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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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Mutations to either GATA or EBox elements alters AICAR-induced increases in GATA/EBox DNA binding and affects PGC-1α promoter activity.A. A schematic of the overlapping EBox/GATA (GATA/EBoxwt) binding sites at −495 and −486 base pairs within the PGC-1α promoter is shown. Mutations to the GATA/EBoxwt oligonucleotide that were introduced by nucleotide exchange to abolish GATA (GATAmt) or EBox (EBoxmt) binding are bolded. B. Representative EMSAs of Vehicle- (−) and AICAR− (+) treated nuclear extracts incubated with GATAmt or EBoxmt radiolabeled probes in the presence or absence of USF-1 Antibody. FP: free probe, 25× CO, and 50CO: 25-fold and 50-fold molar excess of cold oligonucleotide, No Ab: No Antibody, USF-1: USF-1 antibody. C. Relative luciferase activity of the PGC-1α promoter constructs in vehicle- or AICAR-treated cells is shown. Values are means±S.E.M, * p<0.05 versus Vehicle-treated control (n = 3). D. Western blot analysis of GATA-4 and USF-1 protein expression from total proteins extracted from vehicle− (−) and AICAR (+)-treated cells is shown. GAPDH expression was used as a loading control.
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pone-0003614-g004: Mutations to either GATA or EBox elements alters AICAR-induced increases in GATA/EBox DNA binding and affects PGC-1α promoter activity.A. A schematic of the overlapping EBox/GATA (GATA/EBoxwt) binding sites at −495 and −486 base pairs within the PGC-1α promoter is shown. Mutations to the GATA/EBoxwt oligonucleotide that were introduced by nucleotide exchange to abolish GATA (GATAmt) or EBox (EBoxmt) binding are bolded. B. Representative EMSAs of Vehicle- (−) and AICAR− (+) treated nuclear extracts incubated with GATAmt or EBoxmt radiolabeled probes in the presence or absence of USF-1 Antibody. FP: free probe, 25× CO, and 50CO: 25-fold and 50-fold molar excess of cold oligonucleotide, No Ab: No Antibody, USF-1: USF-1 antibody. C. Relative luciferase activity of the PGC-1α promoter constructs in vehicle- or AICAR-treated cells is shown. Values are means±S.E.M, * p<0.05 versus Vehicle-treated control (n = 3). D. Western blot analysis of GATA-4 and USF-1 protein expression from total proteins extracted from vehicle− (−) and AICAR (+)-treated cells is shown. GAPDH expression was used as a loading control.

Mentions: We then tested whether AMPK activation targets the EBox, the GATA sequence, or both sequences to transcriptionally activate the PGC-1α promoter by generating mutations within the EBox/GATA oligonucleotides to abolish either Ebox or GATA binding separately (Fig. 4A). Mutation of the GATA sequence shifted the DNA binding complex to a lower molecular weight, consistent with the absence of GATA-4 (Fig. 4B, lane 4–5 from the left). Although USF-1 was still present in the complex, the AICAR effect on protein-DNA binding was reduced (lane 7). However, when the EBox was mutated, USF-1 binding was prevented (lanes 10, 11), and the AICAR effect was abolished (lane 13). These data suggest that transcription factor binding to the EBox sequence is more important to mediate the effect of AMPK activation, but also suggest the fact that GATA could modulate the AMPK effect on DNA binding in regulating the activity of the promoter. This is suggested by the results showing that GATA-4 overexpression alone increased the transcriptional activity of the PGC-1α promoter (see below). Although the identity of the specific GATA isoform bound to the GATA sequence remains unresolved, GATA-4 is expressed in striated muscle [33], which suggests the possibility that the GATA-4 isoform is the likely candidate to mediate transcription of the PGC-1α promoter at this site.


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)

Mutations to either GATA or EBox elements alters AICAR-induced increases in GATA/EBox DNA binding and affects PGC-1α promoter activity.A. A schematic of the overlapping EBox/GATA (GATA/EBoxwt) binding sites at −495 and −486 base pairs within the PGC-1α promoter is shown. Mutations to the GATA/EBoxwt oligonucleotide that were introduced by nucleotide exchange to abolish GATA (GATAmt) or EBox (EBoxmt) binding are bolded. B. Representative EMSAs of Vehicle- (−) and AICAR− (+) treated nuclear extracts incubated with GATAmt or EBoxmt radiolabeled probes in the presence or absence of USF-1 Antibody. FP: free probe, 25× CO, and 50CO: 25-fold and 50-fold molar excess of cold oligonucleotide, No Ab: No Antibody, USF-1: USF-1 antibody. C. Relative luciferase activity of the PGC-1α promoter constructs in vehicle- or AICAR-treated cells is shown. Values are means±S.E.M, * p<0.05 versus Vehicle-treated control (n = 3). D. Western blot analysis of GATA-4 and USF-1 protein expression from total proteins extracted from vehicle− (−) and AICAR (+)-treated cells is shown. GAPDH expression was used as a loading control.
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Related In: Results  -  Collection

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pone-0003614-g004: Mutations to either GATA or EBox elements alters AICAR-induced increases in GATA/EBox DNA binding and affects PGC-1α promoter activity.A. A schematic of the overlapping EBox/GATA (GATA/EBoxwt) binding sites at −495 and −486 base pairs within the PGC-1α promoter is shown. Mutations to the GATA/EBoxwt oligonucleotide that were introduced by nucleotide exchange to abolish GATA (GATAmt) or EBox (EBoxmt) binding are bolded. B. Representative EMSAs of Vehicle- (−) and AICAR− (+) treated nuclear extracts incubated with GATAmt or EBoxmt radiolabeled probes in the presence or absence of USF-1 Antibody. FP: free probe, 25× CO, and 50CO: 25-fold and 50-fold molar excess of cold oligonucleotide, No Ab: No Antibody, USF-1: USF-1 antibody. C. Relative luciferase activity of the PGC-1α promoter constructs in vehicle- or AICAR-treated cells is shown. Values are means±S.E.M, * p<0.05 versus Vehicle-treated control (n = 3). D. Western blot analysis of GATA-4 and USF-1 protein expression from total proteins extracted from vehicle− (−) and AICAR (+)-treated cells is shown. GAPDH expression was used as a loading control.
Mentions: We then tested whether AMPK activation targets the EBox, the GATA sequence, or both sequences to transcriptionally activate the PGC-1α promoter by generating mutations within the EBox/GATA oligonucleotides to abolish either Ebox or GATA binding separately (Fig. 4A). Mutation of the GATA sequence shifted the DNA binding complex to a lower molecular weight, consistent with the absence of GATA-4 (Fig. 4B, lane 4–5 from the left). Although USF-1 was still present in the complex, the AICAR effect on protein-DNA binding was reduced (lane 7). However, when the EBox was mutated, USF-1 binding was prevented (lanes 10, 11), and the AICAR effect was abolished (lane 13). These data suggest that transcription factor binding to the EBox sequence is more important to mediate the effect of AMPK activation, but also suggest the fact that GATA could modulate the AMPK effect on DNA binding in regulating the activity of the promoter. This is suggested by the results showing that GATA-4 overexpression alone increased the transcriptional activity of the PGC-1α promoter (see below). Although the identity of the specific GATA isoform bound to the GATA sequence remains unresolved, GATA-4 is expressed in striated muscle [33], which suggests the possibility that the GATA-4 isoform is the likely candidate to mediate transcription of the PGC-1α promoter at this site.

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