MUNC, an Enhancer RNA Upstream from the MYOD Gene, Induces a Subgroup of Myogenic Transcripts in trans Independently of MyoD

ABSTRACT MyoD upstream noncoding RNA (MUNC) initiates in the distal regulatory region (DRR) enhancer of MYOD and is formally classified as an enhancer RNA (DRReRNA). MUNC is required for optimal myogenic differentiation, induces specific myogenic transcripts in trans (MYOD, MYOGENIN, and MYH3), and has a functional human homolog. The vast majority of eRNAs are believed to act in cis primarily on their neighboring genes (1, 2), making it likely that MUNC action is dependent on the induction of MYOD RNA. Surprisingly, MUNC overexpression in MYOD−/− C2C12 cells induces many myogenic transcripts in the complete absence of MyoD protein. Genomewide analysis showed that, while many genes are regulated by MUNC in a MyoD-dependent manner, there is a set of genes that are regulated by MUNC, both upward and downward, independently of MyoD. MUNC and MyoD even appear to act antagonistically on certain transcripts. Deletion mutagenesis showed that there are at least two independent functional sites on the MUNC long noncoding RNA (lncRNA), with exon 1 more active than exon 2 and with very little activity from the intron. Thus, although MUNC is an eRNA of MYOD, it is also a trans-acting lncRNA whose sequence, structure, and cooperating factors, which include but are not limited to MyoD, determine the regulation of many myogenic genes.

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MUNC, a Long Noncoding RNA That Facilitates the Function of MyoD in Skeletal Myogenesis

Molecular and Cellular Biology ◽

10.1128/mcb.01079-14 ◽

2014 ◽

Vol 35 (3) ◽

pp. 498-513 ◽

Cited By ~ 85

Author(s):

Adam C. Mueller ◽

Magdalena A. Cichewicz ◽

Bijan K. Dey ◽

Ryan Layer ◽

Brian J. Reon ◽

...

Keyword(s):

Gene Expression ◽

Noncoding Rna ◽

Regulatory Region ◽

Myoblast Differentiation ◽

Content Type ◽

Multiple Promoters ◽

Enhancer Rna ◽

Distal Regulatory Region ◽

Interfering Rna

Anin silicoscreen for myogenic long noncoding RNAs (lncRNAs) revealed nine lncRNAs that are upregulated more than 10-fold in myotubes versus levels in myoblasts. One of these lncRNAs, MyoD upstream noncoding (MUNC, also known as DRReRNA), is encoded 5 kb upstream of the transcription start site ofMyoD, a myogenic transcription factor gene. MUNC is specifically expressed in skeletal muscle and exists as in unspliced and spliced isoforms, and its 5′ end overlaps with thecis-acting distal regulatory region (DRR) ofMyoD. Small interfering RNA (siRNA) of MUNC reduced myoblast differentiation and specifically reduced the association of MyoD to the DRR enhancer and myogenin promoter but not to another MyoD-dependent enhancer. Stable overexpression of MUNC from a heterologous promoter increased endogenousMyoD,Myogenin, andMyh3(myosin heavy chain, [MHC] gene) mRNAs but not the cognate proteins, suggesting that MUNC can act intransto promote gene expression but that this activity does not require an induction of MyoD protein. MUNC also stimulates the transcription of other genes that are not recognized as MyoD-inducible genes. Knockdown of MUNCin vivoimpaired murine muscle regeneration, implicating MUNC in primary satellite cell differentiation in the animal. We also discovered a human MUNC that is induced during differentiation of myoblasts and whose knockdown decreases differentiation, suggesting an evolutionarily conserved role of MUNC lncRNA in myogenesis. Although MUNC overlaps with the DRR enhancer, our results suggest that MUNC is not a classiccis-acting enhancer RNA (e-RNA) acting exclusively by stimulating the neighboringMyoDgene but more like a promyogenic lncRNA that acts directly or indirectly on multiple promoters to increase myogenic gene expression.

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Enhanced growth and myogenic differentiation of spheroid-derived C2C12 cells

Bioscience Biotechnology and Biochemistry ◽

10.1093/bbb/zbab018 ◽

2021 ◽

Author(s):

Guang-Zhen Jin

Keyword(s):

Myogenic Differentiation ◽

Three Dimensional ◽

3D Culture ◽

Stem Cell Differentiation ◽

Culture Conditions ◽

C2c12 Cells ◽

Dependent Manner ◽

Physical Force ◽

Myod Gene ◽

2D And 3D

Abstract Among many factors of controlling stem cell differentiation, the key transcription factor upregulation via physical force is a good strategy on the lineage-specific differentiation of stem cells. The study aimed to compare growth and myogenic potentials between the parental cells (PCs) and the 1-day-old C2C12 spheroid-derived cells (SDCs) in two-dimensional (2D) and three-dimensional (3D) culture conditions through examination of the cell proliferation and the expression of myogenic genes. The data showed that 1-day-old spheroids had more intense expression of MyoD gene with respect to the PCs. The proliferation of the SDCs significantly higher than the PCs in a time dependent manner. The SDCs had also significantly higher myogenic potential than the PCs in 2D and 3D culture conditions. The results suggest that MyoD gene upregulation through cell-cell contacts is the good approach for preparation of seed cells in muscle tissue engineering.

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Inhibition of Drp1-dependent mitochondrial division impairs myogenic differentiation

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.00502.2012 ◽

2013 ◽

Vol 305 (8) ◽

pp. R927-R938 ◽

Cited By ~ 48

Author(s):

Boa Kim ◽

Ji-Seok Kim ◽

Yisang Yoon ◽

Mayra C. Santiago ◽

Michael D. Brown ◽

...

Keyword(s):

Muscle Development ◽

Myogenic Differentiation ◽

Mitochondrial Dynamics ◽

C2c12 Cells ◽

Terminal Deoxynucleotidyl Transferase ◽

Dominant Negative Mutant ◽

Serum Starvation ◽

Dependent Manner ◽

Mitochondrial Division

Mitochondria are dynamic organelles forming a tubular network that is continuously fusing and dividing to control their morphology and functions. Recent literature has shed new light on a potential link between the dynamic behavior of mitochondria and muscle development. In this study, we investigate the role of mitochondrial fission factor dynamin-related protein 1 (Drp1) in myogenic differentiation. We found that differentiation of C2C12 myoblasts induced by serum starvation was accompanied by a gradual increase in Drp1 protein expression (to ∼350% up to 3 days) and a fast reduction of Drp1 phosphorylation at Ser-637 (to ∼30%) resulting in translocation of Drp1 protein from the cytosol to mitochondria. During differentiation, treatment of myoblasts with mitochondrial division inhibitor ( mdivi-1), a specific inhibitor of Drp1 GTPase activity, caused extensive formation of elongated mitochondria, which coincided with increased apoptosis evidenced by both enhanced caspase-3 activity and increased number of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive cells. Furthermore, the mdivi-1-treated myotubes ( day 3 in differentiation media) showed a reduction in mitochondrial DNA content, mitochondrial mass, and membrane potential in a dose-dependent manner indicating defects in mitochondrial biogenesis during myogenic differentiation. Most interestingly, mdivi-1 treatment significantly suppressed myotube formation in both C2C12 cells and primary myoblasts. Likewise, stable overexpression of a dominant negative mutant Drp1 (K38A) dramatically reduced myogenic differentiation. These data suggest that Drp-1-dependent mitochondrial division is a necessary step for successful myogenic differentiation, and perturbation of mitochondrial dynamics hinders normal mitochondrial adaptations during muscle development. Therefore, in the present study, we report a novel physiological role of mitochondrial dynamics in myogenic differentiation.

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Coupled Transcriptional and Translational Control of Cyclin-Dependent Kinase Inhibitor p18INK4cExpression during Myogenesis

Molecular and Cellular Biology ◽

10.1128/mcb.18.4.2334 ◽

1998 ◽

Vol 18 (4) ◽

pp. 2334-2343 ◽

Cited By ~ 56

Author(s):

Dawn E. Phelps ◽

Kuang-Ming Hsiao ◽

Yan Li ◽

Nanpin Hu ◽

David S. Franklin ◽

...

Keyword(s):

Translational Control ◽

Kinase Inhibitor ◽

Myogenic Differentiation ◽

Protein A ◽

Terminal Differentiation ◽

C2c12 Cells ◽

Cdk Inhibitor ◽

Cyclin Dependent Kinase ◽

C2c12 Myoblasts ◽

Exon 1

ABSTRACT Terminal differentiation of many cell types involves permanent withdrawal from the cell division cycle. The p18 INK4c protein, a member of the p16/INK4 cyclin-dependent kinase (CDK) inhibitor family, is induced more than 50-fold during myogenic differentiation of mouse C2C12 myoblasts to become the predominant CDK inhibitor complexed with CDK4 and CDK6 in terminally differentiated myotubes. We have found that the p18 INK4c gene expresses two mRNA transcripts—a 2.4-kb transcript, p18(L), and a 1.2-kb transcript, p18(S). In proliferating C2C12 myoblasts, only the larger p18(L) transcript is expressed from an upstream promoter. As C2C12 cells are induced to differentiate into permanently arrested myotubes, the abundance of the p18(L) transcript decreases. The smaller p18(S) transcript expressed from a downstream promoter becomes detectable by 12 h postinduction and is the predominant transcript expressed in terminally differentiated myotubes. Both transcripts contain coding exons 2 and 3, but p18(L) uniquely contains an additional noncoding 1.2-kb exon, exon 1, corresponding exclusively to the 5′ untranslated region (5′ UTR). The expression pattern of the shorter p18(S) transcript, but not that of the longer p18(L) transcript, correlates with terminal differentiation of muscle, lung, liver, thymus, and eye lens cells during mouse embryo development. The presence of the long 5′ UTR in exon 1 attenuated the translation of p18(L) transcript, while its absence from the shorter p18(S) transcript resulted in significantly more efficient translation of the p18 protein. Our results demonstrate that during terminal muscle cell differentiation, induction of the p18 protein is regulated by promoter switching coupled with translational control.

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Association analyses of polymorphisms in porcine MYF5 and MYOD1 genes with carcass traits

Australian Journal of Agricultural Research ◽

10.1071/ar06420 ◽

2007 ◽

Vol 58 (11) ◽

pp. 1040 ◽

Cited By ~ 1

Author(s):

M. Liu ◽

J. Peng ◽

D. Q. Xu ◽

R. Zheng ◽

F. E. Li ◽

...

Keyword(s):

Myogenic Differentiation ◽

Carcass Traits ◽

Large White ◽

Exon 2 ◽

Association Analyses ◽

Intron 1 ◽

Pcr Rflp ◽

Exon 1 ◽

Myogenic Factor ◽

Fat Thickness

The objective of this study was to assess the effect of polymorphisms of myogenic factor 5 (MYF5) and myogenic differentiation 1 (MYOD1) genes on carcass traits in pigs. PCR-RFLP was used to identify three and one SNP(s) from the MYF5 and the MYOD1 gene, respectively. Association analysis performed on the four polymorphisms in a series of three Large White × Meishan F2 populations totalling near 400 pigs showed: (1) an MYF5 exon 1 Hsp92II polymorphism causing a Met→Leu substitution was significantly associated with fat meat percentage, shoulder fat thickness, thorax-waist fat thickness, average backfat thickness and carcass length to 1st rib (P < 0.05); (2) an MYF5 exon 2 MspI polymorphism and an intron 1 HaeIII polymorphism, which were completely linked, were significantly associated with thorax-waist fat thickness, 6–7th rib fat thickness and carcass length to 1st rib (P < 0.05); (3) an MYOD1 intron 1 DdeI polymorphism was significantly associated with carcass length to 1st rib.

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Coding elements in exons 2 and 3 target c-myc mRNA downregulation during myogenic differentiation.

Molecular and Cellular Biology ◽

10.1128/mcb.17.5.2698 ◽

1997 ◽

Vol 17 (5) ◽

pp. 2698-2707 ◽

Cited By ~ 31

Author(s):

N M Yeilding ◽

W M Lee

Keyword(s):

Myogenic Differentiation ◽

Globin Gene ◽

Regulatory Elements ◽

C2c12 Cells ◽

Regulatory Function ◽

Mrna Levels ◽

Beta Globin ◽

Globin Mrna ◽

Exon 2 ◽

Coding Sequences

Downregulation in expression of the c-myc proto-oncogene is an early molecular event in differentiation of murine C2C12 myoblasts into multinucleated myotubes. During differentiation, levels of c-myc mRNA decrease 3- to 10-fold despite a lack of change in its transcription rate. To identify cis-acting elements that target c-myc mRNA for downregulation during myogenesis, we stably transfected C2C12 cells with mutant myc genes or chimeric genes in which various myc sequences were fused to the human beta-globin gene or to the bacterial chloramphenicol acetyltransferase (CAT) gene. Deletion of coding sequences from myc exon 2 or exon 3 abolished downregulation of myc mRNA during myogenic differentiation, while deletion of introns or sequences in the 5' or 3' untranslated regions (UTRs) did not, demonstrating that coding elements in both exons 2 and 3 are necessary for myc mRNA downregulation. Fusion of coding sequences from either myc exon 2 or 3 to beta-globin mRNA conferred downregulation onto the chimeric mRNA, while fusion of myc 3' UTR sequences or coding sequences from CAT or ribosomal protein L32 did not, demonstrating that coding elements in myc exons 2 and 3 specifically confer downregulation. These results present the apparent paradox that coding elements in either myc exon 2 or myc exon 3 are sufficient to confer downregulation onto beta-globin mRNA, but neither element alone was sufficient for myc mRNA downregulation, suggesting that some feature of beta-globin mRNA may potentiate the regulatory properties of myc exons 2 and 3. A similar regulatory function is not shared by all mRNAs because fusion of either myc exon 2 or myc exon 3 to CAT mRNA did not confer downregulation onto the chimeric mRNA, but fusion of the two elements together did. We conclude from these results that two myc regulatory elements, one exon 2 and one in exon 3, are required for myc mRNA downregulation. Finally, using a highly sensitive and specific PCR-based assay for comparing mRNA levels, we demonstrated that the downregulation mediated by myc exons 2 and 3 results in a decrease in cytoplasmic mRNA levels, but not nuclear mRNA levels, indicating that regulation is a postnuclear event.

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Coupled transcription-splicing regulation of mutually exclusive splicing events at the 5′ exons of protein 4.1R gene

Blood ◽

10.1182/blood-2009-02-206219 ◽

2009 ◽

Vol 114 (19) ◽

pp. 4233-4242 ◽

Cited By ~ 11

Author(s):

Shu-Ching Huang ◽

Aeri Cho ◽

Stephanie Norton ◽

Eva S. Liu ◽

Jennie Park ◽

...

Keyword(s):

Regulatory Elements ◽

Dependent Manner ◽

Exon 2 ◽

Erythroleukemia Cells ◽

Exon 1 ◽

Murine Erythroleukemia ◽

Protein 4.1R ◽

First Time ◽

Splicing Patterns ◽

Murine Erythroleukemia Cells

Abstract The tightly regulated production of distinct erythrocyte protein 4.1R isoforms involves differential splicing of 3 mutually exclusive first exons (1A, 1B, 1C) to the alternative 3′ splice sites (ss) of exon 2′/2. Here, we demonstrate that exon 1 and 2′/2 splicing diversity is regulated by a transcription-coupled splicing mechanism. We also implicate distinctive regulatory elements that promote the splicing of exon 1A to the distal 3′ ss and exon 1B to the proximal 3′ ss in murine erythroleukemia cells. A hybrid minigene driven by cytomegalovirus promoter mimicked 1B-promoter–driven splicing patterns but differed from 1A-promoter–driven splicing patterns, suggesting that promoter identity affects exon 2′/2 splicing. Furthermore, splicing factor SF2/ASF ultraviolet (UV) cross-linked to the exon 2′/2 junction CAGAGAA, a sequence that overlaps the distal U2AF35-binding 3′ ss. Consequently, depletion of SF2/ASF allowed exon 1B to splice to the distal 3′ ss but had no effect on exon 1A splicing. These findings identify for the first time that an SF2/ASF binding site also can serve as a 3′ ss in a transcript-dependent manner. Taken together, our results suggest that 4.1R gene expression involves transcriptional regulation coupled with a complex splicing regulatory network.

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p38 MAPK-induced Nuclear Factor-κB Activity Is Required for Skeletal Muscle Differentiation: Role of Interleukin-6

Molecular Biology of the Cell ◽

10.1091/mbc.e03-08-0585 ◽

2004 ◽

Vol 15 (4) ◽

pp. 2013-2026 ◽

Cited By ~ 161

Author(s):

Bernat Baeza-Raja ◽

Pura Muñoz-Cánoves

Keyword(s):

Signaling Pathways ◽

P38 Mapk ◽

Myogenic Differentiation ◽

Nuclear Factor Κb ◽

Binding Activity ◽

C2c12 Cells ◽

Myoblast Differentiation ◽

Dependent Manner ◽

Nuclear Factor

p38 MAPK and nuclear factor-κB (NF-κB) signaling pathways have been implicated in the control of skeletal myogenesis. However, although p38 is recognized as a potent activator of myoblast differentiation, the role of NF-κB remains controversial. Here, we show that p38 is activated only in differentiating myocytes, whereas NF-κB activity is present both in proliferation and differentiation stages. NF-κB activation was found to be dependent on p38 activity during differentiation, being NF-κB an effector of p38, thus providing a novel mechanism for the promyogenic effect of p38. Activation of p38 in C2C12 cells induced the activity of NF-κB, in a dual way: first, by reducing IκBα levels and inducing NF-κB-DNA binding activity and, second, by potentiating the transactivating activity of p65-NF-κB. Finally, we show that interleukin (IL)-6 expression is induced in C2C12 differentiating myoblasts, in a p38- and NF-κB-dependent manner. Interference of IL-6 mRNA reduced, whereas its overexpression increased, the extent of myogenic differentiation; moreover, addition of IL-6 was able to rescue significantly the negative effect of NF-κB inhibition on this process. This study provides the first evidence of a crosstalk between p38 MAPK and NF-κB signaling pathways during myogenesis, with IL-6 being one of the effectors of this promyogenic mechanism.

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The phosphoenolpyruvate carboxykinase (PEPCK) inhibitor, 3-mercaptopicolinic acid (3-MPA), induces myogenic differentiation in C2C12 cells

Scientific Reports ◽

10.1038/s41598-020-79324-9 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Madelaine C. Brearley ◽

Zoe C. T. R. Daniel ◽

Paul T. Loughna ◽

Tim Parr ◽

John M. Brameld

Keyword(s):

Phosphoenolpyruvate Carboxykinase ◽

Myogenic Differentiation ◽

C2c12 Cell ◽

Creatine Kinase Activity ◽

C2c12 Cells ◽

Dependent Manner ◽

Phosphoserine Aminotransferase ◽

Gluconeogenic Enzyme ◽

High Doses ◽

Dose Dependent

AbstractPhosphoenolpyruvate carboxykinase (PEPCK) is a gluconeogenic enzyme with a cytosolic (Pck1/PEPCK-C) and mitochondrial (Pck2/PEPCK-M) isoform. Here we investigate the effect of 3-mercaptopicolinic acid (3-MPA), a PEPCK inhibitor, on C2C12 muscle cells. We report that Pck2 mRNA is 50–5000-fold higher than Pck1 during C2C12 myogenesis, indicating Pck2 is the predominant PEPCK isoform. C2C12 cell proliferation was inhibited in a dose-dependent manner following 48 h 3-MPA treatment (0.01–1 mM). C2C12 myogenic differentiation was significantly induced following 3-MPA treatment (0.25, 0.5, 1 mM) from day 0 of differentiation, demonstrated by increased creatine kinase activity, fusion index and myotube diameter; likewise, the myosin heavy chain (MyHC)-IIB isoform (encoded by Myh4) is an indicator of hypertrophy, and both porcine MYH4-promoter activity and endogenous Myh4 mRNA were also significantly induced. High doses (0.5 and/or 1 mM) of 3-MPA reduced mRNA expression of Pck2 and genes associated with serine biosynthesis (Phosphoglycerate dehydrogenase, Phgdh; phosphoserine aminotransferase-1, Psat1) following treatment from days 0 and 4. To conclude, as Pck2/PEPCK-M is the predominant isoform in C2C12 cells, we postulate that 3-MPA promoted myogenic differentiation through the inhibition of PEPCK-M. However, we were unable to confirm that 3-MPA inhibited PEPCK-M enzyme activity as 3-MPA interfered with the PEPCK enzyme assay, particularly at 0.5 and 1 mM.

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Transcriptional Pausing and Activation at Exons-1 and -2, Respectively, Mediate the MGMT Gene Expression in Human Glioblastoma Cells

Genes ◽

10.3390/genes12060888 ◽

2021 ◽

Vol 12 (6) ◽

pp. 888

Author(s):

Mohammed A. Ibrahim Al-Obaide ◽

Kalkunte S. Srivenugopal

Keyword(s):

Dna Methyltransferase ◽

Glioblastoma Cells ◽

Rt Pcr ◽

Repair Gene ◽

Exon 2 ◽

Mgmt Gene ◽

Dna Repair Gene ◽

Exon 1 ◽

Transcriptional Pausing ◽

Transcription Assays

Background: The therapeutically important DNA repair gene O6-methylguanine DNA methyltransferase (MGMT) is silenced by promoter methylation in human brain cancers. The co-players/regulators associated with this process and the subsequent progression of MGMT gene transcription beyond the non-coding exon 1 are unknown. As a follow-up to our recent finding of a predicted second promoter mapped proximal to the exon 2 [Int. J. Mol. Sci.2021, 22(5), 2492], we addressed its significance in MGMT transcription. Methods: RT-PCR, RT q-PCR, and nuclear run-on transcription assays were performed to compare and contrast the transcription rates of exon 1 and exon 2 of the MGMT gene in glioblastoma cells. Results: Bioinformatic characterization of the predicted MGMT exon 2 promoter showed several consensus TATA box and INR motifs and the absence of CpG islands in contrast to the established TATA-less, CpG-rich, and GAF-bindable exon 1 promoter. RT-PCR showed very weak MGMT-E1 expression in MGMT-proficient SF188 and T98G GBM cells, compared to active transcription of MGMT-E2. In the MGMT-deficient SNB-19 cells, the expression of both exons remained weak. The RT q-PCR revealed that MGMT-E2 and MGMT-E5 expression was about 80- to 175-fold higher than that of E1 in SF188 and T98G cells. Nuclear run-on transcription assays using bromo-uridine immunocapture followed by RT q-PCR confirmed the exceptionally lower and higher transcription rates for MGMT-E1 and MGMT-E2, respectively. Conclusions: The results provide the first evidence for transcriptional pausing at the promoter 1- and non-coding exon 1 junction of the human MGMT gene and its activation/elongation through the protein-coding exons 2 through 5, possibly mediated by a second promoter. The findings offer novel insight into the regulation of MGMT transcription in glioma and other cancer types.

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