The following alphabetical list represents papers published in 2011 with at least one Whitehead author (in red). Not all of this work was done at the Whitehead Institute. Some of these papers are collaborations with scientists elsewhere. The papers are gathered from PubMed and from Science Citation Index (also known as the Web of Science) Preceding the bibliography is an alphabetical list of the titles of the papers followed by the first author.

P.S. The journal links only work if you have a license to those respective online journals.

-Access to Stem Cells and Data: Persons, Property Rights, and Scientific Progress. Mathews
-Activation of Mir-31 Function in Already-Established Metastases Elicits Metastatic Regression. Valastyan
-Analysis of a Drosophila amplicon in follicle cells highlights the diversity of metazoan replication origins Kim
-Analysis of gene networks in white adipose tissue development reveals a role for ETS2 in adipogenesis. Birsoy
-Ankyrin and band 3 differentially affect expression of membrane glycoproteins but are not required for erythroblast enucleation. Ji
-BAT3 Guides Misfolded Glycoproteins Out of the Endoplasmic Reticulum. Claessen
-BET Bromodomain Inhibition as a Therapeutic Strategy to Target c-Myc. Delmore
-Blimp1 Expression Predicts Embryonic Stem Cell Development In Vitro Chu
-Blood Ties: Chimerism Can Mask Twin Discordance in High-Throughput Sequencing. Erlich
-A Bmp/Admp Regulatory Circuit Controls Maintenance and Regeneration of Dorsal-Ventral Polarity in Planarians.Gavino
-Candida albicans Dicer (CaDcr1) is required for efficient ribosomal and spliceosomal RNA maturation.Bernstein
-CD8 alpha(+) Dendritic Cells Are the Critical Source of Interleukin-12 that Controls Acute Infection by Toxoplasma gondii Tachyzoites Mashayekhi
-Cell Biology. The Tascc of Secretion. Zoncu
-The Cellular Prion Protein Mediates Neurotoxic Signalling of Beta-Sheet-Rich Conformers Independent of Prion Replication. Resenberger
-Characterisation of the Trichinella spiralis Deubiquitinating Enzyme, TsUCH37, an Evolutionarily Conserved Proteasome Interaction Partner. White
-Chemical and Biological Approaches for Adapting Proteostasis to Ameliorate Protein Misfolding and Aggregation Diseases-Progress and Prognosis. Lindquist
-Chromosome Segregation: Keeping Kinetochores in the Loop. Schmidt
-Clonogenic Neoblasts Are Pluripotent Adult Stem Cells That Underlie Planarian Regeneration Wagner
-Common DISC1 Polymorphisms Disrupt Wnt/GSK3 beta Signaling and Brain Development.Singh
-Compatibility with killer explains the rise of RNAi-deficient fungi Drinnenberg
-Competition by Inhibitory Oligonucleotides Prevents Binding of Cpg to C-Terminal Tlr9. Avalos
-Comprehensive analysis of mammalian miRNA* species and their role in myeloid cells. Kuchenbauer
-Connecting Transcriptional Control to Chromosome Structure and Human Disease. Newman
-Conserved Function of lincRNAs in Vertebrate Embryonic Development despite Rapid Sequence Evolution Ulitsky
-Conserved Regulation of p53 Network Dosage by MicroRNA-125b Occurs through Evolving miRNA-Target Gene Pairs. Le
-Constitutive Gene Expression and the Specification of Tissue Identity in Adult Planarian Biology. Reddien
-Control of the Embryonic Stem Cell State. Young
-De Novo DNA Methylation by Dnmt3a and Dnmt3b Is Dispensable for Nuclear Reprogramming of Somatic Cells to a Pluripotent State. Pawlak
-Defective Regulation of Autophagy Upon Leucine Deprivation Reveals a Targetable Liability of Human Melanoma Cells in Vitro and in Vivo. Sheen
-Deletion of the de novo DNA methyltransferase Dnmt3a promotes lung tumor progression. Gao
-Densely Interconnected Transcriptional Circuits Control Cell States in Human Hematopoiesis Novershtern
-Derlin-2 Deficient Mouse Reveals an Essential Role for Protein Dislocation in Chondrocytes. Dougan
-Determinants of GBP Recruitment to Toxoplasma gondii Vacuoles and the Parasitic Factors That Control It. VirreiraWinter
-Developmental control of gene copy number by repression of replication initiation and fork progression Sher
-Developmental Control of Oocyte Maturation and Egg Activation in Metazoan Models VonStetina
-Development of Vaccinia Reporter Viruses for Rapid, High Content Analysis of Viral Function at All Stages of Gene Expression Dower
-Different 8-hydroxyquinolines protect models of TDP-43, alpha-synuclein, and polyglutamine proteotoxicity through distinct mechanisms. Tardiff
-Disc1 regulates both {beta}-catenin-mediated and noncanonical Wnt signaling during vertebrate embryogenesis DeRienzo
-Discovering Regulatory Overlapping Rna Transcripts. Danford
-Discovery and Optimization of Potent and Selective Benzonaphthyridinone Analogs as Small Molecule Mtor Inhibitors with Improved Mouse Microsome Stability. Liu
-Discovery of 9-(6-Aminopyridin-3-Yl)-1-(3-(Trifluoromethyl)-Phenyl)Benzo[H][1,6]Napht Hyridin-2(1h)-One (Torin2) as a Potent, Selective, and Orally Available Mammalian Target of Raparnycin (Mtor) Inhibitor for Treatment of Cancer. Liu
-dlx and sp6-9 Control Optic Cup Regeneration in a Prototypic Eye. Lapan
-Drosophila Inducer of MEiosis 4 (IME4) is required for Notch signaling during oogenesis. Hongay
-The dual role of ancient ubiquitous protein 1 (AUP1) in lipid droplet accumulation and ER protein quality control. Klemm
-Dynamic Changes in the Copy Number of Pluripotency and Cell Proliferation Genes in Human Escs and Ipscs During Reprogramming and Time in Culture.Laurent
-Ebola virus entry requires the cholesterol transporter Niemann-Pick C1. Carette
-Egf Ligand Release by Substrate-Specific Adam Metalloproteases Involves Different Pkc Isoenzymes Depending on the Stimulu Dang
-Elevated Levels of Bmp6 Impair Neurogenesis in Alzheimer's Disease. Choi
-Embryonic stem cell-based mapping of developmental transcriptional programs. Mazzoni
-Enzymatic Blockade of the Ubiquitin-Proteasome Pathway Ernst
-Er Stress Inhibits Mtorc2 and Akt Signaling through Gsk-3 Beta-Mediated Phosphorylation of Rictor. Chen
-Evidence That Gene Activation and Silencing during Stem Cell Differentiation Requires a Transcriptionally Paused Intermediate State. Golob
-Exome Sequencing and Disease-Network Analysis of a Single Family Implicate a Mutation in Kif1a in Hereditary Spastic Paraparesis. Erlich
-A Few Standing for Many: Embryo Receptor-Like Kinases. Nodine
-From stem cell to red cell: regulation of erythropoiesis at multiple levels by multiple proteins, RNAs and chromatin modifications Hattangadi
-Functional Genomics Reveal That the Serine Synthesis Pathway Is Essential in Breast Cancer Possemato
-Functional Heterogeneity of Breast Fibroblasts Is Defined by a Prostaglandin Secretory Phenotype that Promotes Expansion of Cancer-Stem Like Cells Rudnick
-Functional Integration of Dopaminergic Neurons Directly Converted from Mouse Fibroblasts Kim
-Functional Links Between Aβ Toxicity, Endocytic Trafficking, and Alzheimer’s Disease Risk Factors in Yeast Treusch
-Fungal recognition is mediated by the association of dectin-1 and galectin-3 in macrophages Esteban
-Gene induction and repression during terminal erythropoiesis are mediated by distinct epigenetic changes Wong
-Gene Targeting in Human Pluripotent Cells. Hockemeyer.
-Generation of Isogenic Pluripotent Stem Cells Differing Exclusively at Two Early Onset Parkinson Point Mutations. Soldner
-Genes Methylated by DNA Methyltransferase 3b Are Similar in Mouse Intestine and Human Colon Cancer Steine
-Genetic Engineering of Human Pluripotent Cells Using Tale Nucleases Hockemeyer
-Genome-Scale Rnai on Living-Cell Microarrays Identifies Novel Regulators of Drosophila Melanogaster Torc1-S6k Pathway Signaling. Lindquist
-Genome-Wide Detection of Meiotic DNA Double-Strand Break Hotspots Using Single-Stranded DNA. Blitzblau
-Genome-wide Maps of Histone Modifications Unwind In Vivo Chromatin States of the Hair Follicle Lineage Lien
-Genomic Analysis of Parent-of-Origin Allelic Expression in Arabidopsis thaliana Seeds Gehring
-Global Gene Disruption in Human Cells to Assign Genes to Phenotypes by Deep Sequencing. Carette
-Granulin Is a Soluble Cofactor for Toll-Like Receptor 9 Signaling. Park
-Hallmarks of Cancer: The Next Generation Hanahan
-A Haploid Genetic Screen Identifies the Major Facilitator Domain Containing 2a (Mfsd2a) Transporter as a Key Mediator in the Response to Tunicamycin Reiling
-Helicobacter Pylori Cytotoxin-Associated Gene a (Caga) Subverts the Apoptosis-Stimulating Protein of P53 (Aspp2) Tumor Suppressor Pathway of the Host.Buti
-A Hierarchical Combination of Factors Shapes the Genome-Wide Topography of Yeast Meiotic Recombination Initiation. Pan
-HSP90 as a platform for the assembly of more effective cancer chemotherapy Whitesell
-Human Cd34(+) Cd133(+) Hematopoietic Stem Cells Cultured with Growth Factors Including Angptl5 Efficiently Engraft Adult Nod-Scid Il2r Gamma(-/-) (Nsg) Mi.c Drake
-Human Tumors Instigate Granulin-Expressing Hematopoietic Cells That Promote Malignancy by Activating Stromal Fibroblasts in Mice Elkabets
-Identification of host cell factors required for intoxication through use of modified cholera toxin Guimaraes
-Induced Ectopic Kinetochore Assembly Bypasses the Requirement for Cenp-a Nucleosomes Gascoigne
-Influence of Galectin-9/Tim-3 Interaction on Herpes Simplex Virus-1 Latency. Reddy
-The inside-out Mechanism of Dicers from Budding Yeasts. Weinberg
-Integrative Analysis of Gene Amplification in Drosophila Follicle Cells: Parameters of Origin Activation and Repression Kim
-Irreversible inhibitors and activity-based probes as research tools in chemical glycobiology. Witte
-Keeping two animal systems in one lab - a frog plus fish case study. Sive
-Licensing of Gametogenesis, Dependent on Rna Binding Protein Dazl, as a Gateway to Sexual Differentiation of Fetal Germ Cells Gill
-Lipolysis-stimulated lipoprotein receptor (LSR) is the host receptor for the binary toxin Clostridium difficile transferase (CDT) Papatheodorou
-Long noncoding RNA-mediated anti-apoptotic activity in murine erythroid terminal differentiation Hu
-Making and Breaking Peptide Bonds: Protein Engineering Using Sortase. Popp
-Master Transcription Factors Determine Cell-Type-Specific Responses to TGF-β Signaling. Mullen
-Mesenchymal Stem Cells Secreting Angiopoietin-Like-5 Support Efficient Expansion of Human Hematopoietic Stem Cells Without Compromising Their Repopulating Potential. Khoury
-A Method for Probing the Mutational Landscape of Amyloid Structure ODonnell
-Mice with ribosomal protein S19 deficiency develop bone marrow failure and symptoms like patients with Diamond-Blackfan anemia. Jaako
-MicroRNA Destabilization Enables Dynamic Regulation of the miR-16 Family in Response to Cell-Cycle Changes Rissland
-Microrna-15a and -16-1 Act Via Myb to Elevate Fetal Hemoglobin Expression in Human Trisomy 13. Sankaran
-Micrornas in Adipogenesis and as Therapeutic Targets for Obesity Alexander
-Micrornas: The Primary Cause or a Determinant of Progression in Leukemia? Bousquet
-Mir-191 Regulates Mouse Erythroblast Enucleation by Down-Regulating Riok3 and Mxi1. Zhang
-Mir193b-365 Is Essential for Brown Fat Differentiation. Sun
-Mir-290-295 deficiency in mice results in partially penetrant embryonic lethality and germ cell defects.Medeiros
-'Model' or 'Tool'? New Definitions for Translational Research Sive
-The Msx1 Homeoprotein Recruits Polycomb to the Nuclear Periphery during Development Wang
-mTOR Complex 1 Regulates Lipin 1 Localization to Control the SREBP Pathway. Peterson
-Mtor: From Growth Signal Integration to Cancer, Diabetes and Ageing. Zoncu
-mTORC1 senses lysosomal amino acids through an inside-out mechanism that requires the vacuolar H-ATPase Zoncu
-The Mtor-Regulated Phosphoproteome Reveals a Mechanism of Mtorc1-Mediated Inhibition of Growth Factor Signaling. Hsu
-Normal and Neoplastic Nonstem Cells Can Spontaneously Convert to a Stem-Like State. Chaffer
-Ocular Neovascularization Caused by Herpes Simplex Virus Type 1 Infection Results from Breakdown of Binding between Vascular Endothelial Growth Factor a and Its Soluble Receptor. Suryawanshi
-Opposing Effects of Glutamine and Asparagine Govern Prion Formation by Intrinsically Disordered Proteins Halfmann
-Pan-Src Family Kinase Inhibitors Replace Sox2 During the Direct Reprogramming of Somatic Cells. Staerk
-Paracrine and Autocrine Signals Induce and Maintain Mesenchymal and Stem Cell States in the Breast. Scheel
-Peer review of scientific papers in top journals is bogged down by unnecessary demands for extra lab work. Ploegh
-A Perspective on Cancer Cell Metastasis. Chaffer
-Phenotypic plasticity and epithelial-mesenchymal transitions in cancer - and normal stem cells? Scheel
-PIF1 disruption or NBS1 hypomorphism does not affect chromosome healing or fusion resulting from double-strand breaks near telomeres in murine embryonic stem cells. Reynolds
-Piperazinyl quinolines as chemosensitizers to increase fluconazole susceptibility of Candida albicans clinical isolates. Youngsaye
-Polarized Notum Activation at Wounds Inhibits Wnt Function to Promote Planarian Head Regeneration Petersen
-A Portable Rna Sequence Whose Recognition by a Synthetic Antibody Facilitates Structural Determination Koldobskaya
-Postprandial Hepatic Lipid Metabolism Requires Signaling through Akt2 Independent of the Transcription Factors FoxA2, FoxO1, and SREBP1c. Wan
-Progression of Pathogenic Events in Cynomolgus Macaques Infected with Variola Virus WahlJensen
-Protection of repetitive DNA borders from self-induced meiotic instability. Vader
-Protein-Only Mechanism Induces Self-Perpetuating Changes in the Activity of Neuronal Aplysia Cytoplasmic Polyadenylation Element Binding Protein (Cpeb). Heinrich
-Protein quality control in the ER: balancing the ubiquitin checkbook. Claessen
-A Prototype Antifungal Contact Lens.Ciolino
-A Radical Role for Tor in Longevity Lamming
-A regulatory program for excretory system regeneration in planarians.Scimone
-Reprogramming factor stoichiometry influences the epigenetic state and biological properties of induced pluripotent stem cells. Carey
-Reprogramming of Postnatal Neurons into Induced Pluripotent Stem Cells by Defined Factors. Kim
-Research ethics: Treat donors as partners in biobank research. Saha
-Responsenet: Revealing Signaling and Regulatory Networks Linking Genetic and Transcriptomic Screening Data. Lan
-Rna Silencing in Monterey. Rissland
-Road to Ruin: Targeting Proteins for Degradation in the Endoplasmic Reticulum Smith
-Role of autophagy in neurodegenerative diseases. Sarkar
-Role of IL-17 and Th17 Cells in Herpes Simplex Virus-Induced Corneal Immunopathology Suryawanshi
-Role of the Ubiquitin-Like Protein Urm1 as a Noncanonical Lysine-Directed Protein Modifier. VanderVeen
-Roles for Micrornas in the Regulation of Cell Adhesion Molecules. Valastyan
-Sortase A as a tool for high-yield histatin cyclizatin.Bolscher
-Sortase-Catalyzed Transformations That Improve the Properties of Cytokines. Popp
-STITCHER: Dynamic assembly of likely amyloid and prion beta-structures from secondary structure predictions. Bryan
-The structural basis of RNA-catalyzed RNA polymerization. Shechner
-Surface-engineered substrates for improved human pluripotent stem cell culture under fully defined conditions. Saha
-Teaching Bioinformatics at the Secondary School Level. Lewitter
-Ten Simple Rules for Teaching Bioinformatics at the High School Level. Form
-Tet1 is dispensable for maintaining pluripotency and its loss is compatible with embryonic and postnatal development.Dawlaty
-TorsinA and the TorsinA-Interacting Protein Printor Have No Impact on Endoplasmic Reticulum Stress or Protein Trafficking in Yeast Valastyan
-Transcriptional control of embryonic and induced pluripotent stem cells Guenther
-Transgene Excision Has No Impact on In Vivo Integration of Human iPS Derived Neural Precursor. Major
-Translational regulation of the cell cycle: when, where, how and why? Kronja
-Tregs and infections: on the potential value of modifying their function Sehrawat
-Tumor metastasis: molecular insights and evolving paradigms Valastyan
-Ubiquitin-Dependent Control of Class Ii Mhc Localization Is Dispensable for Antigen Presentation and Antibody Production. McGehee
-Unusually effective microRNA targeting within repeat-rich coding regions of mammalian mRNAs SchnallLevin
-Weak seed-pairing stability and high target-site abundance decrease the proficiency of lsy-6 and other microRNA Garcia
-Yap1 Acts Downstream of Alpha-Catenin to Control Epidermal Proliferation. Schlegelmilch
-Yeast: An Experimental Organism for 21st Century Biology Botstein
-A Yeast Model of Fus/Tls-Dependent Cytotoxicit. Ju

Alexander, R., Lodish, H., and Sun, L. (2011). Micrornas in Adipogenesis and as Therapeutic Targets for Obesity. Expert Opinion in Therapeutic Targets.Feb 28. [Epub ahead of print] Introduction: Obesity and obesity-related disease have reached pandemic proportions and are prevalent even in developing countries. Adipose tissue is increasingly being recognized as a key regulator of whole-body energy homeostasis and consequently as a prime therapeutic target for metabolic syndrome. This review discusses the roles of miRNAs, small endogenously expressed RNAs that regulate gene expression at a post-transcriptional level, in the development and function of adipose tissue and other relevant metabolic tissues impacted by obesity. Several high-throughput studies have identified hundreds of miRNAs that are differentially expressed during the development of metabolic tissues or as an indication of pathophysiology. Further investigation has functionalized the regulatory capacity of individual miRNAs and revealed putative targets for these miRNAs. Therefore, as with several other pathologies, miRNAs are emerging as feasible therapeutic targets for metabolic syndrome. Areas covered: This review provides a comprehensive view of miRNAs involved in adipogenesis, from mesenchymal stem cell lineage determination through terminal adipocyte differentiation. We also discuss the differential expression of miRNAs among adipose depots and the dysregulation of miRNAs in other metabolic tissues during metabolic pathophysiology. Finally, we discuss the therapeutic potential of targeting miRNAs in obesity and give a perspective on the challenges and advantages of miRNA-based drugs. Expert opinion: miRNAs are extensive regulators of adipocyte development and function and are viable therapeutic targets for obesity. Despite the broad-spectrum and redundancy of miRNA?target interactions, sophisticated bioinformatic approaches are making it possible to determine the most physiologically relevant miRNAs to target in disease. In vivo delivery of miRNAs for therapeutic purposes is rapidly developing and has been successful in other contexts. Additionally, miRNAs can be used as prognosis markers for disease onset and progression. Ultimately, miRNAs are prime therapeutic targets for obesity and its consequent pathologies in other metabolic tissues.

Avalos, A.M., and Ploegh, H.L. (2011). Competition by Inhibitory Oligonucleotides Prevents Binding of Cpg to C-Terminal Tlr9. European Journal of Immunology .Accepted manuscript online: 18 JUL 2011 TLR9 recognizes unmethylated CpG-containing DNA commonly found in bacteria. Synthetic oligonucleotides containing CpG-motifs (CpG ODNs) recapitulate the activation of TLR9 by microbial DNA, whereas inversion of the CG dinucleotide within the CpG motif to GC (GpC ODN) renders such ODNs inactive. This difference cannot be attributed to binding of ODNs to the full-length TLR9 ectodomain, as both CpG and GpC ODNs bind comparably. Activation of murine TLR9 requires cleavage into an active C-terminal fragment, which binds CpG robustly. We therefore compared the ability of CpG and GpC ODNs to bind to full-length and C-terminal TLR9, and their impact on cleavage of TLR9. We found that CpG binds better to C-terminal TLR9 when compared to GpC, despite comparable and low binding of both ODNs to full-length TLR9. Neither CpG nor GpC ODN affected TLR9 cleavage in murine RAW 264.7 cells stably expressing TLR9-Myc. Inhibitory ODNs (IN-ODNs) block TLR9 signaling, but how they do so remains unclear. We show that IN-ODNs do not impede TLR9 cleavage but bind to C-terminal TLR9 preferentially, and thereby compete for CpG ODN binding both in RAW cells and TLR9-deficient cells transduced with TLR9-Myc. Ligand binding to C-terminal fragment thus determines the outcome of activation through TLR9. PDF

Bernstein, D.A., Vyas, V.K., Weinberg, D.E., Drinnenberg, I.A., Bartel, D.P., and Fink, G.R. (2011). Candida albicans Dicer (CaDcr1) is required for efficient ribosomal and spliceosomal RNA maturation. Proc Natl Acad Sci U S ADec 15. [Epub ahead of print].The generation of mature functional RNAs from nascent transcripts requires the precise and coordinated action of numerous RNAs and proteins. One such protein family, the ribonuclease III (RNase III) endonucleases, includes Rnt1, which functions in fungal ribosome and spliceosome biogenesis, and Dicer, which generates the siRNAs of the RNAi pathway. The recent discovery of small RNAs in Candida albicans led us to investigate the function of C. albicans Dicer (CaDcr1). CaDcr1 is capable of generating siRNAs in vitro and is required for siRNA generation in vivo. In addition, CaDCR1 complements a Dicer knockout in Saccharomyces castellii, restoring RNAi-mediated gene repression. Unexpectedly, deletion of the C. albicans CaDCR1 results in a severe slow-growth phenotype, whereas deletion of another core component of the RNAi pathway (CaAGO1) has little effect on growth, suggesting that CaDCR1 may have an essential function in addition to producing siRNAs. Indeed CaDcr1, the sole functional RNase III enzyme in C. albicans, has additional functions: it is required for cleavage of the 3' external transcribed spacer from unprocessed pre-rRNA and for processing the 3' tail of snRNA U4. Our results suggest two models whereby the RNase III enzymes of a fungal ancestor, containing both a canonical Dicer and Rnt1, evolved through a series of gene-duplication and gene-loss events to generate the variety of RNase III enzymes found in modern-day budding yeasts. PDF

Birsoy, K., Berry, R., Wang, T., Ceyhan, O., Tavazoie, S., Friedman, J.M., and Rodeheffer, M.S. (2011). Analysis of gene networks in white adipose tissue development reveals a role for ETS2 in adipogenesis. Development 138, 4709-4719.Obesity is characterized by an expansion of white adipose tissue mass that results from an increase in the size and the number of adipocytes. However, the mechanisms responsible for the formation of adipocytes during development and the molecular mechanisms regulating their increase and maintenance in adulthood are poorly understood. Here, we report the use of leptinluciferase BAC transgenic mice to track white adipose tissue (WAT) development and guide the isolation and molecular characterization of adipocytes during development using DNA microarrays. These data reveal distinct transcriptional programs that are regulated during murine WAT development in vivo. By using a de novo cis-regulatory motif discovery tool (FIRE), we identify two early gene clusters whose promoters show significant enrichment for NRF2/ETS transcription factor binding sites. We further demonstrate that Ets transcription factors, but not Nrf2, are regulated during early adipogenesis and that Ets2 is essential for the normal progression of the adipocyte differentiation program in vitro. These data identify ETS2 as a functionally important transcription factor in adipogenesis and its possible role in regulating adipose tissue mass in adults can now be tested. Our approach also provides the basis for elucidating the function of other gene networks during WAT development in vivo. Finally these data confirm that although gene expression during adipogenesis in vitro recapitulates many of the patterns of gene expression in vivo, there are additional developmental transitions in pre and post-natal adipose tissue that are not evident in cell culture systems. Full Text.

Blitzbau, H.G., and Hochwagen, A. (2011). Genome-Wide Detection of Meiotic DNA Double-Strand Break Hotspots Using Single-Stranded DNA. Methods Mol Biol 745, 47-63. The controlled fragmentation of chromosomes by DNA double-strand breaks (DSBs) initiates meiotic recombination, which is essential for meiotic chromosome segregation in most eukaryotes. This chapter describes a straightforward microarray-based approach to measure the genome-wide distribution of meiotic DSBs by detecting the single-stranded DNA (ssDNA) that transiently accumulates at DSB sites during recombination. The protocol outlined here has been optimized to detect meiotic DSBs in Saccharomyces cerevisiae. However, because ssDNA is a universal intermediate of homologous recombination, this method can ostensibly be adapted to discover and analyze programmed or damage-induced DSB hotspots in other organisms whose genome sequence is available. Full Text.

Bolscher, J.G.M., Oudhoff, M.J., Nazmi, K., Antos, J.M., Guimaraes, C.P., Spooner, E., Haney, E.F., Vallejo, J.J.G., Vogel, H.J., van't Hof, W, Ploegh, HL, and Veerman ECI.(2011). Sortase A as a tool for high-yield histatin cyclization. Faseb Journal 25, 2650-2658.Cyclic peptides are highly valued tools in biomedical research. In many cases, they show higher receptor affinity, enhanced biological activity, and improved serum stability. Technical difficulties in producing cyclic peptides, especially larger ones, in appreciable yields have precluded a prolific use in biomedical research. Here, we describe a novel and efficient cyclization method that uses the peptidyl-transferase activity of the Staphylococcus aureus enzyme sortase A to cyclize linear synthetic precursor peptides. As a model, we used histatin 1, a 38-mer salivary peptide with motogenic activity. Chemical cyclization of histatin 1 resulted in <= 3% yields, whereas sortase-mediated cyclization provided a yield of > 90%. The sortase-cyclized peptide displayed a maximum wound closure activity at 10 nM, whereas the linear peptide displayed maximal activity at 10 mu M. Circular dichroism and NMR spectroscopic analysis of the linear and cyclic peptide in solution showed no evidence for conformational changes, suggesting that structural differences due to cyclization only became manifest when these peptides were located in the binding domain of the receptor. The sortase-based cyclization technology provides a general method for easy and efficient manufacturing of large cyclic peptides.-Bolscher, J. G. M., Oudhoff, M. J., Nazmi, K., Antos, J. M., Guimaraes, C. P., Spooner, E., Haney, E. F., Garcia-Vallejo, J. J., Vogel, H. J., van't Hof, W., Ploegh, H. L., Veerman. E. C. I. Sortase A as a tool for high-yield histatin cyclization. . Full Text.

Botstein, D., and Fink, G.R. (2011). Yeast: An Experimental Organism for 21st Century Biology. Genetics 189, 695-704.In this essay, we revisit the status of yeast as a model system for biology. We first summarize important contributions of yeast to eukaryotic biology that we anticipated in 1988 in our first article on the subject. We then describe transformative developments that we did not anticipate, most of which followed the publication of the complete genomic sequence of Saccharomyces cerevisiae in 1996. In the intervening 23 years it appears to us that yeast has graduated from a position as the premier model for eukaryotic cell biology to become the pioneer organism that has facilitated the establishment of the entirely new fields of study called "functional genomics" and "systems biology." These new fields look beyond the functions of individual genes and proteins, focusing on how these interact and work together to determine the properties of living cells and organisms. Full Text.

Bousquet, M., and Lodish, H.F. (2011). Micrornas: The Primary Cause or a Determinant of Progression in Leukemia? Expert Reviews of Hematology 4, 121-123.

Bryan, A.W., Jr., O'Donnell, C.W., Menke, M., Cowen, L.J., Lindquist, S., and Berger, B. (2011). STITCHER: Dynamic assembly of likely amyloid and prion beta-structures from secondary structure predictions. Proteins. Sep 23. doi: 10.1002/prot.23203. [Epub ahead of print] The supersecondary structure of amyloids and prions, proteins of intense clinical and biological interest, are difficult to determine by standard experimental or computational means. In addition, significant conformational heterogeneity is known or suspected to exist in many amyloid fibrils. Previous work has demonstrated that probability-based prediction of discrete beta-strand pairs can offer insight into these structures. Here, we devise a system of energetic rules that can be used to dynamically assemble these discrete beta-strand pairs into complete amyloid beta-structures. The STITCHER algorithm progressively 'stitches' strand-pairs into full beta-sheets based on a novel free-energy model, incorporating experimentally observed amino-acid side-chain stacking contributions, entropic estimates, and steric restrictions for amyloidal parallel beta-sheet construction. A dynamic program computes the top 50 structures and returns both the highest scoring structure and a consensus structure taken by polling this list for common discrete elements. Putative structural heterogeneity can be inferred from sequence regions that compose poorly. Predictions show agreement with experimental models of Alzheimer's amyloid beta peptide and the Podospora anserina Het-s prion. Predictions of the HET-s homolog HET-S also reflect experimental observations of poor amyloid formation. We put forward predicted structures for the yeast prion Sup35, suggesting N-terminal structural stability enabled by tyrosine ladders, and C-terminal heterogeneity. Predictions for the Rnq1 prion and alpha-synuclein are also given, identifying a similar mix of homogenous and heterogeneous secondary structure elements. STITCHER provides novel insight into the energetic basis of amyloid structure, provides accurate structure predictions, and can help guide future experimental studies. PDF

Buti, L., Spooner, E., Van der Veen, A.G., Rappuoli, R., Covacci, A., and Ploegh, H.L. (2011). Helicobacter Pylori Cytotoxin-Associated Gene a (Caga) Subverts the Apoptosis-Stimulating Protein of P53 (Aspp2) Tumor Suppressor Pathway of the Host. Proc Natl Acad Sci U S AMay 11. [Epub ahead of print] Type I strains of Helicobacter pylori (Hp) possess a pathogenicity island, cag, that encodes the effector protein cytotoxin-associated gene A (CagA) and a type four secretion system. After translocation into the host cell, CagA affects cell shape, increases cell motility, abrogates junctional activity, and promotes an epithelial to mesenchymal transition-like phenotype. Transgenic expression of CagA enhances gastrointestinal and intestinal carcinomas as well as myeloid and B-cell lymphomas in mice, but the mechanism of the induced cancer formation is not fully understood. Here, we show that CagA subverts the tumor suppressor function of apoptosis-stimulating protein of p53 (ASPP2). Delivery of CagA inside the host results in its association with ASPP2. After this interaction, ASPP2 recruits its natural target p53 and inhibits its apoptotic function. CagA leads to enhanced degradation of p53 and thereby, down-regulates its activity in an ASPP2-dependent manner. Finally, Hp-infected cells treated with the p53-activating drug Doxorubicin are more resistant to apoptosis than uninfected cells, an effect that requires ASPP2. The interaction between CagA and ASPP2 and the consequent degradation of p53 are examples of a bacterial protein that subverts the p53 tumor suppressor pathway in a manner similar to DNA tumor viruses. This finding may contribute to the understanding of the increased risk of gastric cancer in patients infected with Hp CagA+ strains. PDF

Carette, J.E., Raaben, M., Wong, A.C., Herbert, A.S., Obernosterer, G., Mulherkar, N., Kuehne, A.I., Kranzusch, P.J., Griffin, A.M., Ruthel, G .,Cin PD, Dye JM, Whelan SP, Chandran K, Brummelkamp TR. (2011). Ebola virus entry requires the cholesterol transporter Niemann-Pick C1. Nature.Aug 24.[Epub ahead of print] Infections by the Ebola and Marburg filoviruses cause a rapidly fatal haemorrhagic fever in humans for which no approved antivirals are available. Filovirus entry is mediated by the viral spike glycoprotein (GP), which attaches viral particles to the cell surface, delivers them to endosomes and catalyses fusion between viral and endosomal membranes. Additional host factors in the endosomal compartment are probably required for viral membrane fusion; however, despite considerable efforts, these critical host factors have defied molecular identification. Here we describe a genome-wide haploid genetic screen in human cells to identify host factors required for Ebola virus entry. Our screen uncovered 67 mutations disrupting all six members of the homotypic fusion and vacuole protein-sorting (HOPS) multisubunit tethering complex, which is involved in the fusion of endosomes to lysosomes, and 39 independent mutations that disrupt the endo/lysosomal cholesterol transporter protein Niemann-Pick C1 (NPC1). Cells defective for the HOPS complex or NPC1 function, including primary fibroblasts derived from human Niemann-Pick type C1 disease patients, are resistant to infection by Ebola virus and Marburg virus, but remain fully susceptible to a suite of unrelated viruses. We show that membrane fusion mediated by filovirus glycoproteins and viral escape from the vesicular compartment require the NPC1 protein, independent of its known function in cholesterol transport. Our findings uncover unique features of the entry pathway used by filoviruses and indicate potential antiviral strategies to combat these deadly agents. Full Text.

Carette, J.E., Guimaraes, C.P., Wuethrich, I., Blomen, V.A., Varadarajan, M., Sun, C., Bell, G., Yuan, B., Muellner, M.K., Nijman, S.M., Hidde L Ploegh & Thijn R Brummelkamp. (2011). Global Gene Disruption in Human Cells to Assign Genes to Phenotypes by Deep Sequencing. Nat Biotechnol. Insertional mutagenesis in a haploid background can disrupt gene function. We extend our earlier work by using a retroviral gene-trap vector to generate insertions in >98% of the genes expressed in a human cancer cell line that is haploid for all but one of its chromosomes. We apply phenotypic interrogation via tag sequencing (PhITSeq) to examine millions of mutant alleles through selection and parallel sequencing. Analysis of pools of cells, rather than individual clones enables rapid assessment of the spectrum of genes involved in the phenotypes under study. This facilitates comparative screens as illustrated here for the family of cytolethal distending toxins (CDTs). CDTs are virulence factors secreted by a variety of pathogenic Gram-negative bacteria responsible for tissue damage at distinct anatomical sites. We identify 743 mutations distributed over 12 human genes important for intoxication by four different CDTs. Although related CDTs may share host factors, they also exploit unique host factors to yield a profile characteristic for each CDT. Full Text.

Carey, B.W., Markoulaki, S., Hanna, J.H., Faddah, D.A., Buganim, Y., Kim, J., Ganz, K., Steine, E.J., Cassady, J.P., Creyghton, M.P., Welstead GG, Gao Q, and Jaenisch R.(2011). Reprogramming factor stoichiometry influences the epigenetic state and biological properties of induced pluripotent stem cells. Cell Stem Cell 9, 588-598.We compared two genetically highly defined transgenic systems to identify parameters affecting reprogramming of somatic cells to a pluripotent state. Our results demonstrate that the level and stoichiometry of reprogramming factors during the reprogramming process strongly influence the resulting pluripotency of iPS cells. High expression of Oct4 and Klf4 combined with lower expression of c-Myc and Sox2 produced iPS cells that efficiently generated "all-iPSC mice" by tetraploid (4n) complementation, maintained normal imprinting at the Dlk1-Dio3 locus, and did not create mice with tumors. Loss of imprinting (LOI) at the Dlk1-Dio3 locus did not strictly correlate with reduced pluripotency though the efficiency of generating "all-iPSC mice" was diminished. Our data indicate that stoichiometry of reprogramming factors can influence epigenetic and biological properties of iPS cells. This concept complicates efforts to define a "generic" epigenetic state of iPSCs and ESCs and should be considered when comparing different iPS and ES cell lines. Full Text.

ChafferC.L., Brueckmann, I., Scheel, C., Kaestli, A.J., Wiggins, P.A., Rodrigues, L.O., Brooks, M., Reinhardt, F.,Ying c, Kornelia Polyak, Lisa M. Arende,Charlotte Kuperwasser, Brian Bierie, and Robert A. Weinberg. (2011). Normal and Neoplastic Nonstem Cells Can Spontaneously Convert to a Stem-Like State. Proc Natl Acad Sci U S A. 2011 Apr 15. [Epub ahead of print] Current models of stem cell biology assume that normal and neoplastic stem cells reside at the apices of hierarchies and differentiate into nonstem progeny in a unidirectional manner. Here we identify a subpopulation of basal-like human mammary epithelial cells that departs from that assumption, spontaneously dedifferentiating into stem-like cells. Moreover, oncogenic transformation enhances the spontaneous conversion, so that nonstem cancer cells give rise to cancer stem cell (CSC)-like cells in vitro and in vivo. We further show that the differentiation state of normal cells-of-origin is a strong determinant of posttransformation behavior. These findings demonstrate that normal and CSC-like cells can arise de novo from more differentiated cell types and that hierarchical models of mammary stem cell biology should encompass bidirectional interconversions between stem and nonstem compartments. The observed plasticity may allow derivation of patient-specific adult stem cells without genetic manipulation and holds important implications for therapeutic strategies to eradicate cancer. PDF

Chaffer, C.L., and Weinberg, R.A. (2011). A Perspective on Cancer Cell Metastasis. Science 331, 1559-1564. Metastasis causes most cancer deaths, yet this process remains one of the most enigmatic aspects of the disease. Building on new mechanistic insights emerging from recent research, we offer our perspective on the metastatic process and reflect on possible paths of future exploration. We suggest that metastasis can be portrayed as a two-phase process: The first phase involves the physical translocation of a cancer cell to a distant organ, whereas the second encompasses the ability of the cancer cell to develop into a metastatic lesion at that distant site. Although much remains to be learned about the second phase, we feel that an understanding of the first phase is now within sight, due in part to a better understanding of how cancer cell behavior can be modified by a cell-biological program called the epithelial-to-mesenchymal transition. Full Text.

Chen, C.H., Shaikenov, T., Peterson, T.R., Aimbetov, R., Bissenbaev, A.K., Lee, S.W., Wu, J.A., Lin, H.K., and Sarbassov, D. (2011). Er Stress Inhibits Mtorc2 and Akt Signaling through Gsk-3 Beta-Mediated Phosphorylation of Rictor. Science Signaling Vol. 4, Issue 161, p. ra10. In response to environmental cues, cells coordinate a balance between anabolic and catabolic pathways. In eukaryotes, growth factors promote anabolic processes and stimulate cell growth, proliferation, and survival through activation of the phosphoinositide 3-kinase (PI3K)-Akt pathway. Akt-mediated phosphorylation of glycogen synthase kinase-3 beta (GSK-3 beta) inhibits its enzymatic activity, thereby stimulating glycogen synthesis. We show that GSK-3 beta itself inhibits Akt by controlling the mammalian target of rapamycin complex 2 (mTORC2), a key activating kinase for Akt. We found that during cellular stress, GSK-3 beta phosphorylated the mTORC2 component rictor at serine-1235, a modification that interfered with the binding of Akt to mTORC2. The inhibitory effect of GSK-3 beta on mTORC2-Akt signaling and cell proliferation was eliminated by blocking phosphorylation of rictor at serine-1235. Thus, in response to cellular stress, GSK-3 beta restrains mTORC2-Akt signaling by specifically phosphorylating rictor, thereby balancing the activities of GSK-3 beta and Akt, two opposing players in glucose metabolism. Full Text.

Choi, S.H., and Li, Y. (2011). Elevated Levels of Bmp6 Impair Neurogenesis in Alzheimer's Disease. Journal of Neuroscience 31, 371-372. Full Text.

Chu, L.F., Surani, M.A., Jaenisch, R., and Zwaka, T.P. (2011). Blimp1 Expression Predicts Embryonic Stem Cell Development In Vitro. Current Biology 21, 1759-1765.Despite recent critical insights into the pluripotent state of embryonic stem cells (ESCs), there is little agreement over the inaugural and subsequent steps leading to its generation [1-4]. Here we show that inner cell mass (ICM)-generated cells expressing Blimp 1, a key transcriptional repressor of the somatic program during germ cell specification [5, 6], emerge on day 2 of blastocyst culture. Single-cell gene expression profiling indicated that many of these Blimp1-positive cells coexpress other genes typically associated with early germ cell specification. When genetically traced in vitro, these cells acquired properties normally associated with primordial germ cells. Importantly, fate-mapping experiments revealed that ESCs commonly arise from Blimp1-positive precursors; indeed, prospective sorting of such cells from ICM outgrowths increased the rate of ESC derivation more than 9-fold. Finally, using genetic ablation or distinct small molecules [7, 8], we show that epiblast cells can become ESCs without first acquiring Blimp1 positivity. Our findings suggest that the germ cell-like state is facultative for the stabilization of pluripotency in vitro. Thus, the association of Blimp1 expression with ESC development furthers understanding of how the pluripotent state of these cells is established in vitro and suggests a means to enhance the generation of new stem cell lines from blastocysts. Full Text.

Ciolino, J.B., Hudson, S.P., Mobbs, A.N., Hoare, T.R., Iwata, N.G., Fink, G.R., and Kohane, D.S. (2011). A Prototype Antifungal Contact Lens. Investigative Ophthalmology & Visual Science 52, 6286-6291.PURPOSE. To design a contact lens to treat and prevent fungal ocular infections. METHODS. Curved contact lenses were created by encapsulating econazole-impregnated poly(lactic-co-glycolic) acid (PLGA) films in poly(hydroxyethyl methacrylate) (pHEMA) by ultraviolet photopolymerization. Release studies were conducted in phosphate-buffered saline at 37 degrees C with continuous shaking. The contact lenses and their release media were tested in an antifungal assay against Candida albicans. Cross sections of the pre- and postrelease contact lenses were characterized by scanning electron microscopy and by Raman spectroscopy. RESULTS. Econazole-eluting contact lenses provided extended antifungal activity against Candida albicans fungi. Fungicidal activity varied in duration and effectiveness depending on the mass of the econazole-PLGA film encapsulated in the contact lens. CONCLUSIONS. An econazole-eluting contact lens could be used as a treatment for fungal ocular infections.

Claessen, J.H., and Ploegh, H.L. (2011). BAT3 Guides Misfolded Glycoproteins Out of the Endoplasmic Reticulum. PLoS One 6, e28542.Secretory and membrane proteins that fail to acquire their native conformation within the lumen of the Endoplasmic Reticulum (ER) are usually targeted for ubiquitin-dependent degradation by the proteasome. How partially folded polypeptides are kept from aggregation once ejected from the ER into the cytosol is not known. We show that BAT3, a cytosolic chaperone, is recruited to the site of dislocation through its interaction with Derlin2. Furthermore, we observe cytoplasmic BAT3 in a complex with a polypeptide that originates in the ER as a glycoprotein, an interaction that depends on the cytosolic disposition of both, visualized even in the absence of proteasomal inhibition. Cells depleted of BAT3 fail to degrade an established dislocation substrate. We thus implicate a cytosolic chaperone as an active participant in the dislocation of ER glycoproteins. Full Text.

Claessen, J.H., Kundrat, L., and Ploegh, H.L. (2011). Protein quality control in the ER: balancing the ubiquitin checkbook. Trends Cell Biol Nov 3. [Epub ahead of print] .Protein maturation in the endoplasmic reticulum (ER) is subject to stringent quality control. Terminally misfolded polypeptides are usually ejected into the cytoplasm and targeted for destruction by the proteasome. Ubiquitin conjugation is essential for both extraction and proteolysis. We discuss the role of the ubiquitin conjugation machinery in this pathway and focus on the role of ubiquitin ligase complexes as gatekeepers for membrane passage. We then examine the type of ubiquitin modification applied to the misfolded ER protein and the role of de-ubiquitylating enzymes in the extraction of proteins from the ER. Full Text.

Danford, T., Dowell, R., Agarwala, S., Grisafi, P., Fink, G., and Gifford, D. (2011). Discovering Regulatory Overlapping Rna Transcripts. Journal of Computational Biology 18, 295-303. STEREO is a novel algorithm that discovers cis-regulatory RNA interactions by assembling complete and potentially overlapping same-strand RNA transcripts from tiling expression data. STEREO first identifies coherent segments of transcription and then discovers individual transcripts that are consistent with the observed segments given intensity and shape constraints. We used STEREO to identify 1446 regions of overlapping transcription in two strains of yeast, including transcripts that comprise a new form of molecular toggle switch that controls gene variegation. Full Text.

Dang, M., Dubbin, K., D'Aiello, A., Hartmann, M., Lodish, H., and Herrlich, A. (2011). Egf Ligand Release by Substrate-Specific Adam Metalloproteases Involves Different Pkc Isoenzymes Depending on the Stimulu . Journal of Biological Chemistry Papers in Press. Published on March 22, 2011 The dysregulation of EGF family ligand cleavage has severe consequences for the developing as well as the adult organism. Therefore their production is highly regulated. The limiting step is the ectodomain cleavage of membrane-bound precursors by one of several ADAM metalloproteases and understanding the regulation of cleavage is an important goal of current research. We have previously reported that in mouse lung epithelial (MLE) cells, the pro-EGF ligands TGF-alpha, NRG and HB-EGF are differentially cleaved depending on the cleavage stimulus (Herrlich et al., FASEB J. (22) 2008). In our current study in mouse embryonic fibroblasts (MEFs) that lack different ADAMs. we show that induced cleavage of EGF ligands can involve the same substrate-specific metalloprotease but does require different stimulus-dependent signaling pathways. Cleavage was stimulated by phorbol ester (TPA; a mimic of diacylglycerol and PKC activator), hypertonic stress, lysophosphatidic acid (LPA)-induced GPCR activation, or by ionomycin-induced intracellular calcium release (IM). Although ADAMs showed substrate preference (ADAM17: TGFalpha, HB-EGF; ADAM9: NRG), substrate cleavage differed substantially with the stimulus and cleavage of the same substrate depended on the presence of different, sometimes multiple, PKC isoforms. For instance, classical PKC was required for TPA-induced, but not hypertonic stress-induced cleavage of all EGF family ligands. Inhibition of PKCzeta enhanced NRG release upon TPA stimulation, but blocked NRG release in response to hypertonic stress. Our results suggest a model in which substantial regulation of ectodomain cleavage occurs not only on the metalloprotease level but also on the level of the substrate or of a third protein. PDF

Dawlaty, M.M., Ganz, K., Powell, B.E., Hu, Y.C., Markoulaki, S., Cheng, A.W., Gao, Q., Kim, J., Choi, S.W., Page, D.C.and Rudolf Jaenisch. (2011). Tet1 is dispensable for maintaining pluripotency and its loss is compatible with embryonic and postnatal development. Cell Stem Cell 9, 166-175.The Tet family of enzymes (Tet1/2/3) converts 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). Mouse embryonic stem cells (mESCs) highly express Tet1 and have an elevated level of 5hmC. Tet1 has been implicated in ESC maintenance and lineage specification in vitro but its precise function in development is not well defined. To establish the role of Tet1 in pluripotency and development, we have generated Tet1 mutant mESCs and mice. Tet1−/− ESCs have reduced levels of 5hmC and subtle changes in global gene expression, and are pluripotent and support development of live-born mice in tetraploid complementation assay, but display skewed differentiation toward trophectoderm in vitro. Tet1 mutant mice are viable, fertile, and grossly normal, though some mutant mice have a slightly smaller body size at birth. Our data suggest that Tet1 loss leading to a partial reduction in 5hmC levels does not affect pluripotency in ESCs and is compatible with embryonic and postnatal development. Full Text

Delmore, J.E., Issa, G.C., Lemieux, M.E., Rahl, P.B., Shi, J.W., Jacobs, H.M., Kastritis, E., Gilpatrick, T., Paranal, R.M., Qi, J., Marta Chesi, Anna C. Schinzel, Michael R. McKeown, Timothy P. Heffernan, Christopher R. Vakoc, P. Leif Bergsagel Irene M. Ghobrial, Paul G. Richardson , Richard A. Young, William C. Hahn, Kenneth C. Anderson, Andrew L. Kung, James E. Bradner  ,and Constantine S. Mitsiades. (2011). BET Bromodomain Inhibition as a Therapeutic Strategy to Target c-Myc. Cell 146, 903-916. MYC contributes to the pathogenesis of a majority of human cancers, yet strategies to modulate the function of the c-Myc oncoprotein do not exist. Toward this objective, we have targeted MYC transcription by interfering with chromatin-dependent signal transduction to RNA polymerase, specifically by inhibiting the acetyl-lysine recognition domains (bromodomains) of putative coactivator proteins implicated in transcriptional initiation and elongation. Using a selective small-molecule bromodomain inhibitor, JQ1, we identify BET bromodomain proteins as regulatory factors for c-Myc. BET inhibition by JQ1 downregulates MYC transcription, followed by genome-wide downregulation of Myc-dependent target genes. In experimental models of multiple myeloma, a Myc-dependent hematologic malignancy, JQ1 produces a potent antiproliferative effect associated with cell-cycle arrest and cellular senescence. Efficacy of JQ1 in three murine models of multiple myeloma establishes the therapeutic rationale for BET bromodomain inhibition in this disease and other malignancies characterized by pathologic activation of c-Myc. Full Text.

DeRienzo, G., Bishop, J.A., Mao, Y., Pan, L., Ma, T.P., Moens, C.B., Tsai, L.H., and Sive, H. (2011). Disc1 regulates both {beta}-catenin-mediated and noncanonical Wnt signaling during vertebrate embryogenesis. FASEB J. Aug 22. [Epub ahead of print] Disc1 is a schizophrenia risk gene that engages multiple signaling pathways during neurogenesis and brain development. Using the zebrafish as a tool, we analyze the function of zebrafish Disc1 (zDisc1) at the earliest stages of brain and body development. We define a "tool" as a biological system that gives insight into mechanisms underlying a human disorder, although the system does not phenocopy the disorder. A zDisc1 peptide binds to GSK3beta, and zDisc1 directs early brain development and neurogenesis, by promoting beta-catenin-mediated Wnt signaling and inhibiting GSK3beta activity. zDisc1 loss-of-function embryos additionally display a convergence and extension phenotype, demonstrated by abnormal movement of dorsolateral cells during gastrulation, through changes in gene expression, and later through formation of abnormal, U-shaped muscle segments, and a truncated tail. These phenotypes are caused by alterations in the noncanonical Wnt pathway, via Daam and Rho signaling. The convergence and extension phenotype can be rescued by a dominant negative GSK3beta construct, suggesting that zDisc1 inhibits GSK3beta activity during noncanonical Wnt signaling. This is the first demonstration that Disc1 modulates the noncanonical Wnt pathway and suggests a previously unconsidered mechanism by which Disc1 may contribute to the etiology of neuropsychiatric disorders.-De Rienzo, G., Bishop, J. A., Mao, Y., Pan, L., Ma, T. P., Moens, C. B., Tsai, L. H., Sive, H. Disc1 regulates both beta-catenin-mediated and noncanonical Wnt signaling during vertebrate embryogenesis. PDF

Dougan, S.K., Hu, C.C., Paquet, M.E., Greenblatt, M.B., Kim, J., Lilley, B.N., Watson, N., and Ploegh, H.L. (2011). Derlin-2 Deficient Mouse Reveals an Essential Role for Protein Dislocation in Chondrocytes. Mol Cell Biol. Published online ahead of print on 10 January 2011 Protein quality control is a balance between chaperone-assisted folding and removal of misfolded proteins from the ER. Cell-based assays have been used to identify key players of the dislocation machinery, including members of the Derlin family. We generated a conditional knockout mouse to examine the in vivo role of Derlin-2, a component that nucleates cellular dislocation machinery. In most Derlin-2 deficient tissues, we found constitutive upregulation of ER chaperones and IRE-1 mediated induction of the unfolded protein response. The IRE-1/XBP-1 pathway is required for development of highly secretory cells, particularly plasma cells and hepatocytes. However, B lymphocyte development and antibody secretion were normal in the absence of Derlin-2. Likewise, hepatocyte function was unaffected by liver-specific deletion of Derlin-2. Whole body deletion of Derlin-2 results in perinatal death. The few mice that survive to adulthood all developed skeletal dysplasia, likely caused by defects in collagen matrix protein secretion by costal chondrocytes.PDF.

Dower, K., Rubins, K.H., Hensley, L.E., and Connor, J.H. (2011). Development of Vaccinia Reporter Viruses for Rapid, High Content Analysis of Viral Function at All Stages of Gene Expression. Antiviral Research May 5. [Epub ahead of print]. Vaccinia virus is the prototypical orthopoxvirus of Poxviridae, a family of viruses that includes the human pathogens Variola (smallpox) and Monkeypox. Core viral functions are conserved among orthopoxviruses, and consequently Vaccinia is routinely used to study poxvirus biology and screen for novel antiviral compounds. Here we describe the development of a series of fluorescent protein-based reporter Vaccinia viruses that provide unprecedented resolution for tracking viral function. The reporter viruses are divided into two sets: (1) single reporter viruses that utilize temporally regulated early, intermediate, or late viral promoters; and (2) multi-reporter viruses that utilize multiple temporally regulated promoters. Promoter and reporter combinations were chosen that yielded high signal-to-background for stage-specific viral outputs. We provide examples for how these viruses can be used in the rapid and accurate monitoring of Vaccinia function and drug action. Full Text.

Drake, A.C., Khoury, M., Leskov, I., Iliopoulou, B.P., Fragoso, M., Lodish, H., and Chen, J.Z. (2011). Human Cd34(+) Cd133(+) Hematopoietic Stem Cells Cultured with Growth Factors Including Angptl5 Efficiently Engraft Adult Nod-Scid Il2r Gamma(-/-) (Nsg) Mice. PLoS One. 2011 Apr 29;6(4):e18382. Increasing demand for human hematopoietic stem cells (HSCs) in clinical and research applications necessitates expansion of HSCs in vitro. Before these cells can be used they must be carefully evaluated to assess their stem cell activity. Here, we expanded cord blood CD34(+) CD133(+) cells in a defined medium containing angiopoietin like 5 and insulin-like growth factor binding protein 2 and evaluated the cells for stem cell activity in NOD-SCID Il2rg(-/-) (NSG) mice by multi-lineage engraftment, long term reconstitution, limiting dilution and serial reconstitution. The phenotype of expanded cells was characterized by flow cytometry during the course of expansion and following engraftment in mice. We show that the SCID repopulating activity resides in the CD34(+) CD133(+) fraction of expanded cells and that CD34(+) CD133(+) cell number correlates with SCID repopulating activity before and after culture. The expanded cells mediate long-term hematopoiesis and serial reconstitution in NSG mice. Furthermore, they efficiently reconstitute not only neonate but also adult NSG recipients, generating human blood cell populations similar to those reported in mice reconstituted with uncultured human HSCs. These findings suggest an expansion of long term HSCs in our culture and show that expression of CD34 and CD133 serves as a marker for HSC activity in human cord blood cell cultures. The ability to expand human HSCs in vitro should facilitate clinical use of HSCs and large-scale construction of humanized mice from the same donor for research applications. Full Text.

Drinnenberg, I.A., Fink, G.R., and Bartel, D.P. (2011). Compatibility with killer explains the rise of RNAi-deficient fungi. Science 333, 1592. The RNA interference (RNAi) pathway is found in most eukaryotic lineages but curiously is absent in others, including that of Saccharomyces cerevisiae. We show that reconstituting RNAi in S. cerevisiae causes loss of a beneficial double-stranded RNA virus known as killer virus. Incompatibility between RNAi and killer viruses extends to other fungal species in that RNAi is absent in all species known to possess double-stranded RNA killer viruses, whereas killer viruses are absent in closely related species that retained RNAi. Thus, the advantage imparted by acquiring and retaining killer viruses explains the persistence of RNAi-deficient species during fungal evolution. Full Text.

Elkabets, M., Gifford, A.M., Scheel, C., Nilsson, B., Reinhardt, F., Bray, M.A., Carpenter, A.E., Jirstrom, K., Magnusson, K., Ebert, B.L., Fredrik Pontén, Robert A. Weinberg, and Sandra S. McAllister (2011). Human Tumors Instigate Granulin-Expressing Hematopoietic Cells That Promote Malignancy by Activating Stromal Fibroblasts in Mice. Journal of Clinical Investigation 121, 784-799. Systemic instigation is a process by which endocrine signals sent from certain tumors (instigators) stimulate BM cells (BMCs), which are mobilized into the circulation and subsequently foster the growth of otherwise indolent carcinoma cells (responders) residing at distant anatomical sites. The identity of the BMCs and their specific contribution or contributions to responder tumor growth have been elusive. Here, we have demonstrated that Scal(+)cKit(-) hematopoietic BMCs of mouse hosts bearing instigating tumors promote the growth of responding tumors that form with a myofibroblast-rich, desmoplastic stroma. Such stroma is almost always observed in malignant human adenocarcinomas and is an indicator of poor prognosis. We then identified granulin (GRN) as the most upregulated gene in instigating Scal(+)cKit(-) BMCs relative to counterpart control cells. The GRN(+) BMCs that were recruited to the responding tumors induced resident tissue fibroblasts to express genes that promoted malignant tumor progression; indeed, treatment with recombinant GRN alone was sufficient to promote desmoplastic responding tumor growth. Further, analysis of tumor tissues from a cohort of breast cancer patients revealed that high GRN expression correlated with the most aggressive triple-negative, basal-like tumor subtype and reduced patient survival. Our data suggest that GRN and the unique hematopoietic BMCs that produce it might serve as novel therapeutic targets. Full Text.

Erlich , Y., Edvardson, S., Hodges, E., Zenvirt, S., Thekkat, P., Shaag, A., Dor, T., Hannon, G.J., and Elpeleg, O. (2011). Exome Sequencing and Disease-Network Analysis of a Single Family Implicate a Mutation in Kif1a in Hereditary Spastic Paraparesis. Genome Research Published online April 12, 2011. Whole exome sequencing has become a pivotal methodology for rapid and cost-effective detection of pathogenic variations in Mendelian disorders. A major challenge of this approach is determining the causative mutation from a substantial number of bystander variations that do not play any role in the disease etiology. Current strategies to analyze variations have mainly relied on genetic and functional arguments such as mode of inheritance, conservation, and loss of function prediction. Here, we demonstrate that disease-network analysis provides an additional layer of information to stratify variations even in the presence of incomplete sequencing coverage, a known limitation of exome sequencing. We studied a case of Hereditary Spastic Paraparesis (HSP) in a single inbred Palestinian family. HSP is a group of neuropathological disorders that are characterized by abnormal gait and spasticity of the lower limbs. Forty-five loci have been associated with HSP and lesions in 20 genes have been documented to induce the disorder. We used whole exome sequencing and homozygosity mapping to create a list of possible candidates. After exhausting the genetic and functional arguments, we stratified the remaining candidates according to their similarity to the previously known disease genes. Our analysis implicated the causative mutation in the motor domain of KIF1A, a gene that has not yet associated with HSP, which functions in anterograde axonal transportation. Our strategy can be useful for a large class of disorders that are characterized by locus heterogeneity, particularly when studying disorders in single families. PDF.

Erlich, Y. (2011). Blood Ties: Chimerism Can Mask Twin Discordance in High-Throughput Sequencing. Twin Res Hum Genet 14, 137-143. Twin studies have long provided a means to separate the contributions of genetic and environmental factors. A recent pioneering report by Baranzini et al. presented an analysis of the complete genomes and epigenomes of a monozygotic (MZ) twin pair discordant for multiple sclerosis. This failed to find any difference between the twins, raising doubts regarding the value of whole-genome twin studies for defining disease susceptibility alleles. However, the study was carried out with DNA extracted from blood. In many cases, the hematopoietic lineages of MZ twins are chimeric due to twin-to-twin exchange of hematopoietic stem cells during embryogenesis. We therefore wondered how chimerism might impact the ability to identify genetic differences. We inferred the blood chimerism rates and profiles of more than 30 discordant twin cases from a wide variety of medical conditions. We found that the genotype compositions of the twins were highly similar. We then benchmarked the performance of SNP callers to detect discordant variations using high-throughput sequencing data. Our analysis revealed that chimerism patterns, well within the range normally observed in MZ twins, greatly reduce the sensitivity of SNP calls. This raises questions regarding any conclusions of genomic homogeneity that might be drawn from studies of blood-derived twin DNA.

Ernst, R., Claessen, J.H., Mueller, B., Sanyal, S., Spooner, E., van der Veen, A.G., Kirak, O., Schlieker, C.D., Weihofen, W.A., and Ploegh, H.L. (2011). Enzymatic Blockade of the Ubiquitin-Proteasome Pathway. PLoS Biol. 2011 Mar;8(3):e1000605. Epub 2011 Mar 29. Ubiquitin-dependent processes control much of cellular physiology. We show that expression of a highly active, Epstein-Barr virus-derived deubiquitylating enzyme (EBV-DUB) blocks proteasomal degradation of cytosolic and ER-derived proteins by preemptive removal of ubiquitin from proteasome substrates, a treatment less toxic than the use of proteasome inhibitors. Recognition of misfolded proteins in the ER lumen, their dislocation to the cytosol, and degradation are usually tightly coupled but can be uncoupled by the EBV-DUB: a misfolded glycoprotein that originates in the ER accumulates in association with cytosolic chaperones as a deglycosylated intermediate. Our data underscore the necessity of a DUB activity for completion of the dislocation reaction and provide a new means of inhibition of proteasomal proteolysis with reduced cytotoxicity. Full Text.

Esteban, A., Popp, M.W., Vyas, V.K., Strijbis, K., Ploegh, H.L., and Fink, G.R. (2011). Fungal recognition is mediated by the association of dectin-1 and galectin-3 in macrophages. Proc Natl Acad Sci U S A Aug 8. [Epub ahead of print] .Dectin-1, the major beta-glucan receptor in leukocytes, triggers an effective immune response upon fungal recognition. Here we use sortase-mediated transpeptidation, a technique that allows placement of a variety of probes on a polypeptide backbone, to monitor the behavior of labeled functional dectin-1 in live cells with and without fungal challenge. Installation of probes on dectin-1 by sortagging permitted highly specific visualization of functional protein on the cell surface and its subsequent internalization upon ligand presentation. Retrieval of sortagged dectin-1 expressed in macrophages uncovered a unique interaction between dectin-1 and galectin-3 that functions in the proinflammatory response of macrophages to pathogenic fungi. When macrophages expressing dectin-1 are exposed to Candida albicans mutants with increased exposure of beta-glucan, the loss of galectin-3 dramatically accentuates the failure to trigger an appropriate TNF-alpha response. Full Text.

Form, D., and Lewitter, F. (2011). Ten Simple Rules for Teaching Bioinformatics at the High School Level. Plos Computational Biology 7(10):e1002243 Full Text.

Gao, Q., Steine, E.J., Barrasa, M.I., Hockemeyer, D., Pawlak, M., Fu, D., Reddy, S., Bell, G.W., and Jaenisch, R. (2011). Deletion of the de novo DNA methyltransferase Dnmt3a promotes lung tumor progression. Proc Natl Acad Sci U S A Oct 19. [Epub ahead of print].Alterations in DNA methylation have been associated with genome-wide hypomethylation and regional de novo methylation in numerous cancers. De novo methylation is mediated by the de novo methyltransferases Dnmt3a and 3b, but only Dnmt3b has been implicated in promoting cancer by silencing of tumor-suppressor genes. In this study, we have analyzed the role of Dnmt3a in lung cancer by using a conditional mouse tumor model. We show that Dnmt3a deficiency significantly promotes tumor growth and progression but not initiation. Changes in gene expression show that Dnmt3a deficiency affects key steps in cancer progression, such as angiogenesis, cell adhesion, and cell motion, consistent with accelerated and more malignant growth. Our results suggest that Dnmt3a may act like a tumor-suppressor gene in lung tumor progression and may be a critical determinant of lung cancer malignancy. Full Text.

Garcia, D.M., Baek, D., Shin, C., Bell, G.W., Grimson, A., and Bartel, D.P. (2011). Weak seed-pairing stability and high target-site abundance decrease the proficiency of lsy-6 and other microRNAs. Nature Struct Mol Biol. 2011 Sep 11.[Epub ahead of print] Most metazoan microRNAs (miRNAs) target many genes for repression, but the nematode lsy-6 miRNA is much less proficient. Here we show that the low proficiency of lsy-6 can be recapitulated in HeLa cells and that miR-23, a mammalian miRNA, also has low proficiency in these cells. Reporter results and array data indicate two properties of these miRNAs that impart low proficiency: their weak predicted seed-pairing stability (SPS) and their high target-site abundance (TA). These two properties also explain differential propensities of small interfering RNAs (siRNAs) to repress unintended targets. Using these insights, we expand the TargetScan tool for quantitatively predicting miRNA regulation (and siRNA off-targeting) to model differential miRNA (and siRNA) proficiencies, thereby improving prediction performance. We propose that siRNAs designed to have both weaker SPS and higher TA will have fewer off-targets without compromised on-target activity. Full Text.

Gascoigne, K.E., Takeuchi, K., Suzuki, A., Hori, T., Fukagawa, T., and Cheeseman, I.M. (2011). Induced Ectopic Kinetochore Assembly Bypasses the Requirement for Cenp-a Nucleosomes. Cell 145, 410-422. Accurate chromosome segregation requires assembly of the multiprotein kinetochore complex at centromeres. Although prior work identified the centromeric histone H3-variant CENP-A as the important upstream factor necessary for centromere specification, in human cells CENP-A is not sufficient for kinetochore assembly. Here, we demonstrate that two constitutive DNA-binding kinetochore components, CENP-C and CENP-T, function to direct kinetochore formation. Replacing the DNA-binding regions of CENP-C and CENP-T with alternate chromosome-targeting domains recruits these proteins to ectopic loci, resulting in CENP-A-independent kinetochore assembly. These ectopic kinetochore-like foci are functional based on the stoichiometric assembly of multiple kinetochore components, including the microtubule-binding KMN network, the presence of microtubule attachments, the microtubule-sensitive recruitment of the spindle checkpoint protein Mad2, and the segregation behavior of foci-containing chromosomes. We additionally find that CENP-T phosphorylation regulates the mitotic assembly of both endogenous and ectopic kinetochores. Thus, CENP-C and CENP-T form a critical regulated platform for vertebrate kinetochore assembly. Full Text.

Gavino, M.A., and Reddien, P.W. (2011). A Bmp/Admp Regulatory Circuit Controls Maintenance and Regeneration of Dorsal-Ventral Polarity in Planarians. Current Biology. 2011 Feb 22;21(4):294-9. Animal embryos have diverse anatomy and vary greatly in size. It is therefore remarkable that a common signaling pathway, BMP signaling, controls development of the dorsoventral (DV) axis throughout the Bilateria [1-8]. In vertebrates, spatially opposed expression of the BMP family proteins Bmp4 and Admp (antidorsalizing morphogenetic protein) can promote restoration of DV pattern following tissue removal [9-11]. bmp4 orthologs have been identified in all three groups of the Bilateria (deuterostomes, ecdysozoans, and lophotrochozoans) [12]. By contrast, the absence of admp orthologs in ecdysozoans such as Drosophila and C. elegans has suggested that a regulatory circuit of oppositely expressed bmp4 and admp genes represents a deuterostome-specific innovation. Here we describe the existence of spatially opposed bmp and admp expression in a protostome. An admp ortholog (Smed-admp) is expressed ventrally and laterally in adult Schmidtea mediterranea planarians, opposing the dorsal-pole expression of Smed-bmp4. Smed-admp is required for regeneration following parasagittal amputation. Furthermore, Smed-admp promotes Smed-bmp4 expression and Smed-bmp4 inhibits Smed-admp expression, generating a regulatory circuit that buffers against perturbations of Bmp signaling. These results suggest that a Bmp/Admp regulatory circuit is a central feature of the Bilateria, used broadly for the establishment, maintenance, and regeneration of the DV axis. Full Text.

Gehring, M., Missirian, V., and Henikoff, S. (2011). Genomic Analysis of Parent-of-Origin Allelic Expression in Arabidopsis thaliana Seeds PLoS One. 2011;6(8):e23687. Epub 2011 Aug 17.. .Differential expression of maternally and paternally inherited alleles of a gene is referred to as gene imprinting, a form of epigenetic gene regulation common to flowering plants and mammals. In plants, imprinting primarily occurs in the endosperm, a seed tissue that supports the embryo during its growth and development. Previously, we demonstrated that widespread DNA demethylation at remnants of transposable elements accompanies endosperm development and that a subset of these methylation changes are associated with gene imprinting. Here we assay imprinted gene expression genome-wide by performing high-throughput sequencing of RNA derived from seeds of reciprocal intraspecific crosses. We identify more than 200 loci that exhibit parent-of-origin effects on gene expression in the endosperm, including a large number of transcription factors, hormone biosynthesis and response genes, and genes that encode regulators of epigenetic information, such as methylcytosine binding proteins, histone methyltransferases, and chromatin remodelers. The majority of these genes are partially, rather than completely, imprinted, suggesting that gene dosage regulation is an important aspect of imprinted gene expression. Full Text.

Gill, M.E., Hu, Y.C., Lin, Y., and Page, D.C. (2011). Licensing of Gametogenesis, Dependent on Rna Binding Protein Dazl, as a Gateway to Sexual Differentiation of Fetal Germ Cells. Proc Natl Acad Sci U S A. Apr 19. [Epub ahead of print] Mammalian oocytes and spermatozoa derive from fetal cells shared by the sexes. These primordial germ cells (PGCs) migrate to the developing somatic gonad, giving rise to oocytes or spermatozoa. These opposing sexual fates are determined not by the PGCs' own sex chromosome constitution (XX or XY), but by the sexual identity of the fetal gonad that they enter. We asked whether PGCs undergo a developmental transition that enables them to respond to feminizing or masculinizing cues from fetal ovary or testis. We conducted in vivo genetic studies of DAZL, an RNA-binding protein expressed in both ovarian and testicular germ cells. We found that germ cells in C57BL/6 Dazl-deficient fetuses-whether XX or XY-migrate to the gonad but do not develop either male or female features. Instead, they remain in a sexually undifferentiated state similar to that of migrating PGCs. Thus, germ cells in C57BL/6 Dazl-deficient fetuses do not respond to sexual cues from ovary or testis, whereas the earlier processes of germ cell specification and migration are unaffected. We propose that PGCs of both XX and XY fetuses undergo licensing, an active developmental transition that enables the resultant gametogenesis-competent cells to respond to feminizing or masculinizing cues produced by the fetal ovary or testis and hence to embark on oogenesis or spermatogenesis. In C57BL/6 mice, Dazl is required for licensing. Licensing serves as a gateway from the embryonic processes shared between the sexes-germ cell specification and migration-to the sex-specific pathways of oogenesis and spermatogenesis. Full Text.

Golob, J.L., Kumar, R.M., Guenther, M.G., Pabon, L.M., Pratt, G.A., Loring, J.F., Laurent, L.C., Young, R.A., and Murry, C.E. (2011). Evidence That Gene Activation and Silencing during Stem Cell Differentiation Requires a Transcriptionally Paused Intermediate State. PLoS One. 2011;6(8):e22416. Epub 2011 Aug 19..A surprising portion of both mammalian and Drosophila genomes are transcriptionally paused, undergoing initiation without elongation. We tested the hypothesis that transcriptional pausing is an obligate transition state between definitive activation and silencing as human embryonic stem cells (hESCs) change state from pluripotency to mesoderm. Chromatin immunoprecipitation for trimethyl lysine 4 on histone H3 (ChIP-Chip) was used to analyze transcriptional initiation, and 3' transcript arrays were used to determine transcript elongation. Pluripotent and mesodermal cells had equivalent fractions of the genome in active and paused transcriptional states (similar to 48% each), with similar to 4% definitively silenced (neither initiation nor elongation). Differentiation to mesoderm changed the transcriptional state of 12% of the genome, with roughly equal numbers of genes moving toward activation or silencing. Interestingly, almost all loci (98-99%) changing transcriptional state do so either by entering or exiting the paused state. A majority of these transitions involve either loss of initiation, as genes specifying alternate lineages are archived, or gain of initiation, in anticipation of future full-length expression. The addition of chromatin dynamics permitted much earlier predictions of final cell fate compared to sole use of conventional transcript arrays. These findings indicate that the paused state may be the major transition state for genes changing expression during differentiation, and implicate control of transcriptional elongation as a key checkpoint in lineage specification. Full Text.

Guenther, M.G. (2011). Transcriptional control of embryonic and induced pluripotent stem cells. Epigenomics 3, 323-343.Embryonic stem cells (ESCs) have the potential to generate virtually any cell type or tissue type in the body. This remarkable plasticity has yielded great interest in using these cells to understand early development and in treating human disease. In an effort to understand the basis of ESC pluripotency, genetic and genomic studies have revealed transcriptional regulatory circuitry that maintains the pluripotent cell state and poises the genome for downstream activation. Critical components of this circuitry include ESC transcription factors, chromatin regulators, histone modifications, signaling molecules and regulatory RNAs. This article will focus on our current understanding of these components and how they influence ESC and induced pluripotent stem cell states. Emerging themes include regulation of the pluripotent genome by a core set of transcription factors, transcriptional poising of developmental genes by chromatin regulatory complexes and the establishment of multiple layers of repression at key genomic loci.

Guimaraes, C.P., Carette, J.E., Varadarajan, M., Antos, J., Popp, M.W., Spooner, E., Brummelkamp, T.R., and Ploegh, H.L. (2011). Identification of host cell factors required for intoxication through use of modified cholera toxin. J Cell Biol 195, 751-764.We describe a novel labeling strategy to site-specifically attach fluorophores, biotin, and proteins to the C terminus of the A1 subunit (CTA1) of cholera toxin (CTx) in an otherwise correctly assembled and active CTx complex. Using a biotinylated N-linked glycosylation reporter peptide attached to CTA1, we provide direct evidence that approximately 12% of the internalized CTA1 pool reaches the ER. We also explored the sortase labeling method to attach the catalytic subunit of diphtheria toxin as a toxic warhead to CTA1, thus converting CTx into a cytolethal toxin. This new toxin conjugate enabled us to conduct a genetic screen in human cells, which identified ST3GAL5, SLC35A2, B3GALT4, UGCG, and ELF4 as genes essential for CTx intoxication. The first four encode proteins involved in the synthesis of gangliosides, which are known receptors for CTx. Identification and isolation of the ST3GAL5 and SLC35A2 mutant clonal cells uncover a previously unappreciated differential contribution of gangliosides to intoxication by CTx. Full Text.

Halfmann, R., Alberti, S., Krishnan, R., Lyle, N., O'Donnell, C.W., King, O.D., Berger, B., Pappu, R.V., and Lindquist, S. (2011). Opposing Effects of Glutamine and Asparagine Govern Prion Formation by Intrinsically Disordered Proteins. Mol Cell 43, 72-84. Sequences rich in glutamine (Q) and asparagine (N) residues often fail to fold at the monomer level. This, coupled to their unusual hydrogen-bonding abilities, provides the driving force to switch between disordered monomers and amyloids. Such transitions govern processes as diverse as human protein-folding diseases, bacterial biofilm assembly, and the inheritance of yeast prions (protein-based genetic elements). A systematic survey of prion-forming domains suggested that Q and N residues have distinct effects on amyloid formation. Here, we use cell biological, biochemical, and computational techniques to compare Q/N-rich protein variants, replacing Ns with Qs and Qs with Ns. We find that the two residues have strong and opposing effects: N richness promotes assembly of benign self-templating amyloids; Q richness promotes formation of toxic nonamyloid conformers. Molecular simulations focusing on intrinsic folding differences between Qs and Ns suggest that their different behaviors are due to the enhanced turn-forming propensity of Ns over Qs. Full Text.

Hanahan, D., and Weinberg, R.A. (2011). Hallmarks of Cancer: The Next Generation. Cell 144, 646-674. The hallmarks of cancer comprise six biological capabilities acquired during the multistep development of human tumors. The hallmarks constitute an organizing principle for rationalizing the complexities of neoplastic disease. They include sustaining proliferative signaling, evading growth suppressors, resisting cell death, enabling replicative immortality, inducing angiogenesis, and activating invasion and metastasis. Underlying these hallmarks are genome instability, which generates the genetic diversity that expedites their acquisition, and inflammation, which fosters multiple hallmark functions. Conceptual progress in the last decade has added two emerging hallmarks of potential generality to this list-reprogramming of energy metabolism and evading immune destruction. In addition to cancer cells, tumors exhibit another dimension of complexity: they contain a repertoire of recruited, ostensibly normal cells that contribute to the acquisition of hallmark traits by creating the "tumor microenvironment." Recognition of the widespread applicability of these concepts will increasingly affect the development of new means to treat human cancer. Full Text.

Hattangadi, S.M., Wong, P., Zhang, L., Flygare, J., and Lodish, H.F. (2011). From stem cell to red cell: regulation of erythropoiesis at multiple levels by multiple proteins, RNAs and chromatin modifications Blood. 2011 Oct 12. [Epub ahead of print].This article reviews the regulation of production of red blood cells at several levels. We focus on the regulated expansion of BFU-E erythroid progenitors by glucocorticoids and other factors that occurs during chronic anemia, inflammation, and other conditions of stress. We also highlight the rapid production of red cells by the coordinated regulation of terminal proliferation and differentiation of committed erythroid CFU-E progenitors by external signals such as erythropoietin and adhesion to a fibronectin matrix. We discuss the complex intracellular networks of coordinated gene regulation by transcription factors, chromatin modifiers, and miRNAs that regulate the different stages of erythropoiesis. PDF

Heinrich, S.U., and Lindquist, S. (2011). Protein-Only Mechanism Induces Self-Perpetuating Changes in the Activity of Neuronal Aplysia Cytoplasmic Polyadenylation Element Binding Protein (Cpeb). Proc Natl Acad Sci U S A.Jan 26. [Epub ahead of print] Neuronal cytoplasmic polyadenylation element binding protein (CPEB) plays a critical role in maintaining the functional and morphological long-lasting synaptic changes that underlie learning and memory. It can undergo a prion switch, but it remains unclear if this self-templating change in protein conformation is alone sufficient to create a stable change in CPEB activity: a robust "protein-only" biochemical memory. To investigate, we take advantage of yeast cells wherein the neuronal CPEB of Aplysia is expressed in the absence of any neuronal factors and can stably adopt either an active or an inactive state. Reminiscent of well-characterized yeast prions, we find that CPEB can adopt several distinct activity states or "strains." These states are acquired at a much higher spontaneous rate than is typical of yeast prions, but they are extremely stable-perpetuating for years-and have all of the non-Mendelian genetic characteristics of bona fide yeast prions. CPEB levels are too low to allow direct physical characterization, but CPEB strains convert a fusion protein, which shares only the prion-like domain of CPEB, into amyloid in a strain-specific manner. Lysates of CPEB strains seed the purified prion domain to adopt the amyloid conformation with strain-specific efficiencies. Amyloid conformers generated by spontaneous assembly of the purified prion domain (and a more biochemically tractable derivative) transformed cells with inactive CPEB into the full range of distinct CPEB strains. Thus, CPEB employs a prion mechanism to create stable, finely tuned self-perpetuating biochemical memories. These biochemical memories might be used in the local homeostatic maintenance of long-term learning-related changes in synaptic morphology and function. PDF.

Hockemeyer, D., and Jaenisch, R. (2011). Gene Targeting in Human Pluripotent Cells. Cold Spring Harbor Symp Quant Biology. Published online January 5, 2011 Mouse embryonic stem cells (mESCs) have the ability to differentiate into any cell type and can generate chimeric mice when transplanted into a host blastocyst. This remarkable potential, together with the development of robust gene targeting strategies in mESCs, were essential for establishing the mouse as the most widely used model organism in biomedical research. Recent advances have allowed the isolation of human embryonic stem cells and the derivation of induced pluripotent stem cells. Genetic tools similar to those proven routine in the mouse system are needed to realize the full potential of human pluripotent cells as disease models and putative therapeutics. Gene targeting in human cells, however, has proven to be more difficult, more time-consuming, and less robust than in mESCs. In this chapter, we discuss the strategies that have been used to allow specific genetic modifications in human pluripotent cells. We focus on the novel application of custom-engineered zinc-finger nucleases for gene targeting, which has promise to become a robust tool for efficient genetic manipulation of human pluripotent cells. PDF

Hockemeyer, D., Wang, H., Kiani, S., Lai, C.S., Gao, Q., Cassady, J.P., Cost, G.J., Zhang, L., Santiago, Y., Miller, J.C. Bryan Zeitler, Jennifer M Cherone, Xiangdong Meng, Sarah J Hinkley, Edward J Rebar, Philip D Gregory, Fyodor D Urnov & Rudolf Jaenisch. (2011). Genetic Engineering of Human Pluripotent Cells Using Tale Nucleases. Nature Biotechnology .Published online 07 July 2011 Targeted genetic engineering of human pluripotent cells is a prerequisite for exploiting their full potential. Such genetic manipulations can be achieved using site-specific nucleases. Here we engineered transcription activator-like effector nucleases (TALENs) for five distinct genomic loci. At all loci tested we obtained human embryonic stem cell (ESC) and induced pluripotent stem cell (iPSC) clones carrying transgenic cassettes solely at the TALEN-specified location. Our data suggest that TALENs employing the specific architectures described here mediate site-specific genome modification in human pluripotent cells with similar efficiency and precision as do zinc-finger nucleases (ZFNs). Full Text.

Hongay, C.F., and Orr-Weaver, T.L. (2011). Drosophila Inducer of MEiosis 4 (IME4) is required for Notch signaling during oogenesis. Proc Natl Acad Sci U S A Aug 22. [Epub ahead of print] .N(6)-methyladenosine is a nonediting RNA modification found in mRNA of all eukaryotes, from yeast to humans. Although the functional significance of N(6)-methyladenosine is unknown, the Inducer of MEiosis 4 (IME4) gene of Saccharomyces cerevisiae, which encodes the enzyme that catalyzes this modification, is required for gametogenesis. Here we find that the Drosophila IME4 homolog, Dm ime4, is expressed in ovaries and testes, indicating an evolutionarily conserved function for this enzyme in gametogenesis. In contrast to yeast, but as in Arabidopsis, Dm ime4 is essential for viability. Lethality is rescued fully by a wild-type transgenic copy of Dm ime4 but not by introducing mutations shown to abrogate the catalytic activity of yeast Ime4, indicating functional conservation of the catalytic domain. The phenotypes of hypomorphic alleles of Dm ime4 that allow recovery of viable adults reveal critical functions for this gene in oogenesis. Ovarioles from Dm ime4 mutants have fused egg chambers with follicle-cell defects similar to those observed when Notch signaling is defective. Indeed, using a reporter for Notch activation, we find markedly reduced levels of Notch signaling in follicle cells of Dm ime4 mutants. This phenotype of Dm ime4 mutants is rescued by inducing expression of a constitutively activated form of Notch. Our study reveals the function of IME4 in a metazoan. In yeast, this enzyme is responsible for a crucial developmental decision, whereas in Drosophila it appears to target the conserved Notch signaling pathway, which regulates many vital aspects of metazoan development. PDF

Hsu, P.P., Kang, S.A., Rameseder, J., Zhang, Y., Ottina, K.A., Lim, D., Peterson, T.R., Choi, Y., Gray, N.S., Yaffe, M.B., Marto JA, Sabatini DM. (2011). The Mtor-Regulated Phosphoproteome Reveals a Mechanism of Mtorc1-Mediated Inhibition of Growth Factor Signaling. Science 332, 1317-1322. The mammalian target of rapamycin (mTOR) protein kinase is a master growth promoter that nucleates two complexes, mTORC1 and mTORC2. Despite the diverse processes controlled by mTOR, few substrates are known. We defined the mTOR-regulated phosphoproteome by quantitative mass spectrometry and characterized the primary sequence motif specificity of mTOR using positional scanning peptide libraries. We found that the phosphorylation response to insulin is largely mTOR dependent and that mTOR exhibits a unique preference for proline, hydrophobic, and aromatic residues at the +1 position. The adaptor protein Grb10 was identified as an mTORC1 substrate that mediates the inhibition of phosphoinositide 3-kinase typical of cells lacking tuberous sclerosis complex 2 (TSC2), a tumor suppressor and negative regulator of mTORC1. Our work clarifies how mTORC1 inhibits growth factor signaling and opens new areas of investigation in mTOR biology. Full Text.

Hu, W., Yuan, B., Flygare, J., and Lodish, H.F. (2011). Long noncoding RNA-mediated anti-apoptotic activity in murine erythroid terminal differentiation. Genes & Development Dec 15;25(24):2573-8 .Long noncoding RNAs (lncRNAs) are differentially expressed under both normal and pathological conditions, implying that they may play important biological functions. Here we examined the expression of lncRNAs during erythropoiesis and identified an erythroid-specific lncRNA with anti-apoptotic activity. Inhibition of this lncRNA blocks erythroid differentiation and promotes apoptosis. Conversely, ectopic expression of this lncRNA can inhibit apoptosis in mouse erythroid cells. This lncRNA represses expression of Pycard, a proapoptotic gene, explaining in part the inhibition of programmed cell death. These findings reveal a novel layer of regulation of cell differentiation and apoptosis by a lncRNA. Full Text.

Jaako, P., Flygare, J., Olsson, K., Quere, R., Ehinger, M., Henson, A., Ellis, S., Schambach, A., Baum, C., Richter, J.Larsson J, Bryder D ,and Karlsson S. (2011). Mice with ribosomal protein S19 deficiency develop bone marrow failure and symptoms like patients with Diamond-Blackfan anemia. Blood 118, 6087-6096.Diamond-Blackfan anemia (DBA) is a congenital erythroid hypoplasia caused by a functional haploinsufficiency of genes encoding for ribosomal proteins. Among these genes, ribosomal protein S19 (RPS19) is mutated most frequently. Generation of animal models for diseases like DBA is challenging because the phenotype is highly dependent on the level of RPS19 down-regulation. We report the generation of mouse models for RPS19-deficient DBA using transgenic RNAinterference that allows an inducible and graded down-regulation of Rps19. Rps19-deficient mice develop a macrocytic anemia together with leukocytopenia and variable platelet count that with time leads to the exhaustion of hematopoietic stem cells and bone marrow failure. Both RPS19 gene transfer and the loss of p53 rescue the DBA phenotype implying the potential of the models for testing novel therapies. This study demonstrates the feasibility of transgenic RNA interference to generate mouse models for human diseases caused by haploinsufficient expression of a gene. (Blood. 2011;118(23):6087-6096). Full Text.

Ji, P., and Lodish, H.F. (2011). Ankyrin and band 3 differentially affect expression of membrane glycoproteins but are not required for erythroblast enucleation. Biochem Biophys Res Commun Dec 27. [Epub ahead of print].During late stages of mammalian erythropoiesis the nucleus undergoes chromatin condensation, migration to the plasma membrane, and extrusion from the cytoplasm surrounded by a segment of plasma membrane. Since nuclear condensation occurs in all vertebrates, mammalian erythroid membrane and cytoskeleton proteins were implicated as playing important roles in mediating the movement and extrusion of the nucleus. Here we use erythroid ankyrin deficient and band 3 knockout mouse models to show that band 3, but not ankyrin, plays an important role in regulating the level of erythroid cell membrane proteins, as evidenced by decreased cell surface expression of glycophorin A in band 3 knockout mice. However, neither band 3 nor ankyrin are required for enucleation. These results demonstrate that mammalian erythroblast enucleation does not depend on the membrane integrity generated by the ankyrin-band 3 complex. Full Text.

Ju, S.L., Tardiff, D.F., Han, H.S., Divya, K., Zhong, Q., Maquat, L.E., Bosco, D.A., Hayward, L.J., Brown, R.H., Lindquist, S., et al. (2011). A Yeast Model of Fus/Tls-Dependent Cytotoxicity PLoS Biol. 2011 Apr;9(4):e1001052. Epub 2011 Apr 26. FUS/TLS is a nucleic acid binding protein that, when mutated, can cause a subset of familial amyotrophic lateral sclerosis (fALS). Although FUS/TLS is normally located predominantly in the nucleus, the pathogenic mutant forms of FUS/TLS traffic to, and form inclusions in, the cytoplasm of affected spinal motor neurons or glia. Here we report a yeast model of human FUS/TLS expression that recapitulates multiple salient features of the pathology of the disease-causing mutant proteins, including nuclear to cytoplasmic translocation, inclusion formation, and cytotoxicity. Protein domain analysis indicates that the carboxyl-terminus of FUS/TLS, where most of the ALS-associated mutations are clustered, is required but not sufficient for the toxicity of the protein. A genome-wide genetic screen using a yeast over-expression library identified five yeast DNA/RNA binding proteins, encoded by the yeast genes ECM32, NAM8, SBP1, SKO1, and VHR1, that rescue the toxicity of human FUS/TLS without changing its expression level, cytoplasmic translocation, or inclusion formation. Furthermore, hUPF1, a human homologue of ECM32, also rescues the toxicity of FUS/TLS in this model, validating the yeast model and implicating a possible insufficiency in RNA processing or the RNA quality control machinery in the mechanism of FUS/TLS mediated toxicity. Examination of the effect of FUS/TLS expression on the decay of selected mRNAs in yeast indicates that the nonsense-mediated decay pathway is probably not the major determinant of either toxicity or suppression. Full Text.

Khoury, M., Drake, A., Chen, Q.F., Dong, D., Leskov, I., Fragoso, M.F., Li, Y., Iliopoulou, B.P., Hwang, W., Lodish, H.F.,and Jianzhu Chen. (2011). Mesenchymal Stem Cells Secreting Angiopoietin-Like-5 Support Efficient Expansion of Human Hematopoietic Stem Cells Without Compromising Their Repopulating Potential. Stem Cells and Development 20, 1371-1381.Clinical and preclinical applications of human hematopoietic stem cells (HSCs) are often limited by scarcity of cells. Expanding human HSCs to increase their numbers while maintaining their stem cell properties has therefore become an important area of research. Here, we report a robust HSC coculture system wherein cord blood CD34(+) CD133(+) cells were cocultured with mesenchymal stem cells engineered to express angiopoietin-like-5 in a defined medium. After 11 days of culture, SCID repopulating cells were expanded similar to 60-fold by limiting dilution assay in NOD-scid Il2rg(-/-) (NSG) mice. The cultured CD34(+) CD133(+) cells had similar engraftment potential to uncultured CD34(+) CD133(+) cells in competitive repopulation assays and were capable of efficient secondary reconstitution. Further, the expanded cells supported a robust multilineage reconstitution of human blood cells in NSG recipient mice, including a more efficient T-cell reconstitution. These results demonstrate that the expanded CD34(+) CD133(+) cells maintain both short-term and long-term HSC activities. To our knowledge, this similar to 60-fold expansion of SCID repopulating cells is the best expansion of human HSCs reported to date. Further development of this coculture method for expanding human HSCs for clinical and preclinical applications is therefore warranted.

Kim, J., Lengner, C.J., Kirak, O., Hanna, J., Cassady, J.P., Lodato, M.A., Wu, S., Faddah, D.A., Steine, E.J., Gao, Q., et al. (2011). Reprogramming of Postnatal Neurons into Induced Pluripotent Stem Cells by Defined Factors. Stem Cells 29, 992-1000.Pluripotent cells can be derived from different types of somatic cells by nuclear reprogramming through the ectopic expression of four transcription factors, Oct3/4, Sox2, Klf4, and c-Myc. However, it is unclear whether postmitotic neurons are susceptible to direct reprogramming. Here, we show that postnatal cortical neurons, the vast majority of which are postmitotic, are amenable to epigenetic reprogramming. However, ectopic expression of the four canonical reprogramming factors is not sufficient to reprogram postnatal neurons. Efficient reprogramming was only achieved after forced cell proliferation by p53 suppression. Additionally, overexpression of repressor element-1 silencing transcription, a suppressor of neuronal gene activity, increased reprogramming efficiencies in combination with the reprogramming factors. Our findings indicate that terminally differentiated postnatal neurons are able to acquire the pluripotent state by direct epigenetic reprogramming, and this process is made more efficient through the suppression of lineage specific gene expression. STEM CELLS 2011;29:992-1000. Full Text.

Kim, J.C., and Orr-Weaver, T.L. (2011). Analysis of a Drosophila amplicon in follicle cells highlights the diversity of metazoan replication origins. Proc Natl Acad Sci U S ASep 20. [Epub ahead of print].To investigate the properties of metazoan replication origins, recent studies in cell culture have adopted the strategy of identifying origins using genome-wide approaches and assessing correlations with such features as transcription and histone modifications. Drosophila amplicon in follicle cells (DAFCs), genomic regions that undergo repeated rounds of DNA replication to increase DNA copy number, serve as powerful in vivo model replicons. Because there are six DAFCs, compared with thousands of origins activated in the typical S phase, close molecular characterization of all DAFCs is possible. To determine the extent to which the six DAFCs are different or similar, we investigated the developmental and replication properties of the newly identified DAFC-34B. DAFC-34B contains two genes expressed in follicle cells, although the timing and spatial patterns of expression suggest that amplification is not a strategy to promote high expression at this locus. Like the previously characterized DAFC-62D, DAFC-34B displays origin activation at two separate stages of development. However, unlike DAFC-62D, amplification at the later stage is not transcription-dependent. We mapped the DAFC-34B amplification origin to 1 kb by nascent strand analysis and delineated cis requirements for origin activation, finding that a 6-kb region, but not the 1-kb origin alone, is sufficient for amplification. We analyzed the developmental localization of the origin recognition complex (ORC) and the minichromosome maintenance (MCM)2-7 complex, the replicative helicase. Intriguingly, the final round of origin activation at DAFC-34B occurs in the absence of detectable ORC, although MCMs are present, suggesting a new amplification initiation mechanism. PDF

Kim, J.C., Nordman, J., Xie, F., Kashevsky, H., Eng, T., Li, S., Macalpine, D.M., and Orr-Weaver, T.L. (2011). Integrative Analysis of Gene Amplification in Drosophila Follicle Cells: Parameters of Origin Activation and Repression. Genes Dev 25, 1384-1398. In metazoans, how replication origins are specified and subsequently activated is not well understood. Drosophila amplicons in follicle cells (DAFCs) are genomic regions that undergo rereplication to increase DNA copy number. We identified all DAFCs by comparative genomic hybridization, uncovering two new amplicons in addition to four known previously. The complete identification of all DAFCs enabled us to investigate these in vivo replicons with respect to parameters of transcription, localization of the origin recognition complex (ORC), and histone acetylation, yielding important insights into gene amplification as a metazoan replication model. Significantly, ORC is bound across domains spanning 10 or more kilobases at the DAFC rather than at a specific site. Additionally, ORC is bound at many regions that do not undergo amplification, and, in contrast to cell culture, these regions do not correlate with high gene expression. As a developmental strategy, gene amplification is not the predominant means of achieving high expression levels, even in cells capable of amplification. Intriguingly, we found that, in some strains, a new amplicon, DAFC-22B, does not amplify, a consequence of distant repression of ORC binding and origin activation. This repression is alleviated when a fragment containing the origin is placed in different genomic contexts. Full Text.

Kim, J., Su, S.C., Wang, H., Cheng, A.W., Cassady, J.P., Lodato, M.A., Lengner, C.J., Chung, C.Y., Dawlaty, M.M., Tsai, L.H.and R. Jaenisch. (2011). Functional Integration of Dopaminergic Neurons Directly Converted from Mouse Fibroblasts. Cell Stem Cell.Oct 19. [Epub ahead of print] Recent advances in somatic cell reprogramming have highlighted the plasticity of the somatic epigenome, particularly through demonstrations of direct lineage reprogramming of one somatic cell type to another by defined factors. However, it is not clear to what extent this type of reprogramming is able to generate fully functional differentiated cells. In addition, the activity of the reprogrammed cells in cell transplantation assays, such as those envisaged for cell-based therapy of Parkinson's disease (PD), remains to be determined. Here we show that ectopic expression of defined transcription factors in mouse tail tip fibroblasts is sufficient to induce Pitx3+ neurons that closely resemble midbrain dopaminergic (DA) neurons. In addition, transplantation of these induced DA (iDA) neurons alleviates symptoms in a mouse model of PD. Thus, iDA neurons generated from abundant somatic fibroblasts by direct lineage reprogramming hold promise for modeling neurodegenerative disease and for cell-based therapies of PD. Full Text.

Kim, J., Lengner, C.J., Kirak, O., Hanna, J., Cassady, J.P., Lodato, M.A., Wu, S., Faddah, D.A., Steine, E.J., Gao, Q.Fu D, Dawlaty M ,and Jaenisch R.. (2011). Reprogramming of Postnatal Neurons into Induced Pluripotent Stem Cells by Defined Factors. Stem Cells 29 (6) : 992-1000. Pluripotent cells can be derived from different types of somatic cells by nuclear reprogramming through the ectopic expression of four transcription factors, Oct3/4, Sox2, Klf4, and c-Myc. It is however unclear whether post-mitotic neurons are susceptible to direct reprogramming. Here we show that postnatal cortical neurons, the vast majority of which are post-mitotic, are amenable to epigenetic reprogramming. However, ectopic expression of the four canonical reprogramming factors is not sufficient to reprogram postnatal neurons. Efficient reprogramming was only achieved after forced cell proliferation by p53 suppression. Additionally, overexpression of REST, a suppressor of neuronal gene activity, increased reprogramming efficiencies in combination with the reprogramming factors. Our findings indicate that terminally differentiated postnatal neurons are able to acquire the pluripotent state by direct epigenetic reprogramming, and this process is made more efficient through the suppression of lineage specific gene expression. Full Text.

Klemm, E.J., Spooner, E., and Ploegh, H.L. (2011). The dual role of ancient ubiquitous protein 1 (AUP1) in lipid droplet accumulation and ER protein quality control. J Biol Chem Aug 20. [Epub ahead of print] .Quality control of endoplasmic reticulum proteins involves the identification and engagement of misfolded proteins, dislocation of the misfolded protein across the ER membrane, and ubiquitin-mediated targeting to the proteasome for degradation. Ancient ubiquitous protein 1 (AUP1) physically associates with the mammalian HRD1/SEl1L complex and AUP1-depletion impairs degradation of misfolded ER proteins. One of the functions of AUP1 in ER quality control is to recruit the soluble E2-ubiquitin conjugating enzyme UBE2G2. We further show that the CUE domain of AUP1 regulates polyubiquitylation and determines AUP1s interaction with the HRD1 complex and dislocation substrates. AUP1 localizes both to the ER and to lipid droplets. AUP1 expression level affects the abundance of cellular lipid droplets and as such represents the first protein with lipid droplet regulatory activity to be linked to ER quality control. These findings indicate a possible connection between ER protein quality control and lipid droplets. PDF

Koldobskaya, Y., Duguid, E.M., Shechner, D.M., Suslov, N.B., Ye, J.D., Sidhu, S.S., Bartel, D.P., Koide, S., Kossiakoff, A.A., and Piccirilli, J.A. (2011). A Portable Rna Sequence Whose Recognition by a Synthetic Antibody Facilitates Structural Determination. Nature Structural & Molecular Biology 18, 100-106. RNA crystallization and phasing represent major bottlenecks in RNA structure determination. Seeking to exploit antibody fragments as RNA crystallization chaperones, we have used an arginine-enriched synthetic Fab library displayed on phage to obtain Fabs against the class I ligase ribozyme. We solved the structure of a Fab-ligase complex at 3.1-angstrom resolution using molecular replacement with Fab coordinates, confirming the ribozyme architecture and revealing the chaperone's role in RNA recognition and crystal contacts. The epitope resides in the GAAACAC sequence that caps the P5 helix, and this sequence retains high-affinity Fab binding within the context of other structured RNAs. This portable epitope provides a new RNA crystallization chaperone system that easily can be screened in parallel to the U1A RNA-binding protein, with the advantages of a smaller loop and Fabs' high molecular weight, large surface area and phasing power. Full Text.

Kronja, I., and Orr-Weaver, T.L. (2011). Translational regulation of the cell cycle: when, where, how and why? Philos Trans R Soc Lond B Biol Sci 366, 3638-3652.Translational regulation contributes to the control of archetypal and specialized cell cycles, such as the meiotic and early embryonic cycles. Late meiosis and early embryogenesis unfold in the absence of transcription, so they particularly rely on translational repression and activation of stored maternal mRNAs. Here, we present examples of cell cycle regulators that are translationally controlled during different cell cycle and developmental transitions in model organisms ranging from yeast to mouse. Our focus also is on the RNA-binding proteins that affect cell cycle progression by recognizing special features in untranslated regions of mRNAs. Recent research highlights the significance of the cytoplasmic polyadenylation element-binding protein (CPEB). CPEB determines polyadenylation status, and consequently translational efficiency, of its target mRNAs in both transcriptionally active somatic cells as well as in transcriptionally silent mature Xenopus oocytes and early embryos. We discuss the role of CPEB in mediating the translational timing and in some cases spindle-localized translation of critical regulators of Xenopus oogenesis and early embryogenesis. We conclude by outlining potential directions and approaches that may provide further insights into the translational control of the cell cycle. Full Text.

Kuchenbauer, F., Mah, S.M., Heuser, M., McPherson, A., Ruschmann, J., Rouhi, A., Berg, T., Bullinger, L., Argiropoulos, B., Morin, R.D.David LaiDaniel T. Starczynowski,Aly Karsan,Connie J. Eaves,Akira Watahiki,Yuzhuo Wang,Samuel A. Aparicio,Arnold Ganser,Jürgen Krauter,Hartmut Döhner,Konstanze Döhner,Marco A. Marr ,Fernando D Camargo,Lars Palmqvist,9Christian Buske, and R. Keith Humphriesl. (2011). Comprehensive analysis of mammalian miRNA* species and their role in myeloid cells. Blood 118, 3350-3358.Processing of pre-miRNA through Dicer1 generates an miRNA duplex that consists of an miRNA and miRNA* strand. Despite the general view that miRNA*s have no functional role, we further investigated miRNA* species in 10 deep-sequencing libraries from mouse and human tissue. Comparisons of miRNA/miRNA* ratios across the miRNA sequence libraries revealed that 50% of the investigated miRNA duplexes exhibited a highly dominant strand. Conversely, 10% of miRNA duplexes showed a comparable expression of both strands, whereas the remaining 40% exhibited variable ratios across the examined libraries, as exemplified by miR-223/miR-223* in murine and human cell lines. Functional analyses revealed a regulatory role for miR-223* in myeloid progenitor cells, which implies an active role for both arms of the miR-223 duplex. This was further underscored by the demonstration that miR-223 and miR-223* targeted the insulin-like growth factor 1 receptor/phosphatidylinositol 3-kinase axis and that high miR-223* levels were associated with increased overall survival in patients with acute myeloid leukemia. Thus, we found a supporting role for miR-223* in differentiating myeloid cells in normal and leukemic cell states. The fact that the miR-223 duplex acts through both arms extends the complexity of miRNA-directed gene regulation of this myeloid key miRNA. (Blood. 2011;118(12):3350-3358). Full Text.

Lamming, D.W., and Sabatini, D.M. (2011). A Radical Role for Tor in Longevity. Cell Metab 13, 617-618. TOR (target of rapamycin) signaling regulates life span in many organisms, but the mechanism behind the effect is unknown. In this issue of Cell Metabolism, Pan and colleagues (2011) find that reduced TORC1 activity promotes yeast life span via a mechanism that, paradoxically, relies upon the production of normally deleterious reactive oxygen species. Full Text.

Lan, A., Smoly, I.Y., Rapaport, G., Lindquist, S., Fraenkel, E., and Yeger-Lotem, E. (2011). Responsenet: Revealing Signaling and Regulatory Networks Linking Genetic and Transcriptomic Screening Data. Nucleic Acids Research 39, W424-W429. Cellular response to stimuli is typically complex and involves both regulatory and metabolic processes. Large-scale experimental efforts to identify components of these processes often comprise of genetic screening and transcriptomic profiling assays. We previously established that in yeast genetic screens tend to identify response regulators, while transcriptomic profiling assays tend to identify components of metabolic processes. ResponseNet is a network-optimization approach that integrates the results from these assays with data of known molecular interactions. Specifically, ResponseNet identifies a high-probability sub-network, composed of signaling and regulatory molecular interaction paths, through which putative response regulators may lead to the measured transcriptomic changes. Computationally, this is achieved by formulating a minimum-cost flow optimization problem and solving it efficiently using linear programming tools. The ResponseNet web server offers a simple interface for applying ResponseNet. Users can upload weighted lists of proteins and genes and obtain a sparse, weighted, molecular interaction sub-network connecting their data. The predicted sub-network and its gene ontology enrichment analysis are presented graphically or as text. Consequently, the ResponseNet web server enables researchers that were previously limited to separate analysis of their distinct, large-scale experiments, to meaningfully integrate their data and substantially expand their understanding of the underlying cellular response. ResponseNet is available at http://bioinfo.bgu.ac.il/respnet. Full Text.

Lapan, S.W., and Reddien, P.W. (2011). dlx and sp6-9 Control Optic Cup Regeneration in a Prototypic Eye. PLoS Genet 7, e1002226 .Optic cups are a structural feature of diverse eyes, from simple pit eyes to camera eyes of vertebrates and cephalopods. We used the planarian prototypic eye as a model to study the genetic control of optic cup formation and regeneration. We identified two genes encoding transcription factors, sp6-9 and dlx, that were expressed in the eye specifically in the optic cup and not the photoreceptor neurons. RNAi of these genes prevented formation of visible optic cups during regeneration. Planarian regeneration requires an adult proliferative cell population with stem cell-like properties called the neoblasts. We found that optic cup formation occurred only after migration of progressively differentiating progenitor cells from the neoblast population. The eye regeneration defect caused by dlx and sp6-9 RNAi can be explained by a failure to generate these early optic cup progenitors. Dlx and Sp6-9 genes function as a module during the development of diverse animal appendages, including vertebrate and insect limbs. Our work reveals a novel function for this gene pair in the development of a fundamental eye component, and it utilizes these genes to demonstrate a mechanism for total organ regeneration in which extensive cell movement separates new cell specification from organ morphogenesis. Full Text.

Laurent, L.C., Ulitsky, I., Slavin, I., Tran, H., Schork, A., Morey, R., Lynch, C., Harness, J.V., Lee, S., Barrero, M.J., et al. (2011). Dynamic Changes in the Copy Number of Pluripotency and Cell Proliferation Genes in Human Escs and Ipscs During Reprogramming and Time in Culture. Cell Stem Cell 8, 106-118. Genomic stability is critical for the clinical use of human embryonic and induced pluripotent stem cells. We performed high-resolution SNP (single-nucleotide polymorphism) analysis on 186 pluripotent and 119 nonpluripotent samples. We report a higher frequency of subchromosomal copy number variations in pluripotent samples compared to nonpluripotent samples, with variations enriched in specific genomic regions. The distribution of these variations differed between hESCs and hiPSCs, characterized by large numbers of duplications found in a few hESC samples and moderate numbers of deletions distributed across many hiPSC samples. For hiPSCs, the reprogramming process was associated with deletions of tumor-suppressor genes, whereas time in culture was associated with duplications of oncogenic genes. We also observed duplications that arose during a differentiation protocol. Our results illustrate the dynamic nature of genomic abnormalities in pluripotent stem cells and the need for frequent genomic monitoring to assure phenotypic stability and clinical safety. Full Text.

Le, M.T.N., Shyh-Chang, N., Khaw, S.L., Chin, L.Z., Teh, C., Tay, J., O'Day, E., Korzh, V., Yang, H., Lal, A.,Lieberman J, Lodish HF, Lim B. (2011). Conserved Regulation of p53 Network Dosage by MicroRNA-125b Occurs through Evolving miRNA-Target Gene Pairs. Plos Genetics Sep;7(9):e1002242.MicroRNAs regulate networks of genes to orchestrate cellular functions. MiR-125b, the vertebrate homologue of the Caenorhabditis elegans microRNA lin-4, has been implicated in the regulation of neural and hematopoietic stem cell homeostasis, analogous to how lin-4 regulates stem cells in C. elegans. Depending on the cell context, miR-125b has been proposed to regulate both apoptosis and proliferation. Because the p53 network is a central regulator of both apoptosis and proliferation, the dual roles of miR-125b raise the question of what genes in the p53 network might be regulated by miR-125b. By using a gain-and loss-of-function screen for miR-125b targets in humans, mice, and zebrafish and by validating these targets with the luciferase assay and a novel miRNA pull-down assay, we demonstrate that miR-125b directly represses 20 novel targets in the p53 network. These targets include both apoptosis regulators like Bak1, Igfbp3, Itch, Puma, Prkra, Tp53inp1, Tp53, Zac1, and also cell-cycle regulators like cyclin C, Cdc25c, Cdkn2c, Edn1, Ppp1ca, Sel1l, in the p53 network. We found that, although each miRNA-target pair was seldom conserved, miR-125b regulation of the p53 pathway is conserved at the network level. Our results lead us to propose that miR-125b buffers and fine-tunes p53 network activity by regulating the dose of both proliferative and apoptotic regulators, with implications for tissue stem cell homeostasis and oncogenesis. Full Text.

Lewitter, F., and Bourne, P.E. (2011). Teaching Bioinformatics at the Secondary School Level. PLoS Comput Biol 7, e1002242 . We now introduce a subsection of the Education section of PLoS Computational Biology with articles devoted to teaching bioinformatics in secondary schools that is derived from the work of the Education committee of the International Society for Computational Biology (ISCB), who identified a need to address the issue of incorporating bioinformatics into secondary school biology classes. They also recognized the interest among researchers to build and participate in outreach programs at the secondary school level given that many funding agencies worldwide encourage such a component in grant applications. Full Text.

Lien, W.H., Guo, X.Y., Polak, L., Lawton, L.N., Young, R.A., Zheng, D.Y., and Fuchs, E. (2011). Genome-wide Maps of Histone Modifications Unwind In Vivo Chromatin States of the Hair Follicle Lineage. Cell Stem Cell 9, 219-232.Using mouse skin, where bountiful reservoirs of synchronized hair follicle stem cells (HF-SCs) fuel cycles of regeneration, we explore how adult SCs remodel chromatin in response to activating cues. By profiling global mRNA and chromatin changes in quiescent and activated HF-SCs and their committed, transit-amplifying (TA) progeny, we show that polycomb-group (PcG)-mediated H3K27-trimethylation features prominently in HF-lineage progression by mechanisms distinct from embryonic-SCs. In HF-SCs, PcG represses nonskin lineages and HF differentiation. In TA progeny, nonskin regulators remain PcG-repressed, HF-SC regulators acquire H3K27me3-marks, and HF-lineage regulators lose them. Interestingly, genes poised in embryonic stem cells, active in HF-SCs, and PcG-repressed in TA progeny encode not only key transcription factors, but also signaling regulators. We document their importance in balancing HF-SC quiescence, underscoring the power of chromatin mapping in dissecting SC behavior. Our findings explain how HF-SCs cycle through quiescent and activated states without losing stemness and define roles for PcG-mediated repression in governing a fate switch irreversibly. Full Text.

Lindquist, S.L., and Kelly, J.W. (2011). Chemical and Biological Approaches for Adapting Proteostasis to Ameliorate Protein Misfolding and Aggregation Diseases-Progress and Prognosis Cold Spring Harbor Perspect Biology 2011 Sep 7.Maintaining the proteome to preserve the health of an organism in the face of developmental changes, environmental insults, infectious diseases, and rigors of aging is a formidable task. The challenge is magnified by the inheritance of mutations that render individual proteins subject to misfolding and/or aggregation. Maintenance of the proteome requires the orchestration of protein synthesis, folding, degradation, and trafficking by highly conserved/deeply integrated cellular networks. In humans, no less than 2000 genes are involved. Stress sensors detect the misfolding and aggregation of proteins in specific organelles and respond by activating stress-responsive signaling pathways. These culminate in transcriptional and posttranscriptional programs that up-regulate the homeostatic mechanisms unique to that organelle. Proteostasis is also strongly influenced by the general properties of protein folding that are intrinsic to every proteome. These include the kinetics and thermodynamics of the folding, misfolding, and aggregation of individual proteins. We examine a growing body of evidence establishing that when cellular proteostasis goes awry, it can be reestablished by deliberate chemical and biological interventions. We start with approaches that employ chemicals or biological agents to enhance the general capacity of the proteostasis network. We then introduce chemical approaches to prevent the misfolding or aggregation of specific proteins through direct binding interactions. We finish with evidence that synergy is achieved with the combination of mechanistically distinct approaches to reestablish organismal proteostasis

Lindquist, R.A., Ottina, K.A., Wheeler, D.B., Hsu, P.P., Thoreen, C.C., Guertin, D.A., Ali, S.M., Sengupta, S., Shaul, Y.D., Lamprecht, M.R., Katherine L. Madden, Adam R. Papallo,Thouis R. Jones, David M. Sabatini, and Anne E. Carpenter (2011). Genome-Scale Rnai on Living-Cell Microarrays Identifies Novel Regulators of Drosophila Melanogaster Torc1-S6k Pathway Signaling. Genome Research. Jan 14. [Epub ahead of print] The evolutionarily conserved target of rapamycin complex 1 (TORC1) controls cell growth in response to nutrient availability and growth factors. TORC1 signaling is hyperactive in cancer, and regulators of TORC1 signaling may represent therapeutic targets for human diseases. To identify novel regulators of TORC1 signaling, we performed a genome-scale RNA interference screen on microarrays of Drosophila melanogaster cells expressing human RPS6, a TORC1 effector whose phosphorylated form we detected by immunofluorescence. Our screen revealed that the TORC1-S6K-RPS6 signaling axis is regulated by many subcellular components, including the Class I vesicle coat (COPI), the spliceosome, the proteasome, the nuclear pore, and the translation initiation machinery. Using additional RNAi reagents and Western blotting, we confirmed 70 novel genes as significant on-target regulators of RPS6 phosphorylation, and we characterized them with extensive secondary assays probing various arms of the TORC1 pathways, identifying functional relationships among those genes. We conclude that cell-based microarrays are a useful platform for genome-scale and secondary screening in Drosophila, revealing regulators that may represent drug targets for cancers and other diseases of deregulated TORC1 signaling. PDF

Liu, Q.S., Wang, J.H., Kang, S.A., Thoreen, C.C., Hur, W., Choi, H.G., Waller, D.L., Sim, T., Sabatini, D.M., and Gray, N.S. (2011). Discovery and Optimization of Potent and Selective Benzonaphthyridinone Analogs as Small Molecule Mtor Inhibitors with Improved Mouse Microsome Stability. Bioorganic & Medicinal Chemistry Letters 21, 4036-4040. Starting from small molecule mTOR inhibitor Torin1, replacement of the piperazine ring with a phenyl ring resulted in a new series of mTOR inhibitors (as exemplified by 10) that showed superior potency and selectivity for mTOR, along with significantly improved mouse liver microsome stability and a longer in vivo half-life. Full Text

Liu, Q.S., Wang, J.H., Kang, S.A., Thoreen, C.C., Hur, W., Ahmed, T., Sabatini, D.M., and Gray, N.S. (2011). Discovery of 9-(6-Aminopyridin-3-Yl)-1-(3-(Trifluoromethyl)-Phenyl)Benzo[H][1,6]Napht Hyridin-2(1h)-One (Torin2) as a Potent, Selective, and Orally Available Mammalian Target of Raparnycin (Mtor) Inhibitor for Treatment of Cancer. Journal of Medicinal Chemistry 54, 1473-1480. The mTOR mediated PI3K/AKT/mTOR signal transduction pathway has been demonstrated to play a key role in a broad spectrum of cancers. Starting from the mTOR selective inhibitor 1 (Torin1), a focused medicinal chemistry effort led to the discovery of an improved mTOR inhibitor 3 (Torin2), which possesses an EC50 of 0.25 nM for inhibiting cellular mTOR activity. Compound 3 exhibited 800-fold selectivity over PI3K (EC50: 200 nM) and over 100-fold binding selectivity relative to 440 other protein kinases. Compound 3 has significantly improved bioavailability (54%), metabolic stability, and plasma exposure relative to compound 1. Full Text

Major, T., Menon, J., Auyeung, G., Soldner, F., Hockemeyer, D., Jaenisch, R., and Tabar, V. (2011). Transgene Excision Has No Impact on In Vivo Integration of Human iPS Derived Neural Precursors. PLoS One 6(9):e24687.The derivation of induced human pluripotent stem cells (hiPS) has generated significant enthusiasm particularly for the prospects of cell-based therapy. But there are concerns about the suitability of iPS cells for in vivo applications due in part to the introduction of potentially oncogenic transcription factors via viral vectors. Recently developed lentiviral vectors allow the excision of viral reprogramming factors and the development of transgene-free iPS lines. However it is unclear if reprogramming strategy has an impact on the differentiation potential and the in vivo behavior of hiPS progeny. Here we subject viral factor-free, c-myc-free and conventionally reprogrammed four-factor human iPS lines to a further challenge, by analyzing their differentiation potential along the 3 neural lineages and over extended periods of time in vitro, as well as by interrogating their ability to respond to local environmental cues by grafting into the striatum. We demonstrate similar and efficient differentiation into neurons, astrocytes and oligodendrocytes among all hiPS and human ES line controls. Upon intracranial grafting in the normal rat (Sprague Dawley), precursors derived from all hiPS lines exhibited good survival and response to environmental cues by integrating into the subventricular zone, acquiring phenotypes typical of type A, B or C cells and migrating along the rostral migratory stream into the olfactory bulb. There was no teratoma or other tumor formation 12 weeks after grafting in any of the 26 animals used in the study. Thus neither factor excision nor persistence of c-myc impact the behavior of hiPS lines in vivo. Full Text.

Mashayekhi, M., Sandau, M.M., Dunay, I.R., Frickel, E.M., Khan, A., Goldszmid, R.S., Sher, A., Ploegh, H.L., Murphy, T.L., Sibley, L.D.,and Murphy KM(2011). CD8 alpha(+) Dendritic Cells Are the Critical Source of Interleukin-12 that Controls Acute Infection by Toxoplasma gondii Tachyzoites. Immunity 35, 249-259.CD8 alpha(+) dendritic cells (DCs) are important in vivo for cross-presentation of antigens derived from intracellular pathogens and tumors. Additionally, secretion of interleukin-12 (IL-12) by CD8 alpha(+) DCs suggests a role for these cells in response to Toxoplasma gondii antigens, although it remains unclear whether these cells are required for protection against T. gondii infection. Toward this goal, we examined T. gondii infection of Batf3(-/-) mice, which selectively lack only lymphoid-resident CD8 alpha(+) DCs and related peripheral CD103(+) DCs. Batf3(-/-) mice were extremely susceptible to T. gondii infection, with decreased production of IL-12 and interferon-gamma. IL-12 administration restored resistance in Batf3(-/-) mice, and mice in which IL-12 production was ablated only from CD8 alpha(+) DCs failed to control infection. These results reveal that the function of CD8 alpha(+) DCs extends beyond a role in cross-presentation and includes a critical role for activation of innate immunity through IL-12 production during T. gondii infection. Full Text.

Mathews, D.J.H., Graff, G.D., Saha, K., and Winickoff, D.E. (2011). Access to Stem Cells and Data: Persons, Property Rights, and Scientific Progress. Science 331, 725-727. Many fields have struggled to develop strategies, policies, or structures to optimally manage data, materials, and intellectual property rights (IPRs). There is growing recognition that the field of stem cell science, in part because of its complex IPRs landscape and the importance of cell line collections, may require collective action to facilitate basic and translational research. Access to pluripotent stem cell lines and the information associated with them is critical to the progress of stem cell science, but simple notions of access are substantially complicated by shifting boundaries between what is considered information versus material, person versus artifact, and private property versus the public domain. Full Text.

Mazzoni, E.O., Mahony, S., Iacovino, M., Morrison, C.A., Mountoufaris, G., Closser, M., Whyte, W.A., Young, R.A., Kyba, M., Gifford, D.K,and Wichterle H.. (2011). Embryonic stem cell-based mapping of developmental transcriptional programs. Nature Methods 8, 1056-8.The study of developmentally regulated transcription factors by chromatin immunoprecipitation and deep sequencing (ChIP-seq) faces two major obstacles: availability of ChIP-grade antibodies and access to sufficient number of cells. We describe versatile genome-wide analysis of transcription-factor binding sites by combining directed differentiation of embryonic stem cells and inducible expression of tagged proteins. We demonstrate its utility by mapping DNADNADNA-binding sites of transcription factors involved in motor neuron specification. Full Text.

McGehee, A.M., Strijbis, K., Guillen, E., Eng, T., Kirak, O., and Ploegh, H.L. (2011). Ubiquitin-Dependent Control of Class Ii Mhc Localization Is Dispensable for Antigen Presentation and Antibody Production. PLoS One 6(4), e18817. Controlled localization of class II MHC molecules is essential for proper class II MHC-restricted antigen presentation and the subsequent initiation of an adaptive immune response. Ubiquitination of class II MHC molecules on cytosolic lysine (K225) of the beta-chain has been shown to affect localization of the complex. We generated mice in which the endogenous beta-chain locus is replaced with a GFP tagged mutant version that lacks the cytosolic lysine residue (I-A-beta-K225R-EGFP). These mice have elevated levels of class II MHC as compared to I-A-beta-EGFP mice, and immature bone marrow-derived dendritic cells show redistribution of class II MHC to the cell surface. Nonetheless, in these same cells efficiency of antigen presentation is unaffected in I-A-beta-K225R-EGFP mice, as assayed for presentation of ovalbumin to appropriately specific T cells. The I-A-beta-K225R-EGFP animals have normal CD4 T cell populations and are capable of generating antigen-specific antibody in response to model antigens and viral infection. We therefore conclude that in our experimental system modulation of trafficking by ubiquitination of residue K225 of the beta-chain is not essential for the function of class II MHC products in antigen presentation or antibody production. Full Text.

Medeiros, L.A., Dennis, L.M., Gill, M.E., Houbaviy, H., Markoulaki, S., Fu, D., White, A.C., Kirak, O., Sharp, P.A., Page, D.C.,and Rudolf Jaenisch. (2011). Mir-290-295 deficiency in mice results in partially penetrant embryonic lethality and germ cell defects. Proc Natl Acad Sci U S A Aug 15. [Epub ahead of print] Mir-290 through mir-295 (mir-290-295) is a mammalian-specific microRNA (miRNA) cluster that, in mice, is expressed specifically in early embryos and embryonic germ cells. Here, we show that mir-290-295 plays important roles in embryonic development as indicated by the partially penetrant lethality of mutant embryos. In addition, we show that in surviving mir-290-295-deficient embryos, female but not male fertility is compromised. This impairment in fertility arises from a defect in migrating primordial germ cells and occurs equally in male and female mutant animals. Male mir-290-295(-/-) mice, due to the extended proliferative lifespan of their germ cells, are able to recover from this initial germ cell loss and are fertile. Female mir-290-295(-/-) mice are unable to recover and are sterile, due to premature ovarian failure. Full Text.

Mullen, David A., Orlando, Jamie J. Newman, Jakob Lovén, Roshan M. Kumar, Steve Bilodeau, Jessica Reddy, Matthew G. Guenther, Rodney P. DeKoter ,and Richard A. Young  (2011) Master Transcription Factors Determine Cell-Type-Specific Responses to TGF-β Signaling. Cell Volume 147, Issue 3, 28 October 2011, Pages 565-576. Transforming growth factor beta (TGF-β) signaling, mediated through the transcription factors Smad2 and Smad3 (Smad2/3), directs different responses in different cell types. Here we report that Smad3 co-occupies the genome with cell-type-specific master transcription factors. Thus, Smad3 occupies the genome with Oct4 in embryonic stem cells (ESCs), Myod1 in myotubes, and PU.1 in pro-B cells. We find that these master transcription factors are required for Smad3 occupancy and that TGF-β signaling largely affects the genes bound by the master transcription factors. Furthermore, we show that induction of Myod1 in nonmuscle cells is sufficient to redirect Smad3 to Myod1 sites. We conclude that cell-type-specific master transcription factors determine the genes bound by Smad2/3 and are thus responsible for orchestrating the cell-type-specific effects of TGF-β. Full Text

Newman, J.J., and Young, R.A. (2011). Connecting Transcriptional Control to Chromosome Structure and Human Disease. Cold Spring Harbor Symp Quant Biology Published online January 5, 2011 We review key insights into transcriptional regulation of cell state that have emerged from the study of embryonic stem cells. These insights are described in the context of historical studies of the roles of transcription factors, signal transduction pathways, and regulators of chromatin structure. We highlight recent studies that have led to the model that mediator and cohesin physically and functionally connect the enhancers and core promoters of a key subset of active genes in cells, thus generating cell-type-specific DNA loops linked to the gene-expression program of each cell. Mutations in the genes encoding mediator and cohesin components can cause an array of human developmental syndromes and diseases, and we discuss the implications of these findings for the mechanisms involved in these diseases.PDF.

Nodine, M.D., Bryan, A.C., Racolta, A., Jerosky, K.V., and Tax, F.E. (2011). A Few Standing for Many: Embryo Receptor-Like Kinases. Trends in Plant Science.Feb 22. [Epub ahead of print] Development of plant embryos is a complex and highly organized process, and experimental evidence indicates that intercellular signaling plays a major role. The recent identification of Receptor-Like Kinases (RLKs) and related Receptor-Like Cytoplasmic Kinases (RLCKs) with specific roles in Arabidopsis thaliana embryo development suggest important functions of intercellular signaling during embryogenesis. Despite the characterization of only a few RLKs and RLCKs with embryonic roles, expression data indicate that many RLKs and RLCKs with either post-embryonic functions or unknown functions are transcribed in Arabidopsis embryos. The functional characterization of a few members of this large kinase family is likely to represent only the tip of the iceberg, and we predict that many RLKs and RLCKs play major roles throughout embryo development.

Novershtern, N., Subramanian, A., Lawton, L.N., Mak, R.H., Haining, W.N., McConkey, M.E., Habib, N., Yosef, N., Chang, C.Y., Shay, T., Frampton GM, Drake AC, Leskov I, Nilsson B, Preffer F, Dombkowski D, Evans JW, Liefeld T, Smutko JS, Chen J, Friedman N, Young RA, Golub TR, Regev A, and Ebert BL. (2011). Densely Interconnected Transcriptional Circuits Control Cell States in Human Hematopoiesis. Cell 144, 296-309. Though many individual transcription factors are known to regulate hematopoietic differentiation, major aspects of the global architecture of hematopoiesis remain unknown. Here, we profiled gene expression in 38 distinct purified populations of human hematopoietic cells and used probabilistic models of gene expression and analysis of cis-elements in gene promoters to decipher the general organization of their regulatory circuitry. We identified modules of highly coexpressed genes, some of which are restricted to a single lineage but most of which are expressed at variable levels across multiple lineages. We found densely interconnected cis-regulatory circuits and a large number of transcription factors that are differentially expressed across hematopoietic states. These findings suggest a more complex regulatory system for hematopoiesis than previously assumed. Full Text.

ODonnell, C.W., Waldispuhl, J., Lis, M., Halfmann, R., Devadas, S., Lindquist, S., and Berger, B. (2011). A Method for Probing the Mutational Landscape of Amyloid Structure. Bioinformatics 27, i34-i42. MOTIVATION: Proteins of all kinds can self-assemble into highly ordered beta-sheet aggregates known as amyloid fibrils, important both biologically and clinically. However, the specific molecular structure of a fibril can vary dramatically depending on sequence and environmental conditions, and mutations can drastically alter amyloid function and pathogenicity. Experimental structure determination has proven extremely difficult with only a handful of NMR-based models proposed, suggesting a need for computational methods. RESULTS: We present AmyloidMutants, a statistical mechanics approach for de novo prediction and analysis of wild-type and mutant amyloid structures. Based on the premise of protein mutational landscapes, AmyloidMutants energetically quantifies the effects of sequence mutation on fibril conformation and stability. Tested on non-mutant, full-length amyloid structures with known chemical shift data, AmyloidMutants offers roughly 2-fold improvement in prediction accuracy over existing tools. Moreover, AmyloidMutants is the only method to predict complete super-secondary structures, enabling accurate discrimination of topologically dissimilar amyloid conformations that correspond to the same sequence locations. Applied to mutant prediction, AmyloidMutants identifies a global conformational switch between Abeta and its highly-toxic 'Iowa' mutant in agreement with a recent experimental model based on partial chemical shift data. Predictions on mutant, yeast-toxic strains of HET-s suggest similar alternate folds. When applied to HET-s and a HET-s mutant with core asparagines replaced by glutamines (both highly amyloidogenic chemically similar residues abundant in many amyloids), AmyloidMutants surprisingly predicts a greatly reduced capacity of the glutamine mutant to form amyloid. We confirm this finding by conducting mutagenesis experiments. AVAILABILITY: Our tool is publically available on the web at http://amyloid.csail.mit.edu/. CONTACT: lindquist_admin@wi.mit.edu; bab@csail.mit.edu SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online. Full Text.

Pan, J., Sasaki, M., Kniewel, R., Murakami, H., Blitzblau, H.G., Tischfield, S.E., Zhu, X.A., Neale, M.J., Jasin, M., Socci, N.D., Andreas Hochwagen and Scott Keeney (2011). A Hierarchical Combination of Factors Shapes the Genome-Wide Topography of Yeast Meiotic Recombination Initiation. Cell 144, 719-731. The nonrandom distribution of meiotic recombination influences patterns of inheritance and genome evolution, but chromosomal features governing this distribution are poorly understood. Formation of the DNA double-strand breaks (DSBs) that initiate recombination results in the accumulation of Spo11 protein covalently bound to small DNA fragments. By sequencing these fragments, we uncover a genome-wide DSB map of unprecedented resolution and sensitivity. We use this map to explore how DSB distribution is influenced by large-scale chromosome structures, chromatin, transcription factors, and local sequence composition. Our analysis offers mechanistic insight into DSB formation and early processing steps, supporting the view that the recombination terrain is molded by combinatorial and hierarchical interaction of factors that work on widely different size scales. This map illuminates the occurrence of DSBs in repetitive DNA elements, repair of which can lead to chromosomal rearrangements. We also discuss implications for evolutionary dynamics of recombination hot spots. Full Text.

Papatheodorou, P., Carette, J.E., Bell, G.W., Schwan, C., Guttenberg, G., Brummelkamp, T.R., and Aktories, K. (2011). Lipolysis-stimulated lipoprotein receptor (LSR) is the host receptor for the binary toxin Clostridium difficile transferase (CDT). Proceedings of the National Academy of Sciences of the United States of America 108, 16422-16427.Clostridium difficile infection (CDI) causes antibiotic-associated diarrhea and pseudomembranous colitis. Hypervirulent strains of the pathogen, which are responsible for increased morbidity and mortality of CDI, produce the binary actin-ADP ribosylating toxin Clostridium difficile transferase (CDT) in addition to the Rho-glucosylating toxins A and B. CDT depolymerizes the actin cytoskeleton, increases adherence and colonization of Clostridia by induction of microtubule-based cell protrusions and, eventually, causes death of target cells. Using a haploid genetic screen, we identified the lipolysis-stimulated lipoprotein receptor as the membrane receptor for CDT uptake by target cells. Moreover, we show that Clostridium perfringens iota toxin, which is a related binary actin-ADP ribosylating toxin, enters target cells via the lipolysis-stimulated lipoprotein receptor. Identification of the toxin receptors is essential for understanding of the toxin uptake and provides a most valuable basis for antitoxin strategies. Full Text.

Park, B., Buti, L., Lee, S., Matsuwaki, T., Spooner, E., Brinkmann, M.M., Nishihara, M., and Ploegh, H.L. (2011). Granulin Is a Soluble Cofactor for Toll-Like Receptor 9 Signaling. Immunity 34, 505-513. Toll-like receptor (TLR) signaling plays a critical role in innate and adaptive immune responses and must be tightly controlled. TLR4 uses LPS binding protein, MD-2, and CD14 as accessories to respond to LPS. We therefore investigated the presence of an analagous soluble cofactor that might assist in the recruitment of CpG oligonucleotides (CpG-ODNs) to TLR9. We report the identification of granulin as an essential secreted cofactor that potentiates TLR9-driven responses to CpG-ODNs. Granulin, an unusual cysteine-rich protein, bound to CpG-ODNs and interacted with TLR9. Macrophages from granulin-deficient mice showed not only impaired delivery of CpG-ODNs to endolysosomal compartments, but also decreased interaction of TLR9 with CpG-ODNs. As a consequence, granulin-deficient macrophages showed reduced responses to stimulation with CpG-ODNs, a trait corrected by provision of exogenous granulin. Thus, we propose that granulin contributes to innate immunity as a critical soluble cofactor for TLR9 signaling. Full Text.

Pawlak, M., and Jaenisch, R. (2011). De Novo DNA Methylation by Dnmt3a and Dnmt3b Is Dispensable for Nuclear Reprogramming of Somatic Cells to a Pluripotent State. Genes & Development 25, 1035-1040. Induced pluripotent stem cells (iPSCs) are generated from somatic cells by the transduction of defined transcription factors, and this process involves dynamic changes in DNA methylation. While the reprogramming of somatic cells is accompanied by demethylation of pluripotency genes, the functional importance of de novo DNA methylation has not been clarified. Here, using loss-of-function studies, we generated iPSCs from fibroblasts that were deficient in de novo DNA methylation mediated by Dnmt3a and Dnmt3b. These iPSCs reactivated pluripotency genes, underwent self-renewal, and showed restricted developmental potential that was rescued upon reintroduction of Dnmt3a and Dnmt3b. We conclude that de novo DNA methylation by Dnmt3a and Dnmt3b is dispensable for nuclear reprogramming of somatic cells. Full Text.

Petersen, C.P., and Reddien, P.W. (2011). Polarized Notum Activation at Wounds Inhibits Wnt Function to Promote Planarian Head Regeneration. Science 332, 852-855. Regeneration requires initiation of programs tailored to the identity of missing parts. Head-versus-tail regeneration in planarians presents a paradigm for study of this phenomenon. After injury, Wnt signaling promotes tail regeneration. We report that wounding elicits expression of the Wnt inhibitor notum preferentially at anterior-facing wounds. This expression asymmetry occurs at essentially any wound, even if the anterior pole is intact. Inhibition of notum with RNA interference (RNAi) causes regeneration of an anterior-facing tail instead of a head, and double-RNAi experiments indicate that notum inhibits Wnt signaling to promote head regeneration. notum expression is itself controlled by Wnt signaling, suggesting that regulation of feedback inhibition controls the binary head-tail regeneration outcome. We conclude that local detection of wound orientation with respect to tissue axes results in distinct signaling environments that initiate appropriate regeneration responses. Full Text.

Peterson, T.R., Sengupta, S.S., Harris, T.E., Carmack, A.E., Kang, S.A., Balderas, E., Guertin, D.A., Madden, K.L., Carpenter, A.E., Finck, B.N.,and Sabatini DM (2011). mTOR Complex 1 Regulates Lipin 1 Localization to Control the SREBP Pathway. Cell 146, 408-420.The nutrient- and growth factor-responsive kinase mTOR complex 1 (mTORC1) regulates many processes that control growth, including protein synthesis, autophagy, and lipogenesis. Through unknown mechanisms, mTORC1 promotes the function of SREBP, a master regulator of lipo- and sterolgenic gene transcription. Here, we demonstrate that mTORC1 regulates SREBP by controlling the nuclear entry of lipin 1, a phosphatidic acid phosphatase. Dephosphorylated, nuclear, catalytically active lipin 1 promotes nuclear remodeling and mediates the effects of mTORC1 on SREBP target gene, SREBP promoter activity, and nuclear SREBP protein abundance. Inhibition of mTORC1 in the liver significantly impairs SREBP function and makes mice resistant, in a lipin 1-dependent fashion, to the hepatic steatosis and hypercholesterolemia induced by a high-fat and -cholesterol diet. These findings establish lipin 1 as a key component of the mTORC1-SREBP pathway. Full Text

Ploegh, H. (2011). End the Wasteful Tyranny of Reviewer Experiments. Nature 472, 391. Peer review of scientific papers in top journals is bogged down by unnecessary demands for extra lab work. Full Text.

Popp, M.W., and Ploegh, H.L. (2011). Making and Breaking Peptide Bonds: Protein Engineering Using Sortase. Angewandte Chemistry Int Ed Engl.Apr 27 [Epub ahead of print] Sortases are a class of bacterial enzymes that possess transpeptidase activity. It is their ability to site-specifically break a peptide bond and then reform a new bond with an incoming nucleophile that makes sortase an attractive tool for protein engineering. This technique has been adopted for a range of applications, from chemistry-based to cell biology and technology. In this Minireview we provide a brief overview of the biology of sortase enzymes and current applications in protein engineering. We identify areas that lend themselves to further innovation and that suggest new applications. PDF.

Popp, M.W., Dougan, S.K., Chuang, T.Y., Spooner, E., and Ploegh, H.L. (2011). Sortase-Catalyzed Transformations That Improve the Properties of Cytokines. Proc Natl Acad Sci U S A. Feb 4. [Epub ahead of print] Recombinant protein therapeutics often suffer from short circulating half-life and poor stability, necessitating multiple injections and resulting in limited shelf-life. Conjugation to polyethylene glycol chains (PEG) extends the circulatory half-life of many proteins, but the methods for attachment often lack specificity, resulting in loss of biological activity. Using four-helix bundle cytokines as an example, we present a general platform that uses sortase-mediated transpeptidation to facilitate site-specific attachment of PEG to extend cytokine half-life with full retention of biological activity. Covalently joining the N and C termini of proteins to obtain circular polypeptides, again executed using sortase, increases thermal stability. We combined both PEGylation and circularization by exploiting two distinct sortase enzymes and the use of a molecular suture that allows both site-specific PEGylation and covalent closure. The method developed is general, uses a set of easily accessible reagents, and should be applicable to a wide variety of proteins, provided that their termini are not involved in receptor binding or function. Full Text.

Possemato, R., Marks, K.M., Shaul, Y.D., Pacold, M.E., Kim, D., Birsoy, K., Sethumadhavan, S., Woo, H.K., Jang, H.G., Jha, A.K. ,Chen WW, Barrett FG, Stransky N, Tsun ZY, Cowley GS, Barretina J, Kalaany NY, Hsu PP, Ottina K, Chan AM, Yuan B, Garraway LA, Root DE, Mino-Kenudson M, Brachtel EF, Driggers EM, Sabatini DM. (2011). Functional Genomics Reveal That the Serine Synthesis Pathway Is Essential in Breast Cancer. Nature [Epub ahead of print]. Cancer cells adapt their metabolic processes to drive macromolecular biosynthesis for rapid cell growth and proliferation. RNA interference (RNAi)-based loss-of-function screening has proven powerful for the identification of new and interesting cancer targets, and recent studies have used this technology in vivo to identify novel tumour suppressor genes. Here we developed a method for identifying novel cancer targets via negative-selection RNAi screening using a human breast cancer xenograft model at an orthotopic site in the mouse. Using this method, we screened a set of metabolic genes associated with aggressive breast cancer and stemness to identify those required for in vivo tumorigenesis. Among the genes identified, phosphoglycerate dehydrogenase (PHGDH) is in a genomic region of recurrent copy number gain in breast cancer and PHGDH protein levels are elevated in 70% of oestrogen receptor (ER)-negative breast cancers. PHGDH catalyses the first step in the serine biosynthesis pathway, and breast cancer cells with high PHGDH expression have increased serine synthesis flux. Suppression of PHGDH in cell lines with elevated PHGDH expression, but not in those without, causes a strong decrease in cell proliferation and a reduction in serine synthesis. We find that PHGDH suppression does not affect intracellular serine levels, but causes a drop in the levels of alpha-ketoglutarate, another output of the pathway and a tricarboxylic acid (TCA) cycle intermediate. In cells with high PHGDH expression, the serine synthesis pathway contributes approximately 50% of the total anaplerotic flux of glutamine into the TCA cycle. These results reveal that certain breast cancers are dependent upon increased serine pathway flux caused by PHGDH overexpression and demonstrate the utility of in vivo negative-selection RNAi screens for finding potential anticancer targets. Full Text.

Reddien, P.W. (2011). Constitutive Gene Expression and the Specification of Tissue Identity in Adult Planarian Biology .Trends in Genetics 27(7):277-85 . Planarians are flatworms that constitutively maintain adult tissues through cell turnover and can regenerate entire organisms from tiny body fragments. In addition to requiring new cells (from neoblasts), these feats require mechanisms that specify tissue identity in the adult. Crucial roles for Wnt and BMP signaling in the regeneration and maintenance of the body axes have been uncovered, among other regulatory factors. Available data indicate that genes involved in positional identity regulation at key embryonic stages in other animals display persisting regionalized expression in adult planarians. These expression patterns suggest that a constitutively active gene expression map exists for the maintenance of the planarian body. Planarians thus present a fertile ground for the identification of factors regulating the regionalization of the metazoan body plan and for the study of the attributes of these factors that can lead to the maintenance and regeneration of adult tissues. Full Text.

Reddy, P.B.J., Sehrawat, S., Suryawanshi, A., Rajasagi, N.K., Mulik, S., Hirashima, M., and Rouse, B.T. (2011). Influence of Galectin-9/Tim-3 Interaction on Herpes Simplex Virus-1 Latency. Journal of Immunology 187, 5745-5755.After HSV-1 infection, CD8(+) T cells accumulate in the trigeminal ganglion (TG) and participate in the maintenance of latency. However, the mechanisms underlying intermittent virus reactivation are poorly understood. In this study, we demonstrate the role of an inhibitory interaction between T cell Ig and mucin domain-containing molecule 3 (Tim-3) expressing CD8(+) T cells and galectin 9 (Gal-9) that could influence HSV-1 latency and reactivation. Accordingly, we show that most K(b)-gB tetramer-specific CD8(+) T cells in the TG of HSV-1 infected mice express Tim-3, a molecule that delivers negative signals to CD8(+) T cells upon engagement of its ligand Gal-9. Gal-9 was also upregulated in the TG when replicating virus was present as well during latency. This could set the stage for Gal-9/Tim-3 interaction, and this inhibitory interaction was responsible for reduced CD8+ T cell effector function in wild-type mice. Additionally, TG cell cultures exposed to recombinant Gal-9 in the latent phase caused apoptosis of most CD8(+) T cells. Furthermore, Gal-9 knockout TG cultures showed delayed and reduced viral reactivation as compared with wild-type cultures, demonstrating the greater efficiency of CD8(+) T cells to inhibit virus reactivation in the absence of Gal-9. Moreover, the addition of recombinant Gal-9 to ex vivo TG cultures induced enhanced viral reactivation compared with untreated controls. Our results demonstrate that the host homeostatic mechanism mediated by Gal-9/Tim-3 interaction on CD8(+) T cells can influence the outcome of HSV-1 latent infection, and manipulating Gal-9 signals might represent therapeutic means to inhibit HSV-1 reactivation from latency. The Journal of Immunology, 2011, 187: 5745-5755. Full Text.

Reiling J.H., Clish, C.B., Carette, J.E., Varadarajan, M., Brummelkamp, T.R., and Sabatini, D.M. (2011). A Haploid Genetic Screen Identifies the Major Facilitator Domain Containing 2a (Mfsd2a) Transporter as a Key Mediator in the Response to Tunicamycin. Proc Natl Acad Sci U S A [Epub ahead of print]. Tunicamycin (TM) inhibits eukaryotic asparagine-linked glycosylation, protein palmitoylation, ganglioside production, proteoglycan synthesis, 3-hydroxy-3-methylglutaryl coenzyme-A reductase activity, and cell wall biosynthesis in bacteria. Treatment of cells with TM elicits endoplasmic reticulum stress and activates the unfolded protein response. Although widely used in laboratory settings for many years, it is unknown how TM enters cells. Here, we identify in an unbiased genetic screen a transporter of the major facilitator superfamily, major facilitator domain containing 2A (MFSD2A), as a critical mediator of TM toxicity. Cells without MFSD2A are TM-resistant, whereas MFSD2A-overexpressing cells are hypersensitive. Hypersensitivity is associated with increased cellular TM uptake concomitant with an enhanced endoplasmic reticulum stress response. Furthermore, MFSD2A mutant analysis reveals an important function of the C terminus for correct intracellular localization and protein stability, and it identifies transmembrane helical amino acid residues essential for mediating TM sensitivity. Overall, our data uncover a critical role for MFSD2A by acting as a putative TM transporter at the plasma membrane. Full Text.

Resenberger, U.K., Harmeier, A., Woerner, A.C., Goodman, J.L., Muller, V., Krishnan, R., Vabulas, R.M., Kretzschmar, H.A., Lindquist, S., Hartl, F.,. Multhaup G, Winklhofer KF, Tatzelt J.l. (2011). The Cellular Prion Protein Mediates Neurotoxic Signalling of Beta-Sheet-Rich Conformers Independent of Prion Replication. Embo Journal 30, 2057-2070. Formation of aberrant protein conformers is a common pathological denominator of different neurodegenerative disorders, such as Alzheimer's disease or prion diseases. Moreover, increasing evidence indicates that soluble oligomers are associated with early pathological alterations and that oligomeric assemblies of different disease-associated proteins may share common structural features. Previous studies revealed that toxic effects of the scrapie prion protein (PrP(Sc)), a beta-sheet-rich isoform of the cellular PrP (PrP(C)), are dependent on neuronal expression of PrP(C). In this study, we demonstrate that PrP(C) has a more general effect in mediating neurotoxic signalling by sensitizing cells to toxic effects of various beta-sheet-rich (beta) conformers of completely different origins, formed by (i) heterologous PrP, (ii) amyloid beta-peptide, (iii) yeast prion proteins or (iv) designed beta-peptides. Toxic signalling via PrP(C) requires the intrinsically disordered N-terminal domain (N-PrP) and the GPI anchor of PrP. We found that the N-terminal domain is important for mediating the interaction of PrP(C) with beta-conformers. Interestingly, a secreted version of N-PrP associated with beta-conformers and antagonized their toxic signalling via PrP(C). Moreover, PrP(C)-mediated toxic signalling could be blocked by an NMDA receptor antagonist or an oligomer-specific antibody. Our study indicates that PrP(C) can mediate toxic signalling of various beta-sheet-rich conformers independent of infectious prion propagation, suggesting a pathophysiological role of the prion protein beyond of prion diseases. Full Text.

Reynolds, G.E., Gao, Q., Miller, D., Snow, B.E., Harrington, L.A., and Murnane, J.P. (2011). PIF1 disruption or NBS1 hypomorphism does not affect chromosome healing or fusion resulting from double-strand breaks near telomeres in murine embryonic stem cells. DNA Repair 10, 1164-1173.Telomerase serves to maintain telomeric repeat sequences at the ends of chromosomes. However, telomerase can also add telomeric repeat sequences at DNA double-strand breaks (DSBs), a process called chromosome healing. Here, we employed a method of inducing DSBs near telomeres to query the role of two proteins, PIF1 and NBS1, in chromosome healing in mammalian cells. PIF1 was investigated because the PIF1 homolog in Saccharomyces cerevisiae inhibits chromosome healing, as shown by a 1000-fold increase in chromosome in PIF1-deficient cells. NBS1 was investigated because the functional homolog of NBS1 in S. cerevisiae, Xrs2, is part of the Mre11/Rad50/Xrs2 complex that is required for chromosome healing due to its role in the processing of DSBs and recruitment of telomerase. We found that disruption of mPif1 had no detectable effect on the frequency of chromosome healing at DSBs near telomeres in murine embryonic stem cells. Moreover, the Nbs1(Delta B) hypomorph, which is defective in the processing of DSBs, also had no detectable effect on the frequency of chromosome healing. DNA degradation, or gross chromosome rearrangements (GCRs) that result from telomeric DSBs. Although we cannot rule out small changes in chromosome healing using this system, it is clear from our results that knockout of PIF1 or the Nbs1(Delta B) hypomorph does not result in large differences in chromosome healing in murine cells. These results represent the first genetic assessment of the role of these proteins in chromosome healing in mammals, and suggest that murine cells have evolved mechanisms to ensure the functional redundancy of Pif1 or Nbs1 in the regulation of chromosome healing. Full Text.

Rissland, O.S., Hong, S.J., and Bartel, D.P. (2011). MicroRNA Destabilization Enables Dynamic Regulation of the miR-16 Family in Response to Cell-Cycle Changes. Mol Cell 43, 993-1004.The miR-16 family, which targets genes important for the G1-S transition, is a known modulator of the cell cycle, and members of this family are often deleted or downregulated in many types of cancers. Here, we report the reciprocal relationship-that of the cell cycle controlling the miR-16 family. Levels of this family increase rapidly as cells are arrested in G0. Conversely, as cells are released from G0 arrest, levels of the miR-16 family rapidly decrease. Such rapid changes are made possible by the unusual instabilities of several family members. The repression mediated by the miR-16 family is sensitive to these cell-cycle changes, which suggests that the rapid upregulation of the miR-16 family reinforces cell-cycle arrest in G0. Upon cell-cycle re-entry, the rapid decay of several members allows levels of the family to decrease, alleviating repression of target genes and allowing proper resumption of the cell cycle. Full Text.

Rissland, O.S., and Lai, E.C. (2011). Rna Silencing in Monterey. Development 138, 3093-3102. The tenth annual Keystone Symposium on the Mechanism and Biology of Silencing convened in Monterey, California, in March 2011. Those seeking some West Coast sunshine were, unfortunately, met with incessant precipitation throughout the meeting. Nevertheless, attendees were brightened by enlightening and vigorous scientific discussions. Here, we summarize the results presented at the meeting, which inspire and push this expanding field into new territories. Full Text.

Rudnick, J.A., Arendt, L.M., Klebba, I., Hinds, J.W., Iyer, V., Gupta, P.B., Naber, S.P., and Kuperwasser, C. (2011). Functional Heterogeneity of Breast Fibroblasts Is Defined by a Prostaglandin Secretory Phenotype that Promotes Expansion of Cancer-Stem Like Cells. PLoS One 2011;6(9):e24605.Fibroblasts are important in orchestrating various functions necessary for maintaining normal tissue homeostasis as well as promoting malignant tumor growth. Significant evidence indicates that fibroblasts are functionally heterogeneous with respect to their ability to promote tumor growth, but markers that can be used to distinguish growth promoting from growth suppressing fibroblasts remain ill-defined. Here we show that human breast fibroblasts are functionally heterogeneous with respect to tumor-promoting activity regardless of whether they were isolated from normal or cancerous breast tissues. Rather than significant differences in fibroblast marker expression, we show that fibroblasts secreting abundant levels of prostaglandin (PGE2), when isolated from either reduction mammoplasty or carcinoma tissues, were both capable of enhancing tumor growth in vivo and could increase the number of cancer stem-like cells. PGE2 further enhanced the tumor promoting properties of fibroblasts by increasing secretion of IL-6, which was necessary, but not sufficient, for expansion of breast cancer stem-like cells. These findings identify a population of fibroblasts which both produce and respond to PGE2, and that are functionally distinct from other fibroblasts. Identifying markers of these cells could allow for the targeted ablation of tumor-promoting and inflammatory fibroblasts in human breast cancers. Full Text.

Saha, K., Mei, Y., Reisterer, C.M., Pyzocha, N.K., Yang, J., Muffat, J., Davies, M.C., Alexander, M.R., Langer, R., Anderson, D.G.and Rudolph Jaenisch. (2011). Surface-engineered substrates for improved human pluripotent stem cell culture under fully defined conditions. Proc Natl Acad Sci U S A .Nov 7. [Epub ahead of print] The current gold standard for the culture of human pluripotent stem cells requires the use of a feeder layer of cells. Here, we develop a spatially defined culture system based on UV/ozone radiation modification of typical cell culture plastics to define a favorable surface environment for human pluripotent stem cell culture. Chemical and geometrical optimization of the surfaces enables control of early cell aggregation from fully dissociated cells, as predicted from a numerical model of cell migration, and results in significant increases in cell growth of undifferentiated cells. These chemically defined xeno-free substrates generate more than three times the number of cells than feeder-containing substrates per surface area. Further, reprogramming and typical gene-targeting protocols can be readily performed on these engineered surfaces. These substrates provide an attractive cell culture platform for the production of clinically relevant factor-free reprogrammed cells from patient tissue samples and facilitate the definition of standardized scale-up friendly methods for disease modeling and cell therapeutic applications. Full Text.

Saha, K., and Hurlbut, J.B. (2011). Research ethics: Treat donors as partners in biobank research. Nature 478, 312-313 Proposed rules to protect research subjects will impede progress. Instead, give donors more say in how samples are used. Full Text.

Sankaran, V.G., Menne, T.F., Scepanovic, D., Vergilio, J.A., Ji, P., Kim, J., Thiru, P., Orkin, S.H., Lander, E.S., and Lodish, H.F. (2011). Microrna-15a and -16-1 Act Via Myb to Elevate Fetal Hemoglobin Expression in Human Trisomy 13. Proc Natl Acad Sci U S A.Jan 4. [Epub ahead of print] Many human aneuploidy syndromes have unique phenotypic consequences, but in most instances it is unclear whether these phenotypes are attributable to alterations in the dosage of specific genes. In human trisomy 13, there is delayed switching and persistence of fetal hemoglobin (HbF) and elevation of embryonic hemoglobin in newborns. Using partial trisomy cases, we mapped this trait to chromosomal band 13q14; by examining the genes in this region, two microRNAs, miR-15a and -16-1, appear as top candidates for the elevated HbF levels. Indeed, increased expression of these microRNAs in primary human erythroid progenitor cells results in elevated fetal and embryonic hemoglobin gene expression. Moreover, we show that a direct target of these microRNAs, MYB, plays an important role in silencing the fetal and embryonic hemoglobin genes. Thus we demonstrate how the developmental regulation of a clinically important human trait can be better understood through the genetic and functional study of aneuploidy syndromes and suggest that miR-15a, -16-1, and MYB may be important therapeutic targets to increase HbF levels in patients with sickle cell disease and beta-thalassemia PDF

Sarkar, S. (2011). Role of autophagy in neurodegenerative diseases. Current Science 101, 514-519.The growing range of implications of autophagy in a myriad of human physiological and pathological conditions has witnessed an exponential increase in the number of studies published over the last decade. The role of autophagy to function predominantly as a cellular survival mechanism has been widely accepted over the last few years. It is an evolutionarily conserved protein degradation pathway for long-lived proteins and organelles, which contributes to tissue and energy homeostasis. Dysfunction of this process is associated with diverse human diseases, ranging from cancer, infectious diseases and myopathies to neurodegenerative diseases. This review focuses on the role of autophagy in neurodegenerative diseases, where in most instances the mutant aggregate-prone proteins are autophagy substrates. Some of these mutant proteins can impair autophagy and augment neurodegeneration. Stimulation of autophagy by chemical inducers enhances autophagic degradation of aggregate-prone proteins and protects against neurodegeneration in several models of neurodegenerative diseases. The small molecule autophagy enhancers are of paramount importance for future therapeutic studies in other disease conditions beyond neurodegeneration, and also offer great potential in the study of signalling pathways regulating autophagy.PDF.

Scheel, C., and Weinberg, R.A. (2011). Phenotypic plasticity and epithelial-mesenchymal transitions in cancer - and normal stem cells? Int J Cancer.Jul 25. [Epub ahead of print] Cancer stem cells (CSCs) are similar to normal stem cells in their ability to self-renew and to generate large populations of more differentiated descendants. In contrast to the hierarchical organization that is presumed to be the prevalent mode of normal tissue homeostasis, phenotypic plasticity allows cancer cells to dynamically enter into and exit from stem-cell states. The Epithelial-Mesenchymal Transition (EMT) has been closely associated with the acquisition of both invasive and stem-cell properties in cancer cells. Thereby, EMT programs emerge as important regulators of phenotypic plasticity in cancer cells including their entrance into stem-cell states. Much is still to be learned about the regulation of EMTs through epigenetic mechanisms in cancer cells and the contributions that EMT programs make to normal tissue homeostasis. PDF

Scheel, C., Eaton, E.N., Li, S.H., Chaffer, C.L., Reinhardt, F., Kah, K.J., Bell, G., Guo, W., Rubin, J., Richardson, A.L.,and Weinberg RA. (2011). Paracrine and Autocrine Signals Induce and Maintain Mesenchymal and Stem Cell States in the Breast. Cell 145, 926-940. The epithelial-mesenchymal transition (EMT) has been associated with the acquisition of motility, invasiveness, and self-renewal traits. During both normal development and tumor pathogenesis, this change in cell phenotype is induced by contextual signals that epithelial cells receive from their microenvironment. The signals that are responsible for inducing an EMT and maintaining the resulting cellular state have been unclear. We describe three signaling pathways, involving transforming growth factor (TGF)-beta and canonical and noncanonical Wnt signaling, that collaborate to induce activation of the EMT program and thereafter function in an autocrine fashion to maintain the resulting mesenchymal state. Downregulation of endogenously synthesized inhibitors of autocrine signals in epithelial cells enables the induction of the EMT program. Conversely, disruption of autocrine signaling by added inhibitors of these pathways inhibits migration and self-renewal in primary mammary epithelial cells and reduces tumorigenicity and metastasis by their transformed derivatives. Full Text.

Schlegelmilch, K., Mohseni, M., Kirak, O., Pruszak, J., Rodriguez, J.R., Zhou, D.W., Kreger, B.T., Vasioukhin, V., Avruch, J., Brummelkamp, T.R.,and Camargo FD (2011). Yap1 Acts Downstream of Alpha-Catenin to Control Epidermal Proliferation. Cell 144, 782-795. During development and regeneration, proliferation of tissue-specific stem cells is tightly controlled to produce organs of a predetermined size. The molecular determinants of this process remain poorly understood. Here, we investigate the function of Yap1, the transcriptional effector of the Hippo signaling pathway, in skin biology. Using gain- and loss-of-function studies, we show that Yap1 is a critical modulator of epidermal stem cell proliferation and tissue expansion. Yap1 mediates this effect through interaction with TEAD transcription factors. Additionally, our studies reveal that alpha-catenin, a molecule previously implicated in tumor suppression and cell density sensing in the skin, is an upstream negative regulator of Yap1. alpha-catenin controls Yap1 activity and phosphorylation by modulating its interaction with 14-3-3 and the PP2A phosphatase. Together, these data identify Yap1 as a determinant of the proliferative capacity of epidermal stem cells and as an important effector of a "crowd control" molecular circuitry in mammalian skin. FullText

Schmidt, J.C., and Cheeseman, I.M. Chromosome Segregation: Keeping Kinetochores in the Loop. Current Biology 21(3) R110-112. The Ndc80 complex is a key component of the kinetochore-microtubule interface. Two studies now demonstrate that a conserved loop region within the extended coiled-coil of Ndc80 plays an unexpected role in recruiting proteins to the kinetochore. Full Text

SchnallLevin, M., Rissland, O.S., Johnston, W.K., Perrimon, N., Bartel, D.P., and Berger, B. (2011). Unusually effective microRNA targeting within repeat-rich coding regions of mammalian mRNAs. Genome Research 21, 1395-1403.MicroRNAs (miRNAs) regulate numerous biological processes by base-pairing with target messenger RNAs (mRNAs), primarily through sites in 3' untranslated regions (UTRs), to direct the repression of these targets. Although miRNAs have sometimes been observed to target genes through sites in open reading frames (ORFs), large-scale studies have shown such targeting to be generally less effective than 3' UTR targeting. Here, we show that several miRNAs each target significant groups of genes through multiple sites within their coding regions. This ORF targeting, which mediates both predictable and effective repression, arises from highly repeated sequences containing miRNA target sites. We show that such sequence repeats largely arise through evolutionary duplications and occur particularly frequently within families of paralogous C(2)H(2) zinc-finger genes, suggesting the potential for their coordinated regulation. Examples of ORFs targeted by miR-181 include both the well-known tumor suppressor RB1 and RBAK, encoding a C(2)H(2) zinc-finger protein and transcriptional binding partner of RB1. Our results indicate a function for repeat-rich coding sequences in mediating post-transcriptional regulation and reveal circumstances in which miRNA-mediated repression through ORF sites can be reliably predicted. Full Text.

Scimone, M.L., Srivastava, M., Bell, G.W., and Reddien, P.W. (2011). A regulatory program for excretory system regeneration in planarians. Development 138, 4387-4398.Planarians can regenerate any missing body part, requiring mechanisms for the production of organ systems in the adult, including their prominent tubule-based filtration excretory system called protonephridia. Here, we identify a set of genes, Six1/2-2, POU2/3, hunchback, Eya and Sall, that encode transcription regulatory proteins that are required for planarian protonephridia regeneration. During regeneration, planarian stem cells are induced to form a cell population in regeneration blastemas expressing Six1/2-2, POU2/3, Eya, Sall and Osr that is required for excretory system formation. POU2/3 and Six1/2-2 are essential for these precursor cells to form. Eya, Six1/2-2, Sall, Osr and POU2/3-related genes are required for vertebrate kidney development. We determined that planarian and vertebrate excretory cells express homologous proteins involved in reabsorption and waste modification. Furthermore, we identified novel nephridia genes. Our results identify a transcriptional program and cellular mechanisms for the regeneration of an excretory organ and suggest that metazoan excretory systems are regulated by genetic programs that share a common evolutionary origin. Full Text.

Sehrawat, S., and Rouse, B.T. (2011). Tregs and infections: on the potential value of modifying their function. J Leukoc Bio l2011 Sep 13. [Epub ahead of print] .CD4(+) T cells, which express a master transcription factor, Foxp3, have been recognized as bona fide Tregs. These cells are essential to maintain immune homeostasis in healthy as well as infected mice and humans. Extensive investigations in the last decade have provided ways to manipulate the Foxp3(+) Treg response therapeutically so the role of such cells in microbe-induced inflammatory reactions can be evaluated. This review focuses on our current understanding of the mechanisms required for the generation and sustenance of Tregs in vivo and the potential value of modulating Tregs to control microbe-induced immunopathological responses. PDF

Shechner, D.M., and Bartel, D.P. (2011). The structural basis of RNA-catalyzed RNA polymerization. Nat Struct Mol Bio lAug 21. [Epub ahead of print] .Early life presumably required polymerase ribozymes capable of replicating RNA. Known polymerase ribozymes best approximating such replicases use as their catalytic engine an RNA-ligase ribozyme originally selected from random RNA sequences. Here we report 3.15-A crystal structures of this ligase trapped in catalytically viable preligation states, with the 3'-hydroxyl nucleophile positioned for in-line attack on the 5'-triphosphate. Guided by metal- and solvent-mediated interactions, the 5'-triphosphate hooks into the major groove of the adjoining RNA duplex in an unanticipated conformation. Two phosphates and the nucleophile jointly coordinate an active-site metal ion. Atomic mutagenesis experiments demonstrate that active-site nucleobase and hydroxyl groups also participate directly in catalysis, collectively playing a role that in proteinaceous polymerases is performed by a second metal ion. Thus artificial ribozymes can use complex catalytic strategies that differ markedly from those of analogous biological enzymes. Full Text.

Sheen, J.H., Zoncu, R., Kim, D., and Sabatini, D.M. (2011). Defective Regulation of Autophagy Upon Leucine Deprivation Reveals a Targetable Liability of Human Melanoma Cells in Vitro and in Vivo. Cancer Cell 19, 613-628. Autophagy is of increasing interest as a target for cancer therapy. We find that leucine deprivation causes the caspase-dependent apoptotic death of melanoma cells because it fails to appropriately activate autophagy. Hyperactivation of the RAS-MEK pathway, which is common in melanoma, prevents leucine deprivation from inhibiting mTORC1, the main repressor of autophagy under nutrient-rich conditions. In an in vivo tumor xenograft model, the combination of a leucine-free diet and an autophagy inhibitor synergistically suppresses the growth of human melanoma tumors and triggers widespread apoptosis of the cancer cells. Together, our study represents proof of principle that anticancer effects can be obtained with a combination of autophagy inhibition and strategies to deprive tumors of leucine. Full Text.

Sher, N., Bell, G.W., Li, S., Nordman, J., Eng, T., Eaton, M.L., Macalpine, D.M., and Orr-Weaver, T.L. (2011). Developmental control of gene copy number by repression of replication initiation and fork progression. Genome Research Nov 16. [Epub ahead of print].Precise DNA replication is crucial for genome maintenance, yet this process has been inherently difficult to study on a genome-wide level in untransformed differentiated metazoan cells. To determine how metazoan DNA replication can be repressed, we examined regions selectively under-replicated in Drosophila polytene salivary glands and found they are transcriptionally silent and enriched for the repressive H3K27me3 mark. In the first genome-wide analysis of binding of the Origin Recognition Complex (ORC) in a differentiated metazoan tissue, we find that ORC binding is dramatically reduced within these large domains, suggesting reduced initiation as one mechanism leading to under-replication. Inhibition of replication fork progression by the chromatin protein SUUR is an additional repression mechanism to reduce copy number. Although repressive histone marks are removed when SUUR is mutated and copy number restored, neither transcription nor ORC binding are reinstated. Tethering of SUUR protein to a specific site is insufficient to block replication, however. These results establish that developmental control of DNA replication, both at the initiation and elongation stages, is a mechanism to change gene copy number during differentiation. Full Text.

Singh, K.K., De Rienzo, G., Drane, L., Mao, Y.W., Flood, Z., Madison, J., Ferreira, M., Bergen, S., King, C., Sklar, P., Sive H, Tsai LH. (2011). Common DISC1 Polymorphisms Disrupt Wnt/GSK3 beta Signaling and Brain Development. Neuron 72, 545-558. Disrupted in Schizophrenia-1 (DISCI) is a candidate gene for psychiatric disorders and has many roles during brain development. Common DISCI polymorphisms (variants) are associated with neuropsychiatric phenotypes including altered cognition, brain structure, and function; however, it is unknown how this occurs. Here, we demonstrate using mouse, zebrafish, and human model systems that DISCI variants are loss of function in Wnt/GSK3 beta signaling and disrupt brain development. The DISCI variants A83V, R264Q, and L607F, but not S704C, do not activate Wnt signaling compared with wild-type DISC1 resulting in decreased neural progenitor proliferation. In zebrafish, R264Q and L607F could not rescue DISCI knockdown-mediated aberrant brain development. Furthermore, human lymphoblast cell lines endogenously expressing R264Q displayed impaired Wnt signaling. Interestingly, S704C inhibited the migration of neurons in the developing neocortex. Our data demonstrate DISC1 variants impair Wnt signaling and brain development and elucidate a possible mechanism for their role in neuropsychiatric phenotypes. Full Text.

Sive, H. (2011). Keeping two animal systems in one lab - a frog plus fish case study. Methods Mol Biol 770, 571-578.For two decades, my lab has been studying development using two vertebrate animals, the frog Xenopus and the zebrafish, Danio. This has been both productive and challenging. The initial rationale for the choice was to compare the same process in two species, as a means to find commonalities that may carry through all vertebrates. As time progressed, however, each species has become exploited for its specific attributes, more than for comparative studies. Maintaining two species simultaneously has been challenging, as has the division of research between the two and making sure that lab members know both systems well enough to communicate productively. Other significant issues concern funding for disparate research, figuring out how to make contributions to both fish and frog communities, and being accepted as a member of two communities. I discuss whether this dual allegiance has been a good idea. Full Text

Sive, H. (2011). 'Model' or 'Tool'? New Definitions for Translational Research. Dis Model Mech 4, 137-138. The term 'model' often describes non-human biological systems that are used to obtain a better understanding of human disorders. According to the most stringent definition, an animal 'model' would display exactly the same phenotype as seen in the relevant human disorder; however, this precise correspondence is often not present. In this Editorial, I propose the alternative, broader term 'tool' to describe a biological system that does not obviously (or precisely) recapitulate a human disorder, but that nonetheless provides useful insight into the etiology or treatment of that disorder. Applying the term 'tool' to biological systems used in disease-related studies will help to identify those systems that can most effectively address mechanisms underlying human disease. Conversely, differentiating 'models' from 'tools' will help to define more clearly the limitations of biological systems used in preclinical analyses. Full Text.

Smith, M.H., Ploegh, H.L., and Weissman, J.S. (2011). Road to Ruin: Targeting Proteins for Degradation in the Endoplasmic Reticulum. Science 334, 1086-1090.Some nascent proteins that fold within the endoplasmic reticulum (ER) never reach their native state. Misfolded proteins are removed from the folding machinery, dislocated from the ER into the cytosol, and degraded in a series of pathways collectively referred to as ER-associated degradation (ERAD). Distinct ERAD pathways centered on different E3 ubiquitin ligases survey the range of potential substrates. We now know many of the components of the ERAD machinery and pathways used to detect substrates and target them for degradation. Much less is known about the features used to identify terminally misfolded conformations and the broader role of these pathways in regulating protein half-lives. Full Text.

Soldner F., Laganiere, J., Cheng, A.W., Hockemeyer, D., Gao, Q., Alagappan, R., Khurana, V., Golbe, L.I., Myers, R.H., Lindquist, S., Lei Zhang, Dmitry Guschin, Lauren K. Fong, B. Joseph Vu, Xiangdong Meng ,Fyodor D. Urno, Edward J. Rebar, Philip D. Gregory, H. Steve Zhang and Rudolf Jaenisch (2011). Generation of Isogenic Pluripotent Stem Cells Differing Exclusively at Two Early Onset Parkinson Point Mutations. Cell.[Article in Press] Patient-specific induced pluripotent stem cells (iPSCs) derived from somatic cells provide a unique tool for the study of human disease, as well as a promising source for cell replacement therapies. One crucial limitation has been the inability to perform experiments under genetically defined conditions. This is particularly relevant for late age onset disorders in which in vitro phenotypes are predicted to be subtle and susceptible to significant effects of genetic background variations. By combining zinc finger nuclease (ZFN)-mediated genome editing and iPSC technology, we provide a generally applicable solution to this problem, generating sets of isogenic disease and control human pluripotent stem cells that differ exclusively at either of two susceptibility variants for Parkinson's disease by modifying the underlying point mutations in the alpha-synuclein gene. The robust capability to genetically correct disease-causing point mutations in patient-derived hiPSCs represents significant progress for basic biomedical research and an advance toward hiPSC-based cell replacement therapies. Full Text.

Staerk J., Lyssiotis, C.A., Medeiro, L.A., Bollong, M., Foreman, R.K., Zhu, S., Garcia, M., Gao, Q., Bouchez, L.C., Lairson, L.L., Bradley D. Charette, . Lubica Supekova, Jeffrey Janes, Achim Brinker,. Charles Y. Cho, Rudolf Jaenisch, and Peter G. Schultz. Pan-Src Family Kinase Inhibitors Replace Sox2 During the Direct Reprogramming of Somatic Cells. Angew Chem Int Ed Engl. May 5. [Epub ahead of print] Small molecules do the job: Somatic cells are reprogrammed into iPS cells upon ectopic expression of Oct4, Sox2, Klf4 and c-Myc. Application of a cell-based, high-throughput chemical screen led to the identification of Src family kinase (SFK) inhibitors as chemical replacements for retroviral Sox2 delivery. These compounds are used to study the mechanisms underlying direct reprogramming and may ultimately help to bring iPS cell technology one step closer to clinical application. PDF

Steine, E.J., Ehrich, M., Bell, G.W., Raj, A., Reddy, S., van Oudenaarden, A., Jaenisch, R., and Linhart, H.G. (2011). Genes Methylated by DNA Methyltransferase 3b Are Similar in Mouse Intestine and Human Colon Cancer J Clin Invest. 121(5):1748-52 Human cancer cells frequently have regions of their DNA hypermethylated, which results in transcriptional silencing of affected genes and promotion of tumor formation. However, it is still unknown whether cancer-associated aberrant DNA methylation is targeted to specific genomic regions, whether this methylation also occurs in noncancerous cells, and whether these epigenetic events are maintained in the absence of the initiating cause. Here we have addressed some of these issues by demonstrating that transgenic expression of DNA methyltransferase 3b (Dnmt3b) in the mouse colon initiates de novo DNA methylation of genes that are similar to genes that become methylated in human colon cancer. This is consistent with the notion that aberrant methylation in cancer may be attributable to targeting of specific sequences by Dnmt3b rather than to random methylation followed by clonal selection. We also showed that Dnmt3b-induced aberrant DNA methylation was maintained in regenerating tissue, even in the absence of continuous Dnmt3b expression. This supports the concept that transient stressors can cause permanent epigenetic changes in somatic stem cells and that these accumulate over the lifetime of an organism in analogy to DNA mutations. Full Text.

Sun, L., Xie, H., Mori, M.A., Alexander, R., Yuan, B., Hattangadi, S.M., Liu, Q., Kahn, C.R., and Lodish, H.F. (2011). Mir193b-365 Is Essential for Brown Fat Differentiation .Nature Cell biology [Epub ahead of print] Mammals have two principal types of fat. White adipose tissue primarily serves to store extra energy as triglycerides, whereas brown adipose tissue is specialized to burn lipids for heat generation and energy expenditure as a defence against cold and obesity. Recent studies have demonstrated that brown adipocytes arise in vivo from a Myf5-positive, myoblastic progenitor by the action of Prdm16 (PR domain containing 16). Here, we identified a brown-fat-enriched miRNA cluster, MiR-193b-365, as a key regulator of brown fat development. Blocking miR-193b and/or miR-365 in primary brown preadipocytes markedly impaired brown adipocyte adipogenesis by enhancing Runx1t1 (runt-related transcription factor 1; translocated to, 1) expression, whereas myogenic markers were significantly induced. Forced expression of Mir193b and/or Mir365 in C2C12 myoblasts blocked the entire programme of myogenesis, and, in adipogenic conditions, miR-193b induced myoblasts to differentiate into brown adipocytes. Mir193b-365 was upregulated by Prdm16 partially through Pparalpha. Our results demonstrate that Mir193b-365 serves as an essential regulator for brown fat differentiation, in part by repressing myogenesis. Full Text.

Suryawanshi, A., Veiga-Parga, T., Rajasagi, N.K., Reddy, P.B.J., Sehrawat, S., Sharma, S., and Rouse, B.T. (2011). Role of IL-17 and Th17 Cells in Herpes Simplex Virus-Induced Corneal Immunopathology. Journal of Immunology 187, 1919-1930.HSV-1 infection of the cornea leads to a blinding immunoinflammatory lesion of the eye termed stromal keratitis (SK). Recently, IL-17-producing CD4(+) T cells (Th17 cells) were shown to play a prominent role in many autoimmune conditions, but the role of IL-17 and/or of Th17 cells in virus immunopathology is unclear. In this study, we show that, after HSV infection of the cornea, IL-17 is upregulated in a biphasic manner with an initial peak production around day 2 postinfection and a second wave starting from day 7 postinfection with a steady increase until day 21 postinfection, a time point when clinical lesions are fully evident. Further studies demonstrated that innate cells, particularly gamma delta T cells, were major producers of IL-17 early after HSV infection. However, during the clinical phase of SK, the predominant source of IL-17 was Th17 cells that infiltrated the cornea only after the entry of Th1 cells. By ex vivo stimulation, the half fraction of IFN-gamma-producing CD4(+) T cells (Th1 cells) were HSV specific, whereas very few Th17 cells responded to HSV stimulation. The delayed influx of Th17 cells in the cornea was attributed to the local chemokine and cytokine milieu. Finally, HSV infection of IL-17R knockout mice as well as IL-17 neutralization in wild-type mice showed diminished SK severity. In conclusion, our results show that IL-17 and Th17 cells contribute to the pathogenesis of SK, the most common cause of infectious blindness in the Western world. The Journal of Immunology, 2011, 187: 1919-1930. Full Text.

Suryawanshi, A., Mulik, S., Sharma, S., Reddy, P.B.J., Sehrawat, S., and Rouse, B.T. (2011). Ocular Neovascularization Caused by Herpes Simplex Virus Type 1 Infection Results from Breakdown of Binding between Vascular Endothelial Growth Factor a and Its Soluble Receptor. Journal of Immunology 186, 3653-3665. The normal cornea is transparent, which is essential for normal vision, and although the angiogenic factor vascular endothelial growth factor A (VEGF-A) is present in the cornea, its angiogenic activity is impeded by being bound to a soluble form of the VEGF receptor-1 (sVR-1). This report investigates the effect on the balance between VEGF-A and sVR-1 that occurs after ocular infection with HSV, which causes prominent neovascularization, an essential step in the pathogenesis of the vision-impairing lesion, stromal keratitis. We demonstrate that HSV-1 infection causes increased production of VEGF-A but reduces sVR-1 levels, resulting in an imbalance of VEGF-A and sVR-1 levels in ocular tissues. Moreover, the sVR-1 protein made was degraded by the metalloproteinase (MMP) enzymes MMP-2, -7, and -9 produced by infiltrating inflammatory cells that were principally neutrophils. Inhibition of neutrophils, inhibition of sVR-1 breakdown with the MMP inhibitor marimastat, and the provision of exogenous recombinant sVR-1 protein all resulted in reduced angiogenesis. Our results make the novel observation that ocular neovascularization resulting from HSV infection involves a change in the balance between VEGF-A and its soluble inhibitory receptor. Future therapies aimed to increase the production and activity of sVR-1 protein could benefit the management of stromal keratitis, an important cause of human blindness. Full Text.

Tardiff, D.F., Tucci, M.L., Caldwell, K.A., Caldwell, G.A., and Lindquist, S. (2011). Different 8-hydroxyquinolines protect models of TDP-43, alpha-synuclein, and polyglutamine proteotoxicity through distinct mechanisms. J Biol Chem Dec 6. [Epub ahead of print] .No current therapies target the underlying cellular pathologies of age-related neurodegenerative diseases. Model organisms provide a platform for discovering compounds that protect against the toxic, misfolded proteins that initiate these diseases. One such protein, TDP-43, is implicated in multiple neurodegenerative diseases, including Amyotrophic Lateral Sclerosis and Frontotemporal Lobar Degeneration. In yeast, TDP-43 expression is toxic and genetic modifiers first discovered in yeast have proven to modulate TDP-43 toxicity in both neurons and humans. Here, we describe a phenotypic screen for small molecules that reverse TDP-43 toxicity in yeast. One group of hit compounds were 8-hydroxyquinolines (8-OHQ), a class of clinically relevant bioactive metal chelators related to clioquinol. Surprisingly, in otherwise wild-type yeast cells, different 8-OHQs had selectivity for rescuing the distinct toxicities caused by the expression of TDP-43, alpha-synuclein, or polyglutamine proteins. In fact, each 8-OHQ synergized with the other, clearly establishing that they function in different ways. Comparative growth and molecular analyses also revealed that 8-OHQs have distinct metal chelation and ionophore activities. The diverse bioactivity of 8-OHQs indicates that altering different aspects of metal homeostasis and/or metalloprotein activity elicits distinct protective mechanisms against several neurotoxic proteins. Indeed, phase II clinical trials of an 8-OHQ has produced encouraging results in modifying Alzheimer's disease. Our unbiased identification of 8-OHQs in a yeast TDP-43 toxicity model suggests that tailoring 8-OHQ activity to a particular neurodegenerative disease may be a viable therapeutic strategy. PDF

Treusch, S., Hamamichi, S., Goodman, J.L., Matlack, K.E., Chung, C.Y., Baru, V., Shulman, J.M., Parrado, A., Bevis, B.J., Valastyan, J.S., Haesun Han, Malin Lindhagen-Persson, Eric M. Reiman, Denis A. Evans , David A. Bennett , Anders Olofsson , Philip L. DeJager , Rudolph E. Tanzi, Kim A. Caldwell , Guy A. Caldwell, and Susan Lindquist (2011) Functional Links Between Aβ Toxicity, Endocytic Trafficking, and Alzheimer’s Disease Risk Factors in Yeast Science. Published Online October 27 2011 Abeta (beta amyloid peptide) is an important contributor to Alzheimer's disease (AD). We modeled Abeta toxicity in yeast by directing the peptide to the secretory pathway. A genome-wide screen for toxicity modifiers identified the yeast homolog of phosphatidylinositol binding clathrin assembly protein (PICALM) and other endocytic factors connected to AD whose relationship to Abeta was previously unknown. The factors identified in yeast modified Abeta toxicity in glutamatergic neurons of Caenorhabditis elegans and in primary rat cortical neurons. In yeast, Abeta impaired the endocytic trafficking of a plasma membrane receptor, which was ameliorated by endocytic pathway factors identified in the yeast screen. Thus, links between Abeta, endocytosis, and human AD risk factors can be ascertained using yeast as a model system. Full Text.

Ulitsky, I., Shkumatava, A., Jan, C.H., Sive, H., and Bartel, D.P. (2011). Conserved Function of lincRNAs in Vertebrate Embryonic Development despite Rapid Sequence Evolution. Cell 147, 1537-1550.Thousands of long intervening noncoding RNAs (lincRNAs) have been identified in mammals. To better understand the evolution and functions of these enigmatic RNAs, we used chromatin marks, poly(A)-site mapping and RNA-Seq data to identify more than 550 distinct lincRNAs in zebrafish. Although these shared many characteristics with mammalian lincRNAs, only 29 had detectable sequence similarity with putative mammalian orthologs, typically restricted to a single short region of high conservation. Other lincRNAs had conserved genomic locations without detectable sequence conservation. Antisense reagents targeting conserved regions of two zebrafish lincRNAs caused developmental defects. Reagents targeting splice sites caused the same defects and were rescued by adding either the mature lincRNA or its human or mouse ortholog. Our study provides a roadmap for identification and analysis of lincRNAs in model organisms and shows that lincRNAs play crucial biological roles during embryonic development with functionality conserved despite limited sequence conservation. Full Text.

Vader, G., Blitzblau, H.G., Tame, M.A., Falk, J.E., Curtin, L., and Hochwagen, A. (2011). Protection of repetitive DNA borders from self-induced meiotic instability. Nature Published online 07 August 2011.DNA double strand breaks (DSBs) in repetitive sequences are a potent source of genomic instability, owing to the possibility of non-allelic homologous recombination (NAHR). Repetitive sequences are especially at risk during meiosis, when numerous programmed DSBs are introduced into the genome to initiate meiotic recombination. In the repetitive ribosomal DNA (rDNA) array of the budding yeast Saccharomyces cerevisiae, meiotic DSB formation is prevented in part through Sir2-dependent heterochromatin formation. Here we show that the edges of the rDNA array are exceptionally susceptible to meiotic DSBs, revealing an inherent heterogeneity in the rDNA array. We find that this localized DSB susceptibility necessitates a border-specific protection system consisting of the meiotic ATPase Pch2 and the origin recognition complex subunit Orc1. Upon disruption of these factors, DSB formation and recombination increased specifically in the outermost rDNA repeats, leading to NAHR and rDNA instability. Notably, the Sir2-dependent heterochromatin of the rDNA itself was responsible for the induction of DSBs at the rDNA borders in pch2Delta cells. Thus, although the activity of Sir2 globally prevents meiotic DSBs in the rDNA, it creates a highly permissive environment for DSB formation at the junctions between heterochromatin and euchromatin. Heterochromatinized repetitive DNA arrays are abundant in most eukaryotic genomes. Our data define the borders of such chromatin domains as distinct high-risk regions for meiotic NAHR, the protection of which may be a universal requirement to prevent meiotic genome rearrangements that are associated with genomic diseases and birth defects. Full Text.

Valastyan, S., and Weinberg, R.A. (2011). Tumor metastasis: molecular insights and evolving paradigms. Cell 147, 275-292.Metastases represent the end products of a multistep cell-biological process termed the invasion-metastasis cascade, which involves dissemination of cancer cells to anatomically distant organ sites and their subsequent adaptation to foreign tissue microenvironments. Each of these events is driven by the acquisition of genetic and/or epigenetic alterations within tumor cells and the co-option of nonneoplastic stromal cells, which together endow incipient metastatic cells with traits needed to generate macroscopic metastases. Recent advances provide provocative insights into these cell-biological and molecular changes, which have implications regarding the steps of the invasion-metastasis cascade that appear amenable to therapeutic targeting. Full Text.

Valastyan, J.S., and Lindquist, S. (2011). TorsinA and the TorsinA-Interacting Protein Printor Have No Impact on Endoplasmic Reticulum Stress or Protein Trafficking in Yeast. PLoS One. 2011;6(7):e22744 Early-onset torsion dystonia is a severe, life-long disease that leads to loss of motor control and involuntary muscle contractions. While the molecular etiology of the disease is not fully understood, a mutation in an AAA+ ATPase, torsinA, has been linked to disease onset. Previous work on torsinA has shown that it localizes to the endoplasmic reticulum, where there is evidence that it plays roles in protein trafficking, and potentially also protein folding. Given the high level of evolutionary conservation among proteins involved in these processes, the ability of human such proteins to function effectively in yeast, as well as the previous successes achieved in examining other proteins involved in complex human diseases in yeast, we hypothesized that Saccharomyces cerevisiae might represent a useful model system for studying torsinA function and the effects of its mutants. Since torsinA is proposed to function in protein homeostasis, we tested cells for their ability to respond to various stressors, using a fluorescent reporter to measure the unfolded protein response, as well as their rate of protein secretion. TorsinA did not impact these processes, even after co-expression of its recently identified interacting partner, printor. In light of these findings, we propose that yeast may lack an additional cofactor necessary for torsinA function or proteins required for essential post-translational modifications of torsinA. Alternatively, torsinA may not function in endoplasmic reticulum protein homeostasis. The strains and assays we describe may provide useful tools for identifying and investigating these possibilities and are freely available .Full Text

Valastyan, S., Chang, A., Benaich, N., Reinhardt, F., and Weinberg, R.A. (2011). Activation of Mir-31 Function in Already-Established Metastases Elicits Metastatic Regression. Genes Dev 25, 646-659. Distant metastases, rather than the primary tumors from which these lesions arise, are responsible for >90% of carcinoma-associated mortality. Many patients already harbor disseminated tumor cells in their bloodstream, bone marrow, and distant organs when they initially present with cancer. Hence, truly effective anti-metastatic therapeutics must impair the proliferation and survival of already-established metastases. Here, we assess the therapeutic potential of acutely expressing the microRNA miR-31 in already-formed breast cancer metastases. Activation of miR-31 in established metastases elicits metastatic regression and prolongs survival. Remarkably, even brief induction of miR-31 in macroscopic pulmonary metastases diminishes metastatic burden. In contrast, acute miR-31 expression fails to affect primary mammary tumor growth. miR-31 triggers metastatic regression in the lungs by eliciting cell cycle arrest and apoptosis; these responses occur specifically in metastases and can be explained by miR-31-mediated suppression of integrin-alpha5, radixin, and RhoA. Indeed, concomitant re-expression of these three proteins renders already-seeded pulmonary metastases refractory to miR-31-conferred regression. Upon miR-31 activation, Akt-dependent signaling is attenuated and the proapoptotic molecule Bim is induced; these effects occur in a metastasis-specific manner in pulmonary lesions and are abrogated by concurrent re-expression of integrin-alpha5, radixin, and RhoA. Collectively, these findings raise the possibility that intervention strategies centered on restoring miR-31 function may prove clinically useful for combating metastatic disease. Full Text.

Valastyan, S., and Weinberg, R.A. (2011). Roles for Micrornas in the Regulation of Cell Adhesion Molecules. J Cell Sci 124, 999-1006. Maintenance of appropriate cell adhesion is crucial for normal cellular and organismal homeostasis. Certain microRNAs have recently been found capable of regulating molecules that oversee the fundamental cell biological events that drive cellular adhesion. It is now apparent that microRNAs play crucial roles in the great majority of biochemical pathways that contribute to normal cell adhesion. In this Commentary, we describe the latest advances within this still-emerging field, and highlight connections between the deregulation of microRNAs that affect cell-adhesion-associated molecules and the pathogenesis of several human diseases. Current evidence suggests that the ability of certain microRNAs - notably miR-17, miR-29, miR-31, miR-124 and miR-200 - to pleiotropically regulate multiple molecular components of the cell adhesion machinery endows these microRNAs with the capacity to function as key modulators of adhesion-associated processes. This, in turn, holds important implications for our understanding of both the basic biology of cell adhesion and the etiology of multiple pathological conditions. Full Text.

VanderVeen, A.G., Schorpp, K., Schlieker, C., Buti, L., Damon, J.R., Spooner, E., Ploegh, H.L., and Jentsch, S. (2011). Role of the Ubiquitin-Like Protein Urm1 as a Noncanonical Lysine-Directed Protein Modifier. PNAS Published ahead of print January 5, 2011. The ubiquitin (Ub)-related modifier Urm1 functions as a sulfur carrier in tRNA thiolation by means of a mechanism that requires the formation of a thiocarboxylate at the C-terminal glycine residue of Urm1. However, whether Urm1 plays an additional role as a Ub-like protein modifier remains unclear. Here, we show that Urm1 is conjugated to lysine residues of target proteins and that oxidative stress enhances protein urmylation in both Saccharomyces cerevisiae and mammalian cells. Similar to ubiquitylation, urmylation involves a thioester intermediate and results in the formation of a covalent peptide bond between Urm1 and its substrates. In contrast to modification by canonical Ub-like modifiers, however, conjugation of Urm1 involves a C-terminal thiocarboxylate of the modifier. We have confirmed that the peroxiredoxin Ahp1 is such a substrate in S. cerevisiae and found that Urm1 targets a specific lysine residue of Ahp1 in vivo. In addition, we have identified several unique substrates in mammalian cells and show that Urm1 targets at least two pathways on oxidant treatment. First, Urm1 is appended to lysine residues of three components that function in its own pathway (i.e., MOCS3, ATPBD3, and CTU2). Second, Urm1 is conjugated to the nucleocytoplasmic shuttling factor cellular apoptosis susceptibility protein. Thus, Urm1 has a conserved dual role by integrating the functions of prokaryotic sulfur carriers with those of eukaryotic protein modifiers of the Ub family. PDF

VirreiraWinter, S., Niedelman, W., Jensen, K.D., Rosowski, E.E., Julien, L., Spooner, E., Caradonna, K., Burleigh, B.A., Saeij, J.P., Ploegh, H.L., and Frickel, EM (2011). Determinants of GBP Recruitment to Toxoplasma gondii Vacuoles and the Parasitic Factors That Control It. PLoS ONE 6(9): e24434..IFN-gamma is a major cytokine that mediates resistance against the intracellular parasite Toxoplasma gondii. The p65 guanylate-binding proteins (GBPs) are strongly induced by IFN-gamma. We studied the behavior of murine GBP1 (mGBP1) upon infection with T. gondii in vitro and confirmed that IFN-gamma-dependent re-localization of mGBP1 to the parasitophorous vacuole (PV) correlates with the virulence type of the parasite. We identified three parasitic factors, ROP16, ROP18, and GRA15 that determine strain-specific accumulation of mGBP1 on the PV. These highly polymorphic proteins are held responsible for a large part of the strain-specific differences in virulence. Therefore, our data suggest that virulence of T. gondii in animals may rely in part on recognition by GBPs. However, phagosomes or vacuoles containing Trypanosoma cruzi did not recruit mGBP1. Co-immunoprecipitation revealed mGBP2, mGBP4, and mGBP5 as binding partners of mGBP1. Indeed, mGBP2 and mGBP5 co-localize with mGBP1 in T. gondii-infected cells. T. gondii thus elicits a cell-autonomous immune response in mice with GBPs involved. Three parasitic virulence factors and unknown IFN-gamma-dependent host factors regulate this complex process. Depending on the virulence of the strains involved, numerous GBPs are brought to the PV as part of a large, multimeric structure to combat T. gondii. Full Text.

VonStetina, J.R., and Orr-Weaver, T.L. (2011). Developmental Control of Oocyte Maturation and Egg Activation in Metazoan Models Cold Spring Harbor Perspectives in Biology Jun 27;. [Epub ahead of print] Production of functional eggs requires meiosis to be coordinated with developmental signals. Oocytes arrest in prophase I to permit oocyte differentiation, and in most animals, a second meiotic arrest links completion of meiosis to fertilization. Comparison of oocyte maturation and egg activation between mammals, Caenorhabditis elegans, and Drosophila reveal conserved signaling pathways and regulatory mechanisms as well as unique adaptations for reproductive strategies. Recent studies in mammals and C. elegans show the role of signaling between surrounding somatic cells and the oocyte in maintaining the prophase I arrest and controlling maturation. Proteins that regulate levels of active Cdk1/cyclin B during prophase I arrest have been identified in Drosophila. Protein kinases play crucial roles in the transition from meiosis in the oocyte to mitotic embryonic divisions in C. elegans and Drosophila. Here we will contrast the regulation of key meiotic events in oocytes.

Wagner ,D.E., Wang, I.E., and Reddien, P.W. (2011). Clonogenic Neoblasts Are Pluripotent Adult Stem Cells That Underlie Planarian Regeneration. Science 332, 811-816. Pluripotent cells in the embryo can generate all cell types, but lineage-restricted cells are generally thought to replenish adult tissues. Planarians are flatworms and regenerate from tiny body fragments, a process requiring a population of proliferating cells (neoblasts). Whether regeneration is accomplished by pluripotent cells or by the collective activity of multiple lineage-restricted cell types is unknown. We used ionizing radiation and single-cell transplantation to identify neoblasts that can form large descendant-cell colonies in vivo. These clonogenic neoblasts (cNeoblasts) produce cells that differentiate into neuronal, intestinal, and other known postmitotic cell types and are distributed throughout the body. Single transplanted cNeoblasts restored regeneration in lethally irradiated hosts. We conclude that broadly distributed, adult pluripotent stem cells underlie the remarkable regenerative abilities of planarians. Full Text.

WahlJensen, V., Cann, J.A., Rubins, K.H., Huggins, J.W., Fisher, R.W., Johnson, A.J., de Kok-Mercado, F., Larsen, T., Raymond, J.L., Hensley, L.E. ,Jahrling PB.. (2011). Progression of Pathogenic Events in Cynomolgus Macaques Infected with Variola Virus. PLoS One 2011;6(10):e24832.Smallpox, caused by variola virus (VARV), is a devastating human disease that affected millions worldwide until the virus was eradicated in the 1970 s. Subsequent cessation of vaccination has resulted in an immunologically naive human population that would be at risk should VARV be used as an agent of bioterrorism. The development of antivirals and improved vaccines to counter this threat would be facilitated by the development of animal models using authentic VARV. Towards this end, cynomolgus macaques were identified as adequate hosts for VARV, developing ordinary or hemorrhagic smallpox in a dose-dependent fashion. To further refine this model, we performed a serial sampling study on macaques exposed to doses of VARV strain Harper calibrated to induce ordinary or hemorrhagic disease. Several key differences were noted between these models. In the ordinary smallpox model, lymphoid and myeloid hyperplasias were consistently found whereas lymphocytolysis and hematopoietic necrosis developed in hemorrhagic smallpox. Viral antigen accumulation, as assessed immunohistochemically, was mild and transient in the ordinary smallpox model. In contrast, in the hemorrhagic model antigen distribution was widespread and included tissues and cells not involved in the ordinary model. Hemorrhagic smallpox developed only in the presence of secondary bacterial infections - an observation also commonly noted in historical reports of human smallpox. Together, our results support the macaque model as an excellent surrogate for human smallpox in terms of disease onset, acute disease course, and gross and histopathological lesions. Full Text.

Wan, M., Leavens, K.F., Saleh, D., Easton, R.M., Guertin, D.A., Peterson, T.R., Kaestner, K.H., Sabatini, D.M., and Birnbaum, M.J. (2011). Postprandial Hepatic Lipid Metabolism Requires Signaling through Akt2 Independent of the Transcription Factors FoxA2, FoxO1, and SREBP1c. Cell Metabolism 14, 516-527.Under conditions of obesity and insulin resistance, the serine/threonine protein kinase Akt/PKB is required for lipid accumulation in liver. Two forkhead transcription factors, FoxA2 and FoxO1, have been suggested to function downstream of and to be negatively regulated by Akt and are proposed as key determinants of hepatic triglyceride content. In this study, we utilize genetic loss of function experiments to show that constitutive activation of neither FoxA2 nor FoxO1 can account for the protection from steatosis afforded by deletion of Akt2 in liver. Rather, another downstream target positively regulated by Akt, the mTORC1 complex, is required in vivo for de novo lipogenesis and Srebp1c expression. Nonetheless, activation of mTORC1 and SREBP1c is not sufficient to drive postprandial lipogenesis in the absence of Akt2. These data show that insulin signaling through Akt2 promotes anabolic lipid metabolism independent of Foxa2 or FoxO1 and through pathways additional to the mTORC1-dependent activation of SREBP1c. Full Text.

Wang, J., Kumar, R.M., Biggs, V.J., Lee, H., Chen, Y., Kagey, M.H., Young, R.A., and Abate-Shen, C. (2011). The Msx1 Homeoprotein Recruits Polycomb to the Nuclear Periphery during Development. Developmental Cell 21, 575-588.Control of gene expression during development requires the concerted action of sequence-specific transcriptional regulators and epigenetic modifiers, which are spatially coordinated within the nucleus through mechanisms that are poorly understood. Here we show that transcriptional repression by the Msx1 homeoprotein in myoblast cells requires the recruitment of Polycomb to target genes located at the nuclear periphery. Target genes repressed by Msx1 display an Msx1-dependent enrichment of Polycomb-directed trimethylation of lysine 27 on histone H3 (H3K27me3). Association of Msx1 with the Polycomb complex is required for repression and regulation of myoblast differentiation. Furthermore, Msx1 promotes a dynamic spatial redistribution of the H3K27me3 repressive mark to the nuclear periphery in myoblast cells and the developing limb in vivo. Our findings illustrate a hitherto unappreciated spatial coordination of transcription factors with the Polycomb complex for appropriate regulation of gene expression programs during development. Full Text.

Weinberg, D.E., Nakanishi, K., Patel, D.J., and Bartel, D.P. (2011). The inside-out Mechanism of Dicers from Budding Yeasts. Cell 146, 262-276. The Dicer ribonuclease III (RNase III) enzymes process long double-stranded RNA (dsRNA) into small interfering RNAs (siRNAs) that direct RNA interference. Here, we describe the structure and activity of a catalytically active fragment of Kluyveromyces polysporus Dcr1, which represents the noncanonical Dicers found in budding yeasts. The crystal structure revealed a homodimer resembling that of bacterial RNase III but extended by a unique N-terminal domain, and it identified additional catalytic residues conserved throughout eukaryotic RNase III enzymes. Biochemical analyses showed that Dcr1 dimers bind cooperatively along the dsRNA substrate such that the distance between consecutive active sites determines the length of the siRNA products. Thus, unlike canonical Dicers, which successively remove siRNA duplexes from the dsRNA termini, budding-yeast Dicers initiate processing in the interior and work outward. The distinct mechanism of budding-yeast Dicers establishes a paradigm for natural molecular rulers and imparts substrate preferences with ramifications for biological function. Full Text.

White, R.R., Miyata, S., Papa, E., Spooner, E., Gounaris, K., Selkirk, M.E., and Artavanis-Tsakonas, K. (2011). Characterisation of the Trichinella spiralis Deubiquitinating Enzyme, TsUCH37, an Evolutionarily Conserved Proteasome Interaction Partner. Plos Neglected Tropical Diseases 2011 Oct;5(10):e1340 .Background: Trichinella spiralis is a zoonotic parasitic nematode that causes trichinellosis, a disease that has been identified on all continents except Antarctica. During chronic infection, T. spiralis larvae infect skeletal myofibres, severely disrupting their differentiation state. Methodology and Results: An activity-based probe, HA-Ub-VME, was used to identify deubiquitinating enzyme (DUB) activity in lysate of T. spiralis L1 larvae. Results were analysed by immuno-blot and immuno-precipitation, identifying a number of potential DUBs. Immuno-precipitated proteins were subjected to LC/MS/MS, yielding peptides with sequence homology to 5 conserved human DUBs: UCH-L5, UCH-L3, HAUSP, OTU 6B and Ataxin-3. The predicted gene encoding the putative UCH-L5 homologue, TsUCH37, was cloned and recombinant protein was expressed and purified. The deubiquitinating activity of this enzyme was verified by Ub-AMC assay. Co-precipitation of recombinant TsUCH37 showed that the protein associates with putative T. spiralis proteasome components, including the yeast Rpn13 homologue ADRM1. In addition, the UCH inhibitor LDN-57444 exhibited specific inhibition of recombinant TsUCH37 and reduced the viability of cultured L1 larvae. Conclusions: This study reports the identification of the first T. spiralis DUB, a cysteine protease that is putatively orthologous to the human protein, hUCH-L5. Results suggest that the interaction of this protein with the proteasome has been conserved throughout evolution. We show potential for the use of inhibitor compounds to elucidate the role of UCH enzymes in T. spiralis infection and their investigation as therapeutic targets for trichinellosis. Full Text.

Whitesell, L., and Lin, N.U. (2011). HSP90 as a platform for the assembly of more effective cancer chemotherapy. Biochim Biophys Acta.Dec 24. [Epub ahead of print]. Since initial discovery of the first HSP90 inhibitor over a decade and a half ago, tremendous progress has been made in developing potent and selective compounds with which to target this chaperone in the treatment of cancers. These compounds have been invaluable in dissecting how HSP90 supports the dramatic alterations in cellular physiology that constitute the malignant phenotype and give rise to the clinical manifestations of diverse cancers. Unfortunately, single agent activity for HSP90 inhibitors has been disappointingly modest against recurrent, refractory cancers in most of the clinical trials that have been reported to date. This problem could be due to pharmacological limitations of the first-generation inhibitors that have been most extensively studied. But we suggest it may well be intrinsic to the target itself. This review will focus on how the utilization of HSP90 by cancer cells might be targeted to enhance the activity of other anticancer drugs while at the same time limiting the ability of advanced cancers to adapt and evolve drug resistance; the net result being more durable disease control. A better understanding of these fundamental issues will surely make the ongoing clinical development of HSP90 inhibitors as anticancer drugs less empiric, more efficient and hopefully more successful. Full Text.

Witte, M.D., van der Marel, G.A., Aerts, J., and Overkleeft, H.S. (2011). Irreversible inhibitors and activity-based probes as research tools in chemical glycobiology. Organic & Biomolecular Chemistry 9, 5908-5926.In this review, we will discuss the enzymes that are involved in the synthesis and degradation of glycoconjugates and we will give an overview of the inhibitors and activity-based probes (ABPs) that have been used to study these. Following discussion of some general aspects of the biosynthesis and degradation of N-linked glycoproteins, attention is focused on the enzymes that hydrolyze the protein-carbohydrate linkage, peptide N-glycanase and glycosylasparaginase and their mechanism. We then focus on the biosynthesis of O-linked glycoproteins and glycolipids and in particular on the enzymes that hydrolyze the interglycosidic linkages in these, the glycosidases. Some important mechanism-based glycosidase inhibitors that form a covalent bond with the targeted enzyme(s), their corresponding ABPs and their application to study this class of enzymes are highlighted. Finally, alternative pathways for degradation of glycoconjugates and an ABP-based strategy to study these will be discussed. PDF

Wong, P., Hattangadi, S.M., Cheng, A.W., Frampton, G.M., Young, R.A., and Lodish, H.F. (2011). Gene induction and repression during terminal erythropoiesis are mediated by distinct epigenetic changes. Blood Aug 22. [Epub ahead of print] .It is unclear how epigenetic changes regulate the induction of erythroid-specific genes during terminal erythropoiesis. Here we use global mRNA sequencing (mRNA-seq) and chromatin immunoprecipitation coupled to high-throughput sequencing (CHIP-seq) to investigate the changes that occur in mRNA levels, RNA Polymerase II (Pol II) occupancy and multiple post-translational histone modifications when erythroid progenitors differentiate into late erythroblasts. Among genes induced during this developmental transition, there was an increase in the occupancy of Pol II, the activation marks H3K4me2, H3K4me3, H3K9Ac and H4K16Ac, and the elongation methylation mark H3K79me2. In contrast, genes that were repressed during differentiation showed relative decreases in H3K79me2 levels yet had levels of Pol II binding and active histone marks similar to those in erythroid progenitors. We also found that relative changes in histone modification levels-in particular, H3K79me2 and H4K16ac-were most predictive of gene expression patterns. Our results suggest that in terminal erythropoiesis both promoter and elongation-associated marks contribute to the induction of erythroid genes, while gene repression is marked by changes in histone modifications mediating Pol II elongation. Our data maps the epigenetic landscape of terminal erythropoiesis and suggests that control of transcription elongation regulates gene expression during terminal erythroid differentiation. PDF

Young, R.A. (2011). Control of the Embryonic Stem Cell State. Cell 144, 940-954. Embryonic stem cells and induced pluripotent stem cells hold great promise for regenerative medicine. These cells can be propagated in culture in an undifferentiated state but can be induced to differentiate into specialized cell types. Moreover, these cells provide a powerful model system for studies of cellular identity and early mammalian development. Recent studies have provided insights into the transcriptional control of embryonic stem cell state, including the regulatory circuitry underlying pluripotency. These studies have, as a consequence, uncovered fundamental mechanisms that control mammalian gene expression, connect gene expression to chromosome structure, and contribute to human disease. Full Text.

Youngsaye, W., Vincent, B., Hartland, C.L., Morgan, B.J., Buhrlage, S.J., Johnston, S., Bittker, J.A., MacPherson, L., Dandapani, S., Palmer,M Whitesell L, Lindquist S, Schreiber SL, Munoz B.. (2011). Piperazinyl quinolines as chemosensitizers to increase fluconazole susceptibility of Candida albicans clinical isolates. Bioorganic & Medicinal Chemistry Letters 21, 5502-5505.The effectiveness of the potent antifungal drug fluconazole is being compromised by the rise of drug-resistant fungal pathogens. While inhibition of Hsp90 or calcineurin can reverse drug resistance in Candida, such inhibitors also impair the homologous human host protein and fungal-selective chemosensitizers remain rare. The MLPCN library was screened to identify compounds that selectively reverse fluconazole resistance in a Candida albicans clinical isolate, while having no antifungal activity when administered as a single agent. A piperazinyl quinoline was identified as a new small-molecule probe (ML189) satisfying these criteria. Full Text.

Zhang, L.B., Flygare, J., Wong, P., Lim, B., and Lodish, H.F. (2011). Mir-191 Regulates Mouse Erythroblast Enucleation by Down-Regulating Riok3 and Mxi1. Genes & Development 25, 119-124. Using RNA-seq technology, we found that the majority of microRNAs (miRNAs) present in CFU-E erythroid progenitors are down-regulated during terminal erythroid differentiation. Of the developmentally down-regulated miRNAs, ectopic overexpression of miR-191 blocks erythroid enucleation but has minor effects on proliferation and differentiation. We identified two erythroid-enriched and developmentally up-regulated genes, Riok3 and Mxi1, as direct targets of miR-191. Knockdown of either Riok3 or Mxi1 blocks enucleation, and either physiological overexpression of miR-191 or knockdown of Riok3 or Mxi1 blocks chromatin condensation. Thus, down-regulation of miR-191 is essential for erythroid chromatin condensation and enucleation by allowing up-regulation of Riok3 and Mxi1. Full Text.

Zoncu, R., Bar-Peled, L., Efeyan, A., Wang, S., Sancak, Y., and Sabatini, D.M. (2011). mTORC1 senses lysosomal amino acids through an inside-out mechanism that requires the vacuolar H-ATPase. Science 334, 678-683.The mTOR complex 1 (mTORC1) protein kinase is a master growth regulator that is stimulated by amino acids. Amino acids activate the Rag guanosine triphosphatases (GTPases), which promote the translocation of mTORC1 to the lysosomal surface, the site of mTORC1 activation. We found that the vacuolar H(+)-adenosine triphosphatase ATPase (v-ATPase) is necessary for amino acids to activate mTORC1. The v-ATPase engages in extensive amino acid-sensitive interactions with the Ragulator, a scaffolding complex that anchors the Rag GTPases to the lysosome. In a cell-free system, ATP hydrolysis by the v-ATPase was necessary for amino acids to regulate the v-ATPase-Ragulator interaction and promote mTORC1 translocation. Results obtained in vitro and in human cells suggest that amino acid signaling begins within the lysosomal lumen. These results identify the v-ATPase as a component of the mTOR pathway and delineate a lysosome-associated machinery for amino acid sensing. Full Text.

Zoncu, R., and Sabatini, D.M. (2011). Cell Biology. The Tascc of Secretion. Science 332, 923-925.The oncogene-induced activation of signaling pathways involving the tumor suppressor proteins p53 and retinoblastoma is likely an important mechanism for preventing the proliferation of potential cancer cells. Full Text.

Zoncu, R., Efeyan, A., and Sabatini, D.M. (2011). Mtor: From Growth Signal Integration to Cancer, Diabetes and Ageing. Nature Reviews Molecular Cell Biology 12, 21-35. In all eukaryotes, the target of rapamycin (TOR) signalling pathway couples energy and nutrient abundance to the execution of cell growth and division, owing to the ability of TOR protein kinase to simultaneously sense energy, nutrients and stress and, in metazoans, growth factors. Mammalian TOR complex 1 (mTORC1) and mTORC2 exert their actions by regulating other important kinases, such as S6 kinase (S6K) and Akt. In the past few years, a significant advance in our understanding of the regulation and functions of mTOR has revealed the crucial involvement of this signalling pathway in the onset and progression of diabetes, cancer and ageing. Full Text.

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