SirT1 regulation of antioxidant genes is dependent on the formation of a FoxO3a/PGC-1α complex.

UNLABELLED: SirT1 is a class III histone deacetylase that has been implicated in metabolic and reactive oxygen species control. In the vasculature it has been shown to decrease endothelial superoxide production, prevent endothelial dysfunction and atherosclerosis. However, the mechanisms that mediate SirT1 antioxidant functions remain to be characterized. The transcription factor FoxO3a and the transcriptional coactivator peroxisome proliferator activated receptor γ-coactivator 1α (PGC-1α) have been shown to induce the expression of antioxidant genes and to be deacetylated by SirT1. AIMS: Here we investigated SirT1 regulation of antioxidant genes and the roles played by FoxO3a and PGC-1α in this regulation. RESULTS: We found that SirT1 regulates the expression of several antioxidant genes in bovine aortic endothelial cells, including Mn superoxide dismutase (MnSOD), catalase, peroxiredoxins 3 and 5 (Prx3, Prx5), thioredoxin 2 (Trx2), thioredoxin reductase 2 (TR2), and uncoupling protein 2 (UCP-2) and can be localized in the regulatory regions of these genes. We also found that knockdown of either FoxO3a or PGC-1α prevented the induction of antioxidant genes by SirT1 over-expression. Furthermore, SirT1 increased the formation of a FoxO3a/PGC-1α complex as determined by co-immunoprecipitation (IP) assays, concomitantly reducing H2O2-dependent FoxO3a and PGC-1α acetylation. Data showing that FoxO3a knockdown increases PGC-1α acetylation levels and vice versa, suggest that SirT1 activity on FoxO3a and PGC-1α may be dependent of the formation of a FoxO3a/PGC-1α complex. INNOVATION: A unifying mechanism for SirT1 activities is suggested. CONCLUSION: We show that SirT1 regulation of antioxidant genes in vascular endothelial cells depends on the formation of a FoxO3a/PGC-1α complex.

PGC-1α regulates translocated in liposarcoma activity: role in oxidative stress gene expression.

Translocated in liposarcoma (TLS) is a poorly characterized multifunctional protein involved in the genotoxic response. TLS regulates gene expression at several steps, including splicing and mRNA transport, possibly connecting transcriptional and posttranscriptional events. AIMS: In this study we aimed to idenfity molecular targets and regulatory partners of TLS. RESULTS AND INNOVATION: Here we report that TLS transcriptionally regulates the expression of oxidative stress protection genes. This regulation requires interaction with the transcriptional coactivator peroxisome proliferator activated receptor γ-coactivator 1α (PGC-1α), a master regulator of mitochondrial function that coordinately induces the expression of genes involved in detoxification of mitochondrial reactive oxygen species (ROS). Microarray gene expression analysis showed that TLS transcriptional activity is impaired in the absence of PGC-1α, and is thus largely dependent on PGC-1α. CONCLUSION: These results suggest the existence of a regulatory circuit linking the control of ROS detoxification to the coordinated cross-talk between oxidative metabolism and the cellular response to genomic DNA damage.

Inactivation of Foxo3a and subsequent downregulation of PGC-1 alpha mediate nitric oxide-induced endothelial cell migration.

In damaged or proliferating endothelium, production of nitric oxide (NO) from endothelial nitric oxide synthase (eNOS) is associated with elevated levels of reactive oxygen species (ROS), which are necessary for endothelial migration. We aimed to elucidate the mechanism that mediates NO induction of endothelial migration. NO downregulates expression of peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1 alpha), which positively modulates several genes involved in ROS detoxification. We tested whether NO-induced cell migration requires PGC-1 alpha downregulation and investigated the regulatory pathway involved. PGC-1 alpha negatively regulated NO-dependent endothelial cell migration in vitro, and inactivation of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathway, which is activated by NO, reduced NO-mediated downregulation of PGC-1 alpha. Expression of constitutively active Foxo3a, a target for Akt-mediated inactivation, reduced NO-dependent PGC-1 alpha downregulation. Foxo3a is also a direct transcriptional regulator of PGC-1 alpha, and we found that a functional FoxO binding site in the PGC-1 alpha promoter is also a NO response element. These results show that NO-mediated downregulation of PGC-1 alpha is necessary for NO-induced endothelial migration and that NO/protein kinase G (PKG)-dependent downregulation of PGC-1 alpha and the ROS detoxification system in endothelial cells are mediated by the PI3K/Akt signaling pathway and subsequent inactivation of the FoxO transcription factor Foxo3a.

Nucleosome binding and histone methyltransferase activity of Drosophila PRC2.

The Drosophila Polycomb group protein E(z) is a histone methyltransferase (HMTase) that is essential for maintaining HOX gene silencing during development. E(z) exists in a multiprotein complex called Polycomb repressive complex 2 (PRC2) that also contains Su(z)12, Esc and Nurf55. Reconstituted recombinant PRC2 methylates nucleosomes in vitro, but recombinant E(z) on its own shows only poor HMTase activity on nucleosomes. Here, we investigate the function of the PRC2 subunits. We show that PRC2 binds to nucleosomes in vitro but that individual PRC2 subunits alone do not bind to nucleosomes. By analysing PRC2 subcomplexes, we show that Su(z)12-Nurf55 is the minimal nucleosome-binding module of PRC2 and that Esc contributes to high-affinity binding of PRC2 nucleosomes. We find that nucleosome binding of PRC2 is not sufficient for histone methylation and that only complexes that contain Esc protein show robust HMTase activity. These observations suggest that different subunits provide mechanistically distinct functions within the PRC2 HMTase: the nucleosome-binding subunits Su(z)12 and Nurf55 anchor the E(z) enzyme on chromatin substrates, whereas Esc is needed to boost enzymatic activity.

Histone methyltransferase activity of a Drosophila Polycomb group repressor complex.

Polycomb group (PcG) proteins maintain transcriptional repression during development, likely by creating repressive chromatin states. The Extra Sex Combs (ESC) and Enhancer of Zeste [E(Z)] proteins are partners in an essential PcG complex, but its full composition and biochemical activities are not known. A SET domain in E(Z) suggests this complex might methylate histones. We purified an ESC-E(Z) complex from Drosophila embryos and found four major subunits: ESC, E(Z), NURF-55, and the PcG repressor, SU(Z)12. A recombinant complex reconstituted from these four subunits methylates lysine-27 of histone H3. Mutations in the E(Z) SET domain disrupt methyltransferase activity in vitro and HOX gene repression in vivo. These results identify E(Z) as a PcG protein with enzymatic activity and implicate histone methylation in PcG-mediated silencing.