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.

Polycomb response elements (PREs) are specific cis-regulatory sequences needed for transcriptional repression of HOX and other target genes by Polycomb group (PcG) proteins. Among the many PcG proteins known in Drosophila, Pho is the only sequence-specific DNA-binding protein. To gain insight into the function of Pho, we purified Pho protein complexes from Drosophila embryos and found that Pho exists in two distinct protein assemblies: a Pho-dINO80 complex containing the Drosophila INO80 nucleosome-remodeling complex, and a Pho-repressive complex (PhoRC) containing the uncharacterized gene product dSfmbt. Analysis of PhoRC reveals that dSfmbt is a novel PcG protein that is essential for HOX gene repression in Drosophila. PhoRC is bound at HOX gene PREs in vivo, and this targeting strictly depends on Pho-binding sites. Characterization of dSfmbt protein shows that its MBT repeats have unique discriminatory binding activity for methylated lysine residues in histones H3 and H4; the MBT repeats bind mono-and di-methylated H3-K9 and H4-K20 but fail to interact with these residues if they are unmodified or tri-methylated. Our results establish PhoRC as a novel Drosophila PcG protein complex that combines DNA-targeting activity (Pho) with a unique modified histone-binding activity (dSfmbt). We propose that PRE-tethered PhoRC selectively interacts with methylated histones in the chromatin flanking PREs to maintain a Polycomb-repressed chromatin state.[Keywords: Polycomb group; PRE; Pho/dYY1; MBT repeat; histone methylation] Supplemental material is available at http://www.genesdev.org. The regulation of gene expression by Polycomb group (PcG) and trithorax group (trxG) proteins represents a paradigm for understanding the establishment and maintenance of heritable transcriptional states during development. PcG and trxG genes were first genetically identified as regulators that are required for the long-term maintenance of HOX gene expression patterns in Drosophila. PcG proteins keep HOX genes silenced in cells in which they must stay inactive, whereas trxG proteins maintain the active state of these genes in appropriate cells (for review, see Ringrose and Paro 2004). This regulatory relationship is conserved in vertebrates, where PcG and trxG proteins also regulate HOX gene expression. In addition, mammalian PcG and trxG proteins have also been implicated in X-chromosome inactivation, hematopoietic development, control of cell proliferation, and oncogenic processes.Drosophila HOX genes are among the best-studied target genes of the PcG/trxG system. Different studies have led to the identification of specific cis-regulatory sequences in HOX genes that are called Polycomb response elements (PREs) and are required for silencing by PcG proteins. PREs are typically several hundred base pairs in length, and they function as potent transcriptional silencer elements in the context of HOX reporter genes as well as in a variety of other reporter gene assays (e.g., Chan et al. 1994;Zink and Paro 1995;Sengupta et al. 2004). This ope...

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 c-coactivator 1a (PGC-1a) 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-1a 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-1a prevented the induction of antioxidant genes by SirT1 over-expression. Furthermore, SirT1 increased the formation of a FoxO3a/PGC-1a complex as determined by co-immunoprecipitation (IP) assays, concomitantly reducing H 2 O 2 -dependent FoxO3a and PGC-1a acetylation. Data showing that FoxO3a knockdown increases PGC-1a acetylation levels and vice versa, suggest that SirT1 activity on FoxO3a and PGC-1a may be dependent of the formation of a FoxO3a/PGC-1a 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-1a complex. Antioxid. Redox Signal. 19, 1507Signal. 19, -1521

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.

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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 ␥ coactivator 1␣ (PGC-1␣), which positively modulates several genes involved in ROS detoxification. We tested whether NO-induced cell migration requires PGC-1␣ downregulation and investigated the regulatory pathway involved. PGC-1␣ 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␣. Expression of constitutively active Foxo3a, a target for Akt-mediated inactivation, reduced NO-dependent PGC-1␣ downregulation. Foxo3a is also a direct transcriptional regulator of PGC-1␣, and we found that a functional FoxO binding site in the PGC-1␣ promoter is also a NO response element. These results show that NO-mediated downregulation of PGC-1␣ is necessary for NO-induced endothelial migration and that NO/protein kinase G (PKG)-dependent downregulation of PGC-1␣ 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.Nitric oxide (NO) produced by endothelial nitric oxide synthase (eNOS) plays critical roles in the physiology of endothelial cells (5), regulating vascular tone (19), endothelial cell growth (10), migration (22), survival under stress (4), and the levels of reactive oxygen species (ROS) (32). NO-induced angiogenesis requires a NO-dependent increase in ROS (2, 25), and although the molecular mechanisms linking NO and ROS homeostasis have been a matter of intense research, they are still only partly understood (reviewed in reference 20). NOdependent induction of angiogenesis requires activation of the soluble guanylyl cyclase (sGC)/protein kinase G (PKG)/phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) pathway (13). However, whether and how this pathway is associated with the increase in ROS levels are still unclear.It has been proposed that endothelial angiogenesis requires the inactivation of FoxO transcription factors by Akt (11,26). Akt directly phosphorylates and inactivates members of the FoxO family, which are consequently translocated from the nucleus to the cytoplasm (12). The FoxO factors Foxo3a and Foxo4 protect cells from oxidative stress (14), although the significance of this process in normal vascular homeostasis is still unknown.Peroxisome proliferator-activated receptor ␥ coactivator 1␣ (PGC-1␣) is a master regulator of cellular energy metabolism (8) and a positive and direct regulator of a set of genes whose products protect against oxidative stress (29, 31). PGC-1␣ is inactivated by phosphorylation by Akt...

Ascorbic acid is known to influence proliferation and functional properties of several cell types and is therefore widely used in tissue engineering. In this study, the effect of ascorbic acid on the proliferation and functional properties of hyalocytes was evaluated. Hyalocytes were cultured with different amounts of ascorbic acid in classical two-dimensional (2-D) cultures and a three-dimensional (3-D) pellet culture system. Ascorbic acid enhanced hyalocyte proliferation dose-dependently at concentrations between 0.1 and 3 microg/mL; proliferation was constant over a wide concentration range up to 150 microg/mL, concentrations of 500 microg/mL showed toxic effects. In 2-D hyalocyte culture, the accumulation of glycosaminoglycans (GAG) and collagens increased in response to ascorbic acid supplementation of 10 or 200 microg/mL. Normalized to the cell number, GAG production was not influenced, whereas collagen production increased. These results could be verified in a pellet-like 3-D culture system. Ascorbic acid also influenced hyalocytes on the mRNA level; the expression of COL11A1 was clearly enhanced by ascorbic acid. To conclude, ascorbic acid modulates proliferation and collagen accumulation of hyalocytes; it also influences mRNA expression of the cells. Taken together with the fact that ascorbic acid is present in high concentrations in the vitreous body, this vitamin seems to be an important factor for in vitro hyalocyte culture.

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 c-coactivator 1a (PGC-1a), 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-1a, and is thus largely dependent on PGC-1a. 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. Antioxid. Redox Signal. 15, 325-337.