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Monoterpenes have an established use in the food and cosmetic industries and have recently also found application as advanced biofuels. Although metabolic engineering efforts have so far achieved significant yields of larger terpenes, monoterpene productivity is lagging behind. Here, we set out to establish a monoterpene-specific production platform in Saccharomyces cerevisiae and identified the sequential reaction mechanism of the yeast farnesyl diphosphate synthase Erg20p to be an important factor limiting monoterpene yield. To overcome this hurdle, we engineered Erg20p into a geranyl diphosphate synthase and achieved a significant increase in monoterpene titers. To further improve production, we converted the engineered geranyl diphosphate synthase into a dominant negative form, so as to decrease the ability of the endogenous Erg20p to function as a farnesyl diphosphate synthase, without entirely abolishing sterol biosynthesis. Fusion of the synthetic dominant negative Erg20p variant with the terpene synthase, combined with yeast strain engineering, further improved monoterpene yields and achieved an overall 340-fold increase in sabinene yield over the starting strain. The design described here can be readily incorporated to any dedicated yeast strain, while the developed plasmid vectors and heterozygous ERG20 deletion yeast strain can also be used as a plug-and-play system for enzyme characterization and monoterpene pathway elucidation.

SUMMARY The histone H3K27 methyltransferase EZH2 plays an important role in oncogenesis, by mechanisms that are incompletely understood. Here we show that the JmjC domain histone H3 demethylase NDY1 synergizes with EZH2 to silence the EZH2 inhibitor miR-101. NDY1 and EZH2 repress miR-101 by binding its promoter in concert, via a process triggered by upregulation of NDY1. Whereas EZH2 binding depends on NDY1, the latter binds independently of EZH2. However, both are required to repress transcription. NDY1 and EZH2 acting in concert, upregulate EZH2 and stabilize the repression of miR-101 and its outcome. NDY1 is induced by FGF-2 via CREB phosphorylation and activation, downstream of DYRK1A, and mediates the FGF-2 and EZH2 effects on cell proliferation, migration and angiogenesis. The FGF-2-NDY1/EZH2-miR-101-EZH2 axis described here, was found to be active in bladder cancer. These data delineate a novel oncogenic pathway that functionally links FGF-2 with EZH2 via NDY1 and miR-101.

The mammalian inducer of apoptosis Bax is lethal when expressed in yeast and plant cells. To identify potential inhibitors of Bax in plants we transformed yeast cells expressing Bax with a tomato cDNA library and we selected for cells surviving after the induction of Bax. This genetic screen allows for the identification of plant genes, which inhibit either directly or indirectly the lethal phenotype of Bax. Using this method a number of cDNA clones were isolated, the more potent of which encodes a protein homologous to the class glutathione S-transferases. This Bax-inhibiting (BI) protein was expressed in Escherichia coli and found to possess glutathione S-transferase (GST) and weak glutathione peroxidase (GPX) activity. Expression of Bax in yeast decreases the intracellular levels of total glutathione, causes a substantial reduction of total cellular phospholipids, diminishes the mitochondrial membrane potential, and alters the intracellular redox potential. Co-expression of the BI-GST/GPX protein brought the total glutathione levels back to normal and re-established the mitochondrial membrane potential but had no effect on the phospholipid alterations. Moreover, expression of BI-GST/GPX in yeast was found to significantly enhance resistance to H 2 O 2 -induced stress. These results underline the relationship between oxidative stress and Baxinduced death in yeast cells and demonstrate that the yeast-based genetic strategy described here is a powerful tool for the isolation of novel antioxidant and antiapoptotic genes.

DNA gyrase is unique among topoisomerases in its ability to introduce negative supercoils into closed-circular DNA. We have demonstrated that deletion of the C-terminal DNA-binding domain of the A subunit of gyrase gives rise to an enzyme that cannot supercoil DNA but relaxes DNA in an ATP-dependent manner. Novobiocin, a competitive inhibitor of ATP binding by gyrase, inhibits this reaction. The truncated enzyme, unlike gyrase, does not introduce a right-handed wrap when bound to DNA and stabilizes DNA crossovers; characteristics reminiscent of conventional type II topoisomerases. This new enzyme form can decatenate DNA circles with increased efficiency compared with intact gyrase and, as a result, can complement the temperaturesensitive phenotype of a parC ts mutant. Thus these results suggest that the unique properties of DNA gyrase are attributable to the wrapping of DNA around the C-terminal DNAbinding domains of the A subunits and provide an insight into the mechanism of type II topoisomerases.DNA topoisomerases are a ubiquitous class of enzymes responsible for the alteration of the topological state of DNA (1). As they are essential to all cells, topoisomerases are potential targets of antibacterial and antineoplastic drugs (2). The topoisomerization reaction is achieved by the cleavage of DNA and the formation of a transient DNA gate through which a part of the same or another DNA molecule is passed. Mechanistic differences in this reaction distinguish topoisomerases into two types. Type I enzymes introduce a single-stranded break in DNA and facilitate the passage of a second strand through this gate. Type II topoisomerases create a doublestranded break and allow the passage of a double-stranded DNA segment. Covalent linkage between type II enzymes and DNA occurs by a transesterification reaction between two tyrosine residues and a pair of phosphates 4 bp apart in the DNA. This results in the attachment of the 5Ј ends of each cleaved DNA strand to the enzyme and the formation of a double-stranded DNA gate. Another DNA segment is then passed through this gate and the break is resealed. Turnover of type II enzymes in topoisomerization reactions generally requires the hydrolysis of ATP.Type II topoisomerases are able to catalyze a number of reactions, including the relaxation of negative and positive supercoils, the catenation and decatenation of DNA circles, and the knotting and unknotting of DNA (3).

The histone H3 demethylase Not dead yet-1 (Ndy1/KDM2B) is a physiological inhibitor of senescence. Here, we show that Ndy1 is down-regulated during senescence in mouse embryonic fibroblasts (MEFs) and that it represses the Ink4a/Arf locus. Ndy1 counteracts the senescence-associated down-regulation of Ezh2, a component of polycomb-repressive complex (PRC) 2, via a JmjC domain-dependent process leading to the global and Ink4a/Arf locus-specific up-regulation of histone H3K27 trimethylation. The latter promotes the Ink4a/ Arf locus-specific binding of Bmi1, a component of PRC1. Ndy1, which interacts with Ezh2, also binds the Ink4a/Arf locus and demethylates the locus-associated histone H3K36me2 and histone H3K4me3. The combination of histone modifications driven by Ndy1 interferes with the binding of RNA Polymerase II, resulting in the transcriptional silencing of the Ink4a/Arf locus and contributing to the Ndy1 immortalization phenotype. Other studies show that, in addition to inhibiting replicative senescence, Ndy1 inhibits Ras oncogene-induced senescence via a similar molecular mechanism.cancer ͉ chromatin ͉ epigenetics ͉ Fbxl10 ͉ JHDM1B

A common integration site, cloned from MoMuLV-induced rat T cell lymphomas, was mapped immediately upstream of Not dead yet-1 (Ndy1)/KDM2B, a gene expressed primarily in testis, spleen, and thymus, that is also known as FBXL10 or JHDM1B. Ndy1 encodes a nuclear, chromatin-associated protein that harbors Jumonji C (JmjC), CXXC, PHD, proline-rich, F-box, and leucine-rich repeat domains. Ndy1 and its homolog Ndy2/KDM2A (FBXL11 or JHDM1A), which is also a target of provirus integration in retrovirus-induced lymphomas, encode proteins that were recently shown to possess Jumonji C-dependent histone H3 K36 dimethyldemethylase or histone H3 K4 trimethyl-demethylase activities. Here, we show that mouse embryo fibroblasts engineered to express Ndy1 or Ndy2 undergo immortalization in the absence of replicative senescence via a JmjC domain-dependent process that targets the Rb and p53 pathways. Knockdown of endogenous Ndy1 or expression of JmjC domain mutants of Ndy1 promote senescence, suggesting that Ndy1 is a physiological inhibitor of senescence in dividing cells and that inhibition of senescence depends on histone H3 demethylation.cancer ͉ histone demethylase ͉ immortalization ͉ senescence ͉ insertional mutagenesis

The mechanism of type II DNA topoisomerases involves the formation of an enzyme-operated gate in one double-stranded DNA segment and the passage of another segment through this gate. DNA gyrase is the only type II topoisomerase able to introduce negative supercoils into DNA, a feature that requires the enzyme to dictate the directionality of strand passage. Although it is known that this is a consequence of the characteristic wrapping of DNA by gyrase, the detailed mechanism by which the transported DNA segment is captured and directed through the DNA gate is largely unknown. We have addressed this mechanism by probing the topology of the bound DNA segment at distinct steps of the catalytic cycle. We propose a model in which gyrase captures a contiguous DNA segment with high probability, irrespective of the superhelical density of the DNA substrate, setting up an equilibrium of the transported segment across the DNA gate. The overall efficiency of strand passage is determined by the position of this equilibrium, which depends on the superhelical density of the DNA substrate. This mechanism is concerted, in that capture of the transported segment by the ATP-operated clamp induces opening of the DNA gate, which in turn stimulates ATP hydrolysis.The topological state of DNA plays an important role in its biological activity and is maintained by a group of enzymes called DNA topoisomerases (1, 2). These enzymes perform the formidable task of passing one DNA segment through another. This feat is achieved by the cleavage of DNA and the formation of a transient DNA gate through which a segment of the same or another DNA molecule is passed. Mechanistic differences in this reaction distinguish topoisomerases into two types. Type I enzymes introduce a single-stranded break in DNA and facilitate the passage of a second strand through this gate. Type II topoisomerases create a double-stranded break and allow the passage of a double-stranded DNA segment.Type II topoisomerases are able to catalyze a number of reactions, including the negative supercoiling of DNA, the relaxation of negative and positive supercoils, the catenation and decatenation of DNA circles, and the knotting and unknotting of DNA (3). These reactions generally require the hydrolysis of ATP. Most type II enzymes [e.g., prokaryotic topoisomerase (topo) IV and eukaryotic topo II] favor DNA relaxation and decatenation reactions. DNA gyrase is the only topoisomerase able to introduce negative supercoils into DNA in an ATP-dependent manner (3, 4). All type II enzymes share a degree of sequence similarity but tend to differ at their C termini (5). DNA gyrase is composed of two subunits, DNA gyrase A protein (GyrA) (97 kDa in Escherichia coli) and DNA gyrase B protein (GyrB) (90 kDa in E. coli), the active form being an A 2 B 2 heterotetramer. Eukaryotic topo IIs are homodimers where the monomer is equivalent to a fusion of the A and B subunits of gyrase, such that the N terminus is homologous to the B subunit, and the C terminus corresponds approximately ...