MLL1 promotes myogenesis by epigenetically regulating Myf5
Shufang CaiQi ZhuCilin GuoRenqiang YuanXumeng ZhangYaping NieLuxi ChenYing FangKeren ChenJunyan ZhangDelin MoYaosheng Chen
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Abstract Objectives Mixed lineage leukaemia protein‐1 (MLL1) mediates histone 3 lysine 4 (H3K4) trimethylation (me3) and plays vital roles during early embryonic development and hematopoiesis. In our previous study, we found its expression was positively correlated with embryonic myogenic ability in pigs, indicating its potential roles in mammalian muscle development. The present work aimed to explore the roles and regulation mechanisms of MLL1 in myogenesis. Materials and methods The expression of MLL1 in C2C12 cells was experimentally manipulated using small interfering RNAs (siRNA). 5‐ethynyl‐2′‐deoxyuridine (EdU) assay, cell cycle assay, immunofluorescence, qRT‐PCR and Western blot were performed to assess myoblast proliferation and differentiation. Chromatin immunoprecipitation assay was conducted to detect H3K4me3 enrichment on myogenic factor 5 ( Myf5 ) promoter. A cardiotoxin (CTX)‐mediated muscle regeneration model was used to investigate the effects of MLL1 on myogenesis in vivo. Results MLL1 was highly expressed in proliferating C2C12 cells, and expression decreased after differentiation. Knocking down MLL1 suppressed myoblast proliferation and impaired myoblast differentiation. Furthermore, knockdown of MLL1 resulted in the arrest of cell cycle in G1 phase, with decreased expressions of Myf5 and Cyclin D1. Mechanically, MLL1 transcriptionally regulated Myf5 by mediating H3K4me3 on its promoter. In vivo data implied that MLL1 was required for Pax7‐positive satellite cell proliferation and muscle repair. Conclusion MLL1 facilitates proliferation of myoblasts and Pax7‐positive satellite cells by epigenetically regulating Myf5 via mediating H3K4me3 on its promoter.Keywords:
MYF5
H3K4me3
C2C12
Myogenic regulatory factors
Chromatin immunoprecipitation
Muscle regeneration following injury is dependent on the ability of muscle satellite cells to activate, proliferate and fuse with damaged fibres. This process is controlled by the myogenic regulatory factors (MRF). Little is known about the temporal relation of the MRF with the expression of known myogenic growth factors (i.e. IGF-1) in humans following muscle damage. Eight subjects (20.6 +/- 2.1 years; 81.4 +/- 9.8 kg) performed 300 lengthening contractions (180 deg s(-1)) of their knee extensors in one leg on a dynamometer. Blood and muscle samples were collected before and at 4 (T4), 24 (T24), 72 (T72) and 120 h (T120) post-exercise. Mechano growth factor (MGF), IGF-1Ea and IGF-1Eb mRNA were quantified. Serum IGF-1 did not change over the post-exercise time course. IGF-1Ea and IGF-1Eb mRNA increased approximately 4- to 6-fold by T72 (P < 0.01) and MGF mRNA expression peaked at T24 (P = 0.005). MyoD mRNA expression increased approximately 2-fold at T4 (P < 0.05). Myf5 expression peaked at T24 (P < 0.05), while MRF4 and myogenin mRNA expression peaked at T72 (P < 0.05). Myf5 expression strongly correlated with the increase in MGF mRNA (r(2) = 0.83; P = 0.03), while MRF4 was correlated with both IGF-1Ea and -Eb (r(2) = 0.90; r(2) = 0.81, respectively; P < 0.05). Immunofluorescence analysis showed IGF-1 protein expression localized to satellite cells at T24, and to satellite cells and the myofibre at T72 and T120; IGF-1 was not detected at T0 or T4. These results suggest that the temporal response of MGF is probably related to the activation/proliferation phase of the myogenic programme as marked by an increase in both Myf5 and MyoD, while IGF-1Ea and -Eb may be temporally related to differentiation as marked by an increase in MRF4 and myogenin expression following acute muscle damage.
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Myogenic regulatory factors of the Myod family (MRFs) are transcription factors essential for mammalian skeletal myogenesis. However,the roles of each gene in myogenesis remain unclear, owing partly to genetic linkage at the Myf5/Mrf4 locus and to rapid morphogenetic movements in the amniote somite. In mice, Myf5 is essential for the earliest epaxial myogenesis, whereas Myod is required for timely differentiation of hypaxially derived muscle. A second major subdivision of the somite is between primaxial muscle of the somite proper and abaxial somite-derived migratory muscle precursors. Here, we use a combination of mutant and morphant analysis to ablate the function of each of the four conserved MRF genes in zebrafish, an organism that has retained a more ancestral bodyplan. We show that a fundamental distinction in somite myogenesis is into medial versus lateral compartments, which correspond to neither epaxial/hypaxial nor primaxial/abaxial subdivisions. In the medial compartment, Myf5 and/or Myod drive adaxial slow fibre and medial fast fibre differentiation. Myod-driven Myogenin activity alone is sufficient for lateral fast somitic and pectoral fin fibre formation from the lateral compartment, as well as for cranial myogenesis. Myogenin activity is a significant contributor to fast fibre differentiation. Mrf4 does not contribute to early myogenesis in zebrafish. We suggest that the differential use of duplicated MRF paralogues in this novel two-component myogenic system facilitated the diversification of vertebrates.
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Skeletal muscle satellite cells (SC) play an important role in muscle repair following injury. The regulation of SC activity is governed by myogenic regulatory factors (MRF), including MyoD, Myf5, myogenin, and MRF4. The mRNA expression of these MRF in humans following muscle damage has been predominately measured in whole muscle homogenates. Whether the temporal expression of MRF in a whole muscle homogenate reflects SC-specific expression of MRF remains largely unknown. Sixteen young men (23.1 ± 1.0 yr) performed 300 unilateral eccentric contractions (180°/s) of the knee extensors. Percutaneous muscle biopsies from the vastus lateralis were taken before (Pre) and 48 h postexercise. Fluorescence-activated cell sorting analysis was utilized to purify NCAM + muscle SC from the whole muscle homogenate. Forty-eight hours post-eccentric exercise, MyoD, Myf5, and myogenin mRNA expression were increased in the whole muscle homogenate (~1.4-, ~4.0-, ~1.7-fold, respectively, P < 0.05) and in isolated SC (~19.3-, ~17.5-, ~58.9-fold, respectively, P < 0.05). MRF4 mRNA expression was not increased 48 h postexercise in the whole muscle homogenate ( P > 0.05) or in isolated SC ( P > 0.05). In conclusion, our results suggest that the directional changes in mRNA expression of the MRF in a whole muscle homogenate in response to acute eccentric exercise reflects that observed in isolated muscle SC. NEW & NOTEWORTHY The myogenic program is controlled via transcription factors referred to as myogenic regulatory factors (MRF). Previous studies have derived MRF expression from whole muscle homogenates, but little work has examined whether the mRNA expression of these transcripts reflects the pattern of expression in the actual population of satellite cells (SC). We report that MRF expression from an enriched SC population reflects the directional pattern of expression from skeletal muscle biopsy samples following eccentric contractions.
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Although Xenopus is a key model organism in developmental biology, little is known about the myotome formation in this species. Here, we assessed the expression of myogenic regulatory factors of the Myod family (MRFs) during embryonic development and revealed distinct MRF programs.The expression pattern of each MRF during embryonic development highlights three successive myogenic waves. We showed that a first median and lateral myogenesis initiates before dermomyotome formation: the median cell population expresses Myf5, Myod, and Mrf4, whereas the lateral one expresses Myod, moderate levels of Myogenin and Mrf4. The second wave of myoblasts arising from the dermomyotome is characterized by the full MRF program expression, with high levels of Myogenin. The third wave is revealed by Myf5 expression in the myotome and could contribute to the formation of plurinucleated fibers at larval stages. Furthermore, Myf5- or Myod-expressing anlagen are identified in craniofacial myogenesis.The first median and lateral myogenesis and their associated MRF programs have probably disappeared in mammals. However, some aspects of Xenopus myogenesis have been conserved such as the development of somitic muscles by successive myogenic waves and the existence of Myf5-dependent and -independent lineages.
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The important roles of myogenic regulatory factors (MRF) in mammalian skeletal myogenesis have been well studied, but few equivalent studies have been performed in poultry. The expression pattern of MRF during the embryonic development of skeletal muscle in ducks remains unknown. In this study, we identified Myf5, Myf6, MyoD, and myogenin genes in Jinding ducks (Anas platyrhynchos domestica) and quantified their expression levels in breast muscle (BM) and leg muscle (LM) at embryonic d 13, 17, 21, 25, and 27 by real-time reverse-transcription PCR. Body weight and muscle weight show different developmental patterns. The MRF genes were expressed in both BM and LM, but with different expression patterns. The MyoD gene showed lower expression levels in BM before embryonic d 21 compared with LM, whereas the opposite pattern was found later. The higher expression level of MyoD, as well its lagged expression pattern in BM, suggest that the MyoD gene may be involved in maintaining the development of different muscles. Correlation analysis showed that myogenin gene expression levels were significantly negatively correlated with BW and muscle weight in both BM and LM (P < 0.001), and MyoD and Myf6 gene expression levels were more strongly correlated with muscle weight in LM than in BM. The results of this study provide novel evidence for MRF expression in ducks in embryonic stage- and skeletal muscle-dependent manners, and provide a foundation for understanding the molecular control of skeletal muscle growth in duck breeds.
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Satellite cells (SCs) exist amongst other cell types in a unique microenvironment within skeletal muscle. The interactions between SCs and non‐myogenic cells within this niche are poorly characterized. However, most cells behave differently in co‐culture (CC) compared to mono‐culture (MC). Our results indicate that SCs co‐cultured with preadipocytes express less myogenic differentiation factor ( MyoD ) at 24 ( p=0.04 ) and 48 ( p=0.02 ) hours as well as more sprouty1 ( Spry1 ) at 24 ( p=0.04 ) hours post‐differentiation. Thus, SCs induced to differentiate in a CC with preadipocytes exhibit depressed myogenesis when compared with SCs in MC. Polyamines (putrescine, spermidine and spermine) are potent modulators of cell growth. However, no studies have analyzed effects of these compounds on SCs. Administration of polyamines to SCs in MC and CC systems demonstrates that polyamines increase ( p ≤ 0.05 ) expression of MyoD , myogenin ( Myf4 ), myogenic factor 5 ( Myf5 ) and decrease ( p ≤0.05) expression of paired box transcripton factor 7 ( Pax7 ) and Spry1 . Compared with MC, our CC system better reflects the complexity of the SC niche. Furthermore, our observation of a pro‐myogenic effect of polyamines using CC may indicate a potential natural means of enhancing myogenesis in vivo . We gratefully acknowledge the University of Idaho's Student Grant Program, the USDA NC1184 Hatch Grant and the Idaho Beef Council FY 2013 for funding.
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Myogenesis has been a leading model for elucidating the molecular mechanisms that underlie tissue differentiation and development since the discovery of MyoD. During myogenesis, the fate of myogenic precursor cells is first determined by Pax3/Pax7. This is followed by regulation of the myogenic differentiation program by muscle regulatory factors (Myf5, MyoD, Myog, and Mrf4) to form muscle tissues. Recent studies have uncovered a detailed myogenic program that involves the RP58 (Zfp238)-dependent regulatory network, which is critical for repressing the expression of inhibitor of DNA binding (Id) proteins. These novel findings contribute to a comprehensive understanding of the muscle differentiation transcriptional program.
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