ALBERT

Beschreibung: 27 December 2012 Publication year: 2012 Source: Cell Reports, Volume 2, Issue 6 Insulin stimulates glucose uptake through the membrane translocation of GLUT4 and GLUT1. Peroxisome proliferator-activated receptor γ (PPARγ) enhances insulin sensitivity. Here, we demonstrate that insulin stimulates GLUT4 and GLUT1 translocation, and glucose uptake, by activating the signaling pathway involving nicotinic acid adenine dinucleotide phosphate (NAADP), a calcium mobilizer, in adipocytes. We also demonstrate that PPARγ mediates insulin sensitization by enhancing NAADP production through upregulation of CD38, the only enzyme identified for NAADP synthesis. Insulin produced NAADP by both CD38-dependent and -independent pathways, whereas PPARγ produced NAADP by CD38-dependent pathway. Blocking the NAADP signaling pathway abrogated both insulin-stimulated and PPARγ-induced GLUT4 and GLUT1 translocation, thereby inhibiting glucose uptake. CD38 knockout partially inhibited insulin-stimulated glucose uptake. However, CD38 knockout completely blocked PPARγ-induced glucose uptake in adipocytes and PPARγ-mediated amelioration of glucose tolerance in diabetic mice. These results demonstrated that the NAADP signaling pathway is a critical molecular target for PPARγ-mediated insulin sensitization. Graphical abstract Highlights ► Insulin induces glucose uptake via NAADP-mediated calcium increase ► PPARγ mediates insulin sensitization by upregulating the NAADP-producing enzyme CD38 ► PPARγ agonists ameliorate glucose tolerance by CD38 upregulation

Beschreibung: Available online 27 December 2012 Publication year: 2012 Source: Cell Reports Enveloped viruses have developed various adroit mechanisms to invade their host cells. This process requires one or more viral envelope glycoprotein to achieve cell attachment and membrane fusion. Members of the Flaviviridae such as flaviviruses possess only one envelope glycoprotein, E, whereas pestiviruses and hepacivirus encode two glycoproteins, E1 and E2. Although E2 is involved in cell attachment, it has been unclear which protein is responsible for membrane fusion. We report the crystal structures of the homodimeric glycoprotein E2 from the pestivirus bovine viral diarrhea virus 1 (BVDV1) at both neutral and low pH. Unexpectedly, BVDV1 E2 does not have a class II fusion protein fold, and at low pH the N-terminal domain is disordered, similarly to the intermediate postfusion state of E2 from sindbis virus, an alphavirus. Our results suggest that the pestivirus and possibly the hepacivirus fusion machinery are unlike any previously observed. Graphical abstract Highlights ► Structure of the major antigenically dominant protein of BVDV ► The overall fold of BVDV E2 shows no similarity to the class II fusion proteins ► At low pH, BVDV E2 N-terminal domain is disordered ► Entry mechanism of BVDV is probably applicable to hepatitis C virus

Beschreibung: 27 December 2012 Publication year: 2012 Source: Cell Reports, Volume 2, Issue 6 Controversy has recently arisen over the role of sirtuins in metazoan aging. In this issue of Cell Reports , Banerjee et al. demonstrate that Drosophila Sir2 is necessary for life span extension in response to dietary restriction and that its overexpression in the fat body increases the life span.

Beschreibung: 27 December 2012 Publication year: 2012 Source: Cell Reports, Volume 2, Issue 6 Inhibition of sirtuin 2 (SIRT2) deacetylase mediates protective effects in cell and invertebrate models of Parkinson’s disease and Huntington’s disease (HD). Here we report the in vivo efficacy of a brain-permeable SIRT2 inhibitor in two genetic mouse models of HD. Compound treatment resulted in improved motor function, extended survival, and reduced brain atrophy and is associated with marked reduction of aggregated mutant huntingtin, a hallmark of HD pathology. Our results provide preclinical validation of SIRT2 inhibition as a potential therapeutic target for HD and support the further development of SIRT2 inhibitors for testing in humans. Graphical abstract Highlights ► Sirtuin 2 (SIRT2) inhibition is neuroprotective in two HD mouse models ► SIRT2 inhibitor treatment markedly reduces huntingtin aggregates in HD mouse brain ► SIRT2 is a promising therapeutic target for neurological protein aggregation disorders

Beschreibung: 27 December 2012 Publication year: 2012 Source: Cell Reports, Volume 2, Issue 6 Planar cell polarity (PCP) refers to the collective orientation of cells within the epithelial plane. We show that progenitor cells forming the ducts of the embryonic pancreas express PCP proteins and exhibit an active PCP pathway. Planar polarity proteins are acquired at embryonic day 11.5 synchronously to apicobasal polarization of pancreas progenitors. Loss of function of the two PCP core components Celsr2 and Celsr3 shows that they control the differentiation of endocrine cells from polarized progenitors, with a prevalent effect on insulin-producing beta cells. This results in a decreased glucose clearance. Loss of Celsr2 and 3 leads to a reduction of Jun phosphorylation in progenitors, which, in turn, reduces beta cell differentiation from endocrine progenitors. These results highlight the importance of the PCP pathway in cell differentiation in vertebrates. In addition, they reveal that tridimensional organization and collective communication of cells are needed in the pancreatic epithelium in order to generate appropriate numbers of endocrine cells. Graphical abstract Highlights ► PCP proteins are restricted to progenitor cells in the embryonic pancreas ► The PCP core components Celsr2 and Celsr3 control endocrine cell differentiation ► This effect is mediated by the JNK pathway

Beschreibung: 27 December 2012 Publication year: 2012 Source: Cell Reports, Volume 2, Issue 6 MicroRNAs (miRNAs) are small, noncoding RNAs that inhibit translation and promote mRNA decay. The levels of mature miRNAs are the result of different rates of transcription, processing, and turnover. The noncanonical polymerase Gld2 has been implicated in the stabilization of miR-122, possibly through catalyzing 3′ monoadenylation; however, there is little evidence that this relationship is one of cause and effect. Here, we biochemically characterize Gld2’s involvement in miRNA monoadenylation and its effect on miRNA stability. We find that Gld2 directly monoadenylates and stabilizes specific miRNA populations in human fibroblasts and that sensitivity to monoadenylation-induced stability depends on nucleotides in the miRNA 3′ end. These results establish a mechanism of miRNA stability and resulting posttranscriptional gene regulation. Graphical abstract Highlights ► Gld2 monoadenylates specific microRNAs (miRNAs) ► Monoadenylation stabilizes miRNA subpopulations and prolongs their activity ► Sensitivity to monoadenylation and stability depends on nucleotides in the miRNA 3′ end

Beschreibung: 27 December 2012 Publication year: 2012 Source: Cell Reports, Volume 2, Issue 6 In the cytoplasm, the correct delivery of membrane proteins is an essential and highly regulated process. The posttranslational targeting of the important tail-anchor membrane (TA) proteins has recently been under intense investigation. A specialized pathway, called the guided entry of TA proteins (GET) pathway in yeast and the transmembrane domain recognition complex (TRC) pathway in vertebrates, recognizes endoplasmic-reticulum-targeted TA proteins and delivers them through a complex series of handoffs. An early step is the formation of a complex between Sgt2/SGTA, a cochaperone with a presumed ubiquitin-like-binding domain (UBD), and Get5/UBL4A, a ubiquitin-like domain (UBL)-containing protein. We structurally characterize this UBD/UBL interaction for both yeast and human proteins. This characterization is supported by biophysical studies that demonstrate that complex formation is mediated by electrostatics, generating an interface that has high-affinity with rapid kinetics. In total, this work provides a refined model of the interplay of Sgt2 homologs in TA targeting. Graphical abstract Highlights ► Sgt2 homologs contain dimerization motifs that are novel UBDs ► The UBL of Get5 homologs has features that distinguish it from other UBLs ► Complex formation of Sgt2 and Get5 includes a conserved and dynamic interface ► The system can rapidly discriminate between other UBL pathways

Beschreibung: 27 December 2012 Publication year: 2012 Source: Cell Reports, Volume 2, Issue 6 Chimera formation after blastocyst injection or morula aggregation is the principal functional assay of the developmental potential of mouse embryonic stem cells (ESCs). This property, which demonstrates functional equivalence between ESCs and the preimplantation epiblast, is not shared by epiblast stem cell (EpiSC) lines. Here, we show that EpiSCs derived either from postimplantation embryos or from ESCs in vitro readily generate chimeras when grafted to postimplantation embryos in whole embryo culture. EpiSC derivatives integrate and differentiate to derivatives of all three embryonic germ layers and primordial germ cells. In contrast, grafted ESCs seldom proliferate in postimplantation embryos, and fail to acquire the identity of their host-derived neighbors. EpiSCs do not incorporate efficiently into embryonic day 8.5 embryos, a stage by which pluripotency has been lost. Thus, chimera formation by EpiSCs requires a permissive environment, the postimplantation epiblast, and demonstrates functional equivalence between this cell type and EpiSCs. Graphical abstract Highlights ► Epiblast stem cells (EpiSCs) form chimeras when injected into postimplantation epiblast ► Embryonic stem cells do not form postimplantation chimeras ► EpiSCs do not integrate if they are injected after gastrulation

Beschreibung: 27 December 2012 Publication year: 2012 Source: Cell Reports, Volume 2, Issue 6 An increased understanding of antitumor immunity is necessary for improving cell-based immunotherapies against human cancers. Here, we investigated the roles of two immune system-expressed microRNAs (miRNAs), miR-155 and miR-146a, in the regulation of antitumor immune responses. Our results indicate that miR-155 promotes and miR-146a inhibits interferon γ (IFNγ) responses by T cells and reduces solid tumor growth in vivo. Using a double-knockout (DKO) mouse strain deficient in both miR-155 and miR-146a, we have also identified an epistatic relationship between these two miRNAs. DKO mice had defective T cell responses and tumor growth phenotypes similar to miR-155 −/− mice. Further analysis of the T cell compartment revealed that miR-155 modulates IFNγ expression through a mechanism involving repression of Ship1. Our work reveals critical roles for miRNAs in the reciprocal regulation of CD4 + and CD8 + T cell-mediated antitumor immunity and demonstrates the dominant nature of miR-155 during its promotion of immune responses. Graphical abstract Highlights ► miR-155 promotes and miR-146a inhibits both CD4+ and CD8+ T cell antitumor responses ► DKO mice reveal epistasis between miR-155 and miR-146a during tumor immunity ► miR-155 regulation of IFNγ involves repression of its target Ship1 in T cells

Beschreibung: Available online 20 December 2012 Publication year: 2012 Source: Cell Reports In the fission yeast Schizosaccharomyces pombe , a chromosomal imprinting event controls the asymmetric pattern of mating-type switching. The orientation of DNA replication at the mating-type locus is instrumental in this process. However, the factors leading to imprinting are not fully identified and the mechanism is poorly understood. Here, we show that the replication fork pause at the mat1 locus ( MPS1 ), essential for imprint formation, depends on the lysine-specific demethylase Lsd1. We demonstrate that either Lsd1 or Lsd2 amine oxidase activity is required for these processes, working upstream of the imprinting factors Swi1 and Swi3 (homologs of mammalian Timeless and Tipin, respectively). We also show that the Lsd1/2 complex controls the replication fork terminators, within the rDNA repeats. These findings reveal a role for the Lsd1/2 demethylases in controlling polar replication fork progression, imprint formation, and subsequent asymmetric cell divisions. Graphical abstract Highlights ► The lysine-specific demethylase, Lsd1, is a mating-type switching mutant ► Lsd1 is essential for replication fork pausing at the mat1 locus ► Lsd1 is essential for replication fork arrest at the replication fork block at rDNA loci ► Lsd1 and Lsd2 act in a redundant manner for replication fork pausing

Beschreibung: Available online 21 December 2012 Publication year: 2012 Source: Cell Reports Chromatin dynamics play a central role in maintaining genome integrity, but how this is achieved remains largely unknown. Here, we report that microrchidia CW-type zinc finger 2 (MORC2), an uncharacterized protein with a derived PHD finger domain and a conserved GHKL-type ATPase module, is a physiological substrate of p21-activated kinase 1 (PAK1), an important integrator of extracellular signals and nuclear processes. Following DNA damage, MORC2 is phosphorylated on serine 739 in a PAK1-dependent manner, and phosphorylated MORC2 regulates its DNA-dependent ATPase activity to facilitate chromatin remodeling. Moreover, MORC2 associates with chromatin and promotes gamma-H2AX induction in a PAK1 phosphorylation-dependent manner. Consequently, cells expressing MORC2-S739A mutation displayed a reduction in DNA repair efficiency and were hypersensitive to DNA-damaging agent. These findings suggest that the PAK1-MORC2 axis is critical for orchestrating the interplay between chromatin dynamics and the maintenance of genomic integrity through sequentially integrating multiple essential enzymatic processes. Graphical abstract Highlights ► MORC2 is a DNA damage-responsive phosphoprotein activated by PAK1 kinase ► MORC2 regulates phosphorylation-coupled, ATPase-dependent chromatin remodeling ► MORC2 facilitates gamma-H2AX induction independently of PIKK kinases ► MORC2 promotes DSB repair in a PAK1 phosphorylation-dependent manner

Beschreibung: Publication year: 2012 Source: Cell Reports Hong Zan, Clayton A. White, Lisa M. Thomas, Thach Mai, Guideng Li, Zhenming Xu, Jinsong Zhang, Paolo Casali By diversifying the biological effector functions of antibodies, class switch DNA recombination (CSR) plays a critical role in the maturation of the immune response. It is initiated by activation-induced cytidine deaminase (AID)-mediated deoxycytosine deamination, yielding deoxyuridine (dU), and dU glycosylation by uracil DNA glycosylase (Ung) in antibody switch (S) region DNA. Here we showed that the translesion DNA synthesis polymerase Rev1 directly interacted with Ung and targeted in an AID-dependent and Ung-independent fashion the S regions undergoing CSR. Rev1 −/− Ung +/+ B cells reduced Ung recruitment to S regions, DNA-dU glycosylation, and CSR. Together with an S region spectrum of mutations similar to that of Rev1 +/+ Ung −/− B cells, this suggests that Rev1 operates in the same pathway as Ung, as emphasized by further decreased CSR in Rev1 −/− Msh2 −/− B cells. Rescue of CSR in Rev1 −/− B cells by a catalytically inactive Rev1 mutant shows that the important role of Rev1 in CSR is mediated by Rev1’s scaffolding function, not its enzymatic function. Graphical abstract Graphical Abstract Highlights ► Rev1 DNA polymerase plays an important role in class switch DNA recombination ► This role is mediated by Rev1’s scaffolding function, not its enzymatic function ► Rev1 interacts with Ung and recruits it to switch region DNA ► Rev1 enhances Ung-mediated dU glycosylation in DNA

Beschreibung: Publication year: 2012 Source: Cell Reports Michihiko Sugimoto, Masayo Kondo, Michiko Hirose, Misao Suzuki, Kazuyuki Mekada, Takaya Abe, Hiroshi Kiyonari, Atsuo Ogura, Nobuo Takagi, Karen Artzt, Kuniya Abe After implantation, pluripotent epiblasts are converted to embryonic ectoderm through cell–cell interactions that significantly change the transcriptional and epigenetic networks. An entrée to understanding this vital developmental transition is the t w5 mutation of the mouse t complex. This mutation produces highly specific defects in the embryonic ectoderm before gastrulation, leading to death of the embryonic ectoderm. Using a positional cloning approach, we have now identified the mutated gene, completing a decades-long search. The gene, vacuolar protein sorting 52 ( Vps52 ), is a mouse homolog of yeast VPS52 that is involved in the retrograde trafficking of endosomes. Our data suggest that Vps52 acts in extraembryonic tissues to support the growth and differentiation of embryonic ectoderm via cell–cell interactions. It is also required in the formation of embryonic structures at a later stage of development, revealing hitherto unknown functions of Vps52 in the development of a multicellular organism. Graphical abstract Graphical Abstract Highlights ► Vps52 , part of retrograde transport to the Golgi, causes the t w5 lethal phenotype ► Vps52 acts in extraembryonic tissues to dictate embryonic ectoderm differentiation ► VPS52’s essential function in the embryo involves cell–cell interactions ► VPS52 also has an indispensable function later in development

Beschreibung: Publication year: 2012 Source: Cell Reports Myrto Denaxa, Melanie Kalaitzidou, Anna Garefalaki, Angeliki Achimastou, Reena Lasrado, Tamara Maes, Vassilis Pachnis The generation of cortical interneuron subtypes is controlled by genetic programs that are activated in the ventral forebrain and unfold during the prolonged period of inhibitory neuron development. The LIM-homeodomain protein LHX6 is critical for the development of all cortical interneurons originating in the medial ganglionic eminence, but the molecular mechanisms that operate downstream of LHX6 to control the terminal differentiation of somatostatin- and parvalbumin-expressing interneurons within the cortex remain unknown. Here, we provide evidence that the nuclear matrix and genome organizer protein SATB1 is induced by neuronal activity and functions downstream of Lhx6 to control the transition of tangentially migrating immature interneurons into the terminally differentiated Somatostatin (SST)-expressing subtype. Our experiments provide a molecular framework for understanding the genetic and epigenetic mechanisms by which specified but immature cortical interneurons acquire the subtype-defining molecular and morphophysiological characteristics that allow them to integrate and function within cortical circuits. Graphical abstract Graphical Abstract Highlights ► Satb1 is specifically expressed in MGE-derived cortical interneurons ► Satb1 is required for the maturation of MGE-derived cortical interneurons in vivo ► Satb1 promotes the maturation of SST+ cortical interneurons downstream of Lhx6 ► Neuronal activity induces expression of Satb1 in immature cortical interneurons

Beschreibung: Available online 7 December 2012 Publication year: 2012 Source: Cell Reports Periodic somite segmentation is controlled by the cyclic gene Hes7 , whose oscillatory expression depends upon negative feedback with a delayed timing. The mechanism that regulates the pace of segmentation remains to be determined, but mathematical modeling has predicted that negative feedback with shorter delays would give rise to dampened but more rapid oscillations. Here, we show that reducing the number of introns within the Hes7 gene shortens the delay and results in a more rapid tempo of both Hes7 oscillation and somite segmentation, increasing the number of somites and vertebrae in the cervical and upper thoracic region. These results suggest that the number of introns is important for the appropriate tempo of oscillatory expression and that Hes7 is a key regulator of the pace of the segmentation clock. Graphical abstract Highlights ► Reduction of Hes7 intron number accelerates the tempo of Hes7 oscillation ► Reduction of Hes7 intron number accelerates the tempo of somite segmentation ► Hes7 is a key segmentation pacemaker ► The number of introns may be important for normal timing of gene expression

Beschreibung: Available online 13 December 2012 Publication year: 2012 Source: Cell Reports Dopamine contributes to corticostriatal plasticity and motor learning. Dopamine denervation profoundly alters motor performance, as in Parkinson’s disease (PD); however, the extent to which these symptoms reflect impaired motor learning is unknown. Here, we demonstrate a D2 receptor blockade-induced aberrant learning that impedes future motor performance when dopamine signaling is restored, an effect diminished by coadministration of adenosine antagonists during blockade. We hypothesize that an inappropriate corticostriatal potentiation in striatopallidal cells of the indirect pathway underlies aberrant learning. We demonstrate synaptic potentiation in striatopallidal neurons induced by D2 blockade and diminished by application of an adenosine antagonist, consistent with behavioral observations. A neurocomputational model of the basal ganglia recapitulates the behavioral pattern and further links aberrant learning to plasticity in the indirect pathway. Thus, D2-mediated aberrant learning may contribute to motor deficits in PD, suggesting new avenues for the development of therapeutics. Graphical abstract Highlights ► Dopamine blockade induces aberrant learning that impairs future motor performance ► Aberrant learning is mediated by the D2-expressing striatopallidal pathway ► A2A antagonism protects against aberrant learning but impairs recovery ► D2 blockade induces potentiation at striatopallidal corticostriatal synapses

Beschreibung: Available online 13 December 2012 Publication year: 2012 Source: Cell Reports Elimination of aberrantly folded polypeptides from the endoplasmic reticulum (ER) by the ER - a ssociated d egradation (ERAD) system promotes cell survival under stress conditions. This quality control mechanism requires movement of misfolded proteins across the ER membrane for targeting to the cytosolic proteasome, a process facilitated by a “holdase” complex, consisting of Bag6 and the cofactors Ubl4A and Trc35. This multiprotein complex also participates in several other protein quality control processes. Here, we report SGTA as a component of the Bag6 system, which cooperates with Bag6 to channel dislocated ERAD substrates that are prone to aggregation. Using nuclear magnetic resonance spectroscopy and biochemical assays, we demonstrate that SGTA contains a noncanonical ubiquitin-like-binding domain that interacts specifically with an unconventional ubiquitin-like protein/domain in Ubl4A at least in part via electrostatics. This interaction helps recruit SGTA to Bag6, enhances substrate loading to Bag6, and thus prevents the formation of nondegradable protein aggregates in ERAD. Graphical abstract Highlights ► The two UBLs in the Bag6 complex have distinct features that specify their interactors ► The Ubl4A UBL interacts with SGTA to enhance its binding to Bag6 ► The Ubl4A UBL binds SGTA-N via an unconventional means of UBL recognition ► SGTA assists Bag6 in maintaining the solubility of ERAD substrates and promoting ERAD

Beschreibung: Available online 13 December 2012 Publication year: 2012 Source: Cell Reports The formation of the mammalian cortex requires the generation, migration, and differentiation of neurons. The vital role that the microtubule cytoskeleton plays in these cellular processes is reflected by the discovery that mutations in various tubulin isotypes cause different neurodevelopmental diseases, including lissencephaly ( TUBA1A ), polymicrogyria ( TUBA1A , TUBB2B , TUBB3 ), and an ocular motility disorder ( TUBB3 ). Here, we show that Tubb5 is expressed in neurogenic progenitors in the mouse and that its depletion in vivo perturbs the cell cycle of progenitors and alters the position of migrating neurons. We report the occurrence of three microcephalic patients with structural brain abnormalities harboring de novo mutations in TUBB5 (M299V, V353I, and E401K). These mutant proteins, which affect the chaperone-dependent assembly of tubulin heterodimers in different ways, disrupt neurogenic division and/or migration in vivo. Our results provide insight into the functional repertoire of the tubulin gene family, specifically implicating TUBB5 in embryonic neurogenesis and microcephaly. Graphical abstract Highlights ► The β-tubulin Tubb5 is highly expressed in the developing mouse and human cortex ► In vivo knockdown of Tubb5 perturbs the cell cycle and alters neuronal positioning ► Mutations in TUBB5 cause microcephaly with dysmorphic basal ganglia in humans ► TUBB5 mutations affect chaperone-mediated tubulin folding in different ways

Beschreibung: Available online 13 December 2012 Publication year: 2012 Source: Cell Reports Elimination of aberrantly folded polypeptides from the endoplasmic reticulum (ER) by the ER - a ssociated d egradation (ERAD) system promotes cell survival under stress conditions. This quality control mechanism requires movement of misfolded proteins across the ER membrane for targeting to the cytosolic proteasome, a process facilitated by a “holdase” complex, consisting of Bag6 and the cofactors Ubl4A and Trc35. This multiprotein complex also participates in several other protein quality control processes. Here, we report SGTA as a component of the Bag6 system, which cooperates with Bag6 to channel dislocated ERAD substrates that are prone to aggregation. Using nuclear magnetic resonance spectroscopy and biochemical assays, we demonstrate that SGTA contains a noncanonical ubiquitin-like-binding domain that interacts specifically with an unconventional ubiquitin-like protein/domain in Ubl4A at least in part via electrostatics. This interaction helps recruit SGTA to Bag6, enhances substrate loading to Bag6, and thus prevents the formation of nondegradable protein aggregates in ERAD. Graphical abstract Highlights ► The two UBLs in the Bag6 complex have distinct features that specify their interactors ► The Ubl4A UBL interacts with SGTA to enhance its binding to Bag6 ► The Ubl4A UBL binds SGTA-N via an unconventional means of UBL recognition ► SGTA assists Bag6 in maintaining the solubility of ERAD substrates and promoting ERAD

Beschreibung: Available online 7 December 2012 Publication year: 2012 Source: Cell Reports The efficient generation of hematopoietic stem cells from human pluripotent stem cells is dependent on the appropriate specification of the definitive hematopoietic program during differentiation. In this study, we used T lymphocyte potential to track the onset of definitive hematopoiesis from human embryonic and induced pluripotent stem cells differentiated with specific morphogens in serum- and stromal-free cultures. We show that this program develops from a progenitor population with characteristics of hemogenic endothelium, including the expression of CD34, VE-cadherin, GATA2 , LMO2 , and RUNX1 . Along with T cells, these progenitors display the capacity to generate myeloid and erythroid cells. Manipulation of Activin/Nodal signaling during early stages of differentiation revealed that development of the definitive hematopoietic progenitor population is not dependent on this pathway, distinguishing it from primitive hematopoiesis. Collectively, these findings demonstrate that it is possible to generate T lymphoid progenitors from pluripotent stem cells and that this lineage develops from a population whose emergence marks the onset of human definitive hematopoiesis. Graphical abstract Highlights ► Activin/Nodal signaling distinguishes between primitive and definitive hematopoiesis ► T cell development identifies definitive hematopoiesis in human pluripotent cultures ► Primitive hematopoiesis can be distinguished by CD41a and CD235a coexpression ► Demonstration of human T cell development from hiPSCs

Beschreibung: Available online 7 December 2012 Publication year: 2012 Source: Cell Reports DNA ligase IV (LigIV) and Artemis are central components of the nonhomologous end-joining (NHEJ) machinery that is required for V(D)J recombination and the maintenance of genomic integrity in mammalian cells. We report here crystal structures of the LigIV DNA binding domain (DBD) in both its apo form and in complex with a peptide derived from the Artemis C-terminal region. We show that LigIV interacts with Artemis through an extended hydrophobic surface. In particular, we find that the helix α2 in LigIV-DBD is longer than in other mammalian ligases and presents residues that specifically interact with the Artemis peptide, which adopts a partially helical conformation on binding. Mutations of key residues on the LigIV-DBD hydrophobic surface abolish the interaction. Together, our results provide structural insights into the specificity of the LigIV-Artemis interaction and how the enzymatic activities of the two proteins may be coordinated during NHEJ. Graphical abstract Highlights ► Crystal structure of the Ligase-IV-Artemis complex ► Ligase IV DNA binding domain contains a unique hydrophobic cavity ► The structure shows why only Ligase IV can recruit Artemis ► Insights into how activities of Ligase IV and Artemis may be coordinated in NHEJ

Beschreibung: 29 November 2012 Publication year: 2012 Source: Cell Reports, Volume 2, Issue 5 A key feature of RNA polymerase II (Pol II) preinitiation complexes (PICs) is their ability to coordinate transcription initiation with chromatin modification and remodeling. To understand how this coordination is achieved, we employed extensive proteomic and mechanistic analyses to study the composition and assembly of PICs in HeLa cell and mouse embryonic stem cell (ESC) nuclear extracts. Strikingly, most of the machinery that is necessary for transcription initiation on chromatin is part of the PIC. The PIC is nearly identical between ESCs and HeLa cells and contains two major coactivator complexes: Mediator and SAGA. Genome-wide analysis of Mediator reveals that it has a close correlation with Pol II, TATA-binding protein, and messenger RNA levels and thus may play a major role in PIC assembly. Moreover, Mediator coordinates assembly of the Pol II initiation factors and chromatin machinery into a PIC in vitro, whereas SAGA acts after PIC assembly to allow transcription on chromatin. Graphical abstract Highlights ► The composition of mammalian PICs was determined by proteomic analysis ► A wide range of chromatin-modifying and -remodeling factors are recruited to PICs ► Mediator coordinates binding of Pol II initiation, elongation, and chromatin machineries ► SAGA acts after PIC assembly to make chromatin templates transcriptionally competent

Beschreibung: 29 November 2012 Publication year: 2012 Source: Cell Reports, Volume 2, Issue 5 In meiosis, two specialized cell divisions allow the separation of paired chromosomes first, then of sister chromatids. Separase removes the cohesin complex holding sister chromatids together in a stepwise manner from chromosome arms in meiosis I, then from the centromere region in meiosis II. Using mouse oocytes, our study reveals that cyclin A2 promotes entry into meiosis, as well as an additional unexpected role; namely, its requirement for separase-dependent sister chromatid separation in meiosis II. Untimely cyclin A2-associated kinase activity in meiosis I leads to precocious sister separation, whereas inhibition of cyclin A2 in meiosis II prevents it. Accordingly, endogenous cyclin A is localized to kinetochores throughout meiosis II, but not in anaphase I. Additionally, we found that cyclin B1, but not cyclin A2, inhibits separase in meiosis I. These findings indicate that separase-dependent cohesin removal is differentially regulated by cyclin B1 and A2 in mammalian meiosis. Graphical abstract Highlights ► Cyclin A2 is required for meiotic entry and sister chromatid separation in meiosis II ► Constitutive cyclin A2 activity in meiosis I leads to precocious sister separation ► Only cyclin B1, and not cyclin A2, can inhibit separase in mouse oocyte meiosis I ► Endogenous cyclin A is localized to centromeres throughout the second meiotic division

Beschreibung: Available online 6 December 2012 Publication year: 2012 Source: Cell Reports Pathogens that reside in the phagosomes of infected cells persist despite the presence of potent T cell responses. We addressed the mechanism of immune evasion by using a mouse model of Salmonella typhimurium (ST). Recombinants of ST were generated that translocated antigen to the cytosol or phagosomes of infected cells. We find that the kinetics of antigen presentation and CD8 + T cell priming is accelerated by cytosolic antigen delivery, although the magnitude of CD8 + T cell response is not influenced by antigenic location. More importantly, only those targets that readily display antigen on the cell surface, owing to antigenic translocation to the cytosol, are recognized and killed by CD8 + T cells. Thus, vaccination approaches developed to control phagosomal pathogens should incorporate methods for modulating antigen presentation such that infected target cells can be readily recognized by CD8 + T cells. Graphical abstract Highlights ► Intracellular location of antigen governs the duration of infection ► Forcing the recognition of infected cells by CD8 T cells controls infection ► CD8 T cells can convert a chronic infection to an acute infection ► Suppression is not the cause, but the end result, of a chronic infection

Beschreibung: Available online 13 December 2012 Publication year: 2012 Source: Cell Reports Trafficking of proteins specifically to the axonal or somatodendritic membrane allows neurons to establish and maintain polarized compartments with distinct morphology and function. Diverse evidence suggests that an actin-dependent vesicle filter within the axon initial segment (AIS) plays a critical role in polarized trafficking; however, no distinctive actin-based structures capable of comprising such a filter have been found within the AIS. Here, using correlative light and scanning electron microscopy, we visualized networks of actin filaments several microns wide within the AIS of cortical neurons in culture. Individual filaments within these patches are predominantly oriented with their plus ends facing toward the cell body, consistent with models of filter selectivity. Vesicles carrying dendritic proteins are much more likely to stop in regions occupied by the actin patches than in other regions, indicating that the patches likely prevent movement of dendritic proteins to the axon and thereby act as a vesicle filter. Graphical abstract Highlights ► 2 μm diameter actin networks are visualized by SEM and light microscopy in the AIS ► Actin filaments within networks are oriented with their plus ends facing proximally ► Actin network structure suggests a mechanism for selectively halting vesicles ► Vesicles carrying dendritic proteins halt and reverse in actin networks

Beschreibung: Available online 13 December 2012 Publication year: 2012 Source: Cell Reports Dopamine contributes to corticostriatal plasticity and motor learning. Dopamine denervation profoundly alters motor performance, as in Parkinson’s disease (PD); however, the extent to which these symptoms reflect impaired motor learning is unknown. Here, we demonstrate a D2 receptor blockade-induced aberrant learning that impedes future motor performance when dopamine signaling is restored, an effect diminished by coadministration of adenosine antagonists during blockade. We hypothesize that an inappropriate corticostriatal potentiation in striatopallidal cells of the indirect pathway underlies aberrant learning. We demonstrate synaptic potentiation in striatopallidal neurons induced by D2 blockade and diminished by application of an adenosine antagonist, consistent with behavioral observations. A neurocomputational model of the basal ganglia recapitulates the behavioral pattern and further links aberrant learning to plasticity in the indirect pathway. Thus, D2-mediated aberrant learning may contribute to motor deficits in PD, suggesting new avenues for the development of therapeutics. Graphical abstract Highlights ► Dopamine blockade induces aberrant learning that impairs future motor performance ► Aberrant learning is mediated by the D2-expressing striatopallidal pathway ► A2A antagonism protects against aberrant learning but impairs recovery ► D2 blockade induces potentiation at striatopallidal corticostriatal synapses

Beschreibung: Available online 13 December 2012 Publication year: 2012 Source: Cell Reports Sir2, an evolutionarily conserved NAD + -dependent deacetylase, has been implicated as a key factor in mediating organismal life span. However, recent contradictory findings have brought into question the role of Sir2 and its orthologs in regulating organismal longevity. In this study, we report that Drosophila Sir2 (dSir2) in the adult fat body regulates longevity in a diet-dependent manner. We used inducible Gal4 drivers to knock down and overexpress dSir2 in a tissue-specific manner. A diet-dependent life span phenotype of dSir2 perturbations (both knockdown and overexpression) in the fat body, but not muscles, negates the effects of background genetic mutations. In addition to providing clarity to the field, our study contrasts the ability of dSir2 in two metabolic tissues to affect longevity. We also show that dSir2 knockdown abrogates fat-body dFOXO-dependent life span extension. This report highlights the importance of the interplay between genetic factors and dietary inputs in determining organismal life spans. Graphical abstract Highlights ► The dSir2-dependent longevity phenotype is diet specific ► DR-dependent effects are associated with increased dSir2 expression and NAD + levels ► dSir2 in the fat body, but not in muscles, regulates longevity ► dSir2-dFOXO interaction in the fat body affects life-span extension

Beschreibung: Available online 6 December 2012 Publication year: 2012 Source: Cell Reports Deubiquitinating enzymes (DUBs) constitute a large family of cysteine proteases that have a broad impact on numerous biological and pathological processes, including the regulation of genomic stability. DUBs are often assembled onto multiprotein complexes to assist in their localization and substrate selection, yet it remains unclear how the enzymatic activity of DUBs is modulated by intracellular signals. Herein, we show that bursts of reactive oxygen species (ROS) reversibly inactivate DUBs through the oxidation of the catalytic cysteine residue. Importantly, USP1, a key regulator of genomic stability, is reversibly inactivated upon oxidative stress. This, in part, explains the rapid nature of PCNA monoubiquitination-dependent DNA damage tolerance in response to oxidative DNA damage in replicating cells. We propose that DUBs of the cysteine protease family act as ROS sensors in human cells and that ROS-mediated DUB inactivation is a critical mechanism for fine-tuning stress-activated signaling pathways. Graphical abstract Highlights ► USP1 is inactivated upon oxidative stress to fine-tune PCNA monoubiquitination ► The Cys protease family of DUBs are reversibly inactivated by ROS ► The AMSH metalloprotease DUB is refractory to ROS-mediated catalytic inactivation

Beschreibung: Available online 13 December 2012 Publication year: 2012 Source: Cell Reports Genome information, which is three-dimensionally organized within cells as chromatin, is searched and read by various proteins for diverse cell functions. Although how the protein factors find their targets remains unclear, the dynamic and flexible nature of chromatin is likely crucial. Using a combined approach of fluorescence correlation spectroscopy, single-nucleosome imaging, and Monte Carlo computer simulations, we demonstrate local chromatin dynamics in living mammalian cells. We show that similar to interphase chromatin, dense mitotic chromosomes also have considerable chromatin accessibility. For both interphase and mitotic chromatin, we observed local fluctuation of individual nucleosomes (∼50 nm movement/30 ms), which is caused by confined Brownian motion. Inhibition of these local dynamics by crosslinking impaired accessibility in the dense chromatin regions. Our findings show that local nucleosome dynamics drive chromatin accessibility. We propose that this local nucleosome fluctuation is the basis for scanning genome information. Graphical abstract Highlights ► Dense chromatin regions have considerable chromatin accessibility ► Observed local fluctuation of individual nucleosomes in interphase and mitotic chromatin ► Inhibition of nucleoome fluctuation impaired the chromatin accessibility ► Local fluctuation of nucleosomes is the basis for scanning genome information

Beschreibung: 29 November 2012 Publication year: 2012 Source: Cell Reports, Volume 2, Issue 5 Expansions of simple DNA repeats cause numerous hereditary diseases in humans. We analyzed the role of DNA polymerases in the instability of Friedreich’s ataxia (GAA) n repeats in a yeast experimental system. The elementary step of expansion corresponded to ∼160 bp in the wild-type strain, matching the size of Okazaki fragments in yeast. This step increased when DNA polymerase α was mutated, suggesting a link between the scale of expansions and Okazaki fragment size. Expandable repeats strongly elevated the rate of mutations at substantial distances around them, a phenomenon we call repeat-induced mutagenesis (RIM). Notably, defects in the replicative DNA polymerases δ and ε strongly increased rates for both repeat expansions and RIM. The increases in repeat-mediated instability observed in DNA polymerase δ mutants depended on translesion DNA polymerases. We conclude that repeat expansions and RIM are two sides of the same replicative mechanism. Graphical abstract Highlights ► Elementary step of repeat expansion corresponds to size of an Okazaki fragment ► Mutated DNA polymerase α leads to an increase in expansion step ► Mutated DNA polymerases δ and ε lead to elevated expansion rates ► Repeats induce mutagenesis, which is further elevated in polymerase mutants

Beschreibung: 29 November 2012 Publication year: 2012 Source: Cell Reports, Volume 2, Issue 5 Plasma sphingosine-1-phosphate (S1P) regulates vascular permeability, and plasma and lymph S1P guide lymphocyte egress from lymphoid organs. S1P is made intracellularly, and little is known about how S1P is delivered into circulatory fluids. Here, we find that mice without the major facilitator superfamily transporter Spns2 have a profound reduction in lymph S1P, but only a minor decrease in plasma S1P. Spns2-deficient mice have a redistribution of lymphocytes from the spleen to lymph nodes and a loss of circulating lymphocytes, consistent with normal egress from the spleen directed by plasma S1P and blocked egress from lymph nodes directed by lymph S1P. Spns2 is needed in endothelial cells to supply lymph S1P and support lymphocyte circulation. As a differential requirement for lymph and blood S1P, Spns2 may be an attractive target for immune suppressive drugs. Graphical abstract Highlights ► The transporter Spns2 is required to supply lymph, but not plasma, S1P ► Spns2-deficient mice have disrupted peripheral lymphocyte circulation ► Spns2 is required in endothelial cells to secrete lymph S1P and support trafficking

Beschreibung: 29 November 2012 Publication year: 2012 Source: Cell Reports, Volume 2, Issue 5 Elongin A increases the rate of RNA polymerase II (pol II) transcript elongation by suppressing transient pausing by the enzyme. Elongin A also acts as a component of a cullin-RING ligase that can target stalled pol II for ubiquitylation and proteasome-dependent degradation. It is not known whether these activities of Elongin A are functionally interdependent in vivo. Here, we demonstrate that Elongin A-deficient (Elongin A −/− ) embryos exhibit abnormalities in the formation of both cranial and spinal nerves and that Elongin A −/− embryonic stem cells (ESCs) show a markedly decreased capacity to differentiate into neurons. Moreover, we identify Elongin A mutations that selectively inactivate one or the other of the aforementioned activities and show that mutants that retain the elongation stimulatory, but not pol II ubiquitylation, activity of Elongin A rescue neuronal differentiation and support retinoic acid-induced upregulation of a subset of neurogenesis-related genes in Elongin A −/− ESCs. Graphical abstract Highlights ► RA-induced neuronal differentiation is markedly impaired in Elongin A −/− ESCs ► Formation of cranial ganglia and DRG is severely impaired in Elongin A −/− embryos ► Mutations that differentially affect the two activities of Elongin A were identified ► Elongin A’s elongation stimulatory activity is required for a subset of RA-induced genes

Beschreibung: 29 November 2012 Publication year: 2012 Source: Cell Reports, Volume 2, Issue 5 The large Mediator (L-Mediator) is a general coactivator of RNA polymerase II transcription and is formed by the reversible association of the small Mediator (S-Mediator) and the kinase-module-harboring Cdk8. It is not known how the kinase module association/dissociation is regulated. We describe the fission yeast Cdk11-L-type cyclin pombe (Lcp1) complex and show that its inactivation alters the global expression profile in a manner very similar to that of mutations of the kinase module. Cdk11 is broadly distributed onto chromatin and phosphorylates the Med27 and Med4 Mediator subunits on conserved residues. The association of the kinase module and the S-Mediator is strongly decreased by the inactivation of either Cdk11 or the mutation of its target residues on the Mediator. These results show that Cdk11-Lcp1 regulates the association of the kinase module and the S-Mediator to form the L-Mediator complex. Graphical abstract Highlights ► A complex related to metazoan Cdk11-CyclinL is conserved in fission yeast ► Loss of either Cdk8 or Cdk11 similarly affects global expression profile ► Cdk11 phosphorylates two Mediator subunits ► Association of the Cdk8 module with the S-Mediator requires phosphorylation by Cdk11

Beschreibung: 29 November 2012 Publication year: 2012 Source: Cell Reports, Volume 2, Issue 5 Age-related macular degeneration (AMD) is the leading cause of irreversible blindness in the elderly population worldwide. Although recent studies have demonstrated strong genetic associations between AMD and SNPs in a number of genes, other modes of regulation are also likely to play a role in the etiology of this disease. We identified a significantly decreased level of methylation on the IL17RC promoter in AMD patients. Furthermore, we showed that hypomethylation of the IL17RC promoter in AMD patients led to an elevated expression of its protein and messenger RNA in peripheral blood as well as in the affected retina and choroid, suggesting that the DNA methylation pattern and expression of IL17RC may potentially serve as a biomarker for the diagnosis of AMD and likely plays a role in disease pathogenesis. Graphical abstract Highlights ► ∼1.5% of CpG sites are differentially methylated between twins with discordant AMD ► Hypomethylated IL17RC promoter and elevated IL17RC expression are associated with AMD ► IL-17-mediated inflammatory responses contribute to AMD pathogenesis

Beschreibung: 29 November 2012 Publication year: 2012 Source: Cell Reports, Volume 2, Issue 5 RIG-I and MDA5 are cytosolic RNA sensors that play a critical role in innate antiviral responses. Major advances have been made in identifying RIG-I ligands, but our knowledge of the ligands for MDA5 remains restricted to data from transfection experiments mostly using poly(I:C), a synthetic dsRNA mimic. Here, we dissected the IFN-α/β-stimulatory activity of different viral RNA species produced during picornavirus infection, both by RNA transfection and in infected cells in which specific steps of viral RNA replication were inhibited. Our results show that the incoming genomic plus-strand RNA does not activate MDA5, but minus-strand RNA synthesis and production of the 7.5 kbp replicative form trigger a strong IFN-α/β response. IFN-α/β production does not rely on plus-strand RNA synthesis and thus generation of the partially double-stranded replicative intermediate. This study reports MDA5 activation by a natural RNA ligand under physiological conditions. Graphical abstract Highlights ► Viral ssRNA, with or without the 5′ VPg peptide, does not induce IFN-α/β ► Synthesis of minus-strand—but not plus-strand—RNA is crucial for MDA5 activation ► The picornavirus RF activates MDA5 both in vitro and in vivo ► MDA5 activation by viral replicative form is independent of the terminal groups of this RNA

Beschreibung: 29 November 2012 Publication year: 2012 Source: Cell Reports, Volume 2, Issue 5 In mammals, each olfactory bulb (OB) contains a pair of mirror-symmetric glomerular maps organized to reflect odorant receptor identity. The functional implication of maintaining these symmetric medial-lateral maps within each OB remains unclear. Here, using in vivo multielectrode recordings to simultaneously detect odorant-induced activity across the entire OB, we reveal a timing difference in the odorant-evoked onset latencies between the medial and lateral halves. Interestingly, the latencies in the medial and lateral OB decreased at different rates as odorant concentration increased, causing the timing difference between them to also diminish. As a result, output neurons in the medial and lateral OB fired with greater synchrony at higher odorant concentrations. Thus, we propose that temporal differences in activity between the medial and lateral OB can dynamically code odorant concentration, which is subsequently decoded in the olfactory cortex through the integration of synchronous action potentials. Graphical abstract Highlights ► Odorants differentially activate the medial and lateral olfactory bulb (OB) ► An ∼60 ms time window is defined between medial and lateral mitral cell output ► The medial-lateral time window is dynamically modulated by odorant concentrations ► High odorant concentrations synchronize medial-lateral OB output

Beschreibung: 29 November 2012 Publication year: 2012 Source: Cell Reports, Volume 2, Issue 5 MicroRNAs (miRNAs) are essential regulators of development, physiology, and evolution, and their biogenesis is strictly controlled at multiple levels. Regulatory proteins, such as KSRP, modulate rates and timing of enzymatic reactions responsible for maturation of select miRNAs from their primary transcripts in response to specific stimuli. Here, we show that KSRP silencing in mesenchymal C2C12 cells produces a change in the transcriptome largely overlapping that induced by bone morphogenetic protein 2 (BMP2) signaling activation. This induces osteoblastic differentiation while preventing myogenic differentiation. KSRP silencing- and BMP2-dependent myogenic miRNA (myomiR) maturation blockade is required for osteoblastic differentiation of C2C12 cells. Our results demonstrate that phosphorylated R-SMAD proteins, the transducers of BMP2 signal, associate with phosphorylated KSRP and block its interaction with primary myomiRs. This abrogates KSRP-dependent myomiR maturation, with SMAD4, SMAD5, and SMAD9 silencing being able to rescue KSRP function. Thus, SMAD-induced blockade of KSRP-dependent myomiR maturation is critical for orienting C2C12 cell differentiation toward osteoblastic lineage. Graphical abstract Highlights ► KSRP silencing and BMP2 signaling activation block myomiR maturation ► myomiR maturation blockade is required for osteoblastic differentiation of C2C12 cells ► SMAD proteins interact with KSRP and block its ability to promote myomiR maturation

Beschreibung: 29 November 2012 Publication year: 2012 Source: Cell Reports, Volume 2, Issue 5 Senescence is a cellular response preventing tumorigenesis. The Ras oncogene is frequently activated or mutated in human cancers, but Ras activation is insufficient to transform primary cells. In a search for cooperating oncogenes, we identify the lysine demethylase JMJD2A/KDM4A. We show that JMJD2A functions as a negative regulator of Ras-induced senescence and collaborates with oncogenic Ras to promote cellular transformation by negatively regulating the p53 pathway. We find CHD5 , a known tumor suppressor regulating p53 activity, as a target of JMJD2A. The expression of JMJD2A inhibits Ras-mediated CHD5 induction leading to a reduced activity of the p53 pathway. In addition, we show that JMJD2A is overexpressed in mouse and human lung cancers. Depletion of JMJD2A in the human lung cancer cell line A549 bearing an activated K-Ras allele triggers senescence. We propose that JMJD2A is an oncogene that represents a target for Ras-expressing tumors. Graphical abstract Highlights ► JMJD2A contributes to the bypass of Ras-induced senescence ► Genome-wide search for gene promoters regulated by JMJD2A identifies CHD5 ► JMJD2A cooperates with Ras to transform primary cells ► JMJD2A depletion in human lung cancer cells leads to massive senescence

Beschreibung: 29 November 2012 Publication year: 2012 Source: Cell Reports, Volume 2, Issue 5 Mitochondria-originating reactive oxygen species (ROS) control T cell receptor (TCR)-induced gene expression. Here, we show that TCR-triggered activation of ADP-dependent glucokinase (ADPGK), an alternative, glycolytic enzyme typical for Archaea , mediates generation of the oxidative signal. We also show that ADPGK is localized in the endoplasmic reticulum and suggest that its active site protrudes toward the cytosol. The ADPGK-driven increase in glycolytic metabolism coincides with TCR-induced glucose uptake, downregulation of mitochondrial respiration, and deviation of glycolysis toward mitochondrial glycerol-3-phosphate dehydrogenase (GPD) shuttle; i.e., a metabolic shift to aerobic glycolysis similar to the Warburg effect. The activation of respiratory-chain-associated GPD2 results in hyperreduction of ubiquinone and ROS release from mitochondria. In parallel, mitochondrial bioenergetics and ultrastructure are altered. Downregulation of ADPGK or GPD2 abundance inhibits oxidative signal generation and induction of NF-κB-dependent gene expression, whereas overexpression of ADPGK potentiates them. Graphical abstract Highlights ► TCR triggering activates ADPGK, an ER-localized glycolytic enzyme ► TCR induction shifts metabolism toward glycolysis and activates mitochondrial GPD2 ► Activation of GPD2 leads to ubiquinol accumulation and ROS release from mitochondria ► ADPGK- and GPD2-dependent oxidative signal drives NF-κB-regulated gene expression

Beschreibung: 29 November 2012 Publication year: 2012 Source: Cell Reports, Volume 2, Issue 5 Alzheimer's disease (AD) is the most common cause of dementia among older people. There are no effective medications currently available to prevent and treat AD and halt disease progression. Monoacylglycerol lipase (MAGL) is the primary enzyme metabolizing the endocannabinoid 2-arachidonoylglycerol in the brain. We show here that inactivation of MAGL robustly suppressed production and accumulation of β-amyloid (Aβ) associated with reduced expression of β-site amyloid precursor protein cleaving enzyme 1 (BACE1) in a mouse model of AD. MAGL inhibition also prevented neuroinflammation, decreased neurodegeneration, maintained integrity of hippocampal synaptic structure and function, and improved long-term synaptic plasticity, spatial learning, and memory in AD animals. Although the molecular mechanisms underlying the beneficial effects produced by MAGL inhibition remain to be determined, our results suggest that MAGL, which regulates endocannabinoid and prostaglandin signaling, contributes to pathogenesis and neuropathology of AD, and thus is a promising therapeutic target for the prevention and treatment of AD. Graphical abstract Highlights ► Inactivation of MAGL reduces Aβ plaques and BACE1 expression in AD mice ► MAGL inhibition decreases neuroinflammation and neurodegeneration ► MAGL inhibition maintains integrity of hippocampal synaptic structure and function ► MAGL inhibition improves synaptic plasticity and learning and memory in AD mice

Beschreibung: 29 November 2012 Publication year: 2012 Source: Cell Reports, Volume 2, Issue 5 Antagonistic pleiotropy (AP), or genetic tradeoff, is an important concept that is frequently invoked in theories of aging, cancer, genetic disease, and other common phenomena. However, the prevalence of AP, which genes are subject to AP, and to what extent and how AP may be resolved remain unclear. By measuring the fitness difference between the wild-type and null alleles of ∼5,000 nonessential genes in yeast, we found that in any given environment, yeast expresses hundreds of genes that harm rather than benefit the organism, demonstrating widespread AP. Nonetheless, under sufficient selection, AP is often resolvable through regulatory evolution, primarily by trans -acting changes, although in one case we also detected a cis -acting change and localized its causal mutation. However, AP is resolved more slowly in smaller populations, predicting more unresolved AP in multicellular organisms than in yeast. These findings provide an empirical foundation for AP-dependent theories and have broad biomedical and evolutionary implications. Graphical abstract Highlights ► Under any conditions, yeast expresses many genes that are harmful to the cell ► Such problems can often be resolved by regulatory evolution under selection ► Such regulatory evolution tends to occur via trans -acting genetic changes ► Antagonistic pleiotropy is predicted to be more abundant in multicellular organisms

Beschreibung: 29 November 2012 Publication year: 2012 Source: Cell Reports, Volume 2, Issue 5 After implantation, pluripotent epiblasts are converted to embryonic ectoderm through cell–cell interactions that significantly change the transcriptional and epigenetic networks. An entrée to understanding this vital developmental transition is the t w5 mutation of the mouse t complex. This mutation produces highly specific defects in the embryonic ectoderm before gastrulation, leading to death of the embryonic ectoderm. Using a positional cloning approach, we have now identified the mutated gene, completing a decades-long search. The gene, vacuolar protein sorting 52 ( Vps52 ), is a mouse homolog of yeast VPS52 that is involved in the retrograde trafficking of endosomes. Our data suggest that Vps52 acts in extraembryonic tissues to support the growth and differentiation of embryonic ectoderm via cell–cell interactions. It is also required in the formation of embryonic structures at a later stage of development, revealing hitherto unknown functions of Vps52 in the development of a multicellular organism. Graphical abstract Highlights ► Vps52 , part of retrograde transport to the Golgi, causes the t w5 lethal phenotype ► Vps52 acts in extraembryonic tissues to dictate embryonic ectoderm differentiation ► VPS52’s essential function in the embryo involves cell–cell interactions ► VPS52 also has an indispensable function later in development

Beschreibung: 29 November 2012 Publication year: 2012 Source: Cell Reports, Volume 2, Issue 5 Human pluripotent stem cells (hPSCs), including embryonic stem cells and induced pluripotent stem cells, are potentially useful in regenerative therapies for heart disease. For medical applications, clinical-grade cardiac cells must be produced from hPSCs in a defined, cost-effective manner. Cell-based screening led to the discovery of KY02111, a small molecule that promotes differentiation of hPSCs to cardiomyocytes. Although the direct target of KY02111 remains unknown, results of the present study suggest that KY02111 promotes differentiation by inhibiting WNT signaling in hPSCs but in a manner that is distinct from that of previously studied WNT inhibitors. Combined use of KY02111 and WNT signaling modulators produced robust cardiac differentiation of hPSCs in a xeno-free, defined medium, devoid of serum and any kind of recombinant cytokines and hormones, such as BMP4, Activin A, or insulin. The methodology has potential as a means for the practical production of human cardiomyocytes for regeneration therapies. Graphical abstract Highlights ► KY02111 promotes the differentiation of hPSCs to functional cardiomyocytes ► KY02111 acts downstream of APC and GSK3β to inhibit WNT signaling ► KY02111 and WNT inhibitors cooperatively enhance hPSC cardiomyogenesis ► KY02111and WNT modulators permit cytokine and xeno-free hPSC cardiomyogenesis

Beschreibung: 29 November 2012 Publication year: 2012 Source: Cell Reports, Volume 2, Issue 5 Growing evidence suggests that aggregation-prone proteins are both harmful and functional for a cell. How do cellular systems balance the detrimental and beneficial effect of protein aggregation? We reveal that aggregation-prone proteins are subject to differential transcriptional, translational, and degradation control compared to nonaggregation-prone proteins, which leads to their decreased synthesis, low abundance, and high turnover. Genetic modulators that enhance the aggregation phenotype are enriched in genes that influence expression homeostasis. Moreover, genes encoding aggregation-prone proteins are more likely to be harmful when overexpressed. The trends are evolutionarily conserved and suggest a strategy whereby cellular mechanisms specifically modulate the availability of aggregation-prone proteins to (1) keep concentrations below the critical ones required for aggregation and (2) shift the equilibrium between the monomeric and oligomeric/aggregate form, as explained by Le Chatelier’s principle. This strategy may prevent formation of undesirable aggregates and keep functional assemblies/aggregates under control. Graphical abstract Highlights ► mRNA encoding aggregation-prone proteins is complex, suggesting greater translational regulation ► Aggregation-prone proteins are present in low abundance and for short periods of time ► Tight control is evolutionarily conserved and provides robustness against aggregation ► Aggregation-prone proteins are subject to tight regulation

Beschreibung: 29 November 2012 Publication year: 2012 Source: Cell Reports, Volume 2, Issue 5 Variability in signaling pathway activation between neighboring epithelial cells can arise from local differences in the microenvironment, noisy gene expression, or acquired genetic changes. To investigate the consequences of this cell-to-cell variability in signaling pathway activation on coordinated multicellular processes such as morphogenesis, we use DNA-programmed assembly to construct three-dimensional MCF10A microtissues that are mosaic for low-level expression of activated H-Ras. We find two emergent behaviors in mosaic microtissues: cells with activated H-Ras are basally extruded or lead motile multicellular protrusions that direct the collective motility of their wild-type neighbors. Remarkably, these behaviors are not observed in homogeneous microtissues in which all cells express the activated Ras protein, indicating that heterogeneity in Ras activity, rather than the total amount of Ras activity, is critical for these processes. Our results directly demonstrate that cell-to-cell variability in pathway activation within local populations of epithelial cells can drive emergent behaviors during epithelial morphogenesis. Graphical abstract Highlights ► Cell-to-cell variability in Ras activity triggers emergent behaviors ► Homogeneous and low-level Ras activation does not disrupt early morphogenesis ► Programmed assembly precisely controls the composition of mosaic epithelial tissues

Beschreibung: Publication year: 2012 Source: Cell Reports Vadim Shchepachev, Harry Wischnewski, Edoardo Missiaglia, Charlotte Soneson, Claus M. Azzalin Clericuzio-type poikiloderma with neutropenia (PN) is a rare genodermatosis associated with mutations in the C16orf57 gene, which codes for the uncharacterized protein hMpn1. We show here that, in both fission yeasts and humans, Mpn1 processes the spliceosomal U6 small nuclear RNA (snRNA) posttranscriptionally. In Mpn1-deficient cells, U6 molecules carry 3′ end polyuridine tails that are longer than those in normal cells and lack a terminal 2′,3′ cyclic phosphate group. In mpn1 Δ yeast cells, U6 snRNA and U4/U6 di-small nuclear RNA protein complex levels are diminished, leading to precursor messenger RNA splicing defects, which are reverted by expression of either yeast or human Mpn1 and by overexpression of U6. Recombinant hMpn1 is a 3′-to-5′ RNA exonuclease that removes uridines from U6 3′ ends, generating terminal 2′,3′ cyclic phosphates in vitro. Finally, U6 degradation rates increase in mpn1 Δ yeasts and in lymphoblasts established from individuals affected by PN. Our data indicate that Mpn1 promotes U6 stability through 3′ end posttranscriptional processing and implicate altered U6 metabolism as a potential mechanism for PN pathogenesis. Graphical abstract Graphical Abstract Highlights ► Yeast and human cells deficient for Mpn1 fail to process U6 snRNA oligo(U) 3′ ends ► Cellular U6 levels and pre-mRNA splicing are compromised in mpn1 Δ yeasts ► Mpn1 is a 3′-to-5′ RNA exonuclease ► U6 degradation rates are accelerated in yeast and human cells deficient for Mpn1

Beschreibung: Publication year: 2012 Source: Cell Reports Agathe Valluet, Sabine Druillennec, Céline Barbotin, Coralie Dorard, Anne H. Monsoro-Burq, Magalie Larcher, Celio Pouponnot, Manuela Baccarini, Lionel Larue, Alain Eychène B-Raf and C-Raf kinases have emerged as critical players in melanoma. However, little is known about their role during development and homeostasis of the melanocyte lineage. Here, we report that knockout of B-raf and C-raf genes in this lineage results in normal pigmentation at birth with no defect in migration, proliferation, or differentiation of melanoblasts in mouse hair follicles. In contrast, the double raf knockout mice displayed hair graying resulting from a defect in cell-cycle entry of melanocyte stem cells (MSCs) and their subsequent depletion in the hair follicle bulge. Therefore, Raf signaling is dispensable for early melanocyte lineage development, but necessary for MSC maintenance. Graphical abstract Graphical Abstract Highlights ► Direct in vivo evidence of the involvement of Raf proteins in stemness ► B-Raf and C-Raf are required for melanocyte stem cell self-maintenance ► The Raf/MEK/ERK pathway is not essential for melanocyte lineage development ► SCF/Kit and Raf/ERK signaling pathways are uncoupled in the melanocyte lineage

Beschreibung: Publication year: 2012 Source: Cell Reports, Volume 2, Issue 3 Simone Prömel, Marie Frickenhaus, Samantha Hughes, Lamia Mestek, David Staunton, Alison Woollard, Ioannis Vakonakis, Torsten Schöneberg, Ralf Schnabel, Andreas P. Russ, Tobias Langenhan

Beschreibung: Publication year: 2012 Source: Cell Reports Andre L. Samson, Anja S. Knaupp, Maithili Sashindranath, Rachael J. Borg, Amanda E.-L. Au, Elisa J. Cops, Helen M. Saunders, Stephen H. Cody, Catriona A. McLean, Cameron J. Nowell, Victoria A. Hughes, Stephen P. Bottomley, Robert L. Medcalf Cellular injury causes a myriad of processes that affect proteostasis. We describe nucleocytoplasmic coagulation (NCC), an intracellular disulfide-dependent protein crosslinking event occurring upon late-stage cell death that orchestrates the proteolytic removal of misfolded proteins. In vitro and in vivo models of neuronal injury show that NCC involves conversion of soluble intracellular proteins, including tubulin, into insoluble oligomers. These oligomers, also seen in human brain tissue following neurotrauma, act as a cofactor and substrate for the plasminogen-activating system. In plasminogen −/− mice, levels of misfolded β-tubulin were elevated and its clearance delayed following neurotrauma, demonstrating a requirement for plasminogen in the removal of NCC constituents. While additional in vivo studies will further dissect this phenomenon, our study clearly shows that NCC, a process analogous to the formation of thrombi, generates an aggregated protein scaffold that limits release of cellular components and recruits clearance mechanisms to the site of injury. Graphical abstract Graphical Abstract Highlights ► Cell death triggers the process of nucleocytoplasmic coagulation (NCC) ► NCC is the abrupt misfolding and disulfide crosslinking of intracellular proteins ► NCC-aggregated proteins facilitate plasmin formation and subsequent proteolysis ► Thus, NCC limits release and promotes proteolytic clearance of dead cell debris

Beschreibung: Publication year: 2012 Source: Cell Reports Jennifer S. Liu, Justin T. Farlow, Amanda K. Paulson, Mark A. Labarge, Zev J. Gartner Variability in signaling pathway activation between neighboring epithelial cells can arise from local differences in the microenvironment, noisy gene expression, or acquired genetic changes. To investigate the consequences of this cell-to-cell variability in signaling pathway activation on coordinated multicellular processes such as morphogenesis, we use DNA-programmed assembly to construct three-dimensional MCF10A microtissues that are mosaic for low-level expression of activated H-Ras. We find two emergent behaviors in mosaic microtissues: cells with activated H-Ras are basally extruded or lead motile multicellular protrusions that direct the collective motility of their wild-type neighbors. Remarkably, these behaviors are not observed in homogeneous microtissues in which all cells express the activated Ras protein, indicating that heterogeneity in Ras activity, rather than the total amount of Ras activity, is critical for these processes. Our results directly demonstrate that cell-to-cell variability in pathway activation within local populations of epithelial cells can drive emergent behaviors during epithelial morphogenesis. Graphical abstract Graphical Abstract Highlights ► Cell-to-cell variability in Ras activity triggers emergent behaviors ► Homogeneous and low-level Ras activation does not disrupt early morphogenesis ► Programmed assembly precisely controls the composition of mosaic epithelial tissues

Beschreibung: Publication year: 2012 Source: Cell Reports Chandan Shee, Janet L. Gibson, Susan M. Rosenberg Mutation hotspots and showers occur across phylogeny and profoundly influence genome evolution, yet the mechanisms that produce hotspots remain obscure. We report that DNA double-strand breaks (DSBs) provoke mutation hotspots via stress-induced mutation in Escherichia coli . With tet reporters placed 2 kb to 2 Mb (half the genome) away from an I- Sce I site, RpoS/DinB-dependent mutations occur maximally within the first 2 kb and decrease logarithmically to ∼60 kb. A weak mutation tail extends to 1 Mb. Hotspotting occurs independently of I-site/ tet- reporter-pair position in the genome, upstream and downstream in the replication path. RecD, which allows RecBCD DSB-exonuclease activity, is required for strong local but not long-distance hotspotting, indicating that double-strand resection and gap-filling synthesis underlie local hotspotting, and newly illuminating DSB resection in vivo. Hotspotting near DSBs opens the possibility that specific genomic regions could be targeted for mutagenesis, and could also promote concerted evolution (coincident mutations) within genes/gene clusters, an important issue in the evolution of protein functions. Graphical abstract Graphical Abstract Highlights ► Spontaneous mutation pathway in Escherichia coli causes hotpots at double-strand breaks ► Strong local (2–60 kb) hotspot mechanism double-strand resection and gap-fill ► Weak long-distance (1 Mb) mutagenesis by break-induced replication ► Break-induced replication and length of DNA-end resection in natural repair with sister chromosomes

Beschreibung: Publication year: 2012 Source: Cell Reports Aurélie Jory, Carlos Estella, Matt W. Giorgianni, Matthew Slattery, Todd R. Laverty, Gerald M. Rubin, Richard S. Mann Over 6,000 fragments from the genome of Drosophila melanogaster were analyzed for their ability to drive expression of GAL4 reporter genes in the third-instar larval imaginal discs. About 1,200 reporter genes drove expression in the eye, antenna, leg, wing, haltere, or genital imaginal discs. The patterns ranged from large regions to individual cells. About 75% of the active fragments drove expression in multiple discs; 20% were expressed in ventral, but not dorsal, discs (legs, genital, and antenna), whereas ∼23% were expressed in dorsal but not ventral discs (wing, haltere, and eye). Several patterns, for example, within the leg chordotonal organ, appeared a surprisingly large number of times. Unbiased searches for DNA sequence motifs suggest candidate transcription factors that may regulate enhancers with shared activities. Together, these expression patterns provide a valuable resource to the community and offer a broad overview of how transcriptional regulatory information is distributed in the Drosophila genome. Graphical abstract Graphical Abstract Highlights ► Over 6,000 fragments from the Drosophila genome were analyzed for enhancer ► About 1,200 fragments drove expression of GAL4 in at least one of six imaginal discs ► The lines greatly expand the number of GAL4 drivers available to the community

Beschreibung: Publication year: 2012 Source: Cell Reports Rachel Spokoini, Ofer Moldavski, Yaakov Nahmias, Jeremy L. England, Maya Schuldiner, Daniel Kaganovich The division of the S. cerevisiae budding yeast, which produces one mother cell and one daughter cell, is asymmetric with respect to aging. Remarkably, the asymmetry of yeast aging coincides with asymmetric inheritance of damaged and aggregated proteins by the mother cell. Here, we show that misfolded proteins are retained in the mother cell by being sequestered in juxtanuclear quality control compartment (JUNQ) and insoluble protein deposit (IPOD) inclusions, which are attached to organelles. Upon exposure to stress, misfolded proteins accumulate in stress foci that must be disaggregated by Hsp104 in order to be degraded or processed to JUNQ and IPOD. Cells that fail to deliver aggregates to an inclusion pass on aggregates to subsequent generations. Graphical abstract Graphical Abstract Highlights ► Misfolded proteins form stress foci, JUNQ inclusions, or IPOD inclusions ► JUNQs and IPODs are attached to organelles ► JUNQ and IPOD inclusions are asymmetrically inherited during cell division ► Hsp104 is required for disaggregation of stress foci

Beschreibung: Publication year: 2012 Source: Cell Reports Ellen M. van Beek, Julian Alvarez Zarate, Robin van Bruggen, Karin Schornagel, Anton T.J. Tool, Takashi Matozaki, Georg Kraal, Dirk Roos, Timo K. van den Berg The phagocyte NADPH oxidase mediates oxidative microbial killing in granulocytes and macrophages. However, because the reactive oxygen species produced by the NADPH oxidase can also be toxic to the host, it is essential to control its activity. Little is known about the endogenous mechanism(s) that limits NADPH oxidase activity. Here, we demonstrate that the myeloid-inhibitory receptor SIRPα acts as a negative regulator of the phagocyte NADPH oxidase. Phagocytes isolated from SIRPα mutant mice were shown to have an enhanced respiratory burst. Furthermore, overexpression of SIRPα in human myeloid cells prevented respiratory burst activation. The inhibitory effect required interactions between SIRPα and its natural ligand, CD47, as well as signaling through the SIRPα cytoplasmic immunoreceptor tyrosine-based inhibitory motifs. Suppression of the respiratory burst by SIRPα was caused by a selective repression of gp91 phox expression, the catalytic component of the phagocyte NADPH oxidase complex. Thus, SIRPα can limit gp91 phox expression during myeloid development, thereby controlling the magnitude of the respiratory burst in phagocytes. Graphical abstract Graphical Abstract Highlights ► Activity of the phagocyte NADPH oxidase is restricted by SIRPα ► This involves SIRP-CD47 interactions and signaling through the SIRPα immunoreceptor tyrosine-based inhibitory motifs ► SIRPα signaling represses expression of the catalytic NADPH oxidase subunit gp91 phox

Beschreibung: Publication year: 2012 Source: Cell Reports Ping Yin, Dong Deng, Chuangye Yan, Xiaojing Pan, Jianzhong Jeff Xi, Nieng Yan, Yigong Shi The transcription activator-like (TAL) effector targets specific host promoter through its central DNA-binding domain, which comprises multiple tandem repeats (TALE repeats). Recent structural analyses revealed that the TALE repeats form a superhelical structure that tracks along the forward strand of the DNA duplex. Here, we demonstrate that TALE repeats specifically recognize a DNA-RNA hybrid where the DNA strand determines the binding specificity. The crystal structure of a designed TALE in complex with the DNA-RNA hybrid was determined at a resolution of 2.5 Å. Although TALE repeats are in direct contact with only the DNA strand, the phosphodiester backbone of the RNA strand is inaccessible by macromolecules such as RNases. Consistent with this observation, sequence-specific recognition of an HIV-derived DNA-RNA hybrid by an engineered TALE efficiently blocked RNase H-mediated degradation of the RNA strand. Our study broadens the utility of TALE repeats and suggests potential applications in processes involving DNA replication and retroviral infections. Graphical abstract Graphical Abstract Highlights ► TALE repeats specifically bind to DNA-RNA hybrids ► The sequence of DNA determines binding specificity by TALE repeats ► TALE repeats protect DNA-RNA hybrids from RNase H degradation ► TALEs may be used to fight retroviral infection and modulate transcription

Beschreibung: Publication year: 2012 Source: Cell Reports Michael Hiller, Bruce T. Schaar, Vahan B. Indjeian, David M. Kingsley, Lee R. Hagey, Gill Bejerano Genotype-phenotype mapping is hampered by countless genomic changes between species. We introduce a computational “forward genomics” strategy that—given only an independently lost phenotype and whole genomes—matches genomic and phenotypic loss patterns to associate specific genomic regions with this phenotype. We conducted genome-wide screens for two metabolic phenotypes. First, our approach correctly matches the inactivated Gulo gene exactly with the species that lost the ability to synthesize vitamin C. Second, we attribute naturally low biliary phospholipid levels in guinea pigs and horses to the inactivated phospholipid transporter Abcb4. Human ABCB4 mutations also result in low phospholipid levels but lead to severe liver disease, suggesting compensatory mechanisms in guinea pig and horse. Our simulation studies, counts of independent changes in existing phenotype surveys, and the forthcoming availability of many new genomes all suggest that forward genomics can be applied to many phenotypes, including those relevant for human evolution and disease. Graphical abstract Graphical Abstract Highlights ► Matching independent phenotypic losses with ancestral genomic information erosion ► Gulo gene loss uniquely matches to “loss of vitamin C synthesis” in mammals ► Abcb4 gene loss matches “low biliary phospholipid levels” in guinea pig & horse ► Broad applicability of the approach from simulation and phenotype measurements

Beschreibung: Publication year: 2012 Source: Cell Reports Jian Zhu, Gaurav D. Gaiha, Sinu P. John, Thomas Pertel, Christopher R. Chin, Geng Gao, Hongjing Qu, Bruce D. Walker, Stephen J. Elledge, Abraham L. Brass HIV-1 depends on many host factors for propagation. Other host factors, however, antagonize HIV-1 and may have profound effects on viral activation. Curing HIV-1 requires the reduction of latent viral reservoirs that remain in the face of antiretroviral therapy. Using orthologous genetic screens, we identified bromodomain containing 4 (BRD4) as a negative regulator of HIV-1 replication. Antagonism of BRD4, via RNA interference or with a small molecule inhibitor, JQ1, both increased proviral transcriptional elongation and alleviated HIV-1 latency in cell-line models. In multiple instances, JQ1, when used in combination with the NF-κB activators Prostratin or PHA, enhanced the in vitro reactivation of latent HIV-1 in primary T cells. These data are consistent with a model wherein BRD4 competes with the virus for HIV-1 dependency factors (HDFs) and suggests that combinatorial therapies that activate HDFs and antagonize HIV-1 competitive factors may be useful for curing HIV-1 infection. Graphical abstract Graphical Abstract Highlights ► BRD4 depletion or inhibition with JQ1 increases HIV-1 replication and gene expression ► BRD4 inhibition increases Tat-dependent transcriptional elongation and Tat–PTEF-b association ► BRD4 inhibition alleviates HIV-1 latency in cell-line models ► JQ1 with HDF activators enhances HIV-1 replication in primary and latently infected T cells

Beschreibung: Publication year: 2012 Source: Cell Reports Stefanie Reissmann, Lukasz A. Joachimiak, Bryan Chen, Anne S. Meyer, Anthony Nguyen, Judith Frydman The eukaryotic chaperonin TRiC/CCT uses ATP cycling to fold many essential proteins that other chaperones cannot fold. This 1 MDa hetero-oligomer consists of two identical stacked rings assembled from eight paralogous subunits, each containing a conserved ATP-binding domain. Here, we report a dramatic asymmetry in the ATP utilization cycle of this ring-shaped chaperonin, despite its apparently symmetric architecture. Only four of the eight different subunits bind ATP at physiological concentrations. ATP binding and hydrolysis by the low-affinity subunits is fully dispensable for TRiC function in vivo . The conserved nucleotide-binding hierarchy among TRiC subunits is evolutionarily modulated through differential nucleoside contacts. Strikingly, high- and low-affinity subunits are spatially segregated within two contiguous hemispheres in the ring, generating an asymmetric power stroke that drives the folding cycle. This unusual mode of ATP utilization likely serves to orchestrate a directional mechanism underlying TRiC/CCT's unique ability to fold complex eukaryotic proteins. Graphical abstract Graphical Abstract Highlights ► The eight paralogous TRiC subunits display hierarchical ATP occupancy ► Conservation of nucleoside contacts among TRiC orthologs mirrors ATP affinity ► ATP binding and hydrolysis in the low-affinity subunits are dispensable for life ► ATP usage segregates asymmetrically into two hemispheres of the chaperonin ring

Beschreibung: Publication year: 2012 Source: Cell Reports Gil Diamant, Liat Amir-Zilberstein, Yuki Yamaguchi, Hiroshi Handa, Rivka Dikstein NF-κB is central for immune response and cell survival, and its deregulation is linked to chronic inflammation and cancer through poorly defined mechanisms. IκBα and A20 are NF-κB target genes and negative feedback regulators. Upon their activation by NF-κB, DSIF is recruited, P-TEFb is released, and their elongating polymerase II (Pol II) C-terminal domain (CTD) remains hypophosphorylated. We show that upon DSIF knockdown, mRNA levels of a subset of NF-κB targets are not diminished; yet much less IκBα and A20 protein are synthesized, and NF-κB activation is abnormally prolonged. Further analysis of IκBα and A20 mRNA revealed that a significant portion is uncapped, unspliced, and retained in the nucleus. Interestingly, the Spt5 C-terminal repeat (CTR) domain involved in elongation stimulation through P-TEFb is dispensable for IκBα and A20 regulation. These findings assign a function for DSIF in cotranscriptional mRNA processing when elongating Pol II is hypophosphorylated and define DSIF as part of the negative feedback regulation of NF-κB. Graphical abstract Graphical Abstract Highlights ► IκBα and A20, negative feedback regulators of NF-κB, have unusual elongation control ► Upon activation of IκBα and A20, DSIF is recruited, and the Pol II CTD remains hypophosphorylated ► DSIF is selectively required for IκBα and A20 mRNA processing and proper NF-κ B signaling ► DSIF’s CTR domain involved in elongation stimulation is dispensable for this function

Beschreibung: Publication year: 2012 Source: Cell Reports Samantha A. Morris, Yu Guo, Magdalena Zernicka-Goetz Plasticity is a well-known feature of mammalian development, and yet very little is known about its underlying mechanism. Here, we establish a model system to examine the extent and limitations of developmental plasticity in living mouse embryos. We show that halved embryos follow the same strict clock of developmental transitions as intact embryos, but their potential is not equal. We have determine that unless a minimum of four pluripotent cells are established before implantation, development will arrest. This failure can be rescued by modulating Fgf and Wnt signaling to enhance the pluripotent cell number, allowing the generation of monozygotic twins, which is an otherwise rare phenomenon. Knowledge of the minimum pluripotent-cell number required for development to birth, as well as the different potentials of blastomeres, allowed us to establish a protocol for splitting an embryo into one part that develops to adulthood and another that provides embryonic stem cells for that individual. Graphical abstract Graphical Abstract Highlights ► Half embryos follow the same clock as intact embryos, but their potential is not equal ► To support development, four pluripotent cells must be generated before implantation ► Fgf/Wnt signal modulation enhances pluripotency to rescue half-embryo development ► ESCs and a viable mouse can be derived from a single embryo with high efficiency

Beschreibung: Publication year: 2012 Source: Cell Reports Pol Margalef, Vanessa Fernández-Majada, Alberto Villanueva, Ricard Garcia-Carbonell, Mar Iglesias, Laura López, María Martínez-Iniesta, Jordi Villà-Freixa, Mari Carmen Mulero, Montserrat Andreu, Ferran Torres, Marty W. Mayo, Anna Bigas, Lluis Espinosa Nuclear IKKα regulates gene transcription by phosphorylating specific substrates and has been linked to cancer progression and metastasis. However, the mechanistic connection between tumorigenesis and IKKα activity remains poorly understood. We have now analyzed 288 human colorectal cancer samples and found a significant association between the presence of nuclear IKK and malignancy. Importantly, the nucleus of tumor cells contains an active IKKα isoform with a predicted molecular weight of 45 kDa (p45-IKKα) that includes the kinase domain but lacks several regulatory regions. Active nuclear p45-IKKα forms a complex with nonactive IKKα and NEMO that mediates phosphorylation of SMRT and histone H3. Proteolytic cleavage of FL-IKKα into p45-IKKα is required for preventing the apoptosis of CRC cells in vitro and sustaining tumor growth in vivo. Our findings identify a potentially druggable target for treating patients with advance refractory CRC. Graphical abstract Graphical Abstract Highlights ► A truncated active form of IKKα is found in colorectal cancer cells ► Nuclear complex containing p45-IKKα phosphorylates SMRT and histone H3 ► Cleavage of IKK(alpha) into p45-IKKα is required for cancer cell growth

Beschreibung: Publication year: 2012 Source: Cell Reports Adrian W.R. Serohijos, Zilvinas Rimas, Eugene I. Shakhnovich The consistent observation across all kingdoms of life that highly abundant proteins evolve slowly demonstrates that cellular abundance is a key determinant of protein evolutionary rate. However, other empirical findings, such as the broad distribution of evolutionary rates, suggest that additional variables determine the rate of protein evolution. Here, we report that under the global selection against the cytotoxic effects of misfolded proteins, folding stability (Δ G ), simultaneous with abundance, is a causal variable of evolutionary rate. Using both theoretical analysis and multiscale simulations, we demonstrate that the anticorrelation between the premutation Δ G and the arising mutational effect (ΔΔ G ), purely biophysical in origin, is a necessary requirement for abundance–evolutionary rate covariation. Additionally, we predict and demonstrate in bacteria that the strength of abundance–evolutionary rate correlation depends on the divergence time separating reference genomes. Altogether, these results highlight the intrinsic role of protein biophysics in the emerging universal patterns of molecular evolution. Graphical abstract Graphical Abstract Highlights ► Protein stability and abundance mutually determine its evolutionary rate ► Coupling of stability changes with wild-type stability affects rate of evolution ► Abundance–evolutionary rate covariation depends on divergence time

Beschreibung: Publication year: 2012 Source: Cell Reports Byung Ouk Park, Robert Ahrends, Mary N. Teruel Adipogenesis, or the conversion of proliferating preadipocytes into nondividing adipocytes, is an important part of the vertebrate weight-maintenance program. It is not yet understood how and when an irreversible transition occurs into a distinct state capable of accumulating lipid. Here, we use single-cell fluorescence imaging to show that an all-or-none switch is induced before lipid accumulation occurs. Conversion begins by glucocorticoid and cAMP signals raising C/EBPβ levels above a critical threshold, triggering three consecutive positive feedback loops: from PPARγ to C/EBPα, then to C/EBPβ, and last to the insulin receptor. Experiments and modeling show that these feedbacks create a robust, irreversible transition to a terminally differentiated state by rejecting short- and low-amplitude stimuli. After the differentiation switch is triggered, insulin controls fat accumulation in a graded fashion. Altogether, our study introduces a regulatory motif that locks cells in a differentiated state by engaging a sequence of positive feedback loops. Graphical abstract Graphical Abstract Highlights ► A quantitative molecular model of adipogenesis is developed ► Multiple, consecutive feedback loops from PPARγ drive adipogenesis ► The circuit design with sequential, delayed loops prevents accidental triggering ► Insulin-regulated fat accumulation is graded and occurs only after the switch is made

Beschreibung: Publication year: 2012 Source: Cell Reports Yuchen Han, Gena Whitney, Jesse Donovan, Alexei Korennykh 2′,5′-linked oligoadenylates (2-5As) serve as conserved messengers of pathogen presence in the mammalian innate immune system. 2-5As induce self-association and activation of RNase L, which cleaves cytosolic RNA and promotes the production of interferons (IFNs) and cytokines driven by the transcription factors IRF-3 and NF-κB. We report that human RNase L is activated by forming high-order complexes, reminiscent of the mode of activation of the phylogenetically related transmembrane kinase/RNase Ire1 in the unfolded protein response. We describe crystal structures determined at 2.4 Å and 2.8 Å resolution, which show that two molecules of 2-5A at a time tether RNase L monomers via the ankyrin-repeat (ANK) domain. Each ANK domain harbors two distinct sites for 2-5A recognition that reside 50 Å apart. These data reveal a function for the ANK domain as a 2-5A-sensing homo-oligomerization device and describe a nonlinear, ultrasensitive regulation in the 2-5A/RNase L system poised for amplification of the IFN response. Graphical abstract Graphical Abstract Highlights ► Human RNase L undergoes a highly cooperative activation ► Two 2-5A molecules at a time tether RNase L into signaling complexes ► The ANK domain of RNase L is a homo-oligomerization device ► RNase L is poised for ultrasensitive amplification of the interferon response

Beschreibung: Publication year: 2012 Source: Cell Reports Anthony B. Mak, Allison M.L. Nixon, Saranya Kittanakom, Jocelyn M. Stewart, Ginny I. Chen, Jasna Curak, Anne-Claude Gingras, Ralph Mazitschek, Benjamin G. Neel, Igor Stagljar, Jason Moffat The pentaspan membrane glycoprotein CD133 marks lineage-specific cancer progenitor cells and is associated with poor prognosis in a number of tumor types. Despite its utility as a cancer progenitor cell marker, CD133 protein regulation and molecular function remain poorly understood. We find that the deacetylase HDAC6 physically associates with CD133 to negatively regulate CD133 trafficking down the endosomal-lysosomal pathway for degradation. We further demonstrate that CD133, HDAC6, and the central molecule of the canonical Wnt signaling pathway, β-catenin, can physically associate as a ternary complex. This association stabilizes β-catenin via HDAC6 deacetylase activity, which leads to activation of β-catenin signaling targets. Downregulation of either CD133 or HDAC6 results in increased β-catenin acetylation and degradation, which correlates with decreased proliferation in vitro and tumor xenograft growth in vivo. Given that CD133 marks progenitor cells in a wide range of cancers, targeting CD133 may be a means to treat multiple cancer types. Graphical abstract Graphical Abstract Highlights ► HDAC6 negatively regulates CD133 trafficking into endosomes ► CD133 knockdown suppresses colon and ovarian cancer cell differentiation ► CD133, HDAC6, and β-catenin physically associate to form a functional module ► CD133 regulates β-catenin stability through HDAC6 deacetylase activity

Beschreibung: 29 November 2012 Publication year: 2012 Source: Cell Reports, Volume 2, Issue 5 The large Mediator (L-Mediator) is a general coactivator of RNA polymerase II transcription and is formed by the reversible association of the small Mediator (S-Mediator) and the kinase-module-harboring Cdk8. It is not known how the kinase module association/dissociation is regulated. We describe the fission yeast Cdk11-L-type cyclin pombe (Lcp1) complex and show that its inactivation alters the global expression profile in a manner very similar to that of mutations of the kinase module. Cdk11 is broadly distributed onto chromatin and phosphorylates the Med27 and Med4 Mediator subunits on conserved residues. The association of the kinase module and the S-Mediator is strongly decreased by the inactivation of either Cdk11 or the mutation of its target residues on the Mediator. These results show that Cdk11-Lcp1 regulates the association of the kinase module and the S-Mediator to form the L-Mediator complex. Graphical abstract Highlights ► A complex related to metazoan Cdk11-CyclinL is conserved in fission yeast ► Loss of either Cdk8 or Cdk11 similarly affects global expression profile ► Cdk11 phosphorylates two Mediator subunits ► Association of the Cdk8 module with the S-Mediator requires phosphorylation by Cdk11

Beschreibung: 29 November 2012 Publication year: 2012 Source: Cell Reports, Volume 2, Issue 5 Plasma sphingosine-1-phosphate (S1P) regulates vascular permeability, and plasma and lymph S1P guide lymphocyte egress from lymphoid organs. S1P is made intracellularly, and little is known about how S1P is delivered into circulatory fluids. Here, we find that mice without the major facilitator superfamily transporter Spns2 have a profound reduction in lymph S1P, but only a minor decrease in plasma S1P. Spns2-deficient mice have a redistribution of lymphocytes from the spleen to lymph nodes and a loss of circulating lymphocytes, consistent with normal egress from the spleen directed by plasma S1P and blocked egress from lymph nodes directed by lymph S1P. Spns2 is needed in endothelial cells to supply lymph S1P and support lymphocyte circulation. As a differential requirement for lymph and blood S1P, Spns2 may be an attractive target for immune suppressive drugs. Graphical abstract Highlights ► The transporter Spns2 is required to supply lymph, but not plasma, S1P ► Spns2-deficient mice have disrupted peripheral lymphocyte circulation ► Spns2 is required in endothelial cells to secrete lymph S1P and support trafficking

Beschreibung: 29 November 2012 Publication year: 2012 Source: Cell Reports, Volume 2, Issue 5 Elongin A increases the rate of RNA polymerase II (pol II) transcript elongation by suppressing transient pausing by the enzyme. Elongin A also acts as a component of a cullin-RING ligase that can target stalled pol II for ubiquitylation and proteasome-dependent degradation. It is not known whether these activities of Elongin A are functionally interdependent in vivo. Here, we demonstrate that Elongin A-deficient (Elongin A −/− ) embryos exhibit abnormalities in the formation of both cranial and spinal nerves and that Elongin A −/− embryonic stem cells (ESCs) show a markedly decreased capacity to differentiate into neurons. Moreover, we identify Elongin A mutations that selectively inactivate one or the other of the aforementioned activities and show that mutants that retain the elongation stimulatory, but not pol II ubiquitylation, activity of Elongin A rescue neuronal differentiation and support retinoic acid-induced upregulation of a subset of neurogenesis-related genes in Elongin A −/− ESCs. Graphical abstract Highlights ► RA-induced neuronal differentiation is markedly impaired in Elongin A −/− ESCs ► Formation of cranial ganglia and DRG is severely impaired in Elongin A −/− embryos ► Mutations that differentially affect the two activities of Elongin A were identified ► Elongin A’s elongation stimulatory activity is required for a subset of RA-induced genes

Beschreibung: 29 November 2012 Publication year: 2012 Source: Cell Reports, Volume 2, Issue 5 Expansions of simple DNA repeats cause numerous hereditary diseases in humans. We analyzed the role of DNA polymerases in the instability of Friedreich’s ataxia (GAA) n repeats in a yeast experimental system. The elementary step of expansion corresponded to ∼160 bp in the wild-type strain, matching the size of Okazaki fragments in yeast. This step increased when DNA polymerase α was mutated, suggesting a link between the scale of expansions and Okazaki fragment size. Expandable repeats strongly elevated the rate of mutations at substantial distances around them, a phenomenon we call repeat-induced mutagenesis (RIM). Notably, defects in the replicative DNA polymerases δ and ε strongly increased rates for both repeat expansions and RIM. The increases in repeat-mediated instability observed in DNA polymerase δ mutants depended on translesion DNA polymerases. We conclude that repeat expansions and RIM are two sides of the same replicative mechanism. Graphical abstract Highlights ► Elementary step of repeat expansion corresponds to size of an Okazaki fragment ► Mutated DNA polymerase α leads to an increase in expansion step ► Mutated DNA polymerases δ and ε lead to elevated expansion rates ► Repeats induce mutagenesis, which is further elevated in polymerase mutants

Beschreibung: 29 November 2012 Publication year: 2012 Source: Cell Reports, Volume 2, Issue 5 In meiosis, two specialized cell divisions allow the separation of paired chromosomes first, then of sister chromatids. Separase removes the cohesin complex holding sister chromatids together in a stepwise manner from chromosome arms in meiosis I, then from the centromere region in meiosis II. Using mouse oocytes, our study reveals that cyclin A2 promotes entry into meiosis, as well as an additional unexpected role; namely, its requirement for separase-dependent sister chromatid separation in meiosis II. Untimely cyclin A2-associated kinase activity in meiosis I leads to precocious sister separation, whereas inhibition of cyclin A2 in meiosis II prevents it. Accordingly, endogenous cyclin A is localized to kinetochores throughout meiosis II, but not in anaphase I. Additionally, we found that cyclin B1, but not cyclin A2, inhibits separase in meiosis I. These findings indicate that separase-dependent cohesin removal is differentially regulated by cyclin B1 and A2 in mammalian meiosis. Graphical abstract Highlights ► Cyclin A2 is required for meiotic entry and sister chromatid separation in meiosis II ► Constitutive cyclin A2 activity in meiosis I leads to precocious sister separation ► Only cyclin B1, and not cyclin A2, can inhibit separase in mouse oocyte meiosis I ► Endogenous cyclin A is localized to centromeres throughout the second meiotic division

Beschreibung: 29 November 2012 Publication year: 2012 Source: Cell Reports, Volume 2, Issue 5 MicroRNAs (miRNAs) are essential regulators of development, physiology, and evolution, and their biogenesis is strictly controlled at multiple levels. Regulatory proteins, such as KSRP, modulate rates and timing of enzymatic reactions responsible for maturation of select miRNAs from their primary transcripts in response to specific stimuli. Here, we show that KSRP silencing in mesenchymal C2C12 cells produces a change in the transcriptome largely overlapping that induced by bone morphogenetic protein 2 (BMP2) signaling activation. This induces osteoblastic differentiation while preventing myogenic differentiation. KSRP silencing- and BMP2-dependent myogenic miRNA (myomiR) maturation blockade is required for osteoblastic differentiation of C2C12 cells. Our results demonstrate that phosphorylated R-SMAD proteins, the transducers of BMP2 signal, associate with phosphorylated KSRP and block its interaction with primary myomiRs. This abrogates KSRP-dependent myomiR maturation, with SMAD4, SMAD5, and SMAD9 silencing being able to rescue KSRP function. Thus, SMAD-induced blockade of KSRP-dependent myomiR maturation is critical for orienting C2C12 cell differentiation toward osteoblastic lineage. Graphical abstract Highlights ► KSRP silencing and BMP2 signaling activation block myomiR maturation ► myomiR maturation blockade is required for osteoblastic differentiation of C2C12 cells ► SMAD proteins interact with KSRP and block its ability to promote myomiR maturation

Beschreibung: 29 November 2012 Publication year: 2012 Source: Cell Reports, Volume 2, Issue 5 How specific cell types can be directly converted into other distinct cell types is a matter of intense investigation with wide-ranging basic and biomedical implications. Here, we show that removal of the histone 3 lysine 27 (H3K27) methyltransferase Polycomb repressor complex 2 (PRC2) permits ectopically expressed, neuron-type-specific transcription factors (“terminal selectors”) to convert Caenorhabditis elegans germ cells directly into specific neuron types. Terminal-selector-induced germ-cell-to-neuron conversion can be observed not only upon genome-wide loss of H3K27 methylation in PRC2(−) animals but also upon genome-wide redistribution of H3K27 methylation patterns in animals that lack the H3K36 methyltransferase MES-4. Manipulation of the H3K27 methylation status not only permits conversion of germ cells into neurons but also permits hlh-1/MyoD -dependent conversion of germ cells into muscle cells, indicating that PRC2 protects the germline from the aberrant execution of multiple distinct somatic differentiation programs. Taken together, our findings demonstrate that the normally multistep process of development from a germ cell via a zygote to a terminally differentiated somatic cell type can be short-cut by providing an appropriate terminal selector transcription factor and manipulating histone methylation patterns. Graphical abstract Highlights ► Selector-type transcription factors cannot convert germ cells into neurons or muscle ► Loss of H3K27me3 allows direct, selector-driven germ cell conversion to neurons or muscle ► H3K27me3 redistribution in H3K36me(−) animals also permits germ cell conversion ► Mitotic cycling is not required for induced germ cell conversion

Beschreibung: 29 November 2012 Publication year: 2012 Source: Cell Reports, Volume 2, Issue 5 GATA transcription factors regulate transcription during development and differentiation by recognizing distinct GATA sites with a tandem of two conserved zinc fingers, and by mediating long-range DNA looping. However, the molecular basis of these processes is not well understood. Here, we determined three crystal structures of the full DNA-binding domain (DBD) of human GATA3 protein, which contains both zinc fingers, in complex with different DNA sites. In one structure, both zinc fingers wrap around a palindromic GATA site, cooperatively enhancing the binding affinity and kinetic stability. Strikingly, in the other two structures, the two fingers of GATA DBD bind GATA sites on different DNA molecules, thereby bridging two separate DNA fragments. This was confirmed in solution by an in-gel fluorescence resonance energy transfer analysis. These findings not only provide insights into the structure and function of GATA proteins but also shed light on the molecular basis of long-range gene regulation. Graphical abstract Highlights ► Crystal structures of the GATA3 DNA-binding domain are presented ► The two fingers of GATA3 can bridge two separate DNA fragments ► GATA protein may use this mechanism to mediate long-range DNA looping ► GATA3 uses a combination of base and shape readout to bind to its target sites

Beschreibung: Publication year: 2012 Source: Cell Reports Sang Mi Shim, Won Jae Lee, Youngdoo Kim, Jong Wook Chang, Sungmin Song, Yong-Keun Jung The ubiquitin-proteasome system is essential for maintaining protein homeostasis. However, proteasome dysregulation in chronic diseases is poorly understood. Through genome-wide cell-based screening using 5,500 cDNAs, a signaling pathway leading to NFκB activation was selected as an inhibitor of 26S proteasome. TNF-α increased S5b (HGNC symbol PSMD5; hereafter S5b/PSMD5) expression via NFκB, and the surplus S5b/PSMD5 directly inhibited 26S proteasome assembly and activity. Downregulation of S5b/PSMD5 abolished TNF-α-induced proteasome inhibition. TNF-α enhanced the interaction of S5b/PSMD5 with S7/PSMC2 in nonproteasome complexes, and interference of this interaction rescued TNF-α-induced proteasome inhibition. Transgenic mice expressing S5b/PSMD5 exhibited a reduced life span and premature onset of aging-related phenotypes, including reduced proteasome activity in their tissues. Conversely, S5b/PSMD5 deficiency in Drosophila melanogaster ameliorated the tau rough eye phenotype, enhanced proteasome activity, and extended the life span of tau flies. These results reveal the critical role of S5b/PSMD5 in negative regulation of proteasome by TNF-α/NFκB and provide insights into proteasome inhibition in human disease. Graphical abstract Graphical Abstract Highlights ► Proteasome-inhibitory TNF-α/NFκB was found by cell-based functional screening ► TNF-α-induced S5b reduces proteasome activity by interfering with assembly ► Interaction of S5b with S7 is critical for TNF-α-induced proteasome inhibition ► S5b deficiency rescues tauopathy and excess S5b inhibits proteasome in animal models

Beschreibung: Publication year: 2012 Source: Cell Reports Charles A. Easley, Bart T. Phillips, Megan M. McGuire, Jennifer M. Barringer, Hanna Valli, Brian P. Hermann, Calvin R. Simerly, Aleksander Rajkovic, Toshio Miki, Kyle E. Orwig, Gerald P. Schatten Human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) have been shown to differentiate into primordial germ cells (PGCs) but not into spermatogonia, haploid spermatocytes, or spermatids. Here, we show that hESCs and hiPSCs differentiate directly into advanced male germ cell lineages, including postmeiotic, spermatid-like cells, in vitro without genetic manipulation. Furthermore, our procedure mirrors spermatogenesis in vivo by differentiating PSCs into UTF1-, PLZF-, and CDH1-positive spermatogonia-like cells; HIWI- and HILI-positive spermatocyte-like cells; and haploid cells expressing acrosin, transition protein 1, and protamine 1 (proteins that are uniquely found in spermatids and/or sperm). These spermatids show uniparental genomic imprints similar to those of human sperm on two loci: H19 and IGF2 . These results demonstrate that male PSCs have the ability to differentiate directly into advanced germ cell lineages and may represent a novel strategy for studying spermatogenesis in vitro. Graphical abstract Graphical Abstract Highlights ► In vitro culture induces germ cell differentiation of hPSCs ► hPSCs differentiate into spermatogonia, spermatocytes, and haploid spermatids ► Haploid spermatids have uniparental imprints similar to fertile human sperm

Beschreibung: Publication year: 2012 Source: Cell Reports Xin Hui Derryn Chan, Srikanth Nama, Felicia Gopal, Pamela Rizk, Srinivas Ramasamy, Gopinath Sundaram, Ghim Siong Ow, Ivshina Anna Vladimirovna, Vivek Tanavde, Johannes Haybaeck, Vladimir Kuznetsov, Prabha Sampath Malignant gliomas are the most aggressive forms of brain tumors, associated with high rates of morbidity and mortality. Recurrence and tumorigenesis are attributed to a subpopulation of tumor-initiating glioma stem cells (GSCs) that are intrinsically resistant to therapy. Initiation and progression of gliomas have been linked to alterations in microRNA expression. Here, we report the identification of microRNA-138 (miR-138) as a molecular signature of GSCs and demonstrate a vital role for miR-138 in promoting growth and survival of bona fide tumor-initiating cells with self-renewal potential. Sequence-specific functional inhibition of miR-138 prevents tumorsphere formation in vitro and impedes tumorigenesis in vivo. We delineate the components of the miR-138 regulatory network by loss-of-function analysis to identify specific regulators of apoptosis. Finally, the higher expression of miR-138 in GSCs compared to non-neoplastic tissue and association with tumor recurrence and survival highlights the clinical significance of miR-138 as a prognostic biomarker and a therapeutic target for treatment of malignant gliomas. Graphical abstract Graphical Abstract Highlights ► MicroRNA-138 is a molecular signature of glioma stem cells ► Functional inhibition of miR-138 leads to apoptotic death of glioma stem cells ► MicroRNA-138 is a prosurvival oncomiR for glioma stem cells ► MicroRNA-138 is a prognostic biomarker for malignant gliomas

Beschreibung: Publication year: 2012 Source: Cell Reports Tamar Hashimshony, Florian Wagner, Noa Sher, Itai Yanai High-throughput sequencing has allowed for unprecedented detail in gene expression analyses, yet its efficient application to single cells is challenged by the small starting amounts of RNA. We have developed CEL-Seq, a method for overcoming this limitation by barcoding and pooling samples before linearly amplifying mRNA with the use of one round of in vitro transcription. We show that CEL-Seq gives more reproducible, linear, and sensitive results than a PCR-based amplification method. We demonstrate the power of this method by studying early C. elegans embryonic development at single-cell resolution. Differential distribution of transcripts between sister cells is seen as early as the two-cell stage embryo, and zygotic expression in the somatic cell lineages is enriched for transcription factors. The robust transcriptome quantifications enabled by CEL-Seq will be useful for transcriptomic analyses of complex tissues containing populations of diverse cell types. Graphical abstract Graphical Abstract Highlights ► We present CEL-Seq, a single-cell transcriptomics method using in vitro transcription ► We show that CEL-Seq is linear, sensitive, and reproducible ► CEL-Seq is highly multiplexed, allowing for the parallel examination of hundreds of cells ► We describe early C. elegans embryology at the single-cell transcriptomic level

Beschreibung: Publication year: 2012 Source: Cell Reports Chandrama Mukherjee, Deepak P. Patil, Brian A. Kennedy, Baskar Bakthavachalu, Ralf Bundschuh, Daniel R. Schoenberg The notion that decapping leads irreversibly to messenger RNA (mRNA) decay was contradicted by the identification of capped transcripts missing portions of their 5′ ends and a cytoplasmic complex that can restore the cap on uncapped mRNAs. In this study, we used accumulation of uncapped transcripts in cells inhibited for cytoplasmic capping to identify the targets of this pathway. Inhibition of cytoplasmic capping results in the destabilization of some transcripts and the redistribution of others from polysomes to nontranslating messenger ribonucleoproteins, where they accumulate in an uncapped state. Only a portion of the mRNA transcriptome is affected by cytoplasmic capping, and its targets encode proteins involved in nucleotide binding, RNA and protein localization, and the mitotic cell cycle. The 3′ untranslated regions of recapping targets are enriched for AU-rich elements and microRNA binding sites, both of which function in cap-dependent mRNA silencing. These findings identify a cyclical process of decapping and recapping that we term cap homeostasis . Graphical abstract Graphical Abstract Highlights ► mRNAs that undergo cyclical loss and restoration of the cap are identified ► Some recapping targets are destabilized when cytoplasmic capping is inhibited ► Cytoplasmic capping functions in cycling mRNA between polysomes and mRNPs ► In cells inhibited for cytoplasmic capping, nontranslating mRNA is stored uncapped

Beschreibung: Publication year: 2012 Source: Cell Reports Adrienne E. Dubin, Manuela Schmidt, Jayanti Mathur, Matthew J. Petrus, Bailong Xiao, Bertrand Coste, Ardem Patapoutian Heightened nociceptor function caused by inflammatory mediators such as bradykinin (BK) contributes to increased pain sensitivity (hyperalgesia) to noxious mechanical and thermal stimuli. Although it is known that sensitization of the heat transducer TRPV1 largely subserves thermal hyperalgesia, the cellular mechanisms underlying mechanical hyperalgesia have been elusive. The role of the mechanically activated (MA) channel piezo2 (known as FAM38B) present in mammalian sensory neurons is unknown. We test the hypothesis that piezo2 activity is enhanced by BK, an algogenic peptide that induces mechanical hyperalgesia within minutes. Piezo2 current amplitude is increased and inactivation is slowed by bradykinin receptor beta 2 (BDKRB2) activation in heterologous expression systems. Protein kinase A (PKA) and protein kinase C (PKC) agonists enhance piezo2 activity. BDKRB2-mediated effects are abolished by PKA and PKC inhibitors. Finally, piezo2-dependent MA currents in a class of native sensory neurons are enhanced 8-fold by BK via PKA and PKC. Thus, piezo2 sensitization may contribute to PKA- and PKC-mediated mechanical hyperalgesia. Graphical abstract Graphical Abstract Highlights ► Bradykinin (BK) enhances mechanically activated (MA) piezo2 currents ► B2 receptor activation increases piezo2 current amplitude and slows inactivation ► Protein kinase A and protein kinase C inhibition abrogates both BK-induced effects ► MA currents dependent on piezo2 in DRG neurons are similarly modulated by BK

Beschreibung: Publication year: 2012 Source: Cell Reports Eneda Toska, Hayley A. Campbell, Jayasha Shandilya, Sarah J. Goodfellow, Paul Shore, Kathryn F. Medler, Stefan G.E. Roberts The Wilms' tumor 1 protein WT1 is a transcriptional regulator that is involved in cell growth and differentiation. The transcriptional corepressor BASP1 interacts with WT1 and converts WT1 from a transcriptional activator to a repressor. Here, we demonstrate that the N-terminal myristoylation of BASP1 is required in order to elicit transcriptional repression at WT1 target genes. We show that myristoylated BASP1 binds to nuclear PIP2, which leads to the recruitment of PIP2 to the promoter regions of WT1-dependent target genes. BASP1's myristoylation and association with PIP2 are required for the interaction of BASP1 with HDAC1, which mediates the recruitment of HDAC1 to the promoter and elicits transcriptional repression. Our findings uncover a role for myristoylation in transcription, as well as a critical function for PIP2 in gene-specific transcriptional repression through the recruitment of histone deacetylase. Graphical abstract Graphical Abstract Highlights ► Myristoylation of BASP1 is required for its function as a transcriptional repressor ► BASP1 myristoylation facilitates its interaction with nuclear PIP2 ► Myristoylation of BASP1 recruits HDAC1 to gene promoters through PIP2

Beschreibung: Publication year: 2012 Source: Cell Reports Dirk Wohlleber, Hamid Kashkar, Katja Gärtner, Marianne K. Frings, Margarete Odenthal, Silke Hegenbarth, Carolin Börner, Bernd Arnold, Günter Hämmerling, Bernd Nieswandt, Nico van Rooijen, Andreas Limmer, Karin Cederbrant, Mathias Heikenwalder, Manolis Pasparakis, Ulrike Protzer, Hans-Peter Dienes, Christian Kurts, Martin Krönke, Percy A. Knolle Viruses can escape cytotoxic T cell (CTL) immunity by avoiding presentation of viral components via endogenous MHC class I antigen presentation in infected cells. Cross-priming of viral antigens circumvents such immune escape by allowing noninfected dendritic cells to activate virus-specific CTLs, but they remain ineffective against infected cells in which immune escape is functional. Here, we show that cross-presentation of antigen released from adenovirus-infected hepatocytes by liver sinusoidal endothelial cells stimulated cross-primed effector CTLs to release tumor necrosis factor (TNF), which killed virus-infected hepatocytes through caspase activation. TNF receptor signaling specifically eliminated infected hepatocytes that showed impaired anti-apoptotic defense. Thus, CTL immune surveillance against infection relies on two similarly important but distinct effector functions that are both MHC restricted, requiring either direct antigen recognition on target cells and canonical CTL effector function or cross-presentation and a noncanonical effector function mediated by TNF. Graphical abstract Graphical Abstract Highlights ► Endothelial cell cross-presentation in infected tissue facilitates CTL immunity ► A CTL effector function acts independently of MHC recognition on target cells ► Viral infection sensitizes hepatocytes toward TNF-induced death

Beschreibung: Publication year: 2012 Source: Cell Reports María J. Pérez-García, Steven J. Burden Amyotrophic lateral sclerosis (ALS) is a devastating disease that progresses from detachment of motor nerve terminals to complete muscle paralysis and lethal respiratory failure within 5 years of diagnosis. Genetic studies have linked mutations in several genes to ALS, and mice bearing mutations in SOD1 recapitulate hallmark features of the disease. We investigated whether disease symptoms can be ameliorated by co-opting the retrograde signaling pathway that promotes attachment of nerve terminals to muscle. We crossed SOD1G93A mice with transgenic mice that express MuSK, a receptor tyrosine kinase that is required for retrograde signaling, and we used histological and behavioral assays to assess motor innervation and behavior. A 3-fold increase in MuSK expression delayed the onset and reduced the extent of muscle denervation, improving motor function for more than a month without altering survival. These findings suggest that increasing MuSK activity by pharmacological means has the potential to improve motor function in ALS. Graphical abstract Graphical Abstract Highlights ► An increase in MuSK expression delays the onset of muscle denervation in ALS mice ► Increased MuSK expression reduces the extent of denervation in ALS mice ► Increased MuSK expression improves motor function and behavior in ALS mice ► MuSK agonists have the potential to improve motor function in ALS

Beschreibung: Publication year: 2012 Source: Cell Reports Kevin Takaki, Christine L. Cosma, Mark A. Troll, Lalita Ramakrishnan Treatment of tuberculosis, like other infectious diseases, is increasingly hindered by the emergence of drug resistance. Drug discovery efforts would be facilitated by facile screening tools that incorporate the complexities of human disease. Mycobacterium marinum -infected zebrafish larvae recapitulate key aspects of tuberculosis pathogenesis and drug treatment. Here, we develop a model for rapid in vivo drug screening using fluorescence-based methods for serial quantitative assessment of drug efficacy and toxicity. We provide proof-of-concept that both traditional bacterial-targeting antitubercular drugs and newly identified host-targeting drugs would be discovered through the use of this model. We demonstrate the model’s utility for the identification of synergistic combinations of antibacterial drugs and demonstrate synergy between bacterial- and host-targeting compounds. Thus, the platform can be used to identify new antibacterial agents and entirely new classes of drugs that thwart infection by targeting host pathways. The methods developed here should be widely applicable to small-molecule screens for other infectious and noninfectious diseases. Graphical abstract Graphical Abstract Highlights ► A larval zebrafish- M. marium platform for in vivo antitubercular drug discovery ► High-throughput husbandry and reversible anesthesia in 96-well format ► Fluorimetric in situ measurement of drug efficacy and toxicity ► Detection of host-acting compounds and identification of a new drug synergy

Beschreibung: Publication year: 2012 Source: Cell Reports, Volume 1, Issue 4 Chen-Ju Lin, Zeina Nasr, Prem K. Premsrirut, John A. Porco, Yoshitaka Hippo, Scott W. Lowe, Jerry Pelletier The energetically demanding process of translation is linked to multiple signaling events through mTOR-mediated regulation of eukaryotic initiation factor (eIF)4F complex assembly. Disrupting mTOR constraints on eIF4F activity can be oncogenic and alter chemotherapy response, making eIF4F an attractive antineoplastic target. Here, we combine a newly developed inducible RNAi platform and pharmacological targeting of eIF4F activity to define a critical role for endogenous eIF4F in Myc-dependent tumor initiation. We find elevated Myc levels are associated with deregulated eIF4F activity in the prelymphomatous stage of the Eμ-Myc lymphoma model. Inhibition of eIF4F is synthetic lethal with elevated Myc in premalignant pre-B/B cells resulting in reduced numbers of cycling pre-B/B cells and delayed tumor onset. At the organismal level, eIF4F suppression affected a subset of normal regenerating cells, but this was well tolerated and rapidly and completely reversible. Therefore, eIF4F is a key Myc client that represents a tumor-specific vulnerability. Graphical abstract Graphical Abstract Highlights ► Elevated eIF4F levels are a premalignant feature of Eμ-Myc lymphomas ► Transient eIF4E suppression delays tumor initiation in Myc-induced lymphomas ► A novel inducible RNAi platform demonstrates that eIF4F is a client of Myc in vivo ► Elevated Myc levels and eIF4F suppression show a synthetic lethal relationship

Beschreibung: Publication year: 2012 Source: Cell Reports, Volume 1, Issue 4 Markus Knaden, Antonia Strutz, Jawaid Ahsan, Silke Sachse, Bill S. Hansson Brains have to decide whether and how to respond to detected stimuli based on complex sensory input. The vinegar fly Drosophila melanogaster evaluates food sources based on olfactory cues. Here, we performed a behavioral screen using the vinegar fly and established the innate valence of 110 odorants. Our analysis of neuronal activation patterns evoked by attractive and aversive odorants suggests that even though the identity of odorants is coded by the set of activated receptors, the main representation of odorant valence is formed at the output level of the antennal lobe. The topographic clustering within the antennal lobe of valence-specific output neurons resembles a corresponding domain in the olfactory bulb of mice. The basal anatomical structure of the olfactory circuit between insects and vertebrates is known to be similar; our study suggests that the representation of odorant valence is as well. Graphical abstract Graphical Abstract Highlights ► We establish the innate hedonic valence of 110 odors for Drosophila melanogaster ► A clear valence of odors is first represented at the output of the antennal lobe ► Aversive- and attractive-specific glomeruli cluster in separate functional areas

Beschreibung: Publication year: 2012 Source: Cell Reports, Volume 1, Issue 3 Amanda W. Lund, Fernanda V. Duraes, Sachiko Hirosue, Vidya R. Raghavan, Chiara Nembrini, Susan N. Thomas, Amine Issa, Stéphanie Hugues, Melody A. Swartz Tumor expression of the lymphangiogenic factor VEGF-C is correlated with metastasis and poor prognosis, and although VEGF-C enhances transport to the draining lymph node (dLN) and antigen exposure to the adaptive immune system, its role in tumor immunity remains unexplored. Here, we demonstrate that VEGF-C promotes immune tolerance in murine melanoma. In B16 F10 melanomas expressing a foreign antigen (OVA), VEGF-C protected tumors against preexisting antitumor immunity and promoted local deletion of OVA-specific CD8 + T cells. Naive OVA-specific CD8 + T cells, transferred into tumor-bearing mice, were dysfunctionally activated and apoptotic. Lymphatic endothelial cells (LECs) in dLNs cross-presented OVA, and naive LECs scavenge and cross-present OVA in vitro. Cross-presenting LECs drove the proliferation and apoptosis of OVA-specific CD8 + T cells ex vivo. Our findings introduce a tumor-promoting role for lymphatics in the tumor and dLN and suggest that lymphatic endothelium in the local microenvironment may be a target for immunomodulation. Graphical abstract Graphical Abstract Highlights ► VEGF-C protects tumors from preinduced vaccine immunity ► VEGF-C enhances dysfunctional activation of CD8 T cells in tumor-draining lymph nodes ► Lymph node lymphatic endothelial cells scavenge and cross-present tumor antigen ► Lymphatic endothelial cells cross-tolerize antigen-specific CD8 T cells ex vivo

Beschreibung: Publication year: 2012 Source: Cell Reports, Volume 1, Issue 3 Ek Han Tan, Todd Blevins, Thomas S. Ream, Craig S. Pikaard Multisubunit RNA polymerases IV and V (Pol IV and Pol V) evolved as specialized forms of Pol II that mediate RNA-directed DNA methylation (RdDM) and transcriptional silencing of transposons, viruses, and endogenous repeats in plants. Among the subunits common to Arabidopsis thaliana Pols II, IV, and V are 93% identical alternative ninth subunits, NRP(B/D/E)9a and NRP(B/D/E)9b. The 9a and 9b subunit variants are incompletely redundant with respect to Pol II; whereas double mutants are embryo lethal, single mutants are viable, yet phenotypically distinct. Likewise, 9a or 9b can associate with Pols IV or V but RNA-directed DNA methylation is impaired only in 9b mutants. Based on genetic and molecular tests, we attribute the defect in RdDM to impaired Pol V function. Collectively, our results reveal a role for the ninth subunit in RNA silencing and demonstrate that subunit diversity generates functionally distinct subtypes of RNA polymerases II and V. Graphical abstract Graphical Abstract Highlights ► Arabidopsis RNA polymerases II, IV, and V use either of two ninth subunit proteins ► The alternative ninth subunits affect Pol II and Pol V functions ► RNA-directed DNA methylation requires the 9b-containing form of Pol V ► Unlike yeast and archaea, an NRPB9 subunit of Pol II is essential in Arabidopsis

Beschreibung: Publication year: 2012 Source: Cell Reports, Volume 1, Issue 3 Andreas Reiner, Ryan J. Arant, Ehud Y. Isacoff Ionotropic glutamate receptors assemble as homo- or heterotetramers. One well-studied heteromeric complex is formed by the kainate receptor subunits GluK2 and GluK5. Retention motifs prevent trafficking of GluK5 homomers to the plasma membrane, but coassembly with GluK2 yields functional heteromeric receptors. Additional control over GluK2/GluK5 assembly seems to be exerted by the amino-terminal domains, which preferentially assemble into heterodimers as isolated domains. However, the stoichiometry of the full-length GluK2/GluK5 receptor complex has yet to be determined, as is the case for all non-NMDA glutamate receptors. Here, we address this question, using a single-molecule imaging technique that enables direct counting of the number of each GluK subunit type in homomeric and heteromeric receptors in the plasma membranes of live cells. We show that GluK2 and GluK5 assemble with 2:2 stoichiometry. This is an important step toward understanding the assembly mechanism, architecture, and functional consequences of heteromer formation in ionotropic glutamate receptors. Graphical abstract Graphical Abstract Highlights ► Single-molecule imaging directly confirms tetrameric assembly of kainate receptors ► GluK2 and GluK5 subunits form heterotetramers with a fixed 2:2 stoichiometry ► GluK5 devoid of ER retention motifs forms nonfunctional tetramers

Beschreibung: Publication year: 2012 Source: Cell Reports, Volume 1, Issue 5 Romain Mercier, Patricia Domínguez-Cuevas, Jeff Errington The cell wall is a defining structural feature of the bacterial subkingdom. However, most bacteria are capable of mutating into a cell-wall-deficient “L-form” state, requiring remarkable physiological and structural adaptations. L-forms proliferate by an unusual membrane deformation and scission process that is independent of the conserved and normally essential FtsZ based division machinery, and which may provide a model for the replication of primitive cells. Candidate gene screening revealed no requirement for the cytoskeletal systems that might actively drive membrane deformation or scission. Instead, we uncovered a crucial role for branched-chain fatty acid (BCFA) synthesis. BCFA-deficient mutants grow and undergo pulsating shape changes, but membrane scission fails, abolishing the separation of progeny cells. The failure in scission is associated with a reduction in membrane fluidity. The results identify a step in L-form proliferation and demonstrate that purely biophysical processes may have been sufficient for proliferation of primitive cells. Graphical abstract Graphical Abstract Highlights ► The bacterial cytoskeleton is not essential in cell-wall-deficient L-forms ► Membrane fluidity is an important factor for L-form proliferation ► Decreased membrane fluidity blocks the separation of progeny cells

Beschreibung: Publication year: 2012 Source: Cell Reports, Volume 1, Issue 4 Tobias Reichenbach, Aleksandra Stefanovic, Fumiaki Nin, A.J. Hudspeth Sound is detected and converted into electrical signals within the ear. The cochlea not only acts as a passive detector of sound, however, but can also produce tones itself. These otoacoustic emissions are a striking manifestation of the cochlea's mechanical active process. A controversy remains of how these mechanical signals propagate back to the middle ear, from which they are emitted as sound. Here, we combine theoretical and experimental studies to show that mechanical signals can be transmitted by waves on Reissner's membrane, an elastic structure within the cochlea. We develop a theory for wave propagation on Reissner's membrane and its role in otoacoustic emissions. Employing a scanning laser interferometer, we measure traveling waves on Reissner's membrane in the gerbil, guinea pig, and chinchilla. The results are in accord with the theory and thus support a role for Reissner's membrane in otoacoustic emissions. Graphical abstract Graphical Abstract Highlights ► Waves can propagate on Reissner's membrane, an elastic structure in the inner ear ► These waves can be elicited by nonlinear mechanical activity within the cochlea ► Waves on Reissner's membrane can transmit otoacoustic emissions to the outside ► Waves propagate by two modes on the fluid-coupled Reissner's and basilar membranes

Beschreibung: Publication year: 2012 Source: Cell Reports Yue Wu, Di Zhang, Jee Yan Chu, Patrick Boyle, Yong Wang, Ian D. Brindle, Vincenzo De Luca, Charles Després Salicylic acid (SA) is an essential hormone in plant immunity, but its receptor has remained elusive for decades. The transcriptional coregulator NPR1 is central to the activation of SA-dependent defense genes, and we previously found that Cys 521 and Cys 529 of Arabidopsis NPR1's transactivation domain are critical for coactivator function. Here, we demonstrate that NPR1 directly binds SA, but not inactive structural analogs, with an affinity similar to that of other hormone-receptor interactions and consistent with in vivo Arabidopsis SA concentrations. Binding of SA occurs through Cys 521/529 via the transition metal copper. Mechanistically, our results suggest that binding of SA causes a conformational change in NPR1 that is accompanied by the release of the C-terminal transactivation domain from the N-terminal autoinhibitory BTB/POZ domain. While NPR1 is already known as a link between the SA signaling molecule and defense-gene activation, we now show that NPR1 is the receptor for SA. Graphical abstract Graphical Abstract Highlights ► NPR1 is a salicylic acid (SA) receptor, binding specifically to SA via Cys 521/529 ► NPR1 binds copper in vivo via Cys 521/529 , and metals are required for SA binding ► SA directly regulates the conformation of NPR1 by deoligomerizing NPR1 into a dimer ► The NPR1 BTB/POZ domain autoinhibits the function of the NPR1 transactivation domain

Beschreibung: Publication year: 2012 Source: Cell Reports Janai R. Carr, Megan M. Kiefer, Hyun Jung Park, Jing Li, Zebin Wang, Joel Fontanarosa, Danielle DeWaal, Dragana Kopanja, Elizaveta V. Benevolenskaya, Grace Guzman, Pradip Raychaudhuri Elevated expression of FoxM1 in breast cancer correlates with an undifferentiated tumor phenotype and a negative clinical outcome. However, a role for FoxM1 in regulating mammary differentiation was not known. Here, we identify another function of FoxM1, the ability to act as a transcriptional repressor, which plays an important role in regulating the differentiation of luminal epithelial progenitors. Regeneration of mammary glands with elevated levels of FoxM1 leads to aberrant ductal morphology and expansion of the luminal progenitor pool. Conversely, knockdown of FoxM1 results in a shift toward the differentiated state. FoxM1 mediates these effects by repressing the key regulator of luminal differentiation, GATA-3. Through association with DNMT3b, FoxM1 promotes methylation of the GATA-3 promoter in an Rb-dependent manner. This study identifies FoxM1 as a critical regulator of mammary differentiation with significant implications for the development of aggressive breast cancers. Graphical abstract Graphical Abstract Highlights ► Increased FOXM1 leads to an expansion of mammary stem and progenitor pools ► Deletion of FoxM1 in virgin mice causes accumulation of the differentiated luminal cells ► FOXM1 acts as a transcriptional repressor of GATA-3 by promoting DNA methylation ► FOXM1 represses GATA-3 in an Rb-dependent manner

Beschreibung: Publication year: 2012 Source: Cell Reports, Volume 1, Issue 5 Stefan Florian, Thomas U. Mayer During cell division, the molecular motor Eg5 crosslinks overlapping antiparallel microtubules and pushes them apart to separate mitotic spindle poles. Dynein has been proposed as a direct antagonist of Eg5 at the spindle equator, pulling on antiparallel microtubules and favoring spindle collapse. Some of the experiments supporting this hypothesis relied on endpoint quantifications of spindle phenotypes rather than following individual cell fates over time. Here, we present a mathematical model and proof-of-principle experiments to demonstrate that endpoint quantifications can be fundamentally misleading because they overestimate defective phenotypes. Indeed, live-cell imaging reveals that, while depletion of dynein or the dynein binding protein Lis1 enables spindle formation in presence of an Eg5 inhibitor, the activities of dynein and Eg5 cannot be titrated against each other. Thus, dynein most likely antagonizes Eg5 indirectly by exerting force at different spindle locations rather than through a simple push-pull mechanism at the spindle equator. Graphical abstract Graphical Abstract Highlights ► Fixed-cell microscopy images seem to overestimate frequency of defective mitotic spindles ► Live-cell-imaging-based conclusions often contradict results from fixed samples ► Antagonizing mitotic spindle motor activities of dynein and Eg5 are not titratable ► Spindle dynein-Eg5 antagonism is incompatible with a simple push-pull mechanism

Beschreibung: Publication year: 2012 Source: Cell Reports Rafael E. Luna, Haribabu Arthanari, Hiroyuki Hiraishi, Jagpreet Nanda, Pilar Martin-Marcos, Michelle A. Markus, Barak Akabayov, Alexander G. Milbradt, Lunet E. Luna, Hee-Chan Seo, Sven G. Hyberts, Amr Fahmy, Mikhail Reibarkh, David Miles, Patrick R. Hagner, Elizabeth M. O'Day, Tingfang Yi, Assen Marintchev, Alan G. Hinnebusch, Jon R. Lorsch, Katsura Asano, Gerhard Wagner Recognition of the proper start codon on mRNAs is essential for protein synthesis, which requires scanning and involves eukaryotic initiation factors (eIFs) eIF1, eIF1A, eIF2, and eIF5. The carboxyl terminal domain (CTD) of eIF5 stimulates 43S preinitiation complex (PIC) assembly; however, its precise role in scanning and start codon selection has remained unknown. Using nuclear magnetic resonance (NMR) spectroscopy, we identified the binding sites of eIF1 and eIF2β on eIF5-CTD and found that they partially overlapped. Mutating select eIF5 residues in the common interface specifically disrupts interaction with both factors. Genetic and biochemical evidence indicates that these eIF5-CTD mutations impair start codon recognition and impede eIF1 release from the PIC by abrogating eIF5-CTD binding to eIF2β. This study provides mechanistic insight into the role of eIF5-CTD's dynamic interplay with eIF1 and eIF2β in switching PICs from an open to a closed state at start codons. Graphical abstract Graphical Abstract Highlights ► Pinpointing four residues on eIF5-CTD unveils a dynamic interplay with eIF1 and eIF2 ► The eIF5-Quad mutant impairs the ability of PICs to close on start codons in vitro ► eIF5-CTD facilitates the shift of PICs from the open to closed conformation in vivo ► The CTD of eIF5 promotes start codon recognition by interacting with eIF1 and eIF2

Beschreibung: Publication year: 2012 Source: Cell Reports, Volume 1, Issue 5 Zachary T. Campbell, Devesh Bhimsaria, Cary T. Valley, Jose A. Rodriguez-Martinez, Elena Menichelli, James R. Williamson, Aseem Z. Ansari, Marvin Wickens The control and function of RNA are governed by the specificity of RNA binding proteins. Here, we describe a method for global unbiased analysis of RNA-protein interactions that uses in vitro selection, high-throughput sequencing, and sequence-specificity landscapes. The method yields affinities for a vast array of RNAs in a single experiment, including both low- and high-affinity sites. It is reproducible and accurate. Using this approach, we analyzed members of the PUF ( Pu milio and F BF) family of eukaryotic mRNA regulators. Our data identify effects of a specific protein partner on PUF-RNA interactions, reveal subsets of target sites not previously detected, and demonstrate that designer PUF proteins can precisely alter specificity. The approach described here is, in principle, broadly applicable for analysis of any molecule that binds RNA, including proteins, nucleic acids, and small molecules. Graphical abstract Graphical Abstract Highlights ► The SEQRS method is presented for analysis of RNA-protein interactions ► Members of the PUF (Pumilio and FBF) family of eukaryotic mRNA regulators are analyzed ► Cooperative effects on specificity are triggered by binding of a protein partner ► The specificity of an engineered PUF is described

Beschreibung: Publication year: 2012 Source: Cell Reports Hirofumi Toda, Xiaoliang Zhao, Barry J. Dickson Females of many animal species emit chemical signals that attract and arouse males for mating. For example, the major aphrodisiac pheromone of Drosophila melanogaster females, 7,11-heptacosadiene (7,11-HD), is a potent inducer of male-specific courtship and copulatory behaviors. Here, we demonstrate that a set of gustatory sensory neurons on the male foreleg, defined by expression of the ppk23 marker, respond to 7,11-HD. Activity of these neurons is required for males to robustly court females or to court males perfumed with 7,11-HD. Artificial activation of these ppk23 + neurons stimulates male-male courtship even without 7,11-HD perfuming. These data identify the ppk23 + sensory neurons as the primary targets for female sex pheromones in Drosophila . Graphical abstract Graphical Abstract Highlights ► Foreleg gustatory neurons that express ppk23 function in male courtship behavior ► ppk23 + neurons respond to the female aphrodisiac pheromone 7,11-heptacosadiene (7,11-HD) ► Thermogenetic activation of ppk23 + neurons induces male courtship without 7,11-HD

Beschreibung: Publication year: 2012 Source: Cell Reports, Volume 1, Issue 5 Sharvan Sehrawat, Oktay Kirak, Paul-Albert Koenig, Marisa K. Isaacson, Sofia Marques, Gunes Bozkurt, J. Pedro Simas, Rudolph Jaenisch, Hidde L. Ploegh To study the CD8 + T cell response against a mouse γ-herpes virus, we generated K b -MHV-68-ORF8 604–612 RAG −/− CD8 + T cell receptor transnuclear (TN) mice as a source of virus-specific CD8 + T cells. K b -ORF8-Tet + CD8 + T cells, expanded in the course of a resolving MHV-68 infection, served as a source of nucleus donors. Various in vivo and ex vivo assay criteria demonstrated the fine specificity and functionality of TN cells. TN cells proliferated extensively in response to viral infection, helped control viral burden, and exhibited a phenotype similar to that of endogenous K b -ORF8-Tet + cells. When compared to OT-1 cells, TN cells displayed distinct properties in response to lymphopenia and cognate antigen stimulation, which may be attributable to the affinity of the TCR expressed by the TN cells. The availability of MHV-68-specific CD8 + TCR TN mice provides a new tool for investigating aspects of host-pathogen interactions unique to γ-herpes viruses. Graphical abstract Graphical Abstract Highlights ► A mouse model for MHV-68-specific CD8 + T cells is generated using SCNT technology ► Various assays established the fine specificity and functionality of TN CD8+ T cells ► These cells helped control viral burden and behaved like endogenous CD8 + T cells ► A new model for investigating the pathogenesis of γ-herpes viruses is now available

Beschreibung: Publication year: 2012 Source: Cell Reports Joseph D. Coolon, Kraig R. Stevenson, C. Joel McManus, Brenton R. Graveley, Patricia J. Wittkopp Genomic imprinting occurs when expression of an allele differs based on the sex of the parent that transmitted the allele. In D. melanogaster, imprinting can occur, but its impact on allelic expression genome-wide is unclear. Here, we search for imprinted genes in D. melanogaster using RNA-seq to compare allele-specific expression between pools of 7- to 10-day-old adult female progeny from reciprocal crosses. We identified 119 genes with allelic expression consistent with imprinting, and these genes showed significant clustering within the genome. Surprisingly, additional analysis of several of these genes showed that either genomic heterogeneity or high levels of intrinsic noise caused imprinting-like allelic expression. Consequently, our data provide no convincing evidence of imprinting for D. melanogaster genes in their native genomic context. Elucidating sources of false-positive signals for imprinting in allele-specific RNA-seq data, as done here, is critical given the growing popularity of this method for identifying imprinted genes. Graphical abstract Graphical Abstract Highlights ► RNA-seq identified 119 potentially imprinted genes in D. melanogaster ► These potentially imprinted genes were significantly clustered in the genome ► Polymorphic deletions and intrinsic noise caused imprinting-like RNA-seq data ► We conclude that D. melanogaster genes in their native context are not imprinted

Beschreibung: Publication year: 2012 Source: Cell Reports Teruyoshi Hirayama, Etsuko Tarusawa, Yumiko Yoshimura, Niels Galjart, Takeshi Yagi The CCCTC-binding factor (CTCF) is a key molecule for chromatin conformational changes that promote cellular diversity, but nothing is known about its role in neurons. Here, we produced mice with a conditional knockout (cKO) of CTCF in postmitotic projection neurons, mostly in the dorsal telencephalon. The CTCF-cKO mice exhibited postnatal growth retardation and abnormal behavior and had defects in functional somatosensory mapping in the brain. In terms of gene expression, 390 transcripts were expressed at significantly different levels between CTCF-deficient and control cortex and hippocampus. In particular, the levels of 53 isoforms of the clustered protocadherin ( Pcdh ) genes, which are stochastically expressed in each neuron, declined markedly. Each CTCF - deficient neuron showed defects in dendritic arborization and spine density during brain development. Their excitatory postsynaptic currents showed normal amplitude but occurred with low frequency. Our results indicate that CTCF regulates functional neural development and neuronal diversity by controlling clustered Pcdh expression. Graphical abstract Graphical Abstract Highlights ► Loss of CTCF in projection neurons impairs functional somatosensory mapping ► CTCF regulates neuronal diversity elicited by stochastic expression of Pcdh genes ► CTCF-deficient neurons have defects in dendritic arborization and synapse formation ► Synapses of CTCF-deficient neurons show mature structure but low-frequency mEPSCs

Beschreibung: Publication year: 2012 Source: Cell Reports Nicole I. Bieberstein, Fernando Carrillo Oesterreich, Korinna Straube, Karla M. Neugebauer Here, we explore the role of splicing in transcription, employing both genome-wide analysis of human ChIP-seq data and experimental manipulation of exon-intron organization in transgenic cell lines. We show that the activating histone modifications H3K4me3 and H3K9ac map specifically to first exon-intron boundaries. This is surprising, because these marks help recruit general transcription factors (GTFs) to promoters. In genes with long first exons, promoter-proximal levels of H3K4me3 and H3K9ac are greatly reduced; consequently, GTFs and RNA polymerase II are low at transcription start sites (TSSs) and exhibit a second, promoter-distal peak from which transcription also initiates. In contrast, short first exons lead to increased H3K4me3 and H3K9ac at promoters, higher expression levels, accuracy in TSS usage, and a lower frequency of antisense transcription. Therefore, first exon length is predictive for gene activity. Finally, splicing inhibition and intron deletion reduce H3K4me3 levels and transcriptional output. Thus, gene architecture and splicing determines transcription quantity and quality as well as chromatin signatures. Graphical abstract Graphical Abstract Highlights ► First exon length determines H3K4me3 profiles ► Promoter-proximal H3K4me3 profiles are splicing-dependent ► Short first exons act as transcriptional enhancers