Stem cells undergo a shift in metabolic substrate utilization during specification and/or differentiation, a process that has been termed metabolic reprogramming. Here, we report that during the transition from quiescence to proliferation, skeletal muscle stem cells experience a metabolic switch from fatty acid oxidation to glycolysis. This reprogramming of cellular metabolism decreases intracellular NAD + levels and the activity of the histone deacetylase SIRT1, leading to elevated H4K16 acetylation and activation of muscle gene transcription. Selective genetic ablation of the SIRT1 deacetylase domain in skeletal muscle results in increased H4K16 acetylation and deregulated activation of the myogenic program in SCs. Moreover, mice with muscle-specific inactivation of the SIRT1 deacetylase domain display reduced myofiber size, impaired muscle regeneration, and derepression of muscle developmental genes. Overall, these findings reveal how metabolic cues can be mechanistically translated into epigenetic modifications that regulate skeletal muscle stem cell biology.

Satellite cells (SCs) are skeletal muscle stem cells required for muscle growth and tissue repair (). Following intense proliferation associated with mouse postnatal muscle growth, SCs enter a quiescent state, representing 3%–5% of the total number of adult muscle fiber nuclei (). In response to muscle injury, the niche is remodeled and quiescent SCs enter the cell cycle (become activated). Activated SCs are characterized by the presence of the muscle-specific transcription factor MyoD, and give rise to committed proliferating muscle precursors which, upon expression of the myogenic transcription factor Myogenin, differentiate and fuse to repair damaged muscles (). Due to changes in requirements placed on SCs during the transition from quiescence to activation, significant differences in the underlying metabolism of these cellular states are likely to occur. Here we describe a metabolic shift from fatty acid (FA) and pyruvate oxidation in quiescent SCs to increased glycolysis and glutaminolysis during SC activation and proliferation. In addition, we document that this process of SC metabolic reprogramming is associated with a decrease in the intracellular NAD/NADH ratio, reduced SIRT1-mediated deacetylation of H4K16ac, and activation of the myogenic program. SCs derived from mice with muscle-specific inactivation of the Sirt1 deacetylase domain (Sirt1mice) display increased H4K16ac and deregulated activation of the myogenic program. Finally, Sirt1mice have reduced myofiber size, exhibit impaired muscle regeneration, and reveal a derepression of several muscle developmental genes.

The enzymatic activity of sirtuin 1 (SIRT1), a member of the class III deacetylase family (), is regulated by the free concentration of the intermediate metabolite NAD). Whereas a plethora of nonhistone proteins are deacetylated by SIRT1 (), acetylated lysine 16 of histone H4 (H4K16ac) serves as a preferred SIRT1 histone substrate (). Even though regulation of sirtuin enzymology is energy demanding and complex (), it establishes, in principle, a rapid and finely tunable biochemical system through which changes in metabolism can be effectively converted into distinct epigenetic states and gene expression patterns.

Cellular energy is generated via oxidative-phosphorylation (OXPHOS) in the mitochondria and glycolysis in the cytoplasm. In addition to providing a steady supply of energy, the metabolic state of the cell can influence the epigenome and alter gene expression. This flow of information is afforded by intermediate metabolites that directly or indirectly affect the activity of chromatin-modifying enzymes involved in regulating chromatin dynamics and transcription (). Cellular substrate and oxygen availability, as well as energy demand, determine which metabolic pathway is used to generate ATP. Under reduced oxygen tension, ATP is generated via anaerobic glycolysis, whereas in aerobic conditions ATP is produced mainly via OXPHOS, a process involving the breakdown of substrates to acetyl-CoA, and leading to the production of the reduced form of nicotinamide adenine dinucleotide (NAD) NADH via the tricarboxylic cycle (TCA). Compared to OXPHOS, glycolysis is an inefficient method to generate ATP. However, it provides a number of important advantages for cells, including the ability to rapidly generate ATP in response to acute changes in energy demand, as well as generating the necessary glycolytic intermediates for the biosynthesis of new macromolecules essential for proliferating cells ().

In response to injury, quiescent SCs become activated, enter mitosis, giving rise to myogenic progenitor cells, which ultimately differentiate to restore damaged muscles (). To evaluate whether muscle regeneration was altered in Sirt1mice, muscle damage was induced by injecting the tibialis anterior (TA) muscles of 2-month-old WT or Sirt1mice with the myogenic agent cardiotoxin (CTX). Seven days after CTX injection, regenerating muscles of Sirt1mice were composed of myofibers with smaller CSA ( Figures 7 H and 7I), and reduced Pax7 protein ( Figure 7 J, both at 7 and 14 days after CTX injection), compared to littermate controls. Overall, these findings indicate that SIRT1 is required for appropriate postnatal muscle growth and adult muscle regeneration.

To determine the effects of Sirt1 ablation in skeletal muscle, we investigated phenotypic changes in Sirt1mice during both development and regeneration. Sirt1mice were initially smaller than littermate controls ( Figure 7 A) and had reduced Pax7 gene and protein expression ( Figures 7 B and 7C). In addition, at postnatal day 9 (P9), skeletal muscles from Sirt1mice exhibited smaller fiber cross-sectional areas (CSA, Figures 7 D and 7E). By day P14, the body mass of Sirt1mice became indistinguishable from littermate controls (data not shown). Interestingly, it is approximately at P14 that SCs gradually start reducing their proliferation to enter quiescence at P21 (). A microarray assay of RNA derived from the gastrocnemius hindlimb muscles revealed an enrichment of 219 and a reduction of 191 transcripts in the muscles of Sirt1compared to WT mice ( Figure 7 F and Table S5 ). Of the upregulated transcripts in Sirt1muscle, 28 (corresponding to 12% of the total transcripts) were also increased in Sirt1SCs induced to differentiate ( Figure 7 G and Table S5 ). A GO analysis of the transcripts upregulated in both Sirt1muscle and Sirt1SCs returned biological terms related to skeletal muscle development (“myofibril,” “sarcomere,” and “striated muscle development,” Figure 7 H). The most highly upregulated transcript in Sirt1muscles corresponded to the embryonic myosin heavy chain isoform Myh3 (5.8-fold increase, Table S5 ), which is physiologically expressed in fetal muscle and repressed in the adult (). In addition to Myh3, several other transcripts upregulated in both Sirt1muscles and Sirt1SCs induced to differentiate are known to be preferentially expressed during muscle development and/or in regenerating muscles (highlighted in Figure 7 G, Table S5 ).

(J) Quantification of skeletal muscle fiber CSA from adult WT and Sirt1 mKO mice regenerating muscles (7 days after CTX, >1,000 fibers analyzed/muscle). Results are presented as a box-and-whisker plot. (K) Pax7 protein levels in uninjured, and 7 and 14 days regenerating muscles of WT and Sirt1 mKO mice. Data are presented as mean ± SEM, ∗ p < 0.05; ∗∗ p < 0.01.

(G) Heatmap of the microarray results (Log 2 [signal intensity]) for genes upregulated in both Sirt1 mKO SCs induced to differentiate and Sirt1 mKO skeletal muscle, with developmentally regulated muscle genes highlighted in yellow.

(F) Heatmap of the microarray results (Log 2 [signal intensity]) for genes upregulated in Sirt1 mKO skeletal muscle. Each gene listed has a mean fold change of >1.5 and p < 0.05 (n = 3 samples/group).

(B and C) Pax7 mRNA (left) and protein expression (right) were evaluated in the skeletal muscles of P9 WT and Sirt1 mKO mice.

To further examine the link between metabolism, H4K16ac and SC activation, we used isolated single fibers from WT and Sirt1mice and incubated them for 20 hr in either glucose- (25 mM) or galactose- (10 mM) based growth media ( Figure S7 ). Similarly to what observed in C2C12 cells ( Figure 3 ), SCs from WT mice incubated in galactose-supplemented media exhibited a reduction in global H4K16ac and MyoD expression, compared to SCs incubated in glucose media ( Figures S7 A and S7C). In contrast, SCs from Sirt1mice did not exhibit any appreciable difference in global H4K16ac or MyoD expression in either glucose or galactose media ( Figures S7 B and S7D). Overall, these results provide evidence for a direct link between a change in metabolism and SC activation and support a role for SIRT1 in this process, downstream of the change in metabolism.

To directly analyze a link between SIRT1, H4K16ac and gene expression, we performed SIRT1 ChIP-seq in FI and Cul SCs ( Table S4 ). Examples of selected genes are illustrated in genome browser screenviews for SIRT1 ChIP-seq, H4K16ac ChIP-seq as well as RNA-seq traces ( Figures 6 F and 6G; Figures S4 and S5 ). SIRT1 was enriched at the Mylk2, H19, and Myog loci in FI but absent in Cul SCs, its enrichment negatively correlating with H4K16ac and gene transcription ( Figures 6 F and 6G). H4K16 acetylation and SIRT1 enrichment at the Mylk2 and Myog were validated using ChIP-qPCR ( Figure S6 ). These findings are in agreement with our previous results indicating a repressive role of SIRT1 for Myog expression (). Overall, the results described in this paragraph indicate that while SIRT1 regulates the H4K16ac status at several thousand loci, its deletion only permits the expression of a few hundred genes.

Of all histone lysines, acetylation of H4K16 profoundly alters chromatin structure by disrupting formation of the 30 nm chromatin fibers and preventing cross-fiber formation (). Accordingly, subtle enrichment of H4K16ac significantly and positively affects transcription (). To correlate transcription and H4K16ac enrichment, ∼1 × 10SCs derived from seven to eight WT or Sirt1mice were used for chromatin immunoprecipitation sequencing (ChIP-seq) after immunoprecipitation with an H4K16ac antibody. H4K16 acetylation was observed to predominantly occur at and around the transcriptional start site (TSS) and was higher in Cul SCs compared to FI SCs ( Figure 6 A). Moreover, there was a positive correlation between H4K16ac and gene expression, so that the genes with the highest expression were also the most H4K16 acetylated in both Cul SCs ( Figure 6 B) and FI SCs ( Figure S4 A).We addressed the genome-wide role of SIRT1 on H4K16ac and gene expression by correlating H4K16ac ChIP-seq and RNA-seq datasets from WT and Sirt1SCs. Global H4K16ac at the 253 upregulated genes in Cul Sirt1SCs was already increased in FI Sirt1SCs ( Figure 6 C). Similarly, H4K16ac at the 757 genes whose transcription was increased in Sirt1SCs induced to differentiate was also increased in FI Sirt1SCs, being positioned at an intermediate H4K16ac level between that observed in FI and Cul WT SCs ( Figure 6 D). Of the 287 genes upregulated in FI Sirt1SCs, one-third (88/287, 30%, Table S3 ) displayed significantly increased H4K16ac ( Figure 6 E). However, the vast majority of H4K16ac was found to occur at genes whose transcription was not concomitantly increased ( Figure 6 E). In Cul Sirt1SCs, 85 of the 253 upregulated genes (33%, Table S3 ) had increased H4K16ac ( Figure 6 E), and 275 out of the 757 upregulated genes (36%, Table S3 ) in Sirt1SCs were induced to differentiate and manifested increased H4K16ac when assayed in FI Sirt1SCs ( Figure 6 E).

(G) ChIP-seq and RNA-seq profiles of the Myog gene. Bottom to top: SIRT1 ChIP-seq profile in FI and Cul WT SCs (blue signals); H4K16ac profile in WT and Sirt1 mKO FI SCs, and WT Cul SCs (magenta signals); Myog mRNA expression profile in FI WT and Sirt1 mKO SCs (black signals).

(F) ChIP-seq and RNA-seq profiles of the Mylk2 gene. Bottom to top: SIRT1 ChIP-seq profile in FI and Cul WT SCs (blue signals); H4K16ac profile in FI WT and SIRT11 mKO SCs, and WT Cul SCs (magenta signals); Mylk2 mRNA expression profile in FI WT and Sirt1 mKO SCs (black signals).

(F) RNA-seq profiles of the Myog gene in WT (bottom) and Sirt1 mKO SCs (top) induced to differentiate. RNA-seq experiments were done with either three (FI SCs) or two (Cul and Differentiating SCs) biological replicates.

To thoroughly investigate the global impact of inactivating the SIRT1 deacetylase activity on the transcriptome, we performed RNA-seq on SCs derived from WT or Sirt1 mKO mice at several stages of myogenesis, including quiescence (FI), activation (Cul), and early differentiation (Diff).

To investigate whether SIRT1 influences SC biology in vivo, we generated a Pax7-specific Sirt1 knockout mouse (Sirt1) by crossing mice containing the Cre-recombinase under the control of the Pax7 locus (Pax7-Cre,) with mice containing a modified Sirt1 gene where exon 4 (located within the catalytic deacetylase domain) is flanked by loxP sites (SIRT1 flox mice,). The floxed alleles were detected in genomic DNA isolated from skeletal muscle of Cre-negative (WT) mice, while the Δalleles were detected in the presence of Cre-recombinase in Sirt1mice ( Figure 4 A, top). A band corresponding to the SIRT1 protein ( Figure 4 A bottom, arrow) was lost and, concomitantly, a slightly faster migrating band corresponding to SIRT1Δappeared ( Figure 4 A bottom, arrowhead), specifically in the skeletal muscles of Sirt1mice, compared with WT. Freshly isolated single EDL muscle fibers were obtained from WT and Sirt1mice, stained with Pax7 to identify SCs and costained with H4K16ac-specific antibodies. Whereas WT myofibers had a tight distribution of SCs expressing low levels of global H4K16ac, SCs on Sirt1myofibers had a wider distribution of H4K16ac levels, such that there was a 2-fold increase in the median level of global SC H4K16ac ( Figures 4 B and 4C). These results indicate that, in SCs, SIRT1 is required to maintain H4K16 in a deacetylated state. Moreover, the percentage of Pax7/MyoDSCs on Sirt1fibers was increased ( Figures 4 D, 4E, and S3 D). In light of these findings, we asked whether Sirt1SCs may also undergo premature differentiation and, if so, whether this is a cell-autonomous phenomenon. FACS-isolated SCs were cultured for 48 hr in growth conditions, or induced to differentiate for 24 hr. Compared to WT cells, Sirt1SCs mice exhibited an overt spindle-like, elongated morphology, indicative of early differentiation ( Figure 4 F).

(F) DIC images of SCs isolated from WT and Sirt1 mKO mice during proliferation in growth media (GM, 48 hr), or early differentiation in differentiation media (24 hr DM). Note the overt spindle-like, elongated morphology of SCs from Sirt1 mKO mice indicating premature differentiation.

(B and C) In Sirt1 mKO mice, quiescent SCs (identified as Pax7 + ) exhibited a 2-fold increase in global H4K16ac, compared to WT mice, as determined via relative fluorescence (RFU) in SCs labeled for H4K16ac (n = 2 mice, >50 fibers/time point). White scale bar represents 50 μm; inset is magnified by a magnitude of four. Results are presented as box-and-whisker plots (fifth to 95 th percentiles), with a significant difference indicated when the median ± 95% CI does not overlap.

(A) Skeletal muscle from WT mice demonstrated the presence of the Sirt1 floxed allele (fl, top) and detectable levels of SIRT1 protein (arrow, bottom), whereas Sirt1 mKO muscle contained the Sirt1 Δex4 allele (Δ, top) and ablation of SIRT1 protein. A small level of SIRT1 Δex4 protein was detectable in the skeletal muscle of Sirt1 mKO mice (arrowhead, bottom).

The results presented in the preceding paragraphs indicate that a switch from oxidative phosphorylation to glycolysis accompanying SC activation has the potential to modulate the deacetylase activity of SIRT1 via decreased NADavailability. To directly test this possibility, we transduced C2C12 cells with a SIRT1 shRNA retrovirus. The reduced SIRT1 protein levels in these cells were associated with increased MyoD expression, elevated global H4K16ac, and a reduced rate of proliferation ( Figures S3 A and S3B). Consistent with a role of SIRT1 in mediating these phenomena, the addition of increasing concentrations of the SIRT1 inhibitor nicotinamide (NAM) to proliferating C2C12 cells similarly led to an increase in global H4K16ac and MyoD expression ( Figure S3 C). C2C12 cells cultured in galactose (Gal) exhibited a progressive decline in the expression of MyoD protein levels starting at 3 hr and further decreasing at 6 hr ( Figure 3 F, compare lanes 5–6, 9–10). Importantly, the galactose-induced decline in MyoD protein was abrogated by SIRT1 shRNA ( Figure 3 F, compare lanes 6 and 8, and 10 and 12), indicating a role for SIRT1 at the nexus between changes in cellular metabolism and expression of the master regulator MyoD.

In contrast to glucose, for galactose to be used as an energy substrate it must first be converted to glucose-6-phosphate in an ATP-consuming reaction, leading to no net gain in ATP via glycolysis (). Cells incubated with galactose must therefore rely on OXPHOS to generate ATP ( Figure 3 A). To test whether metabolic reprogramming affects the NAD/NADH ratio, H4K16ac, and muscle gene expression, we used myogenic C2C12 cells. Although C2C12 cells do not recapitulate all the features of SCs, they nonetheless retain some shared characteristics. C2C12 cells incubated for 3 hr in growth media containing galactose (10 mM) exhibited significantly reduced glycolysis (as indicated via decreased ECAR) and increased oxygen consumption (measured as OCR) compared to cells incubated in growth media containing glucose (25 mM; Figure 3 B). Culturing C2C12 cells in growth media containing galactose resulted in an increase of NAD, with a concomitant decrease in NADH, and a net 2.5-fold increase in the NAD/NADH ratio of cells ( Figures 3 C and 3D). We confirmed that incubation with galactose did not impair ATP generation (and activate starvation pathways), and in fact resulted in elevated levels of ATP ( Figure 3 E).

(F) Incubating C2C12 cells with galactose instead of glucose for 3 and 6 hr results in a decrease in MyoD in WT (compare lanes 5–6, and 9–10), but not SIRT1 shRNA C2C12 cells (compare lanes 7–8, and 11–12). Data are presented as mean ± SEM. ∗ p < 0.05.

(C and D) Culturing C2C12 cells in galactose-based growth media increases the amount of NAD + , at the expense of NADH such that there was a 3-fold increase in the NAD/NADH ratio (n = 3).

(A) Schematic depicting how glucose is used to preferentially generate ATP via glycolysis. Replacing glucose with galactose forces cells to shift to predominantly use OXPHOS for the generation of ATP.

An increased reliance upon oxidative metabolism has been proposed to lead to elevated SIRT1 deacetylase activity, either via changes in absolute SIRT1 levels or changes in the NAD/NADH ratio ( Figure 2 A). The shift away from FA oxidation in Cul SCs was associated with a 1.5-fold decrease in the expression of both Sirt1 and the NAD-generating enzyme Nampt, as measured by qPCR ( Figure 2 B). However, despite this decline, SIRT1 protein was readily detected and predominantly localized to the nucleus of both FI and Cul SCs ( Figure 2 C). Global H4K16ac, a substrate for SIRT1-mediated deacetylation (), was examined in SCs associated with freshly isolated single extensor digitorum longus (EDL) muscle fibers, and fibers that had been cultured in growth media for 3 or 20 hr ( Figures 2 D and 2E). Single myofiber isolation permits SCs to remain attached to the myofiber and, thus, to maintain their position in the physiological niche between the basal lamina and the sarcolemma (). Myofiber-associated SCs cultured in growth media for 3 or 20 hr displayed a 7- and 16-fold increase in the median level of H4K16ac, compared to SCs on freshly isolated myofibers ( Figures 2 D and 2E), respectively. Thus, while SIRT1 protein was observed in the nucleus of Cul SCs, global H4K16ac was strongly elevated during the early stages of culture, suggesting a possible regulation of SIRT1 activity. Because SIRT1 deacetylase activity is dependent upon NADsupply (), we used FACS-isolated SCs to examine NADlevels. Whereas NADH was undetectable in both FI and Cul SCs, the amount of total NADwas 10-fold higher in FI than Cul SCs ( Figure 2 F). Therefore, H4K16 deacetylation, a proxy for SIRT1 deacetylase activity, and NADwere elevated in FI SCs, and reduced in Cul SCs.

(E) Quantification of relative fluorescence (RFU) in SCs labeled for H4K16ac (n = 2 mice, >50 fibers/time point). Results are presented as box-and-whisker plots, with a significant difference indicated when the median ± 95% CI does not overlap.

Altogether, these results indicate that Cul SCs undergo a shift away from FA oxidation, toward glycolysis, glutaminolysis, and activation of the PPP for macromolecule biosynthesis.

Whereas these results are suggestive of a shift in substrate uptake and utilization during the switch from quiescent to proliferating SCs, to directly confirm that glycolysis was elevated in Cul SCs, we used the Seahorse extracellular flux bioanalyzer to examine both the basal oxygen consumption rate (OCR, an indicator of mitochondrial oxidative activity) and the basal extracellular acidification rate (ECAR, a marker of glycolysis). We observed a 2.5-fold increase in the ECAR (i.e., increased glycolysis) in both Cul-3 hr and Cul-24 hr SCs, compared to FI SCs ( Figure 1 J). However, there was no difference in the basal mitochondrial OCR between FI SCs and the two stages of cultured SCs examined ( Figure 1 K). Because a recent study identified an increase in mitochondria following 60 hr of SC activation in vivo (), we examined mitochondrial content in FI and Cul SCs. Similar to that observed in vivo, there was a progressive increase in Mitotracker fluorescence in Cul SCs during 48 hr of culture ( Figure S2 A) and an enrichment of genes known to regulate the TCA cycle ( Figure S2 B). These results suggest that Cul SCs preferentially increase their glycolytic rate, despite increasing their mitochondrial content. This observation is reminiscent of mouse epiblast stem cells, which have larger and more complex mitochondria than mouse embryonic stem cells, and yet exhibit a lower level of oxygen consumption ().

RNA-seq results from FI and Cul SCs identified a large number of differentially regulated genes ( Figure 1 C and Table S1 ). Gene ontology (GO) analyses of biological processes highlighted the expected changes following the transition from quiescence to proliferation, with adhesion- and homeostatic-related terms enriched in FI SCs ( Figures 1 D and S1 E and Table S1 ), and cell-cycle and nuclear division terms enriched in Cul SCs ( Figures 1 E and S1 F and Table S1 ). Moreover, the two markers of SC quiescence Sprouty 1 (Spry1,) and calcitonin receptor (Calcr,) were enriched in FI SCs ( Table S1 ). In addition, changes in a number of genes encoding for metabolic regulators were observed in the transition from quiescence to proliferation ( Figures 1 C and 1E). The expression of genes corresponding to proteins that regulate FA metabolic processes and lipid catabolic processes was downregulated ( Figure 1 F), whereas that of genes regulating glucose catabolic processes, glutaminolysis, macromolecular biosynthesis (including the pentose phosphate pathway, PPP) and amino-acid transporters was increased in Cul SCs ( Figure 1 G; Figures S1 G and S1H). Indicating that transcriptional activation of the glycolytic program was not simply the result of in vitro culture conditions, transcription of a similar subset of genes was increased in proliferating SCs derived from regenerating skeletal muscle (). In addition to a global upregulation of genes encoding glycolytic enzymes, we observed that the muscle pyruvate kinase 2 (Pkm2) isoform, known to promote “aerobic-glycolysis” (or the Warburg effect), predominated over its alternative spliced isoform Pkm1 in Cul SCs ( Figure 1 H). Consistently, expression of Hnrnpa1 and Srsf3, two mediators of Pkm alternative splicing, was also increased ( Figure 1 I).

To analyze transcriptomes of quiescent and proliferating SCs, we used fluorescence-activated cell sorting (FACS) followed by RNA sequencing (RNA-seq). FACS isolation of SCs from two month-old C57BL/6 mouse hindlimb muscles was based on selection of α7-integrin, Hoechst, PIand Lin(CD11/CD31/CD45/Sca1) cells (modified from Figures S1 A–S1D available online). Freshly isolated (FI) SCs were immediately processed following the completion of sorting, whereas an aliquot of FACS-isolated SCs were cultured (Cul) on collagen-coated plates in growth media for 48 hr. Quiescent SCs are Pax7/MyoD, whereas activated SCs are Pax7/MyoD); therefore, we analyzed aliquots of FI or Cul SCs and confirmed them to be Pax7/MyoDand Pax7/MyoD, respectively ( Figures 1 A and 1B ).

(J and K) Cellular bioenergetics in SCs during culture in growth conditions were evaluated with Seahorse XF96 bioanalyzer. Glycolysis (ECAR) was increased 2.5-fold in Cul-3 hr and Cul-24 hr SCs (J), whereas basal oxygen consumption (OCR) was not different between FI, Cul-3 hr, or Cul-24 hr SCs (K). Data are presented as mean ± SEM. ∗ p < 0.05 and ∗∗ p < 0.01 (FI SCs versus Cul SCs).

(F and G) Heat maps indicating absolute gene expression (Log 2 [FPKM]) of specific metabolic regulators in FI and Cul SCs. Each gene listed had a mean fold change of greater than 1.5.

(D and E) Gene ontology analyses of RNA-seq revealed an enrichment of biological processes specific to FI SCs (D) or Cul SCs (E). The number of genes enriched by greater than 1.5-fold is indicated under the “Count” column.

(C) RNA-seq scatter plot with key metabolic regulators indicated. Each data point represents the mean Log 2 [FPKM] from two independent biological replicates with color indicating the relative fold change in gene expression.

Discussion

+ and SIRT1 deacetylase activity. Ablation of a SIRT1 domain conferring deacetylase activity resulted in widespread H4K16ac and derepression of several hundred genes in SCs. However, such hyperacetylation did not result in a generalized and immediate transcriptional response of all target genes. For instance, enrichment of H4K16ac at the Myog locus was increased in FI Sirt1mKO SCs but its expression was augmented only in Sirt1mKO SCs induced to differentiate. Local chromatin architecture and additional transcriptional events—including histone dynamics and transcription factor availability—likely determine whether, at specific loci, transcriptional derepression is concomitant with SIRT1 removal or will occur at later stages of cell differentiation. Furthermore, that only approximately 30%–40% of the genes derepressed upon Sirt1 ablation acquired H416ac indicates that either additional histone lysines or nonhistone substrates are involved in SIRT1-mediated transcriptional repression ( Houtkooper et al., 2012 Houtkooper R.H.

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Rando T.A. Induction of autophagy supports the bioenergetic demands of quiescent muscle stem cell activation. ERT2xSirt1fl/fl), these authors demonstrated that Sirt1 ablation inhibited autophagic flux and delayed entry into the S phase of the cell cycle. The different timing used to evaluate SC activation, along with the mode of SIRT1 excision (inducible versus constitutive) and the use of MyoD versus EdU as a measure of SC activation, may account for the observed differences between this study and the current results. In this study, SIRT1 was found to lie at the nexus between SC metabolic reprogramming and muscle gene expression. Our findings are consistent with a model wherein the metabolic state influences the gene expression program of SCs via modulation of the metabolite NADand SIRT1 deacetylase activity. Ablation of a SIRT1 domain conferring deacetylase activity resulted in widespread H4K16ac and derepression of several hundred genes in SCs. However, such hyperacetylation did not result in a generalized and immediate transcriptional response of all target genes. For instance, enrichment of H4K16ac at the Myog locus was increased in FI Sirt1SCs but its expression was augmented only in Sirt1SCs induced to differentiate. Local chromatin architecture and additional transcriptional events—including histone dynamics and transcription factor availability—likely determine whether, at specific loci, transcriptional derepression is concomitant with SIRT1 removal or will occur at later stages of cell differentiation. Furthermore, that only approximately 30%–40% of the genes derepressed upon Sirt1 ablation acquired H416ac indicates that either additional histone lysines or nonhistone substrates are involved in SIRT1-mediated transcriptional repression (). It is interesting to note that SIRT1 does not appear to regulate the expression of metabolic regulators. Thus, although SIRT1 responds to the process of SC metabolic reprogramming by influencing H4K16 acetylation and gene expression, it does not play a direct role in the observed switch toward glycolysis in proliferating SCs. Despite the presence of MyoDSCs on Sirt1fibers, the corresponding Myod1 transcripts were not elevated. This observation is consistent with findings reporting that Myod1 transcripts are already present in quiescent SCs, not exhibiting a significant increase during SC activation in vivo (). Analogous to microRNA-mediated Myf5 regulation (), posttranscriptional processing of Myod1 mRNA transcripts may be responsible for the appearance of the MyoD protein in activated SCs. Moreover, while increased Myog transcripts in Cul (24 hr DM) Sirt1SCs correlated with the enrichment of H4K16ac at its locus in quiescent Sirt1SCs, and SIRT1 disengagement in cultured WT SCs, we cannot exclude that additional regulatory mechanisms controlled by SIRT1 also contribute to Myog activation. In this regard, SIRT1 deacetylates the histone acetyltransferases PCAF and p300, and transcription factors MyoD and Foxo3 (), making it plausible that nonhistone substrates relevant to myogenesis are affected in Sirt1SCs. Recently, SIRT1 has been implicated in regulating autophagic flux and SC activation (). In this study, authophagy in fiber-associated SCs was detected after 24–36 hr of culture, during which time SCs had entered the S phase of the cell cycle, as measured via an 5-ethynyl-2′-deoxyuridine (EdU) assay. Interestingly, using an inducible SC-specific SIRT1-KO mouse (Pax7CrexSirt1), these authors demonstrated that Sirt1 ablation inhibited autophagic flux and delayed entry into the S phase of the cell cycle. The different timing used to evaluate SC activation, along with the mode of SIRT1 excision (inducible versus constitutive) and the use of MyoD versus EdU as a measure of SC activation, may account for the observed differences between this study and the current results.

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et al. Glucose and glutamine metabolism regulate human hematopoietic stem cell lineage specification. Altogether, our findings suggest a role for metabolism in the regulation of SC biology, beyond simply providing the building blocks and ATP required for new cell growth. Within this context, it has previously been shown that the generation of nucleocytoplasmic acetyl-CoA is dependent upon glycolytic production of citrate (). In contrast, FA oxidation leads to the production of mitochondrial acetyl-CoA for entry into the TCA cycle (). Thus, any shift toward glycolysis would be expected to lead to both a decrease in SIRT1 deacetylase activity (through reduced NAD), and an increased supply of acetyl-CoA for histone/protein acetylation, and together likely explain the observed increase in global H4K16ac in active SCs. There is previous evidence that the behavior of adult SCs is influenced by regulatory pathways controlled by oxygen tension and metabolic states (). A link between metabolism and SC function has been proposed, with recent studies indicating a link between OXPHOS and SC clonogenic capacity. Elevated mitochondrial abundance and OXPHOS activity, induced by calorie restriction (CR), was found to be associated with an increase in the number of cells capable of initiating myogenic colony formation (). Regulation of MyoD and Myogenin by increased SIRT1 activity elicited by CR would keep SCs in a stem-like state and favor their clonogenicity. Consistent with this, both the SIRT1 level and the metabolic milieu conducive to its activation were augmented in SCs derived from CR animals (). A similar increase in clonogenic capacity was observed in HSC with increased FA oxidation () while, more recently, glucose and glutamine metabolism has been reported to regulate human HSC lineage specification ().

mKO mice. Intriguingly, a subset of the upregulated transcripts was also increased in Sirt1mKO SCs induced to differentiate (mKO muscles derive from the myofiber compartment. The presence of such transcripts in both differentiating Sirt1mKO SCs and Sirt1mKO adult muscles suggests that their deregulation likely originates in SCs and is further maintained in the adult muscle. Finally, our gene expression profiling of whole muscles revealed deregulated transcription in Sirt1mice. Intriguingly, a subset of the upregulated transcripts was also increased in Sirt1SCs induced to differentiate ( Figure 7 G). Because in adult mice SCs have completed their differentiation process to give rise to mature myofibers, we interpret these findings to indicate that the upregulated transcripts identified in Sirt1muscles derive from the myofiber compartment. The presence of such transcripts in both differentiating Sirt1SCs and Sirt1adult muscles suggests that their deregulation likely originates in SCs and is further maintained in the adult muscle.

In summary, our results provide insights into the developing paradigm linking the process of metabolic reprogramming, transcriptional regulation, and acquisition of defined cell states. In the absence of SIRT1, quiescent SCs lose their respective blueprint of carefully regulated H4K16ac and undergo a progressive deregulation of gene expression during activation and differentiation. We conclude that metabolic modifications affect the activity of SIRT1, which acts as a relay able to interpret the rapid change in metabolism (via NAD+) and induce subsequent changes in H4K16 acetylation status and gene expression.