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. 2019 Mar;20(3):e47468.
doi: 10.15252/embr.201847468. Epub 2019 Jan 8.

Myogenin Promoter-Associated lncRNA Myoparr Is Essential for Myogenic Differentiation

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Free PMC article

Myogenin Promoter-Associated lncRNA Myoparr Is Essential for Myogenic Differentiation

Keisuke Hitachi et al. EMBO Rep. .
Free PMC article

Abstract

Promoter-associated long non-coding RNAs (lncRNAs) regulate the expression of adjacent genes; however, precise roles of these lncRNAs in skeletal muscle remain largely unknown. Here, we characterize a promoter-associated lncRNA, Myoparr, in myogenic differentiation and muscle disorders. Myoparr is expressed from the promoter region of the mouse and human myogenin gene, one of the key myogenic transcription factors. We show that Myoparr is essential both for the specification of myoblasts by activating neighboring myogenin expression and for myoblast cell cycle withdrawal by activating myogenic microRNA expression. Mechanistically, Myoparr interacts with Ddx17, a transcriptional coactivator of MyoD, and regulates the association between Ddx17 and the histone acetyltransferase PCAF Myoparr also promotes skeletal muscle atrophy caused by denervation, and knockdown of Myoparr rescues muscle wasting in mice. Our findings demonstrate that Myoparr is a novel key regulator of muscle development and suggest that Myoparr is a potential therapeutic target for neurogenic atrophy in humans.

Keywords: DEAD box protein; chromatin; myogenesis; transcriptional regulation.

Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Figure 1
Figure 1. A promoter‐associated lncRNA, Myoparr, is expressed from the upstream region of the myogenin locus

Occupancies of Pol II at the myogenin locus in C2C12 myoblasts (MB, shown in blue) and myotubes (MT, shown in red). Myoparr is located immediately upstream from the myogenin gene.

Schematic representation of the upstream region of myogenin and amplified regions by RT–PCR (top). RT–PCR for the novel transcripts at the upstream region of myogenin in C2C12 myotubes (bottom). The presence or absence of reverse transcriptase (RT) is shown by (+) or (−), respectively. The templates (cDNA or genomic DNA) are indicated by C or G, respectively.

The primers used for RT–PCR (top). Strand‐specific RT–PCR for the novel transcripts at the upstream region of myogenin in C2C12 myotubes using total RNA (middle and bottom) and poly(A)+ RNA (bottom).

Relative expression of indicated RNAs in differentiating C2C12 cells. The presence or absence of RT is shown by (+) or (−), respectively. PCR products were verified by sequencing. n = 3, mean ± SD. The nuclear/cytoplasmic fractionation is confirmed by the expression of H2B and tubulin, respectively.

Chromatin pellet extract (CPE)/soluble nuclear extract (SNE) ratio of SRA1, Malat1, and Myoparr in C2C12 myotubes on a log scale. n = 3, mean ± SD.

Figure EV1
Figure EV1. Characterization of mouse and human Myoparr

Schematic diagram of the results of 5′‐ and 3′‐RACE analysis of sense and anti‐sense transcripts. The 3′‐ends of several sense transcripts overlap with myogenin mRNA.

Coding potential assessment of the indicated RNAs using a coding potential assessment tool (CPAT). Low coding probabilities for anti‐sense transcript and sense transcript 1 (Long) and 4 (Short) as well as lincRNA‐p21 are shown.

In vitro transcription/translation of pCS2‐Anti‐Sense, pCS2‐Sense (Long), and pCS2‐Sense (Short). The pCS2+ vector was used as a negative control. pCS2‐EGFP and pCS2‐myogenin were used as positive controls.

The sequence of Myoparr cloned from mouse C2C12 cells. The potential RNA nuclear retention signal and putative polyadenylation signal are enclosed in a black and red box, respectively. The LINE‐1‐like sequence is underlined.

Schematic representation of the upstream region of human myogenin and regions amplified by RT–PCR (top). RT–PCR for novel transcripts in human primary myotubes (bottom). The presence or absence of reverse transcriptase (RT) is indicated by (+) or (−), respectively.

The primers used for RT–PCR (top). Strand‐specific RT–PCR for the novel transcripts in the upstream region of human myogenin (bottom).

Schematic diagram of the results of 5′‐ and 3′‐RACE analysis of human Myoparr.

Figure 2
Figure 2. Myoparr expression is correlated with myogenin expression and regulated by MyoD and TGF‐β

Quantitative RT–PCR for myogenin (A) and Myoparr (B) during myogenesis of C2C12 cells, primary mouse myoblasts (growth medium, GM), and mouse embryonic skeletal muscle (S.K.). The x‐axis shows days after differentiation induction or embryonic days. n = 3, mean ± SD.

qRT–PCR showing increased Myoparr expression by MyoD in C3H10T1/2 fibroblasts. n = 4, mean ± SD. **< 0.01 (unpaired two‐tailed Welch's t‐test).

Treatment of recombinant TGF‐β for 24 h decreased Myoparr expression in differentiating C2C12 cells. n = 3, mean ± SD. *< 0.05 (unpaired two‐tailed Student's t‐test).

Schematic diagram of the constructs used for luciferase assays.

Relative luciferase activities of the indicated promoter in differentiating C2C12 cells by exogenous MyoD. n = 3, mean ± SD. **< 0.01 (unpaired two‐tailed Student's t‐test).

Relative luciferase activities of the indicated promoter with/without E‐box mutations in differentiating C2C12 cells. n = 3, mean ± SD. **< 0.01 (unpaired two‐tailed Student's t‐test).

Relative luciferase activities of the indicated promoter with/without E‐box mutations in differentiating C2C12 cells by TGF‐β treatment. n = 3, mean ± SD. *< 0.05. **< 0.01. n.s., not significant (unpaired two‐tailed Student's t‐test).

Figure EV2
Figure EV2. Expression of mouse and human Myoparr

Quantitative RT–PCR for mouse myogenin and Myoparr in embryonic organs. n = 3, mean ± SD. S.K., skeletal muscle.

The expression levels of human myogenin and Myoparr in human primary myoblasts (growth medium, GM) and myotubes (differentiation medium, DM) evaluated by qRT–PCR. n = 4, mean ± SD. **< 0.01. ***< 0.001 (unpaired two‐tailed Welch's t‐test). Preparations from two independent specimens are shown.

Relative luciferase activities of the GAPDH promoters (positive and opposite directions) in C2C12 cells. Bars indicate the average of two independent experiments, and open circles represent the values of each experiment.

Figure 3
Figure 3. Myoparr is required for the activation of myogenin and C2C12 differentiation

C2C12 cells were transfected with 50 nM of indicated siRNAs in growth medium. After additional 24‐h incubation in growth medium, cells were transferred to differentiation medium. The levels of Myoparr and myogenin expression were quantified by qRT–PCR 24 h after differentiation induction. n = 3, mean ± SD. *< 0.05. n.s., not significant. Statistical analyses were performed using unpaired two‐tailed Student's t‐test (Myoparr; Cont1 vs. siRNA‐1, myogenin; Cont1 vs. Cont2). In cases of unequal variances (Myoparr; Cont1 vs. Cont2 and Cont1 vs. siRNA‐2, myogenin; Cont1 vs. siRNA‐1 and Cont1 vs. siRNA‐2), unpaired two‐tailed Welch's t‐test was used.

Western blot showing decreased expression of myogenin in differentiating C2C12 cells 48 h after Myoparr KD. Tubulin expression served as an internal control.

The expression levels of myogenin and Myoparr evaluated by qRT–PCR in myogenin‐depleted C2C12 cells. n = 3, mean ± SD. **< 0.01. ***< 0.001 (unpaired two‐tailed Student's t‐test).

ChIP‐qPCR detection of Pol II occupancy and histone modification status at the myogenin locus in Myoparr‐depleted differentiating C2C12 cells. The data were normalized to input values. n = 3, mean ± SD. *< 0.05. **< 0.01 (unpaired two‐tailed Student's t‐test or unpaired two‐tailed Welch's t‐test).

Retrieval rate of Myoparr using Myoparr‐ChIRP probes from C2C12 cells quantified by qPCR is shown as percent of input values. GAPDH was amplified as a negative control. n = 3, mean ± SD. ND, not detected.

ChIRP‐qPCR detection of the interaction between endogenous Myoparr and the myogenin promoter. The myogenin 3′UTR and GAPDH promoter were amplified as negative controls. The data were normalized to input values. n = 3, mean ± SD. *< 0.05. **< 0.01 (unpaired two‐tailed Welch's t‐test). ND, not detected.

Immunocytochemistry for the detection of myogenin at 24 h (G) or MHC at 72 h (H) in Myoparr‐depleted C2C12 cells after differentiation induction. Nuclei were counterstained with DAPI. Scale bars, 100 μm. The percentage of myogenin‐positive cells or fusion index is shown as percent of the control. n = 3, mean ± SD. **< 0.01 (unpaired two‐tailed Welch's t‐test). ***< 0.001 (unpaired two‐tailed Student's t‐test).

The intersection of genes regulated by Myoparr KD and myogenin KD shows a significant (Fisher's exact test) overlap 25.6 times as large as expected by chance alone.

Genes significantly differentially expressed in both Myoparr‐ and myogenin‐depleted cells show correlated expression (R = 0.63, log 2 ratio scale).

Enrichment of GO categories in genes up‐regulated by Myoparr KD (K), down‐regulated by Myoparr KD (L), down‐regulated by myogenin KD (M), and commonly regulated by both Myoparr KD and myogenin KD (N).

Figure EV3
Figure EV3. Regulation of myogenin expression by Myoparr

Quantitative RT–PCR for Myoparr expression in differentiating C2C12 cells transfected with control or Myoparr anti‐sense oligonucleotides (ASOs). Cells were transfected with 50 nM ASOs, and the levels of Myoparr expression were quantified by qRT–PCR 48 h after knockdowns. n = 3, mean ± SD. *< 0.05 (unpaired two‐tailed Student's t‐test).

qRT–PCR showing decreased myogenin expression in differentiating C2C12 cells transfected with control or Myoparr ASOs. n = 4, mean ± SD. *< 0.05 (unpaired two‐tailed Student's t‐test).

Decreased myogenin protein expression in differentiating C2C12 cells 48 h after Myoparr knockdown using ASOs. Expression of tubulin served as an internal control.

ChIP‐qPCR detection of Pol II occupancy and histone modification status at the GAPDH promoter in Myoparr‐depleted differentiating C2C12 cells. The data were normalized to input values. n = 3, mean ± SD. n.s., not significant. Statistical analyses were performed using an unpaired two‐tailed Student's t‐test (Pol II and H3K4me3). In cases of unequal variances (H3K27ac), an unpaired two‐tailed Welch's t‐test was used.

A schematic diagram of the CpG sites at the myogenin upstream region. Red lines indicate individual CpG sites. The methylation status at the −474/−18 region in Myoparr‐depleted C2C12 cells was examined.

The methylation status at the −474/−18 region is shown. C2C12 cells were transfected with 50 nM siRNAs. The methylation status was evaluated 1 and 3 days after differentiation induction. The day 0 sample is from non‐transfected cells. The CpG sites are indicated by circles (black and white circles indicate methylated and unmethylated cytosine sites, respectively), and each row represents an individual clone.

The methylation status of (F) is shown as a methylated/unmethylated ratio.

Decreased MHC expression by Myoparr knockdowns using siRNAs (H) or ASOs (I) in C2C12 myotubes. Expression of tubulin served as an internal control.

The expression changes of MyoD1 (J), Myf5 (K), and MRF4 (L) quantified by qRT–PCR either in Myoparr‐ or myogenin‐depleted differentiating C2C12 cells. n = 3, mean ± SD. *< 0.05. **< 0.01 (unpaired two‐tailed Student's t‐test).

Decreased Pol II occupancy at the MyoD1 promoter detected by ChIP‐qPCR in Myoparr‐depleted differentiating C2C12 cells. The data were normalized to input values. n = 3, mean ± SD. *< 0.05 (unpaired two‐tailed Student's t‐test).

Figure 4
Figure 4. Myoparr interacts with DEAD box protein Ddx17

Scheme of the identification of Myoparr‐interacting proteins using RiboTrap and differential proteomics analysis.

Following immunoprecipitation, the interaction between Myoparr and endogenous Ddx17 was confirmed by immunoblotting using a Ddx17‐specific antibody. Two different Ddx17 isoforms (p72 and p82) were observed.

Determination of the Ddx17‐binding region of Myoparr by RNA pull‐down analyses. Schematic diagram of full‐length or truncated Myoparr used for RNA pull‐down (top). In vitro‐transcribed/in vitro‐translated Ddx17 protein was pulled down by indicated Myoparr and then detected by Western blot using a Ddx17 antibody (bottom).

Schematic diagram of the constructs used for luciferase assays. The details of all constructs are described in Materials and Methods. p(A) indicates poly(A) site.

Relative luciferase activities of indicated constructs in differentiating C2C12 cells.

Relative luciferase activities of indicated constructs in differentiating C2C12 cells transfected with control or Myoparr shRNA (Myoparr KD).

Data information: In (E‐H), bars indicate the average of two independent experiments, and open circles represent the values of each experiment.
Figure 5
Figure 5. Myoparr promotes the protein–protein interaction between Ddx17 and PCAF

Significantly decreased MHC expression in Ddx17‐depleted C2C12 cells is shown by immunocytochemistry. Nuclei were counterstained with DAPI. Scale bar, 100 μm. Fusion index is shown as percent of the control. n = 3, mean ± SD. **< 0.01 (unpaired two‐tailed Student's t‐test).

Immunocytochemistry for myogenin in Ddx17‐depleted C2C12 cells. Scale bar, 100 μm. Myogenin‐positive cells are shown as percent of the control. n = 4, mean ± SD. **< 0.01 (unpaired two‐tailed Student's t‐test).

Relative luciferase activities of the ‐1650‐Luc by the combination of MyoD and Ddx17 or Ddx17 mutant (K142R). Bars indicate the average of two independent experiments, and open circles represent the values of each experiment.

Ddx17 or Ddx17 mutant (K142R) was pulled down by full‐length Myoparr and then detected by Western blot.

Reduced interaction between endogenous Ddx17 and PCAF in Myoparr‐depleted C2C12 cells. After Myoparr KD, the cell lysates were subjected to immunoprecipitation (IP) with a Ddx17‐specific antibody 36 h after differentiation induction (right panel). Each lysate was loaded as an input (left panel).

Relative quantification of (E) is shown as IP/input ratio. n = 3, mean ± SD. *< 0.05. n.s., not significant (unpaired two‐tailed Student's t‐test).

Increased interaction between Ddx17 and PCAF by Myoparr. PCAF protein was pulled down by 3xFlag‐Ddx17 in the presence or absence of Myoparr using a Flag antibody and then detected by Western blot.

ChIP‐qPCR detection of Ddx17 (H) and PCAF (I) occupancies at the myogenin locus in Myoparr‐depleted differentiating C2C12 cells. The data were normalized to input values. n = 3, mean ± SD. *< 0.05 (unpaired two‐tailed Student's t‐test).

Figure 6
Figure 6. Myoparr and Ddx17 regulate myoblast cell cycle withdrawal in a myogenin‐independent manner

Heatmap displaying expression changes of 754 genes significantly altered either in Myoparr‐ or Ddx17‐depleted cells (log 2 ratio scale).

The intersection of genes regulated by Myoparr KD and Ddx17 KD shows a significant (Fisher's exact test) overlap 26.9 times as large as that expected by chance alone.

Genes significantly differentially expressed in both Myoparr‐ and Ddx17‐depleted cells show correlated expression (R = 0.94, log 2 ratio scale).

Enrichment analysis of GO categories in genes up‐regulated by Ddx17 KD (D), down‐regulated by Ddx17 KD (E), and commonly regulated by both Myoparr KD and Ddx17 KD (F).

Myoparr and Ddx17 are required for C2C12 cell cycle withdrawal. C2C12 cells transfected with each siRNA were cultured in growth medium for 24 h. After differentiation induction, cells were maintained in differentiation medium for 40 h and then treated with EdU for 6 h. EdU‐positive cells are shown as percent of the control. Nuclei were counterstained with Hoechst 33342. n = 3, mean ± SD. **< 0.01 (unpaired two‐tailed Student's t‐test). Scale bar, 100 μm.

Figure 7
Figure 7. Myoparr regulates myoblast cell cycle withdrawal through the activation of miR‐133b, miR‐206, and H19 expression

qRT–PCR for pri‐miR‐133b, pri‐miR‐206, and H19 expression in differentiating C2C12 cells transfected with indicated siRNAs. n = 3, mean ± SD. *< 0.05. **< 0.01.

ChIP‐qPCR detection of Pol II occupancy at the indicated promoters in Myoparr‐depleted C2C12 cells. The data were normalized to input values. n = 3, mean ± SD. *< 0.05. Myoparr KD decreased Pol II occupancy at the miR‐133b promoter with a marginal trend toward significance (= 0.052).

ChIP‐qPCR detection of Ddx17 (C) and PCAF (D) occupancies at the indicated promoters in Myoparr‐depleted C2C12 cells. n = 3, mean ± SD. *< 0.05. **< 0.01.

qRT–PCR showing decreased expression of miR‐133b and miR‐206 in both Myoparr‐ and Ddx17‐depleted C2C12 cells. n = 3, mean ± SD. *< 0.05. **< 0.01.

Western blots showing increased ERK1/2 activity (pERK1/2) and Cdc6 expression in differentiating C2C12 cells 48 h after Myoparr and Ddx17 KD. Tubulin expression served as an internal control.

Increased Pola1 expression detected by qRT–PCR. n = 3, mean ± SD. **< 0.01.

Data information: Statistical analyses were performed using unpaired two‐tailed Student's t‐test. In cases of unequal variances, unpaired two‐tailed Welch's t‐test was used.
Figure 8
Figure 8. Knockdown of Myoparr blocks skeletal muscle atrophy caused by denervation in mice

Seven days after denervation, weights of innervated (−) and denervated (+) tibialis anterior (TA) muscles were measured. n = 4, mean ± SEM. *< 0.05 (unpaired two‐tailed Student's t‐test).

Expression of myogenin in innervated and denervated TA muscles detected by qRT–PCR 7 days after denervation. n = 4, mean ± SD. ***< 0.001 (unpaired two‐tailed Welch's t‐test).

qRT–PCR showing increased Myoparr expression in denervated TA muscles 7 days after denervation. n = 3, mean ± SD. ***< 0.001 (unpaired two‐tailed Student's t‐test).

Immunoblot showing decreased expression of myogenin by Myoparr depletion in denervated TA muscles 7 days after denervation. Expression of tubulin served as an internal control.

Weights of innervated and denervated TA muscles electroporated either with control or Myoparr shRNA. Muscle weights were measured 7 days after denervation. n = 5 for each group, mean ± SEM. ***< 0.001. n.s., not significant (unpaired two‐tailed Student's t‐test).

Representative immunostaining images for laminin (red) and EmGFP (green) of innervated or denervated TA muscles electroporated with control or Myoparr shRNA, both containing EmGFP. Scale bar, 100 μm.

Cross‐sectional area (CSA) of electroporated EmGFP‐positive myofibers (250 fibers per sample) was analyzed 7 days after denervation. n = 5 for each group, mean ± SEM. **< 0.01. n.s., not significant (unpaired two‐tailed Student's t‐test).

Figure EV4
Figure EV4. Myoparr depletion prevents skeletal muscle atrophy

Three days after denervation, weights of innervated (−) and denervated (+) tibialis anterior (TA) muscles were measured. n = 4, mean ± SEM. **< 0.01 (unpaired two‐tailed Student's t‐test).

Expression of myogenin in innervated and denervated TA muscles detected by qRT–PCR 3 days after denervation. n = 4, mean ± SD. ***< 0.001 (unpaired two‐tailed Welch's t‐test).

Quantitative RT–PCR showing increased Myoparr expression in denervated TA muscles 3 days after denervation. n = 4, mean ± SD. ***< 0.001 (unpaired two‐tailed Student's t‐test).

Evaluation of the inhibitory effect of Myoparr expression by Myoparr shRNAs in NIH3T3 cells. n = 3, mean ± SD. **< 0.01. ***< 0.001 (unpaired two‐tailed Welch's t‐test). The results were normalized to Rpl26 expression. Data are shown as percent of the control.

In vivo inhibitory effect of Myoparr shRNA against the expression of Myoparr and myogenin in innervated TA muscles. n = 4, mean ± SD. *< 0.05 (unpaired two‐tailed Student's t‐test).

Distributions of single myofiber areas in innervated (F) and denervated (G) TA muscles in the presence of control or Myoparr shRNA. All EmGFP‐positive myofibers (innervated control shRNA; n = 3,326, innervated Myoparr shRNA; n = 3,035, denervated control shRNA; n = 3,141, denervated Myoparr shRNA; n = 5,417) were counted. The percentage of myofibers with indicated areas per total fibers were plotted. A Mann–Whitney nonparametric test was used for comparisons between each group (innervated control shRNA vs. innervated Myoparr shRNA, < 0.001; denervated control shRNA vs. denervated Myoparr shRNA, < 0.001). Data are shown as mean ± SEM.

Figure 9
Figure 9. Proposed model of Myoparr function during myogenesis
Although Ddx17 and PCAF bind to the myogenin promoter, the interaction between Ddx17 and PCAF is weak in the absence of Myoparr. Thus, occupancies of both Ddx17 and PCAF on the myogenin promoter are not sufficient for maximum Pol II recruitment to the myogenin promoter. After binding to the myogenin promoter, Myoparr interacts with Ddx17 to promote the Ddx17‐PCAF interaction. Enhanced Ddx17‐PCAF interaction by Myoparr may be sufficient for maximum Pol II recruitment to the myogenin promoter. Thus, Myoparr is required for specification of myoblast lineage into myogenic differentiation through the Myoparr (upstream) and myogenin (downstream) pathway. In addition, Myoparr is involved in the regulation of myoblast cell cycle withdrawal by activating the expression of myogenic regulatory miRNAs in a myogenin‐independent manner.

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