrophic muscles(1,2). We show that deacetylase inhibitors increase the size of myofibers in dystrophin-deficient (MDX) and alpha-sarcoglycan (alpha-SG)-deficient mice by inducing the expression of the myostatin antagonist follistatin(3) in satellite cells. Deacetylase inhibitor treatment conferred on dystrophic muscles resistance to contraction-coupled degeneration and alleviated both morphological and functional consequences of the primary genetic defect. These results provide a rationale for using deacetylase inhibitors in the pharmacological therapy of muscular dystrophies. ispartof: Nature Medicine vol:12 issue:10 pages:1147-1150 ispartof: location:United States status: published

Cellular differentiation entails an extensive reprogramming of the genome toward the expression of discrete subsets of genes, which establish the tissue-specific phenotype. This program is achieved by epigenetic marks of the chromatin at particular loci, and is regulated by environmental cues, such as soluble factors and cell-to-cell interactions. How the intracellular cascades convert the myriad of external stimuli into the nuclear information necessary to reprogram the genome toward specific responses is a question of biological and medical interest. The elucidation of the signaling converting cues from outside the cells into chromatin modifications at individual promoters holds the promise to unveil the targets for selective pharmacological interventions to modulate gene expression for therapeutic purposes. Enhancing muscle regeneration and preventing muscle breakdown are important goals in the therapy of muscular diseases, cancer-associated cachexia and aging-associated sarcopenia. We will summarize the recent progress of our knowledge of the regulation of gene expression by intracellular cascades elicited by external cues during skeletal myogenesis. And will illustrate the potential importance of targeting the chromatin signaling in regenerative medicine—e.g. to boost muscle regeneration.

p300 and CBP are general transcriptional coactivators implicated in different cellular processes, including regulation of the cell cycle, differentiation, tumorigenesis, and apoptosis. Posttranslational modifications such as phosphorylation are predicted to select a specific function of p300/CBP in these processes; however, the identification of the kinases that regulate p300/CBP activity in response to individual stimuli and the physiological significance of p300 phosphorylation have not been elucidated. Here we demonstrate that the cardiotoxic anticancer agent doxorubicin (adriamycin) induces the phosphorylation of p300 in primary neonatal cardiomyocytes. Hyperphosphorylation precedes the degradation of p300 and parallels apoptosis in response to doxorubicin. Doxorubicin-activated p38 kinases alpha and beta associate with p300 and are implicated in the phosphorylation-mediated degradation of p300, as pharmacological blockade of p38 prevents p300 degradation. p38 phosphorylates p300 in vitro at both the N and C termini of the protein, and enforced activation of p38 by the constitutively active form of its upstream kinase (MKK6EE) triggers p300 degradation. These data support the conclusion that p38 mitogen-activated protein kinase regulates p300 protein stability and function in cardiomyocytes undergoing apoptosis in response to doxorubicin.

The balance between acetylation and deacetylation of histone and nonhistone proteins controls gene expression in a variety of cellular processes, with transcription being activated by acetyltransferases and silenced by deacetylases. We report here the formation and enzymatic characterization of a complex between the acetyltransferase p300 and histone deacetylases. The C/H3 region of p300 was found to co-purify deacetylase activity from nuclear cell extracts. A prototype of class I histone deacetylases, HDAC1, interacts with p300 C/H3 domain in vitro and in vivo. The p300-binding protein E1A competes with HDAC1 for C/H3 binding; and, like E1A, HDAC1 overexpression interferes with either activation of Gal4p300 fusion protein or p300-dependent co-activation of two C/H3-binding proteins, MyoD and p53. The exposure to deacetylase inhibitors could reverse the dominant-negative effect of a C/H3 fragment insulated from the rest of the molecule, on MyoD- and p53-dependent transcription, whereas inhibition by E1A was resistant to trichostatin A. These data support the hypothesis that association between acetyltransferases and deacetylases can control the expression of genes implicated in cellular growth and differentiation, and suggest that the dominant-negative effect of the p300 C/H3 fragment relies on deacetylase recruitment.

Fusion of undifferentiated myoblasts into multinucleated myotubes is a prerequisite for developmental myogenesis and postnatal muscle growth. We report that deacetylase inhibitors favor the recruitment and fusion of myoblasts into preformed myotubes. Muscle-restricted expression of follistatin is induced by deacetylase inhibitors and mediates myoblast recruitment and fusion into myotubes through a pathway distinct from those utilized by either IGF-1 or IL-4. Blockade of follistatin expression by RNAi-mediated knockdown, functional inactivation with either neutralizing antibodies or the antagonist protein myostatin, render myoblasts refractory to HDAC inhibitors. Muscles from animals treated with the HDAC inhibitor trichostatin A display increased production of follistatin and enhanced expression of markers of regeneration following muscle injury. These data identify follistatin as a central mediator of the fusigenic effects exerted by deacetylase inhibitors on skeletal muscles and establish a rationale for their use to manipulate skeletal myogenesis and promote muscle regeneration.

Background Rhabdomyosarcoma is a common malignancy in children. There are two major types of rhabdomyosarcomas, the embryonal and the alveolar, differing in cytogenetic and morphologic features. The alveolar type of rhabdomyosarcoma is frequently associated with chromosome translocation t(2, 13) and poor clinical prognosis. Pathogenesis of rhabdomyosarcoma remains obscure, and especially it occurs in the location where skeletal muscle is absent. We report here that there is a high frequency of association of rhabdomyosarcoma with ataxia telangiectasia mutated (ATM) gene mutation/deletion. Result Totally 17 cases of rhabdomyosarcoma specimens were studied by immunohistochemical or immunofluorescent staining with ATM antibody and revealed that 7 of the 17 cases were negative for ATM expression (41%). Further analyses of rhabdomyosarcoma cell lines with RT-PCR revealed that in Rh30 cells, an alveolar rhabdomyosarcoma cell line, there are three separate deletions/mutations of the ATM mRNA. Western blotting analysis of the Rh30 cellular extract with anti-ATM antibody showed that there is an aberrant form of ATM protein within the Rh30 cells that are smaller than normal control. Conclusion These results suggest a link of ATM gene deletion/mutation with rhabdomyosarcoma, and since ATM kinase is a crucial regulatory protein in DNA damage repair signaling pathway, and ATM deletion/mutation may contribute to pathogenesis of rhabdomyosarcoma.

Cell-cycle checkpoints help to protect the genomes of proliferating cells under genotoxic stress. In multicellular organisms, cell proliferation is often directed toward differentiation during development and throughout adult homeostasis. To prevent the formation of differentiated cells with genetic instability, we hypothesized that genotoxic stress may trigger a differentiation checkpoint. Here we show that exposure to genotoxic agents causes a reversible inhibition of myogenic differentiation. Muscle-specific gene expression is suppressed by DNA-damaging agents if applied prior to differentiation induction but not after the differentiation program is established. The myogenic determination factor, MyoD (encoded by Myod1), is a target of the differentiation checkpoint in myoblasts. The inhibition of MyoD by DNA damage requires a functional c-Abl tyrosine kinase (encoded by Abl1), but occurs in cells deficient for p53 (transformation-related protein 53, encoded by Trp53) or c-Jun (encoded by the oncogene Jun). These results support the idea that genotoxic stress can regulate differentiation, and identify a new biological function for DNA damage–activated signaling network.

Nuclear acetyltransferases promote and deacetylases inhibit skeletal muscle-gene expression, suggesting the potential effectiveness of deacetylase inhibitors (DIs) in modulating skeletal myogenesis. Surprisingly, previous studies have indicated that DIs suppress myogenesis. The recent observations that histone deacetylases associate with the muscle-regulatory proteins MyoD and MEF2C only in undifferentiated myoblasts prompted us to evaluate the effect of DIs at distinct stages of the myogenic program. We found that exposure of established rodent and human muscle cells to distinct DIs has stage-specific effects. Exposure of undifferentiated skeletal myoblasts to DIs, followed by incubation in differentiation medium, enhanced the expression of muscle-specific reporters and increased the levels of endogenous muscle proteins, leading to a dramatic increase in the formation of multinucleated myotubes. By contrast, simultaneous exposure of muscle cells to differentiation medium and DIs inhibited the myogenic program. Likewise, embryos exposed in utero to nonteratogenic doses of DI at the early stages of somitic myogenesis (embryonic day 8.5) exhibited an increased number of somites and augmented expression of a muscle-specific transgene as well as endogenous muscle genes. The functional effects induced by DIs were mirrored by changes in the state of acetylation of histones present at a muscle-gene enhancer and of MyoD itself. These results represent the first evidence that DIs can enhance muscle differentiation and suggest the rationale for their use in manipulating adult and embryonic skeletal myogenesis.

During development, skeletal muscles adapt to stage-specific functional and metabolic challenges by switching the expression of specific subset of genes. The mechanism that governs these changes is still enigmatic. In a recent issue of Cell, Messina and coworkers shed light on this issue through the identification of a transcription factor--NFix--that coordinates the switch in gene expression at the transition from embryonic to fetal myoblasts.

Abstract —The vascular endothelial growth factor receptor Flk-1/KDR is highly expressed during development and almost disappears in adult tissues. Despite its biological relevance, little is known about the molecular mechanisms controlling its expression. In the present work, it is shown that cAMP response element binding protein (CREB) and nuclear factor-κB (NF-κB)–related antigens bind specific sequences in the Flk-1/KDR promoter. Functional studies demonstrate that cAMP represses whereas tumor necrosis factor-α, an activator of NF-κB, stimulates promoter activity. Histone acetyltransferases (HATs) P/CAF and CBP/p300 together with p65/RelA, the catalytic subunit of NF-κB, increase Flk-1/KDR promoter activity 10- to 20-fold. Consistently, inhibition by cAMP is reverted by increasing intracellular HATs and is completely abolished by site-specific mutagenesis of the cAMP response element. In contrast, specific mutations in the NF-κB response element abolish responsiveness to p65/RelA and HATs without affecting cAMP-dependent repression. These results suggest that opposing signaling pathways, activating NF-κB or CREB and requiring HAT molecules, control Flk-1/KDR promoter activity. The full text of this article is available at http://www.circresaha.org.

In the past decade, much has been learned about the molecular mechanisms that govern myogenesis owing mainly to the discovery of two groups of myogenic transcription factors (4, 45, 62). The first group includes the myogenic regulatory factors (MRFs), which belong to the basic helix-loop-helix (bHLH) protein family. This MRF group consists of four members: Myf5, MyoD, myogenin, and MRF4, all of which are specifically expressed in skeletal muscles. One of the unique features of these MRFs is that when they are ectopically expressed in fibroblasts or certain other nonmuscle cells, each has the ability to initiate the myogenic program and convert nonmuscle cells to myogenic derivatives (9, 59). Myogenic bHLH proteins heterodimerize with other ubiquitous bHLH proteins (like the E2A gene products, E12, and E47) to efficiently bind a consensus DNA site: CANNTG (also called the E box) (4, 33). The second group of transcription factors important in muscle differentiation consists of four different myocyte enhancer binding factor 2 (MEF2) proteins, which belong to the MADS box family (7). The MEF2 proteins (MEF2A, MEF2B, MEF2C, and MEF2D) form homo- or heterodimers which bind to a consensus AT-rich sequence (MEF2 site), found in the promoters of many muscle-specific genes. Myogenic bHLH and MEF2 cooperate to synergistically activate muscle-specific transcription through interactions mediated by the basic region and the MADS domain, respectively (44, 45). The study of muscle differentiation has benefited from the availability of several myogenic cell lines which allow biochemical dissection of the myogenic pathway. These myogenic cell lines (e.g., mouse C2C12 and rat L6) can be induced to differentiate by withdrawal of mitogens, such as serum. Many negative regulators of myogenesis (e.g., Id, Twist, oncogenic Ras, and the viral proteins E1A and simian virus 40 T antigen) have been identified (1, 34). However, little is known about the intracellular components that positively regulate the activities of myogenic transcription factors, especially those that are involved in receiving and transducing extracellular cues. One extracellular signal that positively regulates myogenesis is insulin-like growth factor (IGF). IGF activates the phosphatidylinositol-3 kinase (PI3K) signaling pathway, which is required for myogenesis (11, 29, 30). However, how this signaling pathway influences myogenic transcription remains to be defined. In eukaryotic cells, mitogen-activated protein kinases (MAPKs) are components of several important signaling pathways that relay extracellular cues to transcription factors in the nucleus (24, 25, 32, 41, 51). For mammals, three MAPK pathways, including the extracellular signal-regulated kinases (ERK1 and -2), the Jun–N-terminal kinases (JNK1, -2, and -3), and the p38 isoforms (α, β, γ, and δ) have been characterized (references 51 and 43 and references therein). In general, each group of MAPKs is activated by two homologous MAPK kinases (MKKs [also called MAPKKs]), including MEK1 and -2 for the ERKs, JNK kinase 1 and 2 (JNKK1 and -2) (or MKK4 and -7) for the JNKs, and MKK3 and -6 for the p38s (26, 51). Except for JNKK1, which can also activate p38 in vitro (14, 38), all other MKKs specifically activate their MAPK targets and have little activity on members of other groups. The ERK pathway has been implicated in the control of muscle differentiation, although its role remains controversial, with some reports suggesting a positive function (5, 11) and others suggesting a negative function (18). p38 has also been reported to activate certain MEF2 family members (21, 40, 48, 61, 67) and to stimulate muscle differentiation (12, 64). However, the upstream signals which regulate p38 activation at the onset of muscle differentiation and the mechanism(s) by which p38 activates myogenic transcription remain elusive. Using a combination of different approaches, we found that the p38 kinase is rapidly activated in muscle cells induced to differentiate by serum withdrawal, through a pathway distinct from that activated in response to stress and cytokines. Specific inhibition of p38 prevents differentiation of both established muscle cell lines and human primary myoblasts, while deliberate p38 activation stimulates muscle-specific reporters, accelerates myotube formation, and induces the expression of myogenic markers despite the presence of serum, which otherwise inhibits muscle differentiation. p38 exerts its stimulatory effect on myogenesis by enhancing the transcriptional activities of both MyoD and MEF2A and -C through distinct mechanisms. While MEF2 proteins are activated by direct phosphorylation of residues located within the activation domain, p38-mediated activation of MyoD is likely to occur by an indirect mechanism. The p38 pathway is activated independently of the IGF-PI3K pathway, although the integrity of both these pathways is required to stimulate muscle differentiation. Conversely, the ERK pathway plays a dual role during myogenic differentiation, being inhibitory at early stages and stimulatory at late stages.

MyoD inhibits cell proliferation and promotes muscle differentiation. A paradoxical feature of rhabdomyosarcoma (RMS), a tumor arising from muscle precursors, is the block of the differentiation program and the deregulated proliferation despite MyoD expression. A deficiency in RMS of a factor required for MyoD activity has been implicated by previous studies. We report here that p38 MAP kinase (MAPK) activation, which is essential for muscle differentiation, is deficient in RMS cells. Enforced induction of p38 MAPK by an activated MAPK kinase 6 (MKK6EE) restored MyoD function and enhanced MEF2 activity in RMS deficient for p38 MAPK activation, leading to growth arrest and terminal differentiation. Stress and cytokines could activate the p38 MAPK in RMS cells, however, these stimuli did not promote differentiation, possibly because they activated p38 MAPK only transiently and they also activated JNK, which could antagonize differentiation. Thus, the selective and sustained p38 MAPK activation, which is distinct from the stress-activated response, is required for differentiation and can be disrupted in human tumors.

CDK9 has been recently shown to have increased kinase activity in differentiated cells in culture and a differentiated tissue-specific expression in the developing mouse. In order to identify factors that contribute to CDK9's differentiation-specific function, we screened a mouse embryonic library in the yeast two-hybrid system and found a tumor necrosis factor signal transducer, TRAF2, to be an interacting protein. CDK9 interacts with a conserved domain in the TRAF-C region of TRAF2, a motif that is known to bind other kinases involved in TRAF-mediated signaling. Endogenous interaction between the two proteins appears to be specific to differentiated tissue. TRAF2-mediated signaling may incorporate additional kinases to signal cell survival in myotubes, a cell type that is severely affected in TRAF2 knockout mice.

Doxorubicin (Dox, Adriamicin), a potent broad spectrum anthracycline anticancer drug, selectively inhibits muscle specific gene expression in cardiac cells in vivo and prevents terminal differentiation of skeletal muscle cells in vitro. By inducing the expression of the helix-loop-helix (HLH) transcriptional inhibitor ld2, Dox represses the myogenic function of the MyoD family of muscle regulatory factors (MRFs). In many cell types, terminal differentiation is coupled to an irreversible exit from the cell cycle and MyoD plays a critical role in the permanent cell cycle arrest of differentiating myocytes by upregulating the cyclin dependent kinase inhibitor (cdki) p21. Here, we correlate Dox effects on cell cycle with changes of E2F/DP complexes and activity in differentiating C2C12 myocytes. In Dox-treated quiescent myoblasts, which fail to differentiate into myotubes under permissive culture conditions, serum re-stimulation induces cyclin/cdk re-association on the E2F/DP complexes and this correlates with an evident increase in E2F/DP driven transcription and re-entry of myoblasts into the cell cycle. Despite Dox ability to activate the DNA-damage dependent p53/p21 pathway, when induced in the absence of MyoD or other MRFs, p21 fails to maintain the postmitotic state in Dox-treated myocytes induced to differentiate. Thus, uncoupling p21 induction and MyoD activity results in a serum-reversible cell cycle arrest, indicating that MRF specific activation of cdki(s) is required for permanent cell cycle arrest in differentiating muscle cells.

Withdrawal from the cell cycle of differentiating myocytes is regulated by the myogenic basic helix-loop-helix (bHLH) protein MyoD and the pocket proteins pRb, p107 and pRb2/p130. Downstream effectors of 'pocket' proteins are the components of the E2F family of transcription factors, which regulate the G1/S-phase transition. We analysed by EMSA the composition of E2F complexes in cycling, quiescent undifferentiated and differentiated C2C12 skeletal muscle cells. An E2F complex containing mainly E2F4 and pRb2/p130 (E2F-G0/G1 complex) appears when DNA synthesis arrests, replacing the cyclinA/cdk2 containing E2F complex of proliferating myoblasts (E2F-G1/S complex). Serum stimulation reinduces DNA synthesis and the re-appearance of E2F-G1/S complexes in quiescent myoblasts but not in differentiated C2C12 myotubes. In differentiating C2C12 cells, E2F complexes switch and DNA synthesis in response to serum are prevented when MyoD DNA binding activity and the cdks inhibitor MyoD downstream effector p21 are induced. Thus, during myogenic differentiation, formation of E2F4 and pRb2/p130 containing complexes is an early event, but not enough on its own to prevent the reactivation of DNA synthesis. Using a subclone of C3H10T1/2 mouse fibroblasts stably expressing Estrogen Receptor-MyoD (ER-MyoD) chimerae, we found that estrogen directed MyoD activation prevents the reassociation of cyclinA/cdk2 to the E2F4 containing complex following serum stimulation and this correlates with suppression of E2F activity and the inability of cells to re-enter the cell cycle. Our data indicate that, in differentiating myocytes, one mechanism through which MyoD induces permanent cell cycle arrest involves p21 upregulation and suppression of the proliferation-associated cdks-containing E2F complexes formation.

Previous work has established the existence of dystrophin-nitric oxide (NO) signaling to histone deacetylases (HDACs) that is deregulated in dystrophic muscles. As such, pharmacological interventions that target HDACs (that is, HDAC inhibitors) are of potential therapeutic interest for the treatment of muscular dystrophies. In this study, we explored the effectiveness of long-term treatment with different doses of the HDAC inhibitor givinostat in mdx mice--the mouse model of Duchenne muscular dystrophy (DMD). This study identified an efficacy for recovering functional and histological parameters within a window between 5 and 10 mg/kg/d of givinostat, with evident reduction of the beneficial effects with 1 mg/kg/d dosage. The long-term (3.5 months) exposure of 1.5-month-old mdx mice to optimal concentrations of givinostat promoted the formation of muscles with increased cross-sectional area and reduced fibrotic scars and fatty infiltration, leading to an overall improvement of endurance performance in treadmill tests and increased membrane stability. Interestingly, a reduced inflammatory infiltrate was observed in muscles of mdx mice exposed to 5 and 10 mg/kg/d of givinostat. A parallel pharmacokinetic/pharmacodynamic analysis confirmed the relationship between the effective doses of givinostat and the drug distribution in muscles and blood of treated mice. These findings provide the preclinical basis for an immediate translation of givinostat into clinical studies with DMD patients.