Peroxisome Proliferator-activated Receptor γ Coactivator-1 α Isoforms Selectively Regulate Multiple Splicing Events on Target Genes

Abstract

Endurance and resistance exercise training induces specific and profound changes in the skeletal muscle transcriptome. Peroxisome proliferator-activated receptor γ coactivator-1 α (PGC-1α) coactivators are not only among the genes differentially induced by distinct training methods, but they also participate in the ensuing signaling cascades that allow skeletal muscle to adapt to each type of exercise. Although endurance training preferentially induces PGC-1α1 expression, resistance exercise activates the expression of PGC-1α2, -α3, and -α4. These three alternative PGC-1α isoforms lack the arginine/serine-rich (RS) and RNA recognition motifs characteristic of PGC-1α1. Discrete functions for PGC-1α1 and -α4 have been described, but the biological role of PGC-1α2 and -α3 remains elusive. Here we show that different PGC-1α variants can affect target gene splicing through diverse mechanisms, including alternative promoter usage. By analyzing the exon structure of the target transcripts for each PGC-1α isoform, we were able to identify a large number of previously unknown PGC-1α2 and -α3 target genes and pathways in skeletal muscle. In particular, PGC-1α2 seems to mediate a decrease in the levels of cholesterol synthesis genes. Our results suggest that the conservation of the N-terminal activation and repression domains (and not the RS/RNA recognition motif) is what determines the gene programs and splicing options modulated by each PGC-1α isoform. By using skeletal muscle-specific transgenic mice for PGC-1α1 and -α4, we could validate, in vivo, splicing events observed in in vitro studies. These results show that alternative PGC-1α variants can affect target gene expression both quantitatively and qualitatively and identify novel biological pathways under the control of this system of coactivators.

Keywords: PGC-1α2; PGC-1α3; PGC-1α4; alternative splicing; exercise; exon array; gene regulation; peroxisome proliferator-activated receptor γ coactivator 1-α (PGC-1α) (PPARGC1A); skeletal muscle.

FIGURE 1.

PGC-1α isoform protein dynamics. A, Modular structure of PGC-1α isoforms (amino acid numbers are relative to PGC-1α1). Main domains depicted: activation, repression, RS, and RRM. Leucine-rich motifs (LLXXL and LXXLL) indicate interaction sites for transcription factors and other coactivators. “P” indicates phosphorylation sites for p38 MAPK. C-terminal RS and RRM domains interact with splicing factors and the mediator complex via Med1. AA, amino acids; PP, proximal promoter; AP, alternative promoter. B, β-adrenergic stimulation regulates PGC-1α isoform expression in skeletal muscle. Gene expression in the gastrocnemius muscle was analyzed by qRT-PCR. PBS control was compared with clenbuterol (i.p. 2 mg/kg for 16 h). Error bars represent S.E. *, p < 0.05 versus PBS. C, expression of PGC-1α isoforms in mouse primary myotubes. Differentiated myotubes were transduced with recombinant adenovirus to express GFP alone (control) or GFP and each PGC-1α isoform from independent promoters. Error bars represent S.D. D, protein immunoblot for myotubes transduced with PGC-1α isoforms. E, PGC-1α protein dynamics. 4 h of 10 μm MG132 treatment stabilizes all the PGC-1α isoforms. F, time course for cycloheximide chase and half-life determination for all transduced PGC-1α isoforms. *, at least 240 min as this was the end point of the chase. G, exon/intron structure of PGC-1α isoforms. H, probe signal intensity for transduced PGC-1α isoforms obtained in exon arrays. Arrows and letters indicate regions of interest: a, exon 1a specific for PGC-1α1; b, exon 2 common to all isoforms; c, exon 7-8 region present in PGC-1α1/α2/α3; d, 3′ regions only present in PGC-1α1. * indicates stop codon for each isoform, crossed exons indicate alternative splicing-incomplete exon. I, qRT-PCR validation of the expression of PGC-1α isoforms ectopically expressed in myotubes. Error bars represent S.D. *, p < 0.05 versus GFP.

FIGURE 2.

Global gene expression analysis by exon arrays in myotubes expressing each PGC-1α isoform. A, workflow for the exon array gene/isoform expression, molecular pathway analysis, and alternative splicing determination. Splicing hits were double filtered in Partek (alt.splice attribute ≤0.0001) and EAA (−1 ≥ splicing index ≥ 1). B, principal component analysis (PCA) of the exon array data sets. C, heat map for PGC-1α isoform target gene expression in myotubes transduced plus number of differentially expressed genes (1.2-fold change, p < 0.05) by each PGC-1α isoform versus GFP. Red, induced; green, repressed. D, Venn diagram for gene regulation comparison between each PGC-1α isoform. The union area is divided into two subareas to indicate co-regulated versus contra-regulated genes.

FIGURE 3.

Target gene regulation by PGC-1α isoforms in myotubes. A, top pathways from the Ingenuity Pathway Analysis platform. Gray bars indicate scores for the same pathways from a previous data set (3). B–H, qRT-PCR validation for genes associated with the molecular pathways identified by IPA. Error bars represent S.D. *, p < 0.05 versus GFP, n.s., non-significant statistical differences versus GFP. TCA, tricarboxylic acid; BMP, bone morphogenetic protein; RA, rheumatoid arthritis; ARF, alternative reading frame; AMPK, AMP kinase; HIF-1α, hypoxia-inducible factor-1α.

FIGURE 4.

Global analysis of alternative splicing in myotubes expressing PGC-1α isoforms. A, qRT-PCR analysis of total PGC-1α (exon 2) on myotubes transduced with increasing virus titer (+, single titer; ++, double titer). Error bars represent S.D. B, protein levels of distinct PGC-1α variants upon increasing virus titer plus quantification (ImageLab) normalized by the lower titer in each experiment. Error bars represent S.E. *, p < 0.05 versus titer ×1. C, qRT-PCR analysis of target gene specificity for the different PGC-1α isoforms upon increasing protein expression (+, single titer; ++, double titer). Error bars represent S.D. *, p < 0.05 versus GFP. D, extracellular flux analysis (Seahorse) of myotubes expressing each PGC-1α isoform in medium supplemented with 1 mm pyruvate. Error bars represent S.D. *, p < 0.05 versus GFP control. E, extracellular flux analysis of myotubes expressing each PGC-1α isoform on medium supplemented with 5 mm glucose. Error bars represent S.D. F, left, number of differentially spliced genes between GFP control and each PGC-1α isoform. Right, number of differentially spliced genes between PGC-1α isoforms. G, left, number of differentially expressed exons between GFP control and each PGC-1α isoform. Right, number of differentially expressed exons between PGC-1α isoforms. H, splicing events categorization by AltAnalyze: Alt3′, alternative 3′ splicing; Alt5′, alternative 5′ splicing; Alt C-ter, alternative C-terminal splicing; Alt N-ter, alternative N-terminal splicing; Alt prom, alternative promoter. FCCP, carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone; OCR, oxygen consumption rate.

FIGURE 5.

Alternative splicing of Ddx27 and Ndrg4 induced by distinct PGC-1α isoforms. A, Ddx27 exon array probe set signal intensity. *, differentially expressed exon. B, graphic representation of the mRNA structure of Ddx27 isoforms indicating exons of interest. e8, constitutive exon 8; e8b, alternative exon 8b, Alt 3′-UTR, alternative 3′-UTR); e9, exon9. C, qRT-PCR analysis of specific Ddx27 isoform expression in myotubes expressing each PGC-1α isoform. Error bars represent S.D. *, p < 0.05 versus GFP. D, Ndrg4 exon array probe set signal intensity. *, alternative promoter. E, graphic representation of Ndrg4 isoform mRNA structure indicating exons of interest: e1, constitutive exon 1; e1b, alternative exon 1b; Alt 5′, alternative 5′-UTR; e2–e4, exons 2–4. F, specific Ndrg4 isoform expression in myotubes expressing each PGC-1α isoform was analyzed by qRT-PCR. Error bars represent S.D. *, p < 0.05 versus GFP.

FIGURE 6.

Regulation of Osbpl1a alternative promoter usage by distinct PGC-1α isoforms. A, Osbpl1a exon array probe set signal intensity. *, alternative promoter. B, graphic representation of the mRNA structure of Osbpl1a isoforms: e, exon; Alt3′, alternative 3′-UTR; Alt5′, alternative 5′-UTR. C, qRT-PCR analysis of specific Osbpl1a isoform expression in myotubes expressing each PGC-1α isoform. Error bars represent S.D. *, p < 0.05 versus GFP.

FIGURE 7.

Analysis of Ndrg4 and Osbpl1a alternative promoter usage in skeletal muscle of PGC-1α1 or PGC-1α4 transgenic mice. A and B, qRT-PCR and protein immunoblotting analysis of distinct Osbpl1a isoform expression in gastrocnemius from PGC-1α1 (A) or PGC-1α4 (B) skeletal muscle-specific transgenic mice. C and D, qRT-PCR and protein immunoblotting analysis of distinct Ndrg4 isoform expression in gastrocnemius from PGC-1α1 (C) or PGC-1α4 (D) skeletal muscle-specific transgenic mice (Mck-PGC-1α1 (6) and Myo-PGC-1α4 (4)). E, proposed model for alternative promoter usage upon induction of specific PGC-1α isoforms. In all panels, error bars represent S.E. *, p < 0.05 versus WT; n.s., nonsignificant. Pol, polymerase; Alt5′, alternative 5′-UTR.