Role Of MyoD Research Articles

Characterization of the promoter region of bovine ATP5B: roles of MyoD and GATA1 in the regulation of basal transcription

Intramuscular fat (IMF) content is a key determinant of beef quality, making it a key topic of research interest. ATP5B serves as the catalytic component of the mitochondrial ATP synthase complex and plays essential roles in controlling fat contents and oxidative metabolism in bovine skeletal muscle. In this study, we determined that bovine ATP5B was highly expressed in longissimus thoracis. To elucidate the molecular mechanisms involved in bovine ATP5B regulation, we cloned and characterized the promoter region of ATP5B. Applying 5'-rapid amplification of cDNA end analysis (RACE), we identified two transcriptional start sites (TSSs) in its promoter region. Using a series of 5'-deletion promoter plasmids in luciferase reporter assay, we found that the proximal minimal promoter of ATP5B was located within the region −539/220 relative to the TSS. Site-directed mutation in combination with chromatin immunoprecipitation (ChIP) assays demonstrated that MyoD and GATA1 binding to the promoter region drives bovine ATP5B transcription. Taken together, these results provide new insight into the regulatory mechanisms of ATP5B transcription in mediating the IMF content of beef.

Characterization of the promoter region of the bovine SIX1 gene: Roles of MyoD, PAX7, CREB and MyoG

The SIX1 gene belongs to the family of six homeodomain transcription factors (TFs), that regulates the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway and mediate skeletal muscle growth and regeneration. Previous studies have demonstrated that SIX1 is positively correlated with body measurement traits (BMTs). However, the transcriptional regulation of SIX1 remains unclear. In the present study, we determined that bovine SIX1 was highly expressed in the longissimus thoracis. To elucidate the molecular mechanisms involved in bovine SIX1 regulation, 2-kb of the 5′ regulatory region were obtained. Sequence analysis identified neither a consensus TATA box nor a CCAAT box in the 5′ flanking region of bovine SIX1. However, a CpG island was predicted in the region −235 to +658 relative to the transcriptional start site (TSS). An electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) assay in combination with serial deletion constructs of the 5′ flanking region, site-directed mutation and siRNA interference demonstrated that MyoD, PAX7 and CREB binding occur in region −689/−40 and play important roles in bovine SIX1 transcription. In addition, MyoG drives SIX1 transcription indirectly via the MEF3 motif. Taken together these interactions suggest a key functional role for SIX1 in mediating skeletal muscle growth in cattle.

Abstract 1431: A novel tumor-suppressor role of MyoD, a muscle differentiation factor, in mouse models of medulloblastoma

Abstract Medulloblastoma, an embryonal tumor of the cerebellum, is the most common pediatric brain cancer. For decades, it has been known that a small subset of medulloblastomas present with myogenic differentiation. However, the mechanistic basis and significance of this biological anomaly remain to be determined. We have identified hitherto unknown expression of muscle differentiation factor, MyoD in cerebellar tumors from mouse models of medulloblastoma as well as in a subset of human medulloblastomas. At a cellular level, MyoD is expressed in mitotically active cells and intriguingly, in mouse tumors does not activate its canonical targets involved in the muscle differentiation program. To understand the functional consequence of this finding in medulloblastoma genesis, we have conducted genetic studies to assess tumor formation in the SmoA1 and SmoA2 medulloblastoma models, on a MyoD-deficient background. We demonstrate that MyoD acts as a haploinsufficient tumor suppressor as the MyoD wt/-; SmoA1 and MyoD wt/-; SmoA2 mice have an accelerated onset of tumors compared to MyoD wt/wt; SmoA1 or MyoD wt/wt; SmoA2 mice respectively. Our findings suggest that the expression of MyoD might be an oncogene-induced compensatory response in the cerebellum to suppress tumor growth by restoring balance between proliferation and differentiation, similar to the role of some other bHLH transcription factors. Harnessing this latent tumor suppressor network to induce differentiation or cell cycle arrest holds promise for future therapeutic interventions. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1431. doi:1538-7445.AM2012-1431

Reversible differentiation of myofibroblasts by MyoD

Myofibroblasts participate in tissue repair processes in diverse mammalian organ systems. The deactivation of myofibroblasts is critical for termination of the reparative response and restoration of tissue structure and function. The current paradigm on normal tissue repair is the apoptotic clearance of terminally differentiated myofibroblasts; while, the accumulation of activated myofibroblasts is associated with progressive human fibrotic disorders. The capacity of myofibroblasts to undergo de-differentiation as a potential mechanism for myofibroblast deactivation has not been examined. In this report, we have uncovered a role for MyoD in the induction of myofibroblast differentiation by transforming growth factor-β1 (TGF-β1). Myofibroblasts demonstrate the capacity for de-differentiation and proliferation by modulation of endogenous levels of MyoD. We propose a model of reciprocal signaling between TGF-β1/ALK5/MyoD and mitogen(s)/ERK-MAPK/CDKs that regulate myofibroblast differentiation and de-differentiation, respectively. Our studies provide the first evidence for MyoD in controlling myofibroblast activation and deactivation. Restricted capacity for de-differentiation of myofibroblasts may underlie the progressive nature of recalcitrant human fibrotic disorders.

MyoD expression profile and developmental differences of leg and breast muscle in Peking duck (Anas platyrhynchos Domestica) during embryonic to neonatal stages

In order to investigate the developmental differences between the duck breast muscle and leg muscle tissues during the embryonic stage to neonatal stages, as well as the expression profile of MyoD between the two muscle tissues, the morphologic characteristics in the two muscle tissues during duck embryo stages at E14, E18, E22, E27 and D7 were compared through the muscle paraffin sections. The coding domain sequence of duck MyoD gene was cloned, and then the expression of MyoD in duck leg muscle and breast muscle during embryo stage on E10, E14, E18, E22, E27 and D7 was detected using qRT-PCR method. Results showed that the developmental status of the duck breast muscle in embryonic phrases lag behind that of leg muscle. The CDS of duck MyoD gene consists of 894 nucleotides, and showed relatively high similarity with the gene of other species. The MyoD mRNA expressed in both kinds of muscle tissues and the expression profile had a similar trend, although the expression level of MyoD in the breast muscle was significantly higher than that in the leg muscle at each developmental stages (p<0.05). Results suggested that MyoD might have potential functions in controlling muscle fiber phenotype during the secondary myogenesis of muscle development. These fundamental works may provide some valuable clues for knowing the roles of MyoD in the myogenesis and the muscle fiber type differentiation in birds.

The role of MyoD+ muscle progenitor cells in bone formation and repair

Bone morphogenetic proteins (BMPs) are able to induce osteogenic differentiation in many cells, including muscle cells. However, the actual contribution of muscle cells to bone formation and repair is unclear. Our objective was to examine the capacity of myogenic cells to contribute to BMP-induced ectopic bone formation and fracture repair. Osteogenic gene expression was measured by quantitative PCR in osteoprogenitors, myoblasts, and fibroblasts following BMP-2 treatment. The MyoD-Cre x ROSA26R and MyoD-Cre x Z/AP mouse strains were used to track the fate of MyoD+ cells in vivo. In these double-transgenic mice, MyoD+ progenitors undergo a permanent recombination event to induce reporter gene expression. Ectopic bone was produced by the intramuscular implantation of BMP-7. Closed tibial fractures and open tibial fractures with periosteal stripping were also performed. Cellular contribution was tracked at one, two and three week time points by histological staining. Osteoprogenitors and myoblasts exhibited comparable expression of early and late bone markers; in contrast bone marker expression was considerably less in fibroblasts. The sensitivity of cells to BMP-2 correlated with the expression of BMP receptor-1a (Bmpr1a). Pilot experiments using the MyoD-Cre x Rosa26R mice identified a contribution by MyoD expressing cells in BMP-induced ectopic bone formation. However, false positive LacZ staining in osteoclasts led us to seek alternative systems such as the MyoD-cre x Z/AP mice that have negligible background staining. Initially, a minor contribution from MyoD expressing cells was noted in the ectopic bones in the MyoD-cre x Z/AP mice, but without false positive osteoclast staining. Soft tissue trauma usually precedes the formation of ectopic bone. Hence, to mimic the clinical condition more precisely, physical injury to the muscle was performed. Traumatising the muscle two days prior to BMP-7 implantation: (1) induced MyoD expression in quiescent satellite cells; (2) increased ectopic bone formation; and (3) greatly enhanced the number of MyoD positive cells in the ectopic bone. In open tibial fractures the majority of the initial callus was MyoD+ indicating a significant contribution by myogenic cells. In contrast, closed fractures with the periosteum intact had a negligible myogenic contribution. Myoblasts but not fibroblasts were highly responsive to BMP stimulation and this was associated with BMP receptor expression. Our transgenic mouse models demonstrate for the first time that muscle progenitors can significantly contribute to ectopic bone formation and fracture repair. This may have translational applications for clinical orthopaedic therapies.

Development and application of ChIPSeq: genome‐wide DNA binding by MyoD

ChIPSeq is an application of high throughput direct sequencing (Solexa / Illumina) to assay for factor‐DNA binding sites retrieved by chromatin immunoprecipitation (ChIP). A goal was to achieve reliable identification of true binding sites throughout large genomes, without need for cloning or microarray. The target protein MyoD, a muscle‐specific transcription factor, was chosen for proof‐of‐principle for the large class of factors that bind small and degenerate motifs (e.g. CANNTG) and to acquire new information regarding the role of MyoD in myogenesis. For ChIP optimization, MyoD‐ChIP DNA fragments were assayed by quantitative (real time) PCR using a set of locations known to be positive for MyoD binding. For the direct sequencing assay, MyoD‐ChIP and control normal mouse IgG‐ChIP fragments were cluster‐amplified and sequenced. 32‐nt sequence reads were mapped to the mouse genome. MyoD‐ChIP and control reads were analyzed jointly to identify enriched regions. 6.8M out of 17.4M total sequence reads mapped to single locations. 5,113 genes were identified from 13,194 enriched regions. Data revealed expected and unexpected MyoD binding site locations. The results suggest that the method is broadly applicable for identifying binding locations and thereby identifying binding sequence motifs of any transcription factor in any sequenced genome, large or small. CSUPERB Joint Venture Grant to SBS.

Cluster analysis tests the importance of myogenic gene expression during myofiber hypertrophy in humans

We applied K-means cluster analysis to test the hypothesis that muscle-specific factors known to modulate protein synthesis and satellite cell activity would be differentially expressed during progressive resistance training (PRT, 16 wk) in 66 human subjects experiencing extreme, modest, and failed myofiber hypertrophy. Muscle mRNA expression of IGF-I isoform Ea (IGF-IEa), mechanogrowth factor (MGF, IGF-IEc), myogenin, and MyoD were assessed in muscle biopsies collected at baseline (T1) and 24 h after the first (T2) and last (T3) loading bouts from previously untrained subjects clustered as extreme responders (Xtr, n=17), modest responders (Mod, n=32), and nonresponders (Non, n=17) based on mean myofiber hypertrophy. Myofiber growth averaged 2,475 microm2 in Xtr, 1,111 microm2 in Mod, and -16 microm2 in Non. Main training effects revealed increases in all transcripts (46-83%, P<0.005). For the entire cohort, IGF-IEa, MGF, and myogenin mRNAs were upregulated by T2 (P<0.05), while MyoD did not increase significantly until T3 (P<0.001). Within clusters, MGF and myogenin upregulation was robust in Xtr (126% and 65%) and Mod (73% and 41%) vs. no changes in Non. While significant in all clusters by T3, IGF-IEa increased most in Xtr (105%) and least in Non (44%). Although MyoD expression increased overall, no changes within clusters were detected. We reveal for the first time that MGF and myogenin transcripts are differentially expressed in subjects experiencing varying degrees of PRT-mediated myofiber hypertrophy. The data strongly suggest the load-mediated induction of these genes may initiate important actions necessary to promote myofiber growth during PRT, while the role of MyoD is less clear.

Growth factor responsiveness: Role of MyoD and myogenin

A differentiation-defective variant (DD-1) of the MM14 myoblasts acquired the ability to synthesize DNA in response to treatment with epidermal growth factor (EGF) ( R. W. Lim and S. D. Hauschka, 1984, Dev. Biol. 105, 48) and no longer expressed myogenic determinant genes (i.e., MyoD and myogenin) ( P. R. Mueller, and B. Wold, 1989, Science 246, 780). To determine the effect of expression of MyoD on EGF responsiveness, DD-1 cells were cotransfected with a MyoD expression vector and with pRSVneo. A clone, MyoDD-1 cells, which was G418 resistant, formed multinuclear syncitia, and also expressed MyoD and myogenin, was further characterized. EGF responsiveness, as assessed by DNA synthesis, was decreased 5- to 10-fold in the MyoDD-1 cells from that in G418-resistant control DD-1 cells, despite similar EGF receptor numbers and binding affinities of the receptors. Responsiveness of MyoDD-1 cells to fibroblast growth factor (FGF) was also diminished although to a lesser extent. To determine the effects of decreased myogenic determinant gene expression on mitogen responsiveness, MM14 myoblasts were grown in medium supplemented with 5 μ M 5-bromo-2′-deoxyuridine (BUdR-MM14). BUdR-MM14 cells had decreased expression of MyoD and myogenin, did not fuse, and had an altered morphology, from round to flat. The BUdR effect on fusion and cell shape was reversed by growth in control medium. BUdR-MM14 cells were responsive to EGF and had enhanced responsiveness to FGF. The combined studies support the view that expression of MyoD and/or myogenin contributes to negative regulation of mitogen responsiveness.

MyoD expression in the forming somites is an early response to mesoderm induction in Xenopus embryos.

We describe the cloning, cDNA sequence and embryonic expression of a Xenopus homologue of MyoD, a mouse gene encoding a DNA-binding protein that can activate muscle gene expression in cultured cells. The predicted Xenopus MyoD protein sequence is remarkably similar to mouse MyoD. Zygotic expression of MyoD begins in early gastrulae, but there is a low level of unlocalized maternal message. Northern blot analysis of dissected embryos and in situ hybridization show that MyoD RNA is restricted to the gastrula mesoderm and to the somites of neurulae and tailbud embryos. The time and place of MyoD expression are consistent with a role for MyoD in the activation of other muscle genes in the somites of the frog embryo. However, MyoD is skeletal muscle-specific and is not expressed even in the early embryonic heart, which co-expresses cardiac and skeletal actin isoforms. Striated muscle genes can therefore be activated in some embryonic tissues in the absence of MyoD. The concentration of MyoD in the somites falls once they have formed, suggesting that MyoD may act there transiently to establish muscle gene expression. MyoD transcription is activated following mesoderm induction, and is the earliest muscle-specific response to mesoderm-inducing factors so far described.

MyoD expression in the forming somites is an early response to mesoderm induction in Xenopus embryos

Abstract We describe the cloning, cDNA sequence and embryonic expression of a Xenopus homologue of MyoD, a mouse gene encoding a DNA-binding protein that can activate muscle gene expression in cultured cells. The predicted Xenopus MyoD protein sequence is remarkably similar to mouse MyoD. Zygotic expression of MyoD begins in early gastrulae, but there is a low level of unlocalized maternal message. Northern blot analysis of dissected embryos and in situ hybridization show that MyoD RNA is restricted to the gastrula mesoderm and to the somites of neurulae and tailbud embryos. The time and place of MyoD expression are consistent with a role for MyoD in the activation of other muscle genes in the somites of the frog embryo. However, MyoD is skeletal muscle-specific and is not expressed even in the early embryonic heart, which co-expresses cardiac and skeletal actin isoforms. Striated muscle genes can therefore be activated in some embryonic tissues in the absence of MyoD. The concentration of MyoD in the somites falls once they have formed, suggesting that MyoD may act there transiently to establish muscle gene expression. MyoD transcription is activated following mesoderm induction, and is the earliest muscle-specific response to mesoderm-inducing factors so far described.