Distinct Transcriptional Networks in Quiescent Myoblasts: A Role for Wnt Signaling in Reversible vs. Irreversible Arrest

<div><p>Most cells in adult mammals are non-dividing: differentiated cells exit the cell cycle permanently, but stem cells exist in a state of reversible arrest called quiescence. In damaged skeletal muscle, quiescent satellite stem cells re-enter the cell cycle, proliferate and subsequently execute divergent programs to regenerate both post-mitotic myofibers and quiescent stem cells. The molecular basis for these alternative programs of arrest is poorly understood. In this study, we used an established myogenic culture model (C2C12 myoblasts) to generate cells in alternative states of arrest and investigate their global transcriptional profiles. Using cDNA microarrays, we compared G₀ myoblasts with post-mitotic myotubes. Our findings define the transcriptional program of quiescent myoblasts in culture and establish that distinct gene expression profiles, especially of tumour suppressor genes and inhibitors of differentiation characterize reversible arrest, distinguishing this state from irreversibly arrested myotubes. We also reveal the existence of a tissue-specific quiescence program by comparing G₀ C2C12 myoblasts to isogenic G₀ fibroblasts (10T1/2). Intriguingly, in myoblasts but not fibroblasts, quiescence is associated with a signature of Wnt pathway genes. We provide evidence that different levels of signaling via the canonical Wnt pathway characterize distinct cellular states (proliferation vs. quiescence vs. differentiation). Moderate induction of Wnt signaling in quiescence is associated with critical properties such as clonogenic self-renewal. Exogenous Wnt treatment subverts the quiescence program and negatively affects clonogenicity. Finally, we identify two new quiescence-induced regulators of canonical Wnt signaling, Rgs2 and Dkk3, whose induction in G₀ is required for clonogenic self-renewal. These results support the concept that active signal-mediated regulation of quiescence contributes to stem cell properties, and have implications for pathological states such as cancer and degenerative disease.</p></div

Chromatin-IP analysis of Myf5 and MyoG promoters in different cellular states.

<p>Antibodies against β-cat or HBP were used to assess the association of these Wnt-regulated transcription factors with chromatin in different cellular states as described in Materials and Methods. Control pulldowns used IgG and all values shown represent fold enrichment of the specific transcription factor after normalization against control IgG values. (<b>A</b>)<b>.</b> The Wnt target transcription factor β-cat associates with the known Wnt-responsive site in Myf5 enhancer preferentially in G₀ (G₀) but shows low enrichment in either proliferating (MB) or differentiating muscle cells (MT) (Blue bars-MB; pink bars-G₀; green bars-MT). This observation is consistent with the hypothesis that Wnt signaling is active in quiescent myoblasts. Comparison of β-cat association on another myogenic promoter (Myogenin promoter) shows greater enrichment in MT. Taken together, these observations suggest that Wnt/β-cat regulates different genes in different cellular states. (<b>B</b>)<b>.</b> ChIP analysis shows that HBP1 (a Wnt-induced repressor) co-associates with the Myf5 enhancer only in G₀ (Blue bars-Mb; pink bars-G₀; green bars-MT) and does not associate with this element in either proliferating or differentiated muscle cells. This observation suggests that fine-tuning of Myf5 expression by both activating and repressive mechanisms may occur in quiescent cells by association of two types of Wnt-responsive transcription factors. Taken together, this observation would account for the absence of induction of Myf5 mRNA in quiescent myoblasts despite the association of the transcriptional activator β-cat.</p

Selected classes of genes up-regulated in quiescence.

<p>Selected classes of genes up-regulated in quiescence.</p

Moderate induction of Wnt signaling in two models of quiescence.

<p>(<b>A</b>) Wnt signaling revealed by TCF-dependent luciferase activity in stabily transfected TOPflash myoblast clone TFC1. Asynchronously proliferating myoblasts (As), suspension-arrested quiescent myoblasts (G₀), suspension-synchronized myoblasts after re-activatation for 2–24 hrs (R2–R24), or myoblasts induced to differentiate for 72 hrs (MT). Transcriptional activity of the Wnt responsive TCF reporter is induced as MB enter quiescence and rapidly suppressed upon re-activation into the cell cycle. TCF-dependent luciferase activity (rlu/µg protein) is moderately induced in G₀ arrest but strongly induced in differentiation-associated arrest (MT, inset). Data represents mean +SE from atleast 3 independent experiments. (<b>B</b>) Conditioned medium (CM) from G₀ MB cultures contains more Wnt/TCF reporter-inducing activity than CM from proliferating or differentiated cultures. TOPflash reporter cells were exposed to CM derived from G₀ cultures (G₀-CM), or proliferating cultures (Mb-CM), or differentiated cultures (Mt-CM). Fresh growth medium (GM) and differentiation medium (DM) were used as controls. Data represents mean +SE from 3 independent experiments. (<b>C</b>) Secreted Wnt agonist R-spondin expression is strongly induced in G₀. RNA isolated from growing (Mb), arrested (G₀) and differentiated (Mt) muscle cells was analysed by Q-RT-PCR (n = 3). (<b>D–F</b>) Wnt signaling is induced in an independent culture model of G₀ MB (reserve cells). (D) Phase contrast photographs of quiescent mononucleated undifferentiated reserve cells (RC) after differential trypsinization specifically removed myotubes (MT) in 5-day differentiated TFC1cultures. (E) RC isolated from away from MT do not induce MyoG (QRT-PCR analysis), confirming their undifferentiated state. (F) TOPflash TCF reporter activity is induced in purified quiescent reserve cells (G₀rc) and its decline in reserve cells that have been reactivated by the addition of GM for 2–24 hrs (R2–R24). Inset shows TOPflash activity in G₀rc compared to purified MT cultures depleted of reserve cells [MT(-rc)]. Data represents mean +SE from 3 independent experiments.</p

Microarray analysis reveals distinct genetic programs in reversible and irreversible arrest.

<p>(<b>A</b>) Schematic of microarray loop experiment. RNA isolated from G₀ MB was analyzed by 2-color competitive hybridization to NIA15K mouse cDNA arrays. Pair-wise analysis: G₀ MB vs. either proliferating MB, differentiated MT or G₀ FB. In each pair, genes consistently up- or down-regulated (>1.6 fold, normalized log ratio (NLR) 0.67, false discovery rate 4%) were selected for further analysis. The numbers of genes regulated between each pair of samples are shown: induced (red), suppressed (green). Expression of key regulators characteristic of the different samples is indicated, confirming the identity of each cellular state. (B) Hierarchical clustering (MEV software, TIGR) shows the relatedness of the myoblast and fibroblast sample pairs drawn from independent biological experiments including a dye reversal test. Based on the total transcriptional response, genes up-regulated in G₀ MB fall into 3 broad categories-genes specifically induced in G₀ MB (the largest number), genes induced in both G₀ MB and MT but down-regulated in G₀ FB, and genes induced in G₀ MB and G₀ FB but not in MT. (<b>C</b>) In muscle cells, common features of two types of cell cycle exit (reversible vs irreversible) are overshadowed by differences. Venn diagram reveals minimal overlap between the three myogenic sample pairs (MB vs G₀ MB, MB vs MT, G₀ MB vs MT), and show that distinct transcriptional programs characterize reversible arrest vs. differentiation-coupled irreversible arrest. (D) Validation of microarray analysis using Northern blots. RNA samples used for microarray analysis were also interrogated with ³²P-labelled probes representing genes selected to test the range of quantitative changes in expression (i) genes that showed no change (log ratio of 0), (ii) genes that were down-regulated in G₀ (negative log ratio) or (iii) genes that were up regulated in G₀ (positive log ratio). Numbers on the left of blots represent log ratios derived from G₀ vs. asynchronous myoblast array. Gene symbols & transcript sizes are shown on the right of each blot. 28S rRNA was used as a loading control. Rfg, a gene expressed equally in all conditions. Cofilin, a regulator of microfilament dynamics - down-regulated in both G₀ Mb and in Mt. Of the 12 transcripts selected from the array as enriched in G₀, all 12 showed G0 induced expression. Of these, 8 (FGFR1, BMP1, GSTa4, TM7SF1, IGFBP5, TIMP3, Cathepsin L, Col3A1) were specifically induced in G₀ MB, and not in differentiated MT. The other 4 genes, IGF2, Rho GAP, LAMP2 and Map1LC3b were induced in both G₀ MB and MT. Histone 2B and MyoD transcripts (not derived from array analysis, marked by *) were detected to confirm the cellular state. E. Functional diversity of genes specifically enriched in G₀ MB indicates a signature of <b><i>recycling</i></b> (autophagy, ubiquitin pathway), <b><i>resistance</i></b> (anti-apoptosis) and <b><i>repair</i></b> (DNA repair). Gene ontology classification of 663 annotated gene from the list of genes upregulated in quiescent (G0) MB suggests that enhanced survival mechanisms may compensate for depressed metabolic functions in quiescence (see Supplementary Information for altered cell cycle and metabolic pathways). In addition, there is evidence of altered expression of genome reprogramming factors (chromatin and transcriptional regulators), signaling (growth factors, GF receptors, signaling adaptors) and developmental regulators [Wnt (indicated by arrow), Hh, BMP, Notch pathways)]. The surprising increase in induction of Wnt genes in quiescence was further investigated.</p

C2C12 myoblasts enter alternate states of arrest.

<p>(<b>A</b>) Asynchronous proliferating myoblasts (MB) were held in suspension in mitogen-rich media to induce quiescence (arrest in an undifferentiated mono-nucleated state; G₀ MB), or shifted to mitogen-poor medium for 96 hrs to induce differentiation (arrest coupled with fusion into multinucleated myotubes; MT). Actin staining (Oregon Green-Phalloidin, top panel) reveals the distinct morphology of these 3 states. Nuclei are detected with Hoechst 33352. DNA synthesis was analyzed by detection of BrdU incorporated in a 15-minute pulse (middle panel)- ∼40% of cycling MB are labeled, while less than 1% of G₀ MB synthesize DNA. In differentiated cultures (MT), only residual mono-nucleated cycling cells (5–10%) incorporate label whereas myotube nuclei do not. Expression of determination factor MyoD can be detected in ∼50% of proliferating MB, is lost in G₀ but sustained in MT (lower panel). (<b>B</b>) FACS analysis of DNA content of cycling (Asynchronous MB) and 48-hr suspension-cultured populations confirms that >97% of cells in suspension (G₀ MB) possess a 2C DNA content while >40% of cells in an asynchronous culture have replicated their genomes (representative graphs from 4 independent experiments). (<b>C</b>) Muscle transcription factor expression distinguishes alternate states of arrest. Q-RT-PCR analysis of specification/survival factor Pax7, determination factor MyoD, and early differentiation marker Myogenin (MyoG) in asynchronous proliferating myoblasts (As), suspension-arrested MB (G₀) and differentiated MT (MT). Values represent normalized fold differences between GAPDH (control) mRNA and myogenic mRNAs in each sample (n = 3). (<b>D</b>) Cell cycle inhibitor expression distinguishes alternate states of arrest-G₀ MB and MT express different combinations of cyclin-dependent kinase inhibitors p21/p27 and Rb-related p130. Immuno-detection of p21, p130 or p27 in MT and G₀; p21 (green) co-localizes with MyoG (red) in all nuclei of differentiated MT but neither factor is expressed in G₀; p130 is specific to G₀ and p27 is expressed in both G₀ and MT. Data depicted is representative of three independent experiments. (<b>E</b>) G₀ arrest is reversible. DNA synthesis in asynchronous MB (Mb), 48-hour suspension cultures (G₀) and G₀ MB replated for 6 or 24 hours (R6, R24). BrdU detected as in Fig. 1A. Values represent mean<u>+</u>SEM (n = 3). (<b>F</b>) MyoD expression is suppressed in G₀ and restored during early reactivation. Asynchronous cultures (A) were arrested in G₀ (48 hrs) and reactivated by replating for 2 to 24 hrs (R2–R24). MyoD expression detected as in Fig. 1A. Values represent mean<u>+</u>SEM (n = 3).</p

Exogenous Wnt treatment compromises a G0-induced program that promotes clonogenic potential.

<p>(A) Wnt3A treatment of MB reduces clonogenic potential. Colony formation was measured after 48 hrs in control culture conditions (either in proliferating conditions-Mb, or in suspension culture-G₀), or in the presence of 50 ng/ml of rWnt3A. Cloning efficiency (a measure of self-renewal) was strongly reduced by Wnt3A supplementation and restored by simultaneous addition of 50ng/ml sFRP2. Values represent the mean±SEM from three independent experiments, <i>p</i><0.05 (denoted by asterisk *). (B) Knockdown of Rgs2 and Dkk3 transcripts using siRNAs. siRNAs were designed against the putative Wnt regulators Rgs2, Dkk3 or an irrelevant gene (GAPDH) or a control scrambled siRNA sequence and transfected into C2C12 myoblasts along with a GFP plasmid. GFP<b>⁺</b> transfected cells were enriched by FACS, RNA isolated and analysed by Q-RT-PCR and the relative mRNA levels calculated. In each pair, the mRNA level is depicted of cells transfected with scrambled siRNA (blue bars) and cells transfected with the targeting siRNA (pink bars). Values represent the mean and SEM of 3 independent experiments. In each case, modest but reproducible reduction of the target transcript level is observed. (C) Reduction of Rgs2 and Dkk3 protein expression by siRNA-mediated knockdown. Western blot analysis of total protein isolated from control and knockdown C2C12 muscle cells probed with antibodies against Rgs2 (top) and Dkk3 (bottom). GAPDH protein levels indicate equal loading. Data depicted is representative of 3 independent experiments. (D) Rgs2 and Dkk3 expression is necessary for Wnt signaling. Knockdown of either Rgs2 or Dkk3 in growing or quiescent MB leads to suppression of TOPflash activity. Cells were treated and enriched as described in (B) and luciferase activity measured. Despite modest reduction of protein levels, strong reduction in TOPflash activity are seen, indicating a critical role for Rgs2 and Dkk3 in Wnt-βcat signaling. Values represent the mean and SEM of 3 independent experiments. (E,F) Knockdown cells (‘Rgs sh’ and ‘Dkk sh’) were enriched as described in (B) cultured in quiescence-inducing conditions, recovered from suspension culture and plated at clonogenic density for assessment of self-renewal (colony formation). Controls include untransfected cells (‘UT’) and control shRNA transfected cells (‘Con sh’). Typical plates with colony assays are shown in (E) and data are quantified as CFU (colony forming units) in (F). Values represent the mean and SEM of 3 independent experiments.</p

Wnt pathway genes are enriched in quiescent myoblasts.

<p>(A) Microarray analysis identifies a Wnt signature in quiescence. Cluster analysis of the 4 sample pairs analysed in Fig. 2 reveals induction of Wnt pathway genes in G₀ MB-many of these genes are also induced in G₀ FB but not in MT, indicating greater transcriptomal relatedness between the quiescent state of different cell types (G₀ FB vs G₀ MB) than between reversibly and irreversibly arrested cells of the same cell type (G₀ MB vs MT). (B) Schematic of Wnt signaling depicting genes induced in G₀ Mb. Genes identified as enriched in G₀ by microarray analysis are shown in black, Wnt2 and R-spondin whose expression was directly tested are in grey. Dvl, an important Wnt signaling node is depicted for clarity (was not recovered in array). Note that components of both canonical and planar cell polarity (PCP) pathways were induced as well as some components that cross-talk with Rho/Jnk pathways. (<b>C</b>) Northern blot analysis of selected Wnt-related genes on RNA isolated from proliferating (MB), quiescent (G₀ MB) and differentiated cells (MT). Numbers to the left of blots represent log ratios derived from comparison of G₀ MB to asynchronous MB. Gene symbols and mRNA sizes are shown on the right. All genes tested in this independent assay show expression patterns that support their recovery in the microarray experiment. (<b>D</b>) Q-RT-PCR analysis of two putative Wnt pathway genes Rgs2 (top graph) and Dkk3 (bottom graph) in proliferating (A), quiescent (G₀) and differentiated (MT) cells. Values represent normalized fold differences between GAPDH (control) mRNA and Rgs2/Dkk3 mRNAs in each sample calculated from cycle thresholds [2^{−(−ΔΔCt)}] (n = 3, p<0.05). (<b>E</b>) Wnt pathway genes are rapidly induced in G₀, rapidly suppressed during G1. Northern analysis of selected Wnt pathway genes during a time course of entry and exit from quiescence. Asynchronous MB (MB), cells in suspension culture for 6, 12, 24 and 48 hrs (S6, S12, S24, S48) or reactivated into the cell cycle for 20 min to 24 hrs (R20’, R1–R24). The suspended population is completely arrested by 48 hours (S48); thus ‘S48’ time point corresponds to ‘G₀ MB’ depicted in all other figures. S-phase specific (replication-dependent) histone H2B expression is suppressed as cells arrest in suspension culture, reactivated at 24 hrs after replating when cells re-enter DNA synthesis. L7 and 28S are loading controls. Wnt pathway regulatory genes- LEF1, Groucho, Dab2 as well as putative Wnt pathway genes Dkk3, Rgs2 are more tightly quiescence-dependent (expression lost by 2–6 hrs after reactivation) than Wnt target gene Ecm1 (expression down-regulated but still detected at 24 hrs after reactivation). Arrow shows a smaller LEF1 transcript seen only in G₀.</p

Enhancement of Wnt signaling subverts the quiescence program.

<p>(A) Exposure of adherent MB to rWnt3a (50 ng/ml) leads to β-cat nuclear localization, TOPflash activation and suppression of MyoD protein as compared to control cells. (B) rWnt 3a (50 ng/ml) does not enhance proliferation (BrdU incorporated in a 30′ pulse) in muscle cells: Asynchronous MB, G₀ MB, MB reactivated after synchronization in (R18) or differentiated myotubes (MT) [Note: all BrdU+ nuclei in myotube cultures were in residual mono-nucleated myobalsts]. Values represent the mean±SEM from three independent experiments. (C) Exogenous Wnt3a alters the quiescence program: Q-RTPCR analysis of control (blue bars) and Wnt-treated (pink bars) cells held in suspension for 48 hrs shows repression of MyoD and MyoG but induction of Myf5, indicating differential response of MRFs; repression of p21 and induction of CyclinD1 collectively suggesting a shift to a proliferative gene expression program; and finally, repression of quiescence-induced genes Rgs2 and Dkk3, consistent with this shift. Values represent the mean±SEM from three independent experiments. (D) Context-dependent response to Wnt enhancement. Cells in three different states (MB, G₀ or MT) were treated for 48 hours with 50ng/ml of rWnt3a. Of the MRFs, Myf5 mRNA is only induced by Wnt3a if the target cells are in G₀. Values represent the mean±SEM from three independent experiments.</p

Expression profile of Rgs2 and Dkk3 in different cell states.

<p>(<b>A</b>) Quiescence-induced genes Rgs2 and Dkk3 are not expressed throughout myogenic differentiation, confirming the distinct induction pattern in reversible arrest and not in irreversible arrest. Northern analysis of growing MB (Mb) shifted to DM for 12–96 hrs. G₀ Mb are shown for comparison. (<b>B</b>) Quiescence-induced expression of Rgs2 and Dkk3 is cell-type specific – these putative Wnt pathway genes are differentially expressed in MB and FB. Northern analysis of growing C3H10T1/2 FB (Fb), suspension-arrested FB (S48) and G₀ FB reactivated for 20 minutes to 24 hrs. Myoblast samples are shown for comparison. Rgs2 transcript is mildly induced when C3H10T1/2 FB exit from proliferation into G₀ [compare with strong G₀ induction in C2C12], but Dkk3 mRNA is not detected in FB, suggesting muscle-specific activation restricted to the quiescent state. (C,D) Induction of Rgs2 and Dkk3 protein expression in quiescent myoblasts. Western blot analysis of total protein isolated from growing (MB), quiescent (G₀) and differentiated (MT) C2C12 muscle cells and probed with antibodies against Rgs2 (C) and Dkk3 (D). In both cases the induction of protein levels is modest compared to the strong induction of the respective transcripts, potentially reflecting the strong translational repression typical of G₀. Data depicted are representative of 3 independent experiments.</p