MyoD Target Research Articles

Dual-specificity phosphatases 13 and 27 as key switches in muscle stem cell transition from proliferation to differentiation.

Muscle regeneration depends on muscle stem cell (MuSC) activity. Myogenic regulatory factors, including myoblast determination protein 1 (MyoD), regulate the fate transition of MuSCs. However, the direct target of MYOD in the process is not completely clear. Using previously established MyoD knock-in (MyoD-KI) mice, we revealed that MyoD targets dual-specificity phosphatase (Dusp) 13 and Dusp27. In Dusp13:Dusp27 double knock-out mice, the ability for muscle regeneration after injury was reduced. Moreover, single-cell RNA sequencing of MyoD-high expressing MuSCs from MyoD-KI mice revealed that Dusp13 and Dusp27 are expressed only in specific populations within MyoD-high MuSCs, which also express Myogenin. Overexpressing Dusp13 in MuSCs causes premature muscle differentiation. Thus, we propose a model where DUSP13 and DUSP27 contribute to the fate transition of MuSCs from proliferation to differentiation during myogenesis.

Tumor necrosis factor α deficiency promotes myogenesis and muscle regeneration.

Tumor necrosis factor α (TNFα) exhibits diverse biological functions; however, its regulatory roles in myogenesis are not fully understood. In the present study, we explored the function of TNFα in myoblast proliferation, differentiation, migration, and myotube fusion in primary myoblasts and C2C12 cells. To this end, we constructed TNFα muscle-conditional knockout ( TNFα-CKO) mice and compared them with flox mice to assess the effects of TNFα knockout on skeletal muscles. Results indicated that TNFα-CKO mice displayed phenotypes such as accelerated muscle development, enhanced regenerative capacity, and improved exercise endurance compared to flox mice, with no significant differences observed in major visceral organs or skeletal structure. Using label-free proteomic analysis, we found that TNFα-CKO altered the distribution of several muscle development-related proteins, such as Hira, Casz1, Casp7, Arhgap10, Gas1, Diaph1, Map3k20, Cfl2, and Igf2, in the nucleus and cytoplasm. Gene set enrichment analysis (GSEA) further revealed that TNFα deficiency resulted in positive enrichment in oxidative phosphorylation and MyoD targets and negative enrichment in JAK-STAT signaling. These findings suggest that TNFα-CKO positively regulates muscle growth and development, possibly via these newly identified targets and pathways.

Regulation of Dystroglycan Gene Expression in Early Myoblast Differentiation.

Dystroglycan, a component of the dystrophin-associated glycoprotein complex, connects the extracellular matrix and cytoskeleton to maintain muscle membrane integrity. As such, abnormalities of dystroglycan are linked to different types of muscular dystrophies. In an effort to develop therapeutic approaches to re-establish signal integration for muscle repair and homeostasis, we have previously determined that a clinically approved agonist of retinoid X receptor enhances myoblast differentiation through direct regulation of gene expression of the muscle master regulator MyoD. Using comprehensive omics and molecular analyses, we found that dystroglycan gene expression is responsive to retinoid X receptor-selective signaling in early myoblast differentiation. In addition, the dystroglycan gene is a MyoD target, and residue-specific histone acetylation coincides with the occupancy of histone acetyltransferase p300 at the MyoD binding sites. Consequently, the p300 function is important for rexinoid-augmented dystroglycan gene expression. Finally, dystroglycan plays a role in myoblast differentiation. Our study sheds new light on dystroglycan regulation and function in myoblast differentiation and presents a potential avenue for re-establishing signal integration of a specific chromatin state pharmacologically to overcome muscle pathology and identify additional myogenic interactions for therapeutic applications.

MyoD induces ARTD1 and nucleoplasmic poly-ADP-ribosylation during fibroblast to myoblast transdifferentiation

SummaryWhile protein ADP-ribosylation was reported to regulate differentiation and dedifferentiation, it has so far not been studied during transdifferentiation. Here, we found that MyoD-induced transdifferentiation of fibroblasts to myoblasts promotes the expression of the ADP-ribosyltransferase ARTD1. Comprehensive analysis of the genome architecture by Hi-C and RNA-seq analysis during transdifferentiation indicated that ARTD1 locally contributed to A/B compartmentalization and coregulated a subset of MyoD target genes that were however not sufficient to alter transdifferentiation. Surprisingly, the expression of ARTD1 was accompanied by the continuous synthesis of nuclear ADP ribosylation that was neither dependent on the cell cycle nor induced by DNA damage. Conversely to the H2O2-induced ADP-ribosylation, the MyoD-dependent ADP-ribosylation was not associated to chromatin but rather localized to the nucleoplasm. Together, these data describe a MyoD-induced nucleoplasmic ADP-ribosylation that is observed particularly during transdifferentiation and thus potentially expands the plethora of cellular processes associated with ADP-ribosylation.

Poly(ADP-ribose) Polymerase 1 (PARP1) restrains MyoD-dependent gene expression during muscle differentiation

The myogenic factor MyoD regulates skeletal muscle differentiation by interacting with a variety of chromatin-modifying complexes. Although MyoD can induce and maintain chromatin accessibility at its target genes, its binding and trans-activation ability can be limited by some types of not fully characterized epigenetic constraints. In this work we analysed the role of PARP1 in regulating MyoD-dependent gene expression. PARP1 is a chromatin-associated enzyme, playing a well recognized role in DNA repair and that is implicated in transcriptional regulation. PARP1 affects gene expression through multiple mechanisms, often involving the Poly(ADP-ribosyl)ation of chromatin proteins. In line with PARP1 down-regulation during differentiation, we observed that PARP1 depletion boosts the up-regulation of MyoD targets, such as p57, myogenin, Mef2C and p21, while its re-expression reverts this effect. We also found that PARP1 interacts with some MyoD-binding regions and that its presence, independently of the enzymatic activity, interferes with MyoD recruitment and gene induction. We finally suggest a relationship between the binding of PARP1 and the loss of the activating histone modification H3K4me3 at MyoD-binding regions. This work highlights not only a novel player in the epigenetic control of myogenesis, but also a repressive and catalytic-independent mechanisms by which PARP1 regulates transcription.

Maternal Selenium Supplementation Enhanced Skeletal Muscle Development Through Increasing Protein Synthesis and SelW mRNA Levels of their Offspring.

The present study aimed to investigate the influence of maternal selenium supplementation on the skeletal muscle development of the offspring. A total of 720 Ross 308 broiler breeders at 24-week-old were allocated into 3 treatments with 6 replicates of 40 hens each and fed with 0mg/kg-(group Se/C), 0.5mg/kg organic-(group Se/O), and 0.5mg/kg inorganic-(group Se/I) selenium, respectively for 8weeks. The male offspring from each nutritional treatment were divided and housed into 8 cages of 12 birds each and fed with a commercial diet supplemented with selenium from Na2SeO3 at 0.15mg/kg. Results showed that Se/O group had the highest selenium deposition (P < 0.05) in the egg yolk and albumen. Furthermore, maternal selenium supplementation promoted breast muscle yield; increased serum insulin and IGF-I concentration; upregulated AKT, mammalian target of rapamycin (mTOR), P70S6K, Myf5, MyoD, MyoG, and SelW mRNA levels; and improved the phosphorylation of AKT at Serine 473 residue, mTOR at Serine 2448 residue, and FOXO at Serine 256 residue in skeletal muscles of the offspring. In contrast, the hens' diet supplemented with selenium could result in reduction of uric acid level in serum and downregulation of Atrogin-1 and MuRF1 mRNA levels in the skeletal muscle of the offspring. Additionally, no significant effect on the skeletal muscle development post-hatch was observed between organic and inorganic selenium supplementation. In conclusion, maternal organic selenium supplementation improved selenium deposition in egg; however, no significant effect has been detected on the breast muscle development of the offspring of broiler breeder compared with inorganic selenium supplementation.

G9a promotes proliferation and inhibits cell cycle exit during myogenic differentiation.

Differentiation of skeletal muscle cells, like most other cell types, requires a permanent exit from the cell cycle. The epigenetic programming underlying these distinct cellular states is not fully understood. In this study, we provide evidence that the lysine methyltransferase G9a functions as a central axis to regulate proliferation and differentiation of skeletal muscle cells. Transcriptome analysis of G9a knockdown cells revealed deregulation of many cell cycle regulatory genes. We demonstrate that G9a enhances cellular proliferation by two distinct mechanisms. G9a blocks cell cycle exit via methylation-dependent transcriptional repression of the MyoD target genes p21Cip/Waf1 and Rb1. In addition, it activates E2F1-target genes in a methyltransferase activity-independent manner. We show that G9a is present in the E2F1/PCAF complex, and enhances PCAF occupancy and histone acetylation marks at E2F1-target promoters. Interestingly, G9a preferentially associates with E2F1 at the G1/S phase and with MyoD at the G2/M phase. Our results provide evidence that G9a functions both as a co-activator and a co-repressor to enhance cellular proliferation and inhibit myogenic differentiation.

Identification of MyoD Interactome Using Tandem Affinity Purification Coupled to Mass Spectrometry.

Skeletal muscle terminal differentiation starts with the commitment of pluripotent mesodermal precursor cells to myoblasts. These cells have still the ability to proliferate or they can differentiate and fuse into multinucleated myotubes, which maturate further to form myofibers. Skeletal muscle terminal differentiation is orchestrated by the coordinated action of various transcription factors, in particular the members of the Muscle Regulatory Factors or MRFs (MyoD, Myogenin, Myf5, and MRF4), also called the myogenic bHLH transcription factors family. These factors cooperate with chromatin-remodeling complexes within elaborate transcriptional regulatory network to achieve skeletal myogenesis. In this, MyoD is considered the master myogenic transcription factor in triggering muscle terminal differentiation. This notion is strengthened by the ability of MyoD to convert non-muscle cells into skeletal muscle cells. Here we describe an approach used to identify MyoD protein partners in an exhaustive manner in order to elucidate the different factors involved in skeletal muscle terminal differentiation. The long-term aim is to understand the epigenetic mechanisms involved in the regulation of skeletal muscle genes, i.e., MyoD targets. MyoD partners are identified by using Tandem Affinity Purification (TAP-Tag) from a heterologous system coupled to mass spectrometry (MS) characterization, followed by validation of the role of relevant partners during skeletal muscle terminal differentiation. Aberrant forms of myogenic factors, or their aberrant regulation, are associated with a number of muscle disorders: congenital myasthenia, myotonic dystrophy, rhabdomyosarcoma and defects in muscle regeneration. As such, myogenic factors provide a pool of potential therapeutic targets in muscle disorders, both with regard to mechanisms that cause disease itself and regenerative mechanisms that can improve disease treatment. Thus, the detailed understanding of the intermolecular interactions and the genetic programs controlled by the myogenic factors is essential for the rational design of efficient therapies.

MyoD Is a Novel Activator of Porcine FIT1 Gene by Interacting with the Canonical E-Box Element during Myogenesis

Fat-induced transcript 1 (FIT1/FITM1) gene is a member of the conserved gene family important for triglyceride-rich lipid droplet accumulation. FIT1 gene displays a similar muscle-specific expression across pigs, mice, and humans. Thus pigs can act as a useful model of many human diseases resulting from misexpression of FIT1 gene. Triglyceride content in skeletal muscle plays a key role in pork meat quality and flavors. An insertion/deletion mutation in porcine FIT1 coding region shows a high correlation with a series of fat traits. To gain better knowledge of the potential role of FIT1 gene in human diseases and the correlations with pork meat quality, our attention is given to the region upstream of the porcine FIT1 coding sequence. We cloned ~1 kb of the 5′-flanking region of porcine FIT1 gene to define the role of this sequence in modulating the myogenic expression. A canonical E-box element that activated porcine FIT1 promoter activity during myogenesis was identified. Further analysis demonstrated that promoter activity was induced by overexpression of MyoD1, which bound to this canonical E-box during C2C12 differentiation. This is the first evidence that FIT1 as the direct novel target of MyoD is involved in muscle development.

Jmjd2C increases MyoD transcriptional activity through inhibiting G9a-dependent MyoD degradation

Skeletal muscle cell differentiation requires a family of proteins called myogenic regulatory factors (MRFs) to which MyoD belongs. The activity of MyoD is under epigenetic regulation, however, the molecular mechanism by which histone KMTs and KDMs regulate MyoD transcriptional activity through methylation remains to be determined. Here we provide evidence for a unique regulatory mechanism of MyoD transcriptional activity through demethylation by Jmjd2C demethylase whose level increases during muscle differentiation. G9a decreases MyoD stability via methylation-dependent MyoD ubiquitination. Jmjd2C directly associates with MyoD in vitro and in vivo to demethylate and stabilize MyoD. The hypo-methylated MyoD due to Jmjd2C is significantly more stable than hyper-methylated MyoD by G9a. Cul4/Ddb1/Dcaf1 pathway is essential for the G9a-mediated MyoD degradation in myoblasts. By the stabilization of MyoD, Jmjd2C increases myogenic conversion of mouse embryonic fibroblasts and MyoD transcriptional activity with erasing repressive H3K9me3 level at the promoter of MyoD target genes. Collectively, Jmjd2C increases MyoD transcriptional activity to facilitate skeletal muscle differentiation by increasing MyoD stability through inhibiting G9a-dependent MyoD degradation.

DNA damage-activated ABL-MyoD signaling contributes to DNA repair in skeletal myoblasts

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.

Proteomic and Functional Analyses Reveal the Role of Chromatin Reader SFMBT1 in Regulating Epigenetic Silencing and the Myogenic Gene Program*

SFMBT1 belongs to the malignant brain tumor domain-containing chromatin reader family that recognizes repressive histone marks and represses transcription. The biological functions and molecular basis underlying SFMBT1-mediated transcriptional repression are poorly elucidated. Here, our proteomic analysis revealed that SFMBT1 is associated with multiple transcriptional corepressor complexes, including CtBP/LSD1/HDAC complexes, polycomb repressive complexes, and malignant brain tumor family proteins, that collectively contribute to SFMBT1 repressor activity. During myogenesis, Sfmbt1 represses myogenic differentiation of cultured and primary myoblasts. Mechanistically, Sfmbt1 interacts with MyoD and mediates epigenetic silencing of MyoD target genes via recruitment of its associated corepressors and subsequent induction of epigenetic modifications and chromatin compaction. Therefore, our study identified novel mechanisms accounting for SFMBT1-mediated transcription repression and revealed an essential role of Sfmbt1 in regulating MyoD-mediated transcriptional silencing that is required for the maintenance of undifferentiated states of myogenic progenitor cells.

CCAAT/Enhancer Binding Protein Beta is Expressed in Satellite Cells and Controls Myogenesis

Upon injury, muscle satellite cells become activated and produce skeletal muscle precursors that engage in myogenesis. We demonstrate that the transcription factor CCAAT/enhancer binding protein beta (C/EBPβ) is expressed in the satellite cells of healthy muscle. C/EBPβ expression is regulated during myogenesis such that C/EBPβ is rapidly and massively downregulated upon induction to differentiate. Furthermore, persistent expression of C/EBPβ in myoblasts potently inhibits differentiation at least in part through the inhibition of MyoD protein function and stability. As a consequence, myogenic factor expression, myosin heavy chain expression, and fusogenic activity were reduced in C/EBPβ-overexpressing cells. Using knockout models, we demonstrate that loss of Cebpb expression in satellite cells results in precocious differentiation of myoblasts in growth conditions and greater cell fusion upon differentiation. In vivo, loss of Cebpb expression in satellite cells resulted in larger muscle fiber cross-sectional area and improved repair after muscle injury. Our results support the notion that C/EBPβ inhibits myogenic differentiation and that its levels must be reduced to allow for activation of MyoD target genes and the progression of differentiation.

Early transcriptional targets of MyoD link myogenesis and somitogenesis

In order to identify early transcriptional targets of MyoD prior to skeletal muscle differentiation, we have undertaken a transcriptomic analysis on gastrula stage Xenopus embryos in which MyoD has been knocked-down. Our validated list of genes transcriptionally regulated by MyoD includes Esr1 and Esr2, which are known targets of Notch signalling, and Tbx6, mesogenin, and FoxC1; these genes are all are known to be essential for normal somitogenesis but are expressed surprisingly early in the mesoderm. In addition we found that MyoD is required for the expression of myf5 in the early mesoderm, in contrast to the reverse relationship of these two regulators in amniote somites. These data highlight a role for MyoD in the early mesoderm in regulating a set of genes that are essential for both myogenesis and somitogenesis.

Snail Regulates MyoD Binding-Site Occupancy to Direct Enhancer Switching and Differentiation-Specific Transcription in Myogenesis

In skeletal myogenesis, the transcription factor MyoD activates distinct transcriptional programs in progenitors compared to terminally differentiated cells. Using ChIP-Seq and gene expression analyses, we show that in primary myoblasts, Snail-HDAC1/2 repressive complex binds and excludes MyoD from its targets. Notably, Snail binds E box motifs that are G/C rich in their central dinucleotides, and such sites are almost exclusively associated with genes expressed during differentiation. By contrast, Snail does not bind the A/T-rich E boxes associated with MyoD targets in myoblasts. Thus, Snai1-HDAC1/2 prevent MyoD occupancy on differentiation-specific regulatory elements, and the change from Snail to MyoD binding often results in enhancer switching during differentiation. Furthermore, we show that a regulatory network involving myogenic regulatory factors (MRFs), Snai1/2, miR-30a, and miR-206 acts as a molecular switch that controls entry into myogenic differentiation. Together, these results reveal a regulatory paradigm that directs distinct gene expression programs in progenitors versus terminally differentiated cells.

Lysine methyltransferase G9a methylates the transcription factor MyoD and regulates skeletal muscle differentiation

Skeletal muscle cells have served as a paradigm for understanding mechanisms leading to cellular differentiation. The proliferation and differentiation of muscle precursor cells require the concerted activity of myogenic regulatory factors including MyoD. In addition, chromatin modifiers mediate dynamic modifications of histone tails that are vital to reprogramming cells toward terminal differentiation. Here, we provide evidence for a unique dimension to epigenetic regulation of skeletal myogenesis. We demonstrate that the lysine methyltransferase G9a is dynamically expressed in myoblasts and impedes differentiation in a methyltransferase activity-dependent manner. In addition to mediating histone H3 lysine-9 di-methylation (H3K9me2) on MyoD target promoters, endogenous G9a interacts with MyoD in precursor cells and directly methylates it at lysine 104 (K104) to constrain its transcriptional activity. Mutation of K104 renders MyoD refractory to inhibition by G9a and enhances its myogenic activity. Interestingly, MyoD methylation is critical for G9a-mediated inhibition of myogenesis. These findings provide evidence of an unanticipated role for methyltransferases in cellular differentiation states by direct posttranslational modification of a transcription factor.

MyoD Directly Up-regulates Premyogenic Mesoderm Factors during Induction of Skeletal Myogenesis in Stem Cells

Gain- and loss-of-function experiments have illustrated that the family of myogenic regulatory factors is necessary and sufficient for the formation of skeletal muscle. Furthermore, MyoD required cellular aggregation to induce myogenesis in P19 embryonal carcinoma stem cells. To determine the mechanism by which stem cells can be directed into skeletal muscle, a time course of P19 cell differentiation was examined in the presence and absence of exogenous MyoD. By quantitative PCR, the first MyoD up-regulated transcripts were the premyogenic mesoderm factors Meox1, Pax7, Six1, and Eya2 on day 4 of differentiation. Subsequently, the myoblast markers myogenin, MEF2C, and Myf5 were up-regulated, leading to skeletal myogenesis. These results were corroborated by Western blot analysis, showing up-regulation of Pax3, Six1, and MEF2C proteins, prior to myogenin protein expression. To determine at what stage a dominant-negative MyoD/EnR mutant could inhibit myogenesis, stable cell lines were created and examined. Interestingly, the premyogenic mesoderm factors, Meox1, Pax3/7, Six1, Eya2, and Foxc1, were down-regulated, and as expected, skeletal myogenesis was abolished. Finally, to identify direct targets of MyoD in this system, chromatin immunoprecipitation experiments were performed. MyoD was observed associated with regulatory regions of Meox1, Pax3/7, Six1, Eya2, and myogenin genes. Taken together, MyoD directs stem cells into the skeletal muscle lineage by binding and activating the expression of premyogenic mesoderm genes, prior to activating myoblast genes.

The RNA-binding protein Seb4/RBM24 is a direct target of MyoD and is required for myogenesis during Xenopus early development

RNA-binding proteins play an important role to post-transcriptionally regulate gene expression. During early development they exhibit temporally and spatially regulated expression pattern. The expression of Xenopus laevis Seb4 gene, also known as RBM24 in other vertebrates, is restricted to the lateral and ventral mesoderm during gastrulation and then localized to the somitic mesoderm, in a similar pattern as XMyoD gene. Using a hormone-inducible form of MyoD to identify potential direct MyoD target genes, we find that Seb4 expression is directly regulated by MyoD at the gastrula stage. We further show that a 0.65 kb X. tropicalis RBM24 regulatory region contains multiple E boxes (CANNTG), which are potential binding sites for MyoD and other bHLH proteins. By injecting a RBM24 reporter construct into the animal pole of X. laevis embryos, we find that this reporter gene is indeed specifically activated by MyoD and repressed by a dominant negative MyoD mutant. Knockdown of Seb4 produces similar effects as those obtained by the dominant negative MyoD mutant, indicating that it is required for the expression of myogenic genes and myogenesis in the embryo. In cultured ectodermal explants, although overexpression of Seb4 has no obvious effect on myogenesis, knockdown of Seb4 inhibits the expression of myogenic genes and myogenesis induced by MyoD. These results reveal that Seb4 is a target of MyoD during myogenesis and is required for myogenic gene expression.

The Core Binding Factor CBF Negatively Regulates Skeletal Muscle Terminal Differentiation

BackgroundCore Binding Factor or CBF is a transcription factor composed of two subunits, Runx1/AML-1 and CBF beta or CBFβ. CBF was originally described as a regulator of hematopoiesis.Methodology/Principal FindingsHere we show that CBF is involved in the control of skeletal muscle terminal differentiation. Indeed, downregulation of either Runx1 or CBFβ protein level accelerates cell cycle exit and muscle terminal differentiation. Conversely, overexpression of CBFβ in myoblasts slows terminal differentiation. CBF interacts directly with the master myogenic transcription factor MyoD, preferentially in proliferating myoblasts, via Runx1 subunit. In addition, we show a preferential recruitment of Runx1 protein to MyoD target genes in proliferating myoblasts. The MyoD/CBF complex contains several chromatin modifying enzymes that inhibits MyoD activity, such as HDACs, Suv39h1 and HP1β. When overexpressed, CBFβ induced an inhibition of activating histone modification marks concomitant with an increase in repressive modifications at MyoD target promoters.Conclusions/SignificanceTaken together, our data show a new role for Runx1/CBFβ in the control of the proliferation/differentiation in skeletal myoblasts.

A Systems Approach Reveals that the Myogenesis Genome Network Is Regulated by the Transcriptional Repressor RP58

We 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.