Limits...
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.

Show MeSH
The human PGC-1α promoter.The nucleotide sequence +28 to −2190 corresponding to the proximal 2-kb hPGC-1α promoter is shown. The arrows indicate the transcription start sites, which have been previously described [14]. Putative binding sites for transcription factors are either underlined or overlined. Also included are binding sites for transcription factors that have previously been characterized [12;13;19]. Numbers enclosed in circles represent the 5′- deletions of the PGC-1α promoter reporter constructs  p 2215,  p 1164,  p 851,  p501,  p 191 as shown in Fig. 2.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2570798&req=5

pone-0003614-g001: The human PGC-1α promoter.The nucleotide sequence +28 to −2190 corresponding to the proximal 2-kb hPGC-1α promoter is shown. The arrows indicate the transcription start sites, which have been previously described [14]. Putative binding sites for transcription factors are either underlined or overlined. Also included are binding sites for transcription factors that have previously been characterized [12;13;19]. Numbers enclosed in circles represent the 5′- deletions of the PGC-1α promoter reporter constructs p 2215, p 1164, p 851, p501, p 191 as shown in Fig. 2.

Mentions: The mechanism(s) regulating PGC-1α transcription were first investigated by cloning the proximal 2 kb sequence of the human PGC-1α promoter. This sequence contains +28 to −2190 nucleotides relative to the first transcriptional start site (GenBank Accession No. BD103728; [24]. Inspection of this sequence for the presence of consensus transcription factor binding sites was performed by high stringency searches using PATCH (Pattern search for transcription factor binding sites) and TRANSFAC 6.0. The odds of identifying false positives were minimized by excluding non-canonical sequences, or sequences that contained nucleotide mismatches. The putative DNA binding sites that were found within the hPGC-1α promoter are identified in Fig. 1. Although the promoter does not appear to contain a TATA box, our search identified a putative GC Box within the first intron that has been shown to bind Sp1 [25]. This finding is also in line with those of Esterbauer et al [24]. In addition to the putative Sp1 site, there is one consensus cAMP Response Element (CRE) at position −133, three insulin response sequences (IRS) at −354, −589, and −979, and one Muscle Enhancer Factor-2 (MEF2) site at −1335. These sequences have recently been shown to bind the cAMP Response Element Binding protein (CREB) and a CREB-related family member, Activating Transcription Factor 2 (ATF2), the forkhead transcription factor (FKHR) and MEF2, respectively [15], [16], [26]. Our search also identified three GATA sequences at −493, −1605, and −1961, as well as a consensus sequence representing the serum response element (SRE) at −705 upstream from the transcriptional start site. Putative binding sites for p53 at −1237 and NF-kB at −1575, as well as multiple EBoxes at −23, −362, −495, −785, −1083, −1190, −1927, −1995 and −2185 were also found within the PGC-1α promoter.


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)

The human PGC-1α promoter.The nucleotide sequence +28 to −2190 corresponding to the proximal 2-kb hPGC-1α promoter is shown. The arrows indicate the transcription start sites, which have been previously described [14]. Putative binding sites for transcription factors are either underlined or overlined. Also included are binding sites for transcription factors that have previously been characterized [12;13;19]. Numbers enclosed in circles represent the 5′- deletions of the PGC-1α promoter reporter constructs  p 2215,  p 1164,  p 851,  p501,  p 191 as shown in Fig. 2.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0003614-g001: The human PGC-1α promoter.The nucleotide sequence +28 to −2190 corresponding to the proximal 2-kb hPGC-1α promoter is shown. The arrows indicate the transcription start sites, which have been previously described [14]. Putative binding sites for transcription factors are either underlined or overlined. Also included are binding sites for transcription factors that have previously been characterized [12;13;19]. Numbers enclosed in circles represent the 5′- deletions of the PGC-1α promoter reporter constructs p 2215, p 1164, p 851, p501, p 191 as shown in Fig. 2.
Mentions: The mechanism(s) regulating PGC-1α transcription were first investigated by cloning the proximal 2 kb sequence of the human PGC-1α promoter. This sequence contains +28 to −2190 nucleotides relative to the first transcriptional start site (GenBank Accession No. BD103728; [24]. Inspection of this sequence for the presence of consensus transcription factor binding sites was performed by high stringency searches using PATCH (Pattern search for transcription factor binding sites) and TRANSFAC 6.0. The odds of identifying false positives were minimized by excluding non-canonical sequences, or sequences that contained nucleotide mismatches. The putative DNA binding sites that were found within the hPGC-1α promoter are identified in Fig. 1. Although the promoter does not appear to contain a TATA box, our search identified a putative GC Box within the first intron that has been shown to bind Sp1 [25]. This finding is also in line with those of Esterbauer et al [24]. In addition to the putative Sp1 site, there is one consensus cAMP Response Element (CRE) at position −133, three insulin response sequences (IRS) at −354, −589, and −979, and one Muscle Enhancer Factor-2 (MEF2) site at −1335. These sequences have recently been shown to bind the cAMP Response Element Binding protein (CREB) and a CREB-related family member, Activating Transcription Factor 2 (ATF2), the forkhead transcription factor (FKHR) and MEF2, respectively [15], [16], [26]. Our search also identified three GATA sequences at −493, −1605, and −1961, as well as a consensus sequence representing the serum response element (SRE) at −705 upstream from the transcriptional start site. Putative binding sites for p53 at −1237 and NF-kB at −1575, as well as multiple EBoxes at −23, −362, −495, −785, −1083, −1190, −1927, −1995 and −2185 were also found within the PGC-1α promoter.

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