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AMP-activated protein kinase-regulated activation of the PGC-1alpha promoter in skeletal muscle cells.

Irrcher I, Ljubicic V, Kirwan AF, Hood DA - PLoS ONE (2008)

Bottom Line: The effect of AICAR on transcriptional activation was mediated by an overlapping GATA/EBox binding site at -495 within the PGC-1alpha promoter based on gel shift analyses that revealed increases in GATA/EBox DNA binding.Mutation of the EBox within the GATA/EBox binding site in the promoter reduced basal promoter activity and completely abolished the AICAR effect.Our data identify a novel AMPK-mediated regulatory pathway that regulates PGC-1alpha gene expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, York University, Toronto, Ontario, Canada.

ABSTRACT
The mechanisms by which PGC-1alpha gene expression is controlled in skeletal muscle remains largely undefined. Thus, we sought to investigate the transcriptional regulation of PGC-1alpha using AICAR, an activator of AMPK, that is known to increase PGC-1alpha expression. A 2.2 kb fragment of the human PGC-1alpha promoter was cloned and sequence analysis revealed that this TATA-less sequence houses putative consensus sites including a GC-box, a CRE, several IRSs, a SRE, binding sites for GATA, MEF2, p 53, NF-kappaB, and EBox binding proteins. AMPK activation for 24 hours increased PGC-1alpha promoter activity with concomitant increases in mRNA expression. The effect of AICAR on transcriptional activation was mediated by an overlapping GATA/EBox binding site at -495 within the PGC-1alpha promoter based on gel shift analyses that revealed increases in GATA/EBox DNA binding. Mutation of the EBox within the GATA/EBox binding site in the promoter reduced basal promoter activity and completely abolished the AICAR effect. Supershift analyses identified USF-1 as a DNA binding transcription factor potentially involved in regulating PGC-1alpha promoter activity, which was confirmed in vivo by ChIP. Overexpression of either GATA-4 or USF-1 alone increased the p851 PGC-1alpha promoter activity by 1.7- and 2.0-fold respectively, while co-expression of GATA-4 and USF-1 led to an additive increase in PGC-1alpha promoter activity. The USF-1-mediated increase in PGC-1alpha promoter activation led to similar increases at the mRNA level. Our data identify a novel AMPK-mediated regulatory pathway that regulates PGC-1alpha gene expression. This could represent a potential therapeutic target to control PGC-1alpha expression in skeletal muscle.

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AMPK activation increases GATA/EBox DNA binding.A. Representative EMSAs of nuclear extracts from Vehicle− (−AIC) and AICAR− (+AIC) treated cells that were incubated with radiolabeled oligonucleotides corresponding to the EBox, SRE and IRS sequences found within the AICAR-responsive region of the PGC-1α promoter (as shown in Fig. 1). B. A representative EMSA (Left panel) and a summary of multiple experiments (Right panel) showing the effect of AICAR (A) on GATA/EBox-DNA binding. Values are represented as means±S.E.M (n = 8) relative to vehicle-treated (V) cells. C. Representative EMSAs of nuclear extracts that were incubated with radiolabeled oligonucleotides corresponding to GATA/EBox wt. Vehicle-treated cells were incubated with radiolabeled oligonucleotides corresponding to GATA/EBox wt as well as antibodies against MyoD, USF-1 and c-Myc which are known to bind to the EBox sequence. FP: free probe, 25×, 50×, 100× CO: 25-fold, 50-fold, 100-fold molar excess of cold oligo, No Ab: No Antibody, SRF: SRF antibody, FKHR: Forkhead antibody, GATA-4: GATA-4 antibody, c-Myc: c-Myc Antibody, USF-1: USF-1 antibody. **The representative blot of IRS/DNA binding was made from parts of the same gel. D. Representative chromatin immunoprecipitation from cells treated with or without 1 mM AICAR for 24 hours. Protein/DNA complexes were immunoprecipitated with USF-1 antibody, or with non-specific IgG. Primers encompassing the region between −473 and −823 were used to analyze USF-1 binding to the PGC-1α promoter. The representative blot on the left was made from parts of the same gel. At right is a graphical summary of repeated experiments. Values are representative of means±S.E.M (n = 6).
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pone-0003614-g003: AMPK activation increases GATA/EBox DNA binding.A. Representative EMSAs of nuclear extracts from Vehicle− (−AIC) and AICAR− (+AIC) treated cells that were incubated with radiolabeled oligonucleotides corresponding to the EBox, SRE and IRS sequences found within the AICAR-responsive region of the PGC-1α promoter (as shown in Fig. 1). B. A representative EMSA (Left panel) and a summary of multiple experiments (Right panel) showing the effect of AICAR (A) on GATA/EBox-DNA binding. Values are represented as means±S.E.M (n = 8) relative to vehicle-treated (V) cells. C. Representative EMSAs of nuclear extracts that were incubated with radiolabeled oligonucleotides corresponding to GATA/EBox wt. Vehicle-treated cells were incubated with radiolabeled oligonucleotides corresponding to GATA/EBox wt as well as antibodies against MyoD, USF-1 and c-Myc which are known to bind to the EBox sequence. FP: free probe, 25×, 50×, 100× CO: 25-fold, 50-fold, 100-fold molar excess of cold oligo, No Ab: No Antibody, SRF: SRF antibody, FKHR: Forkhead antibody, GATA-4: GATA-4 antibody, c-Myc: c-Myc Antibody, USF-1: USF-1 antibody. **The representative blot of IRS/DNA binding was made from parts of the same gel. D. Representative chromatin immunoprecipitation from cells treated with or without 1 mM AICAR for 24 hours. Protein/DNA complexes were immunoprecipitated with USF-1 antibody, or with non-specific IgG. Primers encompassing the region between −473 and −823 were used to analyze USF-1 binding to the PGC-1α promoter. The representative blot on the left was made from parts of the same gel. At right is a graphical summary of repeated experiments. Values are representative of means±S.E.M (n = 6).

Mentions: The responsiveness of the PGC-1α promoter to AICAR suggested that AMPK activation could mediate at least part of the increase PGC-1α mRNA expression via transcriptional activation. Since the largest portion of the activation effect occurred between −474 and −823 bp of the promoter (Fig. 2C), we evaluated the DNA binding activities of proteins bound to the putative consensus sequences within this region using gel shift analyses. The binding sites for the transcription factors found within this AICAR-responsive region of the promoter are shown in Fig. 1. They include an overlapping EBox/GATA sequence, an IRS sequence that binds FKHR [16], as well as an SRE and an EBox. AICAR treatment had no effect on the binding of proteins bound to the EBox, SRE, or the IRS sequences (Fig. 3A). However, AICAR treatment led to an ∼2.0-fold increase (p<0.05; Fig. 3B) in GATA/Ebox DNA binding. A number of possible candidate proteins exist which could bind to this overlapping sequence, a GATA isoform (i.e. GATA-4), c-myc, Upstream Stimulatory Factor (USF-1) or MyoD [30]–[32]. Analyses using specific antibodies against GATA-4, c-myc and MyoD did not reveal a supershift in the GATA/EBox-DNA complex (Fig. 3B and C). However, a strong supershifted complex was apparent in the presence of the USF-1 Ab (Fig. 3C), suggesting that USF-1 is a protein bound to the EBox within this sequence. The physiological relevance of this finding in vivo was evaluated with the use of ChIP analysis in which we found USF-1 bound to the PGC-1α promoter in non-stimulated conditions (Fig. 3D, lane 3). Following 24 hours of AICAR treatment, the amount of USF-1 bound to the PGC-1α promoter in vivo was increased by 2.3-fold (Fig. 3D, lane 5), consistent with the gel shift analysis above.


AMP-activated protein kinase-regulated activation of the PGC-1alpha promoter in skeletal muscle cells.

Irrcher I, Ljubicic V, Kirwan AF, Hood DA - PLoS ONE (2008)

AMPK activation increases GATA/EBox DNA binding.A. Representative EMSAs of nuclear extracts from Vehicle− (−AIC) and AICAR− (+AIC) treated cells that were incubated with radiolabeled oligonucleotides corresponding to the EBox, SRE and IRS sequences found within the AICAR-responsive region of the PGC-1α promoter (as shown in Fig. 1). B. A representative EMSA (Left panel) and a summary of multiple experiments (Right panel) showing the effect of AICAR (A) on GATA/EBox-DNA binding. Values are represented as means±S.E.M (n = 8) relative to vehicle-treated (V) cells. C. Representative EMSAs of nuclear extracts that were incubated with radiolabeled oligonucleotides corresponding to GATA/EBox wt. Vehicle-treated cells were incubated with radiolabeled oligonucleotides corresponding to GATA/EBox wt as well as antibodies against MyoD, USF-1 and c-Myc which are known to bind to the EBox sequence. FP: free probe, 25×, 50×, 100× CO: 25-fold, 50-fold, 100-fold molar excess of cold oligo, No Ab: No Antibody, SRF: SRF antibody, FKHR: Forkhead antibody, GATA-4: GATA-4 antibody, c-Myc: c-Myc Antibody, USF-1: USF-1 antibody. **The representative blot of IRS/DNA binding was made from parts of the same gel. D. Representative chromatin immunoprecipitation from cells treated with or without 1 mM AICAR for 24 hours. Protein/DNA complexes were immunoprecipitated with USF-1 antibody, or with non-specific IgG. Primers encompassing the region between −473 and −823 were used to analyze USF-1 binding to the PGC-1α promoter. The representative blot on the left was made from parts of the same gel. At right is a graphical summary of repeated experiments. Values are representative of means±S.E.M (n = 6).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2570798&req=5

pone-0003614-g003: AMPK activation increases GATA/EBox DNA binding.A. Representative EMSAs of nuclear extracts from Vehicle− (−AIC) and AICAR− (+AIC) treated cells that were incubated with radiolabeled oligonucleotides corresponding to the EBox, SRE and IRS sequences found within the AICAR-responsive region of the PGC-1α promoter (as shown in Fig. 1). B. A representative EMSA (Left panel) and a summary of multiple experiments (Right panel) showing the effect of AICAR (A) on GATA/EBox-DNA binding. Values are represented as means±S.E.M (n = 8) relative to vehicle-treated (V) cells. C. Representative EMSAs of nuclear extracts that were incubated with radiolabeled oligonucleotides corresponding to GATA/EBox wt. Vehicle-treated cells were incubated with radiolabeled oligonucleotides corresponding to GATA/EBox wt as well as antibodies against MyoD, USF-1 and c-Myc which are known to bind to the EBox sequence. FP: free probe, 25×, 50×, 100× CO: 25-fold, 50-fold, 100-fold molar excess of cold oligo, No Ab: No Antibody, SRF: SRF antibody, FKHR: Forkhead antibody, GATA-4: GATA-4 antibody, c-Myc: c-Myc Antibody, USF-1: USF-1 antibody. **The representative blot of IRS/DNA binding was made from parts of the same gel. D. Representative chromatin immunoprecipitation from cells treated with or without 1 mM AICAR for 24 hours. Protein/DNA complexes were immunoprecipitated with USF-1 antibody, or with non-specific IgG. Primers encompassing the region between −473 and −823 were used to analyze USF-1 binding to the PGC-1α promoter. The representative blot on the left was made from parts of the same gel. At right is a graphical summary of repeated experiments. Values are representative of means±S.E.M (n = 6).
Mentions: The responsiveness of the PGC-1α promoter to AICAR suggested that AMPK activation could mediate at least part of the increase PGC-1α mRNA expression via transcriptional activation. Since the largest portion of the activation effect occurred between −474 and −823 bp of the promoter (Fig. 2C), we evaluated the DNA binding activities of proteins bound to the putative consensus sequences within this region using gel shift analyses. The binding sites for the transcription factors found within this AICAR-responsive region of the promoter are shown in Fig. 1. They include an overlapping EBox/GATA sequence, an IRS sequence that binds FKHR [16], as well as an SRE and an EBox. AICAR treatment had no effect on the binding of proteins bound to the EBox, SRE, or the IRS sequences (Fig. 3A). However, AICAR treatment led to an ∼2.0-fold increase (p<0.05; Fig. 3B) in GATA/Ebox DNA binding. A number of possible candidate proteins exist which could bind to this overlapping sequence, a GATA isoform (i.e. GATA-4), c-myc, Upstream Stimulatory Factor (USF-1) or MyoD [30]–[32]. Analyses using specific antibodies against GATA-4, c-myc and MyoD did not reveal a supershift in the GATA/EBox-DNA complex (Fig. 3B and C). However, a strong supershifted complex was apparent in the presence of the USF-1 Ab (Fig. 3C), suggesting that USF-1 is a protein bound to the EBox within this sequence. The physiological relevance of this finding in vivo was evaluated with the use of ChIP analysis in which we found USF-1 bound to the PGC-1α promoter in non-stimulated conditions (Fig. 3D, lane 3). Following 24 hours of AICAR treatment, the amount of USF-1 bound to the PGC-1α promoter in vivo was increased by 2.3-fold (Fig. 3D, lane 5), consistent with the gel shift analysis above.

Bottom Line: The effect of AICAR on transcriptional activation was mediated by an overlapping GATA/EBox binding site at -495 within the PGC-1alpha promoter based on gel shift analyses that revealed increases in GATA/EBox DNA binding.Mutation of the EBox within the GATA/EBox binding site in the promoter reduced basal promoter activity and completely abolished the AICAR effect.Our data identify a novel AMPK-mediated regulatory pathway that regulates PGC-1alpha gene expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, York University, Toronto, Ontario, Canada.

ABSTRACT
The mechanisms by which PGC-1alpha gene expression is controlled in skeletal muscle remains largely undefined. Thus, we sought to investigate the transcriptional regulation of PGC-1alpha using AICAR, an activator of AMPK, that is known to increase PGC-1alpha expression. A 2.2 kb fragment of the human PGC-1alpha promoter was cloned and sequence analysis revealed that this TATA-less sequence houses putative consensus sites including a GC-box, a CRE, several IRSs, a SRE, binding sites for GATA, MEF2, p 53, NF-kappaB, and EBox binding proteins. AMPK activation for 24 hours increased PGC-1alpha promoter activity with concomitant increases in mRNA expression. The effect of AICAR on transcriptional activation was mediated by an overlapping GATA/EBox binding site at -495 within the PGC-1alpha promoter based on gel shift analyses that revealed increases in GATA/EBox DNA binding. Mutation of the EBox within the GATA/EBox binding site in the promoter reduced basal promoter activity and completely abolished the AICAR effect. Supershift analyses identified USF-1 as a DNA binding transcription factor potentially involved in regulating PGC-1alpha promoter activity, which was confirmed in vivo by ChIP. Overexpression of either GATA-4 or USF-1 alone increased the p851 PGC-1alpha promoter activity by 1.7- and 2.0-fold respectively, while co-expression of GATA-4 and USF-1 led to an additive increase in PGC-1alpha promoter activity. The USF-1-mediated increase in PGC-1alpha promoter activation led to similar increases at the mRNA level. Our data identify a novel AMPK-mediated regulatory pathway that regulates PGC-1alpha gene expression. This could represent a potential therapeutic target to control PGC-1alpha expression in skeletal muscle.

Show MeSH
Related in: MedlinePlus