SIRT1 controls the transcription of the peroxisome proliferator-activated receptor-gamma Co-activator-1alpha (PGC-1alpha) gene in skeletal muscle through the PGC-1alpha autoregulatory loop and interaction with MyoD

Abstract

Peroxisome proliferator activated receptor-gamma co-activator-1alpha (PGC-1alpha) is a transcriptional co-activator that coordinately regulates the expression of distinct sets of metabolism-related genes in different tissues. Here we show that PGC-1alpha expression is reduced in skeletal muscles from mice lacking the sirtuin family deacetylase SIRT1. Conversely, SIRT1 activation or overexpression in differentiated C2C12 myotubes increased PGC-1alpha mRNA expression. The transcription-promoting effects of SIRT1 occurred through stimulation of PGC-1alpha promoter activity and were enhanced by co-transfection of myogenic factors, such as myocyte enhancer factor 2 (MEF2) and, especially, myogenic determining factor (MyoD). SIRT1 bound to the proximal promoter region of the PGC-1alpha gene, an interaction potentiated by MEF2C or MyoD, which also interact with this region. In the presence of MyoD, SIRT1 promoted a positive autoregulatory PGC-1alpha expression loop, such that overexpression of PGC-1alpha increased PGC-1alpha promoter activity in the presence of co-expressed MyoD and SIRT1. Chromatin immunoprecipitation showed that SIRT1 interacts with PGC-1alpha promoter and increases PGC-1alpha recruitment to its own promoter region. Immunoprecipitation assays further showed that SIRT1-PGC-1alpha interactions are enhanced by MyoD. Collectively, these data indicate that SIRT1 controls PGC-1alpha gene expression in skeletal muscle and that MyoD is a key mediator of this action. The involvement of MyoD in SIRT1-dependent PGC-1alpha expression may help to explain the ability of SIRT1 to drive muscle-specific gene expression and metabolism. Autoregulatory control of PGC-1alpha gene transcription seems to be a pivotal mechanism for conferring a transcription-activating response to SIRT1 in skeletal muscle.

FIGURE 1.

Expression of PGC-1α, UCP3, and myogenin mRNA in skeletal muscles from SIRT1-null mice. Gastrocnemius, tibialis anterior, and soleus muscles from 30-day-old SIRT1 homozygote-null mice (−/−), SIRT1 heterozygote-null mice (+/−), and wild-type littermates (+/+) were analyzed by quantitative reverse transcription-PCR. Data are presented as means ± S.E. of the relative abundance of transcript (8 mice/group). Statistically significant differences between SIRT1 −/− or SIRT1 +/− and SIRT1 +/+ are denoted by the asterisk (p < 0.05).

FIGURE 2.

Effects of SIRT1 overexpression and SIRT1 activity modulators on PGC-1α gene expression in muscle cells. C2C12 myotubes were transduced with Ad-SIRT1 or Ad-GFP (control) and treated with 50 μm resveratrol (resveratrol) or 10 mm nicotinamide (NAM) for 24 h before harvesting. a, data are presented as means ± S.E. of relative transcript levels from three to five independent experiments. Statistically significant differences (p < 0.05) are denoted by the asterisk (SIRT1 versus GFP) and # (drug treatment versus absence). b, representative immunoblot showing Ad-SIRT1-mediated overexpression of SIRT1.

FIGURE 3.

Effects of SIRT1 on PGC-1α gene promoter activity. C2C12 cells were transfected with a luciferase reporter construct driven by 2 kb of the mouse PGC-1α promoter region (PGC-1α -Luc). Data show the -fold induction of luciferase activity with respect to basal promoter values, and represent the means ± S.E. from at least three independent experiments performed in triplicate. a, cells were co-transfected with an expression vector for SIRT1 and treated with 50 μm resveratrol (RSV) or 10 mm NAM for 24 h, as indicated. Statistically significant differences (p < 0.05) due to drugs with respect to basal promoter values are denoted by the asterisk, and those due to the effects of SIRT1 are denoted by #. b, cells were co-transfected with expression vectors for PPARβ/δ, Foxo1, MEF2A, MEF2C, MEF2D, or MyoD, as indicated, as well as an expression vector for SIRT1 or the mutant form, SIRT1-H363Y. Cells transfected with PPARβ/δ were also treated with the specific agonist, 10 μm GW501516. Data show the -fold induction of luciferase activity with respect to basal promoter values, and represent the means ± S.E. from at least three independent experiments performed in triplicate. Statistically significant differences (p < 0.05) for each transfection condition with respect to basal promoter values are denoted by an asterisk, and those due to the effects of SIRT1 in the absence or presence of a given expression vector are denoted by #.

FIGURE 4.

SIRT1 action on the myogenic factor-responsive region of the PGC-1α promoter. a, C2C12 cells were transfected with a promoter-reporter construct in which luciferase expression is driven by the proximal region (140 bp) of the PGC-1α gene promoter. Where indicated, cells were co-transfected with expression vectors for SIRT1, SIRT1-H363Y, MyoD, and/or MEF2C. Data show the -fold induction of luciferase activity with respect to basal promoter values and represent the means ± S.E. from at least three independent experiments performed in triplicate. Statistically significant differences (p < 0.05) for each transfection condition with respect to basal promoter values are denoted by an asterisk, and those due to the effects of SIRT1 in the absence or presence of a given expression vector are denoted by #. b, representative ChIP analysis of SIRT1 binding to the proximal region of the mouse PGC-1α promoter. Experiments were performed in the presence of a transfected 140-bp mPGC-1α-Luc construct and co-transfected expression vectors for HA-tagged SIRT1, MEF2C, and/or MyoD, as indicated. The arrow indicates the 177-bp PCR product from the mouse PGC-1α promoter. c, quantitative analysis of ChIP amplification. Data are expressed as the means ± S.E. of the -fold induction in relative intensity of the amplified PCR product from three independent experiments. Statistically significant differences (p < 0.05) are denoted by the asterisk (SIRT1-HA, MEF2C, or MyoD versus vector control) and # (MEF2c + SIRT1-HA or MyoD + SIRT1-HA versus SIRT1-HA, only).

FIGURE 5.

Effects of SIRT1 on a MyoD response-defective mutant form of the human PGC-1α promoter. C2C12 cells were transfected with a luciferase construct containing 49 bp of the proximal human PGC-1α promoter core region (hPGC-1α-LUC wt) or a version in which the two E boxes (which mediate MyoD responsiveness) have been mutated (hPGC-1α-LUC Mt3). Cells were co-transfected with expression vectors for SIRT1 and/or MyoD as indicated. The results are expressed as -fold induction of luciferase activity with respect to basal promoter values, and represent the means ± S.E. of at least three independent experiments performed in triplicate. Statistically significant differences are denoted by the asterisk (MyoD versus vector control, p < 0.01), # (SIRT1 versus control vector, p < 0.01), and † (wt versus Mt3, p < 0.005).

FIGURE 6.

Effects of a non acetylable form of MyoD on mediating the responsiveness of the PGC-1α promoter to SIRT1. C2C12 cells were transfected with the 2-kb mouse PGC-1α-Luc promoter construct and co-transfected with expression vectors for MyoD or MyoD-RRR (a non acetylable mutant form of MyoD) and SIRT1, as indicated. Results are presented as means ± S.E. of the -fold induction of relative luciferase activity with respect to basal activity. Statistically significant differences (p < 0.05) are denoted by the asterisk (MyoD versus vector control) and # (SIRT1 versus vector control).

FIGURE 7.

Effects of P/CAF on the functional interaction of SIRT1 and MyoD on PGC-1α gene transcription. a, C2C12 cells were transfected with a luciferase reporter construct containing 2 kb of the mouse PGC-1α promoter region (mPGC-1α -Luc), and were co-transfected with expression vectors for MyoD, P/CAF, or P/CAFΔHAT (a mutated form of P/CAF devoid of acetyltransferase activity) and SIRT1, as indicated. Results are expressed as means ± S.E. of the -fold induction of relative luciferase activity with respect to basal activity. Statistically significant differences (p < 0.05) are denoted by the asterisk (MyoD versus vector control) and # (SIRT1 versus vector control). b, C2C12 cells were transfected with pTK-Luc (a luciferase expression vector driven by the thymidine kinase promoter) and co-transfected with pcDNA3-e2F1. DP1, P/CAF, and/or P/CAFΔHAT expression vectors were co-transfected, as indicated. Results are presented as means ± S.E. of at least three independent experiments. Statistically significant differences (p < 0.05) due to DP1 are denoted by the asterisk, and those due to the co-transfection of P/CAF expression vectors are denoted by #.

FIGURE 8.

Effects of PGC-1α on the action of SIRT1 and MyoD at the PGC-1α gene promoter. a, CV1 cells were transfected with a promoter-reporter construct in which luciferase expression is driven by the proximal region (140 bp) of the PGC-1α gene promoter. Cells were co-transfected with expression vectors for PGC-1α, SIRT1, and/or MyoD, as indicated. Data are presented as -fold induction of luciferase activity with respect to basal promoter values, and represent the means ± S.E. of at least three independent experiments performed in triplicate. Statistically significant differences (p < 0.05) for each transfection condition with respect to basal promoter values are denoted by the asterisk, and those due to the effects of PGC-1α in the absence or presence of a given expression vector are denoted by #. b, representative ChIP analysis of PGC-1α binding to the proximal region of its own promoter. Experiments were performed in the presence of a transfected 140bpPGC-1α-Luc construct and co-transfected expression vectors for PGC-1α, SIRT1, and/or MyoD, as indicated. A representative ChIP analysis is shown. The arrow indicates the 177-bp PCR product from the mouse PGC-1α promoter. c, quantitative analysis of ChIP amplification. Data are expressed as the means ± S.E. of the -fold induction in relative intensity of the amplified PCR product from three independent experiments. Statistical significance of differences is denoted by: *, p < 0.05; **, p < 0.005.

FIGURE 9.

Effect of MyoD on the interaction of PGC-1α with SIRT1. a, immunoprecipitation analysis of the interaction of PGC-1α with SIRT1. C2C12 cells were transduced with adenoviral vectors for PGC-1α and/or MyoD (left panel), and SIRT1 (right panel), as indicated. Cell extracts were immunoprecipitated (IP) using an antibody directed against PGC-1α and immunoblotted (IB) with an anti-SIRT1 antibody (top panel). Whole cell extracts were probed with the anti-SIRT1 antibody to confirm equivalent SIRT1 input before immunoprecipitation (bottom panels). b, C2C12 cells were transduced with adenoviral vectors for PGC-1α and/or MyoD, and SIRT1, as indicated. Cell extracts were immunoprecipitated using an antibody directed against SIRT1 and immunoblotted with an anti-PGC-1α antibody (top panel). Whole cell extracts were probed with the anti-PGC-1α antibody to confirm equivalent PGC-1α input before immunoprecipitation (bottom panel). c, C2C12 cells were transduced with adenoviral vectors for PGC-1α, MyoD, with or without co-transduction with the SIRT1 vector, as indicated. Cell extracts were immunoprecipitated using an antibody directed against MyoD and immunoblotted with an anti-PGC-1α antibody (top panel). Whole cell extracts were probed with the anti-PGC-1α antibody to confirm equivalent PGC-1α input before immunoprecipitation (bottom panel).

FIGURE 10.

Schematic representation of the possible mechanism-of-action of SIRT1 on PGC-1α gene transcription. Schematic overview of the interaction of SIRT1 with MyoD and involvement of the PGC-1α positive autoregulatory loop.