G1/S Inhibitors and the SWI/SNF Complex Control Cell-Cycle Exit during Muscle Differentiation.

The transition from proliferating precursor cells to post-mitotic differentiated cells is crucial for development, tissue homeostasis, and tumor suppression. To study cell-cycle exit during differentiation in vivo, we developed a conditional knockout and lineage-tracing system for Caenorhabditis elegans. Combined lineage-specific gene inactivation and genetic screening revealed extensive redundancies between previously identified cell-cycle inhibitors and the SWI/SNF chromatin-remodeling complex. Muscle precursor cells missing either SWI/SNF or G1/S inhibitor function could still arrest cell division, while simultaneous inactivation of these regulators caused continued proliferation and a C. elegans tumor phenotype. Further genetic analyses support that SWI/SNF acts in concert with hlh-1 MyoD, antagonizes Polycomb-mediated transcriptional repression, and suppresses cye-1 Cyclin E transcription to arrest cell division of muscle precursors. Thus, SWI/SNF and G1/S inhibitors provide alternative mechanisms to arrest cell-cycle progression during terminal differentiation, which offers insight into the frequent mutation of SWI/SNF genes in human cancers.

MEF2 is an in vivo immune-metabolic switch.

Infections disturb metabolic homeostasis in many contexts, but the underlying connections are not completely understood. To address this, we use paired genetic and computational screens in Drosophila to identify transcriptional regulators of immunity and pathology and their associated target genes and physiologies. We show that Mef2 is required in the fat body for anabolic function and the immune response. Using genetic and biochemical approaches, we find that MEF2 is phosphorylated at a conserved site in healthy flies and promotes expression of lipogenic and glycogenic enzymes. Upon infection, this phosphorylation is lost, and the activity of MEF2 changes--MEF2 now associates with the TATA binding protein to bind a distinct TATA box sequence and promote antimicrobial peptide expression. The loss of phosphorylated MEF2 contributes to loss of anabolic enzyme expression in Gram-negative bacterial infection. MEF2 is thus a critical transcriptional switch in the adult fat body between metabolism and immunity.

The transcription factor MEF2 directs developmental visually driven functional and structural metaplasticity.

Natural sensory input shapes both structure and function of developing neurons, but how early experience-driven morphological and physiological plasticity are interrelated remains unclear. Using rapid time-lapse two-photon calcium imaging of network activity and single-neuron growth within the unanesthetized developing brain, we demonstrate that visual stimulation induces coordinated changes to neuronal responses and dendritogenesis. Further, we identify the transcription factor MEF2A/2D as a major regulator of neuronal response to plasticity-inducing stimuli directing both structural and functional changes. Unpatterned sensory stimuli that change plasticity thresholds induce rapid degradation of MEF2A/2D through a classical apoptotic pathway requiring NMDA receptors and caspases-9 and -3/7. Knockdown of MEF2A/2D alone is sufficient to induce a metaplastic shift in threshold of both functional and morphological plasticity. These findings demonstrate how sensory experience acting through altered levels of the transcription factor MEF2 fine-tunes the plasticity thresholds of brain neurons during neural circuit formation.

Multiple autism-linked genes mediate synapse elimination via proteasomal degradation of a synaptic scaffold PSD-95.

The activity-dependent transcription factor myocyte enhancer factor 2 (MEF2) induces excitatory synapse elimination in mouse neurons, which requires fragile X mental retardation protein (FMRP), an RNA-binding protein implicated in human cognitive dysfunction and autism. We report here that protocadherin 10 (Pcdh10), an autism-spectrum disorders gene, is necessary for this process. MEF2 and FMRP cooperatively regulate the expression of Pcdh10. Upon MEF2 activation, PSD-95 is ubiquitinated by the ubiquitin E3 ligase murine double minute 2 (Mdm2) and then binds to Pcdh10, which links it to the proteasome for degradation. Blockade of the Pcdh10-proteasome interaction inhibits MEF2-induced PSD-95 degradation and synapse elimination. In FMRP-lacking neurons, elevated protein levels of eukaryotic translation elongation factor 1 α (EF1α), an Mdm2-interacting protein and FMRP target mRNA, sequester Mdm2 and prevent MEF2-induced PSD-95 ubiquitination and synapse elimination. Together, our findings reveal roles for multiple autism-linked genes in activity-dependent synapse elimination.

A long noncoding RNA controls muscle differentiation by functioning as a competing endogenous RNA.

Recently, a new regulatory circuitry has been identified in which RNAs can crosstalk with each other by competing for shared microRNAs. Such competing endogenous RNAs (ceRNAs) regulate the distribution of miRNA molecules on their targets and thereby impose an additional level of post-transcriptional regulation. Here we identify a muscle-specific long noncoding RNA, linc-MD1, which governs the time of muscle differentiation by acting as a ceRNA in mouse and human myoblasts. Downregulation or overexpression of linc-MD1 correlate with retardation or anticipation of the muscle differentiation program, respectively. We show that linc-MD1 "sponges" miR-133 and miR-133 [corrected] to regulate the expression of MAML1 and MEF2C, transcription factors that activate muscle-specific gene expression. Finally, we demonstrate that linc-MD1 exerts the same control over differentiation timing in human myoblasts, and that its levels are strongly reduced in Duchenne muscle cells. We conclude that the ceRNA network plays an important role in muscle differentiation.

Structural insights into the HDAC4-MEF2A-DNA complex and its implication in long-range transcriptional regulation.

Class IIa Histone deacetylases (HDACs), including HDAC4, 5, 7 and 9, play key roles in multiple important developmental and differentiation processes. Recent studies have shown that class IIa HDACs exert their transcriptional repressive function by interacting with tissue-specific transcription factors, such as members of the myocyte enhancer factor 2 (MEF2) family of transcription factors. However, the molecular mechanism is not well understood. In this study, we determined the crystal structure of an HDAC4-MEF2A-DNA complex. This complex adopts a dumbbell-shaped overall architecture, with a 2:4:2 stoichiometry of HDAC4, MEF2A and DNA molecules. In the complex, two HDAC4 molecules form a dimer through the interaction of their glutamine-rich domain (GRD) to form the stem of the 'dumbbell'; while two MEF2A dimers and their cognate DNA molecules are bridged by the HDAC4 dimer. Our structural observations were then validated using biochemical and mutagenesis assays. Further cell-based luciferase reporter gene assays revealed that the dimerization of HDAC4 is crucial in its ability to repress the transcriptional activities of MEF2 proteins. Taken together, our findings not only provide the structural basis for the assembly of the HDAC4-MEF2A-DNA complex but also shed light on the molecular mechanism of HDAC4-mediated long-range gene regulation.

MEF2B mutations lead to deregulated expression of the oncogene BCL6 in diffuse large B cell lymphoma.

MEF2B encodes a transcriptional activator and is mutated in ∼11% of diffuse large B cell lymphomas (DLBCLs) and ∼12% of follicular lymphomas (FLs). Here we found that MEF2B directly activated the transcription of the proto-oncogene BCL6 in normal germinal-center (GC) B cells and was required for DLBCL proliferation. Mutation of MEF2B resulted in enhanced transcriptional activity of MEF2B either through disruption of its interaction with the corepressor CABIN1 or by rendering it insensitive to inhibitory signaling events mediated by phosphorylation and sumoylation. Consequently, the transcriptional activity of Bcl-6 was deregulated in DLBCLs with MEF2B mutations. Thus, somatic mutations of MEF2B may contribute to lymphomagenesis by deregulating BCL6 expression, and MEF2B may represent an alternative target for blocking Bcl-6 activity in DLBCLs.

PGRP-SD, an Extracellular Pattern-Recognition Receptor, Enhances Peptidoglycan-Mediated Activation of the Drosophila Imd Pathway.

Activation of the innate immune response in Metazoans is initiated through the recognition of microbes by host pattern-recognition receptors. In Drosophila, diaminopimelic acid (DAP)-containing peptidoglycan from Gram-negative bacteria is detected by the transmembrane receptor PGRP-LC and by the intracellular receptor PGRP-LE. Here, we show that PGRP-SD acted upstream of PGRP-LC as an extracellular receptor to enhance peptidoglycan-mediated activation of Imd signaling. Consistent with this, PGRP-SD mutants exhibited impaired activation of the Imd pathway and increased susceptibility to DAP-type bacteria. PGRP-SD enhanced the localization of peptidoglycans to the cell surface and hence promoted signaling. Moreover, PGRP-SD antagonized the action of PGRP-LB, an extracellular negative regulator, to fine-tune the intensity of the immune response. These data reveal that Drosophila PGRP-SD functions as an extracellular receptor similar to mammalian CD14 and demonstrate that, comparable to lipopolysaccharide sensing in mammals, Drosophila relies on both intra- and extracellular receptors for the detection of bacteria.

Direct reprogramming of fibroblasts into functional cardiomyocytes by defined factors.

The reprogramming of fibroblasts to induced pluripotent stem cells (iPSCs) raises the possibility that a somatic cell could be reprogrammed to an alternative differentiated fate without first becoming a stem/progenitor cell. A large pool of fibroblasts exists in the postnatal heart, yet no single "master regulator" of direct cardiac reprogramming has been identified. Here, we report that a combination of three developmental transcription factors (i.e., Gata4, Mef2c, and Tbx5) rapidly and efficiently reprogrammed postnatal cardiac or dermal fibroblasts directly into differentiated cardiomyocyte-like cells. Induced cardiomyocytes expressed cardiac-specific markers, had a global gene expression profile similar to cardiomyocytes, and contracted spontaneously. Fibroblasts transplanted into mouse hearts one day after transduction of the three factors also differentiated into cardiomyocyte-like cells. We believe these findings demonstrate that functional cardiomyocytes can be directly reprogrammed from differentiated somatic cells by defined factors. Reprogramming of endogenous or explanted fibroblasts might provide a source of cardiomyocytes for regenerative approaches.

Nfix regulates fetal-specific transcription in developing skeletal muscle.

Skeletal myogenesis, like hematopoiesis, occurs in successive developmental stages that involve different cell populations and expression of different genes. We show here that the transcription factor nuclear factor one X (Nfix), whose expression is activated by Pax7 in fetal muscle, in turn activates the transcription of fetal specific genes such as MCK and beta-enolase while repressing embryonic genes such as slow myosin. In the case of the MCK promoter, Nfix forms a complex with PKC theta that binds, phosphorylates, and activates MEF2A. Premature expression of Nfix activates fetal and suppresses embryonic genes in embryonic muscle, whereas muscle-specific ablation of Nfix prevents fetal and maintains embryonic gene expression in the fetus. Therefore, Nfix acts as a transcriptional switch from embryonic to fetal myogenesis.

Single nucleotide polymorphisms in the MRFs gene family associated with growth in Nile tilapia.

BACKGROUND: Muscle occupies most of the fish body, promoting the proliferation of fish muscle fibers can facilitate rapid growth and increase the body weight of fish. Some studiesSeveral previous suggest that Myogenic regulatory factors (MRFs) play an important role in the growth of fish. OBJECTIVE: To investigate the association between the polymorphism of MRFs gene family and growth traits in Nile tilapia (Oreochromis niloticus), get more molecular markers for growth. METHODS: Amplified the Nile tilapia MRFs family gene, including Myogenic determination 1 (Myod1), Myogenic determination 2 (Myod2), Myogenin (Myog), Myogenic factor 5 (Myf5), and Myogenic factor 6 (Myf6), single nucleotide polymorphism (SNP) were screened by Sanger sequencing. RESULTS: A total of 16 SNP loci were screened, including six for Myf5, six for Myf6, one for Myog, one for Myod1 and two for Myod2. The growth traits were analyzed in relation to these 16 SNP loci, and the results indicated significant associations between all 16 SNP loci and the growth traits (P < 0.05). The linkage disequilibrium analysis revealed that D1 and D2 diplotypes of Myf5 gene, E1, E2, E3 and E4 of Myf6 gene, and F1 diplotype of Myod2 gene were significantly associated with superior growth traits. CONCLUSION: There were 6, 6, 1, 1 and 2 growth-related molecular markers in Myf5, Myf6, Myog, Myod1 and Myod2 genes, respectively, which could be applied to the breeding of Nile tilapia.

Myogenic regulatory factors Myod and Myf5 are required for dorsal aorta formation and angiogenic sprouting.

The vertebrate embryonic midline vasculature forms in close proximity to the developing skeletal muscle, which originates in the somites. Angioblasts migrate from bilateral positions along the ventral edge of the somites until they meet at the midline, where they sort and differentiate into the dorsal aorta and the cardinal vein. This migration occurs at the same time that myoblasts in the somites are beginning to differentiate into skeletal muscle, a process which requires the activity of the basic helix loop helix (bHLH) transcription factors Myod and Myf5. Here we examined vasculature formation in myod and myf5 mutant zebrafish. In the absence of skeletal myogenesis, angioblasts migrate normally to the midline but form only the cardinal vein and not the dorsal aorta. The phenotype is due to the failure to activate vascular endothelial growth factor ligand vegfaa expression in the somites, which in turn is required in the adjacent angioblasts for dorsal aorta specification. Myod and Myf5 cooperate with Hedgehog signaling to activate and later maintain vegfaa expression in the medial somites, which is required for angiogenic sprouting from the dorsal aorta. Our work reveals that the early embryonic skeletal musculature in teleosts evolved to organize the midline vasculature during development.

Regulation of myogenic gene expression.

Skeletal muscle development and regeneration is governed by the combined action of Myf5, MyoD, Mrf4 and MyoG, also known as the myogenic regulatory factors (MRFs). These transcription factors are expressed in a highly spatio-temporal restricted manner, ensuring the significant functional and metabolic diversity observed between the different muscle groups. In this review, we will discuss the multiple layers of regulation that contribute to the control of the exquisite expression patterns of the MRFs in particular, and of myogenic genes in general. We will highlight all major regulatory processes that play a role in myogenesis: from those that modulate chromatin status and transcription competence, such as DNA methylation, histone modification, chromatin remodeling, or non-coding RNAs, to those that control transcript and protein processing and modification, such as alternative splicing, polyadenylation, other mRNA modifications, or post-translational protein modifications. All these processes are exquisitely and tightly coordinated to ensure the proper activation, maintenance and termination of the myogenic process.

Diversification of muscle types in Drosophila embryos.

Drosophila embryonic somatic muscles represent a simple and tractable model system to study the gene regulatory networks that control diversification of cell types. Somatic myogenesis in Drosophila is initiated by intrinsic action of the mesodermal master gene twist, which activates a cascade of transcriptional outputs including myogenic differentiation factor Mef2, which triggers all aspects of the myogenic differentiation program. In parallel, the expression of a combinatorial code of identity transcription factors (iTFs) defines discrete particular features of each muscle fiber, such as number of fusion events, and specific attachment to tendon cells or innervation, thus ensuring diversification of muscle types. Here, we take the example of a subset of lateral transverse (LT) muscles and discuss how the iTF code and downstream effector genes progressively define individual LT properties such as fusion program, attachment and innervation. We discuss new challenges in the field including the contribution of posttranscriptional and epitranscriptomic regulation of gene expression in the diversification of cell types.

Satellite cells deficiency and defective regeneration in dynamin 2-related centronuclear myopathy.

Dynamin 2 (DNM2) is a ubiquitously expressed protein involved in many functions related to trafficking and remodeling of membranes and cytoskeleton dynamics. Mutations in the DNM2 gene cause the autosomal dominant centronuclear myopathy (AD-CNM), characterized mainly by muscle weakness and central nuclei. Several defects have been identified in the KI-Dnm2R465W/+ mouse model of the disease to explain the muscle phenotype, including reduction of the satellite cell pool in muscle, but the functional consequences of this depletion have not been characterized until now. Satellite cells (SC) are the main source for muscle growth and regeneration of mature tissue. Here, we investigated muscle regeneration in the KI-Dnm2R465W/+ mouse model for AD-CNM. We found a reduced number of Pax7-positive SCs, which were also less activated after induced muscle injury. The muscles of the KI-Dnm2R465W/+ mouse regenerated more slowly and less efficiently than wild-type ones, formed fewer new myofibers, and did not recover its normal mass 15 days after injury. Altogether, our data provide evidence that the muscle regeneration is impaired in the KI-Dnm2R465W/+ mouse and contribute with one more layer to the comprehension of the disease, by identifying a new pathomechanism linked to DNM2 mutations which may be involved in the muscle-specific impact occurring in AD-CNM.

Effect of eccentric and concentric contraction mode on myogenic regulatory factors expression in human vastus lateralis muscle.

Skeletal muscle contractions are caused to release myokines by muscle fiber. This study investigated the myogenic regulatory factors, as MHC I, IIA, IIX, Myo-D, MRF4, Murf, Atrogin-1, Decorin, Myonection, and IL-15 mRNA expression in the response of eccentric vs concentric contraction. Eighteen healthy men were randomly divided into two eccentric and concentric groups, each of 9 persons. Isokinetic contraction protocols included maximal single-leg eccentric or concentric knee extension tasks at 60°/s with the dominant leg. Contractions consisted of a maximum of 12 sets of 10 reps, and the rest time between each set was 30 s. The baseline biopsy was performed 4 weeks before the study, and post-test biopsies were taken immediately after exercise protocols from the vastus lateralis muscle. The gene expression levels were evaluated using Real-Time PCR methods. The eccentric group showed a significantly lower RPE score than the concentric group (P ≤ 0.05). A significant difference in MyoD, MRF4, Myonection, and Decorin mRNA, were observed following eccentric or concentric contractions (P ≤ 0.05). The MHC I, MHC IIA, IL-15 mRNA has been changed significantly compared to the pre-exercise in the concentric group (P ≤ 0.05). While only MHC IIX and Atrogin-1 mRNA changed significantly in the eccentric group (P ≤ 0.05). Additionally, the results showed a significant difference in MyoD, MRF4, IL-15, and Decorin at the follow-up values between eccentric or concentric groups (P ≤ 0.05). Our findings highlight the growing importance of elucidating the different responses of muscle growth factors associated with a myogenic activity such as MHC IIA, Decorin, IL-15, Myonectin, Decorin, MuRF1, and MHC IIX mRNA in following various types of exercise.

Myf6/MRF4 is a myogenic niche regulator required for the maintenance of the muscle stem cell pool.

The function and maintenance of muscle stem cells (MuSCs) are tightly regulated by signals originating from their niche environment. Skeletal myofibers are a principle component of the MuSC niche and are in direct contact with the muscle stem cells. Here, we show that Myf6 establishes a ligand/receptor interaction between muscle stem cells and their associated muscle fibers. Our data show that Myf6 transcriptionally regulates a broad spectrum of myokines and muscle-secreted proteins in skeletal myofibers, including EGF. EGFR signaling blocks p38 MAP kinase-induced differentiation of muscle stem cells. Homozygous deletion of Myf6 causes a significant reduction in the ability of muscle to produce EGF, leading to a deregulation in EGFR signaling. Consequently, although Myf6-knockout mice are born with a normal muscle stem cell compartment, they undergo a progressive reduction in their stem cell pool during postnatal life due to spontaneous exit from quiescence. Taken together, our data uncover a novel role for Myf6 in promoting the expression of key myokines, such as EGF, in the muscle fiber which prevents muscle stem cell exhaustion by blocking their premature differentiation.

Inhibition of MDFI attenuates proliferation and glycolysis of Helicobacter pylori-infected gastric cancer cells by inhibiting Wnt/ß-catenin pathway.

MyoD family inhibitor (MDFI) is a myogenic transcription factor regulatory protein. MDFI has been proven to be upregulated and to promote cell proliferation in colorectal cancer. However, the role of MDFI in gastric cancer (GC) is unclear. In this study, MDFI expression in GC tissues and cell lines was examined by quantitative real-time PCR and western blot. Cell Counting Kit-8 assay, clone formation assay, and 5-ethynyl-2'-deoxyuridine assay were used to evaluate GC cell proliferation. Glycolysis was assessed by measuring glucose consumption and lactate and ATP production using commercial assay kits. Western blot was used to detect the expression levels of glycolytic key proteins and Wnt/ß-catenin pathway proteins. To activate Wnt/ß-catenin signaling, GC cells were treated with CHIR-99021. We found that MDFI expression was increased in GC tumor tissues and cells with a positive correlation with poor survival. Knockdown of MDFI inhibited the increase in GC cell proliferation and glycolysis induced by Helicobacter pylori. Helicobacter pylori infection promoted MDFI expression and activated Wnt/ß-catenin signaling. What is more, activation of the Wnt/ß-catenin pathway remarkably reversed the effect of knocking down MDFI on GC cells. Further studies found that MDFI participated in GC cell proliferation and glycolysis by regulating the Wnt/ß-catenin pathway, thereby affecting the development of GC. In conclusion, we demonstrated for the first time that knockdown of MDFI inhibited the increase in GC cell proliferation and glycolysis by regulating the Wnt/ß-catenin pathway. MDFI may be a new target for the clinical treatment of GC.

Insight into the role of myokines and myogenic regulatory factors under hypobaric hypoxia induced skeletal muscle loss.

BACKGROUND: The present study aimed to analyse the role of myokines and the regeneration capacity of skeletal muscle during chronic hypobaric hypoxia (CHH). METHODS: Male Sprague-Dawley rats were exposed to hypobaric hypoxia (HH) for 1d, 3d and 7d. RESULTS: Exposure to HH enhanced the levels of decorin, irisin, IL-6 and IL-15 till 3 days of hypoxia and on 7 day of exposure, no significant changes were observed in relation to control. A significant upregulation in myostatin, activated protein kinase, SMAD3, SMAD4, FOXO-1, MURF-1 expression was observed with prolonged HH exposure as compared to normoxic control. Further, myogenesis-related markers, PAX-7, Cyclin D1 and myogenin were downregulated during CHH exposure in comparison to control. Energy metabolism regulators such as Sirtuin 1, proliferator-activated receptor gamma coactivator-1α and GLUT-4, were also increased on 1-d HH exposure that showed a declining trend on CHH exposure. CONCLUSIONS: These results indicated the impairment in the levels of myokines and myogenesis during prolonged hypoxia. CHH exposure enhanced the levels of myostatin and reduced the regeneration or repair capacity of the skeletal muscles. Myokine levels could be a predictive biomarker for evaluating skeletal muscle performance and loss at high altitudes.

Establishment of primary chicken embryo myoblast cell culture, antigenic epitopes prediction and production of anti activin receptor type IIB polyclonal antibody in chicken.

The detection of activin receptor typeIIB (ACTRIIB) protein, a prominent negative muscle growth regulator has paramount value in augmenting growth traits through molecular breeding schemes in chicken. The study was formulated to establish primary chicken embryo myoblast culture (CEM) using 9th and 18th day chick embryos and to develop antibodies for immunodetection of ACTRIIB protein. The physicochemical and structural attributes of the ACTRIIB sequence were evaluated to identify substantial antigenic regions. The ACTRIIB sequence was transfected into CEM and expressed protein was injected subcutaneously into rats to produce hyperimmune serum. The average propensity of protein sequence for beta turns, surface accessibility, chain flexibility, antigenicity, hydrophilicity and linear epitopes was 0.978, 1.000, 0.991, 1.038, 1.258 and 0.512, respectively. The 9th day CEM exhibited confluency (80-90%) earlier than the 18th day. The expression of myogenic regulatory factors in 9th day myoblasts was higher than the 18th day by 7.28, 5.16, 6.28 and 6.93 folds for MYF5, MRF4, MYOG and MYOD, respectively. The ACTRIIB mRNA was downregulated by 2.54 folds on the 9th day compared to the 18th day myoblasts and protein varied significantly between 9th and 18th day myoblasts. The CEM culture can be harnessed unequivocally to investigate molecular mechanisms underlying muscle growth besides raising antibodies.