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Multi-transcriptome analysis following an acute skeletal muscle growth stimulus yields tools for discerning global and MYC regulatory networks

  • Kevin A. Murach
    Correspondence
    For correspondence: Kevin A. Murach; Ivan J. Vechetti; Ferdinand von Walden
    Affiliations
    Department of Health, Human Performance, and Recreation, Exercise Science Research Center, University of Arkansas, Fayetteville, Arkansas, USA

    Cell and Molecular Biology Graduate Program, University of Arkansas, Fayetteville, Arkansas, USA
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  • Zhengye Liu
    Affiliations
    Department of Physiology and Pharmacology, Karolinska Institute, Solna, Sweden
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  • Baptiste Jude
    Affiliations
    Department of Physiology and Pharmacology, Karolinska Institute, Solna, Sweden

    Department of Women’s and Children’s Health, Karolinska Institute, Solna, Sweden
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  • Vandre C. Figueiredo
    Affiliations
    Center for Muscle Biology, University of Kentucky, Lexington, Kentucky, USA

    Department of Physiology, University of Kentucky, Lexington, Kentucky, USA
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  • Yuan Wen
    Affiliations
    Center for Muscle Biology, University of Kentucky, Lexington, Kentucky, USA

    Department of Physical Therapy, University of Kentucky, Lexington, Kentucky, USA
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  • Sabin Khadgi
    Affiliations
    Department of Health, Human Performance, and Recreation, Exercise Science Research Center, University of Arkansas, Fayetteville, Arkansas, USA
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  • Seongkyun Lim
    Affiliations
    Department of Health, Human Performance, and Recreation, Exercise Science Research Center, University of Arkansas, Fayetteville, Arkansas, USA

    Cachexia Research Laboratory, University of Arkansas, Fayetteville, Arkansas, USA
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  • Francielly Morena da Silva
    Affiliations
    Department of Health, Human Performance, and Recreation, Exercise Science Research Center, University of Arkansas, Fayetteville, Arkansas, USA

    Cachexia Research Laboratory, University of Arkansas, Fayetteville, Arkansas, USA
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  • Nicholas P. Greene
    Affiliations
    Department of Health, Human Performance, and Recreation, Exercise Science Research Center, University of Arkansas, Fayetteville, Arkansas, USA

    Cell and Molecular Biology Graduate Program, University of Arkansas, Fayetteville, Arkansas, USA

    Cachexia Research Laboratory, University of Arkansas, Fayetteville, Arkansas, USA
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  • Johanna T. Lanner
    Affiliations
    Department of Physiology and Pharmacology, Karolinska Institute, Solna, Sweden
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  • John J. McCarthy
    Affiliations
    Center for Muscle Biology, University of Kentucky, Lexington, Kentucky, USA

    Department of Physiology, University of Kentucky, Lexington, Kentucky, USA
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  • Author Footnotes
    ‡ Co-Senior.
    Ivan J. Vechetti
    Correspondence
    For correspondence: Kevin A. Murach; Ivan J. Vechetti; Ferdinand von Walden
    Footnotes
    ‡ Co-Senior.
    Affiliations
    Department of Nutrition and Health Sciences, University of Nebraska-Lincoln, Nebraska, USA
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  • Author Footnotes
    ‡ Co-Senior.
    Ferdinand von Walden
    Correspondence
    For correspondence: Kevin A. Murach; Ivan J. Vechetti; Ferdinand von Walden
    Footnotes
    ‡ Co-Senior.
    Affiliations
    Department of Women’s and Children’s Health, Karolinska Institute, Solna, Sweden
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  • Author Footnotes
    ‡ Co-Senior.
Open AccessPublished:September 20, 2022DOI:https://doi.org/10.1016/j.jbc.2022.102515
      Myc is a powerful transcription factor implicated in epigenetic reprogramming, cellular plasticity, and rapid growth as well as tumorigenesis. Cancer in skeletal muscle is extremely rare despite marked and sustained Myc induction during loading-induced hypertrophy. Here, we investigated global, actively transcribed, stable, and myonucleus-specific transcriptomes following an acute hypertrophic stimulus in mouse plantaris. With these datasets, we define global and Myc-specific dynamics at the onset of mechanical overload-induced muscle fiber growth. Data collation across analyses reveals an under-appreciated role for the muscle fiber in extracellular matrix remodeling during adaptation, along with the contribution of mRNA stability to epigenetic-related transcript levels in muscle. We also identify Runx1 and Ankrd1 (Marp1) as abundant myonucleus-enriched loading-induced genes. We observed that a strong induction of cell cycle regulators including Myc occurs with mechanical overload in myonuclei. Additionally, in vivo Myc-controlled gene expression in the plantaris was defined using a genetic muscle fiber-specific doxycycline-inducible Myc-overexpression model. We determined Myc is implicated in numerous aspects of gene expression during early-phase muscle fiber growth. Specifically, brief induction of Myc protein in muscle represses Reverbα, Reverbβ, and Myh2 while increasing Rpl3, recapitulating gene expression in myonuclei during acute overload. Experimental, comparative, and in silico analyses place Myc at the center of a stable and actively transcribed, loading-responsive, muscle fiber–localized regulatory hub. Collectively, our experiments are a roadmap for understanding global and Myc-mediated transcriptional networks that regulate rapid remodeling in postmitotic cells. We provide open webtools for exploring the five RNA-seq datasets as a resource to the field.

      Keywords

      Abbreviations:

      ChIP-seq (chromatin immunoprecipitation sequencing), DEG (differentially expressed gene), ECM (extracellular matrix), EU (5-Ethenyl uridine), FANS (fluorescent activated nuclear-sorting), Lisa (Landscape In Silico deletion analysis), RNA-seq (RNA-sequencing)
      Myc is a transcription factor known to drive cellular plasticity (
      • Santoro A.
      • Vlachou T.
      • Luzi L.
      • Melloni G.
      • Mazzarella L.
      • D'Elia E.
      • et al.
      p53 loss in breast cancer leads to Myc activation, increased cell plasticity, and expression of a mitotic signature with prognostic value.
      ,
      • Hurlin P.J.
      Control of vertebrate development by MYC.
      ), epigenetic reprogramming toward stemness as a Yamanaka factor (
      • Brenner C.
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      • Didelot C.
      • Loriot A.
      • Viré E.
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      Myc represses transcription through recruitment of DNA methyltransferase corepressor.
      ,
      • Lin C.-H.
      • Lin C.
      • Tanaka H.
      • Fero M.L.
      • Eisenman R.N.
      Gene regulation and epigenetic remodeling in murine embryonic stem cells by c-Myc.
      ,
      • Takahashi K.
      • Yamanaka S.
      Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors.
      ,
      • Nakagawa M.
      • Takizawa N.
      • Narita M.
      • Ichisaka T.
      • Yamanaka S.
      Promotion of direct reprogramming by transformation-deficient Myc.
      ), and rapid growth via proliferative and nonproliferative mechanisms (
      • Kim S.
      • Li Q.
      • Dang C.V.
      • Lee L.A.
      Induction of ribosomal genes and hepatocyte hypertrophy by adenovirus-mediated expression of c-Myc in vivo.
      ,
      • Dang C.V.
      MYC on the path to cancer.
      ,
      • Gabay M.
      • Li Y.
      • Felsher D.W.
      MYC activation is a hallmark of cancer initiation and maintenance.
      ). A proto-oncogene that dimerizes with MAX and interacts and/or complexes with numerous other proteins, (
      • Kalkat M.
      • Wasylishen A.R.
      • Kim S.S.
      • Penn L.
      More than MAX: discovering the Myc interactome.
      ,
      • Conacci-Sorrell M.
      • McFerrin L.
      • Eisenman R.N.
      An overview of MYC and its interactome.
      ) MYC protein is a “universal amplifier” and “supermanager” of transcription with intricate and multifaceted functionality (
      • Nie Z.
      • Hu G.
      • Wei G.
      • Cui K.
      • Yamane A.
      • Resch W.
      • et al.
      c-Myc is a universal amplifier of expressed genes in lymphocytes and embryonic stem cells.
      ,
      • Nie Z.
      • Guo C.
      • Das S.K.
      • Chow C.C.
      • Batchelor E.
      • Jnr S.S.S.
      • et al.
      Dissecting transcriptional amplification by MYC.
      ,
      • Das S.K.
      • Lewis B.A.
      • Levens D.
      MYC: a complex problem.
      ). Evidence for a role of MYC in syncytial muscle fiber growth emerged 20 to 30 years ago (
      • Alway S.E.
      Overload-induced C-Myc oncoprotein is reduced in aged skeletal muscle.
      ,
      • Whitelaw P.F.
      • Hesketh J.E.
      Expression of c-myc and c-fos in rat skeletal muscle. Evidence for increased levels of c-myc mRNA during hypertrophy.
      ,
      • Chen Y.W.
      • Nader G.A.
      • Baar K.R.
      • Fedele M.J.
      • Hoffman E.P.
      • Esser K.A.
      Response of rat muscle to acute resistance exercise defined by transcriptional and translational profiling.
      ). A decade ago, MYC was hypothesized to be a key aspect of skeletal muscle adaptation to exercise (
      • Gohil K.
      • Brooks G.A.
      Exercise tames the wild side of the Myc network: a hypothesis.
      ). We recently found that the promoter region of Myc was hypomethylated in myonuclei following short-term mechanical overload of the mouse plantaris muscle (
      • von Walden F.
      • Rea M.
      • Mobley C.B.
      • Fondufe-Mittendorf Y.
      • McCarthy J.J.
      • Peterson C.A.
      • et al.
      The myonuclear DNA methylome in response to an acute hypertrophic stimulus.
      ). MYC protein binds the ribosomal DNA promoter during muscle overload (
      • von Walden F.
      • Casagrande V.
      • Östlund Farrants A.-K.
      • Nader G.A.
      Mechanical loading induces the expression of a Pol I regulon at the onset of skeletal muscle hypertrophy.
      ), consistent with its influence on ribosome biogenesis and protein synthesis in striated muscle (
      • Mori T.
      • Ato S.
      • Knudsen J.R.
      • Henriquez-Olguin C.
      • Li Z.
      • Wakabayashi K.
      • et al.
      c-Myc overexpression increases ribosome biogenesis and protein synthesis independent of mTORC1 activation in mouse skeletal muscle.
      ,
      • West D.W.
      • Baehr L.M.
      • Marcotte G.R.
      • Chason C.M.
      • Tolento L.
      • Gomes A.V.
      • et al.
      Acute resistance exercise activates rapamycin-sensitive and-insensitive mechanisms that control translational activity and capacity in skeletal muscle.
      ). We also report that MYC-associated areas of ribosomal DNA are differentially methylated in murine myonuclei and human muscle tissue after acute loading (
      • Figueiredo V.C.
      • Wen Y.
      • Alkner B.
      • Fernandez-Gonzalo R.
      • Norrbom J.
      • Vechetti Jr., I.J.
      • et al.
      Genetic and epigenetic regulation of skeletal muscle ribosome biogenesis with exercise.
      ). Dysregulation of Myc results in tumor development and maintenance in mononuclear cells (
      • Dang C.V.
      MYC on the path to cancer.
      ,
      • Gabay M.
      • Li Y.
      • Felsher D.W.
      MYC activation is a hallmark of cancer initiation and maintenance.
      ). Even transiently elevated MYC can cause tumors in some cells (
      • Felsher D.W.
      • Bishop J.M.
      Transient excess of MYC activity can elicit genomic instability and tumorigenesis.
      ); however, Myc transcript and protein (MYC) may be elevated for up to 2 weeks during continuous mechanical overload in mouse muscle without an overt deleterious effect (
      • Figueiredo V.C.
      • Wen Y.
      • Alkner B.
      • Fernandez-Gonzalo R.
      • Norrbom J.
      • Vechetti Jr., I.J.
      • et al.
      Genetic and epigenetic regulation of skeletal muscle ribosome biogenesis with exercise.
      ,
      • Kirby T.J.
      • Patel R.M.
      • McClintock T.S.
      • Dupont-Versteegden E.E.
      • Peterson C.A.
      • McCarthy J.J.
      Myonuclear transcription is responsive to mechanical load and DNA content but uncoupled from cell size during hypertrophy.
      ,
      • Chaillou T.
      • Lee J.D.
      • England J.H.
      • Esser K.A.
      • McCarthy J.J.
      Time course of gene expression during mouse skeletal muscle hypertrophy.
      ,
      • Armstrong D.D.
      • Esser K.A.
      Wnt/β-catenin signaling activates growth-control genes during overload-induced skeletal muscle hypertrophy.
      ,
      • Goodman C.A.
      • Dietz J.M.
      • Jacobs B.L.
      • McNally R.M.
      • You J.-S.
      • Hornberger T.A.
      Yes-associated protein is up-regulated by mechanical overload and is sufficient to induce skeletal muscle hypertrophy.
      ). MYC protein abundance in human muscle after resistance training also associates with the magnitude of hypertrophic adaptation (
      • Stec M.J.
      • Kelly N.A.
      • Many G.M.
      • Windham S.T.
      • Tuggle S.C.
      • Bamman M.M.
      Ribosome biogenesis may augment resistance training-induced myofiber hypertrophy and is required for myotube growth in vitro.
      ). The unique multinuclear, terminally differentiated postmitotic nature of muscle fibers likely explains how muscle is resistant to developing cancer (
      • Seely S.
      Possible reasons for the high resistance of muscle to cancer.
      ,
      • Keckesova Z.
      • Donaher J.L.
      • De Cock J.
      • Freinkman E.
      • Lingrell S.
      • Bachovchin D.A.
      • et al.
      LACTB is a tumour suppressor that modulates lipid metabolism and cell state.
      ,
      • Sridhar K.S.
      • Rao R.K.
      • Kunhardt B.
      Skeletal muscle metastases from lung cancer.
      ,
      • Crist S.B.
      • Nemkov T.
      • Dumpit R.F.
      • Dai J.
      • Tapscott S.J.
      • True L.D.
      • et al.
      Unchecked oxidative stress in skeletal muscle prevents outgrowth of disseminated tumour cells.
      ) and why sustained MYC is tolerated in this tissue. Although the role of MYC in proliferative cells is well studied, its function as a transcription factor in postmitotic myonuclei during muscle growth is incompletely defined.
      In the current investigation, we generated four interrelated murine muscle RNA-sequencing (RNA-seq) datasets using a proven hypertrophic loading stimulus (
      • Kirby T.J.
      • McCarthy J.J.
      • Peterson C.A.
      • Fry C.S.
      Synergist ablation as a rodent model to study satellite cell dynamics in adult skeletal muscle.
      ,
      • Murach K.A.
      • McCarthy J.J.
      • Peterson C.A.
      • Dungan C.M.
      Making mice mighty: recent advances in translational models of load-induced muscle hypertrophy.
      ) to understand muscle tissue and myonucleus-specific transcriptional dynamics at the onset of rapid muscle growth. We focused on the effects of Myc given its (1) established role in driving nonproliferative tissue growth and overall cellular plasticity in nonmuscle cell types (
      • Santoro A.
      • Vlachou T.
      • Luzi L.
      • Melloni G.
      • Mazzarella L.
      • D'Elia E.
      • et al.
      p53 loss in breast cancer leads to Myc activation, increased cell plasticity, and expression of a mitotic signature with prognostic value.
      ,
      • Kim S.
      • Li Q.
      • Dang C.V.
      • Lee L.A.
      Induction of ribosomal genes and hepatocyte hypertrophy by adenovirus-mediated expression of c-Myc in vivo.
      ) and (2) proposed role in human (
      • Figueiredo V.C.
      • Wen Y.
      • Alkner B.
      • Fernandez-Gonzalo R.
      • Norrbom J.
      • Vechetti Jr., I.J.
      • et al.
      Genetic and epigenetic regulation of skeletal muscle ribosome biogenesis with exercise.
      ,
      • Stec M.J.
      • Kelly N.A.
      • Many G.M.
      • Windham S.T.
      • Tuggle S.C.
      • Bamman M.M.
      Ribosome biogenesis may augment resistance training-induced myofiber hypertrophy and is required for myotube growth in vitro.
      ,
      • Figueiredo V.C.
      • Roberts L.A.
      • Markworth J.F.
      • Barnett M.P.G.
      • Coombes J.S.
      • Raastad T.
      • et al.
      Impact of resistance exercise on ribosome biogenesis is acutely regulated by post-exercise recovery strategies.
      ) and rodent (
      • Chen Y.W.
      • Nader G.A.
      • Baar K.R.
      • Fedele M.J.
      • Hoffman E.P.
      • Esser K.A.
      Response of rat muscle to acute resistance exercise defined by transcriptional and translational profiling.
      ,
      • von Walden F.
      • Casagrande V.
      • Östlund Farrants A.-K.
      • Nader G.A.
      Mechanical loading induces the expression of a Pol I regulon at the onset of skeletal muscle hypertrophy.
      ,
      • West D.W.
      • Baehr L.M.
      • Marcotte G.R.
      • Chason C.M.
      • Tolento L.
      • Gomes A.V.
      • et al.
      Acute resistance exercise activates rapamycin-sensitive and-insensitive mechanisms that control translational activity and capacity in skeletal muscle.
      ,
      • Kirby T.J.
      • Patel R.M.
      • McClintock T.S.
      • Dupont-Versteegden E.E.
      • Peterson C.A.
      • McCarthy J.J.
      Myonuclear transcription is responsive to mechanical load and DNA content but uncoupled from cell size during hypertrophy.
      ,
      • Chaillou T.
      • Lee J.D.
      • England J.H.
      • Esser K.A.
      • McCarthy J.J.
      Time course of gene expression during mouse skeletal muscle hypertrophy.
      ,
      • Armstrong D.D.
      • Esser K.A.
      Wnt/β-catenin signaling activates growth-control genes during overload-induced skeletal muscle hypertrophy.
      ,
      • Goodman C.A.
      • Dietz J.M.
      • Jacobs B.L.
      • McNally R.M.
      • You J.-S.
      • Hornberger T.A.
      Yes-associated protein is up-regulated by mechanical overload and is sufficient to induce skeletal muscle hypertrophy.
      ) loading-induced skeletal muscle hypertrophy. We then performed a genetically driven muscle fiber–specific in vivo Myc overexpression experiment along with in silico chromatin immunoprecipitation sequencing (ChIP-seq) analysis to provide focused insight on how Myc may contribute to global gene expression during rapid muscle remodeling.

      Results

      Figure 1A is a study design schematic. Experiment 1 defines the global transcriptome using RNA-seq after 72 h of synergist ablation mechanical overload of the mouse plantaris muscle. Experiments 2 and 3 used the tissue from Experiment 1 to provide information on the contribution of active transcription versus mRNA stability to global gene expression with overload. Experiment 4 details the myonucleus-specific transcriptome during overload to identify muscle fiber–enriched genes, which is further informed by Experiments 2 and 3. Myonuclei only comprise ∼30% of all nuclei after short-term mechanical overload (
      • von Walden F.
      • Rea M.
      • Mobley C.B.
      • Fondufe-Mittendorf Y.
      • McCarthy J.J.
      • Peterson C.A.
      • et al.
      The myonuclear DNA methylome in response to an acute hypertrophic stimulus.
      ), so defining the transcriptome specifically in myonuclei is critical for understanding muscle fiber adaptation. The transcriptional regulation and localization of Myc and its impact on muscle gene expression were explored using data from Experiments 1 to 4. Experiment 5 utilized a doxycycline-inducible muscle fiber–specific in vivo Myc pulse in the plantaris to understand what genes Myc controls within muscle fibers and how this relates to myonuclear gene expression during overload (Experiment 4); we corroborated the results from these analyses using computational ChIP-seq (
      • Qin Q.
      • Fan J.
      • Zheng R.
      • Wan C.
      • Mei S.
      • Wu Q.
      • et al.
      Lisa: inferring transcriptional regulators through integrative modeling of public chromatin accessibility and ChIP-seq data.
      ). All transcriptome data are publicly available for browsing:
      Figure thumbnail gr1
      Figure 1Experiment 1: RNA-sequencing of total RNA from plantaris muscle after 72 h of mechanical overload. A, study design schematic showing the conditions of Experiments 1 to 5. B, pathway analysis of upregulated genes in OV versus sham. C, pathway analysis of downregulated genes in OV versus sham. D, Myc mRNA levels in sham and overload determined by RNA-seq. E, digital deconvolution of muscle overload data using CIBERSORTx (
      • Newman A.M.
      • Steen C.B.
      • Liu C.L.
      • Gentles A.J.
      • Chaudhuri A.A.
      • Scherer F.
      • et al.
      Determining cell type abundance and expression from bulk tissues with digital cytometry.
      ) and data from Oprescu et al. (
      • Oprescu S.N.
      • Yue F.
      • Qiu J.
      • Brito L.F.
      • Kuang S.
      Temporal dynamics and heterogeneity of cell populations during skeletal muscle regeneration.
      ) to delineate cellular contributions to the global transcriptome. Normalized gene count, gene counts normalized using DESeq2. EU, 5-Ethenyl uridine; OV, overload.

      Experiment 1: The global plantaris transcriptome after 72 h of mechanical overload

      Pathway analysis of differentially regulated genes (false discovery rate adjusted p value [adj. p] < 0.05) in plantaris tissue after overload revealed extracellular matrix (ECM), inflammatory, histone (RNA Pol I), and RHO-GTPase gene expression were higher relative to sham (Fig. 1B) (Table S1 and S2). A large proportion of downregulated genes were related to oxidative metabolism (Fig. 1C) (Table S3). This repressed gene signature could contribute to a “Warburg effect” that occurs during rapid overload-induced muscle hypertrophy, marking a shift toward “aerobic glycolysis” for rapid biomass accumulation (
      • Valentino T.
      • Figueiredo V.C.
      • Mobley C.B.
      • McCarthy J.J.
      • Vechetti Jr., I.J.
      Evidence of myomiR regulation of the pentose phosphate pathway during mechanical load-induced hypertrophy.
      ,
      • Wackerhage H.
      • Vechetti I.J.
      • Baumert P.
      • Gehlert S.
      • Becker L.
      • Jaspers R.T.
      • et al.
      Does a hypertrophying muscle fibre reprogramme its metabolism similar to a cancer cell?.
      ,
      • Verbrugge S.A.
      • Gehlert S.
      • Stadhouders L.E.
      • Jacko D.
      • Aussieker T.
      • MJ de Wit G.
      • et al.
      PKM2 determines myofiber hypertrophy in vitro and increases in response to resistance exercise in human skeletal muscle.
      ,
      • Vazquez A.
      • Liu J.
      • Zhou Y.
      • Oltvai Z.N.
      Catabolic efficiency of aerobic glycolysis: the Warburg effect revisited.
      ). Consistent with our prior murine studies (
      • von Walden F.
      • Rea M.
      • Mobley C.B.
      • Fondufe-Mittendorf Y.
      • McCarthy J.J.
      • Peterson C.A.
      • et al.
      The myonuclear DNA methylome in response to an acute hypertrophic stimulus.
      ,
      • Kirby T.J.
      • Patel R.M.
      • McClintock T.S.
      • Dupont-Versteegden E.E.
      • Peterson C.A.
      • McCarthy J.J.
      Myonuclear transcription is responsive to mechanical load and DNA content but uncoupled from cell size during hypertrophy.
      ,
      • Chaillou T.
      • Lee J.D.
      • England J.H.
      • Esser K.A.
      • McCarthy J.J.
      Time course of gene expression during mouse skeletal muscle hypertrophy.
      ) and human resistance exercise time course data (
      • Figueiredo V.C.
      • Wen Y.
      • Alkner B.
      • Fernandez-Gonzalo R.
      • Norrbom J.
      • Vechetti Jr., I.J.
      • et al.
      Genetic and epigenetic regulation of skeletal muscle ribosome biogenesis with exercise.
      ), Myc was higher after overload (Log2FC = 2.0, adj. p = 0.006) (Fig. 1D).
      To provide insight on what cell types contribute to global gene expression profiles in sham and overload, we conducted digital deconvolution analysis with CIBERSORTx using Experiment 1 transcriptome data (
      • Newman A.M.
      • Steen C.B.
      • Liu C.L.
      • Gentles A.J.
      • Chaudhuri A.A.
      • Scherer F.
      • et al.
      Determining cell type abundance and expression from bulk tissues with digital cytometry.
      ). The analysis algorithm was trained using single cell RNA-seq data from a 10 days muscle regeneration dataset (
      • Oprescu S.N.
      • Yue F.
      • Qiu J.
      • Brito L.F.
      • Kuang S.
      Temporal dynamics and heterogeneity of cell populations during skeletal muscle regeneration.
      ) (Fig. 1E). The interstitial cell proportion in muscle increases at the onset of overload, outnumbering myonuclei (
      • von Walden F.
      • Rea M.
      • Mobley C.B.
      • Fondufe-Mittendorf Y.
      • McCarthy J.J.
      • Peterson C.A.
      • et al.
      The myonuclear DNA methylome in response to an acute hypertrophic stimulus.
      ). Despite this shift, the largest contribution to gene expression in muscle was predicted to be from muscle fibers (i.e., myonuclei) after 72 h of overload. The second largest contributions were from muscle stem cells (satellite cells) and fibro-adipogenic progenitors (Fig. 1E). We recently reported that successful ECM remodeling during the first 96 h of overload determines the long-term hypertrophic response (
      • Murach K.A.
      • Peck B.D.
      • Policastro R.A.
      • Vechetti I.J.
      • Van Pelt D.W.
      • Dungan C.M.
      • et al.
      Early satellite cell communication creates a permissive environment for long-term muscle growth.
      ). Early stage ECM remodeling is strongly influenced by satellite cells and fibro-adipogenic progenitors (
      • Murach K.A.
      • Peck B.D.
      • Policastro R.A.
      • Vechetti I.J.
      • Van Pelt D.W.
      • Dungan C.M.
      • et al.
      Early satellite cell communication creates a permissive environment for long-term muscle growth.
      ,
      • Fry C.S.
      • Kirby T.J.
      • Kosmac K.
      • McCarthy J.J.
      • Peterson C.A.
      Myogenic progenitor cells control extracellular matrix production by fibroblasts during skeletal muscle hypertrophy.
      ); it follows that these cell types are major contributors to early-phase gene expression during growth.

      Experiments 2 and 3: Nascent and stable mRNA transcriptomes after 72 h of mechanical overload

      Most transcription in skeletal muscle is rRNA (
      • Zak R.
      • Rabinowitz M.
      • Platt C.
      Ribonucleic acids associated with myofibrils.
      ), and rRNA levels are further augmented specifically in myonuclei during overload (
      • Kirby T.J.
      • Patel R.M.
      • McClintock T.S.
      • Dupont-Versteegden E.E.
      • Peterson C.A.
      • McCarthy J.J.
      Myonuclear transcription is responsive to mechanical load and DNA content but uncoupled from cell size during hypertrophy.
      ). We conducted mRNA profiling to understand global transcriptional dynamics in the non-rRNA pool during growth using 5-Ethenyl uridine (EU) metabolic labeling (
      • Kirby T.J.
      • Patel R.M.
      • McClintock T.S.
      • Dupont-Versteegden E.E.
      • Peterson C.A.
      • McCarthy J.J.
      Myonuclear transcription is responsive to mechanical load and DNA content but uncoupled from cell size during hypertrophy.
      ) (Tables S4–S9). In the EU (nascent, Experiment 2) and non-EU (not actively transcribed and presumably stable, Experiment 3) mRNA fractions, ECM remodeling was among the most upregulated processes during the last 5 h of overload versus sham (Fig. 2, A and B) (Tables S4–S6, and S8). Myc was also significantly higher in nascent and stable fractions relative to sham (Fig. 2C). Active Myc transcription during overload could be facilitated by hypomethylation of its promoter in myonuclei (
      • von Walden F.
      • Rea M.
      • Mobley C.B.
      • Fondufe-Mittendorf Y.
      • McCarthy J.J.
      • Peterson C.A.
      • et al.
      The myonuclear DNA methylome in response to an acute hypertrophic stimulus.
      ). Thus, ECM and Myc gene expression are highly regulated during the acute phase of muscle loading. In addition, RHO-GTPase genes were higher in both fractions with overload relative to sham (Fig. 2, A and B). RHO-GTPases are implicated in muscle mass regulation, but their role in load-induced hypertrophy is still being defined (
      • Rodríguez-Fdez S.
      • Bustelo X.R.
      Rho GTPases in skeletal muscle development and homeostasis.
      ). Genes related to epigenetic control of gene expression (specifically histones and Dnmt1) were enriched in the non-EU fraction with overload (Fig. 2, D and E). Myonuclear histone turnover (
      • Ohsawa I.
      • Kawano F.
      Chronic exercise training activates histone turnover in mouse skeletal muscle fibers.
      ) and dynamic regulation of DNA methylation in myonuclei (
      • von Walden F.
      • Rea M.
      • Mobley C.B.
      • Fondufe-Mittendorf Y.
      • McCarthy J.J.
      • Peterson C.A.
      • et al.
      The myonuclear DNA methylome in response to an acute hypertrophic stimulus.
      ,
      • Figueiredo V.C.
      • Wen Y.
      • Alkner B.
      • Fernandez-Gonzalo R.
      • Norrbom J.
      • Vechetti Jr., I.J.
      • et al.
      Genetic and epigenetic regulation of skeletal muscle ribosome biogenesis with exercise.
      ,
      • Murach K.A.
      • Dungan C.M.
      • von Walden F.
      • Wen Y.
      Epigenetic evidence for distinct contributions of resident and acquired myonuclei during long-term exercise adaptation using timed in vivo myonuclear labeling.
      ) likely facilitates hypertrophic gene expression and adaptation in muscle fibers.
      Figure thumbnail gr2
      Figure 2Experiments 2 and 3: RNA-sequencing of EU- and Non-EU–labeled mRNA from plantaris after 72 h of mechanical overload. A, pathway analysis of genes upregulated in the EU-labeled fraction in OV versus sham. B, pathway analysis of genes upregulated in the Non-EU–labeled fraction in OV versus sham. C, Myc mRNA levels in the EU and Non-EU fractions. D, histone genes elevated in the Non-EU fraction during overload. E, Dnmt1 mRNA levels in EU and Non-EU fractions. F, pathway analysis of genes downregulated in the EU-labeled fraction in OV versus sham. G, pathway analysis of genes downregulated in the Non-EU–labeled fraction in OV versus sham. EU, 5-Ethenyl uridine; OV, overload.
      In the EU and non-EU fractions, oxidative metabolism-related gene expression was lower during overload (Fig. 2, F and G) (Tables S7 and S9); these data inform the findings from Experiment 1. Pgc1α (Ppargc1a), a core regulator of mitochondrial biogenesis (
      • Lin J.
      • Wu H.
      • Tarr P.T.
      • Zhang C.-Y.
      • Wu Z.
      • Boss O.
      • et al.
      Transcriptional co-activator PGC-1α drives the formation of slow-twitch muscle fibres.
      ,
      • Puigserver P.
      • Wu Z.
      • Park C.W.
      • Graves R.
      • Wright M.
      • Spiegelman B.M.
      A cold-inducible coactivator of nuclear receptors linked to adaptive thermogenesis.
      ), was among genes that were lower in both fractions during overload (Tables S4 and S5). Apart from epigenetic-related genes, most mRNA differences between sham and overload at the pathway level were attributable to differences in both nascent transcription and, presumably, enhanced mRNA stability.

      Experiment 4: The myonuclear transcriptome after 72 h of mechanical overload

      To understand what genes were specifically regulated in muscle fibers during rapid muscle growth, we conducted RNA-seq on fluorescent activated nuclear-sorted (FANS)-purified myonuclei using the HSA-GFP mouse (Fig. 3A) (
      • von Walden F.
      • Rea M.
      • Mobley C.B.
      • Fondufe-Mittendorf Y.
      • McCarthy J.J.
      • Peterson C.A.
      • et al.
      The myonuclear DNA methylome in response to an acute hypertrophic stimulus.
      ,
      • Iwata M.
      • Englund D.A.
      • Wen Y.
      • Dungan C.M.
      • Murach K.A.
      • Vechetti I.J.
      • et al.
      A novel tetracycline-responsive transgenic mouse strain for skeletal muscle-specific gene expression.
      ). Relative to sham, genes related to ECM remodeling (primarily collagens, matrix metalloproteinases, and secreted factors) and immune signaling (namely chemokines) were most highly enriched in myonuclei from overloaded muscle (Fig. 3B) (Tables S10 and S11). We and others have reported that matrix metalloproteinase 9 (Mmp9) is responsive to loading in skeletal muscle and is a key component of muscle growth (
      • Murach K.A.
      • Vechetti Jr., I.J.
      • Van Pelt D.W.
      • Crow S.E.
      • Dungan C.M.
      • Figueiredo V.C.
      • et al.
      Fusion-independent satellite cell communication to muscle fibers during load-induced hypertrophy.
      ,
      • Player D.
      • Martin N.
      • Passey S.
      • Sharples A.
      • Mudera V.
      • Lewis M.
      Acute mechanical overload increases IGF-I and MMP-9 mRNA in 3D tissue-engineered skeletal muscle.
      ,
      • Dahiya S.
      • Bhatnagar S.
      • Hindi S.M.
      • Jiang C.
      • Paul P.K.
      • Kuang S.
      • et al.
      Elevated levels of active matrix metalloproteinase-9 cause hypertrophy in skeletal muscle of normal and dystrophin-deficient mdx mice.
      ). We confirm here that Mmp9 is enriched in myonuclei during overload in vivo (Fig. 3C). Interstitial cells of the muscle microenvironment are generally viewed as the primary contributors to ECM deposition and turnover. Emerging evidence suggests that muscle fibers also play a major role in ECM remodeling (
      • Murach K.A.
      • Vechetti Jr., I.J.
      • Van Pelt D.W.
      • Crow S.E.
      • Dungan C.M.
      • Figueiredo V.C.
      • et al.
      Fusion-independent satellite cell communication to muscle fibers during load-induced hypertrophy.
      ,
      • Accornero F.
      • Kanisicak O.
      • Tjondrokoesoemo A.
      • Attia A.C.
      • McNally E.M.
      • Molkentin J.D.
      Myofiber-specific inhibition of TGFβ signaling protects skeletal muscle from injury and dystrophic disease in mice.
      ,
      • Petrosino J.M.
      • Leask A.
      • Accornero F.
      Genetic manipulation of CCN2/CTGF unveils cell-specific ECM-remodeling effects in injured skeletal muscle.
      ,
      • Brightwell C.R.
      • Latham C.M.
      • Thomas N.T.
      • Keeble A.R.
      • Murach K.A.
      • Fry C.S.
      A glitch in the matrix: the pivotal role for extracellular matrix remodeling during muscle hypertrophy.
      ), which the current data reinforces.
      Figure thumbnail gr3
      Figure 3Experiment 4: RNA-sequencing of myonuclear RNA from plantaris muscle after 72 h of mechanical overload. A, image showing myonuclear GFP labeling, DNA (DAPI), and dystrophin via histochemistry in a doxycycline-treated HSA-GFP mouse (see refs. (
      • von Walden F.
      • Rea M.
      • Mobley C.B.
      • Fondufe-Mittendorf Y.
      • McCarthy J.J.
      • Peterson C.A.
      • et al.
      The myonuclear DNA methylome in response to an acute hypertrophic stimulus.
      ) and (
      • DePinho R.
      • Mitsock L.
      • Hatton K.
      • Ferrier P.
      • Zimmerman K.
      • Legouy E.
      • et al.
      Myc family of cellular oncogenes.
      )); the scale bar represents 100 μm. B, pathway analysis of upregulated genes specifically in FANS-isolated myonuclei in OV versus sham. C, myonuclear Mmp9 mRNA levels in sham and overload determined by RNA-seq. D, Myc mRNA levels in sham and overload determined by RNA-seq. E, Runx1 mRNA levels in sham and overload determined by RNA-seq. F, Ankrd1 (Marp1) mRNA levels in sham and overload determined by RNA-seq. G, pathway analysis of downregulated genes specifically in FANS-isolated myonuclei in OV versus sham. FANS, fluorescent activated nuclear-sorted; OV, overload.
      Numerous cell cycle regulators were enriched in myonuclei after overload (Fig. 3B) which included Myc (top 30 upregulated gene, adj. p = 0.0136 × 10−7) (Fig. 3C) (Table S11). Runx1, another transcription factor, was highly abundant and enriched in myonuclei by overload (Log2FC = 4.1, adj. p = 0.05 × 10−14) (Fig. 3E); it was also markedly higher in total RNA, EU, and non-EU fractions (Tables S1, S4 and S5). Runx1 induction during overload is intuitive since it regulates muscle mass, myofibrillar organization, and autophagy in myofibers (
      • Wang X.
      • Blagden C.
      • Fan J.
      • Nowak S.J.
      • Taniuchi I.
      • Littman D.R.
      • et al.
      Runx1 prevents wasting, myofibrillar disorganization, and autophagy of skeletal muscle.
      ). RUNX1 is also known to interact and complex with MYC (
      • Agrawal P.
      • Yu K.
      • Salomon A.R.
      • Sedivy J.M.
      Proteomic profiling of Myc-associated proteins.
      ,
      • Pippa R.
      • Dominguez A.
      • Malumbres R.
      • Endo A.
      • Arriazu E.
      • Marcotegui N.
      • et al.
      MYC-dependent recruitment of RUNX1 and GATA2 on the SET oncogene promoter enhances PP2A inactivation in acute myeloid leukemia.
      ). Ankrd1 (Marp1, also Carp1) was the most upregulated gene with overload in myonuclei (Log2FC = 6.0, adj. p = 0.0127 × 10−62) as well as the EU fraction (Log2FC = 5.1, adj. p = 0.038 × 10−60) (Fig. 3F) (Tables S4, S5, and S10); however, it was only the 1495th most differentially regulated gene in the total RNA dataset (Table S1). Ankrd10 was also among the most upregulated genes in myonuclei and the EU fraction. Ankrd1 localizes in myotendinous junction myonuclei (
      • Petrany M.J.
      • Swoboda C.O.
      • Sun C.
      • Chetal K.
      • Chen X.
      • Weirauch M.T.
      • et al.
      Single-nucleus RNA-seq identifies transcriptional heterogeneity in multinucleated skeletal myofibers.
      ) and is induced by eccentric exercise in rodent and human muscle (
      • Barash I.A.
      • Mathew L.
      • Ryan A.F.
      • Chen J.
      • Lieber R.L.
      Rapid muscle-specific gene expression changes after a single bout of eccentric contractions in the mouse.
      ,
      • Mahoney D.J.
      • Safdar A.
      • Parise G.
      • Melov S.
      • Fu M.
      • MacNeil L.
      • et al.
      Gene expression profiling in human skeletal muscle during recovery from eccentric exercise.
      ). Perhaps Ankrd1 upregulation during mechanical overload is partially explained by muscle lengthening and/or myotendinous junction remodeling (
      • Murach K.A.
      • McCarthy J.J.
      • Peterson C.A.
      • Dungan C.M.
      Making mice mighty: recent advances in translational models of load-induced muscle hypertrophy.
      ,
      • Armstrong R.
      • Marum P.
      • Tullson P.
      • Saubert 4th, C.
      Acute hypertrophic response of skeletal muscle to removal of synergists.
      ). These results highlight the power of EU-labeling and myonucleus-specific transcriptomics for identifying potentially important genes for muscle growth. Our findings also provide impetus for further investigation of Ankrd1 during muscle hypertrophy. The category of genes most downregulated during overload in myonuclei was oxidative metabolism (Fig. 3G) (Table S12). Thus, the total mRNA and EU results are likely driven by changes within the myofiber.

      Experiment 5: The Myc-controlled transcriptome in plantaris muscle fibers

      We generated a doxycycline-inducible HSA-Myc mouse to experimentally define the MYC regulatory network in plantaris muscle fibers. A pulse of Myc was driven via doxycycline in water overnight followed by a 12-h period without doxycycline. Principal component analysis revealed stark differences between control and Myc overexpression (Fig. 4A). At the pathway level, ribosome biogenesis-related genes such as ribosomal proteins and eukaryotic initiation factors were most upregulated relative to controls (Fig. 4B) (Table S13). A MYC pulse induced gene expression of the large ribosomal subunit protein Rpl3 (Log2FC = 1.95, adj. p = 0.00053) (Fig. 4C). Rpl3 was also higher in myonuclei during overload, and its muscle-specific paralog Rpl3l was lower (adj. p < 0.05) (Table S10). Upregulation of Rpl3 has been implicated in robust hypertrophy in mouse (
      • Chaillou T.
      • Lee J.D.
      • England J.H.
      • Esser K.A.
      • McCarthy J.J.
      Time course of gene expression during mouse skeletal muscle hypertrophy.
      ) and human muscle (
      • Stec M.J.
      • Kelly N.A.
      • Many G.M.
      • Windham S.T.
      • Tuggle S.C.
      • Bamman M.M.
      Ribosome biogenesis may augment resistance training-induced myofiber hypertrophy and is required for myotube growth in vitro.
      ). Rpl3 may influence growth via ribosome specialization (
      • Chaillou T.
      Ribosome specialization and its potential role in the control of protein translation and skeletal muscle size.
      ), but more work is needed in this area. The induction of Rpl3 by overload and Myc alongside increased levels of the rRNA transcription-associated genes Bop1 (
      • Strezoska Z.A.
      • Pestov D.G.
      • Lau L.F.
      Bop1 is a mouse WD40 repeat nucleolar protein involved in 28S and 5.8 S rRNA processing and 60S ribosome biogenesis.
      ,
      • Lapik Y.R.
      • Fernandes C.J.
      • Lau L.F.
      • Pestov D.G.
      Physical and functional interaction between Pes1 and Bop1 in mammalian ribosome biogenesis.
      ), Ftsj3 (
      • Morello L.G.
      • Coltri P.P.
      • Quaresma A.J.
      • Simabuco F.M.
      • Silva T.C.
      • Singh G.
      • et al.
      The human nucleolar protein FTSJ3 associates with NIP7 and functions in pre-rRNA processing.
      ), Polr3g (
      • Madan B.
      • Harmston N.
      • Nallan G.
      • Montoya A.
      • Faull P.
      • Petretto E.
      • et al.
      Temporal dynamics of Wnt-dependent transcriptome reveal an oncogenic Wnt/MYC/ribosome axis.
      ), Rpl10a (
      • Wang H.
      • Wang L.
      • Wang Z.
      • Dang Y.
      • Shi Y.
      • Zhao P.
      • et al.
      The nucleolar protein NOP2 is required for nucleolar maturation and ribosome biogenesis during preimplantation development in mammals.
      ), and Rps19 (
      • Idol R.A.
      • Robledo S.
      • Du H.-Y.
      • Crimmins D.L.
      • Wilson D.B.
      • Ladenson J.H.
      • et al.
      Cells depleted for RPS19, a protein associated with Diamond Blackfan Anemia, show defects in 18S ribosomal RNA synthesis and small ribosomal subunit production.
      ), could also be a sign of enhanced ribosome biogenesis. In total, 31 upregulated genes were common to Myc overexpression in muscle fibers and myonuclei with overload (Fig. 4D).
      Figure thumbnail gr4
      Figure 4Experiment 5: RNA-sequencing of total RNA from plantaris muscle of HSA-Myc mice following a single pulse of Myc. A, PCA plots from doxycycline-treated HSA-Myc versus littermate HSA-rtTA (Control) mice (generated using DESeq2 normalized gene counts). B, pathway analysis of upregulated genes after Myc overexpression. C, Rpl3 mRNA levels after Myc overexpression. D, genes upregulated by Myc in muscle and also enriched in myonuclei during 72 h of overload. E, Myh2 mRNA levels after Myc overexpression. F, Reverbα (Nr1d1) and Reverbβ (Nr1d2) after Myc overexpression. G, genes upregulated by Myc in muscle also enriched in myonuclei during 72 h of overload. OV, overload; PCA, principal component analysis.
      Approximately, 100 genes were downregulated (adj. p < 0.05) by MYC in the plantaris (Table S13). MYC strongly regulates microRNA expression (
      • Dang C.V.
      MYC on the path to cancer.
      ,
      • O'Donnell K.A.
      • Wentzel E.A.
      • Zeller K.I.
      • Dang C.V.
      • Mendell J.T.
      c-Myc-regulated microRNAs modulate E2F1 expression.
      ,
      • Wang X.
      • Zhao X.
      • Gao P.
      • Wu M.
      c-Myc modulates microRNA processing via the transcriptional regulation of Drosha.
      ,
      • Lin C.H.
      • Jackson A.L.
      • Guo J.
      • Linsley P.S.
      • Eisenman R.N.
      Myc-regulated microRNAs attenuate embryonic stem cell differentiation.
      ,
      • Bui T.V.
      • Mendell J.T.
      Myc: maestro of microRNAs.
      ,
      • Luo W.
      • Chen J.
      • Li L.
      • Ren X.
      • Cheng T.
      • Lu S.
      • et al.
      c-Myc inhibits myoblast differentiation and promotes myoblast proliferation and muscle fibre hypertrophy by regulating the expression of its target genes, miRNAs and lincRNAs.
      ). Repressed genes with MYC induction are potentially attributable to MYC-controlled microRNA-mediated mRNA destabilization and degradation. MYC may also repress gene expression via regulating DNA methylation and chromatin remodeling (
      • Brenner C.
      • Deplus R.
      • Didelot C.
      • Loriot A.
      • Viré E.
      • De Smet C.
      • et al.
      Myc represses transcription through recruitment of DNA methyltransferase corepressor.
      ,
      • Lin C.-H.
      • Lin C.
      • Tanaka H.
      • Fero M.L.
      • Eisenman R.N.
      Gene regulation and epigenetic remodeling in murine embryonic stem cells by c-Myc.
      ,
      • Nie Z.
      • Hu G.
      • Wei G.
      • Cui K.
      • Yamane A.
      • Resch W.
      • et al.
      c-Myc is a universal amplifier of expressed genes in lymphocytes and embryonic stem cells.
      ), as well as through specific protein-protein interactions (
      • Kalkat M.
      • Wasylishen A.R.
      • Kim S.S.
      • Penn L.
      More than MAX: discovering the Myc interactome.
      ,
      • Conacci-Sorrell M.
      • McFerrin L.
      • Eisenman R.N.
      An overview of MYC and its interactome.
      ). The abundant myosin heavy chain type 2a gene Myh2 was repressed by MYC (Log2FC = −0.75, adj. p = 0.013) (Fig. 4E), similar to what occurred in myonuclei during overload (Table S10). Type 2a myosin is associated with oxidative metabolism in murine muscle (
      • Bloemberg D.
      • Quadrilatero J.
      Rapid determination of myosin heavy chain expression in rat, mouse, and human skeletal muscle using multicolor immunofluorescence analysis.
      ,
      • Dungan C.M.
      • Brightwell C.
      • Wen Y.
      • Zdunek C.J.
      • Latham C.
      • Thomas N.T.
      • et al.
      Muscle-specific cellular and molecular adaptations to late-life voluntary concurrent exercise.
      ). Lower Myh2 may be part of a Warburg-like program that accompanies rapid muscle growth (
      • Valentino T.
      • Figueiredo V.C.
      • Mobley C.B.
      • McCarthy J.J.
      • Vechetti Jr., I.J.
      Evidence of myomiR regulation of the pentose phosphate pathway during mechanical load-induced hypertrophy.
      ,
      • Wackerhage H.
      • Vechetti I.J.
      • Baumert P.
      • Gehlert S.
      • Becker L.
      • Jaspers R.T.
      • et al.
      Does a hypertrophying muscle fibre reprogramme its metabolism similar to a cancer cell?.
      ). Reverbα (Nr1d1, Log2FC = −1.36, adj. p = 0.065) and Reverbβ (Nr1d2, Log2FC = −1.31, adj. p = 0.044) mRNA levels were lower with MYC overexpression (Fig. 4F) and in myonuclei with overload (Table S10). In cancer cells, MYC promotes Reverbα and Reverbβ expression (
      • Altman B.J.
      • Hsieh A.L.
      • Sengupta A.
      • Krishnanaiah S.Y.
      • Stine Z.E.
      • Walton Z.E.
      • et al.
      MYC disrupts the circadian clock and metabolism in cancer cells.
      ) which affects the core clock gene Bmal1 (
      • Altman B.J.
      • Hsieh A.L.
      • Sengupta A.
      • Krishnanaiah S.Y.
      • Stine Z.E.
      • Walton Z.E.
      • et al.
      MYC disrupts the circadian clock and metabolism in cancer cells.
      ,
      • Shostak A.
      • Ruppert B.
      • Ha N.
      • Bruns P.
      • Toprak U.H.
      • Eils R.
      • et al.
      MYC/MIZ1-dependent gene repression inversely coordinates the circadian clock with cell cycle and proliferation.
      ), circadian rhythm, and cell metabolism (
      • Altman B.J.
      • Hsieh A.L.
      • Sengupta A.
      • Krishnanaiah S.Y.
      • Stine Z.E.
      • Walton Z.E.
      • et al.
      MYC disrupts the circadian clock and metabolism in cancer cells.
      ). Thus, MYC control of Reverbs could be unique in muscle fibers. In total, 35 downregulated genes were common to MYC overexpression in muscle fibers and myonuclei with overload (Fig. 4G).
      To corroborate MYC regulation of target genes in muscle, we compared our overload myonuclear and Myc overexpression RNA-seq data to published MYC ChIP-seq data from myogenic cells (
      • Luo W.
      • Chen J.
      • Li L.
      • Ren X.
      • Cheng T.
      • Lu S.
      • et al.
      c-Myc inhibits myoblast differentiation and promotes myoblast proliferation and muscle fibre hypertrophy by regulating the expression of its target genes, miRNAs and lincRNAs.
      ). Of genes regulated by both overload and MYC (Fig. 4, D and G), Anp32b, Aqp4, Atp1a1, Cdkn1b, Cntfr, Epas1, Ftsj3, Jak2, Ncl, Nr1d2/Reverbβ, P2ry1, Pcmtd1, Rpl3, and Slc7a5 featured MYC occupancy in myogenic cells. After differentiation, MYC-binding peaks on all these genes except Aqp4, Atp1a1, and P2ry1 were altered, indicating regulation by MYC in dynamic conditions. Of note, MYC binding to Ankrd1 increased during myotube formation (
      • Luo W.
      • Chen J.
      • Li L.
      • Ren X.
      • Cheng T.
      • Lu S.
      • et al.
      c-Myc inhibits myoblast differentiation and promotes myoblast proliferation and muscle fibre hypertrophy by regulating the expression of its target genes, miRNAs and lincRNAs.
      ). Brief Myc overexpression in the soleus did not induce Ankrd1, so MYC may function cooperatively with another factor that is induced during overload to regulate this gene (
      • Das S.K.
      • Lewis B.A.
      • Levens D.
      MYC: a complex problem.
      ,
      • Li Z.
      • Ivanov A.A.
      • Su R.
      • Gonzalez-Pecchi V.
      • Qi Q.
      • Liu S.
      • et al.
      The OncoPPi network of cancer-focused protein–protein interactions to inform biological insights and therapeutic strategies.
      ). To confirm MYC transcription factor binding of myonuclear DNA during mechanical overload, we utilized our RNA-seq data to perform epigenetic Landscape In Silico deletion Analysis (Lisa) (
      • Qin Q.
      • Fan J.
      • Zheng R.
      • Wan C.
      • Mei S.
      • Wu Q.
      • et al.
      Lisa: inferring transcriptional regulators through integrative modeling of public chromatin accessibility and ChIP-seq data.
      ). Lisa incorporates transcriptome input data with an extensive library of publicly available transcription factor ChIP-seq and global chromatin accessibility profiles to infer transcriptional regulators. Leveraging our MYC overexpression RNA-seq data as a control (Experiment 5), MYC/MYCN was the highest ranked transcription factor driving upregulated genes (Table S14), confirming the accuracy of Lisa. Using the first 500 differentially regulated genes in each direction from Experiment 4, MYC was in the top 5% of transcription factors controlling upregulated genes (p = 1.7 × 10−24) in myonuclei during overload and the top 10% for controlling downregulated genes (p = 2.4 × 10−11) (Table S15). Motif target prediction suggested that MYC regulates Rpl3 in myonuclei during overload (top 45% of genes targeted by MYC) (Table S16). Ribosome biogenesis-associated Bop1 (top 15%), Ftsj3 (top 15%), Polr3g (top 1%), and Rps19 (top 15%) had high regulatory potential by MYC during overload according to H3k27ac ChIP-seq (promoter/enhancer) information; Ncl was also in the top 1% (Table S17). MYC had regulatory potential for Nr1d2/Reverbβ during overload (top 35% of gene targets) (Table S18). All together, these data suggest that MYC controls gene expression in myonuclei during loading-induced hypertrophy.

      Discussion

      Interrelated RNA-seq datasets define the early phase of growth processes in differentiated muscle fibers (Fig. 5). Lower oxidative metabolism-related gene expression during the onset of rapid muscle growth is due to changes in mRNA transcription and stability and occurs specifically in myonuclei. The overload datasets also revealed an under-appreciated role for muscle fibers in ECM remodeling during adaptation. Regulation of several collagens and remodeling enzymes such as Mmps by active transcription, transcript stability, and in myonuclei emphasizes the importance of ECM dynamics for muscle hypertrophy (
      • Accornero F.
      • Kanisicak O.
      • Tjondrokoesoemo A.
      • Attia A.C.
      • McNally E.M.
      • Molkentin J.D.
      Myofiber-specific inhibition of TGFβ signaling protects skeletal muscle from injury and dystrophic disease in mice.
      ). The non-EU RNA-seq data suggests elevated epigenetic-related gene expression during overload reflects greater mRNA stability; the mechanism underlying the change in mRNA stability in muscle during hypertrophy deserves further investigation. We identified key genes that are actively transcribed in muscle and enriched in myonuclei during in vivo muscle growth, such as Runx1 and Ankrd1. RUNX1 regulates muscle mass (
      • Wang X.
      • Blagden C.
      • Fan J.
      • Nowak S.J.
      • Taniuchi I.
      • Littman D.R.
      • et al.
      Runx1 prevents wasting, myofibrillar disorganization, and autophagy of skeletal muscle.
      ) and participates in ribosome biogenesis (
      • Cai X.
      • Gao L.
      • Teng L.
      • Ge J.
      • Oo Z.M.
      • Kumar A.R.
      • et al.
      Runx1 deficiency decreases ribosome biogenesis and confers stress resistance to hematopoietic stem and progenitor cells.
      ), as well as interacts with MYC (
      • Agrawal P.
      • Yu K.
      • Salomon A.R.
      • Sedivy J.M.
      Proteomic profiling of Myc-associated proteins.
      ,
      • Pippa R.
      • Dominguez A.
      • Malumbres R.
      • Endo A.
      • Arriazu E.
      • Marcotegui N.
      • et al.
      MYC-dependent recruitment of RUNX1 and GATA2 on the SET oncogene promoter enhances PP2A inactivation in acute myeloid leukemia.
      ). ANKRD1 associates with titin’s N2A element, a major mechanosensory and signaling hub in skeletal muscle (
      • Nishikawa K.
      • Lindstedt S.L.
      • Hessel A.
      • Mishra D.
      N2A titin: signaling hub and mechanical switch in skeletal muscle.
      ,
      • van der Pijl R.J.
      • Domenighetti A.A.
      • Sheikh F.
      • Ehler E.
      • Ottenheijm C.A.
      • Lange S.
      The titin N2B and N2A regions: biomechanical and metabolic signaling hubs in cross-striated muscles.
      ). It also locks titin to the thin filament, regulates passive force, and protects the sarcomere from mechanical damage (
      • van der Pijl R.J.
      • van den Berg M.
      • van de Locht M.
      • Shen S.
      • Bogaards S.J.
      • Conijn S.
      • et al.
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      ). Some evidence suggests ANKRD1 inhibits TNFα-induced NFκB signaling (
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      ANKRD1 modulates inflammatory responses in C2C12 myoblasts through feedback inhibition of NF-κB signaling activity.
      ) and affects androgen receptor signaling (
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      ) in myogenic cell culture. Given these functions, ANKRD1 may be critical for successful muscle hypertrophy. Ankrd1 was not among the most differentially expressed genes in the total RNA dataset but emerged in the nascent and myonuclear RNA-seq as the most highly responsive gene to mechanical overload. This mismatch highlights the utility of evaluating transcriptional dynamics and myonuclear-specific gene expression for understanding muscle adaptation. Furthermore, Runx1 and Ankrd1 are typically upregulated after muscle denervation (
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      ANKRD1 modulates inflammatory responses in C2C12 myoblasts through feedback inhibition of NF-κB signaling activity.
      ), suggesting a compensatory response to counteract atrophy in that condition.
      Figure thumbnail gr5
      Figure 5Summary of key findings from Experiments 1 to 5.
      MYC protein localizes in myonuclei during loading-induced skeletal muscle growth (
      • Alway S.E.
      Overload-induced C-Myc oncoprotein is reduced in aged skeletal muscle.
      ,
      • Armstrong D.D.
      • Esser K.A.
      Wnt/β-catenin signaling activates growth-control genes during overload-induced skeletal muscle hypertrophy.
      ). Genetic Myc induction recapitulates diverse aspects of the loading response in muscle fibers. These changes include downregulation of Reverbα, Reverbβ, and Myh2, along with increased Rpl3. Published ChIP-seq data in myogenic cells (
      • Luo W.
      • Chen J.
      • Li L.
      • Ren X.
      • Cheng T.
      • Lu S.
      • et al.
      c-Myc inhibits myoblast differentiation and promotes myoblast proliferation and muscle fibre hypertrophy by regulating the expression of its target genes, miRNAs and lincRNAs.
      ) as well as in silico transcriptional regulator analysis (
      • Qin Q.
      • Fan J.
      • Zheng R.
      • Wan C.
      • Mei S.
      • Wu Q.
      • et al.
      Lisa: inferring transcriptional regulators through integrative modeling of public chromatin accessibility and ChIP-seq data.
      ) using our myonuclear RNA-sequencing data corroborates the association of MYC with Reverbβ and Rpl3, along with numerous other genes. The ChIP-seq also revealed a potential interaction with Ankrd1 (
      • Luo W.
      • Chen J.
      • Li L.
      • Ren X.
      • Cheng T.
      • Lu S.
      • et al.
      c-Myc inhibits myoblast differentiation and promotes myoblast proliferation and muscle fibre hypertrophy by regulating the expression of its target genes, miRNAs and lincRNAs.
      ) that did not emerge in our muscle-specific MYC overexpression experiment. Lower levels of Reverbs during overload could have implications for circadian regulation and metabolism in muscle fibers (
      • Altman B.J.
      • Hsieh A.L.
      • Sengupta A.
      • Krishnanaiah S.Y.
      • Stine Z.E.
      • Walton Z.E.
      • et al.
      MYC disrupts the circadian clock and metabolism in cancer cells.
      ); this is salient since MYC is exercise-responsive and exercise shifts the circadian rhythm in skeletal muscle (
      • Zambon A.C.
      • McDearmon E.L.
      • Salomonis N.
      • Vranizan K.M.
      • Johansen K.L.
      • Adey D.
      • et al.
      Time-and exercise-dependent gene regulation in human skeletal muscle.
      ,
      • Wolff G.
      • Esser K.A.
      Scheduled exercise phase shifts the circadian clock in skeletal muscle.
      ,
      • Casanova-Vallve N.
      • Duglan D.
      • Vaughan M.E.
      • Pariollaud M.
      • Handzlik M.K.
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      Daily running enhances molecular and physiological circadian rhythms in skeletal muscle.
      ,
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      • et al.
      Contraction influences Per2 gene expression in skeletal muscle through a calcium-dependent pathway.
      ). Perhaps an exercise-mediated shift in muscle circadian rhythm is controlled by MYC, but more work is needed in this area, especially with respect to resistance exercise. Altered Rpl3 by mechanical overload and MYC expression could facilitate muscle-specific growth via ribosome specialization (
      • Chaillou T.
      Ribosome specialization and its potential role in the control of protein translation and skeletal muscle size.
      ,
      • Gerst J.E.
      Pimp my ribosome: ribosomal protein paralogs specify translational control.
      ). MYC is also known to be a potent driver of ribosome biogenesis in muscle (
      • Mori T.
      • Ato S.
      • Knudsen J.R.
      • Henriquez-Olguin C.
      • Li Z.
      • Wakabayashi K.
      • et al.
      c-Myc overexpression increases ribosome biogenesis and protein synthesis independent of mTORC1 activation in mouse skeletal muscle.
      ,
      • West D.W.
      • Baehr L.M.
      • Marcotte G.R.
      • Chason C.M.
      • Tolento L.
      • Gomes A.V.
      • et al.
      Acute resistance exercise activates rapamycin-sensitive and-insensitive mechanisms that control translational activity and capacity in skeletal muscle.
      ). We previously reported that ribosome biogenesis increases in total and nascent RNA pools following 72 h of synergist ablation in the mouse (
      • von Walden F.
      • Casagrande V.
      • Östlund Farrants A.-K.
      • Nader G.A.
      Mechanical loading induces the expression of a Pol I regulon at the onset of skeletal muscle hypertrophy.
      ,
      • Figueiredo V.C.
      • Wen Y.
      • Alkner B.
      • Fernandez-Gonzalo R.
      • Norrbom J.
      • Vechetti Jr., I.J.
      • et al.
      Genetic and epigenetic regulation of skeletal muscle ribosome biogenesis with exercise.
      ,
      • Kirby T.J.
      • Patel R.M.
      • McClintock T.S.
      • Dupont-Versteegden E.E.
      • Peterson C.A.
      • McCarthy J.J.
      Myonuclear transcription is responsive to mechanical load and DNA content but uncoupled from cell size during hypertrophy.
      ,
      • Kirby T.J.
      • Lee J.D.
      • England J.H.
      • Chaillou T.
      • Esser K.A.
      • McCarthy J.J.
      Blunted hypertrophic response in aged skeletal muscle is associated with decreased ribosome biogenesis.
      ). Thus, MYC appears central to the regulation of rRNA synthesis and ribosome assembly, processes hypothesized to be necessary for sustained hypertrophy in response to loading (
      • Wen Y.
      • Alimov A.P.
      • McCarthy J.J.
      Ribosome biogenesis is necessary for skeletal muscle hypertrophy.
      ,
      • von Walden F.
      Ribosome biogenesis in skeletal muscle: coordination of transcription and translation.
      ,
      • Figueiredo V.C.
      • McCarthy J.J.
      Regulation of ribosome biogenesis in skeletal muscle hypertrophy.
      ). MYH2 protein and oxidative fiber proportion increases after prolonged muscle overload (
      • Fry C.S.
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      • Jackson J.R.
      • Kirby T.J.
      • Stasko S.A.
      • Liu H.
      • et al.
      Regulation of the muscle fiber microenvironment by activated satellite cells during hypertrophy.
      ). MYC-mediated and early Myh2 downregulation during overload may relate to an acute glycolytic preference during rapid hypertrophy (
      • Valentino T.
      • Figueiredo V.C.
      • Mobley C.B.
      • McCarthy J.J.
      • Vechetti Jr., I.J.
      Evidence of myomiR regulation of the pentose phosphate pathway during mechanical load-induced hypertrophy.
      ).
      The induction of MYC in instances of attenuated muscle plasticity such as aging, where MYC activity may be blunted during hypertrophy (
      • Alway S.E.
      Overload-induced C-Myc oncoprotein is reduced in aged skeletal muscle.
      ), could restore adaptive potential and increase muscle mass. Future experiments involving simultaneous muscle-specific inducible knockout of c-Myc and its several analogous family members (
      • DePinho R.
      • Mitsock L.
      • Hatton K.
      • Ferrier P.
      • Zimmerman K.
      • Legouy E.
      • et al.
      Myc family of cellular oncogenes.
      ), myonuclear MYC ChIP-seq, and MYC protein–protein interactome analysis during overload will provide more granular insight on the MYC regulatory network during muscle hypertrophy. Since differentiated myofibers can sustain high levels of oncogene expression without tumor formation, we suggest that MYC in muscle fibers induced by loading is a core component of rapid yet functional adaptive remodeling. Collectively, our data are a rich resource for understanding transcriptional dynamics and MYC regulation during the onset of loading-induced muscle fiber growth.

      Experimental procedures

      Animals and animal procedures

      All animal procedures were approved by the University of Kentucky and the University of Arkansas IACUC. Mice were housed in a temperature and humidity-controlled room, maintained on a 14:10-h light-dark cycle, and food and water were provided ad libitum throughout experimentation. Animals were sacrificed via a lethal dosage of sodium pentobarbital injected intraperitoneally or CO2 asphyxiation followed by cervical dislocation.
      Female C57BL6/J mice were obtained from the Jackson Laboratory for Experiments 1 to 3. Female HSA-rtTA+/−-;TRE-H2B-GFP+/− (HSA-GFP) mice were generated as previously described by us (
      • von Walden F.
      • Rea M.
      • Mobley C.B.
      • Fondufe-Mittendorf Y.
      • McCarthy J.J.
      • Peterson C.A.
      • et al.
      The myonuclear DNA methylome in response to an acute hypertrophic stimulus.
      ,
      • Iwata M.
      • Englund D.A.
      • Wen Y.
      • Dungan C.M.
      • Murach K.A.
      • Vechetti I.J.
      • et al.
      A novel tetracycline-responsive transgenic mouse strain for skeletal muscle-specific gene expression.
      ) for Experiment 4. The TRE-H2B-GFP mouse was originally obtained from the Jackson Laboratory (005104, bred to homozygosity by our laboratory) (
      • Tumbar T.
      • Guasch G.
      • Greco V.
      • Blanpain C.
      • Lowry W.E.
      • Rendl M.
      • et al.
      Defining the epithelial stem cell niche in skin.
      ). For Experiment 5, male HSA-rtTA+/−-;TRE-Myc+/− (HSA-Myc) were generated by crossing homozygous HSA-rtTA mice (
      • Iwata M.
      • Englund D.A.
      • Wen Y.
      • Dungan C.M.
      • Murach K.A.
      • Vechetti I.J.
      • et al.
      A novel tetracycline-responsive transgenic mouse strain for skeletal muscle-specific gene expression.
      ) with heterozygous TRE-Myc mice (
      • Felsher D.W.
      • Bishop J.M.
      Reversible tumorigenesis by MYC in hematopoietic lineages.
      ); HSA-rtTA littermate mice were used as controls. All mice were genotyped as described (
      • Iwata M.
      • Englund D.A.
      • Wen Y.
      • Dungan C.M.
      • Murach K.A.
      • Vechetti I.J.
      • et al.
      A novel tetracycline-responsive transgenic mouse strain for skeletal muscle-specific gene expression.
      ,
      • Rutledge E.A.
      • Lindström N.O.
      • Michos O.
      • McMahon A.P.
      Genetic manipulation of ureteric bud tip progenitors in the mammalian kidney through an Adamts18 enhancer driven tet-on inducible system.
      ). HSA-GFP mice were treated with low-dose doxycycline (0.5 mg/ml doxycycline in drinking water with 2% sucrose) for 5 days to label myonuclei with GFP. HSA-Myc and littermate control mice were treated with doxycycline water overnight, and this water was replaced with unsupplemented water for 12 h prior to being euthanized. All mice were at least 2 months of age at the time of experimentation.
      For Experiments 1 to 4, synergist ablation overload of the plantaris was performed as described (
      • von Walden F.
      • Rea M.
      • Mobley C.B.
      • Fondufe-Mittendorf Y.
      • McCarthy J.J.
      • Peterson C.A.
      • et al.
      The myonuclear DNA methylome in response to an acute hypertrophic stimulus.
      ,
      • Kirby T.J.
      • McCarthy J.J.
      • Peterson C.A.
      • Fry C.S.
      Synergist ablation as a rodent model to study satellite cell dynamics in adult skeletal muscle.
      ). Synergist ablation involves removal of ∼50% of the gastrocnemius–soleus complex while under anesthesia, followed by ambulatory cage behavior for 72 h. Sham surgery (control condition) involved all the steps of synergist ablation but without removal of muscle. For Experiments 1 to 3, mice were injected with EU 5 h prior to being euthanized (
      • Kirby T.J.
      • Patel R.M.
      • McClintock T.S.
      • Dupont-Versteegden E.E.
      • Peterson C.A.
      • McCarthy J.J.
      Myonuclear transcription is responsive to mechanical load and DNA content but uncoupled from cell size during hypertrophy.
      ). Briefly, mice were given an intraperitoneal injection of 2 mg of EU (Jena Biosciences) suspended in sterile PBS. For Experiment 4, myonuclei were isolated via FANS (
      • von Walden F.
      • Rea M.
      • Mobley C.B.
      • Fondufe-Mittendorf Y.
      • McCarthy J.J.
      • Peterson C.A.
      • et al.
      The myonuclear DNA methylome in response to an acute hypertrophic stimulus.
      ). Plantaris muscles were harvested immediately after being euthanized. Muscle was minced and homogenized via Dounce in a sucrose buffer with RNAse inhibitors. After straining through 40 μm filters, the nuclear suspension was pulsed with propidium iodide to label DNA. GFP+/PI+ myonuclei were sorted using FANS into TRIzol LS for RNA isolation.

      RNA isolation and sequencing

      For Experiments 1, 2, 3, and 5, plantaris RNA was isolated using TRI Reagent (Sigma-Aldrich). Tissue was homogenized using beads and the Bullet Blender Tissue Homogenizer (Next Advance) or the Fisher Bead Mill (Fisher). Following homogenization, RNA was isolated via phase separation by addition of bromochloropropane or chloroform and then by centrifugation. The aqueous phase was transferred to a new tube and further processed on columns using the Direct-zol Kit (Zymo Research) (
      • Figueiredo V.C.
      • Wen Y.
      • Alkner B.
      • Fernandez-Gonzalo R.
      • Norrbom J.
      • Vechetti Jr., I.J.
      • et al.
      Genetic and epigenetic regulation of skeletal muscle ribosome biogenesis with exercise.
      ). For Experiment 4, RNA was isolated by Norgen. Experiments 1, 4, and 5 utilized Poly-A enrichment. For Experiments 2 and 3, RNA was depleted of rRNA using the NEBNext rRNA Depletion Kit (New England Biolabs) prior to isolation of EU- and non-EU–labeled RNA. A total of 4 × 1 μg RNA reactions were used per sample for rRNA depletion and pooled for the EU pulldown. The EU- and non-EU–labeled RNA fractions were isolated using the Click-iT Nascent RNA Capture kit (ThermoFisher) per the manufacturers protocol. cDNA libraries were constructed using NEBNext Ultra# II RNA Library Prep kit with NEBNext Multiplex Oligos for Illumina (New England Biolabs). EU pulldowns were unsuccessful for one sham experiment, so sequencing for that group was n = 2. Library preparation for Experiment 4 was low input and utilized the SMARTer pico kit (TaKaRa), as described by the National Genomics Infrastructure at SciLifeLab. RNA for Experiments 1, 2, 3, and 5 were sequenced by Novogene on an Illumina HiSeq using 150 bp paired-end sequencing, as we have done previously (
      • Dungan C.M.
      • Brightwell C.
      • Wen Y.
      • Zdunek C.J.
      • Latham C.
      • Thomas N.T.
      • et al.
      Muscle-specific cellular and molecular adaptations to late-life voluntary concurrent exercise.
      ,
      • Wen Y.
      • Dungan C.M.
      • Mobley C.B.
      • Valentino T.
      • von Walden F.
      • Murach K.A.
      Nucleus type-specific DNA methylomics reveals epigenetic “memory” of prior adaptation in skeletal muscle.
      ). Experiment 4 was sequenced by the SciLifeLab on a NovaSeq 6000 (150 bp paired-end).

      Transcriptomic analyses

      Raw counts from RNA-seq were used as inputs into R (Version 4.1.0) or Partek Flow. Alignment was performed using STAR with mmu39. After filtering low-expressed genes, DESeq2 (Version 1.34.0) was used for normalization and differential analyses of RNA-seq data to identify differentially expressed genes (DEGs) with pairwise comparisons (
      • Love M.I.
      • Huber W.
      • Anders S.
      Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2.
      ). DEGs were identified with a false discovery rate (Benjamini-Hochberg method) adjusted p-value <0.05. DEGs with a log2 fold change (Log2FC) over 1 and adj. p < 0.05 were used for downstream functional analysis in Experiments 1 to 4. A fold-change cut-off was not used for Experiment 5. Kyoto Encyclopedia of Genes and Genomes and Reactome (https://reactome.org/) were utilized for pathway analysis. We utilized clusterProfiler (Version 4.4.1), ReactomePA, ggplot2 (3.15), and ConcensusPathDB (
      • Kamburov A.
      • Herwig R.
      ConsensusPathDB 2022: molecular interactions update as a resource for network biology.
      ) with mouse as the reference organism.

      Digital cell sorting with CIBERSORTx

      CIBERSORTx (https://cibersortx.stanford.edu/) is a machine learning method that enables prediction of cell type proportions from bulk tissue analysis using single cell RNA-seq data (
      • Newman A.M.
      • Steen C.B.
      • Liu C.L.
      • Gentles A.J.
      • Chaudhuri A.A.
      • Scherer F.
      • et al.
      Determining cell type abundance and expression from bulk tissues with digital cytometry.
      ). We used skeletal muscle single-cell data from 10 days muscle regeneration data from Oprescu et al. (
      • Oprescu S.N.
      • Yue F.
      • Qiu J.
      • Brito L.F.
      • Kuang S.
      Temporal dynamics and heterogeneity of cell populations during skeletal muscle regeneration.
      ). The datasets (10X Genomics) were reanalyzed with Seurat, and cell clusters were identified with a resolution of 0.8 (
      • Hao Y.
      • Hao S.
      • Andersen-Nissen E.
      • Mauck III, W.M.
      • Zheng S.
      • Butler A.
      • et al.
      Integrated analysis of multimodal single-cell data.
      ). Normalized gene expression matrices of individual cells were used to create a signature matrix of all cell types using default settings, and cell proportions were predicted by CIBERSORTx with 1000 permutations.

      Transcriptional regulator analysis using Lisa

      Lisa was run according to recommended procedures (
      • Qin Q.
      • Fan J.
      • Zheng R.
      • Wan C.
      • Mei S.
      • Wu Q.
      • et al.
      Lisa: inferring transcriptional regulators through integrative modeling of public chromatin accessibility and ChIP-seq data.
      ). In brief, DEG lists (adj. p < 0.05) from Experiments 4 and 5 were input into the online graphical user interface. Output files were downloaded and the strength of MYC regulation was determined by ranking of regulatory potential in H3k27ac ChIP-seq files. The Cauchy combination p-value test was used to determine overall influence of MYC.

      Data availability

      Raw data are available in Gene Expression Omnibus GEO213406, and all processed data are provided in Supporting information and online webtools.

      Supporting information

      This article contains supporting information.

      Conflict of interest

      Y. W. is the founder of MyoAnalytics LLC. The authors have no other conflicts to declare.

      Acknowledgments

      The TRE-Myc mouse was a generous gift from Dr Andrew McMahon at the University of Southern California. The authors thank Dr Christopher Sundberg of Marquette University for his thoughtful comments on our work, Dr Charlotte Peterson and the University Kentucky Center for Muscle Biology for resources and support, Dr C. Brooks Mobley for assistance with mouse colony management, and Dr Qian (Alvin) Qin of Harvard University for input on Lisa. Figures 1A and 5 were created using BioRender.

      Author contributions

      K. A. M., I. J. V., and F. v. W. conceptualization; K. A. M., N. P. G., J. T. L., J. J. M., I. J. V., and F. v. W. funding acquisition; K. A. M., B. J., V. C. F., Y. W., S. K., S. L., F. M. d. S., I. J. V., and F. v. W. investigation; K. A. M., Z. L., B. J., V. C. F., Y. W., S. K., S. L., F. M. d. S., I. J. V., and F. v. W. formal analysis; K. A. M., N. P. G., J. T. L., J. J. M., I. J. V., and F. v. W. supervision; K. A. M., Z. L., B. J., V. C. F., Y. W., S. K., S. L., F. M. d. S., N. P. G., J. T. L., J. J. M., I. J. V., and F. v. W. writing–review and editing; Z. L. data curation; K. A. M. and Z. L. methodology; K. A. M. writing–original draft.

      Funding and additional information

      This work was supported by NIH R00 AG063994 and startup funds from the University of Arkansas Vice Chancellor for Research and Innovation to K. A. M., support from NIH P20GM104320-07 to I. J. V., and support from AFM-Telethon 23137 , SMDF , Åke Wiberg , Swedish Medical Association , and the Swedish Research Council for Sport Science to F. v. W., Z. L. was supported by the Chinese Scholarship Council . The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

      Supporting information

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