Hot off the presses! Nov 01 njobs

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Latest Articles Include:

• Guide to Authors: calling all authors!
  - njobs 12(11):1023 (2010)
  The 'Guide to Authors' provides detailed information on manuscript preparation and submission, the editorial process, and editorial and publishing policies. Awareness of these requirements can help avoid delays in the review and publication process.
• Nascent nitrosylases
  - njobs 12(11):1024-1026 (2010)
  Protein S-nitrosylation is thought to be mediated primarily by nitric oxide synthases. S-nitrosylated GAPDH is now shown to function within signal transduction cascades as a nuclear nitrosylase. Along with other recent demonstrations of regulated protein–protein transnitrosylation, these findings point to a new mechanism of signal transduction with transformative implications for nitric oxide biology and redox signalling.
• Opening ahead: early steps in lumen formation revealed
  - njobs 12(11):1026-1028 (2010)
  The contribution and order of polarity complexes and vesicular trafficking events during lumen formation remains obscure. Now, lumenogenesis in MDCK cell cysts is shown to require a Rab11a–Rabin8–Rab8a network that recruits Sec15A and Cdc42 and that promotes apical exocytosis by enlisting the Par complex and Sec8–Sec10 to an early apical membrane initiation site.
• An age of fewer histones
  - njobs 12(11):1029-1031 (2010)
  Changes in chromatin structure are a conserved hallmark of ageing, and the mechanism driving these changes, as well as their functional significance, are heavily investigated. Loss of core histones is now observed in aged cells and may contribute to this phenomenon. Histone loss is coupled to cell division and seems to be triggered by telomeric DNA damage.
• Bending the path to TOR
  - njobs 12(11):1031-1033 (2010)
  Cells sense and respond to physical stresses through mechanotransduction, a process that converts mechanical stimuli into biochemical signals. The bending of primary cilia has now been shown to modulate TOR signalling to negatively regulate cell size.
• Research highlights
  - njobs 12(11):1034 (2010)
  Adjusting kinetochore–microtubule attachments Neutral drift in intestinal stem cells Calcium activates Aurora-A kinase Myosin II muscles in on asymmetric cell division
• A molecular network for de novo generation of the apical surface and lumen
  - njobs 12(11):1035-1045 (2010)
  Adjusting kinetochore–microtubule attachments Neutral drift in intestinal stem cells Calcium activates Aurora-A kinase Myosin II muscles in on asymmetric cell division
• Angiocrine factors from Akt-activated endothelial cells balance self-renewal and differentiation of haematopoietic stem cells
  - njobs 12(11):1046-1056 (2010)
  
• Rickettsia Sca2 is a bacterial formin-like mediator of actin-based motility
  - njobs 12(11):1057-1063 (2010)
  
• Microtubule and katanin-dependent dynamics of microtubule nucleation complexes in the acentrosomal Arabidopsis cortical array
  - njobs 12(11):1064-1070 (2010)
  
• Epithelial septate junction assembly relies on melanotransferrin iron binding and endocytosis in Drosophila
  - njobs 12(11):1071-1077 (2010)
  
• SUMOylation of the GTPase Rac1 is required for optimal cell migration
  - njobs 12(11):1078-1085 (2010)
  
• The histone H4 Lys 20 methyltransferase PR-Set7 regulates replication origins in mammalian cells
  - njobs 12(11):1086-1093 (2010)
  
• GAPDH mediates nitrosylation of nuclear proteins
  - njobs 12(11):1094-1100 (2010)
  Nature Cell Biology | Letter Epithelial septate junction assembly relies on melanotransferrin iron binding and endocytosis in Drosophila * Katarína Tiklová1 Search for this author in: * NPG journals * PubMed * Google Scholar * Kirsten-André Senti1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Shenqiu Wang1 Search for this author in: * NPG journals * PubMed * Google Scholar * Astrid Gräslund3 Search for this author in: * NPG journals * PubMed * Google Scholar * Christos Samakovlis1christos@devbio.su.se Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature Cell BiologyVolume: 12 ,Pages:1071–1077Year published:(2010)DOI:doi:10.1038/ncb2111Received04 May 2010Accepted02 September 2010Published online10 October 2010 Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Iron is an essential element in many biological processes. In vertebrates, serum transferrin is the major supplier of iron to tissues, but the function of additional transferrin-like proteins remains poorly understood. Melanotransferrin (MTf) is a phylogenetically conserved, iron-binding epithelial protein. Elevated MTf levels have been implicated in melanoma pathogenesis. Here, we present a functional analysis of MTf in Drosophila melanogaster. Similarly to its human homologue, Drosophila MTf is a lipid-modified, iron-binding protein attached to epithelial cell membranes, and is a component of the septate junctions that form the paracellular permeability barrier in epithelial tissues. We demonstrate that septate junction assembly during epithelial maturation relies on endocytosis and apicolateral recycling of iron-bound MTf. Mouse MTf complements the defects of Drosophila MTf mutants. Drosophila provides the first genetic model for the functional dissection of MTf in epithe! lial junction assembly and morphogenesis. View full text Figures at a glance * Figure 1: MTf is a septate junction component. () Schematic representation of the MTf protein domains. The iron-binding domains of Drosophila MTf show 28% and 35% similarity to the mouse MTf domains. SS; signal peptide. D, Y, H and N indicate potential iron-binding residues. () Wild-type () and MTf mutant () embryos at stage 16, stained with 2A12 antibody to visualize trachea. Scale bar, 25 μm. () Confocal microscopy section of a wild-type () and an MTf embryo () immunostained for Nrx. Scale bar, 10 μm. () Transmission electron micrographs of wild-type () and MTf mutant () embryos at stage 16. Brackets indicate paracellular septa. Arrows indicate adherens junctions. Scale bar, 0.2 μm. () Representative confocal microscopy image of embryonic tracheal cells immunostained to indicate localization of MTf. () Merged confocal microscopy images of embryonic tracheal cells immunostained for MTf and Nrx. () Confocal microscopy images of ATPα () and cora () mutant embryos immunostained to indicate localization of MTf. Scale ba! rs, 10 μm. () Cell extracts from wild-type embryos at stage 14–16 were immunoprecipitated with anti-MTf (left) or anti-Nrx (right). Co-immunoprecipitated proteins were resolved and identified by western blotting using the indicated antibodies. Pre-immune sera were used as negative controls. Input; 5% of extract that was used for immunoprecipitation. * Figure 2: MTf co-localizes with early and recycling endosomes. () Confocal microscopy section of trachea () and hindgut () tissues immunostained with anti-MTf. MTf is localized along the basolateral membrane and in intracellular puncta at stage 13 in tracheal () and hindgut () cells. At stage 16, MTf accumulates predominantly at the apicolateral membrane in the trachea () and hindgut () cells. () Confocal microscopy section of tracheal cells from a stage 13 embryo, stained with anti-MTf and expressing GFP–2×FYVE () or GFP–Rab5 (). () Confocal microscopy of hindgut sections from stage 13 embryos, immunostained with anti-MTf and anti-Rab5() and anti-MTf and anti-Rab11 (). Scale bar , 10 μm. Images on the right of show localization of individual proteins (top, middle) and a merge of these images (bottom) from a region of the image on left. () Immunoblots of fractions (1; top–9; bottom) from an equilibrium density gradient of wild-type membrane extracts. () Membrane extracts from wild-type embryos at stage 10–13 and stage 14–17 ! expressing GFP–Rab5, GFP–Rab11 and GFP–GPI were immunoprecipitated with anti-GFP. Co-immunoprecipitated proteins were resolved and identified by western blotting using the indicated antibodies. Input; 25% of extract that was used for immunoprecipitation. * Figure 3: Endocytosis is required for MTf localization. () Confocal microscopy of hindgut sections from stage 13 () and stage 16 () wild-type embryos immunostained with anti-MTf. () Confocal microscopy of hindgut sections, immunostained with anti-MTf, from stage 16 embryos expressing dominant-negative rab5 () and dominant-negative rab11 constructs (). () Confocal microscopy of hindgut sections, immunostained with anti-MTf, from stage 16 chc1- and shits-mutant embryos. Scale bar, 10 μm. * Figure 4: MTf is a GPI-anchored iron-binding protein. () Schematic representation of the MTf protein. Arrows indicate the residues at which substitution mutations were made (Y231F and Y533F), and the GPI domain, which was deleted in the MTfΔGPI mutant. () Proteins in membrane and supernatant fractions of lysed wild-type embryos treated with PI-PLCγ, as indicated, were resolved and identified by western blotting. The GPI-anchored protein Knk and the transmembrane protein Syx1A were used as controls. () Confocal microscopy images of the trachea in an MTf mutant (), and in MTf mutants expressing MTf (), MTfΔGPI () MTf-TM (), MTfY231F () and MTfY533F () under the control of the btl–Gal4 driver. Cells were immunostained with antibodies specific to MTf and Nrx; images on the left indicate localization of Nrx and images on the right indicate co-localization of Nrx and MTf. Scale bar, 10 μm. () EPR spectra of MTf, MTf Y533F, mouse MTf (mMTf) and vermiform (Verm). A g-factor signal of 4.3 indicates a high-spin Fe(III) atom is boun! d to the protein. The signal was absent in the preparations containing the mutated MTf or the unrelated protein vermiform. * Figure 5: Uptake of MTf induces septate junction assembly in MTf mutant cells. () Confocal microscopy x–y images of embryonic ectodermal cells from en>GFP–CAAX, MTf; MTf (), en>GFP–CAAX, MTf ΔGPI; MTf () and en>GFP–CAAX, MTfY533FΔGPI; MTf () embryos. Cells were immunostained with antibodies specific to MTf and Nrx, as indicated. In all of the imaged embryos, engrailed cells were expressing GFP, as indicated. , and show the epidermis from above, whereas and show cross sections. Image on the right is a merge of the images on the left. () Confocal microscopy x–y images of en>GFP–CAAX, MTf ΔGPI; MTf embryos immunostained with antibodies specific to MTf and Rab11. en–GAL4 cells are expressing GFP. () Confocal microscopy x–z images of cells shown in Co-localization of MTf and Rab11 is visualized in white (right). GFP–expressing cells are marked by a blue bracket (right). In all x–y sections the apical cell surface is up. Scale bar, 10 μm. () Model of septate junction maturation during embryogenesis. Septate junction components at stag! e 13 are deposited along the whole lateral and at the basal membranes. Fe(III)–MTf induces endocytosis and redistribution of septate junction complexes from the basolateral to the apicolateral membrane. After septate junction assembly by stage15, Fe(III)–MTf may also promote junction stability. Author information * Author information * Supplementary information Affiliations * Department of Developmental Biology, Wenner-Gren Institute, Stockholm University, S-10691 Stockholm, Sweden. * Katarína Tiklová, * Kirsten-André Senti, * Shenqiu Wang & * Christos Samakovlis * Current address: IMBA, Dr. Bohr-Gasse 3, 1030 Vienna, Austria. * Kirsten-André Senti * Department of Biochemistry and Biophysics, Stockholm University, S-10691 Stockholm, Sweden. * Astrid Gräslund Contributions C.S., K.S. and K.T. designed the experiments. K.T. performed the experiments. S.W. identified the tracheal overelongation phenotype of the MTf P-element mutant. A.G. analysed the EPR data. C.S., K.T. and K.S. wrote the paper. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Christos Samakovlis (christos@devbio.su.se) Supplementary information * Author information * Supplementary information PDF files * Supplementary Information (1M) Supplementary Information Additional data
• Ascl1a regulates Müller glia dedifferentiation and retinal regeneration through a Lin-28-dependent, let-7 microRNA signalling pathway
  - njobs 12(11):1101-1107 (2010)
  Nature Cell Biology | Letter SUMOylation of the GTPase Rac1 is required for optimal cell migration * Sonia Castillo-Lluva1 Search for this author in: * NPG journals * PubMed * Google Scholar * Michael H. Tatham2 Search for this author in: * NPG journals * PubMed * Google Scholar * Richard C. Jones3, 4 Search for this author in: * NPG journals * PubMed * Google Scholar * Ellis G. Jaffray2 Search for this author in: * NPG journals * PubMed * Google Scholar * Ricky D. Edmondson3, 5 Search for this author in: * NPG journals * PubMed * Google Scholar * Ronald T. Hay2 Search for this author in: * NPG journals * PubMed * Google Scholar * Angeliki Malliri1amalliri@picr.man.ac.uk Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature Cell BiologyVolume: 12 ,Pages:1078–1085Year published:(2010)DOI:doi:10.1038/ncb2112Received12 July 2010Accepted09 September 2010Published online10 October 2010 Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg The Rho-like GTPase, Rac1, induces cytoskeletal rearrangements required for cell migration. Rac activation is regulated through a number of mechanisms, including control of nucleotide exchange and hydrolysis, regulation of subcellular localization or modulation of protein-expression levels1, 2, 3. Here, we identify that the small ubiquitin-like modifier (SUMO) E3-ligase, PIAS3, interacts with Rac1 and is required for increased Rac activation and optimal cell migration in response to hepatocyte growth factor (HGF) signalling. We demonstrate that Rac1 can be conjugated to SUMO-1 in response to hepatocyte growth factor treatment and that SUMOylation is enhanced by PIAS3. Furthermore, we identify non-consensus sites within the polybasic region of Rac1 as the main location for SUMO conjugation. We demonstrate that PIAS3-mediated SUMOylation of Rac1 controls the levels of Rac1–GTP and the ability of Rac1 to stimulate lamellipodia, cell migration and invasion. The finding that a ! Ras superfamily member can be SUMOylated provides an insight into the regulation of these critical mediators of cell behaviour. Our data reveal a role for SUMO in the regulation of cell migration and invasion. View full text Figures at a glance * Figure 1: PIAS3 is a Rac1-binding protein required for optimal Rac activation and cell migration in response to HGF treatment. () GST protein or GST–Rac1 were incubated with cell extracts from cells expressing Flag–PIAS3, and GST pulldowns were analysed by western blot. () Epitope-tagged proteins were expressed in HEK293T cells and GFP immunoprecipitation was performed. Purified proteins were detected by western blot. () GST–Rac1 beads divided into two samples were incubated with GDP or γGTP and then with equal amounts of cell lysate containing Flag–PIAS3. PIAS3 interaction was detected by western blot. Fold-change represents increase in band intensity for γGTP treatment, compared with GDP treatment. () HEK293T cells were transfected with plasmids expressing the indicated proteins, and subjected to immunoprecipitation with anti-GFP. Samples were analysed by western blot. () Cells were treated with doxycycline to induce control scrambled-sequence shRNA or shRNA specific to pias3 (two independent targets, #1 and #2). The migration speed of individual cells was tracked for at least 24 h in th! e presence of HGF. Data shown are means ± s.d. from four independent experiments (90 cells in total). Asterisk indicates P < 0.001, double asterisk indicates P < 0.05, NS indicates no significant difference. () MDCKII cells inducibly expressing pias3 or control shRNAs were treated with doxycycline and HGF and activation of Rac and the ERK MAPK pathway assessed (top). Rac–GTP bands were quantified and normalized intensities were calculated relative to control (bottom). Values are means ± s.d. (n = 3). () MDCKII cells were treated with doxycycline to induce expression of pias3 or control shRNAs, and Rac activation at indicated times was assessed (top). Bands were quantified and normalized intensities were calculated relative to control (bottom). () COS7 cells transfected with control empty vector or plasmids expressing Myc–PIAS3 were treated with HGF, and Rac activation was assessed. () Quantification of the normalized relative amounts of Rac–GTP in determined by scan! ning densitometry. Data are means ± s.d. of three independent! experiments. Asterisks indicate P < 0.001. Uncropped images of blots are shown in Supplementary Information, Fig. S6. * Figure 2: PIAS3 regulates Rac1 activation in the cytoplasm. () Schematic representation of full-length PIAS3 and CPIAS3. SAP domain; scaffold attachment factor-A/B/acinus/PIAS. () Representative images of PIAS3 and CPIAS3 localization in COS7 cells. Scale bar, 20 μm. () COS7 cells transfected with empty vector, or plasmids expressing Myc–PIAS3 or CPIAS3 were treated with HGF as indicated, and Rac activation assessed. () Quantification of the normalized relative amounts of Rac–GTP determined by scanning densitometry of (). Data are means ± s.d. for 3 independent experiments. Asterisk indicates P < 0.05, NS indicates no significant difference. Uncropped images of blots are shown in Supplementary Information, Fig. S6. * Figure 3: Rac1 is SUMOylated in vitro and in vivo. () GST–Rac1 protein was incubated at 37 °C in vitro in the presence of complete SUMOylation assay components (E1, E2, SUMO-1; S1, or SUMO-2; S2) and resolved by SDS–PAGE. Unmodified sample (Um) contains all the assay components but without incubation at 37 °C. Rac1–S1/Rac1–S2 indicates Rac1 modified by SUMO-1 or SUMO-2. () GFP–Rac1 was immunoprecipitated from HeLa cells in the presence or absence of PIAS3 and in vitro SUMOylation assay was performed, as indicated. Rac1 was detected by western blot. Fold-change represents increase in band intensity of SUMOylated GFP–Rac1 in presence of Flag–PIAS3, compared with SUMOylated GFP–Rac1 in absence of Flag–PIAS3. () Immunoprecipitated GFP–Rac1 or GFP–Rac1V12 was subjected to in vitro SUMOylation as indicated and Rac1 was detected by western blot. Fold-change represents increase in band intensity for SUMOylated GFP–Rac1V12, compared with SUMOylated GFP–Rac1. () HeLa cells, transfected as indicated, were ly! sed in the presence of NEM, and GFP was immunoprecipitated. The presence in immunoprecipitates of exogenous Rac1 or GFP was detected by western blot. () GFP–SUMO-1 was immunoprecipitated from HeLa cells (transfected as indicated), divided into two and incubated in the presence or absence of SENP1. () HeLa cells stably expressing 6His–SUMO-1 were transfected with Tpr-Met , and 6His–SUMO-1-modified proteins were purified from lysates. Endogenous Rac1–SUMO-1 was detected by western blot. () MDCKII cells stably expressing control shRNA or pias3 shRNA were treated with HGF as indicated, in the presence of the crosslinker DSS, and Rac–GTP immunoprecipitation was performed. GTP–Rac1–SUMO-1 bands were detected by western blot. () MDCKII cells were grown to confluency and incubated in low calcium medium. Rac activation was measured after calcium re-addition for the indicated times. Pulldowns of activated Rac were analysed by western blot for Rac and SUMO-1. Blots at th! e bottom represent part of the top blot and show the unmodifie! d Rac–GTP band at lower exposure, and levels of total Rac. Quantification of the Rac–GTP bands, both SUMO-modified and unmodified, are represented in the histogram, normalized to total Rac. Rac–nS1 in (–) is multi-mono-SUMOylated Rac1. Uncropped images of blots are shown in Supplementary Information, Fig. S6. * Figure 4: Rac1 is SUMOylated in the polybasic region and SUMOylation affects its GTP levels. () GST–Rac1 was incubated in vitro in the presence or absence of complete SUMOylation assay components and resolved by SDS–PAGE. () The GST–Rac1–SUMO-1 band from was excised and subjected to in-gel trypsin digestion. The resultant peptides were analysed by mass spectrometry and MaxQuant data processing to detect modified and unmodified peptides. Column chart indicates the absolute intensity of SUMO–Rac1 branched peptides detected. Asterisk indicates that discrimination between lysines is not possible. () Schematic representation of putative Rac1 SUMOylation sites and the mutations generated in this study. () HeLa cells stably expressing 6His–SUMO-1 were transfected with plasmids expressing the indicated proteins, lysed in the presence of NEM, GFP immunoprecipitated and analysed by western blot using anti-GFP. Rac1–nS1 indicates multi-mono-SUMOylated Rac1. () HeLa cells were transfected with plasmids expressing the indicating proteins, lysed in the presence of N! EM, GFP immunoprecipitated and analysed by western blot. GFP–Rac1–nUb indicates Rac1 modified by poly-ubiquitin. (, COS7 cells were transfected with plasmids to express the indicated proteins and treated with HGF. Activation of exogenous Rac1 was assessed (top). Band intensities were quantified by scanning densitometry (bottom). Bar graphs depict quantification of the normalized relative amounts of Rac–GTP, determined from at least three independent experiments. Data are means ± s.e.m. Asterisk indicates P < 0.05. Uncropped images of blots are shown in Supplementary Information, Fig. S6. * Figure 5: SUMOylation of the polybasic region is required for optimal cell migration and invasion. () Representative western blot showing expression levels of wild-type GFP–Rac1 or GFP–Rac1ΔSUMO in Rac1-depleted cells, transfected with plasmids to express the indicated proteins. () Representative images of Rac1-depleted MEFs reconstituted with wild-type GFP–Rac1 or GFP–Rac1ΔSUMO. Images were taken 24 h later. Scale bar, 20 μm. () Fraction of Rac1−/− cells expressing the indicated constructs with lamellipodia-membrane ruffles 24 h after transfection. Data are mean ± s.d., n = 110–150 cells from three independent experiments. () Cells with the indicated genotypes were grown on glass-bottom dishes, transfected with empty, control vector or vectors to express the indicated proteins, and were then tracked for at least 24 h. Box-whisker plots of cell velocities of n = 90–100 cells from three independent experiments are shown. () Cells treated as in were analysed for invasion at 40 μm in the presence of HGF. Data are means ± s.d., n = 5 wells. A representat! ive from three independent experiments is shown. () Representative western blot of Rac1-depleted cells transfected with plasmids to express the indicated proteins and stably expressing control, scrambled-sequence shRNA or pias3 shRNA, as indicated. There are lower levels of expression of exogenously expressed wild-type GFP–Rac1, compared with endogenous levels of Rac1 in control Rac1+/+ MEFs. () Individual cells expressing the indicated shRNA and transfected with empty control vectors, or plasmids expressing the indicated proteins, were tracked for 24 h. Data shown are average speed of migration of at least 50 cells. A representative experiment is shown here. (–, ) Asterisk indicates P < 0.001, double asterisks indicate P < 0.05 and NS indicates no significant difference. Uncropped images of blots are shown in Supplementary Information, Fig. S6. Author information * Author information * Supplementary information Affiliations * Cell Signalling Group, Cancer Research UK Paterson Institute for Cancer Research, The University of Manchester, Manchester, M20 4BX, UK. * Sonia Castillo-Lluva & * Angeliki Malliri * Wellcome Trust Centre for Gene Regulation and Expression, Sir James Black Centre College of Life Sciences, University of Dundee, Dundee, DD1 5EH, UK. * Michael H. Tatham, * Ellis G. Jaffray & * Ronald T. Hay * National Center for Toxicological Research (NCTR), Food and Drug Administration (FDA), Jefferson, AR 72079, USA. * Richard C. Jones & * Ricky D. Edmondson * Current address: MS Bioworks, 3950 Varsity Drive, Ann Arbor, MI 48108, USA. * Richard C. Jones * Current address: Myeloma Institute for Research and Therapy, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA. * Ricky D. Edmondson Contributions S.C.L. co-wrote the manuscript and designed and executed all the experiments apart from the mass spectrometry of the sites of SUMO modification, which was performed by M.H.T. R.C.J. and R.D.E. performed the mass spectrometry analysis of the TAP–Rac1 purification. E.G.J. purified all the components required for the in vitro SUMOylation and helped with the in vitro SUMOylation experiments. R.T.H. provided expertise and help with the SUMOylation experiments. A.M. provided team leadership, project management and wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Angeliki Malliri (amalliri@picr.man.ac.uk) Supplementary information * Author information * Supplementary information PDF files * Supplementary Information (3M) Supplementary Information Additional data
• Cyclin-dependent kinases regulate epigenetic gene silencing through phosphorylation of EZH2
  - njobs 12(11):1108-1114 (2010)
  Nature Cell Biology | Letter The histone H4 Lys 20 methyltransferase PR-Set7 regulates replication origins in mammalian cells * Mathieu Tardat1, 2, 3, 7 Search for this author in: * NPG journals * PubMed * Google Scholar * Julien Brustel1, 2, 3, 7 Search for this author in: * NPG journals * PubMed * Google Scholar * Olivier Kirsh1, 2, 3 Search for this author in: * NPG journals * PubMed * Google Scholar * Christine Lefevbre4, 5, 6 Search for this author in: * NPG journals * PubMed * Google Scholar * Mary Callanan4, 5, 6 Search for this author in: * NPG journals * PubMed * Google Scholar * Claude Sardet1, 2, 3claude.sardet@igmm.cnrs.fr Search for this author in: * NPG journals * PubMed * Google Scholar * Eric Julien1, 2, 3eric.julien@igmm.cnrs.fr Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorsJournal name:Nature Cell BiologyVolume: 12 ,Pages:1086–1093Year published:(2010)DOI:doi:10.1038/ncb2113Received05 July 2010Accepted30 September 2010Published online17 October 2010 Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg The initiation of DNA synthesis is governed by the licensing of replication origins, which consists of assembling a pre-replication complex (pre-RC) on origins during late M- and G1-phases1, 2. In metazoans, functional replication origins do not show defined DNA consensus sequences, thus evoking the involvement of chromatin determinants in the selection of these origins3. Here, we show that the onset of licensing in mammalian cells coincides with an increase in histone H4 Lys 20 monomethylation (H4K20me1) at replication origins by the methyltransferase PR-Set7 (also known as Set8 or KMT5A). Indeed, tethering PR-Set7 methylase activity to a specific genomic locus promotes the loading of pre-RC proteins on chromatin. In addition, we demonstrate that PR-Set7 undergoes a PCNA- and Cul4–Ddb1-driven degradation during S phase that contributes to the disappearance of H4K20me1 at origins and the inhibition of replication licensing. Strikingly, expression of a PR-Set7 mutant insens! itive to this degradation causes the maintenance of H4K20me1 and repeated DNA replication at origins. These results elucidate a critical role for PR-Set7 and H4K20me1 in the chromatin events that regulate replication origins. View full text Figures at a glance * Figure 1: Cell-cycle changes in PR-Set7 levels are regulated by the ubiquitin–proteasome pathway. () PR-Set7 is modified by ubiquitylation. U2OS cells were transfected with expression plasmids encoding HA–tagged PR-Set7 and a 6His-tagged ubiquitin (His–Ub), followed by treatment with MG132. Similar amounts of cellular extracts were probed for HA–PR-Set7 (input) and loaded onto a nickel-(Ni+)–NTA column. Ubiquitylated proteins purified by nickel chromatography were then subjected to immunoblot analysis using a HA–tag antiserum (bottom). () PR-Set7 stability in asynchronous U2OS cells treated with cycloheximide (CHX) alone or in combination with the proteasome inhibitor MG132. Cells were collected at the indicated times and analysed by immunoblotting with antibodies against PR-Set7 and β-actin. () Immunoblot analysis of PR-Set7, β-actin and H4K20me1 levels in U2OS cells synchronized in M (nocodazole block), early G1 (45 min after release from nocodazole), G1/S (thymidine block), and in S and G2 phase (3 h and 8 h after release from thymidine, respectively). Bef! ore harvesting, cells were treated with CHX alone or in combination with MG132, as indicated. To ensure equal loading of histone H4, total histones were stained with Ponceau red. Bottom left: cell-cycle phases of U2OS cells were confirmed by FACS analysis. () PR-Set7 stability 3 h after release from thymidine block and at the indicated times after treatment with CHX, and MG132, as indicated. PR-Set7 levels in the cells were analysed by immunoblotting with antibodies against PR-Set7, using β-actin as an internal control. Uncropped images of blots are shown in Supplementary Information, Fig. S6. * Figure 2: PCNA binding is required for S-phase degradation of PR-Set7 by Cul4–Ddb1-mediated ubiquitylation. () PR-Set7 half-life is increased in PCNA-depleted cells. Immunoblot analysis of U2OS cells using antibodies against PR-Set7, PCNA and β-actin proteins, 4 days after either luciferase (control) or PCNA shRNA expression. () PR-Set7 ubiquitylation is reduced in PCNA-depleted cells. Cells stably expressing control and PCNA shRNA were transfected with expression vectors encoding HA-tagged PR-Set7 and 6His–ubiquitin and, 24 h later were treated with MG132 for 1 h. Ubiquitylated proteins were purified as in Figure 1a and subjected to immunoblot analysis with anti-HA antiserum. () Mutation prevents PR-Set7 interaction with PCNA. Wild-type recombinant GST–PR-Set7, mutated GST–PR-Set7 (F184A/Y158A and Y57A/M58A mutants), or recombinant GST, were incubated with whole-cell extracts and glutathione beads. After washing, bound proteins were analysed by immunoblot with anti-PCNA antiserum. Bottom: Coomassie staining indicates equal loading of GST–PR-Set7 proteins. () F184A/Y158A ! mutation inhibits PR-Set7 ubiquitylation. Cells expressing HA–PR-Set7WT, HA–PR-Set7Y57A/M58A or HA–PR-Set7F184A/Y158A were established after transduction with retroviral vectors. Cells were then transfected with expression vectors encoding 6His–ubiquitin and were treated with MG132. Ubiquitylated proteins were purified as in Figure 1a, followed by immunoblotting with anti-HA antiserum. () F184A/Y158A mutation prevents PR-Set7 degradation during S phase. Cells expressing HA–PR-Set7WT, HA–PR-Set7Y57A/M58A or HA–PR-Set7F184A/Y158A were established after transduction with retroviral vectors. Cells were released into S phase after thymidine block, and protein levels were analysed by immunoblot with anti-HA and anti-β-actin antisera at indicated times after addition of CHX. () Depletion of Ddb1 and Cul4 proteins by siRNA stabilizes PR-Set7 protein levels during S phase. U2OS cells were treated with control, Ddb1 or Cul4a and Cul4b siRNA, released into S phase after! thymidine block and treated with CHX, as indicated. Endogenou! s PR-Set7 and β-actin protein levels were examined by immunoblot with anti-PR-Set7 and anti-β-actin antisera. () Co-immunoprecipitation of PR-Set7 with Ddb1. Whole-cell extracts from U2OS cells expressing the indicated constructs were immunoprecipitated with anti-HA antiserum and analysed by immunoblot with anti-HA and anti-Flag antisera. * Figure 3: Neutralization of PCNA-mediated PR-Set7 degradation causes a G2 arrest with re-replicated DNA. () Representative phase-contrast images of U2OS cells 3 days after expression of HA–PR-Set7WT and HA–PR-Set7F184A/Y158A had been established in cells by transduction with retroviral vectors. Scale bars, 10 μm. () The DNA content of cells expressing PR-Set7WT (top) and PR-Set7F184A/Y158A (bottom) was assessed by FACS analysis. Left: quantification of cells with the indicated DNA content. Middle: incorporation of BrdU was assessed by treating cells with BrdU followed by BrdU-specific and FITC-conjugated antibodies. Right: cyclin B1 levels were assessed by treating cells with cyclin B1-specific and FITC-conjugated antibodies. The percentage of cells with DNA content >4 N and either incorporating BrdU (middle) or with high-levels of cyclin B1 (right) are indicated. () Immunofluorescence microscopy of EdU and cyclin B1 localization in cells expressing HA–PR-Set7WT (left) and HA–PR-Set7F184A/Y158A (right). Cells were pulse-labelled with EdU. DNA was stained with DAPI. Arr! owheads (right) indicate enlarged EdU-positive nuclei (re-replicating) of PR-Set7F184A/Y158A cells that display a G2-phase cyclin B1 staining15 (perinuclear signal). Scale bars, 10 μm. () FACS analysis of γH2AX signal in cells expressing HA–PR-Set7WT and HA–PR-Set7F184A/Y158A. DNA content of the cells was assessed by 7-AAD staining. Ultraviolet treatment was used to induce γH2AX in cells expressing HA–PR-Set7WT as a reference. Percentage of γH2AX-positive cells (red) is indicated. Bottom: comparison of number of cells with the indicated DNA content (white) versus number of cells with a γH2AX–positive signal (red). () Density gradient centrifugation of BrdU-labelled genomic DNA from U2OS cells expressing PR-Set7WT and PR-Set7F184A/Y158A. Fractions were collected from the bottom and DNA concentration in each fraction was determined by spectrometry. The position of re-replicated (heavy-heavy, H/H), replicated (heavy-light, H/L), and unreplicated (light-light, L/L)! DNA species is indicated. Each value represents the mean of t! wo independent experiments. () FACS analysis of DNA content and incorporation of BrdU in PR-Set7WT- and PR-Set7F184A/Y158A-expressing cells during the indicated cell-cycle phases. Cells were arrested at G1/S transition before re-replication occurred (left). After release DNA content was assessed (middle) and cells were pulse-labelled with BrdU (right). Percentage of re-replicated cells is indicated. * Figure 4: PR-Set7-induced H4K20me1 at replication origins is associated with the onset of replication licensing. () PR-Set7F184A/Y158A methylase activity contributes to DNA re-replication. BrdU incorporation and DNA content were measured by FACS in cells expressing PR-Set7 or the indicated mutants. The percentage of cells with DNA content >4 N and incorporating BrdU is indicated. () H4K20me1 is found at replication origins in M- and G1-phases. H4K20me1 and H4 acetylation (H4Ac) at the MCM4 (top left), β-globin (bottom left), TOP1 (top middle) and LaminB2 (bottom middle) replication origins and flanking regions (indicated by the distance from the origin; kb) were measured by ChIP–qPCR analyses in M-, G1- and S-phase-synchronized U2OS cells. Protein phosphate 1 (PP1) antiserum was used as ChIP control. The y axis is the ratio of DNA in the immunoprecipitate to that in the input (data are means ± s.d., n = 5). Cell-cycle synchronization was verified by FACS (right). () Expression of the PR-Set7F184A/Y158A mutant leads to the maintenance of high levels of H4K20me1 at replication origin! s during S phase, without affecting H4 acetylation levels. Quantification of H4K20me1 and H4 acetylation levels by ChIP–qPCR at the indicated origins in U2OS cells expressing PR-Set7WT or the indicated mutants, 4 h after release from thymidine block. ChIP–qPCR analyses were performed as in Results are normalized to levels in cells expressing HA–PR-Set7WT (data are means ± s.d., n = 5). () LaminB2 and β-globin origins display multiple replication initiation in PR-Set7F184A/Y158A-expressing cells. Representative images from FISH experiment on U2OS cells, expressing PR-Set7WT or PR-Set7F184A/Y158A retrovirus. BAC probes corresponding to laminB2 (green) and β-globin (red) origins containing segment chromosomes were labelled and used for hybridization. Chromosomal DNA was stained with DAPI. Note that control U2OS cells contain two copies of the chromosomal locus 19p13.3 (LaminB2) and three of the chromosomal locus 11p15.4 (β-globin). Scale bars, 5 μm. * Figure 5: PR-Set7 contributes to the assembly of pre-RC complexes on chromatin. () Chromatin loading of ORC2, CDC6 and MCM proteins in control- and PR-Set7-shRNA-treated U2OS cells. Two days after shRNA treatment, cells were synchronized at G1/S transition (thymidine block) before harvest and were subjected to biochemical fractionation. Cytosolic (S1) and nuclear (S2) supernatants and a chromatin-enriched fraction (P3) were separated by SDS–PAGE and analysed by immunoblot. MEK1 is used to ensure that P3 is free of soluble components, whereas the chromatin-bound protein HCF-1 is used as a control for chromatin localization. () Schematic representation of the method used to test the recruitment of pre-RC proteins by the Gal4–PR-Set7 fusion protein at the 5×Gal4 site integrated in the first intron of the NCOA5 gene on chromosome 20 in HEK293 cells (HEK293Gal4 cells). Relative positions of Gal4 and Luc regions amplified by quantitative PCR, and the distance between them, are indicated. () ChIP–qPCR analysis of the Gal4 site and the downstream flankin! g region (Luc) with anti-Gal4, anti-H4K20me1 or anti-Flag antisera (filled bars), and using chromatin of HEK293 cells expressing the indicated Flag–tagged and Gal4 fusion proteins (Supplementary information, Fig. S5b). ChIP–qPCR control with beads only is shown in each panel (white bars). The y axis is the ratio of DNA in immunoprecipitate to that in input (data are means ± s.d, n = 5). () ChIP–qPCR analysis of the Gal4 site and the downstream flanking region (Luc) with anti-Gal4, anti-H4K20me1 or anti-Flag antisera (filled bars) and using chromatin of HEK293 cells expressing the indicated Flag–tagged and Gal4 fusion proteins (Supplementary information, Fig. S5c). ChIP–qPCR control with anti-PP1 antiserum is shown in each panel (white bars). The y axis is the ratio of DNA in immunoprecipitate to that in input (data are means ± s.d. n = 5). Of note, the higher enrichment (approximately 2-fold) of Gal4–PR-Set7WT probably reflects the fact that, in contrast to Ga! l4–JunD and Gal4–PR-Set7ΔSET, this fusion protein efficie! ntly forms homodimer as does endogenous PR-Set7 (unpublished results), which indirectly increases its amounts at the Gal4 site. Author information * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Mathieu Tardat & * Julien Brustel Affiliations * Institut de Génétique Moléculaire de Montpellier (IGMM), UMR 5535 CNRS, Montpellier, France. * Mathieu Tardat, * Julien Brustel, * Olivier Kirsh, * Claude Sardet & * Eric Julien * Université Montpellier 2, Montpellier, France. * Mathieu Tardat, * Julien Brustel, * Olivier Kirsh, * Claude Sardet & * Eric Julien * Université Montpellier 1, Montpellier, France. * Mathieu Tardat, * Julien Brustel, * Olivier Kirsh, * Claude Sardet & * Eric Julien * INSERM U823, Institut Albert Bonniot, Grenoble, France. * Christine Lefevbre & * Mary Callanan * Université Joseph-Fourier, Grenoble, France. * Christine Lefevbre & * Mary Callanan * Onco-Hematology Unit, Department of Hematology, Onco-genetics and Immunology, Pôle de Biologie, CHU-Grenoble, Grenoble, France. * Christine Lefevbre & * Mary Callanan Contributions E.J. conceived the project and designed and supervised the study. M.T., J.B., O.K. and E.J. performed and analysed experiments. C.L. and M.C. performed and analysed FISH experiments. C.S. provided conceptual advice on study design and the interpretation of the results. The manuscript was written by E.J. and edited by C.S. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Claude Sardet (claude.sardet@igmm.cnrs.fr) or * Eric Julien (eric.julien@igmm.cnrs.fr) Supplementary information * Author information * Supplementary information PDF files * Supplementary Information (1M) Supplementary Information Additional data
• Primary cilia regulate mTORC1 activity and cell size through Lkb1
  - njobs 12(11):1115-1122 (2010)
  Nature Cell Biology | Letter GAPDH mediates nitrosylation of nuclear proteins * Michael D. Kornberg1 Search for this author in: * NPG journals * PubMed * Google Scholar * Nilkantha Sen1 Search for this author in: * NPG journals * PubMed * Google Scholar * Makoto R. Hara1, 4 Search for this author in: * NPG journals * PubMed * Google Scholar * Krishna R. Juluri1 Search for this author in: * NPG journals * PubMed * Google Scholar * Judy Van K. Nguyen1 Search for this author in: * NPG journals * PubMed * Google Scholar * Adele M. Snowman1 Search for this author in: * NPG journals * PubMed * Google Scholar * Lindsey Law1 Search for this author in: * NPG journals * PubMed * Google Scholar * Lynda D. Hester1 Search for this author in: * NPG journals * PubMed * Google Scholar * Solomon H. Snyder1, 2, 3ssnyder@jhmi.edu Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature Cell BiologyVolume: 12 ,Pages:1094–1100Year published:(2010)DOI:doi:10.1038/ncb2114Received09 August 2010Accepted01 September 2010Published online24 October 2010 Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg S-nitrosylation of proteins by nitric oxide is a major mode of signalling in cells1. S-nitrosylation can mediate the regulation of a range of proteins, including prominent nuclear proteins, such as HDAC2 (ref. 2) and PARP1 (ref. 3). The high reactivity of the nitric oxide group with protein thiols, but the selective nature of nitrosylation within the cell, implies the existence of targeting mechanisms. Specificity of nitric oxide signalling is often achieved by the binding of nitric oxide synthase (NOS) to target proteins, either directly4 or through scaffolding proteins such as PSD-95 (ref. 5) and CAPON6. As the three principal isoforms of NOS—neuronal NOS (nNOS), endothelial NOS (eNOS) and inducible NOS (iNOS) —are primarily non-nuclear, the mechanisms by which nuclear proteins are selectively nitrosylated have been elusive. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is physiologically nitrosylated at its Cys 150 residue. Nitrosylated GAPDH (SNO–GAPDH) binds to! Siah1, which possesses a nuclear localization signal, and is transported to the nucleus7. Here, we show that SNO–GAPDH physiologically transnitrosylates nuclear proteins, including the deacetylating enzyme sirtuin-1 (SIRT1), histone deacetylase-2 (HDAC2) and DNA-activated protein kinase (DNA-PK). Our findings reveal a novel mechanism for targeted nitrosylation of nuclear proteins and suggest that protein–protein transfer of nitric oxide groups may be a general mechanism in cellular signal transduction. View full text Figures at a glance * Figure 1: SNO–GAPDH interacts with SIRT1 near its nitrosylated Cys 150 residue. () Endogenous co-immunoprecipitation of SIRT1 and GAPDH in HEK293 cells treated with nitric oxide donor. Cells were treated with GSH or GSNO before lysis. Lysates were immunoprecipitated with immunoglobulin G (IgG) or antibodies specific to SIRT1. SIRT1 and GAPDH were resolved from the immunoprecipitate by SDS–PAGE and identified by western blotting. GAPDH levels in the input are also shown (bottom). () Nitrosylated GAPDH (SNO–GAPDH) binds directly to SIRT1 in vitro. GST and GST–GAPDH were pre-treated with GSH or GSNO. Recombinant SIRT1 was added, and binding was assessed by GST pulldown with GSH–agarose beads and identification of proteins by western blotting. () A small peptide corresponding to the region of GAPDH that spans Cys 150 (Peptide-C150) blocks the interaction between SNO–GAPDH and SIRT1. GST–GAPDH was treated with GSH or GSNO, before addition of recombinant SIRT1. Peptide-C150 and a scrambled peptide were also added, as indicated. GAPDH binding of SI! RT1 was detected by GST pulldown and western blotting. Bottom: sequences of the two peptides used in the assay. The residue corresponding to Cys 150 of GAPDH is indicated in the Peptide-C150 sequence. () Mutation of GAPDH Thr 152 abolishes binding to SIRT1. After transfection with wild-type HA-tagged GAPDH or the indicated point mutants, HEK293 cells were treated with GSH or GSNO. Cell lysates were immunoprecipitated with anti-SIRT1 antibody and analysed by western blotting with anti-HA or anti-SIRT1 antibodies. Uncropped images of blots are shown in Supplementary Information, Fig. S5. * Figure 2: Nuclear SNO–GAPDH mediates nitrosylation of SIRT1 through transnitrosylation. () HEK293–nNOS cells were treated with A23187 for 2 h, lysed, and the supernatants were then incubated with ascorbate (which initiates the biotin switch), as indicated. Biotinylated SIRT1 was detected by pulldown with neutravidin–agarose and western blotting with anti-SIRT1 antibodies. Bottom: presence of SIRT1 in the input supernatant was detected by western blotting. () Mouse cortical neurons were treated with L-VNIO and NMDA, lysed, and the supernatants were treated with ascorbate, as indicated. Biotinylated SIRT1 was detected as in . Bottom: presence of SIRT1 in the input supernatant was detected by western blotting. () In vitro transnitrosylation assay. Recombinant SIRT1 was incubated with or without recombinant GAPDH (wild type, or T152A or C150S mutants) that had been pre-treated with GSH, GSNO or GNSO desalted to remove excess small molecules. A biotin switch assay was then performed, followed by pulldown of biotinylated proteins and western blotting to identify ! the indicated proteins. () HEK293–nNOS cells overexpressing Flag–HA–SIRT1 and co-overexpressing GAPDH, were lysed, and supernatants were treated with ascorbate, as indicated. Proteins, after pulldown from the biotin switch assay, and in the input supernatant, were detected by western blotting. () Experiments performed as in , except HEK293–nNOS cells overexpressing Flag–HA–SIRT1 were co-overexpressing wild-type GAPDH or GAPDH mutants. () HEK293–nNOS cells were treated with the indicated shRNA and biotinylation of SIRT1 and β-tubulin was assessed by biotin switch assay and western blotting. () HEK293–nNOS cells, or HEK293–nNOS cells overexpressing GAPDH or GAPDHT152A were treated with GAPDH shRNA, as indicated. Biotinylation was assessed by biotin switch assay and western blotting. () HEK293T cells were transfected with plasmids encoding HA or HA–Siah1ΔNLS and treated with or without GSNO, followed by nuclear fractionation. The indicated proteins were d! etected by western blotting. () HEK293–nNOS cells were trans! fected with plasmid encoding Siah1ΔNLS or empty control vector. All cells were treated with A23187, and assayed by biotin switch, followed by western blotting. Results from the western blots in this figure are quantified in Supplementary Information, Fig. S2. Uncropped images of blots are shown in Supplementary Information, Fig. S5. * Figure 3: SNO–GAPDH mediates inhibition of SIRT1 enzymatic activity by nitric oxide. () In vitro histone deacetylation assay. SIRT1 was pre-treated with the indicated concentration of GSH or GSNO and was then incubated with acetylated histones. Reactions were resolved by SDS–PAGE and western blotting. () In vitro histone deacetylation assay. Left: SIRT1 was incubated with GAPDH or GAPDHT152A pre-treated with GSH or GSNO, as indicated, before the addition of acetylated histones. Reactions were resolved by SDS–PAGE and western blotting. Right: the percentage of acetylated histone H3 was quantified from the western blots, compared with the control. Asterisk indicates P < 0.05, one-way ANOVA; n = 3. Data are means ± s.e.m. () HEK293 and HEK293–nNOS cells were transfected with plasmids encoding Flag–PGC1α. The cells were then treated with A23187 as indicated, before lysis and immunoprecipitation with anti-Flag antibody. Acetylation of Flag–PGC1α in the immunoprecipitate was identified by western blotting. () Left: HEK293–nNOS cells, transfected wit! h plasmids encoding Flag–PGC1α, were treated with the indicated shRNA, and A23187, before lysis and immunoprecipitation with anti-Flag antibody. Acetylation of Flag–PGC1α in the immunoprecipitate was identified by western blotting. Right: Quantification of acetylated PGC1α, from bands on the western blot. Asterisk indicates P < 0.01, student's t-test, n = 3. Data are means ± s.e.m. () HEK293–nNOS cells were transfected with plasmids encoding the HNF4α luciferase reporter construct. The cells were then transduced with lentiviral shRNA specific to GAPDH and transfected with empty control plasmids, or plasmids encoding wild-type GAPDH or GAPDHT152A. All cells were treated with A23187. Asterisks indicate P < 0.05, student's t-test, n = 3. Data are means ± s.e.m. Uncropped images of blots are shown in Supplementary Information, Fig. S5. * Figure 4: Identification of HDAC2 and DNA-PK as nuclear targets of SNO–GAPDH mediated transnitrosylation. () Co-immunoprecipitation of endogenous HDAC2 and GAPDH in HEK293 cells treated with nitric oxide donor. Cells were treated with GSH or GSNO before lysis. () Cells were treated with control, scrambled-sequence shRNA and GAPDH shRNA and nitrosylation of HDAC2 was assessed by biotin switch assay, pulldown and western blotting. Bottom: quantification of bands from western blot. () Cells were transfected with empty control vector or vector encoding Siah1ΔNLS and nitrosylation of HDAC2 was assessed by biotin switch assay, pulldown and western blotting. Bottom: quantification of bands from western blot. () Experiments were performed as in , but nitrosylation of DNA-PK was evaluated instead of HDAC2. () Experiments were performed as in , but nitrosylation of DNA-PK was evaluated instead of HDAC2. Asterisks in indicate P < 0.05, student's t-test; n = 3. Data are means ± s.e.m. Uncropped images of blots are shown in Supplementary Information, Fig. S5. Author information * Author information * Supplementary information Affiliations * Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA. * Michael D. Kornberg, * Nilkantha Sen, * Makoto R. Hara, * Krishna R. Juluri, * Judy Van K. Nguyen, * Adele M. Snowman, * Lindsey Law, * Lynda D. Hester & * Solomon H. Snyder * Department of Psychiatry and Behavioral Sciences, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA. * Solomon H. Snyder * Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA. * Solomon H. Snyder * Present address: Department of Medicine, Duke University Medical Center, Durham, NC 27710, USA. * Makoto R. Hara Contributions M.D.K. designed and performed most of the experiments, analysed the data, prepared the figures, helped write the manuscript and contributed to project design. N.S. performed experiments investigating the effects of GAPDH mutants on SIRT1 nitrosylation in intact cells, performed the GAPDH glycolytic activity assay and the luciferase reporter assay, and analysed the data and prepared the figures from these experiments. M.R.H. identified the physical interaction between GAPDH and SIRT1. K.R.J. identified S-nitrosylation of SIRT1, helped with SIRT1 assay design and prepared constructs. J.V.K.N. performed some in vitro binding and enzyme activity assays. A.M.S. performed site-directed mutagenesis and prepared plasmids. L.L. helped perform some experiments. L.D.H. generated neuronal cultures. S.H.S. designed and supervised the project, wrote the manuscript and provided financial support. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Solomon H. Snyder (ssnyder@jhmi.edu) Supplementary information * Author information * Supplementary information PDF files * Supplementary Information (801K) Supplementary Information Additional data
• Topoisomerase I suppresses genomic instability by preventing interference between replication and transcription
  - njobs 12(11):1122 (2010)
  Nature Cell Biology | Letter Ascl1a regulates Müller glia dedifferentiation and retinal regeneration through a Lin-28-dependent, let-7 microRNA signalling pathway * Rajesh Ramachandran1 Search for this author in: * NPG journals * PubMed * Google Scholar * Blake V. Fausett1 Search for this author in: * NPG journals * PubMed * Google Scholar * Daniel Goldman1neuroman@umich.edu Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature Cell BiologyVolume: 12 ,Pages:1101–1107Year published:(2010)DOI:doi:10.1038/ncb2115Received22 February 2010Accepted27 August 2010Published online10 October 2010 Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Unlike mammals, teleost fish mount a robust regenerative response to retinal injury that culminates in restoration of visual function1, 2. This regenerative response relies on dedifferentiation of Müller glia into a cycling population of progenitor cells. However, the mechanism underlying this dedifferentiation is unknown. Here, we report that genes encoding pluripotency factors are induced following retinal injury. Interestingly, the proneural transcription factor, Ascl1a, and the pluripotency factor, Lin-28, are induced in Müller glia within 6 h following retinal injury and are necessary for Müller glia dedifferentiation. We demonstrate that Ascl1a is necessary for lin-28 expression and that Lin-28 suppresses let-7 microRNA (miRNA) expression. Furthermore, we demonstrate that let-7 represses expression of regeneration-associated genes such as, ascl1a, hspd1, lin-28, oct4, pax6b and c-myc. hspd1, oct4 and c-myca exhibit basal expression in the uninjured retina and let-7 ! may inhibit this expression to prevent premature Müller glia dedifferentiation. The opposing actions of Lin-28 and let-7 miRNAs on Müller glia differentiation and dedifferentiation are similar to that of embryonic stem cells3 and suggest novel targets for stimulating Müller glia dedifferentiation and retinal regeneration in mammals. View full text Figures at a glance * Figure 1: ascl1a and lin-28 mRNAs are rapidly induced in dedifferentiating Müller glia following retinal injury. () RT–PCR analysis of the indicated pluripotency-factor mRNAs from retina at the indicated times after injury. Expression of actin mRNA was used as an internal control. () Real-time PCR quantification of pluripotency factor mRNA levels during regeneration of retina. Data represent means ± s.d. (n = 3 individual fish; compared with uninjured retina, P = 0.0001 or less for lin-28, c-mycb and ascl1a expression at all times after retinal injury; P = 0.0001 for klf4 at 15 hpi, and 2- and 4-dpi; P = 0.02 for klf4 at 7 dpi; P = 0.0001 or less for sox2, c-myca and nanog at 2- and 4-dpi; P = 0.0425 for c-myca at 7 dpi; P = 0.0178 and 0.0069 for oct4 at 2- and 4-dpi, respectively). Note, y axis is fold-induction in log scale and normalized to 0 h time that is assigned a value of 1. () RT–PCR demonstrates ascl1a and lin-28 mRNA levels following retinal injury. Expression of actin mRNA was used as an internal control. () Combined in situ hybridization and immunofluorescence microsc! opy images of a retinal section at 4 dpi from a 1016 tuba1a:gfp transgenic fish. Retina were treated with probes specific to lin-28 mRNA and antibodies specific to GFP and glutamine synthetase. Arrows indicate co-localization of lin-28 mRNA and GFP in cells expressing glutamine synthetase. Scale bar, 10 μm. () Combined in situ hybridization and immunofluorescence microscopy images of a retinal section at 4 dpi. Retina were treated with probes specific to lin-28 and ascl1a mRNA. The fish received an intraperitoneal injection of BrdU 3 h before being killed. Arrows indicate co-localization of lin-28 and ascl1a mRNA in proliferating BrdU-positive cells. Scale bar, 10 μm. ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. * Figure 2: Ascl1a and Lin-28 knockdown inhibit 1016 tuba1a:gfp transgene expression and Müller glia-derived progenitor proliferation. () Control, ascl1a or lin-28 lissamine-tagged morpholino oligonucleotides were electroporated into the retina of 1016 tuba1a:gfp transgenic fish at the time of retinal injury and 3 h before killing, at 4 dpi, fish received an intraperitoneal injection of BrdU. Arrows indicate cells harbouring lissamine-tagged morpholino oligonucleotide. Asterisk indicates autofluorescence in ONL. Scale bar, 10 μm and applies to all photomicrographs. () Quantification of the total number of morpholino oligonucleotide-positive cells that are in S phase as indicated by BrdU uptake. Data represent means ± s.d. (n = 3 individual fish; compared with control morpholino oligonucleotide, lin-28 morpholino oligonucleotide and ascl1a morpholino oligonucleotide P = 0.000178, or less). * Figure 3: Ascl1a regulates lin-28 expression. () Lissamine-labelled control (; top), lin-28- (; bottom) or ascl1a-targeting (; bottom) morpholino oligonucleotides were electroporated into injured retinas of 1016 tuba1a:gfp transgenic zebrafish. At 4 dpi, expression of GFP, and ascl1a and lin-28 mRNA were detected by immunofluorescence microscopy and in situ hybridization, respectively. Control morpholino oligonucleotide-treated retinas retain GFP, ascl1a and lin-28 expression (arrows), whereas Lin-28 knockdown suppresses GFP expression (arrows) and knockdown of Ascl1a suppresses both GFP and lin-28 expression. Asterisks indicate autofluorescence in ONL. Scale bar, 10 μm. () RT–PCR of zebrafish retina at 2 dpi. Retina were electroporated with lin-28 () or ascl1a () morpholino oligonucleotides. Control lane is RT–PCR from uninjured retina. Expression of actin mRNA was used as an internal control. () Real-time PCR quantification of lin-28 and ascl1a mRNA levels in injured retina at 2 dpi. Retina were electroporated wi! th two different lin-28 () or ascl1a () morpholino oligonucleotides at the indicated concentrations. Data are normalized to uninjured retinas and represent means ± s.d. of 3 replicates from a single experiment. () Ascl1a regulates lin-28 promoter activity. HEK293 cells were transfected with lin-28:luciferase vector, the indicated amounts of cmv:ascl1a vector , and for normalization, SV40:Renilla luciferase vector. Normalized promoter activity data are means ± s.d. (n = 3; compared with control, P = 0.0123 for 50 ng Ascl1a expression vector and P = 0.0001 for 200 ng of Ascl1a expression vector). () Ascl1a binds to regions of the lin-28 promoter that harbour multiple E-boxes. Top: ChIP analysis of zebrafish embryos injected with either myc RNA or myc–ascl1a mRNA. Chromatin, immunoprecipitated with antibodies specific to Myc, was assayed by PCR using primers flanking putative Ascl1a-binding sites 1 and 2 in the lin-28 promoter (ethidium-bromide-stained gel shown). The pred! icted fragments sizes of 287 bp and 323 bp were amplified. Bot! tom: schematic representation of the 3.1 kb lin-28 promoter indicates consensus E-box binding sites (ovals). Orange ovals are putative Ascl1-binding sites. Arrows indicate location of the primers used in the ChIP assay. * Figure 4: Lin-28 regulates let-7 miRNA levels in Müller glia-derived progenitors. () RT–PCR of let-7a and let-7f miRNA, and lin-28 mRNA, in Müller glia purified from uninjured retina (control), and injured retinas 4 days dpi. Expression of L-24 mRNA was used as an internal control. () Real-time PCR quantification of lin-28 mRNA, and let-7a and let-7f miRNA levels in purified Müller glia and Müller glia-derived progenitors at 4 dpi. Data are normalized to uninjured retina and represent means ± s.d. A single sample, consisting of Müller glia purified from two uninjured fish and Müller glia-derived progenitors purified from ten injured fish, was assayed in triplicate. () Images from combined let-7a in situ hybridization (LNA probe; left) and BrdU immunofluorescence microscopy (middle) of injured (top) and uninjured retina at 4 dpi (bottom). Arrows indicate let-7a miRNA suppression in BrdU-positive cells. Scale bar, 20 μm. () Real-time PCR quantification of let-7 miRNA levels in uninjured retinas (control) and injured retinas at the indicated times a! fter injury. Data represent means ± s.d. (n = 3 fish; compared with control uninjured retina, P = 0.0001 or less for let-7a and let-7f at all times). () RT–PCR of let-7a and let-7f miRNA in uninjured retina (control), or in injured retina at 4 dpi that were injected with control morpholino oligonucleotides or with two different types of lin-28 morpholino oligonucleotides. Expression of L-24 mRNA was used as an internal control. () Real-time PCR quantification of let-7a and let-7f miRNA levels in uninjured retina (control),or in injured retina at 4 dpi injected with control morpholino oligonucleotides or with two different lin-28 morpholino oligonucleotides. Data represent means ± s.d. from three replicates of a single experiment. * Figure 5: let-7 miRNAs suppress expression of regeneration- and pluripotency-associated genes. () Images from combined hspd1 in situ hybridization and glutamine synthetase immunofluorescence microscopy of uninjured retina. Arrows indicate localization of hspd1 mRNA and glutamine synthetase-expressing Müller glia. () RT–PCR of hspd1 and pax6b mRNA in uninjured retina (control) and injured retina, 4 dpi. Expression of actin mRNA was used as an internal control. () Real-time PCR quantification of hspd1, pax6a and pax6b mRNA levels in uninjured retina and injured retina 4 dpi. Data represent means ± s.d. (n = 3 fish; compared to uninjured retina, P = 0.0001 or less for hspd1, pax6a and pax6b. () let-7-dependent suppression of zebrafish proteins that are necessary for retinal regeneration or associated with pluripotency. HEK293 cells were transfected with plasmids expressing Flag- or Myc-tagged proteins that are associated with regeneration or pluripotency and with increasing concentrations (0, 50, 200 and 500 ng) of the ubC:let-7a,let-7f expression vector. pCS2:mCherr! y was co-transfected for normalization. After 48 h, cells were harvested and proteins resolved by denaturing SDS–PAGE. Western blots were probed with anti-mCherry, anti-Flag or anti-Myc antibodies to identify the indicated proteins. Experiments were repeated three times with similar results. Control lane indicates untransfected cells. Actin was used as an internal control. () Schematic representation of the constructs used to overexpress let-7 and investigate the function of putative let-7 miRNA-binding sites in the hspd1 3′ UTR. Mutations in the let-7-binding-site seed sequence are indicated. () The hspd1 3′ UTR let-7-binding site at position 1970 confers let-7-dependent regulation on luciferase expression. Luciferase activity from 24 hpf zebrafish embryos that were co-injected at the single-cell stage with the luciferase reporter and the let-7 miRNA expression vectors shown in , along with a vector expressing capped Renilla luciferase mRNA for normalization. Data re! present means ± s.d. (n = 3 fish; compared with the wild-type! 3′ UTR construct in absence of pri-let-7 miRNA or the mutant 3′ UTR construct with pri-let-7 miRNA, P = 0.0001 or less for wild-type (WT) 3′ UTR activity, treated with 1, 5 and 20 ng of the ubC:let-7a,let-7f construct). Uncropped images of blots are shown in Supplementary Information, Fig. S9. Author information * Author information * Supplementary information Affiliations * Molecular and Behavioral Neuroscience Institute and Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48109, USA. * Rajesh Ramachandran, * Blake V. Fausett & * Daniel Goldman Contributions D.G. and R.R. designed and analyzed the research and wrote the paper. D.G. generated the 1016 tuba1a:gfp transgenic fish. R.R. performed all experiments except for Fig. 4a, e where B.V.F. assayed let-7a and let-7f miRNA levels by RT–PCR. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Daniel Goldman (neuroman@umich.edu) Supplementary information * Author information * Supplementary information PDF files * Supplementary Information (1M) Supplementary Information Additional data