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. 2005 Jun 15;19(12):1466-73.
doi: 10.1101/gad.1295005.

Activation of nuclear receptor coactivator PGC-1alpha by arginine methylation

Catherine Teyssier  1 , Han Ma, Roger Emter, Anastasia Kralli, Michael R Stallcup
Affiliations

Activation of nuclear receptor coactivator PGC-1alpha by arginine methylation

Catherine Teyssier et al. Genes Dev. .

Abstract

Peroxisome proliferator-activated receptor gamma coactivator 1alpha (PGC-1alpha), a tissue-specific and inducible transcriptional coactivator for several nuclear receptors, plays a key role in energy metabolism. We report here that PGC-1alpha coactivator activity is potentiated by arginine methylation by protein arginine methyltransferase 1 (PRMT1), another nuclear receptor coactivator. Mutation of three substrate arginines in the C-terminal region of PGC-1alpha abolished the cooperative coactivator function of PGC-1alpha and PRMT1, and compromised the ability of PGC-1alpha to induce endogenous target genes. Finally, endogenous PRMT1 contributes to PGC-1alpha coactivator activity, and to the induction of genes important for mitochondrial biogenesis.

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Figures

Figure 1.
Figure 1.
Specific and cooperative coactivator function between PGC-1α and PRMT1. CV-1 cells were transiently transfected with MMTV(ERE)-LUC reporter plasmid (250 ng) and expression vectors encoding ERα (0.1 ng), PGC-1α (50 ng), and PRMT1, PRMT1(E153Q) mutant or CARM1 (250 ng). Luciferase activity was measured after growth in 20 nM estradiol. (Bottom of figure) Immunoblots of extracts from COS7 cells transfected with HA-tagged PRMT1 expression vectors were performed with antibodies against HA.
Figure 2.
Figure 2.
PGC-1α N-terminal domains required for functional synergy with PRMT1. (A) Schematic representation of PGC-1α mutants used in B. The mutant proteins are expressed at wild-type levels (data not shown). (B) CV-1 cells were transiently transfected with MMTV(ERE)-LUC reporter plasmid (250 ng) and expression vectors encoding ERα (0.1 ng) and PGC-1α wild type or mutants as indicated (50 ng) in the absence (white boxes) or presence (black boxes) of PRMT1 (200 ng). Transfected cells were grown in culture medium with 20 nM estradiol, and extracts of the harvested cells were tested for luciferase activity.
Figure 3.
Figure 3.
Methylation of PGC-1α by PRMT1. (A) Full-length GST-PGC-1α was incubated with GST-CARM1 or GST-PRMT1 and [3H]AdoMet for 1 h at 30°C. Products were analyzed by SDS-PAGE and fluorography. (B) Methylation of PGC-1α fragments was analyzed by SDS-PAGE (lower panels show Coomassie blue staining) and fluorography (upper panels). Diagram shows amino acid numbers. Asterisks show the stained fragments (lower panels) that were methylated (upper panels); positions of molecular weight markers are indicated beside the panels. (C) Mutant forms of the PGC-1α E fragment (sequence shown) were methylated by PRMT1 and analyzed by fluorography (left panel) and staining (right panel). In the R4K and R3K mutants, the bracketed Arg residues (R) were changed to Lys. The results presented are from a single experiment representative of three independent experiments.
Figure 4.
Figure 4.
Role of PGC-1α C-terminal domains in synergy with PRMT1. (A) Schematic representation of PGC-1α deletion mutants used in B. (B) CV-1 cells were transiently transfected with MMTV(ERE)-LUC reporter plasmid (250 ng) and expression vectors encoding ERα (0.1 ng) and PGC-1α wild type or mutants as indicated (50 ng), in the absence (white boxes) or presence (black boxes) of PRMT1 (200 ng). Transfected cells were grown in culture medium with 20 nM estradiol, and extracts of the harvested cells were tested for luciferase activity.
Figure 5.
Figure 5.
Role of methylated Arg residues of PGC-1α E region in coactivator function. (A) CV-1 cells were transfected with MMTV(ERE)-LUC reporter plasmid (250 ng) and plasmids encoding ERα (0.1 ng), PGC-1α wild type or R3K mutant (50 ng), and PRMT1 (200 ng) as indicated, and grown with 20 nM estradiol. (B) CV-1 cells were transfected with pERRα-Luc reporter plasmid (250 ng) and plasmids encoding PRMT1 (250 ng) and PGC-1α wild type or R3K mutant (50 ng).
Figure 6.
Figure 6.
Role of endogenous PRMT1 in PGC-1α coactivator function. COS7 cells in 24-well plates were transfected with 20, 40, or 60 pmol of two different siRNAs against PRMT1 (si#1 and si#2). Two days later, cells were transfected with pERRα-Luc reporter plasmid (125 ng), a PGC-1α expression vector (25 ng), and a Renilla luciferase reporter plasmid driven by a cytomegalovirus promoter (12.5 ng). Luciferase activities were quantified 48 h later; firefly luciferase activity was normalized by Renilla luciferase activity. Protein levels were determined by immunoblots from cell extracts of a fourth siRNA-transfected well. Results shown are from a single experiment that is representative of three independent experiments.
Figure 7.
Figure 7.
Role of Arg methylation in induction of endogenous genes important for the biogenesis and function of mitochondria. (A) SAOS2 cells in six-well plates were infected with adenoviruses expressing GFP, PGC-1α wild type, or PGC-1α R3K mutant (multiplicity of infection = 40) and harvested 24 h later. mRNA levels for the ERRα and cyt c genes were determined by quantitative PCR and normalized to the level of GAPDH mRNA. (B) SAOS2 cells in six-well plates were transfected with siRNA#1 against PRMT1 (100 pmol/well) two times, separated by 72 h. Four hours after the second siRNA transfection, cells were infected with adenovirus expressing GFP or PGC-1α and harvested 24 h later. mRNA levels for the indicated genes were determined by quantitative PCR and normalized to the GAPDH mRNA level. The mean and range of variation shown are from duplicate transfections in a single experiment that is representative of five independent experiments, each with duplicate transfected cell cultures. In all five experiments, the multiplicity of infection was 10-40, and the reduction in PRMT1 mRNA levels caused by siRNA was at least 50%. A paired, two-tailed t-test performed on the values from the five experiments indicated that the siRNA against PRMT1 caused significant decreases in the PGC-1α-induced levels of Cyt c mRNA (p = 0.014) and ERRα mRNA (p = 0.008). For the five experiments, the mean decrease and 95% confidence interval was 35 ± 15% for Cyt c mRNA, 26 ± 5% for ERRα mRNA, and 75 ± 9% for PRMT1 mRNA.
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