Molecular biology: RNA interference hangs by a thread - PowerPoint Presentation

Molecular biology: RNA interference hangs by a thread Mikel Zaratiegui The Paf1 protein complex in fission yeast has been found to protect protein-coding genes from inhibition by RNA-mediated silencing of transcription, by stimulating the release of nascent transcripts from DNA.  See Letter p.248 The Paf1 complex represses small-RNA-mediated epigenetic gene silencing Katarzyna Maria Kowalik , Yukiko Shimada, Valentin Flury , Michael Beda Stadler , Julia Batki & Marc Bühler RNA interference ( RNAi ) refers to the ability of exogenously introduced double-stranded RNA to silence expression of homologous sequences. Silencing is initiated when the enzyme Dicer processes the double-stranded RNA into small interfering RNAs (siRNAs). Small RNA molecules are incorporated into Argonaute -protein-containing effector complexes, which they guide to complementary targets to mediate different types of gene silencing, specifically post-transcriptional gene silencing and chromatin-dependent gene silencing 1 . Although endogenous small RNAs have crucial roles in chromatin-mediated processes across kingdoms, efforts to initiate chromatin modifications  in trans  by using siRNAs have been inherently difficult to achieve in all eukaryotic cells. Using fission yeast, here we show that RNAi -directed heterochromatin formation is negatively controlled by the highly conserved RNA polymerase-associated factor 1 complex (Paf1C). Temporary expression of a synthetic hairpin RNA in Paf1C mutants triggers stable heterochromatin formation at homologous loci, effectively silencing genes  in trans . This repressed state is propagated across generations by the continual production of secondary siRNAs, independently of the synthetic hairpin RNA. Our data support a model in which Paf1C prevents targeting of nascent transcripts by the siRNA-containing RNA-induced transcriptional silencing complex and thereby epigenetic gene silencing, by promoting efficient transcription termination and rapid release of the RNA from the site of transcription. We show that although compromised transcription termination is sufficient to initiate the formation of bi-stable heterochromatin by  trans -acting siRNAs, impairment of both transcription termination and nascent transcript release is imperative to confer stability to the repressed state. Our work uncovers a novel mechanism for small-RNA-mediated epigenome regulation and highlights fundamental roles for Paf1C and the RNAi machinery in building epigenetic memory.

Metabolism: Growth in the fat lane Robert A. Egnatchik & Ralph J. DeBerardinisAnalysis of endothelial cells, which are involved in blood-vessel formation, unexpectedly reveals that proliferation in this cell type depends on fatty-acid oxidation to support DNA synthesis.  See Article  p.192 Fatty acid carbon is essential for dNTP synthesis in endothelial cells. Schoors S1, Bruning U1, Missiaen R1, Queiroz KC1, Borgers G1, Elia I2, Zecchin A1, Cantelmo AR1, Christen S2, Goveia J1, Heggermont W3, Goddé L1, Vinckier S1, Van Veldhoven PP4, Eelen G1, Schoonjans L1, Gerhardt H5, Dewerchin M1, Baes M6, De Bock K7, Ghesquière B1, Lunt SY8, Fendt SM2, Carmeliet P1. The metabolism of endothelial cells during vessel sprouting remains poorly studied. Here we report that endothelial loss of CPT1A, a rate-limiting enzyme of fatty acid oxidation (FAO), causes vascular sprouting defects due to impaired proliferation, not migration, of human and murine endothelial cells. Reduction of FAO in endothelial cells did not cause energy depletion or disturb redox homeostasis, but impaired de novo nucleotide synthesis for DNA replication. Isotope labelling studies in control endothelial cells showed that fatty acid carbons substantially replenished the Krebs cycle, and were incorporated into aspartate (a nucleotide precursor), uridine monophosphate (a precursor of pyrimidine nucleoside triphosphates) and DNA. CPT1A silencing reduced these processes and depleted endothelial cell stores of aspartate and deoxyribonucleoside triphosphates. Acetate (metabolized to acetyl-CoA, thereby substituting for the depleted FAO-derived acetyl-CoA) or a nucleoside mix rescued the phenotype of CPT1A-silenced endothelial cells. Finally, CPT1 blockade inhibited pathological ocular angiogenesis in mice, suggesting a novel strategy for blocking angiogenesis.

EZH2 inhibition sensitizes BRG1 and EGFR mutant lung tumours to TopoII inhibitors.Fillmore CM1, Xu C2, Desai PT3, Berry JM3, Rowbotham SP1, Lin YJ4, Zhang H2, Marquez VE5, Hammerman PS6, Wong KK2, Kim CF1. Non-small-cell lung cancer is the leading cause of cancer-related death worldwide. Chemotherapies such as the topoisomerase II ( TopoII ) inhibitor etoposide effectively reduce disease in a minority of patients with this cancer; therefore, alternative drug targets, including epigenetic enzymes, are under consideration for therapeutic intervention. A promising potential epigenetic target is the methyltransferase EZH2, which in the context of the polycomb repressive complex 2 (PRC2) is well known to tri-methylate histone H3 at lysine 27 (H3K27me3) and elicit gene silencing. Here we demonstrate that EZH2 inhibition has differential effects on the TopoII inhibitor response of non-small-cell lung cancers in vitro and in vivo. EGFR and BRG1 mutations are genetic biomarkers that predict enhanced sensitivity to TopoII inhibitor in response to EZH2 inhibition. BRG1 loss-of-function mutant tumours respond to EZH2 inhibition with increased S phase, anaphase bridging, apoptosis and TopoII inhibitor sensitivity. Conversely, EGFR and BRG1 wild-type tumours upregulate BRG1 in response to EZH2 inhibition and ultimately become more resistant to TopoII inhibitor. EGFR gain-of-function mutant tumours are also sensitive to dual EZH2 inhibition and TopoII inhibitor, because of genetic antagonism between EGFR and BRG1. These findings suggest an opportunity for precision medicine in the genetically complex disease of non-small-cell lung cancer.

Cardiology: Race for healthy hearts Marc-Phillip Hitz & Gregor AndelfingerTransplantation experiments in mice reveal that the increased risk of congenital heart disease in the pups of older mothers is not conferred by ageing eggs, but by the mothers' age, and can be mitigated by exercise.  See Letter  p.230 The maternal-age-associated risk of congenital heart disease is modifiable Claire E. Schulkey , Suk D. Regmi , Rachel A. Magnan , Megan T. Danzo , Herman Luther, Alayna K. Hutchinson, Adam A. Panzer, Mary M. Grady, David B. Wilson & Patrick Y. Jay

Scientific Reports

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12 Dopamine Modulates Egalitarian (all people d eserve equal rights and opportunities) Behavior in Humans Helen Wills Neuroscience Institute, University of California, Berkeley, 175 Li Ka Shing Center, Berkeley, CA 94720, USA Summary Egalitarian motives form a powerful force in promoting prosocial behavior and enabling large-scale cooperation in the human species [ 1 ]. At the neural level, there is substantial, albeit correlational, evidence suggesting a link between dopamine and such behavior [ 2, 3 ]. However, important questions remain about the specific role of dopamine in setting or modulating behavioral sensitivity to prosocial concerns. Here, using a combination of pharmacological tools and economic games, we provide critical evidence for a causal involvement of dopamine in human egalitarian tendencies. Specifically, using the brain penetrant catechol-O-methyl transferase (COMT) inhibitor tolcapone [ 4, 5 ], we investigated the causal relationship between dopaminergic mechanisms and two prosocial concerns at the core of a number of widely used economic games: (1) the extent to which individuals directly value the material payoffs of others, i.e., generosity, and (2) the extent to which they are averse to differences between their own payoffs and those of others, i.e., inequity. We found that dopaminergic augmentation via COMT inhibition increased egalitarian tendencies in participants who played an extended version of the dictator game [ 6 ]. Strikingly, computational modeling of choice behavior [ 7 ] revealed that tolcapone exerted selective effects on inequity aversion, and not on other computational components such as the extent to which individuals directly value the material payoffs of others. Together, these data shed light on the causal relationship between neurochemical systems and human prosocial behavior and have potential implications for our understanding of the complex array of social impairments accompanying neuropsychiatric disorders involving dopaminergic dysregulation .

13 Brain Regeneration in Drosophila Involves Comparison of Neuronal Fitness Institute of Cell Biology (IZB), University of Bern, Bern 3012, Switzerland Summary Darwinian-like cell selection has been studied during development and cancer [ 1–11 ]. Cell selection is often mediated by direct intercellular comparison of cell fitness, using “fitness fingerprints” [ 12–14 ]. In Drosophila, cells compare their fitness via several isoforms of the transmembrane protein Flower [ 12, 13 ]. Here, we studied the role of intercellular fitness comparisons during regeneration. Regeneration-competent organisms are traditionally injured by amputation [ 15, 16 ], whereas in clinically relevant injuries such as local ischemia or traumatic injury, damaged tissue remains within the organ [ 17–19 ]. We reasoned that “Darwinian” interactions between old and newly formed tissues may be important in the elimination of damaged cells. We used a model of adult brain regeneration in Drosophila in which mechanical puncture activates regenerative neurogenesis based on damage-responsive stem cells [ 20 ]. We found that apoptosis after brain injury occurs in damage-exposed tissue located adjacent to zones of de novo neurogenesis. Injury-affected neurons start to express isoforms of the Flower cell fitness indicator protein not found on intact neurons. We show that this change in the neuronal fitness fingerprint is required to recognize and eliminate such neurons. Moreover, apoptosis is inhibited if all neurons express “low-fitness” markers, showing that the availability of new and healthy cells drives tissue replacement. In summary, we found that elimination of impaired tissue during brain regeneration requires comparison of neuronal fitness and that tissue replacement after brain damage is coordinated by injury-modulated fitness fingerprints. Intercellular fitness comparisons between old and newly formed tissues could be a general mechanism of regenerative tissue replacement.

Nature reviews Mol. Cell Biol.