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. 2004 Apr 27;101(17):6570-5.
doi: 10.1073/pnas.0401401101. Epub 2004 Apr 20.

Erralpha and Gabpa/b specify PGC-1alpha-dependent oxidative phosphorylation gene expression that is altered in diabetic muscle

Vamsi K Mootha  1 , Christoph Handschin, Dan Arlow, Xiaohui Xie, Julie St Pierre, Smita Sihag, Wenli Yang, David Altshuler, Pere Puigserver, Nick Patterson, Patricia J Willy, Ira G Schulman, Richard A Heyman, Eric S Lander, Bruce M Spiegelman
Affiliations

Erralpha and Gabpa/b specify PGC-1alpha-dependent oxidative phosphorylation gene expression that is altered in diabetic muscle

Vamsi K Mootha et al. Proc Natl Acad Sci U S A. .

Erratum in

  • Proc Natl Acad Sci U S A. 2005 Jul 19;102(29):10405

Abstract

Recent studies have shown that genes involved in oxidative phosphorylation (OXPHOS) exhibit reduced expression in skeletal muscle of diabetic and prediabetic humans. Moreover, these changes may be mediated by the transcriptional coactivator peroxisome proliferator-activated receptor gamma coactivator-1alpha (PGC-1alpha). By combining PGC-1alpha-induced genome-wide transcriptional profiles with a computational strategy to detect cis-regulatory motifs, we identified estrogen-related receptor alpha (Erralpha) and GA repeat-binding protein alpha as key transcription factors regulating the OXPHOS pathway. Interestingly, the genes encoding these two transcription factors are themselves PGC-1alpha-inducible and contain variants of both motifs near their promoters. Cellular assays confirmed that Erralpha and GA-binding protein a partner with PGC-1alpha in muscle to form a double-positive-feedback loop that drives the expression of many OXPHOS genes. By using a synthetic inhibitor of Erralpha, we demonstrated its key role in PGC-1alpha-mediated effects on gene regulation and cellular respiration. These results illustrate the dissection of gene regulatory networks in a complex mammalian system, elucidate the mechanism of PGC-1alpha action in the OXPHOS pathway, and suggest that Erralpha agonists may ameliorate insulin-resistance in individuals with type 2 diabetes mellitus.

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Figures

Fig. 1.
Fig. 1.
Schematic overview of motifADE and application to the PGC-1α time course. (A) The motifADE strategy. It begins with a list of genes ordered on the basis of differential expression across two conditions. Each gene is then annotated for the presence of a given motif in the promoter region. A nonparametric statistic is used to assess whether genes with the motif tend to rank high on this list. In this example, genes with Motif 1 are randomly distributed on the list, whereas genes with Motif 2 tend to rank high, suggesting an association between Motif 2 and the differential expression. (B) C2C12 cells were infected with an adenovirus expressing either GFP (control) or with PGC-1α and were profiled over a 3-day period. Experiments were performed in duplicate and relative gene expression measures are shown. Genes are ranked according to the difference in expression between PGC-1α and GFP on day 3. Mouse genes having a perfect Errα motif (5′-TGACCTTG-3′), a perfect Gabpa/b motif (5′-CTTCCG-3′), or both motifs are labeled with a black bar on the right side of the correlogram.
Fig. 2.
Fig. 2.
Proposed model of mechanism of action of PGC-1α. PGC-1α is a highly regulated gene that responds to external stimuli, e.g., reduced in diabetes and increased after exercise. When PGC-1α levels rise, the expression of Errα and Gabpa are immediately induced by means of a double-positive-feedback loop. These levels rise, and over the course of 2–3 days, these factors couple with PGC-1α to induce the expression of NRF-1 and hundreds of downstream targets, such as OXPHOS and other mitochondrial genes that are enriched for these transcription factor-binding sites.
Fig. 3.
Fig. 3.
Errα and Gabpa cooperate with PGC-1α to induce their own expression. (A) Putative Errα and Gabpa motifs 1 kb upstream and downstream of the Errα and the Gabpa TSSs. (B–D) C2C12 cells were transfected with a reporter gene plasmid containing 2 kb of the Errα promoter (B), Gabpa promoter (C), or Gabpa intron 1 (D), together with expression plasmids for Errα, Gabpa, Gabpb1, and PGC-1α. Forty-eight hours after transfection, reporter gene levels were determined and were normalized to β-galactosidase levels.
Fig. 4.
Fig. 4.
A small-molecule inhibitor of Errα inhibits the response to PGC-1α. (A) C2C12 myoblasts transfected with wild-type Errα promoter and the respective expression plasmids were treated with vehicle (0.1% DMSO) or 1 μM XCT790 for 48 h before reporter gene levels were measured. (B) C2C12 cells were transfected with wild-type or Errα-promoter harboring a mutated Errα motif (mutant), together with expression plasmids for Errα, Gabpa, Gabpb1, and PGC-1α. After 48 h, reporter gene levels were determined and normalized to β-galactosidase levels. (C) C2C12 myotubes were infected with GFP- or PGC-1α adenovirus and were treated with vehicle (0.1% DMSO) or 1 μM XCT790 for 1 day. Relative expression levels of several genes were then determined by semiquantitative real-time PCR and were normalized against 18S rRNA levels. (D) Fao rat hepatoma cells were infected with adenoviral GFP or PGC-1α and were treated with vehicle (0.1% DMSO) or 1 μM XCT790 for 1 day before relative gene expression levels were measured by real-time PCR, and were normalized against 18S rRNA levels. *, P < 0.05. NS, not significant.
Fig. 5.
Fig. 5.
Total and uncoupled mitochondrial respiration are inhibited by the synthetic Errα-inverse agonist XCT790. (A and B) C2C12 myotubes were infected with GFP or PGC-1α adenovirus and were treated with vehicle (0.1% DMSO) or 1 μM XCT790 for 2 days before total mitochondrial respiration (A) and uncoupled respiration (B) was determined. *, P < 0.05 in paired t test.
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