The myogenic regulatory factors, determinants of muscle development, cell identity and regeneration.

The Myogenic Regulatory Factors (MRFs) Myf5, MyoD, myogenin and MRF4 are members of the basic helix-loop-helix family of transcription factors that control the determination and differentiation of skeletal muscle cells during embryogenesis and postnatal myogenesis. The dynamics of their temporal and spatial expression as well as their biochemical properties have allowed the identification of a precise and hierarchical relationship between the four MRFs. This relationship establishes the myogenic lineage as well as the maintenance of the terminal myogenic phenotype. The application of genome-wide technologies has provided important new information as to how the MRFs function to activate muscle gene expression. Application of combined functional genomics technologies along with single cell lineage tracing strategies will allow a deeper understanding of the mechanisms mediating myogenic determination, cell differentiation and muscle regeneration.

Contrasting roles for MyoD in organizing myogenic promoter structures during embryonic skeletal muscle development.

BACKGROUND: Among the complexities of skeletal muscle differentiation is a temporal distinction in the onset of expression of different lineage-specific genes. The lineage-determining factor MyoD is bound to myogenic genes at the onset of differentiation whether gene activation is immediate or delayed. How temporal regulation of differentiation-specific genes is established remains unclear. RESULTS: Using embryonic tissue, we addressed the molecular differences in the organization of the myogenin and muscle creatine kinase (MCK) gene promoters by examining regulatory factor binding as a function of both time and spatial organization during somitogenesis. At the myogenin promoter, binding of the homeodomain factor Pbx1 coincided with H3 hyperacetylation and was followed by binding of co-activators that modulate chromatin structure. MyoD and myogenin binding occurred subsequently, demonstrating that Pbx1 facilitates chromatin remodeling and modification before myogenic regulatory factor binding. At the same time, the MCK promoter was bound by HDAC2 and MyoD, and activating histone marks were largely absent. The association of HDAC2 and MyoD was confirmed by co-immunoprecipitation, proximity ligation assay (PLA), and sequential ChIP. CONCLUSIONS: MyoD differentially promotes activated and repressed chromatin structures at myogenic genes early after the onset of skeletal muscle differentiation in the developing mouse embryo.

The Scaffold attachment factor b1 (Safb1) regulates myogenic differentiation by facilitating the transition of myogenic gene chromatin from a repressed to an activated state.

The regulation of skeletal muscle gene expression during myogenesis is mediated by lineage-specific transcription factors in combination with numerous cofactors, many of which modify chromatin structure. However, the involvement of scaffolding proteins that organize chromatin and chromatin-associated regulatory proteins has not extensively been explored in myogenic differentiation. Here, we report that Scaffold attachment factor b1 (Safb1), primarily associated with transcriptional repression, functions as a positive regulator of myogenic differentiation. Knockdown of Safb1 inhibited skeletal muscle marker gene expression and differentiation in cultured C2C12 myoblasts. In contrast, over-expression resulted in the premature expression of critical muscle structural proteins and formation of enlarged thickened myotubes. Safb1 co-immunoprecipitated with MyoD and was co-localized on myogenic promoters. Upon Safb1 knockdown, the repressive H3K27me3 histone mark and binding of the Polycomb histone methyltransferase Ezh2 persisted at differentiation-dependent gene promoters. In contrast, the appearance of histone marks and regulators associated with myogenic gene activation, such as myogenin and the SWI/SNF chromatin remodelling enzyme ATPase, Brg1, was blocked. These results indicate that the scaffold protein Safb1 contributes to the activation of skeletal muscle gene expression during myogenic differentiation by facilitating the transition of promoter sequences from a repressive chromatin structure to one that is transcriptionally permissive.

Wnt3 function in the epiblast is required for the maintenance but not the initiation of gastrulation in mice.

The formation of the anteroposterior axis in mice requires a Wnt3-dependent symmetry-breaking event that leads to the formation of the primitive streak and gastrulation. Wnt3 is expressed sequentially in two distinct areas of the mouse embryo before the appearance of the primitive streak; first in the posterior visceral endoderm and soon after in the adjacent posterior epiblast. Hence, although an axial requirement for Wnt3 is well established, its temporal and tissue specific requirements remain an open question. Here, we report the conditional inactivation of Wnt3 in the epiblast of developing mouse embryos. Contrary to previous studies, our data shows that embryos lacking Wnt3 specifically in the epiblast are able to initiate gastrulation and advance to late primitive streak stages but fail to thrive and are resorbed by E9.5. At the molecular level, we provide evidence that Wnt3 regulates its own expression and that of other primitive streak markers via activation of the canonical Wnt signaling pathway.

(-)-Epicatechin modulates the expression of myomiRs implicated in exercise response in mouse skeletal muscle.

The flavanol (-)-epicatechin has exercise-mimetic properties. Besides, several miRNAs play a role in modulating the adaptation of the muscle to different training protocols. However, notwithstanding all information, few studies aimed to determine if (-)-epicatechin can modify the expression of miRNAs related to skeletal muscle development and regeneration. Mice were treated for fifteen days by oral gavage with the flavanol (-)-epicatechin. After treatment, the quadriceps of the mice was dissected, and total RNA was extracted. The expression level of miR-133, -204, -206, -223, -486, and -491 was analyzed by qRT-PCR. We also used bioinformatic analysis to predict the participation of these miRNAs in different skeletal muscle signal transduction pathways. Additionally, we analyzed the level of the myogenic proteins MyoD and myogenin by Western blot and measured the cross-sectional area of muscle fibers stained with E&H. (-)-Epicatechin upregulated the expression of miR-133, -204, -206, -223, and -491 significantly, which was associated with an increase in the level of the myogenic proteins MyoD and Myogenin and an augment in the fiber size. The bioinformatics analysis showed that the studied miRNAs might participate in different signal transduction pathways related to muscle development and adaptation. Our results showed that (-)-epicatechin upregulated miRNAs that participate in skeletal exercise muscle adaptation, induced muscle hypertrophy, and increased the level of myogenic proteins MyoD and MyoG.

Differential histological features and myogenic protein levels in distinct muscles of d-sarcoglycan null muscular dystrophy mouse model.

Skeletal muscle (SkM) comprises slow and fast-twitch fibers, which differ in molecular composition, function, and systemic energy consumption. In addition, muscular dystrophies (DM), a group of diverse hereditary diseases, present different patterns of muscle involvement, progression, and severity, suggesting that the regeneration-degeneration process may differ depending on the muscle type. Therefore, the study aimed to explore the expression of proteins involved in the repair process in different muscles at an early stage of muscular dystrophy in the δ-sarcoglycan null mice (Sgcd-null), a limb-girdle muscular dystrophy 2 F model. Hematoxylin & Eosin (H&E) Staining showed a high number of central nuclei in soleus (Sol), tibialis (Ta), gastrocnemius (Gas), and extensor digitorum longus (Edl) from four months Sgcd-null mice. However, fibrosis, determined by trichrome of Gomori modified staining, was only observed in Sgcd-null Sol. In addition, the number of Type I and II fibers variated differentially in the Sgcd-null muscles vs. wild-type muscles. Besides, the protein expression level of ß-catenin, myomaker, MyoD, and myogenin also presented different expression levels in all the Sgcd-null muscles studied. In summary, our study reveals that muscles with different metabolic characteristics showed distinct expression patterns of proteins involved in the muscle regeneration process. These results could be relevant in designing therapies for genetic and acquired myopathy.

RNA Foci Formation in a Retinal Glial Model for Spinocerebellar Ataxia Type 7.

Spinocerebellar ataxia type 7 (SCA7) is a neurodegenerative disorder characterized by cerebellar ataxia and retinopathy. SCA7 is caused by a CAG expansion in the ATXN7 gene, which results in an extended polyglutamine (polyQ) tract in the encoded protein, the ataxin-7. PolyQ expanded ataxin-7 elicits neurodegeneration in cerebellar Purkinje cells, however, its impact on the SCA7-associated retinopathy remains to be addressed. Since Müller glial cells play an essential role in retinal homeostasis, we generate an inducible model for SCA7, based on the glial Müller MIO-M1 cell line. The SCA7 pathogenesis has been explained by a protein gain-of-function mechanism, however, the contribution of the mutant RNA to the disease cannot be excluded. In this direction, we found nuclear and cytoplasmic foci containing mutant RNA accompanied by subtle alternative splicing defects in MIO-M1 cells. RNA foci were also observed in cells from different lineages, including peripheral mononuclear leukocytes derived from SCA7 patient, suggesting that this molecular mark could be used as a blood biomarker for SCA7. Collectively, our data showed that our glial cell model exhibits the molecular features of SCA7, which makes it a suitable model to study the RNA toxicity mechanisms, as well as to explore therapeutic strategies aiming to alleviate glial dysfunction.

Transcriptome Analysis Reveals Altered Inflammatory Pathway in an Inducible Glial Cell Model of Myotonic Dystrophy Type 1.

Myotonic dystrophy type 1 (DM1), the most frequent inherited muscular dystrophy in adults, is caused by the CTG repeat expansion in the 3'UTR of the DMPK gene. Mutant DMPK RNA accumulates in nuclear foci altering diverse cellular functions including alternative splicing regulation. DM1 is a multisystemic condition, with debilitating central nervous system alterations. Although a defective neuroglia communication has been described as a contributor of the brain pathology in DM1, the specific cellular and molecular events potentially affected in glia cells have not been totally recognized. Thus, to study the effects of DM1 mutation on glial physiology, in this work, we have established an inducible DM1 model derived from the MIO-M1 cell line expressing 648 CUG repeats. This new model recreated the molecular hallmarks of DM1 elicited by a toxic RNA gain-of-function mechanism: accumulation of RNA foci colocalized with MBNL proteins and dysregulation of alternative splicing. By applying a microarray whole-transcriptome approach, we identified several gene changes associated with DM1 mutation in MIO-M1 cells, including the immune mediators CXCL10, CCL5, CXCL8, TNFAIP3, and TNFRSF9, as well as the microRNAs miR-222, miR-448, among others, as potential regulators. A gene ontology enrichment analyses revealed that inflammation and immune response emerged as major cellular deregulated processes in the MIO-M1 DM1 cells. Our findings indicate the involvement of an altered immune response in glia cells, opening new windows for the study of glia as potential contributor of the CNS symptoms in DM1.

Chromatin Landscape During Skeletal Muscle Differentiation.

Cellular commitment and differentiation involve highly coordinated mechanisms by which tissue-specific genes are activated while others are repressed. These mechanisms rely on the activity of specific transcription factors, chromatin remodeling enzymes, and higher-order chromatin organization in order to modulate transcriptional regulation on multiple cellular contexts. Tissue-specific transcription factors are key mediators of cell fate specification with the ability to reprogram cell types into different lineages. A classic example of a master transcription factor is the muscle specific factor MyoD, which belongs to the family of myogenic regulatory factors (MRFs). MRFs regulate cell fate determination and terminal differentiation of the myogenic precursors in a multistep process that eventually culminate with formation of muscle fibers. This developmental progression involves the activation and proliferation of muscle stem cells, commitment, and cell cycle exit and fusion of mononucleated myoblast to generate myotubes and myofibers. Although the epigenetics of muscle regeneration has been extensively addressed and discussed over the recent years, the influence of higher-order chromatin organization in skeletal muscle regeneration is still a field of development. In this review, we will focus on the epigenetic mechanisms modulating muscle gene expression and on the incipient work that addresses three-dimensional genome architecture and its influence in cell fate determination and differentiation to achieve skeletal myogenesis. We will visit known alterations of genome organization mediated by chromosomal fusions giving rise to novel regulatory landscapes, enhancing oncogenic activation in muscle, such as alveolar rhabdomyosarcomas (ARMS).

Identification of two E-boxes that negatively modulate the activity of MyoD on the alpha-sarcoglycan core promoter.

The alpha-SG promoter is composed of a plethora of cis-regulatory elements, whose individual contribution to alpha-SG gene expression modulation remains unknown. We have identified a negative regulatory element in the alpha-SG distal promoter including two conserved E-boxes (E1 and E2), which interact with MyoD. We found that E1 and E2 negatively modulate the transactivation potential of MyoD on the alpha-SG core promoter. Moreover, such negative effect is mainly mediated by E2, which is surrounded by conserved nucleotides conferring MyoD binding capacity. Our results suggest that modulation of MyoD activity by E1, and particularly E2, contributes to the negative regulation of alpha-SG gene expression during myogenic differentiation.

WITHDRAWN: Identification of two E-boxes that negatively modulate the activity of MyoD on the alpha-sarcoglycan core promoter.

The Publisher regrets that this article is an accidental duplication of an article that has already been published in Biochem. Biophys. Acta, doi:10.1016/j.bbagrm.2007.09.002. The duplicate article has therefore been withdrawn.

Chromatin Immunoprecipitation in Early Mouse Embryos.

Epigenetic regulation is achieved at many levels by different factors such as tissue-specific transcription factors, members of the basal transcriptional apparatus, chromatin-binding proteins, and noncoding RNAs. Importantly, chromatin structure dictates the availability of a specific genomic locus for transcriptional activation as well as the efficiency with which transcription can occur. Chromatin immunoprecipitation (ChIP) is a method that allows elucidating gene regulation at the molecular level by assessing if chromatin modifications or proteins are present at a specific locus. Initially, the majority of ChIP experiments were performed on cultured cell lines and more recently this technique has been adapted to a variety of tissues in different model organisms. Using ChIP on mouse embryos, it is possible to document the presence or absence of specific proteins and chromatin modifications at genomic loci in vivo during mammalian development and to get biological meaning from observations made on tissue culture analyses. We describe here a ChIP protocol on freshly isolated mouse embryonic somites for in vivo analysis of muscle specific transcription factor binding on chromatin. This protocol has been easily adapted to other mouse embryonic tissues and has also been successfully scaled up to perform ChIP-Seq.

Sox9 represses alpha-sarcoglycan gene expression in early myogenic differentiation.

Alpha sarcoglycan (alpha-SG) is highly expressed in differentiated striated muscle, and its disruption causes limb-girdle muscular dystrophy. Accordingly, the myogenic master regulator MyoD finely modulates its expression. However, the mechanisms preventing alpha-SG gene expression at early stages of myogenic differentiation remain unknown. In this study, we uncovered Sox9, which was not previously known to directly bind muscle gene promoters, as a negative regulator of alpha-SG gene expression. Reporter gene and chromatin immunoprecipitation assays revealed three functional Sox-binding sites that mediate alpha-SG promoter activity repression during early myogenic differentiation. In addition, we show that Sox9-mediated inhibition of alpha-SG gene expression is independent of MyoD. Moreover, we provide evidence suggesting that Smad3 enhances the repressive activity of Sox9 over alpha-SG gene expression in a transforming growth factor-beta-dependent manner. On the basis of these results, we propose that Sox9 and Smad3 are responsible for preventing precocious activation of alpha-SG gene expression during myogenic differentiation.