The following alphabetical list represents papers published in 2013 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.
2013 Titles :
-Acidic Amino Acid Residues in the Juxtamembrane Region of the Nucleotide-Sensing TLRs Are Important for UNC93B1 Binding and Signaling .Kim
-Against storytelling of scientific results. Katz
-Amino Acid Copolymers That Alleviate Experimental Autoimmune Encephalomyelitis In Vivo Interact with Heparan Sulfates and Glycoprotein 96 in APC Koenig
-Analysis of In Vitro Insulin-Resistance Models and Their Physiological Relevance to In Vivo Diet-Induced Adipose Insulin Resistance. Lo
-Antigen-specific B-cell receptor sensitizes B cells to infection by influenza virus.
Dougan
-ATR/Mec1 prevents lethal meiotic recombination initiation on partially replicated chromosomes in budding yeast.
Blitzblau
-Autoregulation of the Mechanistic Target of Rapamycin (mTOR) Complex 2 Integrity Is Controlled by an ATP-dependent Mechanism.
Chen3
-Beyond Secondary Structure: Primary-Sequence Determinants License Pri-miRNA Hairpins for Processing. Auyeung
-Blessings in disguise: biological benefits of prion-like mechanisms. Newby
-The bromodomain protein Brd4 insulates chromatin from DNA damage signalling. Floyd
-Bruton's Tyrosine Kinase (BTK) and Vav1 Contribute to Dectin1-Dependent Phagocytosis of Candida albicans in Macrophages. Strijbis
-Calorie restriction in humans inhibits the PI3K/AKT pathway and induces a younger transcription profile.
Mercken
-Candida albicans induces pro-inflammatory and anti-apoptotic signals in macrophages as revealed by quantitative proteomics and phosphoproteomics.
RealesCalderon
-A catalytically inactive mutant of the deubiquitylase YOD-1 enhances antigen cross-presentation.
Sehrawat
-Catch-and-Release Probes Applied to Semi-Intact Cells Reveal Ubiquitin-Specific Protease Expression in Chlamydia trachomatis Infection Claessen
-CDK-dependent phosphorylation and nuclear exclusion coordinately control kinetochore assembly state.
Gascoigne
-CENP-T provides a structural platform for outer kinetochore assembly.
Nishino
-A Central Role for mTOR in Lipid Homeostasis.
Lamming3
-Centromere proteins CENP-C and CAL1 functionally interact in meiosis for centromere clustering, pairing, and chromosome segregation. Unhavaithaya2
-Chaperones as thermodynamic sensors of drug-target interactions reveal kinase inhibitor specificities in living cells.
Taipale
-Characterization of Torin2, an ATP-Competitive Inhibitor of mTOR, ATM, and ATR .Liu
-The chlamydial OTU domain-containing protein ChlaOTU is an early type III secretion effector targeting ubiquitin and NDP5.
Furtado
-Cinnamides as selective small-molecule inhibitors of a cellular model of breast cancer stem cells. Germain
-Clinical experience with fetal hemoglobin induction therapy in patients with beta-thalassemia. Musallam
-Cloning Expeditions: Risky but Rewarding.
Lodish
-Combined Deficiency of Tet1 and Tet2 Causes Epigenetic Abnormalities but Is Compatible with Postnatal Development.
Dawlaty
-A comparative perspective on lipid storage in animals.
Birsoy
-Compartmentalization of metabolic pathways in yeast mitochondria improves the production of branched-chain alcohols. Avalos
-Comprehensive analysis of imprinted genes in maize reveals allelic variation for imprinting and limited conservation with other species. Waters
-Cortical Dynein and asymmetric membrane elongation coordinately position the spindle in anaphase.
Kiyomitsu
-A CREB3-ARF4 signalling pathway mediates the response to Golgi stress and susceptibility to pathogens. Reiling
-Cryptic variation in morphological evolution: HSP90 as a capacitor for loss of eyes in cavefish.
Rohner
-Deletion of microRNA-155 reduces autoantibody responses and alleviates lupus-like disease in the Fas(lpr) mouse.
Thai
-Different Roles for Tet1 and Tet2 Proteins in Reprogramming-Mediated Erasure of Imprints Induced by EGC Fusion.
Piccolo
-Discovery and characterization of super-enhancer-associated dependencies in diffuse large B cell lymphoma .Chapuy
-Divergent transcription of long noncoding RNA/mRNA gene pairs in embryonic stem cells. Sigova
-Dose-dependent roles for canonical Wnt signalling in de novo crypt formation and cell cycle properties of the colonic epithelium. Hirata
-Drosophila Embryonic Cell Cycle Mutants. Unhavaithaya
-ElaD Peptidase.
Ploegh3
-Efficiency of siRNA delivery by lipid nanoparticles is limited by endocytic recycling.
Sahay
-Elegant biochemistry, chaotic origin.
Weng2
-EpCAM regulates cell cycle progression via control of cyclin D1 expression.
ChavesPerez
-The epigenetics of epithelial-mesenchymal plasticity in cancer. Tam2
-Escherichia coli Virulence Protein NleH1 Interaction with the v-Crk Sarcoma Virus CT10 Oncogene-like Protein (CRKL) Governs NleH1 Inhibition of the Ribosomal Protein 53 (RPS3)/Nuclear Factor kappa B (NF-kappa B) Pathway. Pham
-Esperanto for histones: CENP-A, not CenH3, is the centromeric histone H3 variant.
Earnshaw
-The evolutionary paths towards complexity: a metabolic perspective.
Weng
-Fetal Hepatic Progenitors Support Long-term Expansion of Hematopoietic Stem Cells.
Chou
-Financing drug discovery for orphan disease.
Fagnan
-Fitness Trade-offs Restrict the Evolution of Resistance to Amphotericin B.
Vincent
-The Folliculin Tumor Suppressor Is a GAP for the RagC/D GTPases That Signal Amino Acid Levels to mTOCR1. Tsun
-The functions and consequences of force at kinetochores.
Rago.
-Fundamental differences in endoreplication in mammals and Drosophila revealed by analysis of endocycling and endomitotic cells. Sher
-Gata4 is required for formation of the genital ridge in mice. Hu2
-Genome-wide association studies of hematologic phenotypes: a window into human hematopoiesis.
Sankaran
-Genome-wide localization of small molecules.
Anders
-Genome-Wide Methylation Analysis and Epigenetic Unmasking Identify Tumor Suppressor Genes in Hepatocellular Carcinoma.
Revill
-A Genome-wide siRNA Screen Identifies Proteasome Addiction as a Vulnerability of Basal-like Triple-Negative Breast Cancer Cells. Petrocca
-Genomic Imprinting: Parental Lessons from Plants.
Gehring
-Global discovery of erythroid long non-coding RNAs reveals novel regulators of red cell maturation.
AlvarezDominguez
-Global Transcriptional and Translational Repression in Human-Embryonic-Stem-Cell-Derived Rett Syndrome Neurons.
Li
-Hepatic signaling by the mechanistic target of rapamycin complex 2 (mTORC2).
Lamming4
-Heritable remodeling of yeast multicellularity by an environmentally responsive prion.
Holmes
-High-Resolution Mapping Reveals a Conserved, Widespread, Dynamic mRNA Methylation Program in Yeast Meiosis.Schwartz
-Identification and Rescue of alpha-Synuclein Toxicity in Parkinson Patient-Derived Neurons.
Chung
-Identifying personal genomes by surname inference. Gymrek
-Impaired Autophagy in the Lipid-Storage Disorder Niemann-Pick Type C1 Disease. Sarkar2
-Independent specialization of the human and mouse X chromosomes for the male germ line. Mueller
-Induced dicentric chromosome formation promotes genomic rearrangements and tumorigenesis.
Gascoigne2
-Intact sphingomyelin biosynthetic pathway is essential for intracellular transport of influenza virus glycoproteins.
Tafesse
-An Integrin-Linked Machinery of Cytoskeletal Regulation that Enables Experimental Tumor Initiation and Metastatic Colonization.
Shibue
-Intrachromosomal homologous recombination between inverted amplicons on opposing Y-chromosome arms. Lange
-Isogenic Human iPSC Parkinson's Model Shows Nitrosative Stress-Induced Dysfunction in MEF2-PGC1alpha Transcription. Ryan
-Keratins control intercellular adhesion involving PKC-alpha-mediated desmoplakin phosphorylation.
Kroger
-Let-7 represses Nr6a1 and a mid-gestation developmental program in adult fibroblast
. Gurtan
-The Ligand Binding Domain of GCNF Is Not Required for Repression of Pluripotency Genes in Mouse Fetal Ovarian Germ Cells.
Okumura
-lincRNAs: Genomics, Evolution, and Mechanisms.
Ulitsky
-Lin28: time for tissue repair.
Reddien2
-Logic of the immune system.
Ploegh4
-Long noncoding RNAs regulate adipogenesis.
Sun
-LoQAtE--Localization and Quantitation ATlas of the yeast proteomE. A new tool for multiparametric dissection of single-protein behavior in response to biological perturbations in yeast.
Breker2
-Loss of the Keratin Cytoskeleton Is Not Sufficient to Induce Epithelial Mesenchymal Transition in a Novel KRAS Driven Sporadic Lung Cancer Mouse Model. Konig
-Mapping differential interactomes by affinity purification coupled with data-independent mass spectrometry acquisition. Lambert
-Master transcription factors and mediator establish super-enhancers at key cell identity gene. Whyte
-Mechanisms and models of somatic cell reprogramming.
Buganim
-MicroRNA networks regulate development of brown adipocyte.
Trajkovski
-MicroRNA-126-mediated control of cell fate in B-cell myeloid progenitors as a potential alternative to transcriptional factors.
Okuyama
-MiR-150 Blocks MLL-AF9 Associated Leukemia By Repressing Multiple Oncogenes.
Bousquet
-miR-221 redirects precursor B cells to the bone marrow and regulates their residence.
Knoll
-ML212: A small-molecule probe for investigating fluconazole resistance mechanisms in Candida albicans. Youngsaye
-Monovalent and Multivalent Ligation of the B Cell Receptor Exhibit Differential Dependence upon Syk and Src Family Kinases. Mukherjee
-Moving education forward, again!
Lewitter
-mTORC1 phosphorylation sites encode their sensitivity to starvation and rapamycin.
Kang
-Multiple Structural Maintenance of Chromosome Complexes at Transcriptional Regulatory Elements.
Dowen
-Multiplexed activation of endogenous genes by CRISPR-on, an RNA-guided transcriptional activator system . Cheng
-Muscle Cells Provide Instructions for Planarian Regeneration. Witchley
-No bull : Upholding community standards in public sharing of biological datasets. Hughes
-A novel single-cell screening platform reveals proteome plasticity during yeast stress responses.
Breker
-Nutrients and growth factors in mTORC1 activation. Efeyan
-One-Step Generation of Mice Carrying Mutations in Multiple Genes by CRISPR/Cas-Mediated Genome Engineering. Wang
-One-Step Generation of Mice Carrying Reporter and Conditional Alleles by CRISPR/Cas-Mediated Genome Engineering.
Yang
-Online on-ramps.
Sive
-Oocyte differentiation is genetically dissociable from meiosis in mice.
Dokshin
-Orthogonal Labeling of M13 Minor Capsid Proteins with DNA to Self-Assemble End-to-End Multiphage Structure.
Hess
-PAX8: a sensitive and specific marker to identify cancer cells of ovarian origin for patients prior to neoadjuvant chemotherapy. Wang3
-Pbx is required for pole and eye regeneration in planarians. Chen
-Phenotypic screens for compounds that target the cellular pathologies underlying Parkinson's Disease. Tardiff
-PIKfyve, a Class III PI Kinase, Is the Target of the Small Molecular IL-12/IL-23 Inhibitor Apilimod and a Player in Toll-like Receptor Signaling.
Cai
-Poised Chromatin at the ZEB1 Promoter Enables Breast Cancer Cell Plasticity and Enhances Tumorigenicity. Chaffer
-Poised with purpose: Cell plasticity enhances tumorigenicity
. Marjanovic
-Production of unnaturally linked chimeric proteins using a combination of sortase-catalyzed transpeptidation and click chemistry.
Witte
-Profiling Short Tandem Repeats from Short Reads.
Gymrek2
-Profoundly different prion diseases in knock-in mice carrying single PrP codon substitutions associated with human diseases.
Jackson
-Protein Kinase C alpha Is a Central Signaling Node and Therapeutic Target for Breast Cancer Stem Cells Tam
-Pten-Null Tumors Cohabiting the Same Lung Display Differential AKT Activation and Sensitivity to Dietary Restriction. Curry
-Rapalogs and mTOR inhibitors as anti-aging therapeutics.
Lamming
-Rapamycin doses sufficient to extend lifespan do not compromise muscle mitochondrial content or endurance.
Ye
-Regulated ADAM17-dependent EGF family ligand release by substrate-selecting signaling pathways.
Dang
-Regulation of autophagy by mTOR-dependent and mTOR-independent pathways: autophagy dysfunction in neurodegenerative diseases and therapeutic application of autophagy enhancers.
Sarkar
-Regulation of mTORC1 and its impact on gene expression at a glance.
Laplante
-Remodeling a beta-peptide bundle .
Molski
-A Reporter Screen in a Human Haploid Cell Line Identifies CYLD as a Constitutive Inhibitor of NF-kappaB.
LeeCC
-Rictor regulates cell migration by suppressing RhoGDI2.
Agarwal
-The Role of Adipocyte XBP1 in Metabolic Regulation during Lactation.
Gregor
-Role of SWI/SNF in acute leukemia maintenance and enhancer-mediated Myc regulation. Shi
-Secretory cell expansion with aging: Risk for pelvic serous carcinogenesis. Li2
-Selective inhibition of tumor oncogenes by disruption of super-enhancers. Loven
-Sequester Impact on Biomedical Research.
Collins "
-A set of genes critical to development is epigenetically poised in mouse germ cells from fetal stages through completion of meiosis .Lesch
-Sialylneolacto-N-tetraose c (LSTc)-bearing Liposomal Decoys Capture Influenza A Virus. Hendricks
-Single-Cell Analysis Reveals that Expression of Nanog Is Biallelic and Equally Variable as that of Other Pluripotency Factors in Mouse ESCs.
Faddah
-SIRT1 collaborates with ATM and HDAC1 to maintain genomic stability in neuron.
Dobbin
-Site-specific C-terminal and internal loop labeling of proteins using sortase-mediated reactions.
Guimaraes
-Site-specific N-terminal labeling of proteins using sortase-mediated reactions.
Theile
-Smc5/6 coordinates formation and resolution of joint molecules with chromosome morphology to ensure meiotic divisions.
Copsey
-Snx3 Regulates Recycling of the Transferrin Receptor and Iron Assimilation.
Chen2
-Sortase-mediated modification of alphaDEC205 affords optimization of antigen presentation and immunization against a set of viral epitopes.
Swee
-SOX2 Co-Occupies Distal Enhancer Elements with Distinct POU Factors in ESCs and NPCs to Specify Cell State. Lodato
-Specialized progenitors and regeneration . Reddien
-Spindle assembly checkpoint robustness requires Tpr-mediated regulation of Mad1/Mad2 proteostasis. Schweizer
-Stalled Spliceosomes Are a Signal for RNAi-Mediated Genome Defense. Dumesic
-Stochastic cytokine expression induces mixed T helper cell States. Fang
-Stop, go, and evolve. Victora
-Super-Enhancers in the Control of Cell Identity and Disease.Hnisz
-The Switch from Fetal to Adult Hemoglobin. Sankaran2
-Synthetic shuffling and in vitro selection reveal the rugged adaptive fitness landscape of a kinase ribozyme. Curtis
-T follicular helper cell dynamics in germinal centers. Shulman
-The TAL1 complex targets the FBXW7 tumor suppressor by activating miR-223 in human T cell acute lymphoblastic leukemia. Mansour
-TALEN-mediated editing of the mouse Y chromosome. Wang2
-Tat engagement of p38 MAP kinase and IRF7 pathways leads to activation of interferon-stimulated genes in antigen-presenting cells. Kim2
-Tet1 is critical for neuronal activity-regulated gene expression and memory extinction. Rudenko
-TET1 plays an essential oncogenic role in MLL-rearranged leukemia. Huang
-3 ' UTR-isoform choice has limited influence on the stability and translational efficiency of most mRNAs in mouse fibroblasts. Spies
-Tight Coordination of Protein Translation and HSF1 Activation Supports the Anabolic Malignant State.Santagata
-Tissue absence initiates regeneration through Follistatin-mediated inhibition of Activin signaling. Gavino
-Toward eliminating HLA class I expression to generate universal cells from allogeneic donor. Torikai
-Transcriptional regulation and its misregulation in disease. Lee
-Transgenerational epigenetic inheritance: how important is it? Grossniklaus
-Transnuclear TRP1-specific CD8 T cells with high or low affinity TCRs show equivalent anti-tumor activity. Dougan2
-The TSC-mTOR pathway regulates macrophage polarization. Byles
-A Tumor suppressor complex with GAP activity for the Rag GTPases that signal amino acid sufficiency to mTORC1. BarPeled
-Tumor suppressor gene Rb is required for self-renewal of spermatogonial stem cells in mice. Hu
-Type I Interferon Imposes a TSG101/ISG15 Checkpoint at the Golgi for Glycoprotein Trafficking during Influenza Virus Infection. Sanyal
-Ubiquitin-specific Peptidases 14 and 6. Ploegh1
-UL36 Deubiquitinylating Peptidase .Ploegh2
-Widespread regulation of translation by elongation pausing in heat shock.Shalgi
-Yeast Reveal a "Druggable" Rsp5/Nedd4 Network that Ameliorates alpha-Synuclein Toxicity in Neurons. Tardiff2
-Young and old genetically heterogeneous HET3 mice on a rapamycin diet are glucose intolerant but insulin sensitive. Lamming2
-ZFP36L2 is required for self-renewal of early burst-forming unit erythroid progenitors. Zhang
2013 References:
Agarwal, N.K., Chen, C.H., Cho, H., Boulbes, D.R., Spooner, E., and Sarbassov, D.D. (2013). Rictor regulates cell migration by suppressing RhoGDI2. Oncogene 32, 2521-2526.Rictor and its binding partner Sin1 are indispensable components of mTORC2 (mammalian target of rapamycin complex 2). The mTORC2 signaling complex functions as the regulatory kinase of the distinct members of AGC kinase family known to regulate cell proliferation and survival. In the early chemotaxis studies in Dictyostelium, the rictor's ortholog has been identified as a regulator of cell migration. How rictor regulates cell migration is poorly characterized. Here we show that rictor regulates cell migration by controlling a potent inhibitor of Rho proteins known as the Rho-GDP dissociation inhibitor 2 (RhoGDI2). On the basis of on our proteomics study we identified that the rictor-dependent deficiency in cell migration is caused by upregulation of RhoGDI2 leading to a low activity of Rac and Cdc42. We found that a suppression of RhoGDI2 by rictor is not related to the Sin1 or raptor function that excludes a role of mTORC2 or mTORC1 in regulation of RhoGDI2. Our study reveals that rictor by suppressing RhoGDI2 promotes activity of the Rho proteins and cell migration. Full Text
AlvarezDominguez, J.R., Hu, W., Yuan, B., Shi, J., Park, S.S., Gromatzky, A.A., van Oudenaarden, A., and Lodish, H.F. (2013). Global discovery of erythroid long non-coding RNAs reveals novel regulators of red cell maturation. Blood November 7, 2013[Epub ahead of print] Erythropoiesis is regulated at multiple levels to ensure the proper generation of mature red cells under multiple physiological conditions. To probe the contribution of long non-coding RNAs (lncRNAs) to this process, we examined >1 billion RNA-Seq reads of polyadenylated and non-polyadenylated RNA from differentiating mouse fetal liver red blood cells, and identified 655 lncRNA genes including not only intergenic, antisense and intronic but also pseudogene and enhancer loci. Over 100 of these genes are previously unrecognized and highly erythroid-specific. By integrating genome-wide surveys of chromatin states, transcription factor occupancy, and tissue expression patterns, we identify multiple lncRNAs that are dynamically expressed during erythropoiesis, show epigenetic regulation and are targeted by key erythroid transcription factors GATA1, TAL1 or KLF1. We focus on 12 such candidates and find that they are nuclear-localized and exhibit complex developmental expression patterns. Depleting them severely impaired erythrocyte maturation, inhibiting cell size reduction and subsequent enucleation. One of them, alncRNA-EC7, is transcribed from an enhancer and is specifically needed for activation of the neighboring gene encoding BAND3. Our study provides an annotated catalog of erythroid lncRNAs, readily available through an online resource, and shows that diverse types of lncRNAs participate in the regulatory circuitry underlying erythropoiesis. Full Text
Anders, L., Guenther, M.G., Qi, J., Fan, Z.P., Marineau, J.J., Rahl, P.B., Loven, J., Sigova, A.A., Smith, W.B., Lee, T.I., James E Bradner
& Richard A Young. (2013). Genome-wide localization of small molecules. Nature Biotechnology Published online15 December 2013 .A vast number of small-molecule ligands, including therapeutic drugs under development and in clinical use, elicit their effects by binding specific proteins associated with the genome. An ability to map the direct interactions of a chemical entity with chromatin genome-wide could provide important insights into chemical perturbation of cellular function. Here we describe a method that couples ligand-affinity capture and massively parallel DNA sequencing (Chem-seq) to identify the sites bound by small chemical molecules throughout the human genome. We show how Chem-seq can be combined with ChIP-seq to gain unique insights into the interaction of drugs with their target proteins throughout the genome of tumor cells. These methods will be broadly useful to enhance understanding of therapeutic action and to characterize the specificity of chemical entities that interact with DNA or genome-associated proteins. Full Text
Auyeung, V.C., Ulitsky, I., McGeary, S.E., and Bartel, D.P. (2013). Beyond Secondary Structure: Primary-Sequence Determinants License Pri-miRNA Hairpins for Processing. Cell 152, 844-858.To use microRNAs to downregulate mRNA targets, cells must first process these approximately 22 nt RNAs from primary transcripts (pri-miRNAs). These transcripts form RNA hairpins important for processing, but additional determinants must distinguish pri-miRNAs from the many other hairpin-containing transcripts expressed in each cell. Illustrating the complexity of this recognition, we show that most Caenorhabditis elegans pri-miRNAs lack determinants required for processing in human cells. To find these determinants, we generated many variants of four human pri-miRNAs, sequenced millions that retained function, and compared them with the starting variants. Our results confirmed the importance of pairing in the stem and revealed three primary-sequence determinants, including an SRp20-binding motif (CNNC) found downstream of most pri-miRNA hairpins in bilaterian animals, but not in nematodes. Adding this and other determinants to C. elegans pri-miRNAs imparted efficient processing in human cells, thereby confirming the importance of primary-sequence determinants for distinguishing pri-miRNAs from other hairpin-containing transcripts. Full Text
Avalos, J.L., Fink, G.R., and Stephanopoulos, G. (2013). Compartmentalization of metabolic pathways in yeast mitochondria improves the production of branched-chain alcohols. Nature Biotechnology .Published online17 February 2013 Efforts to improve the production of a compound of interest in Saccharomyces cerevisiae have mainly involved engineering or overexpression of cytoplasmic enzymes. We show that targeting metabolic pathways to mitochondria can increase production compared with overexpression of the enzymes involved in the same pathways in the cytoplasm. Compartmentalization of the Ehrlich pathway into mitochondria increased isobutanol production by 260%, whereas overexpression of the same pathway in the cytoplasm only improved yields by 10%, compared with a strain overproducing enzymes involved in only the first three steps of the biosynthetic pathway. Subcellular fractionation of engineered strains revealed that targeting the enzymes of the Ehrlich pathway to the mitochondria achieves greater local enzyme concentrations. Other benefits of compartmentalization may include increased availability of intermediates, removing the need to transport intermediates out of the mitochondrion and reducing the loss of intermediates to competing pathways. Full Text
BarPeled, L., Chantranupong, L., Cherniack, A.D., Chen, W.W., Ottina, K.A., Grabiner, B.C., Spear, E.D., Carter, S.L., Meyerson, M., and Sabatini, D.M. (2013). A Tumor suppressor complex with GAP activity for the Rag GTPases that signal amino acid sufficiency to mTORC1. Science 340, 1100-1106.The mTOR complex 1 (mTORC1) pathway promotes cell growth in response to many cues, including amino acids, which act through the Rag guanosine triphosphatases (GTPases) to promote mTORC1 translocation to the lysosomal surface, its site of activation. Although progress has been made in identifying positive regulators of the Rags, it is unknown if negative factors also exist. Here, we identify GATOR as a complex that interacts with the Rags and is composed of two subcomplexes we call GATOR1 and -2. Inhibition of GATOR1 subunits (DEPDC5, Nprl2, and Nprl3) makes mTORC1 signaling resistant to amino acid deprivation. In contrast, inhibition of GATOR2 subunits (Mios, WDR24, WDR59, Seh1L, and Sec13) suppresses mTORC1 signaling, and epistasis analysis shows that GATOR2 negatively regulates DEPDC5. GATOR1 has GTPase-activating protein (GAP) activity for RagA and RagB, and its components are mutated in human cancer. In cancer cells with inactivating mutations in GATOR1, mTORC1 is hyperactive and insensitive to amino acid starvation, and such cells are hypersensitive to rapamycin, an mTORC1 inhibitor. Thus, we identify a key negative regulator of the Rag GTPases and reveal that, like other mTORC1 regulators, Rag function can be deregulated in cancer. Full Text
Birsoy, K., Festuccia, W.T., and Laplante, M. (2013). A comparative perspective on lipid storage in animals. J Cell Sci 126, 1541-1552.Lipid storage is an evolutionary conserved process that exists in all organisms from simple prokaryotes to humans. In Metazoa, long-term lipid accumulation is restricted to specialized cell types, while a dedicated tissue for lipid storage (adipose tissue) exists only in vertebrates. Excessive lipid accumulation is associated with serious health complications including insulin resistance, type 2 diabetes, cardiovascular diseases and cancer. Thus, significant advances have been made over the last decades to dissect out the molecular and cellular mechanisms involved in adipose tissue formation and maintenance. Our current understanding of adipose tissue development comes from in vitro cell culture and mouse models, as well as recent approaches to study lipid storage in genetically tractable lower organisms. This Commentary gives a comparative insight into lipid storage in uni- and multi-cellular organisms with a particular emphasis on vertebrate adipose tissue. We also highlight the molecular mechanisms and nutritional signals that regulate the formation of mammalian adipose tissue. Full Text
Blitzblau, H.G., and Hochwagen, A. (2013). ATR/Mec1 prevents lethal meiotic recombination initiation on partially replicated chromosomes in budding yeast. Elife 2, e00844.During gamete formation, crossover recombination must occur on replicated DNA to ensure proper chromosome segregation in the first meiotic division. We identified a Mec1/ATR- and Dbf4-dependent replication checkpoint in budding yeast that prevents the earliest stage of recombination, the programmed induction of DNA double-strand breaks (DSBs), when pre-meiotic DNA replication was delayed. The checkpoint acts through three complementary mechanisms: inhibition of Mer2 phosphorylation by Dbf4-dependent Cdc7 kinase, preclusion of chromosomal loading of Rec114 and Mre11, and lowered abundance of the Spo11 nuclease. Without this checkpoint, cells formed DSBs on partially replicated chromosomes. Importantly, such DSBs frequently failed to be repaired and impeded further DNA synthesis, leading to a rapid loss in cell viability. We conclude that a checkpoint-dependent constraint of DSB formation to duplicated DNA is critical not only for meiotic chromosome assortment, but also to protect genome integrity during gametogenesis. DOI:http://dx.doi.org/10.7554/eLife.00844.001. Full Text
Bousquet, M., Zhuang, G., Meng, C., Ying, W., Cheruku, P.S., Shie, A.T., Wang, S., Ge, G., Wong, P .Wang, G, Stephen S, , and Beiyan Zhoul. (2013). MiR-150 Blocks MLL-AF9 Associated Leukemia By Repressing Multiple Oncogenes. Mol Cancer Res.Published OnlineFirst on April 19, 2013. The microRNA miR-150, a critical regulator of hematopoiesis, is downregulated in MLL-associated leukemias. In this study, we demonstrate that miR-150 acts as a potent leukemic tumor suppressor by blocking the oncogenic properties of leukemic cells. By using murine MLL-AF9 transformed cells, we demonstrate that miR-150 ectopic expression inhibits blast colony formation and cell growth and increases apoptosis in vitro. More importantly, ectopic expression of miR-150 in transformed MLL-AF9 cells completely blocked the development of myeloid leukemia in transplanted mice. Gene expression profiles demonstrated that miR-150 altered the expression levels of more than 30 "stem cell signature" genes and many others that are involved in cancer pathways. Furthermore, we identified Cbl and Egr2, additional to the known target Myb, as bona fide miR-150 targets, and knockdown of these genes by shRNAs recapitulated the pro-apoptotic effects observed in leukemic cells with miR-150 ectopic expression. In conclusion, we demonstrate that miR-150 is a potent leukemic tumor suppressor that regulates multiple oncogenes, providing new key players for the development of therapeutic strategies to treat MLL-AF9 related leukemia. Full Text
Breker2, M., Gymrek, M., Moldavski, O., and Schuldiner, M. (2013). LoQAtE--Localization and Quantitation ATlas of the yeast proteomE. A new tool for multiparametric dissection of single-protein behavior in response to biological perturbations in yeast. Nucleic Acids Res.Published online: October 22, 2013 Living organisms change their proteome dramatically to sustain a stable internal milieu in fluctuating environments. To study the dynamics of proteins during stress, we measured the localization and abundance of the Saccharomyces cerevisiae proteome under various growth conditions and genetic backgrounds using the GFP collection. We created a database (DB) called 'LoQAtE' (Localizaiton and Quantitation Atlas of the yeast proteomE), available online at http://www.weizmann.ac.il/molgen/loqate/, to provide easy access to these data. Using LoQAtE DB, users can get a profile of changes for proteins of interest as well as querying advanced intersections by either abundance changes, primary localization or localization shifts over the tested conditions. Currently, the DB hosts information on 5330 yeast proteins under three external perturbations (DTT, H2O2 and nitrogen starvation) and two genetic mutations [in the chaperonin containing TCP1 (CCT) complex and in the proteasome]. Additional conditions will be uploaded regularly. The data demonstrate hundreds of localization and abundance changes, many of which were not detected at the level of mRNA. LoQAtE is designed to allow easy navigation for non-experts in high-content microscopy and data are available for download. These data should open up new perspectives on the significant role of proteins while combating external and internal fluctuations. Full Text
Breker, M., Gymrek, M., and Schuldiner, M. (2013). A novel single-cell screening platform reveals proteome plasticity during yeast stress responses. Journal of Cell Biology 200, 839-850.Uncovering the mechanisms underlying robust responses of cells to stress is crucial for our understanding of cellular physiology. Indeed, vast amounts of data have been collected on transcriptional responses in Saccharomyces cerevisiae. However, only a handful of pioneering studies describe the dynamics of proteins in response to external stimuli, despite the fact that regulation of protein levels and localization is an essential part of such responses. Here we characterized unprecedented proteome plasticity by systematically tracking the localization and abundance of 5,330 yeast proteins at single-cell resolution under three different stress conditions (DTT, H2O2, and nitrogen starvation) using the GFP-tagged yeast library. We uncovered a unique "fingerprint" of changes for each stress and elucidated a new response arsenal for adapting to radical environments. These include bet-hedging strategies, organelle rearrangement, and redistribution of protein localizations. All data are available for download through our online database, LOQATE (localization and quantitation atlas of yeast proteome). Full Text
Buganim, Y., Faddah, D.A., and Jaenisch, R. (2013). Mechanisms and models of somatic cell reprogramming. Nature Review Genetics 14, 427-439. Conversion of somatic cells to pluripotency by defined factors is a long and complex process that yields embryonic-stem-cell-like cells that vary in their developmental potential. To improve the quality of resulting induced pluripotent stem cells (iPSCs), which is important for potential therapeutic applications, and to address fundamental questions about control of cell identity, molecular mechanisms of the reprogramming process must be understood. Here we discuss recent discoveries regarding the role of reprogramming factors in remodelling the genome, including new insights into the function of MYC, and describe the different phases, markers and emerging models of reprogramming. Full Text
Byles, V., Covarrubias, A.J., Ben-Sahra, I., Lamming, D.W., Sabatini, D.M., Manning, B.D., and Horng, T. (2013). The TSC-mTOR pathway regulates macrophage polarization. Nature Communications 4, Article number: 2834 .Macrophages are able to polarize to proinflammatory M1 or alternative M2 states with distinct phenotypes and physiological functions. How metabolic status regulates macrophage polarization remains not well understood, and here we examine the role of mTOR (mechanistic target of rapamycin), a central metabolic pathway that couples nutrient sensing to regulation of metabolic processes. Using a mouse model in which myeloid lineage-specific deletion of Tsc1 (Tsc1(Delta/Delta)) leads to constitutive mTOR complex 1 (mTORC1) activation, we find that Tsc1(Delta/Delta) macrophages are refractory to IL-4-induced M2 polarization, but produce increased inflammatory responses to proinflammatory stimuli. Moreover, mTORC1-mediated downregulation of Akt signalling critically contributes to defective polarization. These findings highlight a key role for the mTOR pathway in regulating macrophage polarization, and suggest how nutrient sensing and metabolic status could be 'hard-wired' to control of macrophage function, with broad implications for regulation of type 2 immunity, inflammation and allergy. Full Text
Cai, X.M., Xu, Y.Y., Cheung, A.K., Tomlinson, R.C., Alcazar-Roman, A., Murphy, L., Billich, A., Zhang, B.L., Feng, Y., Klumpp, M, .Hidde L. Ploegh, et al. (2013). PIKfyve, a Class III PI Kinase, Is the Target of the Small Molecular IL-12/IL-23 Inhibitor Apilimod and a Player in Toll-like Receptor Signaling. Chemistry & Biology 20, 912-921.Toll-like receptor (TLR) signaling is a key component of innate immunity. Aberrant TLR activation leads to immune disorders via dysregulation of cytokine production, such as IL-12/IL-23. Herein, we identify and characterize PIKfyve, a lipid kinase, as a critical player in TLR signaling using apilimod as an affinity tool. Apilimod is a potent small molecular inhibitor of IL-12/IL-23 with an unknown target and has been evaluated in clinical trials for patients with Crohn's disease or rheumatoid arthritis. Using a chemical genetic approach, we show that it binds to PIKfyve and blocks its phosphotransferase activity, leading to selective inhibition of IL-12/IL-23p40. Pharmacological or genetic inactivation of PIKfyve is necessary and sufficient for suppression of IL-12/IL-23p40 expression. Thus, we have uncovered a phosphoinositide-mediated regulatory mechanism that controls TLR signaling. Full Text
Chaffer, C.L., Marjanovic, N.D., Lee, T., Bell, G., Kleer, C.G., Reinhardt, F., D'Alessio, A.C., Young, R.A., and Weinberg, R.A. (2013). Poised Chromatin at the ZEB1 Promoter Enables Breast Cancer Cell Plasticity and Enhances Tumorigenicity. Cell 154, 61-74.The recent discovery that normal and neoplastic epithelial cells re-enter the stem cell state raised the intriguing possibility that the aggressiveness of carcinomas derives not from their existing content of cancer stem cells (CSCs) but from their proclivity to generate new CSCs from non-CSC populations. Here, we demonstrate that non-CSCs of human basal breast cancers are plastic cell populations that readily switch from a non-CSC to CSC state. The observed cell plasticity is dependent on ZEB1, a key regulator of the epithelial-mesenchymal transition. We find that plastic non-CSCs maintain the ZEB1 promoter in a bivalent chromatin configuration, enabling them to respond readily to microenvironmental signals, such as TGFbeta. In response, the ZEB1 promoter converts from a bivalent to active chromatin configuration, ZEB1 transcription increases, and non-CSCs subsequently enter the CSC state. Our findings support a dynamic model in which interconversions between low and high tumorigenic states occur frequently, thereby increasing tumorigenic and malignant potential. Full Text
Chapuy, B., McKeown, M.R., Lin, C.Y., Monti, S., Roemer, M.G., Qi, J., Rahl, P.B., Sun, H.H., Yeda, K.T., Doench, J.G., Elaine Reichert, Andrew L. Kung ,Scott J. Rodig ,Richard A. Young ,Margaret A. Shipp ,and James E. Bradner (2013). Discovery and characterization of super-enhancer-associated dependencies in diffuse large B cell lymphoma. Cancer Cell 24, 777-790.Diffuse large B cell lymphoma (DLBCL) is a biologically heterogeneous and clinically aggressive disease. Here, we explore the role of bromodomain and extra-terminal domain (BET) proteins in DLBCL, using integrative chemical genetics and functional epigenomics. We observe highly asymmetric loading of bromodomain 4 (BRD4) at enhancers, with approximately 33% of all BRD4 localizing to enhancers at 1.6% of occupied genes. These super-enhancers prove particularly sensitive to bromodomain inhibition, explaining the selective effect of BET inhibitors on oncogenic and lineage-specific transcriptional circuits. Functional study of genes marked by super-enhancers identifies DLBCLs dependent on OCA-B and suggests a strategy for discovering unrecognized cancer dependencies. Translational studies performed on a comprehensive panel of DLBCLs establish a therapeutic rationale for evaluating BET inhibitors in this disease. Full Text
ChavesPerez, A., Mack, B., Maetzel, D., Kremling, H., Eggert, C., Harreus, U., and Gires, O. (2013). EpCAM regulates cell cycle progression via control of cyclin D1 expression. Oncogene 32, 641-650.The epithelial cell adhesion molecule (EpCAM) is an integral transmembrane protein that is frequently overexpressed in embryonic stem cells, tissue progenitors, carcinomas and cancer-initiating cells. In cancer cells, expression of EpCAM is associated with enhanced proliferation and upregulation of target genes including c-myc. However, the exact molecular mechanisms underlying the observed EpCAM-dependent cell proliferation remained unexplored. Here, we show that EpCAM directly affects cell cycle progression via its capacity to regulate the expression of cyclin D1 at the transcriptional level and depending on the direct interaction partner FHL2 (four-and-a-half LIM domains protein 2). As a result, downstream events such as phosphorylation of the retinoblastoma protein (Rb) and expression of cyclins E and A are similarly affected. In vivo, EpCAM expression strength and pattern are both positively correlated with the proliferation marker Ki67, high expression and nuclear localisation of cyclin D1, and Rb phosphorylation. Thus, EpCAM enhances cell cycle progression via the classical cyclin-regulated pathway. Full Text
Chen3, C.H., Kiyan, V., Zhylkibayev, A.A., Kazyken, D., Bulgakova, O., Page, K.E., Bersimbaev, R.I., Spooner, E., and Sarbassov, D.D. (2013). Autoregulation of the Mechanistic Target of Rapamycin (mTOR) Complex 2 Integrity Is Controlled by an ATP-dependent Mechanism. Journal of Biological Chemistry 288, 27019-27030.Nutrients are essential for living organisms because they fuel biological processes in cells. Cells monitor nutrient abundance and coordinate a ratio of anabolic and catabolic reactions. Mechanistic target of rapamycin (mTOR) signaling is the essential nutrient-sensing pathway that controls anabolic processes in cells. The central component of this pathway is mTOR, a highly conserved and essential protein kinase that exists in two distinct functional complexes. The nutrient-sensitive mTOR complex 1 (mTORC1) controls cell growth and cell size by phosphorylation of the regulators of protein synthesis S6K1 and 4EBP1, whereas its second complex, mTORC2, regulates cell proliferation by functioning as the regulatory kinase of Akt and other members of the AGC kinase family. The regulation of mTORC2 remains poorly characterized. Our study shows that the cellular ATP balance controls a basal kinase activity of mTORC2 that maintains the integrity of mTORC2 and phosphorylation of Akt on the turn motif Thr-450 site. We found that mTOR stabilizes SIN1 by phosphorylation of its hydrophobic and conserved Ser-260 site to maintain the integrity of mTORC2. The optimal kinase activity of mTORC2 requires a concentration of ATP above 1.2 mM and makes this kinase complex highly sensitive to ATP depletion. We found that not amino acid but glucose deprivation of cells or acute ATP depletion prevented the mTOR-dependent phosphorylation of SIN1 on Ser-260 and Akt on Thr-450. In a low glucose medium, the cells carrying a substitution of SIN1 with its phosphomimetic mutant show an increased rate of cell proliferation related to a higher abundance of mTORC2 and phosphorylation of Akt. Thus, the homeostatic ATP sensor mTOR controls the integrity of mTORC2 and phosphorylation of Akt on the turn motif site. PDF
Chen2, C., Garcia-Santos, D., Ishikawa, Y., Seguin, A., Li, L., Fegan, K.H., Hildick-Smith, G.J., Shah, D.I., Cooney, J.D., Chen, W., Harvey F. Lodish. et al. (2013). Snx3 Regulates Recycling of the Transferrin Receptor and Iron Assimilation. Cell Metabolism .Feb 13. [Epub ahead of print].Sorting of endocytic ligands and receptors is critical for diverse cellular processes. The physiological significance of endosomal sorting proteins in vertebrates, however, remains largely unknown. Here we report that sorting nexin 3 (Snx3) facilitates the recycling of transferrin receptor (Tfrc) and thus is required for the proper delivery of iron to erythroid progenitors. Snx3 is highly expressed in vertebrate hematopoietic tissues. Silencing of Snx3 results in anemia and hemoglobin defects in vertebrates due to impaired transferrin (Tf)-mediated iron uptake and its accumulation in early endosomes. This impaired iron assimilation can be complemented with non-Tf iron chelates. We show that Snx3 and Vps35, a component of the retromer, interact with Tfrc to sort it to the recycling endosomes. Our findings uncover a role of Snx3 in regulating Tfrc recycling, iron homeostasis, and erythropoiesis. Thus, the identification of Snx3 provides a genetic tool for exploring erythropoiesis and disorders of iron metabolism. Full Text
Chen, C.C., Wang, I.E., and Reddien, P.W. (2013). pbx is required for pole and eye regeneration in planarians. Development 140, 719-729.Planarian regeneration involves regionalized gene expression that specifies the body plan. After amputation, planarians are capable of regenerating new anterior and posterior poles, as well as tissues polarized along the anterior-posterior, dorsal-ventral and medial-lateral axes. Wnt and several Hox genes are expressed at the posterior pole, whereas Wnt inhibitory genes, Fgf inhibitory genes, and prep, which encodes a TALE-family homeodomain protein, are expressed at the anterior pole. We found that Smed-pbx (pbx for short), which encodes a second planarian TALE-family homeodomain transcription factor, is required for restored expression of these genes at anterior and posterior poles during regeneration. Moreover, pbx(RNAi) animals gradually lose pole gene expression during homeostasis. By contrast, pbx was not required for initial anterior-posterior polarized responses to wounds, indicating that pbx is required after wound responses for development and maintenance of poles during regeneration and homeostatic tissue turnover. Independently of the requirement for pbx in pole regeneration, pbx is required for eye precursor formation and, consequently, eye regeneration and eye replacement in homeostasis. Together, these data indicate that pbx promotes pole formation of body axes and formation of regenerative progenitors for eyes. Full Text
Cheng, A.W., Wang, H., Yang, H., Shi, L., Katz, Y., Theunissen, T.W., Rangarajan, S., Shivalila, C.S., Dadon, D.B., and Jaenisch, R. (2013). Multiplexed activation of endogenous genes by CRISPR-on, an RNA-guided transcriptional activator system .Cell Research advance online publication 27 Aug 2013. Technologies allowing for specific regulation of endogenous genes are valuable for the study of gene functions and have great potential in therapeutics. We created the CRISPR-on system, a two-component transcriptional activator consisting of a nuclease-dead Cas9 (dCas9) protein fused with a transcriptional activation domain and single guide RNAs (sgRNAs) with complementary sequence to gene promoters. We demonstrate that CRISPR-on can efficiently activate exogenous reporter genes in both human and mouse cells in a tunable manner. In addition, we show that robust reporter gene activation in vivo can be achieved by injecting the system components into mouse zygotes. Furthermore, we show that CRISPR-on can activate the endogenous IL1RN, SOX2, and OCT4 genes. The most efficient gene activation was achieved by clusters of 3-4 sgRNAs binding to the proximal promoters, suggesting their synergistic action in gene induction. Significantly, when sgRNAs targeting multiple genes were simultaneously introduced into cells, robust multiplexed endogenous gene activation was achieved. Genome-wide expression profiling demonstrated high specificity of the system.; doi:10.1038/cr.2013.122. Full Text
Chou, S., Flygare, J., and Lodish, H.F. (2013). Fetal Hepatic Progenitors Support Long-term Expansion of Hematopoietic Stem Cells. Experimental Hematology Published online Feb 13.We have developed a co-culture system that establishes DLK+ fetal hepatic progenitors as the authentic supportive cells for expansion of hematopoietic stem (HSCs) and progenitor cells. In one - week cultures supplemented with serum and supportive cytokines, both co-cultured DLK+ fetal hepatic progenitors and their conditioned medium supported rapid expansion of hematopoietic progenitors and a small increase of HSC numbers. In 2- and 3- week cultures DLK+ cells, but not their conditioned medium, continuously and significantly (>20 fold) expanded both hematopoietic stem and progenitor cells; physical contact between HSCs and DLK+ cells was crucial to maintaining this long-term expansion. Similar HSC expansion ( approximately 7 fold) was achieved in co-cultures using a serum-free, low cytokine- containing medium. In contrast, DLK- cells are incapable of expanding hematopoietic cells, demonstrating that hepatic progenitors are the principle supportive cells for HSC expansion in the fetal liver. Full Text
Chung, C.Y., Khurana, V., Auluck, P.K., Tardiff, D.F., Mazzulli, J.R., Soldner, F., Baru, V., Lou, Y., Freyzon, Y., Cho, S. ,Mungenast AE, Muffat J, Mitalipova M, Pluth MD, Jui NT, Schüle B, Lippard SJ, Tsai LH, Krainc D, Buchwald SL, Jaenisch R, Lindquist S.. (2013). Identification and Rescue of alpha-Synuclein Toxicity in Parkinson Patient-Derived Neurons. Science.Oct 24. [Epub ahead of print] The induced pluripotent stem (iPS) cell field holds promise for in vitro disease modeling. However, identifying innate cellular pathologies, particularly for age-related neurodegenerative diseases, has been challenging. Here, we exploited mutation correction of iPS cells and conserved proteotoxic mechanisms from yeast to humans to discover and reverse phenotypic responses to alpha-Synuclein (alphaSyn), a key protein involved in Parkinson's disease (PD). We generated cortical neurons from iPS cells of patients harboring alphaSyn mutations, who are at high risk of developing PD dementia. Genetic modifiers from unbiased screens in a yeast model of alphaSyn toxicity led to identification of early pathogenic phenotypes in patient neurons. These included nitrosative stress, accumulation of endoplasmic reticulum (ER)-associated degradation (ERAD) substrates and ER stress. A small molecule identified in a yeast screen, and the ubiquitin ligase Nedd4 it affects, reversed pathologic phenotypes in these neurons. Full Text
Claessen, J.H., Witte, M.D., Yoder, N.C., Zhu, A.Y., Spooner, E., and Ploegh, H.L. (2013). Catch-and-Release Probes Applied to Semi-Intact Cells Reveal Ubiquitin-Specific Protease Expression in Chlamydia trachomatis Infection. Chembiochem Jan 18. [Epub ahead of print].Protein ubiquitylation controls many cellular pathways, and timely removal of ubiquitin by deubiquitylating enzymes (DUBs) is essential to govern these different functions. To map endogenous expression of individual DUBs as well as that of any interacting proteins, we developed a catch-and-release ubiquitin probe. Ubiquitin was equipped with an activity-based warhead and a cleavable linker attached to a biotin affinity-handle through tandem site-specific modification, in which we combined intein chemistry with sortase-mediated ligation. The resulting probe is cell-impermeable and was therefore delivered to the cytosol of perfringolysin O (PFO)-permeabilized cells. This allowed us to retrieve and identify 34 DUBs and their interacting partners. We also noted the expression, in host cells infected with Chlamydia trachomatis, of two additional DUBs. Furthermore, we retrieved and identified chlamydial DUB1 (ChlaDUB1) and DUB2 (ChlaDUB2), demonstrating by experiment that ChlaDUB2, the presence and activity of which had not been detected in infected cells, is in fact expressed during the course of infection. PDF
Collins, F.S., Li, R., Tjian, R., Weinberg, R., Pollard, T.D., and Johnson, E.B. (2013). Sequester Impact on Biomedical Research. Cell 154, 14-15. The research system that led to the powerful engine of American biomedical development is in a state of rapid implosion, being deconstructed one brick at a time with prospects for reconstruction being remote at best. The current sequester only adds insult to injury. Full Text
Copsey, A., Tang, S., Jordan, P.W., Blitzblau, H.G., Newcombe, S., Chan, A.C., Newnham, L., Li, Z., Gray, S., Herbert, A.D., et al. (2013). Smc5/6 coordinates formation and resolution of joint molecules with chromosome morphology to ensure meiotic divisions. PLoS Genet 9, e1004071.DURING MEIOSIS, STRUCTURAL MAINTENANCE OF CHROMOSOME (SMC) COMPLEXES UNDERPIN TWO FUNDAMENTAL FEATURES OF MEIOSIS: homologous recombination and chromosome segregation. While meiotic functions of the cohesin and condensin complexes have been delineated, the role of the third SMC complex, Smc5/6, remains enigmatic. Here we identify specific, essential meiotic functions for the Smc5/6 complex in homologous recombination and the regulation of cohesin. We show that Smc5/6 is enriched at centromeres and cohesin-association sites where it regulates sister-chromatid cohesion and the timely removal of cohesin from chromosomal arms, respectively. Smc5/6 also localizes to recombination hotspots, where it promotes normal formation and resolution of a subset of joint-molecule intermediates. In this regard, Smc5/6 functions independently of the major crossover pathway defined by the MutLgamma complex. Furthermore, we show that Smc5/6 is required for stable chromosomal localization of the XPF-family endonuclease, Mus81-Mms4(Eme1). Our data suggest that the Smc5/6 complex is required for specific recombination and chromosomal processes throughout meiosis and that in its absence, attempts at cell division with unresolved joint molecules and residual cohesin lead to severe recombination-induced meiotic catastrophe. Full Text
Curry N.L., Mino-Kenudson, M., Oliver, T.G., Yilmaz, O.H., Yilmaz, V.O., Moon, J.Y., Jacks, T., Sabatini, D.M., and Kalaany, N.Y. (2013). Pten-Null Tumors Cohabiting the Same Lung Display Differential AKT Activation and Sensitivity to Dietary Restriction. Cancer Discovery 3, 908-921.PTEN loss is considered a biomarker for activated phosphoinositide 3-kinase (PI3K)/AKT, a pathway frequently mutated in cancer, and was recently shown to confer resistance to dietary restriction. Here, we show that Pten loss is not sufficient to drive AKT activation and resistance to dietary restriction in tumors with low growth factor receptor levels. We describe a murine Pten-null Kras-driven lung cancer model that harbors both dietary restriction-resistant, higher-grade, bronchiolar tumors with high AKT activity, and dietary restriction-sensitive, lower-grade, alveolar tumors with low AKT activity. We find that this phenotype is cell autonomous and that normal bronchiolar cells express higher levels of insulin-like growth factor-I receptor (IGF-IR) and of ectonucleoside triphosphate diphosphohydrolase 5 (ENTPD5), an endoplasmic reticulum enzyme known to modulate growth factor receptor levels. Suppression of ENTPD5 is sufficient to decrease IGF-IR levels and sensitize bronchiolar tumor cells to serum in vitro and to dietary restriction in vivo. Furthermore, we find that a significant percentage of human non-small cell lung carcinomas (NSCLC) have low AKT activity despite PTEN loss.. Full Text
Curtis, E.A., and Bartel, D.P. (2013). Synthetic shuffling and in vitro selection reveal the rugged adaptive fitness landscape of a kinase ribozyme. RNA.Published online June 24, 2013 The relationship between genotype and phenotype is often described as an adaptive fitness landscape. In this study, we used a combination of recombination, in vitro selection, and comparative sequence analysis to characterize the fitness landscape of a previously isolated kinase ribozyme. Point mutations present in improved variants of this ribozyme were recombined in vitro in more than 1014 different arrangements using synthetic shuffling, and active variants were isolated by in vitro selection. Mutual information analysis of 65 recombinant ribozymes isolated in the selection revealed a rugged fitness landscape in which approximately one-third of the 91 pairs of positions analyzed showed evidence of correlation. Pairs of correlated positions overlapped to form densely connected networks, and groups of maximally connected nucleotides occurred significantly more often in these networks than they did in randomized control networks with the same number of links. The activity of the most efficient recombinant ribozyme isolated from the synthetically shuffled pool was 30-fold greater than that of any of the ribozymes used to build it, which indicates that synthetic shuffling can be a rich source of ribozyme variants with improved properties. Full Text
Dang, M., Armbruster, N., Miller, M.A., Cermeno, E., Hartmann, M., Bell, G.W., Root, D.E., Lauffenburger, D.A., Lodish, H.F., and Herrlich, A. (2013). Regulated ADAM17-dependent EGF family ligand release by substrate-selecting signaling pathways. Proc Natl Acad Sci U S A May 29. [Epub ahead of print] .Ectodomain cleavage of cell-surface proteins by A disintegrin and metalloproteinases (ADAMs) is highly regulated, and its dysregulation has been linked to many diseases. ADAM10 and ADAM17 cleave most disease-relevant substrates. Broad-spectrum metalloprotease inhibitors have failed clinically, and targeting the cleavage of a specific substrate has remained impossible. It is therefore necessary to identify signaling intermediates that determine substrate specificity of cleavage. We show here that phorbol ester or angiotensin II-induced proteolytic release of EGF family members may not require a significant increase in ADAM17 protease activity. Rather, inducers activate a signaling pathway using PKC-alpha and the PKC-regulated protein phosphatase 1 inhibitor 14D that is required for ADAM17 cleavage of TGF-alpha, heparin-binding EGF, and amphiregulin. A second pathway involving PKC-delta is required for neuregulin (NRG) cleavage, and, indeed, PKC-delta phosphorylation of serine 286 in the NRG cytosolic domain is essential for induced NRG cleavage. Thus, signaling-mediated substrate selection is clearly distinct from regulation of enzyme activity, an important mechanism that offers itself for application in disease. Full Text
Dawlaty, M.M., Breiling, A., Le, T., Raddatz, G., Barrasa, M.I., Cheng, A.W., Gao, Q., Powell, B.E., Li, Z., Xu, M., and Rudolf Jaenisch. (2013). Combined Deficiency of Tet1 and Tet2 Causes Epigenetic Abnormalities but Is Compatible with Postnatal Development. Developmental Cell. Jan 22. [Epub ahead of print] Tet enzymes (Tet1/2/3) convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) in various embryonic and adult tissues. Mice mutant for either Tet1 or Tet2 are viable, raising the question of whether these enzymes have overlapping roles in development. Here we have generated Tet1 and Tet2 double-knockout (DKO) embryonic stem cells (ESCs) and mice. DKO ESCs remained pluripotent but were depleted of 5hmC and caused developmental defects in chimeric embryos. While a fraction of double-mutant embryos exhibited midgestation abnormalities with perinatal lethality, viable and overtly normal Tet1/Tet2-deficient mice were also obtained. DKO mice had reduced 5hmC and increased 5mC levels and abnormal methylation at various imprinted loci. Nevertheless, animals of both sexes were fertile, with females having smaller ovaries and reduced fertility. Our data show that loss of both enzymes is compatible with development but promotes hypermethylation and compromises imprinting. The data also suggest a significant contribution of Tet3 to hydroxylation of 5mC during development. PDF
Dobbin, M.M., Madabhushi, R., Pan, L., Chen, Y., Kim, D., Gao, J., Ahanonu, B., Pao, P.C., Qiu, Y., Zhao, Y.M., et al. (2013). SIRT1 collaborates with ATM and HDAC1 to maintain genomic stability in neurons. Nature Neuroscience 16, 1008-U1054.Defects in DNA repair have been linked to cognitive decline with age and neurodegenerative disease, yet the mechanisms that protect neurons from genotoxic stress remain largely obscure. We sought to characterize the roles of the NAD+-dependent deacetylase SIRT1 in the neuronal response to DNA double-strand breaks (DSBs). We found that SIRT1 was rapidly recruited to DSBs in postmitotic neurons, where it showed a synergistic relationship with ataxia telangiectasia mutated (ATM). SIRT1 recruitment to breaks was ATM dependent; however, SIRT1 also stimulated ATM autophosphorylation and activity and stabilized ATM at DSB sites. After DSB induction, SIRT1 also bound the neuroprotective class I histone deacetylase HDAC1. We found that SIRT1 deacetylated HDAC1 and stimulated its enzymatic activity, which was necessary for DSB repair through the nonhomologous end-joining pathway. HDAC1 mutations that mimic a constitutively acetylated state rendered neurons more susceptible to DNA damage, whereas pharmacological SIRT1 activators that promoted HDAC1 deacetylation also reduced DNA damage in two mouse models of neurodegeneration. We propose that SIRT1 is an apical transducer of the DSB response and that SIRT1 activation offers an important therapeutic avenue in neurodegeneration. Full Text
Dokshin, G.A., Baltus, A.E., Eppig, J.J., and Page, D.C. (2013). Oocyte differentiation is genetically dissociable from meiosis in mice. Nature Genetics. Published online16 June 2013 Oogenesis is the process by which ovarian germ cells undertake meiosis and differentiate to become eggs. In mice, Stra8 is required for the chromosomal events of meiosis to occur, but its role in differentiation remains unknown. Here we report Stra8-deficient ovarian germ cells that grow and differentiate into oocyte-like cells that synthesize zonae pellucidae, organize surrounding somatic cells into follicles, are ovulated in response to hormonal stimulation, undergo asymmetric cell division to produce a polar body and cleave to form two-cell embryos upon fertilization. These events occur without premeiotic chromosomal replication, sister chromatid cohesion, synapsis or recombination. Thus, oocyte growth and differentiation are genetically dissociable from the chromosomal events of meiosis. These findings open to study the independent contributions of meiosis and oocyte differentiation to the making of a functional egg. Full Text
Dougan2, S.K., Dougan, M., Kim, J., Turner, J.A., Ogata, S., Cho, H.I., Jaenisch, R., Celis, E., and Ploegh, H.L. (2013). Transnuclear TRP1-specific CD8 T cells with high or low affinity TCRs show equivalent anti-tumor activity. Cancer Immunol Res 1, 99-111.We have generated, via somatic cell nuclear transfer, two independent lines of transnuclear (TN) mice, using as nuclear donors CD8 T cells, sorted by tetramer staining, that recognize the endogenous melanoma antigen TRP1. These two lines of nominally identical specificity differ greatly in their affinity for antigen (TRP1high or TRP1low) as inferred from tetramer dissociation and peptide responsiveness. Ex vivo-activated CD8 T cells from either TRP1high or TRP1low mice show cytolytic activity in 3D tissue culture and in vivo, and slow the progression of subcutaneous B16 melanoma. Although naive TRP1low CD8 T cells do not affect tumor growth, upon activation these cells function indistinguishably from TRP1high cells in vivo, limiting tumor cell growth and increasing mouse survival. The anti-tumor effect of both TRP1high and TRP1low CD8 T cells is enhanced in RAG-deficient hosts. However, tumor outgrowth eventually occurs, likely due to T cell exhaustion. The TRP1 TN mice are an excellent model for examining the functional attributes of T cells conferred by TCR affinity, and they may serve as a platform for screening immunomodulatory cancer therapies. Full Text
Dougan, S.K., Ashour, J., Karssemeijer, R.A., Popp, M.W., Avalos, A.M., Barisa, M., Altenburg, A.F., Ingram, J.R., Cragnolini, J.J,.Guo,C , Frederick W. Alt ,Rudolf Jaenisch& Hidde L. Ploegh. (2013). Antigen-specific B-cell receptor sensitizes B cells to infection by influenza virus. Nature. Published online 20 October 2013 Influenza A virus-specific B lymphocytes and the antibodies they produce protect against infection. However, the outcome of interactions between an influenza haemagglutinin-specific B cell via its receptor (BCR) and virus is unclear. Through somatic cell nuclear transfer we generated mice that harbour B cells with a BCR specific for the haemagglutinin of influenza A/WSN/33 virus (FluBI mice). Their B cells secrete an immunoglobulin gamma 2b that neutralizes infectious virus. Whereas B cells from FluBI and control mice bind equivalent amounts of virus through interaction of haemagglutinin with surface-disposed sialic acids, the A/WSN/33 virus infects only the haemagglutinin-specific B cells. Mere binding of virus is not sufficient for infection of B cells: this requires interactions of the BCR with haemagglutinin, causing both disruption of antibody secretion and FluBI B-cell death within 18 h. In mice infected with A/WSN/33, lung-resident FluBI B cells are infected by the virus, thus delaying the onset of protective antibody release into the lungs, whereas FluBI cells in the draining lymph node are not infected and proliferate. We propose that influenza targets and kills influenza-specific B cells in the lung, thus allowing the virus to gain purchase before the initiation of an effective adaptive response. Full Text
Dowen, J.M., Bilodeau, S., Orlando, D.A., Hubner, M.R., Abraham, B.J., Spector, D.L., and Young, R.A. (2013). Multiple Structural Maintenance of Chromosome Complexes at Transcriptional Regulatory Elements. Stem Cell Reports 1, 371-378.Transcription factors control cell-specific gene expression programs by binding regulatory elements and recruiting cofactors and the transcription apparatus to the initiation sites of active genes. One of these cofactors is cohesin, a structural maintenance of chromosomes (SMC) complex that is necessary for proper gene expression. We report that a second SMC complex, condensin II, is also present at transcriptional regulatory elements of active genes during interphase and is necessary for normal gene activity. Both cohesin and condensin II are associated with genes in euchromatin and not heterochromatin. The two SMC complexes and the SMC loading factor NIPBL are particularly enriched at super-enhancers, and the genes associated with these regulatory elements are especially sensitive to reduced levels of these complexes. Thus, in addition to their well-established functions in chromosome maintenance during mitosis, both cohesin and condensin II make important contributions to the functions of the key transcriptional regulatory elements during interphase. Full Text
Dumesic, P.A., Natarajan, P., Chen, C.B., Drinnenberg, I.A., Schiller, B.J., Thompson, J., Moresco, J.J., Yates, J.R., Bartel, D.P., and Madhani, H.D. (2013). Stalled Spliceosomes Are a Signal for RNAi-Mediated Genome Defense. Cell 152, 957-968.Using the yeast Cryptococcus neoformans, we describe a mechanism by which transposons are initially targeted for RNAi-mediated genome defense. We show that intron-containing mRNA precursors template siRNA synthesis. We identify a Spliceosome-Coupled And Nuclear RNAi (SCANR) complex required for siRNA synthesis and demonstrate that it physically associates with the spliceosome. We find that RNAi target transcripts are distinguished by suboptimal introns and abnormally high occupancy on spliceosomes. Functional investigations demonstrate that the stalling of mRNA precursors on spliceosomes is required for siRNA accumulation. Lariat debranching enzyme is also necessary for siRNA production, suggesting a requirement for processing of stalled splicing intermediates. We propose that recognition of mRNA precursors by the SCANR complex is in kinetic competition with splicing, thereby promoting siRNA production from transposon transcripts stalled on spliceosomes. Disparity in the strength of expression signals encoded by transposons versus host genes offers an avenue for the evolution of genome defense. Full Text
Earnshaw, W.C., Allshire, R.C., Black, B.E., Bloom, K., Brinkley, B.R., Brown, W., Cheeseman, I.M., Choo, K.H.A., Copenhaver, G.P., DeLuca, J.G., et al. (2013). Esperanto for histones: CENP-A, not CenH3, is the centromeric histone H3 variant. Chromosome Research 21, 101-106.The first centromeric protein identified in any species was CENP-A, a divergent member of the histone H3 family that was recognised by autoantibodies from patients with scleroderma-spectrum disease. It has recently been suggested to rename this protein CenH3. Here, we argue that the original name should be maintained both because it is the basis of a long established nomenclature for centromere proteins and because it avoids confusion due to the presence of canonical histone H3 at centromeres. PDF
Efeyan, A., and Sabatini, D.M. (2013). Nutrients and growth factors in mTORC1 activation. Biochem Soc Trans 41, 902-905.Growth factors and nutrients regulate the mTORC1 [mammalian (or mechanistic) target of rapamycin complex 1] by different mechanisms. The players that link growth factors and mTORC1 activation have been known for several years and mouse models have validated its relevance for human physiology and disease. In contrast with the picture for growth factor signalling, the means by which nutrient availability leads to mTORC1 activation have remained elusive until recently, with the discovery of the Rag GTPases upstream of mTORC1. The Rag GTPases recruit mTORC1 to the outer lysosomal surface, where growth factor signalling and nutrient signalling converge on mTORC1 activation. A mouse model of constitutive RagA activity has revealed qualitative differences between growth-factor- and nutrient-dependent regulation of mTORC1. Regulation of mTORC1 activity by the Rag GTPases in vivo is key for enduring early neonatal starvation, showing its importance for mammalian physiology. Full Text
Faddah, D.A., Wang, H., Cheng, A.W., Katz, Y., Buganim, Y., and Jaenisch, R. (2013). Single-Cell Analysis Reveals that Expression of Nanog Is Biallelic and Equally Variable as that of Other Pluripotency Factors in Mouse ESCs. Cell Stem Cell 13, 23-29.The homeodomain transcription factor Nanog is a central part of the core pluripotency transcriptional network and plays a critical role in embryonic stem cell (ESC) self-renewal. Several reports have suggested that Nanog expression is allelically regulated and that transient downregulation of Nanog in a subset of pluripotent cells predisposes them toward differentiation. Using single-cell gene expression analyses combined with different reporters for the two alleles of Nanog, we show that Nanog is biallelically expressed in ESCs independently of culture condition. We also show that the overall variation in endogenous Nanog expression in ESCs is very similar to that of several other pluripotency markers. Our analysis suggests that reporter-based studies of gene expression in pluripotent cells can be significantly influenced by the gene-targeting strategy and genetic background employed. Full Text
Fagnan, D.E., Gromatzky, A.A., Stein, R.M., Fernandez, J.M., and Lo, A.W. (2013). Financing drug discovery for orphan diseases. Drug Discov Today Available online 20 November 2013 .Recently proposed 'megafund' financing methods for funding translational medicine and drug development require billions of dollars in capital per megafund to de-risk the drug discovery process enough to issue long-term bonds. Here, we demonstrate that the same financing methods can be applied to orphan drug development but, because of the unique nature of orphan diseases and therapeutics (lower development costs, faster FDA approval times, lower failure rates and lower correlation of failures among disease targets) the amount of capital needed to de-risk such portfolios is much lower in this field. Numerical simulations suggest that an orphan disease megafund of only US$575 million can yield double-digit expected rates of return with only 10-20 projects in the portfolio. Full Text
Fang, M., Xie, H., Dougan, S.K., Ploegh, H., and van Oudenaarden, A. (2013). Stochastic cytokine expression induces mixed T helper cell States. PLoS Biol 11, e1001618.During eukaryotic development, the induction of a lineage-specific transcription factor typically drives differentiation of multipotent progenitor cells, while repressing that of alternative lineages. This process is often mediated by some extracellular signaling molecules, such as cytokines that can bind to cell surface receptors, leading to activation and/or repression of transcription factors. We explored the early differentiation of naive CD4 T helper (Th) cells into Th1 versus Th2 states by counting single transcripts and quantifying immunofluorescence in individual cells. Contrary to mutually exclusive expression of antagonistic transcription factors, we observed their ubiquitous co-expression in individual cells at high levels that are distinct from basal-level co-expression during lineage priming. We observed that cytokines are expressed only in a small subpopulation of cells, independent from the expression of transcription factors in these single cells. This cell-to-cell variation in the cytokine expression during the early phase of T helper cell differentiation is significantly larger than in the fully differentiated state. Upon inhibition of cytokine signaling, we observed the classic mutual exclusion of antagonistic transcription factors, thus revealing a weak intracellular network otherwise overruled by the strong signals that emanate from extracellular cytokines. These results suggest that during the early differentiation process CD4 T cells acquire a mixed Th1/Th2 state, instructed by extracellular cytokines. The interplay between extracellular and intracellular signaling components unveiled in Th1/Th2 differentiation may be a common strategy for mammalian cells to buffer against noisy cytokine expression. Full Text
Floyd, S.R., Pacold, M.E., Huang, Q.Y., Clarke, S.M., Lam, F.C., Cannell, I.G., Bryson, B.D., Rameseder, J., Lee, M.J., Blake, E.J., David M. Sabatini, et al. (2013). The bromodomain protein Brd4 insulates chromatin from DNA damage signalling. Nature 498, 246-250.DNA damage activates a signalling network that blocks cell-cycle progression, recruits DNA repair factors and/or triggers senescence or programmed cell death(1). Alterations in chromatin structure are implicated in the initiation and propagation of the DNA damage response(2). Here we further investigate the role of chromatin structure in the DNA damage response by monitoring ionizing-radiation-induced signalling and response events with a high-content multiplex RNA-mediated interference screen of chromatin-modifying and -interacting genes. We discover that an isoform of Brd4, a bromodomain and extra-terminal (BET) family member, functions as an endogenous inhibitor of DNA damage response signalling by recruiting the condensin II chromatin remodelling complex to acetylated histones through bromodomain interactions. Loss of this isoform results in relaxed chromatin structure, rapid cell-cycle checkpoint recovery and enhanced survival after irradiation, whereas functional gain of this isoform compacted chromatin, attenuated DNA damage response signalling and enhanced radiation-induced lethality. These data implicate Brd4, previously known for its role in transcriptional control, as an insulator of chromatin that can modulate the signalling response to DNA damage. Full Text
Furtado, A.R., Essid, M., Perrinet, S., Balana, M.E., Yoder, N., Dehoux, P., and Subtil, A. (2013). The chlamydial OTU domain-containing protein ChlaOTU is an early type III secretion effector targeting ubiquitin and NDP52. Cellular Microbiology 15, 2064-2079.Chlamydia are obligate intracellular pathogens. Upon contact with the host, they use type III secretion to deliver proteins into the cell, thereby triggering actin-dependent entry and establishing the infection. We observed that Chlamydia caviae elicited a local and transient accumulation of ubiquitinated proteins at the entry sites, which disappeared within 20min. We investigated the mechanism for the rapid clearance of ubiquitin. We showed that the OTU-like domain containing protein CCA00261, predicted to have deubiquitinase activity, was detected in infectious particles and was a type III secretion effector. This protein is present in several Chlamydia strains, including the human pathogen Chlamydia pneumoniae, and we further designate it as ChlaOTU. We demonstrated that ChlaOTU bound ubiquitin and NDP52, and we mapped these interactions to distinct domains. NDP52 was recruited to Chlamydia entry sites and was dispensable for infection and for bacterial growth. ChlaOTU functioned as a deubiquitinase in vitro. Heterologousexpression of ChlaOTU reduced ubiquitin accumulation at the entry sites, while a catalytic mutant of the deubiquitinase activity had the opposite effect. Altogether, we have identified a novel secreted protein of chlamydiae. ChlaOTU targets both ubiquitin and NDP52 and likely participates in the clearance of ubiquitin at the invasion sites. Full Text
Gascoigne2, K.E., and Cheeseman, I.M. (2013). Induced dicentric chromosome formation promotes genomic rearrangements and tumorigenesis. Chromosome Research.Published online 22 June 2013. Chromosomal rearrangements can radically alter gene products and their function, driving tumor formation or progression. However, the molecular origins and evolution of such rearrangements are varied and poorly understood, with cancer cells often containing multiple, complex rearrangements. One mechanism that can lead to genomic rearrangements is the formation of a "dicentric" chromosome containing two functional centromeres. Indeed, such dicentric chromosomes have been observed in cancer cells. Here, we tested the ability of a single dicentric chromosome to contribute to genomic instability and neoplastic conversion in vertebrate cells. We developed a system to transiently and reversibly induce dicentric chromosome formation on a single chromosome with high temporal control. We find that induced dicentric chromosomes are frequently damaged and mis-segregated during mitosis, and that this leads to extensive chromosomal rearrangements including translocations with other chromosomes. Populations of pre-neoplastic cells in which a single dicentric chromosome is induced acquire extensive genomic instability and display hallmarks of cellular transformation including anchorage-independent growth in soft agar. Our results suggest that a single dicentric chromosome could contribute to tumor initiation. PDF
Gascoigne, K.E., and Cheeseman, I.M. (2013). CDK-dependent phosphorylation and nuclear exclusion coordinately control kinetochore assembly state. J Cell Biol. Published online March 25, 2013 Accurate chromosome segregation requires assembly of the multiprotein kinetochore complex. Prior work has identified more than 100 different kinetochore components in human cells. However, little is known about the regulatory processes that specify their assembly upon mitotic entry and disassembly at mitotic exit. In this paper, we used a live-cell imaging-based assay to quantify kinetochore disassembly kinetics and systematically analyze the role of potential regulatory mechanisms in controlling kinetochore assembly state. We find that kinetochore assembly and disassembly was driven primarily by mitotic phosphorylation downstream of cyclin-dependent kinase (CDK). In addition, we demonstrate that nuclear exclusion of the Ndc80 complex helped restrict kinetochore formation to mitosis. Combining constitutive CDK-dependent phosphorylation of CENP-T and forced nuclear localization of the Ndc80 complex partially prevented kinetochore disassembly at mitotic exit and led to chromosome segregation defects in subsequent divisions. In total, we find that the coordinated temporal regulation of outer kinetochore assembly is essential for accurate cell division. Full Text
Gavino, M.A., Wenemoser, D., Wang, I.E., and Reddien, P.W. (2013). Tissue absence initiates regeneration through Follistatin-mediated inhibition of Activin signaling. Elife 2, e00247.Regeneration is widespread, but mechanisms that activate regeneration remain mysterious. Planarians are capable of whole-body regeneration and mount distinct molecular responses to wounds that result in tissue absence and those that do not. A major question is how these distinct responses are activated. We describe a follistatin homolog (Smed-follistatin) required for planarian regeneration. Smed-follistatin inhibition blocks responses to tissue absence but does not prevent normal tissue turnover. Two activin homologs (Smed-activin-1 and Smed-activin-2) are required for the Smed-follistatin phenotype. Finally, Smed-follistatin is wound-induced and expressed at higher levels following injuries that cause tissue absence. These data suggest that Smed-follistatin inhibits Smed-Activin proteins to trigger regeneration specifically following injuries involving tissue absence and identify a mechanism critical for regeneration initiation, a process important across the animal kingdom. Full Text
Gehring, M. (2013). Genomic Imprinting: Parental Lessons from Plants. Annual Review of Genetics Aug 30. [Epub ahead of print].Imprinted gene expression-the biased expression of alleles dependent on their parent of origin-is an important type of epigenetic gene regulation in flowering plants and mammals. In plants, genes are imprinted primarily in the endosperm, the triploid placenta-like tissue that surrounds and nourishes the embryo during its development. Differential allelic expression is correlated with active DNA demethylation by DNA glycosylases and repressive targeting by the Polycomb group proteins. Imprinted gene expression is one consequence of a large-scale remodeling to the epigenome, primarily directed at transposable elements, that occurs in gametes and seeds. This remodeling could be important for maintaining the epigenome in the embryo as well as for establishing gene imprinting. Expected final online publication date for the Annual Review of Genetics Volume 47 is November 23, 2013. Please see http://www.annualreviews.org/catalog/pubdates.aspx for revised estimates. Full Text
Germain, A.R., Carmody, L.C., Nag, P.P., Morgan, B., VerPlank, L., Fernandez, C., Donckele, E., Feng, Y.X., Perez, J.R., Dandapani, S,.Palmer M, Lander ES, Gupta PB, Schreiber SL, and Munoz B. (2013). Cinnamides as selective small-molecule inhibitors of a cellular model of breast cancer stem cells. Bioorganic & Medicinal Chemistry Letters 23, 1834-1838.A high-throughput screen (HTS) was conducted against stably propagated cancer stem cell (CSC)-enriched populations using a library of 300,718 compounds from the National Institutes of Health (NIH) Molecular Libraries Small Molecule Repository (MLSMR). A cinnamide analog displayed greater than 20-fold selective inhibition of the breast CSC-like cell line (HMLE_sh_Ecad) over the isogenic control cell line (HMLE_sh_eGFP). Herein, we report structure-activity relationships of this class of cinnamides for selective lethality towards CSC-enriched populations. Full Text
Gregor, M.F., Misch, E.S., Yang, L., Hummasti, S., Inouye, K.E., Lee, A.H., Bierie, B., and Hotamisligil, G.S. (2013). The Role of Adipocyte XBP1 in Metabolic Regulation during Lactation. Cell Reports 3, 1430-1439.The adipocyte is central to organismal metabolism and exhibits significant functional and morphological plasticity during its formation and lifespan. Remarkable transformations of this cell occur during obesity and lactation, and thus it is essential to gain a better understanding of adipocyte function in these two metabolic processes. Considering the critical importance of the cellular organelle endoplasmic reticulum (ER) in adapting to fluctuations in synthetic processes, we explored the role of XBP1, a central regulator of ER adaptive responses, in adipocyte formation and function. Unexpectedly, deletion of adipocyte-XBP1 in vivo in mice (XBP1(Delta Ad)) had no effect on adipocyte formation or on systemic homeostatic metabolism in mice fed a a regular or high-fat diet. However, during lactation, XBP1(Delta Ad) dams displayed increased adiposity, decreased milk production, and decreased litter growth as compared with control dams. Moreover, we demonstrate that XBP1 is regulated during lactation and responds to prolactin to alter lipogenic gene expression. These results demonstrate a role for adipocyte-XBP1 in the regulation of lactational metabolism. Full Text
Grossniklaus, U., Kelly, B., Ferguson-Smith, A.C., Pembrey, M., and Lindquist, S. (2013). Transgenerational epigenetic inheritance: how important is it? Nat Rev Genet 14, 228-235.Much attention has been given to the idea of transgenerational epigenetic inheritance, but fundamental questions remain regarding how much takes place and the impact that this might have on organisms. We asked five leading researchers in this area - working on a range of model organisms and in human disease - for their views on these topics. Their responses highlight the mixture of excitement and caution that surrounds transgenerational epigenetic inheritance and the wide gulf between species in terms of our knowledge of the mechanisms that may be involved. Full Text
Guimaraes, C.P., Witte, M.D., Theile, C.S., Bozkurt, G., Kundrat, L., Blom, A.E., and Ploegh, H.L. (2013). Site-specific C-terminal and internal loop labeling of proteins using sortase-mediated reactions. Nature Protocols 8, 1787-1799.Methods for site-specific modification of proteins should be quantitative and versatile with respect to the nature and size of the biological or chemical targets involved. They should require minimal modification of the target, and the underlying reactions should be completed in a reasonable amount of time under physiological conditions. Sortase-mediated transpeptidation reactions meet these criteria and are compatible with other labeling methods. Here we describe the expression and purification conditions for two sortase A enzymes that have different recognition sequences. We also provide a protocol that allows the functionalization of any given protein at its C terminus, or, for select proteins, at an internal site. The target protein is engineered with a sortase-recognition motif (LPXTG) at the place where modification is desired. Upon recognition, sortase cleaves the protein between the threonine and glycine residues, facilitating the attachment of an exogenously added oligoglycine peptide modified with the functional group of choice (e.g., fluorophore, biotin, protein or lipid). Expression and purification of sortase takes approximately 3 d, and sortase-mediated reactions take only a few minutes, but reaction times can be extended to increase yields. Full Text
Gurtan, A.M., Ravi, A., Rahl, P.B., Bosson, A.D., JnBaptiste, C.K., Bhutkar, A., Whittaker, C.A., Young, R.A., and Sharp, P.A. (2013). Let-7 represses Nr6a1 and a mid-gestation developmental program in adult fibroblasts. Genes & Development 27, 941-954.MicroRNAs (miRNAs) are critical to proliferation, differentiation, and development. Here, we characterize gene expression in murine Dicer-null adult mesenchymal stem cell lines, a fibroblast cell type. Loss of Dicer leads to derepression of let-7 targets at levels that exceed 10-fold to 100-fold with increases in transcription. Direct and indirect targets of this miRNA belong to a mid-gestation embryonic program that encompasses known oncofetal genes as well as oncogenes not previously associated with an embryonic state. Surprisingly, this mid-gestation program represents a distinct period that occurs between the pluripotent state of the inner cell mass at embryonic day 3.5 (E3.5) and the induction of let-7 upon differentiation at E10.5. Within this mid-gestation program, we characterize the let-7 target Nr6a1, an embryonic transcriptional repressor that regulates gene expression in adult fibroblasts following miRNA loss. In total, let-7 is required for the continual suppression of embryonic gene expression in adult cells, a mechanism that may underlie its tumor-suppressive function. Full Text
Gymrek2, M., and Erlich, Y. (2013). Profiling Short Tandem Repeats from Short Reads. Deep Sequencing Data Analysis, pp. 113-135. Short tandem repeats (STRs), also known as microsatellites, have a wide range of applications, including medical genetics, forensics, and population genetics. High-throughput sequencing has the potential to profile large numbers of STRs, but cumbersome gapped alignment and STR-specific noise patterns hamper this task. We recently developed an algorithm, called lobSTR, to overcome these challenges and to accurately profile STRs from short reads. Here we describe how to use lobSTR to call STR variations from high-throughput sequencing datasets and to diagnose the quality of the calls. PDF
Gymrek, M., McGuire, A.L., Golan, D., Halperin, E., and Erlich, Y. (2013). Identifying personal genomes by surname inference. Science 339, 321-324.Sharing sequencing data sets without identifiers has become a common practice in genomics. Here, we report that surnames can be recovered from personal genomes by profiling short tandem repeats on the Y chromosome (Y-STRs) and querying recreational genetic genealogy databases. We show that a combination of a surname with other types of metadata, such as age and state, can be used to triangulate the identity of the target. A key feature of this technique is that it entirely relies on free, publicly accessible Internet resources. We quantitatively analyze the probability of identification for U.S. males. We further demonstrate the feasibility of this technique by tracing back with high probability the identities of multiple participants in public sequencing projects. Full Text
Hendricks, G.L., Weirich, K.L., Viswanathan, K., Li, J., Shriver, Z.H., Ashour, J., Ploegh, H.L., Kurt-Jones, E.A., Fygenson, D.K., Finberg, R.W., et al. (2013). Sialylneolacto-N-tetraose c (LSTc)-bearing Liposomal Decoys Capture Influenza A Virus. Journal of Biological Chemistry 288, 8061-8073.Influenza is a severe disease in humans and animals with few effective therapies available. All strains of influenza virus are prone to developing drug resistance due to the high mutation rate in the viral genome. A therapeutic agent that targets a highly conserved region of the virus could bypass resistance and also be effective against multiple strains of influenza. Influenza uses many individually weak ligand binding interactions for a high avidity multivalent attachment to sialic acid-bearing cells. Polymerized sialic acid analogs can form multivalent interactions with influenza but are not ideal therapeutics due to solubility and toxicity issues. We used liposomes as a novel means for delivery of the glycan sialylneolacto-N-tetraose c (LSTc). LSTc-bearing decoy liposomes form multivalent, polymer-like interactions with influenza virus. Decoy liposomes competitively bind influenza virus in hemagglutination inhibition assays and inhibit infection of target cells in a dose-dependent manner. Inhibition is specific for influenza virus, as inhibition of Sendai virus and respiratory syncytial virus is not observed. In contrast, monovalent LSTc does not bind influenza virus or inhibit infectivity. LSTc decoy liposomes prevent the spread of influenza virus during multiple rounds of replication in vitro and extend survival of mice challenged with a lethal dose of virus. LSTc decoy liposomes co-localize with fluorescently tagged influenza virus, whereas control liposomes do not. Considering the conservation of the hemagglutinin binding pocket and the ability of decoy liposomes to form high avidity interactions with influenza hemagglutinin, our decoy liposomes have potential as a new therapeutic agent against emerging influenza strains. Full Text
Hess, G.T., Guimaraes, C.P., Spooner, E., Ploegh, H.L., and Belcher, A.M. (2013). Orthogonal Labeling of M13 Minor Capsid Proteins with DNA to Self-Assemble End-to-End Multiphage Structures. ACS Synthetic Biology 2, 490-496.M13 bacteriophage has been used as a scaffold to organize :materials for various applications. Building more complex multiphage devices requires precise control of interactions between, the M13 capsid proteins. Toward we engineered a loop structure onto the pIII capsid protein of M13 bacteriophage to enable sortase-me labeling reactions for C-terminal display. Combining with N-terminal sortase-mediated labeling, we thusc created a phage scaffold that can he labeled orthogonally on three capsid proteins: the body and both ends. We show that covalent attachment of attachment different DNA ohgonucleotides at the ends of the new phage structure enables formation of multiphage particles oriented in a specific order. These have potential as nanascale scaffolds for multi-material devices. Full Text
Hirata, A., Utikal, J., Yamashita, S., Aoki, H., Watanabe, A., Yamamoto, T., Okano, H., Bardeesy, N., Kunisada, T., Ushijima, T, Akira Hara, Rudolf Jaenisch, Konrad Hochedlinger and Yasuhiro Yamada (2013). Dose-dependent roles for canonical Wnt signalling in de novo crypt formation and cell cycle properties of the colonic epithelium. Development 140, 66-75.There is a gradient of beta-catenin expression along the colonic crypt axis with the highest levels at the crypt bottom. In addition, colorectal cancers show a heterogeneous subcellular pattern of beta-catenin accumulation. However, it remains unclear whether different levels of Wnt signalling exert distinct roles in the colonic epithelium. Here, we investigated the dose-dependent effect of canonical Wnt activation on colonic epithelial differentiation by controlling the expression levels of stabilised beta-catenin using a doxycycline-inducible transgenic system in mice. We show that elevated levels of Wnt signalling induce the amplification of Lgr5+ cells, which is accompanied by crypt fission and a reduction in cell proliferation among progenitor cells. By contrast, lower levels of beta-catenin induction enhance cell proliferation rates of epithelial progenitors without affecting crypt fission rates. Notably, slow-cycling cells produced by beta-catenin activation exhibit activation of Notch signalling. Consistent with the interpretation that the combination of Notch and Wnt signalling maintains crypt cells in a low proliferative state, the treatment of beta-catenin-expressing mice with a Notch inhibitor turned such slow-cycling cells into actively proliferating cells. Our results indicate that the activation of the canonical Wnt signalling pathway is sufficient for de novo crypt formation, and suggest that different levels of canonical Wnt activations, in cooperation with Notch signalling, establish a hierarchy of slower-cycling stem cells and faster-cycling progenitor cells characteristic for the colonic epithelium. Full Text
Hnisz, D., Abraham, B.J., Lee, T.I., Lau, A., Saint-Andre, V., Sigova, A.A., Hoke, H.A., and Young, R.A. (2013). Super-Enhancers in the Control of Cell Identity and Disease. Cell Online 10 Oct. 2013 .Super-enhancers are large clusters of transcriptional enhancers that drive expression of genes that define cell identity. Improved understanding of the roles that super-enhancers play in biology would be afforded by knowing the constellation of factors that constitute these domains and by identifying super-enhancers across the spectrum of human cell types. We describe here the population of transcription factors, cofactors, chromatin regulators, and transcription apparatus occupying super-enhancers in embryonic stem cells and evidence that super-enhancers are highly transcribed. We produce a catalog of super-enhancers in a broad range of human cell types and find that super-enhancers associate with genes that control and define the biology of these cells. Interestingly, disease-associated variation is especially enriched in the super-enhancers of disease-relevant cell types. Furthermore, we find that cancer cells generate super-enhancers at oncogenes and other genes important in tumor pathogenesis. Thus, super-enhancers play key roles in human cell identity in health and in disease. Full Text
Holmes, D.L., Lancaster, A.K., Lindquist, S., and Halfmann, R. (2013). Heritable remodeling of yeast multicellularity by an environmentally responsive prion. Cell 153, 153-165.Prion proteins undergo self-sustaining conformational conversions that heritably alter their activities. Many of these proteins operate at pivotal positions in determining how genotype is translated into phenotype. But the breadth of prion influences on biology and their evolutionary significance are just beginning to be explored. We report that a prion formed by the Mot3 transcription factor, [MOT3(+)], governs the acquisition of facultative multicellularity in the budding yeast Saccharomyces cerevisiae. The traits governed by [MOT3(+)] involved both gains and losses of Mot3 regulatory activity. [MOT3(+)]-dependent expression of FLO11, a major determinant of cell-cell adhesion, produced diverse lineage-specific multicellular phenotypes in response to nutrient deprivation. The prions themselves were induced by ethanol and eliminated by hypoxia-conditions that occur sequentially in the natural respiro-fermentative cycles of yeast populations. These data demonstrate that prions can act as environmentally responsive molecular determinants of multicellularity and contribute to the natural morphological diversity of budding yeast. . Full Text
Hu2, Y.C., Okumura, L.M., and Page, D.C. (2013). Gata4 is required for formation of the genital ridge in mice. PLoS Genet 9, e1003629.In mammals, both testis and ovary arise from a sexually undifferentiated precursor, the genital ridge, which first appears during mid-gestation as a thickening of the coelomic epithelium on the ventromedial surface of the mesonephros. At least four genes (Lhx9, Sf1, Wt1, and Emx2) have been demonstrated to be required for subsequent growth and maintenance of the genital ridge. However, no gene has been shown to be required for the initial thickening of the coelomic epithelium during genital ridge formation. We report that the transcription factor GATA4 is expressed in the coelomic epithelium of the genital ridge, progressing in an anterior-to-posterior (A-P) direction, immediately preceding an A-P wave of epithelial thickening. Mouse embryos conditionally deficient in Gata4 show no signs of gonadal initiation, as their coelomic epithelium remains a morphologically undifferentiated monolayer. The failure of genital ridge formation in Gata4-deficient embryos is corroborated by the absence of the early gonadal markers LHX9 and SF1. Our data indicate that GATA4 is required to initiate formation of the genital ridge in both XX and XY fetuses, prior to its previously reported role in testicular differentiation of the XY gonad. Full Text
Hu, Y.C., de Rooij, D.G., and Page, D.C. (2013). Tumor suppressor gene Rb is required for self-renewal of spermatogonial stem cells in mice. Proc Natl Acad Sci U S A .Jul 15. [Epub ahead of print] The retinoblastoma tumor suppressor gene Rb is essential for maintaining the quiescence and for regulating the differentiation of somatic stem cells. Inactivation of Rb in somatic stem cells typically leads to their overexpansion, often followed by increased apoptosis, defective terminal differentiation, and tumor formation. However, Rb's roles in germ-line stem cells have not been explored. We conditionally disrupted the Rb gene in mouse germ cells in vivo and discovered unanticipated consequences for GFRa1-protein-expressing Asingle (GFRa1+ As) spermatogonia, the major source of male germ-line stem cells. Rb-deficient GFRa1+ As spermatogonia were present at normal density in testes 5 d after birth, but they lacked the capacity for self-renewal, resulting in germ cell depletion by 2 mo of age. Rb deficiency did not affect the proliferative activity of GFRa1+ As spermatogonia, but their progeny were exclusively transit-amplifying progenitor spermatogonia and did not include GFRa1+ As spermatogonia. In addition, Rb deficiency caused prolonged proliferation of progenitor spermatogonia, transiently enlarging this population. Despite these defects, Rb deficiency did not block terminal differentiation into functional sperm; offspring were readily obtained from young males whose germ cell pool was not yet depleted. We conclude that Rb is required for self-renewal of germ-line stem cells, but contrary to its critical roles in somatic stem cells, it is dispensable for their proliferative activity and terminal differentiation. Thus, this study identifies an unexpected function for Rb in maintaining the stem cell pool in the male germ line. PDF
Huang, H., Jiang, X., Li, Z.J., Li, Y.Y., Song, C.X., He, C.J., Sun, M., Chen, P., Gurbuxani, S., Wang, J.P., Meelad Dawlaty, Rudolf Jaenisch, et al. (2013). TET1 plays an essential oncogenic role in MLL-rearranged leukemia. Proceedings of the National Academy of Sciences of the United States of America 110, 11994-11999.The ten-eleven translocation 1 (TET1) gene is the founding member of the TET family of enzymes (TET1/2/3) that convert 5-methylcytosine to 5-hydroxymethylcytosine. Although TET1 was first identified as a fusion partner of the mixed lineage leukemia (MLL) gene in acute myeloid leukemia carrying t(10,11), its definitive role in leukemia is unclear. In contrast to the frequent down-regulation (or loss-of-function mutations) and critical tumor-suppressor roles of the three TET genes observed in various types of cancers, here we show that TET1 is a direct target of MLL-fusion proteins and is significantly up-regulated in MLL-rearranged leukemia, leading to a global increase of 5-hydroxymethylcytosine level. Furthermore, our both in vitro and in vivo functional studies demonstrate that Tet1 plays an indispensable oncogenic role in the development of MLL-rearranged leukemia, through coordination with MLL-fusion proteins in regulating their critical cotargets, including homeobox A9 (Hoxa9)/myeloid ecotropic viral integration 1 (Meis1)/pre-B-cell leukemia homeobox 3 (Pbx3) genes. Collectively, our data delineate an MLL-fusion/Tet1/Hoxa9/Meis1/Pbx3 signaling axis in MLL-rearranged leukemia and highlight TET1 as a potential therapeutic target in treating this presently therapy-resistant disease. Full Text
Hughes, J.F., Skaletsky, H., Bellott, D.W., Chowdhary, B.P., Warren, W.C., Worley, K.C., Wilson, R.K., Gibbs, R.A., and Page, D.C. (2013). No bull: Upholding community standards in public sharing of biological datasets. Proc Natl Acad Sci U S A Vol. 110 no. 46 Jennifer F. Hughes, E4277 Full Text
Jackson, W.S., Borkowski, A.W., Watson, N.E., King, O.D., Faas, H., Jasanoff, A., and Lindquist, S. (2013). Profoundly different prion diseases in knock-in mice carrying single PrP codon substitutions associated with human diseases. Proc Natl Acad Sci U S Aug 19. [Epub ahead of print] .In man, mutations in different regions of the prion protein (PrP) are associated with infectious neurodegenerative diseases that have remarkably different clinical signs and neuropathological lesions. To explore the roots of this phenomenon, we created a knock-in mouse model carrying the mutation associated with one of these diseases [Creutzfeldt-Jakob disease (CJD)] that was exactly analogous to a previous knock-in model of a different prion disease [fatal familial insomnia (FFI)]. Together with the WT parent, this created an allelic series of three lines, each expressing the same protein with a single amino acid difference, and with all native regulatory elements intact. The previously described FFI mice develop neuronal loss and intense reactive gliosis in the thalamus, as seen in humans with FFI. In contrast, CJD mice had the hallmark features of CJD, spongiosis and proteinase K-resistant PrP aggregates, initially developing in the hippocampus and cerebellum but absent from the thalamus. A molecular transmission barrier protected the mice from any infectious prion agents that might have been present in our mouse facility and allowed us to conclude that the diseases occurred spontaneously. Importantly, both models created agents that caused a transmissible neurodegenerative disease in WT mice. We conclude that single codon differences in a single gene in an otherwise normal genome can cause remarkably different neurodegenerative diseases and are sufficient to create distinct protein-based infectious elements. Full Text
Kang, S.A., Pacold, M.E., Cervantes, C.L., Lim, D., Lou, H.J., Ottina, K., Gray, N.S., Turk, B.E., Yaffe, M.B., and Sabatini, D.M. (2013). mTORC1 phosphorylation sites encode their sensitivity to starvation and rapamycin. Science 341, 1236566.The mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) protein kinase promotes growth and is the target of rapamycin, a clinically useful drug that also prolongs life span in model organisms. A persistent mystery is why the phosphorylation of many bona fide mTORC1 substrates is resistant to rapamycin. We find that the in vitro kinase activity of mTORC1 toward peptides encompassing established phosphorylation sites varies widely and correlates strongly with the resistance of the sites to rapamycin, as well as to nutrient and growth factor starvation within cells. Slight modifications of the sites were sufficient to alter mTORC1 activity toward them in vitro and to cause concomitant changes within cells in their sensitivity to rapamycin and starvation. Thus, the intrinsic capacity of a phosphorylation site to serve as an mTORC1 substrate, a property we call substrate quality, is a major determinant of its sensitivity to modulators of the pathway. Our results reveal a mechanism through which mTORC1 effectors can respond differentially to the same signals. Full Text
Katz, Y. (2013). Against storytelling of scientific results. Nature Methods 10, 1045 Full Text
Kim, J., Huh, J., Hwang, M., Kwon, E.H., Jung, D.J., Brinkmann, M.M., Jang, M.H., Ploegh, H.L., and Kim, Y.M. (2013). Acidic Amino Acid Residues in the Juxtamembrane Region of the Nucleotide-Sensing TLRs Are Important for UNC93B1 Binding and Signaling. Journal of Immunology 190, 5287-5295.TLRs are divided into two groups based on their subcellular localization patterns. TLR1, 2, 4, 5, and 6 are expressed on the cell surface, whereas the nucleotide-sensing TLRs, such as TLR3, 7, 8, and 9 stay mainly inside cells. The polytopic membrane protein UNC93B1 physically interacts with the nucleotide-sensing TLRs and delivers them from the endoplasmic reticulum to endolysosomes, where the TLRs recognize their ligands and initiate signaling. In cells with nonfunctional UNC93B1, the nucleic acid-sensing TLRs fail to exit the endoplasmic reticulum and consequently do not signal. However, the detailed molecular mechanisms that underlie the UNC93B1-mediated TLR trafficking remain to be clarified. All nucleotide-sensing TLRs contain acidic amino acid residues in the juxtamembrane region between the leucine-rich repeat domain and the transmembrane segment. We show that the D812 and E813 residues of TLR9 and the D699 and E704 residues of TLR3 help to determine the interaction of these TLRs with UNC93B1. Mutation of the acidic residues in TLR3 and TLR9 prevents UNC93B1 binding, as well as impairs TLR trafficking and renders the mutant receptors incapable of transmitting signals. Therefore, the acidic residues in the juxtamembrane region of the nucleotide-sensing TLRs have important functional roles. Full Text
Kim2 N., Kukkonen, S., Martinez-Viedma, M.D., Gupta, S., and Aldovini, A. (2013). Tat engagement of p38 MAP kinase and IRF7 pathways leads to activation of interferon-stimulated genes in antigen-presenting cells. Blood 121, 4090-4100.As a result of its interaction with transcription factors, HIV type 1 (HIV-1) Tat can modulate the expression of both HIV and cellular genes. In antigen-presenting cells Tat induces the expression of a subset of interferon (IFN)-stimulated genes (ISGs) in the absence of IFNs. We investigated the genome-wide Tat association with promoters in immature dendritic cells and inmonocyte-derived macrophages. Among others, Tat associated with the MAP2K6, MAP2K3, and IRF7 promoters that are functionally part of IL-1 and p38 mitogen-activated protein kinase (MAPK) signaling pathways. The association correlated with their increased gene expression, increased activation of p38 MAPK and of phosphorylated signal transducer and activator of transcription 1 (STAT1), and consequent induction of ISGs. Probing these pathways with RNA interference, pharmacological p38 MAPK inhibition, and in cell lines lacking STAT1s or the type I IFN receptor chain confirmed the role of MAPKKs and IRF7 in Tat-mediated modulation of ISGs and excluded the involvement of IFNs in this modulation. Tat interaction with the 2 MAPKK and IRF7 promoters in HIV-1-infected cells and the resulting persistent activation of ISGs, which include inflammatory cytokines and chemokines, can contribute to the increased immune activation that characterizes HIV infection. Full Text
Kiyomitsu, T., and Cheeseman, I.M. (2013). Cortical Dynein and asymmetric membrane elongation coordinately position the spindle in anaphase. Cell 154, 391-402. Mitotic spindle position defines the cell-cleavage site during cytokinesis. However, the mechanisms that control spindle positioning to generate equal-sized daughter cells remain poorly understood. Here, we demonstrate that two mechanisms act coordinately to center the spindle during anaphase in symmetrically dividing human cells. First, the spindle is positioned directly by the microtubule-based motor dynein, which we demonstrate is targeted to the cell cortex by two distinct pathways: a Galphai/LGN/NuMA-dependent pathway and a 4.1G/R and NuMA-dependent, anaphase-specific pathway. Second, we find that asymmetric plasma membrane elongation occurs in response to spindle mispositioning to alter the cellular boundaries relative to the spindle. Asymmetric membrane elongation is promoted by chromosome-derived Ran-GTP signals that locally reduce Anillin at the growing cell cortex. In asymmetrically elongating cells, dynein-dependent spindle anchoring at the stationary cell cortex ensures proper spindle positioning. Our results reveal the anaphase-specific spindle centering systems that achieve equal-sized cell division. Full Text
Knoll, M., Simmons, S., Bouquet, C., Grun, J.R., and Melchers, F. (2013). miR-221 redirects precursor B cells to the bone marrow and regulates their residence. European J Immunology Accepted manuscript online: 29 MAY 2013 .Pluripotent hematopoietic stem cells and multipotent myeloid/lymphoid progenitors express miR-221 and miR-222. When Pax5 expression commits these progenitors to monopotent pre-B-lymphocytes the two miRNAs are downregulated. Upon transplantation, stem cells and progenitors can home to the bone marrow, while pre-B cells, after their commitment, no longer do so. Retrovirally transduced, doxycycline-induced overexpression of either miR-221 or miR-222 in pre-B-I-cells does not revert their monopotency to multipotency. However, upon transplantation miR-221, but not miR-222, transduced pre-B-I cells regain the capacity to home to the bone marrow. Upon subsequent termination of miR-221-expression by removal of doxycycline, the transplanted cells leave the bone marrow again. Microarray analyses identified 25 downregulated miR-221-target genes, which could function to localize phases of B-lymphocyte development in bone marrow before and after commitment. PDF
Koenig, P.A., Spooner, E., Kawamoto, N., Strominger, J.L., and Ploegh, H.L. (2013). Amino Acid Copolymers That Alleviate Experimental Autoimmune Encephalomyelitis In Vivo Interact with Heparan Sulfates and Glycoprotein 96 in APCs. J Immunology Jun 5. [Epub ahead of print] .Multiple sclerosis (MS) is an autoimmune disease that affects the CNS. One approved treatment for relapsing forms of MS is YEAK, a random copolymer of the amino acids tyrosine, glutamic acid, alanine, and lysine. YFAK, a second-generation copolymer composed of tyrosine, phenylalanine, alanine, and lysine, is more successful in treating experimental autoimmune encephalomyelitis, a mouse model of MS. Although originally designed and optimized based on the autoantigen myelin basic protein (MBP) and the MBP-derived peptide MBP85-99 presented to the MS-associated class II MHC molecule HLA-DR2, YEAK and YFAK also stimulate cytokine and chemokine production in APCs that lack class II MHC products. How YEAK and YFAK copolymers interact with APCs remains enigmatic. We used biotinylated YFAK to affinity-purify YFAK-interacting proteins from RAW264.7 cells and tested APCs from mice deficient in several of the newly identified interactors for their capacity to secrete CCL22 in response to YEAK and YFAK. We propose that initial contact of YFAK with cells is mediated mainly by electrostatic interactions, and find that interaction of YFAK with host proteins is strongly dependent on ionic strength. Cells deficient in enzymes involved in sulfation of proteins and proteoglycans showed strongly reduced binding of biotinylated YFAK. Lastly, cells stimulated with YFAK in the presence of heparin, structurally similar to heparan sulfates, failed to produce CCL22. We conclude that charge-dependent interactions of copolymers that alleviate MS/experimental autoimmune encephalomyelitis are critical for their effects exerted on APCs and may well be the main initial mediators of these therapeutically active copolymers. Full Text
Konig, K., Meder, L., Kroger, C., Diehl, L., Florin, A., Rommerscheidt-Fuss, U., Kahl, P., Wardelmann, E., Magin, T.M., Buettner, R., et al. (2013). Loss of the Keratin Cytoskeleton Is Not Sufficient to Induce Epithelial Mesenchymal Transition in a Novel KRAS Driven Sporadic Lung Cancer Mouse Model. PLoS ONE 8(3): e57996. Epithelial-to-mesenchymal transition (EMT), the phenotypical change of cells from an epithelial to a mesenchymal type, is thought to be a key event in invasion and metastasis of adenocarcinomas. These changes involve loss of keratin expression as well as loss of cell polarity and adhesion. We here aimed to determine whether the loss of keratin expression itself drives increased invasion and metastasis in adenocarcinomas and whether keratin loss leads to the phenotypic changes associated with EMT. Therefore, we employed a recently described murine model in which conditional deletion of the Keratin cluster II by Cre-recombinase leads to the loss of the entire keratinmultiprotein family. These mice were crossed into a newly generated Cre-recombinase inducible KRAS-driven murine lung cancer model to examine the effect of keratin loss on morphology, invasion and metastasis as well as expression of EMT related genes in the resulting tumors. We here clearly show that loss of a functional keratin cytoskeleton did not significantly alter tumor morphology or biology in terms of invasion, metastasis, proliferation or tumor burden and did not lead to induction of EMT. Further, tumor cells did not induce synchronously expression of vimentin, which is often seen in EMT, to compensate for keratin loss. In summary, our data suggest that changes in cell shape and migration that underlie EMT are dependent on changes in signaling pathways that cause secondary changes in keratin expression and organization. Thus, we conclude that loss of the keratin cytoskeleton per se is not sufficient to causally drive EMT in this tumor model. Full Text
Kroger, C., Loschke, F., Schwarz, N., Windoffer, R., Leube, R.E., and Magin, T.M. (2013). Keratins control intercellular adhesion involving PKC-alpha-mediated desmoplakin phosphorylation. Journal of Cell Biology Published Online May 20, 2013 .Maintenance of epithelial cell adhesion is crucial for epidermal morphogenesis and homeostasis and relies predominantly on the interaction of keratins with desmosomes. Although the importance of desmosomes to epidermal coherence and keratin organization is well established, the significance of keratins in desmosome organization has not been fully resolved. Here, we report that keratinocytes lacking all keratins show elevated, PKC-alpha-mediated desmoplakin phosphorylation and subsequent destabilization of desmosomes. We find that PKC-alpha activity is regulated by Rack1-keratin interaction. Without keratins, desmosomes assemble but are endocytosed at accelerated rates, rendering epithelial sheets highly susceptible to mechanical stress. Re-expression of the keratin pair K5/14, inhibition of PKC-alpha activity, or blocking of endocytosis reconstituted both desmosome localization at the plasma membrane and epithelial adhesion. Our findings identify a hitherto unknown mechanism by which keratins control intercellular adhesion, with potential implications for tumor invasion and keratinopathies, settings in which diminished cell adhesion facilitates tissue fragility and neoplastic growth. Full Text
Lambert, J.P., Ivosev, G., Couzens, A.L., Larsen, B., Taipale, M., Lin, Z.Y., Zhong, Q., Lindquist, S., Vidal, M., Aebersold, R., et al. (2013). Mapping differential interactomes by affinity purification coupled with data-independent mass spectrometry acquisition. Nature Methods 10, 1239-+.Characterizing changes in protein-protein interactions associated with sequence variants (e. g., disease-associated mutations or splice forms) or following exposure to drugs, growth factors or hormones is critical to understanding how protein complexes are built, localized and regulated. Affinity purification (AP) coupled with mass spectrometry permits the analysis of protein interactions under near-physiological conditions, yet monitoring interaction changes requires the development of a robust and sensitive quantitative approach, especially for large-scale studies in which cost and time are major considerations. We have coupled AP to data-independent mass spectrometric acquisition (sequential window acquisition of all theoretical spectra, SWATH) and implemented an automated data extraction and statistical analysis pipeline to score modulated interactions. We used AP-SWATH to characterize changes in protein-protein interactions imparted by the HSHSP90 inhibitor NVP-AUY922 or melanoma-associated mutations in the human kinase CDCDK4. We show that AP-SWATH is a robust label-free approach to characterize such changes and propose a scalable pipeline for systems biology studies. Full Text
Lamming3, D.W., and Sabatini, D.M. (2013). A Central Role for mTOR in Lipid Homeostasis. Cell Metabolism 2013 Aug 20.0820. [Epub ahead of print].The mechanistic target of rapamycin (mTOR) signaling pathway regulates many fundamental metabolic and physiological processes, including lipid metabolism. We explore recent findings on the role of mTOR in lipid homeostasis, with an emphasis on recent findings from in vivo models regarding the role of mTORC2 in lipolysis, lipogenesis, and adipogenesis. Full Text
Lamming4, D.W., Demirkan, G., Boylan, J.M., Mihaylova, M.M., Peng, T., Ferreira, J., Neretti, N., Salomon, A., Sabatini, D.M., and Gruppuso, P.A. (2013). Hepatic signaling by the mechanistic target of rapamycin complex 2 (mTORC2). FASEB J.Published online September 27, 2013. The mechanistic target of rapamycin (mTOR) exists in two complexes that regulate diverse cellular processes. mTOR complex 1 (mTORC1), the canonical target of rapamycin, has been well studied, whereas the physiological role of mTORC2 remains relatively uncharacterized. In mice in which the mTORC2 component Rictor is deleted in liver [Rictor-knockout (RKO) mice], we used genomic and phosphoproteomic analyses to characterize the role of hepatic mTORC2 in vivo. Overnight food withdrawal followed by refeeding was used to activate mTOR signaling. Rapamycin was administered before refeeding to specify mTORC2-mediated events. Hepatic mTORC2 regulated a complex gene expression and post-translational network that affects intermediary metabolism, ribosomal biogenesis, and proteasomal biogenesis. Nearly all changes in genes related to intermediary metabolic regulation were replicated in cultured fetal hepatocytes, indicating a cell-autonomous effect of mTORC2 signaling. Phosphoproteomic profiling identified mTORC2-related signaling to 144 proteins, among which were metabolic enzymes and regulators. A reduction of p38 MAPK signaling in the RKO mice represents a link between our phosphoproteomic and gene expression results. We conclude that hepatic mTORC2 exerts a broad spectrum of biological effects under physiological conditions. Our findings provide a context for the development of targeted therapies to modulate mTORC2 signaling.-Lamming, D. W., Demirkan, G., Boylan, J. M., Mihaylova, M. M., Peng, T., Ferreira, J., Neretti, N., Salomon, A., Sabatini, D. M., Gruppuso, P. A. Hepatic signaling by the mechanistic target of rapamycin complex 2 (mTORC2). Full Text
Lamming2, D.W., Ye, L., Astle, M.C., Baur, J.A., Sabatini, D.M., and Harrison, D.E. (2013). Young and old genetically heterogeneous HET3 mice on a rapamycin diet are glucose intolerant but insulin sensitive. Aging Cell May 4. [Epub ahead of print] Rapamycin, an inhibitor of the mechanistic target of rapamycin (mTOR) signaling pathway, extends the lifespan of yeast, worms, flies, and mice. Interventions that promote longevity are often correlated with increased insulin sensitivity, and it therefore is surprising that chronic rapamycin treatment of mice, rats and humans is associated with insulin resistance (Johnston et al. 2008; Houde et al. 2010; Lamming et al. 2012). We examined the effect of dietary rapamycin treatment on glucose homeostasis and insulin resistance in the genetically heterogeneous HET3 mouse strain, a strain in which dietary rapamycin robustly extends mean and maximum lifespan. We find that rapamycin treatment leads to glucose intolerance in both young and old HET3 mice, but in contrast to the previously reported effect of injected rapamycin in C57BL/6 mice, HET3 mice treated with dietary rapamycin responded normally in an insulin tolerance test. To gauge the overall consequences of rapamycin treatment on average blood glucose levels, we measured HBA1c. Dietary rapamycin increased HBA1c over the first three weeks of treatment in young animals, but the effect was lost by three months, and no effect was detected in older animals. Our results demonstrate that the extended lifespan of HET3 mice on a rapamycin diet occurs in the absence of major changes in insulin sensitivity, and highlight the importance of strain background and delivery method in testing effects of longevity interventions. .PDF
Lamming, D.W., Ye, L., Sabatini, D.M., and Baur, J.A. (2013). Rapalogs and mTOR inhibitors as anti-aging therapeutics. Journal of Clinical Investigation 123, 980-989.Rapamycin, an inhibitor of mechanistic target of rapamycin (mTOR), has the strongest experimental support to date as a potential anti-aging therapeutic in mammals. Unlike many other compounds that have been claimed to influence longevity, rapamycin has been repeatedly tested in long-lived, genetically heterogeneous mice, in which it extends both mean and maximum life spans. However, the mechanism that accounts for these effects is far from clear, and a growing list of side effects make it doubtful that rapamycin would ultimately be beneficial in humans. This Review discusses the prospects for developing newer, safer anti-aging therapies based on analogs of rapamycin (termed rapalogs) or other approaches targeting mTOR signaling. Full Text
Lange, J., Noordam, M.J., van Daalen, S.K., Skaletsky, H., Clark, B.A., Macville, M.V., Page, D.C., and Repping, S. (2013). Intrachromosomal homologous recombination between inverted amplicons on opposing Y-chromosome arms. Genomics. Available online 2 May 2013 Amplicons - large, nearly identical repeats in direct or inverted orientation - are abundant in the male-specific region of the human Y chromosome (MSY) and provide targets for intrachromosomal non-allelic homologous recombination (NAHR). Thus far, NAHR events resulting in deletions, duplications, inversions, or isodicentric chromosomes have been reported only for amplicon pairs located exclusively on the short arm (Yp) or the long arm (Yq). Here we report our finding of four men with Y chromosomes that evidently formed by intrachromosomal NAHR between inverted repeat pairs comprising one amplicon on Yp and one amplicon on Yq. In two men with spermatogenic failure, sister-chromatid crossing-over resulted in pseudoisoYp chromosome formation and loss of distal Yq. In two men with normal spermatogenesis, intrachromatid crossing-over generated pericentric inversions. These findings highlight the recombinogenic nature of the MSY, as intrachromosomal NAHR occurs for nearly all Y-chromosome amplicon pairs, even those located on opposing chromosome arms. Full Text
Laplante, M., and Sabatini, D.M. (2013). Regulation of mTORC1 and its impact on gene expression at a glance. Journal of Cell Science 126, 1713-1719.The mechanistic (or mammalian) target of rapamycin (mTOR) is a kinase that regulates key cellular functions linked to the promotion of cell growth and metabolism. This kinase, which is part of two protein complexes termed mTOR complex 1 (mTORC1) and 2 (mTORC2), has a fundamental role in coordinating anabolic and catabolic processes in response to growth factors and nutrients. Of the two mTOR complexes, mTORC1 is by far the best characterized. When active, mTORC1 triggers cell growth and proliferation by promoting protein synthesis, lipid biogenesis, and metabolism, and by reducing autophagy. The fact that mTORC1 deregulation is associated with several human diseases, such as type 2 diabetes, cancer, obesity and neurodegeneration, highlights its importance in the maintenance of cellular homeostasis. Over the last years, several groups observed that mTORC1 inhibition, in addition to reducing protein synthesis, deeply affects gene transcription. Here, we review the connections between mTORC1 and gene transcription by focusing on its impact in regulating the activation of specific transcription factors including including STAT3, SREBPs, PPAR gamma, PPAR alpha, HIF1 alpha, YY1-PGC1 alpha and TFEB. We also discuss the importance of these transcription factors in mediating the effects of mTORC1 on various cellular processes in physiological and pathological contexts. PDF
LeeCC, Carette, J.E., Brummelkamp, T.R., and Ploegh, H.L. (2013). A Reporter Screen in a Human Haploid Cell Line Identifies CYLD as a Constitutive Inhibitor of NF-kappaB. Plos One 8, e70339.The development of forward genetic screens in human haploid cells has the potential to transform our understanding of the genetic basis of cellular processes unique to man. So far, this approach has been limited mostly to the identification of genes that mediate cell death in response to a lethal agent, likely due to the ease with which this phenotype can be observed. Here, we perform the first reporter screen in the near-haploid KBM7 cell line to identify constitutive inhibitors of NF-kappaB. CYLD was the only currently known negative regulator of NF-kappaB to be identified, thus uniquely distinguishing this gene. Also identified were three genes with no previous known connection to NF-kappaB. Our results demonstrate that reporter screens in haploid human cells can be applied to investigate the many complex signaling pathways that converge upon transcription factors. Full Text
Lee, T.I., and Young, R.A. (2013). Transcriptional regulation and its misregulation in disease. Cell 152, 1237-1251.The gene expression programs that establish and maintain specific cell states in humans are controlled by thousands of transcription factors, cofactors, and chromatin regulators. Misregulation of these gene expression programs can cause a broad range of diseases. Here, we review recent advances in our understanding of transcriptional regulation and discuss how these have provided new insights into transcriptional misregulation in disease. PDF
Lesch, B.J., Dokshin, G.A., Young, R.A., McCarrey, J.R., and Page, D.C. (2013). A set of genes critical to development is epigenetically poised in mouse germ cells from fetal stages through completion of meiosis. Proc Natl Acad Sci U S ASep 16. [Epub ahead of print].In multicellular organisms, germ cells carry the hereditary material from one generation to the next. Developing germ cells are unipotent gamete precursors, and mature gametes are highly differentiated, specialized cells. However, upon gamete union at fertilization, their genomes drive a totipotent program, giving rise to a complete embryo as well as extraembryonic tissues. The biochemical basis for the ability to transition from differentiated cell to totipotent zygote is unknown. Here we report that a set of developmentally critical genes is maintained in an epigenetically poised (bivalent) state from embryonic stages through the end of meiosis. We performed ChIP-seq and RNA-seq analysis on flow-sorted male and female germ cells during embryogenesis at three time points surrounding sexual differentiation and female meiotic initiation, and then extended our analysis to meiotic and postmeiotic male germ cells. We identified a set of genes that is highly enriched for regulators of differentiation and retains a poised state (high H3K4me3, high H3K27me3, and lack of expression) across sexes and across developmental stages, including in haploid postmeiotic cells. The existence of such a state in embryonic stem cells has been well described. We now demonstrate that a subset of genes is maintained in a poised state in the germ line from the initiation of sexual differentiation during fetal development and into postmeiotic stages. We propose that the epigenetically poised condition of these developmental genes is a fundamental property of the mammalian germ-line nucleus, allowing differentiated gametes to unleash a totipotent program following fertilization. Full Text
Lewitter, F. (2013). Moving education forward, again! PLoS Comput Biol 9, e1003390 Educating biologists and computational biologists in methods and analyses is an ever-growing challenge. The amount of data and tools available to the scientific community continue to grow, and with that, there is a growing need to teach how to get the most out of this information. Full Text
Li2J., Ning, Y., Abushahin, N., Yuan, Z., Wang, Y.Y., Wang, Y., Yuan, B.B., Cragun, J.M., Chambers, S.K., Hatch, K., et al. (2013). Secretory cell expansion with aging: Risk for pelvic serous carcinogenesis. Gynecologic Oncology 131, 555-560.Objective. Recent advances suggest that precancerous lesions of pelvic serous carcinoma (PSC) originate from tubal secretory cells. The purpose of our study was to determine if increased number of secretory cells shows difference in age and location and to examine their association with serous neoplasia. Materials and methods. Three groups (benign control, high-risk, and PSC) of patients with matched ages were studied. The age data was stratified into 10-year intervals ranging from age 20 to older than 80. The number of secretory and ciliated cells from both tubal fimbria and ampulla segments was counted by microscopy and immunohistochemical staining methods. The data was analyzed by standard contingency table and Poisson distribution methods after age justification. Results. We found that the absolute number of tubal secretory cells increased significantly with age within each age group. Age remained a significant risk factor for serous neoplasia after age adjustment. In addition, a dramatic increase of secretory cells was observed in high-risk and PSC patients. Further, secretory cell expansion (SCE) was more prevalent than secretory cell outgrowth in both fimbria and ampulla tubal segments and was significantly associated with serous neoplasia (p < 0.001). Conclusions. These findings suggest that SCE could potentially serve as a sensitive biomarker for early serous carcinogenesis within the fallopian tube. Findings support a relationship between serous neoplasia and increased secretory to ciliated cell ratios. Findings also support a relationship between frequency of SCE and increasing age, presence of high-risk factors and co-existing serous cancers.
Li, Y., Wang, H., Muffat, J., Cheng, A.W., Orlando, D.A., Loven, J., Kwok, S.M., Feldman, D.A., Bateup, H.S., Gao, Q,.Hockemeyer D, Mitalipova M, Lewis CA, Vander Heiden MG, Sur M, Young RA, Jaenisch R Global Transcriptional and Translational Repression in Human-Embryonic-Stem-Cell-Derived Rett Syndrome Neurons. Cell Stem Cell 13, 446-458.Rett syndrome (RTT) is caused by mutations of MECP2, a methyl CpG binding protein thought to act as a global transcriptional repressor. Here we show, using an isogenic human embryonic stem cell model of RTT, that MECP2 mutant neurons display key molecular and cellular features of this disorder. Unbiased global gene expression analyses demonstrate that MECP2 functions as a global activator in neurons but not in neural precursors. Decreased transcription in neurons was coupled with a significant reduction in nascent protein synthesis and lack of MECP2 was manifested as a severe defect in the activity of the AKT/mTOR pathway. Lack of MECP2 also leads to impaired mitochondrial function in mutant neurons. Activation of AKT/mTOR signaling by exogenous growth factors or by depletion of PTEN boosted protein synthesis and ameliorated disease phenotypes in mutant neurons. Our findings indicate a vital function for MECP2 in maintaining active gene transcription in human neuronal cells. Full Text
Liu, Q., Xu, C., Kirubakaran, S., Zhang, X., Hur, W., Liu, Y., Kwiatkowski, N.P., Wang, J., Westover, K.D., Gao, P, Seong A. Kang ,Matthew P. Patricelli,Yuchuan Wang,Tanya Tupper,Abigail Altabef,Hidemasa Kawamura,Kathryn D. Held,Danny M. Chou,Stephen J. Elledge,Pasi A. Janne,Kwok-Kin Wong, David M. Sabatini, and Nathanael S. Gray. (2013). Characterization of Torin2, an ATP-Competitive Inhibitor of mTOR, ATM, and ATR. Cancer Res 73, 2574-2586 .mTOR is a highly conserved serine/threonine protein kinase that serves as a central regulator of cell growth, survival, and autophagy. Deregulation of the PI3K/Akt/mTOR signaling pathway occurs commonly in cancer and numerous inhibitors targeting the ATP-binding site of these kinases are currently undergoing clinical evaluation. Here, we report the characterization of Torin2, a second-generation ATP-competitive inhibitor that is potent and selective for mTOR with a superior pharmacokinetic profile to previous inhibitors. Torin2 inhibited mTORC1-dependent T389 phosphorylation on S6K (RPS6KB1) with an EC50 of 250 pmol/L with approximately 800-fold selectivity for cellular mTOR versus phosphoinositide 3-kinase (PI3K). Torin2 also exhibited potent biochemical and cellular activity against phosphatidylinositol-3 kinase-like kinase (PIKK) family kinases including ATM (EC50, 28 nmol/L), ATR (EC50, 35 nmol/L), and DNA-PK (EC50, 118 nmol/L; PRKDC), the inhibition of which sensitized cells to Irradiation. Similar to the earlier generation compound Torin1 and in contrast to other reported mTOR inhibitors, Torin2 inhibited mTOR kinase and mTORC1 signaling activities in a sustained manner suggestive of a slow dissociation from the kinase. Cancer cell treatment with Torin2 for 24 hours resulted in a prolonged block in negative feedback and consequent T308 phosphorylation on Akt. These effects were associated with strong growth inhibition in vitro. Single-agent treatment with Torin2 in vivo did not yield significant efficacy against KRAS-driven lung tumors, but the combination of Torin2 with mitogen-activated protein/extracellular signal-regulated kinase (MEK) inhibitor AZD6244 yielded a significant growth inhibition. Taken together, our findings establish Torin2 as a strong candidate for clinical evaluation in a broad number of oncologic settings where mTOR signaling has a pathogenic role. Full Text
Lo, K.A., Labadorf, A., Kennedy, N.J., Han, M.S., Yap, Y.S., Matthews, B., Xin, X., Sun, L., Davis, R.J., Lodish, H.F., et al. (2013). Analysis of In Vitro Insulin-Resistance Models and Their Physiological Relevance to In Vivo Diet-Induced Adipose Insulin Resistance. Cell Reports 5 (1) : 259-270. Diet-induced obesity (DIO) predisposes individuals to insulin resistance, and adipose tissue has a major role in the disease. Insulin resistance can be induced in cultured adipocytes by a variety of treatments, but what aspects of the in vivo responses are captured by these models remains unknown. We use global RNA sequencing to investigate changes induced by TNF-alpha, hypoxia, dexamethasone, high insulin, and a combination of TNF-alpha and hypoxia, comparing the results to the changes in white adipose tissue from DIO mice. We found that different in vitro models capture distinct features of DIO adipose insulin resistance, and a combined treatment of TNF-alpha and hypoxia is most able to mimic the in vivo changes. Using genome-wide DNase I hypersensitivity followed by sequencing, we further examined the transcriptional regulation of TNF-alpha-induced insulin resistance, and we found that C/EPBbeta is a potential key regulator of adipose insulin resistance. Full Text
Lodato, M.A., Ng, C.W., Wamstad, J.A., Cheng, A.W., Thai, K.K., Fraenkel, E., Jaenisch, R., and Boyer, L.A. (2013). SOX2 Co-Occupies Distal Enhancer Elements with Distinct POU Factors in ESCs and NPCs to Specify Cell State. PLoS Genet 9, e1003288.SOX2 is a master regulator of both pluripotent embryonic stem cells (ESCs) and multipotent neural progenitor cells (NPCs); however, we currently lack a detailed understanding of how SOX2 controls these distinct stem cell populations. Here we show by genome-wide analysis that, while SOX2 bound to a distinct set of gene promoters in ESCs and NPCs, the majority of regions coincided with unique distal enhancer elements, important cis-acting regulators of tissue-specific gene expression programs. Notably, SOX2 bound the same consensus DNA motif in both cell types, suggesting that additional factors contribute to target specificity. We found that, similar to its association with OCT4 (Pou5f1) in ESCs, the related POU family member BRN2 (Pou3f2) co-occupied a large set of putative distal enhancers with SOX2 in NPCs. Forced expression of BRN2 in ESCs led to functional recruitment of SOX2 to a subset of NPC-specific targets and to precocious differentiation toward a neural-like state. Further analysis of the bound sequences revealed differences in the distances of SOX and POU peaks in the two cell types and identified motifs for additional transcription factors. Together, these data suggest that SOX2 controls a larger network of genes than previously anticipated through binding of distal enhancers and that transitions in POU partner factors may control tissue-specific transcriptional programs. Our findings have important implications for understanding lineage specification and somatic cell reprogramming, where SOX2, OCT4, and BRN2 have been shown to be key factors. Full Text
Lodish, H. (2013). Cloning Expeditions: Risky but Rewarding. Mol Cell Biol.(published online ahead of print on 23 September 2013). In the 1980's a good part of my lab was using the then new recombinant DNA techniques to clone and characterize many important cell surface membrane proteins: GLUT1 (the red cell glucose transporter) and then GLUT2 and GLUT4, the red cell anion exchange protein (Band 3), asialoglycoprotein receptor subunits, sucrase-isomaltase, the erythropoietin receptor, and two of the subunits of the TGFss receptor. These cloned genes opened many new fields of basic research, including membrane insertion and trafficking of transmembrane proteins, signal transduction by many members of the cytokine and TGF-ss family of receptors, and the cellular physiology of glucose and anion transport. They also led to many insights into the molecular biology of several cancers, hematopoietic disorders, and diabetes. This work was done by an exceptional group of postdocs and students who took exceptionally large risks in developing and using novel cloning technologies. Unsurprisingly, all have gone on to become leaders in molecular cell biology and molecular medicine. Full Text
Loven, J., Hoke, H.A., Lin, C.Y., Lau, A., Orlando, D.A., Vakoc, C.R., Bradner, J.E., Lee, T.I., and Young, R.A. (2013). Selective inhibition of tumor oncogenes by disruption of super-enhancers. Cell 153, 320-334.Chromatin regulators have become attractive targets for cancer therapy, but it is unclear why inhibition of these ubiquitous regulators should have gene-specific effects in tumor cells. Here, we investigate how inhibition of the widely expressed transcriptional coactivator BRD4 leads to selective inhibition of the MYC oncogene in multiple myeloma (MM). BRD4 and Mediator were found to co-occupy thousands of enhancers associated with active genes. They also co-occupied a small set of exceptionally large super-enhancers associated with genes that feature prominently in MM biology, including the MYC oncogene. Treatment of MM tumor cells with the BET-bromodomain inhibitor JQ1 led to preferential loss of BRD4 at super-enhancers and consequent transcription elongation defects that preferentially impacted genes with super-enhancers, including MYC. Super-enhancers were found at key oncogenic drivers in many other tumor cells. These observations have implications for the discovery of cancer therapeutics directed at components of super-enhancers in diverse tumor types. Full Text
Mansour, M.R., Sanda, T., Lawton, L.N., Li, X.Y., Kreslavsky, T., Novina, C.D., Brand, M., Gutierrez, A., Kelliher, M.A., Jamieson, C.H.M ,.Richard A. Young, et al. (2013). The TAL1 complex targets the FBXW7 tumor suppressor by activating miR-223 in human T cell acute lymphoblastic leukemia. Journal of Experimental Medicine 210, 1545-1557.The oncogenic transcription factor TAL1/SCL is aberrantly expressed in 60% of cases of human T cell acute lymphoblastic leukemia (T-ALL) and initiates T-ALL in mouse models. By performing global microRNA (miRNA) expression profiling after depletion of TAL1, together with genome-wide analysis of TAL1 occupancy by chromatin immunoprecipitation coupled to massively parallel DNA sequencing, we identified the miRNA genes directly controlled by TAL1 and its regulatory partners HEB, E2A, LMO1/2, GATA3, and RUNX1. The most dynamically regulated miRNA was miR-223, which is bound at its promoter and up-regulated by the TAL1 complex. miR-223 expression mirrors TAL1 levels during thymic development, with high expression in early thymocytes and marked down-regulation after the double-negative-2 stage of maturation. We demonstrate that aberrant miR-223 up-regulation by TAL1 is important for optimal growth of TAL1-positive T-ALL cells and that sustained expression of miR-223 partially rescues T-ALL cells after TAL1 knockdown. Overexpression of miR-223 also leads to marked down-regulation of FBXW7 protein expression, whereas knockdown of TAL1 leads to up-regulation of FBXW7 protein levels, with a marked reduction of its substrates MYC, MYB, NOTCH1, and CYCLIN E. We conclude that TAL1-mediated up-regulation of miR-223 promotes the malignant phenotype in T-ALL through repression of the FBXW7 tumor suppressor. Full Text
Marjanovic, N.D., Weinberg, R.A., and Chaffer, C.L. (2013). Poised with purpose: Cell plasticity enhances tumorigenicity. Cell Cycle 12: 17, 2713-2714. In light of current findings, therapies targeting non-CSC-to-CSC plasticity should offer improved clinical outcome for cancer patients. PDF
Mercken, E.M., Crosby, S.D., Lamming, D.W., JeBailey, L., Krzysik-Walker, S., Villareal, D.T., Capri, M., Franceschi, C., Zhang, Y.Q., Becker, K.,Sabatini DM, de Cabo R, Fontana L. (2013). Calorie restriction in humans inhibits the PI3K/AKT pathway and induces a younger transcription profile. Aging Cell 12, 645-651.Caloric restriction (CR) and down-regulation of the insulin/IGF pathway are the most robust interventions known to increase longevity in lower organisms. However, little is known about the molecular adaptations induced by CR in humans. Here, we report that long-term CR in humans inhibits the IGF-1/insulin pathway in skeletal muscle, a key metabolic tissue. We also demonstrate that CR induces dramatic changes of the skeletal muscle transcriptional profile that resemble those of younger individuals. Finally, in both rats and humans, CR evoked similar responses in the transcriptional profiles of skeletal muscle. This common signature consisted of three key pathways typically associated with longevity: IGF-1/insulin signaling, mitochondrial biogenesis, and inflammation. Furthermore, our data identify promising pathways for therapeutic targets to combat age-related diseases and promote health in humans. Full Text
Molski, M.A., Goodman, J.L., Chou, F.C., Baker, D., Das, R., and Schepartz, A. (2013). Remodeling a beta-peptide bundle. Chemical Science 4, 319-324. Natural biopolymers fold with fidelity, burying diverse side chains into well-packed cores and protecting their backbones from solvent. Certain beta-peptide oligomers assemble into bundles of defined octameric stoichiometry that resemble natural proteins in many respects. These beta-peptide bundles are thermostable, fold cooperatively, exchange interior amide N-H protons slowly, exclude hydrophobic dyes, and can be characterized at high resolution using X-ray crystallography - just like many proteins found in nature. But unlike natural proteins, all octameric beta-peptide bundles contain a sequence-uniform hydrophobic core composed of 32 leucine side chains. Here we apply rational design principles, including the Rosetta computational design methodology, to introduce sequence diversity into the bundle core while retaining the characteristic beta-peptide bundle fold. Using circular dichroism spectroscopy and analytical ultracentrifugation, we confirmed the prediction that an octameric bundle still assembles upon a major remodelling of its core: the mutation of sixteen core beta-homo-leucine side chains into sixteen beta-homo-phenylalanine side chains. Nevertheless, the bundle containing a partially beta-homo-phenylalanine core poorly protects interior amide protons from exchange, suggesting molten-globule-like properties. We further improve stability by the incorporation of eight beta-homo-pentafluorophenyalanine side chains, giving an assembly with amide protection factors comparable to prior well-structured bundles. By demonstrating that their cores tolerate significant sequence variation, the beta-peptide bundles reported here represent a starting point for the "bottom-up" construction of beta-peptide assemblies possessing both structure and sophisticated function. Full Text
Mueller, J.L., Skaletsky, H., Brown, L.G., Zaghlul, S., Rock, S., Graves, T., Auger, K., Warren, W.C., Wilson, R.K., and Page, D.C. (2013). Independent specialization of the human and mouse X chromosomes for the male germ line .Nature Genetics Jul 21. [Epub ahead of print] .We compared the human and mouse X chromosomes to systematically test Ohno's law, which states that the gene content of X chromosomes is conserved across placental mammals. First, we improved the accuracy of the human X-chromosome reference sequence through single-haplotype sequencing of ampliconic regions. The new sequence closed gaps in the reference sequence, corrected previously misassembled regions and identified new palindromic amplicons. Our subsequent analysis led us to conclude that the evolution of human and mouse X chromosomes was bimodal. In accord with Ohno's law, 94-95% of X-linked single-copy genes are shared by humans and mice; most are expressed in both sexes. Notably, most X-ampliconic genes are exceptions to Ohno's law: only 31% of human and 22% of mouse X-ampliconic genes had orthologs in the other species. X-ampliconic genes are expressed predominantly in testicular germ cells, and many were independently acquired since divergence from the common ancestor of humans and mice, specializing portions of their X chromosomes for sperm production. Full Text
Mukherjee, S., Zhu, J., Zikherman, J., Parameswaran, R., Kadlecek, T.A., Wang, Q., Au-Yeung, B., Ploegh, H., Kuriyan, J., Das, J., et al. (2013). Monovalent and Multivalent Ligation of the B Cell Receptor Exhibit Differential Dependence upon Syk and Src Family Kinases. Science Signaling 6(256) : ra1. The Src and Syk families of kinases are two distinct sets of kinases that play critical roles in initiating membrane-proximal B cell receptor (BCR) signaling. However, unlike in other lymphocytes, such as T cells, the "division of labor" between Src family kinases (SFKs) and Syk in B cells is not well separated because both Syk and SFKs can phosphorylate immunoreceptor tyrosine-based activation motifs (ITAMs) present in proteins comprising the BCR. To understand why B cells require both SFKs and Syk for activation, we investigated the roles of both families of kinases in BCR signaling with computational modeling and in vitro experiments. Our computational model suggested that positive feedback enabled Syk to substantially compensate for the absence of SFKs when spatial clustering of BCRs was induced by multimeric ligands. We confirmed this prediction experimentally. In contrast, when B cells were stimulated by monomeric ligands that failed to produce BCR clustering, both Syk and SFKs were required for complete and rapid BCR activation. Our data suggest that SFKs could play a pivotal role in increasing BCR sensitivity to monomeric antigens of pathogens and in mediating a rapid response to soluble multimeric antigens of pathogens that can induce spatial BCR clustering. Full Text
Musallam K.M., Taher, A.T., Cappellini, M.D., and Sankaran, V.G. (2013). Clinical experience with fetal hemoglobin induction therapy in patients with beta-thalassemia. Blood 121, 2199-2212.Recent molecular studies of fetal hemoglobin (HbF) regulation have reinvigorated the field and shown promise for the development of clinical HbF inducers to be used in patients with beta-thalassemia and sickle cell disease. However, while numerous promising inducers of HbF have been studied in the past in beta-thalassemia patient populations, with limited success in some cases, no universally effective agents have been found. Here we examine the clinical studies of such inducers in an attempt to systematically review the field. We examine trials of agents, including 5-azacytidine, hydroxyurea, and short-chain fatty acids. This review highlights the heterogeneity of clinical studies done on these agents, including both the patient populations examined and the study end points. By examining the published studies of these agents, we hope to provide a resource that will be valuable for the design of future studies of HbF inducers in beta-thalassemia patient populations. Full Text
Newby, G.A., and Lindquist, S. (2013). Blessings in disguise: biological benefits of prion-like mechanisms. Trends Cell Biol .Feb 25. [Epub ahead of print] Prions and amyloids are often associated with disease, but related mechanisms provide beneficial functions in nature. Prion-like mechanisms (PriLiMs) are found from bacteria to humans, where they alter the biological and physical properties of prion-like proteins. We have proposed that prions can serve as heritable bet-hedging devices for diversifying microbial phenotypes. Other, more dynamic proteinaceous complexes may be governed by similar self-templating conformational switches. Additional PriLiMs continue to be identified and many share features of self-templating protein structure (including amyloids) and dependence on chaperone proteins. Here, we discuss several PriLiMs and their functions, intending to spur discussion and collaboration on the subject of beneficial prion-like behaviors. Full Text
Nishino, T., Rago, F., Hori, T., Tomii, K., Cheeseman, I.M., and Fukagawa, T. (2013). CENP-T provides a structural platform for outer kinetochore assembly. EMBO J 32, 424-436.The kinetochore forms a dynamic interface with microtubules from the mitotic spindle during mitosis. The Ndc80 complex acts as the key microtubule-binding complex at kinetochores. However, it is unclear how the Ndc80 complex associates with the inner kinetochore proteins that assemble upon centromeric chromatin. Here, based on a high-resolution structural analysis, we demonstrate that the N-terminal region of vertebrate CENP-T interacts with the 'RWD' domain in the Spc24/25 portion of the Ndc80 complex. Phosphorylation of CENP-T strengthens a cryptic hydrophobic interaction between CENP-T and Spc25 resulting in a phospho-regulated interaction that occurs without direct recognition of the phosphorylated residue. The Ndc80 complex interacts with both CENP-T and the Mis12 complex, but we find that these interactions are mutually exclusive, supporting a model in which two distinct pathways target the Ndc80 complex to kinetochores. Our results provide a model for how the multiple protein complexes at kinetochores associate in a phospho-regulated manner. PDF
Okumura, L.M., Lesch, B.J., and Page, D.C. (2013). The Ligand Binding Domain of GCNF Is Not Required for Repression of Pluripotency Genes in Mouse Fetal Ovarian Germ Cells. Plos One 8, e66062.In mice, successful development and reproduction require that all cells, including germ cells, transition from a pluripotent to a differentiated state. This transition is associated with silencing of the pluripotency genes Oct4 and Nanog. Interestingly, these genes are repressed at different developmental timepoints in germ and somatic cells. Ovarian germ cells maintain their expression until about embryonic day (E) 14.5, whereas somatic cells silence them much earlier, at about E8.0. In both somatic cells and embryonic stem cells, silencing of Oct4 and Nanog requires the nuclear receptor GCNF. However, expression of the Gcnf gene has not been investigated in fetal ovarian germ cells, and whether it is required for silencing Oct4 and Nanog in that context is not known. Here we demonstrate that Gcnf is expressed in fetal ovarian germ cells, peaking at E14.5, when Oct4 and Nanog are silenced. However, conditional ablation of the ligand-binding domain of Gcnf using a ubiquitous, tamoxifen-inducible Cre indicates that Gcnf is not required for the down-regulation of pluripotency genes in fetal ovarian germ cells, nor is it required for initiation of meiosis and oogenesis. These results suggest that the silencing of Oct4 and Nanog in germ cells occurs via a different mechanism from that operating in somatic cells during gastrulation. Full Text
Okuyama, K., Ikawa, T., Gentner, B., Hozumi, K., Harnprasopwat, R., Lu, J., Yamashita, R., Ha, D.O., Toyoshima, T., Chanda, B. , Harvey F. Lodish, et al. (2013). MicroRNA-126-mediated control of cell fate in B-cell myeloid progenitors as a potential alternative to transcriptional factors. Proceedings of the National Academy of Sciences of the United States of America 110, 13410-13415.Lineage specification is thought to be largely regulated at the level of transcription, where lineage-specific transcription factors drive specific cell fates. MicroRNAs (miR), vital to many cell functions, act posttranscriptionally to decrease the expression of target mRNAs. MLL-AF4 acute lymphocytic leukemia exhibits both myeloid and B-cell surface markers, suggesting that the transformed cells are B-cell myeloid progenitor cells. Through gain- and loss-of-function experiments, we demonstrated that microRNA 126 (miR-126) drives B-cell myeloid biphenotypic leukemia differentiation toward B cells without changing expression of E2A immunoglobulin enhancer-binding factor E12/E47 (E2A), early B-cell factor 1 (EBF1), or paired box protein 5, which are critical transcription factors in B-lymphopoiesis. Similar induction of B-cell differentiation by miR-126 was observed in normal hematopoietic cells in vitro and in vivo in uncommitted murine c-Kit(+)Sca1(+)Lineage(-) cells, with insulin regulatory subunit-1 acting as a target of miR-126. Importantly, in EBF1-deficient hematopoietic progenitor cells, which fail to differentiate into B cells, miR-126 significantly up-regulated B220, and induced the expression of B-cell genes, including recombination activating genes-1/2 and CD79a/b. These data suggest that miR-126 can at least partly rescue B-cell development independently of EBF1. These experiments show that miR-126 regulates myeloid vs. B-cell fate through an alternative machinery, establishing the critical role of miRNAs in the lineage specification of multipotent mammalian cells. Full Text
Petrocca, F., Altschuler, G., Tan, S.M., Mendillo, M.L., Yan, H.H., Jerry, D.J., Kung, A.L., Hide, W., Ince, T.A., and Lieberman, J. (2013). A Genome-wide siRNA Screen Identifies Proteasome Addiction as a Vulnerability of Basal-like Triple-Negative Breast Cancer Cells. Cancer Cell 24, 182-196.Basal-like triple-negative breast cancers (TNBCs) have poor prognosis. To identify basal-like TNBC dependencies, a genome-wide siRNA lethality screen compared two human breast epithelial cell lines transformed with the same genes: basal-like BPLER and myoepithelial HMLER. Expression of the screen's 154 BPLER dependency genes correlated with poor prognosis in breast, but not lung or colon, cancer. Proteasome genes were overrepresented hits. Basal-like TNBC lines were selectively sensitive to proteasome inhibitor drugs relative to normal epithelial, luminal, and mesenchymal TNBC lines. Proteasome inhibition reduced growth of established basal-like TNBC tumors in mice and blocked tumor-initiating cell function and macronnetastasis. Proteasome addiction in basal-like TNBCs was mediated by NOXA and linked to MCL-1 dependence .PDF
Pham, T.H., Gao, X.F., Singh, G., and Hardwidge, P.R. (2013). Escherichia coli Virulence Protein NleH1 Interaction with the v-Crk Sarcoma Virus CT10 Oncogene-like Protein (CRKL) Governs NleH1 Inhibition of the Ribosomal Protein 53 (RPS3)/Nuclear Factor kappa B (NF-kappa B) Pathway. Journal of Biological Chemistry 288, 34567-34574.Background: Bacterial pathogens use virulence proteins to inhibit the host innate immune system. Results: The Escherichia coli O157:H7 NleH1 protein interacts with the host CRKL protein. Conclusion: CRKL may recruit NleH1 to a host kinase on which NleH1 performs its inhibitory function. Significance: These data clarify a mechanism by which E. coli inhibits innate immunity. Enterohemorrhagic Escherichia coli and other attaching/effacing bacterial pathogens cause diarrhea in humans. These pathogens use a type III secretion system to inject virulence proteins (effectors) into host cells, some of which inhibit the innate immune system. The enterohemorrhagic E. coli NleH1 effector prevents the nuclear translocation of RPS3 (ribosomal protein S3) to inhibit its participation as a nuclear specifier of NF-B binding to target gene promoters. NleH1 binds to RPS3 and inhibits its phosphorylation on Ser-209 by IB kinase- (IKK). However, the precise mechanism of this inhibition is unclear. NleH1 possesses a Ser/Thr protein kinase activity that is essential both for its ability to inhibit the RPS3/NF-B pathway and for full virulence of the attaching/effacing mouse pathogen Citrobacter rodentium. However, neither RPS3 nor IKK is a substrate of NleH1 kinase activity. We therefore screened approximate to 9,000 human proteins to identify NleH1 kinase substrates and identified CRKL (v-Crk sarcoma virus CT10 oncogene-like protein), a substrate of the BCR/ABL kinase. Knockdown of CRKL abundance prevented NleH1 from inhibiting RPS3 nuclear translocation and NF-B activity. CRKL residues Tyr-198 and Tyr-207 were required for interaction with NleH1. Lys-159, the kinase-active site of NleH1, was necessary for its interaction with CRKL. We also identified CRKL as an IKK interaction partner, mediated by CRKL Tyr-198. We propose that the CRKL interaction with IKK recruits NleH1 to the IKK complex, where NleH1 then inhibits the RPS3/NF-B pathway. Full Text
Piccolo, F.M., Bagci, H., Brown, K.E., Landeira, D., Soza-Ried, J., Feytout, A., Mooijman, D., Hajkova, P., Leitch, H.G., Tada, T.,Kriaucionis S, Dawlaty MM, Jaenisch R, Merkenschlager M, Fisher AG. (2013). Different Roles for Tet1 and Tet2 Proteins in Reprogramming-Mediated Erasure of Imprints Induced by EGC Fusion. Molecular Cell 49, 1023-1033.Genonnic imprinting directs the allele-specific marking and expression of loci according to their parental origin. Differential DNA methylation at imprinted control regions (ICRs) is established in gametes and, although largely preserved through development, can be experimentally reset by fusing somatic cells with embryonic germ cell (EGG) lines. Here, we show that the Ten-Eleven Translocation proteins Tet1 and Tet2 participate in the efficient erasure of imprints in this model system. The fusion of B cells with EGCs initiates pluripotent reprogramming, in which rapid re-expression of Oct4 is accompanied by an accumulation of 5-hydroxymethylcytosine (5hmC) at several ICRs. Tet2 was required for the efficient reprogramming capacity of EGCs, whereas Tet1 was necessary to induce 5-methylcytosine oxidation specifically at ICRs. These data show that the Tet1 and Tet2 proteins have discrete roles in cell-fusion-mediated pluripotent reprogramming and imprint erasure in somatic cells. Full Text
Ploegh1, H. (2013). Ubiquitin-specific Peptidases 14 and 6. In Handbook of Proteolytic Enzymes, Vol. 2, 3rd Edition, Chapter 470, pp. 2086-2089.
Ploegh2, H. (2013). UL36 Deubiquitinylating Peptidase. In Handbook of Proteolytic Enzymes, Vol. 2 , 3rd Edition,Chapter 480, pp. 2131-2134.
Ploegh3, H. (2013). ElaD Peptidase (Escherichia coli). In Handbook of Proteolytic Enzymes, Vol. 2, 3rd Edition,Chapter 536, pp. 2392-2396.
Ploegh4, H.L. (2013). Logic of the immune system. Cancer Immunol Res 1, 5-10.Our immune system evolves under continuous selection to protect us against pathogens, a trait that can be used to deploy the immune system or its component parts for diagnostic and therapeutic purposes in cancer. What follows is a thumbnail sketch of the logic that underlies immune recognition, so that those not directly active in the field of immunology may grasp both possibilities and pitfalls when considering applying the tools and concepts of immunology to cancer and cancer therapy. Full Text
Rago, F., and Cheeseman, I.M. (2013). The functions and consequences of force at kinetochores. J Cell Biol 200, 557-565.Chromosome segregation requires the generation of force at the kinetochore-the multiprotein structure that facilitates attachment of chromosomes to spindle microtubules. This force is required both to move chromosomes and to signal the formation of proper bioriented attachments. To understand the role of force in these processes, it is critical to define how force is generated at kinetochores, the contributions of this force to chromosome movement, and how the kinetochore is structured and organized to withstand and respond to force. Classical studies and recent work provide a framework to dissect the mechanisms, functions, and consequences of force at kinetochores. Full Text
RealesCalderon, J.A., Sylvester, M., Strijbis, K., Jensen, O.N., Nombela, C., Molero, G., and Gil, C. (2013). Candida albicans induces pro-inflammatory and anti-apoptotic signals in macrophages as revealed by quantitative proteomics and phosphoproteomics. Journal of Proteomics 91, 106-135.Macrophages play a pivotal role in the prevention of Candida albicans infections. Yeast recognition and phagocytosis by macrophages is mediated by Pattern Recognition Receptors (PRRs) that initiate downstream signal transduction cascades by protein phosphorylation and dephosphorylation. We exposed RAW 264.7 macrophages to C. albicans for 3 h and used SILAC to quantify macrophage proteins and phosphoproteins by mass spectrometry to study the effects of infection. We identified 53 macrophage up-regulated proteins and 15 less abundant in the presence of C. albicans out of a total of 2071 identified proteins. 922 unique protein phosphorylation sites were identified by phosphopeptide enrichment and mass spectrometry, including 327 previously unidentified mouse protein phosphorylation sites. 126 peptides showed an increase and 70 a decrease in their phosphorylation level. The majority of the differentially expressed and phosphorylated proteins are receptors, mitochondrial ribosomal proteins, cytoskeletal proteins, and transcription factor activators involved in inflammatory and oxidative responses. In addition, we identified 22 proteins and phosphoproteins related to apoptosis. The analysis of apoptotic markers revealed that anti-apoptotic signals prevailed during the interaction of the yeast. Our proteomics study suggests that besides inflammation, apoptosis is a central pathway in the immune defense against C. albicans infection. Biological significance This work uses SILAC and SIMAC methodology combined with CPP (+ TiO2) to study protein and phosphopeptide changes in RAW 264.7 macrophages in response to coincubation with Candida albicans for 3 h. We show that the presence of C. albicans induces inflammatory responses and inhibits apoptosis in the macrophages. Our phosphoproteomic analysis identified 327 new mouse protein phosphorylation sites.
Reddien, P.W. (2013). Lin28: time for tissue repair. Cell 155, 738-739.Embryos and juveniles in many organisms repair tissue injuries better than adults. In this issue, Shyh-Chang et al. find that postnatal activation of Lin28a, a gene typically active in embryonic development, promotes better than normal tissue repair in mice, including following ear and digit injuries. Full Text
Reddien, P.W. (2013). Specialized progenitors and regeneration. Development 140, 951-957.Planarians are flatworms capable of regenerating all body parts. Planarian regeneration requires neoblasts, a population of dividing cells that has been studied for over a century. Neoblast progeny generate new cells of blastemas, which are the regenerative outgrowths at wounds. If the neoblasts comprise a uniform population of cells during regeneration (e.g. they are all uncommitted and pluripotent), then specialization of new cell types should occur in multipotent, non-dividing neoblast progeny cells. By contrast, recent data indicate that some neoblasts express lineage-specific transcription factors during regeneration and in uninjured animals. These observations raise the possibility that an important early step in planarian regeneration is the specialization of neoblasts to produce specified rather than naive blastema cells. Full Text
Reiling, J.H., Olive, A.J., Sanyal, S., Carette, J.E., Brummelkamp, T.R., Ploegh, H.L., Starnbach, M.N., and Sabatini, D.M. (2013). A CREB3-ARF4 signalling pathway mediates the response to Golgi stress and susceptibility to pathogens. Nature Cell Biology. Published online 03 November 2013 Treatment of cells with brefeldin A (BFA) blocks secretory vesicle transport and causes a collapse of the Golgi apparatus. To gain more insight into the cellular mechanisms mediating BFA toxicity, we conducted a genome-wide haploid genetic screen that led to the identification of the small G protein ADP-ribosylation factor 4 (ARF4). ARF4 depletion preserves viability, Golgi integrity and cargo trafficking in the presence of BFA, and these effects depend on the guanine nucleotide exchange factor GBF1 and other ARF isoforms including ARF1 and ARF5. ARF4 knockdown cells show increased resistance to several human pathogens including Chlamydia trachomatis and Shigella flexneri. Furthermore, ARF4 expression is induced when cells are exposed to several Golgi-disturbing agents and requires the CREB3 (also known as Luman or LZIP) transcription factor, whose downregulation mimics ARF4 loss. Thus, we have uncovered a CREB3-ARF4 signalling cascade that may be part of a Golgi stress response set in motion by stimuli compromising Golgi capacity. Full Text
Revill, K., Wang, T., Lachenmayer, A., Kojima, K., Harrington, A., Li, J.Y., Hoshida, Y., Llovet, J.M., and Powers, S. (2013). Genome-Wide Methylation Analysis and Epigenetic Unmasking Identify Tumor Suppressor Genes in Hepatocellular Carcinoma. Gastroenterology 145, 1424-+.BACKGROUND & AIMS: Epigenetic silencing of tumor suppressor genes contributes to the pathogenesis of hepatocellular carcinoma (HCC). To identify clinically relevant tumor suppressor genes silenced by DNA methylation in HCC, we integrated DNA methylation data from human primary HCC samples with data on up-regulation of gene expression after epigenetic unmasking. METHODS: We performed genome-wide methylation analysis of 71 human HCC samples using the Illumina HumanBeadchip27K array; data were combined with those from microarray analysis of gene re-expression in 4 liver cancer cell lines after their exposure to reagents that reverse DNA methylation (epigenetic unmasking). RESULTS: Based on DNA methylation in primary HCC and gene re-expression in cell lines after epigenetic unmasking, we identified 13 candidate tumor suppressor genes. Subsequent validation led us to focus on functionally characterizing 2 candidates, sphingomyelin phosphodiesterase 3 (SMPD3) and neurofilament, heavy polypeptide (NEFH), which we found to behave as tumor suppressor genes in HCC. Overexpression of SMPD3 and NEFH by stable transfection of inducible constructs into an HCC cell line reduced cell proliferation by 50% and 20%, respectively (SMPD3, P = .003 and NEFH, P = .003). Conversely, knocking down expression of these genes with small hairpin RNA promoted cell invasion and migration in vitro (SMPD3, P = .0001 and NEFH, P = .022), and increased their ability to form tumors after subcutaneous injection or orthotopic transplantation into mice, confirming their role as tumor suppressor genes in HCC. Low levels of SMPD3 were associated with early recurrence of HCC after curative surgery in an independent patient cohort (P = .001; hazard ratio = 3.22; 95% confidence interval: 1.6-6.5 in multivariate analysis). CONCLUSIONS: Integrative genomic analysis identified SMPD3 and NEFH as tumor suppressor genes in HCC. We provide evidence that SMPD3 is a potent tumor suppressor gene that could affect tumor aggressiveness; a reduced level of SMPD3 is an independent prognostic factor for early recurrence of HCC. Full Text
Rohner, N., Jarosz, D.F., Kowalko, J.E., Yoshizawa, M., Jeffery, W.R., Borowsky, R.L., Lindquist, S., and Tabin, C.J. (2013). Cryptic variation in morphological evolution: HSP90 as a capacitor for loss of eyes in cavefish. Science 342, 1372-1375.In the process of morphological evolution, the extent to which cryptic, preexisting variation provides a substrate for natural selection has been controversial. We provide evidence that heat shock protein 90 (HSP90) phenotypically masks standing eye-size variation in surface populations of the cavefish Astyanax mexicanus. This variation is exposed by HSP90 inhibition and can be selected for, ultimately yielding a reduced-eye phenotype even in the presence of full HSP90 activity. Raising surface fish under conditions found in caves taxes the HSP90 system, unmasking the same phenotypic variation as does direct inhibition of HSP90. These results suggest that cryptic variation played a role in the evolution of eye loss in cavefish and provide the first evidence for HSP90 as a capacitor for morphological evolution in a natural setting. Full Text
Rudenko, A., Dawlaty, M.M., Seo, J., Cheng, A.W., Meng, J., Le, T., Faull, K.F., Jaenisch, R., and Tsai, L.H. (2013). Tet1 is critical for neuronal activity-regulated gene expression and memory extinction. Neuron 79, 1109-1122.The ten-eleven translocation (Tet) family of methylcytosine dioxygenases catalyze oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) and promote DNA demethylation. Despite the abundance of 5hmC and Tet proteins in the brain, little is known about the functions of the neuronal Tet enzymes. Here, we analyzed Tet1 knockout mice (Tet1KO) and found downregulation of multiple neuronal activity-regulated genes, including Npas4, c-Fos, and Arc. Furthermore, Tet1KO animals exhibited abnormal hippocampal long-term depression and impaired memory extinction. Analysis of the key regulatory gene, Npas4, indicated that its promoter region, containing multiple CpG dinucleotides, is hypermethylated in both naive Tet1KO mice and after extinction training. Such hypermethylation may account for the diminished expression of Npas4 itself and its downstream targets, impairing transcriptional programs underlying cognitive processes. In summary, we show that neuronal Tet1 regulates normal DNA methylation levels, expression of activity-regulated genes, synaptic plasticity, and memory extinction. Full Text
Ryan, S.D., Dolatabadi, N., Chan, S.F., Zhang, X., Akhtar, M.W., Parker, J., Soldner, F., Sunico, C.R., Nagar, S., Talantova, M.R, Rudolph Jaenisch, et al. (2013). Isogenic Human iPSC Parkinson's Model Shows Nitrosative Stress-Induced Dysfunction in MEF2-PGC1alpha Transcription. Cell .Nov 23. [Epub ahead of print] Parkinson's disease (PD) is characterized by loss of A9 dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc). An association has been reported between PD and exposure to mitochondrial toxins, including environmental pesticides paraquat, maneb, and rotenone. Here, using a robust, patient-derived stem cell model of PD allowing comparison of A53T alpha-synuclein (alpha-syn) mutant cells and isogenic mutation-corrected controls, we identify mitochondrial toxin-induced perturbations in A53T alpha-syn A9 DA neurons (hNs). We report a pathway whereby basal and toxin-induced nitrosative/oxidative stress results in S-nitrosylation of transcription factor MEF2C in A53T hNs compared to corrected controls. This redox reaction inhibits the MEF2C-PGC1alpha transcriptional network, contributing to mitochondrial dysfunction and apoptotic cell death. Our data provide mechanistic insight into gene-environmental interaction (GxE) in the pathogenesis of PD. Furthermore, using small-molecule high-throughput screening, we identify the MEF2C-PGC1alpha pathway as a therapeutic target to combat PD. Full Text
Sahay, G., Querbes, W., Alabi, C., Eltoukhy, A., Sarkar, S., Zurenko, C., Karagiannis, E., Love, K., Chen, D.L., Zoncu, R.,Buganim Y, Schroeder A, Langer R, Anderson DG. (2013). Efficiency of siRNA delivery by lipid nanoparticles is limited by endocytic recycling. Nature Biotechnology 31, 653-U119.Despite efforts to understand the interactions between nanoparticles and cells, the cellular processes that determine the efficiency of intracellular drug delivery remain unclear. Here we examine cellular uptake of short interfering RNA (siRNA) delivered in lipid nanoparticles (LNPs) using cellular trafficking probes in combination with automated high-throughput confocal microscopy. We also employed defined perturbations of cellular pathways paired with systems biology approaches to uncover protein-protein and protein-small molecule interactions. We show that multiple cell signaling effectors are required for initial cellular entry of LNPs through macropinocytosis, including proton pumps, mTOR and cathepsins. siRNA delivery is substantially reduced as congruent to 70% of the internalized siRNA undergoes exocytosis through egress of LNPs from late endosomes/lysosomes. Niemann-Pick type C1 (NPC1) is shown to be an important regulator of the major recycling pathways of LNP-delivered siRNAs. NPC1-deficient cells show enhanced cellular retention of LNPs inside late endosomes and lysosomes, and increased gene silencing of the target gene. Our data suggest that siRNA delivery efficiency might be improved by designing delivery vehicles that can escape the recycling pathways. Full Text
Sankaran2, V.G., and Orkin, S.H. (2013). The Switch from Fetal to Adult Hemoglobin. Cold Spring Harbor Perspectives in Medicine 3(1) : a01164. The fetal-to-adult hemoglobin switch and silencing of fetal hemoglobin (HbF) have been areas of long-standing interest among hematologists, given the fact that clinical induction of HbF production holds tremendous promise to ameliorate the clinical symptoms of sickle cell disease (SCD) and beta-thalassemia. In this article, we discuss historic attempts to induce HbF that have resulted in some therapeutic approaches to manage SCD and beta-thalassemia. We then go on to discuss how more recent molecular studies that have identified regulators, including BCL11A, MYB, and KLF1, hold great promise to develop targeted and more effective approaches for HbF induction. We go on to discuss strategies by which such approaches may be developed. Older studies in this field can provide important lessons for future studies aimed at developing more effective strategies for HbF induction, and we therefore chronologically cover the work accomplished as this field has evolved over the course of the past four decades. Full Text
Sankaran, V.G., and Orkin, S.H. (2013). Genome-wide association studies of hematologic phenotypes: a window into human hematopoiesis. Curr Opin Genet Dev. Available online 7 March 2013 The study of human hematopoiesis is often limited by the inability to manipulate this process in vivo and differences that exist between humans and commonly employed model organisms. However, human genetics provides a way to gain insight into natural variation in a variety of hematologic phenotypes and creates an opportunity to better understand hematopoiesis. In this review, we discuss how genome-wide association studies are revealing common genetic variation that is associated with hematologic traits and diseases. We discuss how the resulting insight from these studies promises to increase our understanding of human hematopoiesis and outline the challenges that lay ahead in this field. PDF
Santagata, S., Mendillo, M.L., Tang, Y.C., Subramanian, A., Perley, C.C., Roche, S.P., Wong, B., Narayan, R., Kwon, H., Koeva, M.,Amon A, Golub TR, Porco JA Jr, Whitesell L, and Lindquist S. (2013). Tight Coordination of Protein Translation and HSF1 Activation Supports the Anabolic Malignant State. Science 341, 250-+.The ribosome is centrally situated to sense metabolic states, but whether its activity, in turn, coherently rewires transcriptional responses is unknown. Here, through integrated chemical-genetic analyses, we found that a dominant transcriptional effect of blocking protein translation in cancer cells was inactivation of heat shock factor 1 (HSF1), a multifaceted transcriptional regulator of the heat-shock response and many other cellular processes essential for anabolic metabolism, cellular proliferation, and tumorigenesis. These analyses linked translational flux to the regulation of HSF1 transcriptional activity and to the modulation of energy metabolism. Targeting this link with translation initiation inhibitors such as rocaglates deprived cancer cells of their energy and chaperone armamentarium and selectively impaired the proliferation of both malignant and premalignant cells with early-stage oncogenic lesions. Full Text
Sanyal S., Ashour, J., Maruyama, T., Altenburg, A.F., Cragnolini, J.J., Bilate, A., Avalos, A.M., Kundrat, L., Garcia-Sastre, A., and Ploegh, H.L. (2013). Type I Interferon Imposes a TSG101/ISG15 Checkpoint at the Golgi for Glycoprotein Trafficking during Influenza Virus Infection. Cell Host Microbe 14, 510-521.Several enveloped viruses exploit host pathways, such as the cellular endosomal sorting complex required for transport (ESCRT) machinery, for their assembly and release. The influenza A virus (IAV) matrix protein binds to the ESCRT-I complex, although the involvement of early ESCRT proteins such as Tsg101 in IAV trafficking remain to be established. We find that Tsg101 can facilitate IAV trafficking, but this is effectively restricted by the interferon (IFN)-stimulated protein ISG15. Cytosol from type I IFN-treated cells abolished IAV hemagglutinin (HA) transport to the cell surface in infected semi-intact cells. This inhibition required Tsg101 and could be relieved with deISGylases. Tsg101 is itself ISGylated in IFN-treated cells. Upon infection, intact Tsg101-deficient cells obtained by CRISPR-Cas9 genome editing were defective in the surface display of HA and for infectious virion release. These data support the IFN-induced generation of a Tsg101- and ISG15-dependent checkpoint in the secretory pathway that compromises influenza virus release. Full Text
Sarkar2, S., Carroll, B., Buganim, Y., Maetzel, D., Ng, A.H., Cassady, J.P., Cohen, M.A., Chakraborty, S., Wang, H., Spooner, E, Hidde Ploegh, Rudolph Jaenisch, et al. (2013). Impaired Autophagy in the Lipid-Storage Disorder Niemann-Pick Type C1 Disease. Cell Reports Nov 23 [Epub ahead of print].Autophagy dysfunction has been implicated in misfolded protein accumulation and cellular toxicity in several diseases. Whether alterations in autophagy also contribute to the pathology of lipid-storage disorders is not clear. Here, we show defective autophagy in Niemann-Pick type C1 (NPC1) disease associated with cholesterol accumulation, where the maturation of autophagosomes is impaired because of defective amphisome formation caused by failure in SNARE machinery, whereas the lysosomal proteolytic function remains unaffected. Expression of functional NPC1 protein rescues this defect. Inhibition of autophagy also causes cholesterol accumulation. Compromised autophagy was seen in disease-affected organs of Npc1 mutant mice. Of potential therapeutic relevance is that HP-beta-cyclodextrin, which is used for cholesterol-depletion treatment, impedes autophagy, whereas stimulating autophagy restores its function independent of amphisome formation. Our data suggest that a low dose of HP-beta-cyclodextrin that does not perturb autophagy, coupled with an autophagy inducer, may provide a rational treatment strategy for NPC1 disease. Full Text
Sarkar, S. (2013). Regulation of autophagy by mTOR-dependent and mTOR-independent pathways: autophagy dysfunction in neurodegenerative diseases and therapeutic application of autophagy enhancers. Biochem Soc Trans 41, 1103-1130.Autophagy is an intracellular degradation pathway essential for cellular and energy homoeostasis. It functions in the clearance of misfolded proteins and damaged organelles, as well as recycling of cytosolic components during starvation to compensate for nutrient deprivation. This process is regulated by mTOR (mammalian target of rapamycin)-dependent and mTOR-independent pathways that are amenable to chemical perturbations. Several small molecules modulating autophagy have been identified that have potential therapeutic application in diverse human diseases, including neurodegeneration. Neurodegeneration-associated aggregation-prone proteins are predominantly degraded by autophagy and therefore stimulating this process with chemical inducers is beneficial in a wide range of transgenic disease models. Emerging evidence indicates that compromised autophagy contributes to the aetiology of various neurodegenerative diseases related to protein conformational disorders by causing the accumulation of mutant proteins and cellular toxicity. Combining the knowledge of autophagy dysfunction and the mechanism of drug action may thus be rational for designing targeted therapy. The present review describes the cellular signalling pathways regulating mammalian autophagy and highlights the potential therapeutic application of autophagy inducers in neurodegenerative disorders. Full Text
Schwartz, S., Agarwala, S.D., Mumbach, M.R., Jovanovic, M., Mertins, P., Shishkin, A., Tabach, Y., Mikkelsen, T.S., Satija, R., Ruvkun, G.,Gerald R Fink, et al. (2013). High-Resolution Mapping Reveals a Conserved, Widespread, Dynamic mRNA Methylation Program in Yeast Meiosis. Cell 155, 1409-1421.N-6-methyladenosine (m(6)A) is the most ubiquitous mRNA base modification, but little is known about its precise location, temporal dynamics, and regulation. Here, we generated genomic maps of m(6)A sites in meiotic yeast transcripts at nearly single-nucleotide resolution, identifying 1,308 putatively methylated sites within 1,183 transcripts. We validated eight out of eight methylation sites in different genes with direct genetic analysis, demonstrated that methylated sites are significantly conserved in a related species, and built a model that predicts methylated sites directly from sequence. Sites vary in their methylation profiles along a dense meiotic time course and are regulated both locally, via predictable methylatability of each site, and globally, through the core meiotic circuitry. The methyltransferase complex components localize to the yeast nucleolus, and this localization is essential for mRNA methylation. Our data illuminate a conserved, dynamically regulated methylation program in yeast meiosis and provide an important resource for studying the function of this epitranscriptomic modification. Full Text
Schweizer, N., Ferras, C., Kern, D.M., Logarinho, E., Cheeseman, I.M., and Maiato, H. (2013). Spindle assembly checkpoint robustness requires Tpr-mediated regulation of Mad1/Mad2 proteostasis. Journal of Cell Biology 203, 883-893.Tpr is a conserved nuclear pore complex (NPC) protein implicated in the spindle assembly checkpoint (SAC) by an unknown mechanism. Here, we show that Tpr is required for normal SAC response by stabilizing Mad1 and Mad2 before mitosis. Tpr coimmunoprecipitated with Mad1 and Mad2 (hereafter designated as Tpr/Mad1/Mad2 or TM2 complex) during interphase and mitosis, and is required for Mad1-c-Mad2 recruitment to NPCs. Interestingly, Tpr was normally undetectable at kinetochores and dispensable for Mad1, but not for Mad2, kinetochore localization, which suggests that SAC robustness depends on Mad2 levels at kinetochores. Protein half-life measurements demonstrate that Tpr stabilizes Mad1 and Mad2, ensuring normal Mad1-c-Mad2 production in an mRNA-and kinetochore-independent manner. Overexpression of GFP-Mad2 restored normal SAC response and Mad2 kinetochore levels in Tpr-depleted cells. Mechanistically, we provide evidence that Tpr might spatially regulate SAC proteostasis through the SUMO-isopeptidases SENP1 and SENP2 at NPCs. Thus, Tpr is a kinetochore-independent, rate-limiting factor required to mount and sustain a robust SAC response. Full Text
Sehrawat, S., Koenig, P.-A., Kirak, O., Schlieker, C., Fankhauser, M., and Ploegh, H.L. (2013). A catalytically inactive mutant of the deubiquitylase YOD-1 enhances antigen cross-presentation. Blood 121, 1145-1156 Antigen presenting cells (APCs) that express a catalytically inactive version of the deubiquitylase YOD1 (YOD1-C160S) present exogenous antigens more efficiently to CD8(+) T cells, both in vitro and in vivo. Compared with controls, immunization of YOD1-C160S mice led to greater expansion of specific CD8(+) T cells and showed improved control of infection with a recombinant -herpes virus, MHV-68, engineered to express SIINFEKL peptide, the ligand for the ovalbumin-specific TCR transgenic OT-I cells. Enhanced expansion of specific CD8(+) T cells was likewise observed on infection of YOD1-C160S mice with a recombinant influenza A virus expressing SIINFEKL. YOD1-C160S APCs retained antigen longer than did control APCs. Enhanced crosspresentation by YOD1-C160S APCs was transporter associated with antigen processing (TAP1)-independent but sensitive to inclusion of inhibitors of acidification and of the proteasome. The activity of deubiquitylating enzymes may thus help control antigenspecific CD8(+) T-cell responses during immunization. Full Text
Shalgi R., Hurt, J.A., Krykbaeva, I., Taipale, M., Lindquist, S., and Burge, C.B. (2013). Widespread regulation of translation by elongation pausing in heat shock. Mol Cell 49, 439-452.Global repression of protein synthesis is a hallmark of the cellular stress response and has been attributed primarily to inhibition of translation initiation, although this mechanism may not always explain the full extent of repression. Here, using ribosome footprinting, we show that 2 hr of severe heat stress triggers global pausing of translation elongation at around codon 65 on most mRNAs in both mouse and human cells. The genome-wide nature of the phenomenon, its location, and features of protein N termini suggested the involvement of ribosome-associated chaperones. After severe heat shock, Hsp70's interactions with the translational machinery were markedly altered and its association with ribosomes was reduced. Pretreatment with mild heat stress or overexpression of Hsp70 protected cells from heat shock-induced elongation pausing, while inhibition of Hsp70 activity triggered elongation pausing without heat stress. Our findings suggest that regulation of translation elongation in general, and by chaperones in particular, represents a major component of cellular stress responses. PDF
Sher, N., Von Stetina, J.R., Bell, G.W., Matsuura, S., Ravid, K., and Orr-Weaver, T.L. (2013). Fundamental differences in endoreplication in mammals and Drosophila revealed by analysis of endocycling and endomitotic cells. PNAS.Published online 23 April 2013 Throughout the plant and animal kingdoms specific cell types become polyploid, increasing their DNA content to attain a large cell size. In mammals, megakaryocytes (MKs) become polyploid before fragmenting into platelets. The mammalian trophoblast giant cells (TGCs) exploit their size to form a barrier between the maternal and embryonic tissues. The mechanism of polyploidization has been investigated extensively in Drosophila, in which a modified cell cycle-the endocycle, consisting solely of alternating S and gap phases-produces polyploid tissues. During S phase in the Drosophila endocycle, heterochromatin and specific euchromatic regions are underreplicated and reduced in copy number. Here we investigate the properties of polyploidization in murine MKs and TGCs. We induced differentiation of primary MKs and directly microdissected TGCs from embryonic day 9.5 implantation sites. The copy number across the genome was analyzed by array-based comparative genome hybridization. In striking contrast to Drosophila, the genome was uniformly and integrally duplicated in both MKs and TGCs. This was true even for heterochromatic regions analyzed by quantitative PCR. Underreplication of specific regions in polyploid cells is proposed to be due to a slower S phase, resulting from low expression of S-phase genes, causing failure to duplicate late replicating genomic intervals. We defined the transcriptome of TGCs and found robust expression of S-phase genes. Similarly, S-phase gene expression is not repressed in MKs, providing an explanation for the distinct endoreplication parameters compared with Drosophila. Consistent with TGCs endocycling rather than undergoing endomitosis, they have low expression of M-phase genes. Full Text
Shi, J., Whyte, W.A., Zepeda-Mendoza, C.J., Milazzo, J.P., Shen, C., Roe, J.S., Minder, J.L., Mercan, F., Wang, E., Eckersley-Maslin, M.A.,Richard A. Young, et al. (2013). Role of SWI/SNF in acute leukemia maintenance and enhancer-mediated Myc regulation. Genes & Development 27, 2648-2662.Cancer cells frequently depend on chromatin regulatory activities to maintain a malignant phenotype. Here, we show that leukemia cells require the mammalian SWI/SNF chromatin remodeling complex for their survival and aberrant self-renewal potential. While Brg1, an ATPase subunit of SWI/SNF, is known to suppress tumor formation in several cell types, we found that leukemia cells instead rely on Brg1 to support their oncogenic transcriptional program, which includes Myc as one of its key targets. To account for this context-specific function, we identify a cluster of lineage-specific enhancers located 1.7 Mb downstream from Myc that are occupied by SWI/SNF as well as the BET protein Brd4. Brg1 is required at these distal elements to maintain transcription factor occupancy and for long-range chromatin looping interactions with the Myc promoter. Notably, these distal Myc enhancers coincide with a region that is focally amplified in similar to 3% of acute myeloid leukemias. Together, these findings define a leukemia maintenance function for SWI/SNF that is linked to enhancer-mediated gene regulation, providing general insights into how cancer cells exploit transcriptional coactivators to maintain oncogenic gene expression programs. Full Text
Shibue, T., Brooks, M.W., and Weinberg, R.A. (2013). An Integrin-Linked Machinery of Cytoskeletal Regulation that Enables Experimental Tumor Initiation and Metastatic Colonization. Cancer Cell Sep 11. [Epub ahead of print] .Recently extravasated metastatic cancer cells use the Rif/mDia2 actin-nucleating/polymerizing machinery in order to extend integrin beta1-containing, filopodium-like protrusions (FLPs), which enable them to interact productively with the surrounding extracellular matrix; this process governs the initial proliferation of these cancer cells. Here, we identify the signaling pathway governing FLP lifetime, which involves integrin-linked kinase (ILK) and beta-parvin, two integrin:actin-bridging proteins that block cofilin-mediated actin-filament severing. Notably, the combined actions of Rif/mDia2 and ILK/beta-parvin/cofilin pathways on FLPs are required not only for metastatic outgrowth but also for primary tumor formation following experimental implantation. This provides one mechanistic explanation for how the epithelial-mesenchymal transition (EMT) program imparts tumor-initiating powers to carcinoma cells, since it enhances FLP formation through the activation of ILK/beta-parvin/cofilin pathway. Full Text
Shulman, Z., Gitlin, A.D., Targ, S., Jankovic, M., Pasqual, G., Nussenzweig, M.C., and Victora, G.D. (2013). T follicular helper cell dynamics in germinal centers. Science 341, 673-677.T follicular helper (TFH) cells are a specialized subset of effector T cells that provide help to and thereby select high-affinity B cells in germinal centers (GCs). To examine the dynamic behavior of TFH cells in GCs in mice, we used two-photon microscopy in combination with a photoactivatable fluorescent reporter. Unlike GC B cells, which are clonally restricted, TFH cells distributed among all GCs in lymph nodes and continually emigrated into the follicle and neighboring GCs. Moreover, newly activated TFH cells invaded preexisting GCs, where they contributed to B cell selection and plasmablast differentiation. Our data suggest that the dynamic exchange of TFH cells between GCs ensures maximal diversification of T cell help and that their ability to enter ongoing GCs accommodates antigenic variation during the immune response. Full Text
Sigova, A.A., Mullen, A.C., Molinie, B., Gupta, S., Orlando, D.A., Guenther, M.G., Almada, A.E., Lin, C., Sharp, P.A., Giallourakis, C.C, and Richard A. Young. (2013). Divergent transcription of long noncoding RNA/mRNA gene pairs in embryonic stem cells. Proc Natl Acad Sci U S A. Feb 4. [Epub ahead of print] Many long noncoding RNA (lncRNA) species have been identified in mammalian cells, but the genomic origin and regulation of these molecules in individual cell types is poorly understood. We have generated catalogs of lncRNA species expressed in human and murine embryonic stem cells and mapped their genomic origin. A surprisingly large fraction of these transcripts (>60%) originate from divergent transcription at promoters of active protein-coding genes. The divergently transcribed lncRNA/mRNA gene pairs exhibit coordinated changes in transcription when embryonic stem cells are differentiated into endoderm. Our results reveal that transcription of most lncRNA genes is coordinated with transcription of protein-coding genes. Full Text
Sive, H., and Sarma, S. (2013). Online on-ramps. Nature 499, 277-278 Giving scientists greater access to conceptual and technical tuition through massive open online courses will aid interdisciplinary research, say Hazel Sive and Sanjay Sarma. Full Text
Spies, N., Burge, C.B., and Bartel, D.P. (2013). 3 ' UTR-isoform choice has limited influence on the stability and translational efficiency of most mRNAs in mouse fibroblasts. Genome Research 23, 2078-2090.Variation in protein output across the genome is controlled at several levels, but the relative contributions of different regulatory mechanisms remain poorly understood. Here, we obtained global measurements of decay and translation rates for mRNAs with alternative 3' untranslated regions (3' UTRs) in murine 3T3 cells. Distal tandem isoforms had slightly but significantly lower mRNA stability and greater translational efficiency than proximal isoforms on average. The diversity of alternative 3' UTRs also enabled inference and evaluation of both positively and negatively acting cis-regulatory elements. The 3' UTR elements with the greatest implied influence were microRNA complementary sites, which were associated with repression of 32% and 4% at the stability and translational levels, respectively. Nonetheless, both the decay and translation rates were highly correlated for proximal and distal 3' UTR isoforms from the same genes, implying that in 3T3 cells, alternative 3' UTR sequences play a surprisingly small regulatory role compared to other mRNA regions. Full Text
Strijbis, K., Tafesse, F.G., Fairn, G.D., Witte, M.D., Dougan, S.K., Watson, N., Spooner, E., Esteban, A., Vyas, V.K., Fink, G.R.,Grinstein S, & Ploegh H Ll. (2013). Bruton's Tyrosine Kinase (BTK) and Vav1 Contribute to Dectin1-Dependent Phagocytosis of Candida albicans in Macrophages. PLoS Pathog 9, e1003446.Phagocytosis of the opportunistic fungal pathogen Candida albicans by cells of the innate immune system is vital to prevent infection. Dectin-1 is the major phagocytic receptor involved in anti-fungal immunity. We identify two new interacting proteins of Dectin-1 in macrophages, Bruton's Tyrosine Kinase (BTK) and Vav1. BTK and Vav1 are recruited to phagocytic cups containing C. albicans yeasts or hyphae but are absent from mature phagosomes. BTK and Vav1 localize to cuff regions surrounding the hyphae, while Dectin-1 lines the full length of the phagosome. BTK and Vav1 colocalize with the lipid PI(3,4,5)P3 and F-actin at the phagocytic cup, but not with diacylglycerol (DAG) which marks more mature phagosomal membranes. Using a selective BTK inhibitor, we show that BTK contributes to DAG synthesis at the phagocytic cup and the subsequent recruitment of PKCepsilon. BTK- or Vav1-deficient peritoneal macrophages display a defect in both zymosan and C. albicans phagocytosis. Bone marrow-derived macrophages that lack BTK or Vav1 show reduced uptake of C. albicans, comparable to Dectin1-deficient cells. BTK- or Vav1-deficient mice are more susceptible to systemic C. albicans infection than wild type mice. This work identifies an important role for BTK and Vav1 in immune responses against C. albicans. Full Text
Sun, L., Goff, L.A., Trapnell, C., Alexander, R., Lo, K.A., Hacisuleyman, E., Sauvageau, M., Tazon-Vega, B., Kelley, D.R., Hendrickson, D.G, Bingbing Yuan,.Kellis M, Lodish HF,and Rinn JL.. (2013). Long noncoding RNAs regulate adipogenesis.Proc Natl Acad Sci U S A. 2013 Feb 11. [Epub ahead of print].The prevalence of obesity has led to a surge of interest in understanding the detailed mechanisms underlying adipocyte development. Many protein-coding genes, mRNAs, and microRNAs have been implicated in adipocyte development, but the global expression patterns and functional contributions of long noncoding RNA (lncRNA) during adipogenesis have not been explored. Here we profiled the transcriptome of primary brown and white adipocytes, preadipocytes, and cultured adipocytes and identified 175 lncRNAs that are specifically regulated during adipogenesis. Many lncRNAs are adipose-enriched, strongly induced during adipogenesis, and bound at their promoters by key transcription factors such as peroxisome proliferator-activated receptor gamma (PPARgamma) and CCAAT/enhancer-binding protein alpha (CEBPalpha). RNAi-mediated loss of function screens identified functional lncRNAs with varying impact on adipogenesis. Collectively, we have identified numerous lncRNAs that are functionally required for proper adipogenesis PDF
Swee, L.K., Guimaraes, C.P., Sehrawat, S., Spooner, E., Barrasa, M.I., and Ploegh, H.L. (2013). Sortase-mediated modification of alphaDEC205 affords optimization of antigen presentation and immunization against a set of viral epitopes. PNAS Jan 7. [Epub ahead of print] .A monoclonal antibody against the C-type lectin DEC205 (alphaDEC205) is an effective vehicle for delivery of antigens to dendritic cells through creation of covalent alphaDEC205-antigen adducts. These adducts can induce antigen-specific T-cell immune responses or tolerance. We exploit the transpeptidase activity of sortase to install modified peptides and protein-sized antigens onto the heavy chain of alphaDEC205, including linkers that contain nonnatural amino acids. We demonstrate stoichiometric site-specific labeling on a scale not easily achievable by genetic fusions (49 distinct fusions in this report). We conjugated a biotinylated version of a class I MHC-restricted epitope to unlabeled alphaDEC205 and monitored epitope generation upon binding of the adduct to dendritic cells. Our results show transfer of alphaDEC205 heavy chain to the cytoplasm, followed by proteasomal degradation. Introduction of a labile dipeptide linker at the N terminus of a T-cell epitope improves proteasome-dependent class I MHC-restricted peptide cross-presentation when delivered by alphaDEC205 in vitro and in vivo. We also conjugated alphaDEC205 with a linker-optimized peptide library of known CD8 T-cell epitopes from the mouse gamma-herpes virus 68. Animals immunized with such conjugates displayed a 10-fold reduction in viral load. PDF
Tafesse, F.G., Sanyal, S., Ashour, J., Guimaraes, C.P., Hermansson, M., Somerharju, P., and Ploegh, H.L. (2013). Intact sphingomyelin biosynthetic pathway is essential for intracellular transport of influenza virus glycoproteins. Proc Natl Acad Sci U S A Published online 1 April 2013..Cells genetically deficient in sphingomyelin synthase-1 (SGMS1) or blocked in their synthesis pharmacologically through exposure to a serine palmitoyltransferase inhibitor (myriocin) show strongly reduced surface display of influenza virus glycoproteins hemagglutinin (HA) and neuraminidase (NA). The transport of HA to the cell surface was assessed by accessibility of HA on intact cells to exogenously added trypsin and to HA-specific antibodies. Rates of de novo synthesis of viral proteins in wild-type and SGMS1-deficient cells were equivalent, and HA negotiated the intracellular trafficking pathway through the Golgi normally. We engineered a strain of influenza virus to allow site-specific labeling of HA and NA using sortase. Accessibility of both HA and NA to sortase was blocked in SGMS1-deficient cells and in cells exposed to myriocin, with a corresponding inhibition of the release of virus particles from infected cells. Generation of influenza virus particles thus critically relies on a functional sphingomyelin biosynthetic pathway, required to drive influenza viral glycoproteins into lipid domains of a composition compatible with virus budding and release. Full Text
Taipale, M., Krykbaeva, I., Whitesell, L., Santagata, S., Zhang, J., Liu, Q., Gray, N.S., and Lindquist, S. (2013). Chaperones as thermodynamic sensors of drug-target interactions reveal kinase inhibitor specificities in living cells. Nature Biotechnology 31, 630–637(2013).The interaction between the HSP90 chaperone and its client kinases is sensitive to the conformational status of the kinase, and stabilization of the kinase fold by small molecules strongly decreases chaperone interaction. Here we exploit this observation and assay small-molecule binding to kinases in living cells, using chaperones as 'thermodynamic sensors'. The method allows determination of target specificities of both ATP-competitive and allosteric inhibitors in the kinases' native cellular context in high throughput. We profile target specificities of 30 diverse kinase inhibitors against >300 kinases. Demonstrating the value of the assay, we identify ETV6-NTRK3 as a target of the FDA-approved drug crizotinib (Xalkori). Crizotinib inhibits proliferation of ETV6-NTRK3-dependent tumor cells with nanomolar potency and induces the regression of established tumor xenografts in mice. Finally, we show that our approach is applicable to other chaperone and target classes by assaying HSP70/steroid hormone receptor and CDC37/kinase interactions, suggesting that chaperone interactions will have broad application in detecting drug-target interactions in vivo. Full Text
Tam2, W.L., and Weinberg, R.A. (2013). The epigenetics of epithelial-mesenchymal plasticity in cancer. Nature Medicine 19, 1438-1449.During the course of malignant cancer progression, neoplastic cells undergo dynamic and reversible transitions between multiple phenotypic states, the extremes of which are defined by the expression of epithelial and mesenchymal phenotypes. This plasticity is enabled by underlying shifts in epigenetic regulation. A small cohort of pleiotropically acting transcription factors is widely recognized to effect these shifts by controlling the expression of a constituency of key target genes. These master regulators depend on complex epigenetic regulatory mechanisms, notably the induction of changes in the modifications of chromatin-associated histones, in order to achieve the widespread changes in gene expression observed during epithelial-mesenchymal transitions (EMTs). These associations indicate that an understanding of the functional interactions between such EMT-inducing transcription factors and the modulators of chromatin configuration will provide crucial insights into the fundamental mechanisms underlying cancer progression and may, in the longer term, generate new diagnostic and therapeutic modalities for treating high-grade malignancies. Full Text
Tam, W.L., Lu, H., Buikhuisen, J., Soh, B.S., Lim, E., Reinhardt, F., Wu, Z.J., Krall, J.A., Bierie, B., Guo, W.,Chen X, Liu XS, Brown M, Lim B, and Weinberg RA.. (2013). Protein Kinase C alpha Is a Central Signaling Node and Therapeutic Target for Breast Cancer Stem Cells. Cancer Cell 24, 347-364.The epithelial-mesenchymal transition program becomes activated during malignant progression and can enrich for cancer stem cells (CSCs). We report that inhibition of protein kinase C alpha (PKCalpha) specifically targets CSCs but has little effect on non-CSCs. The formation of CSCs from non-stem cells involves a shift from EGFR to PDGFR signaling and results in the PKCalpha-dependent activation of FRA1. We identified an AP-1 molecular switch in which c-FOS and FRA1 are preferentially utilized in non-CSCs and CSCs, respectively. PKCalpha and FRA1 expression is associated with the aggressive triple-negative breast cancers, and the depletion of FRA1 results in a mesenchymal-epithelial transition. Hence, identifying molecular features that shift between cell states can be exploited to target signaling components critical to CSCs. Full Text
Tardiff2, D.F., Jui, N.T., Khurana, V., Tambe, M.A., Thompson, M.L., Chung, C.Y., Kamadurai, H.B., Kim, H.T., Lancaster, A.K., Caldwell, K.A.,Caldwell GA, Rochet JC, Buchwald SL, Lindquist S.. (2013). Yeast Reveal a "Druggable" Rsp5/Nedd4 Network that Ameliorates alpha-Synuclein Toxicity in Neurons. Science. Oct 24. [Epub ahead of print] alpha-synuclein (alpha-syn) is a small lipid binding protein implicated in several neurodegenerative diseases, including Parkinson's disease, whose pathobiology is conserved from yeast to man. There are no therapies targeting these underlying cellular pathologies, or indeed those of any major neurodegenerative disease. Using unbiased phenotypic screens as an alternative to target-based approaches, we discovered an N-aryl benzimidazole (NAB) that strongly and selectively protected diverse cell-types from alpha-syn toxicity. Three chemical genetic screens in wild-type yeast cells established that NAB promoted endosomal transport events dependent on the E3 ubiquitin ligase, Rsp5/Nedd4. These same steps were perturbed by alpha-syn itself. Thus, NAB identifies a druggable node in the biology of alpha-syn that can correct multiple aspects of its underlying pathology, including dysfunctional endosomal and ER-to-Golgi vesicle trafficking. Full Text
Tardiff, D.F., and Lindquist, S. (2013). Phenotypic screens for compounds that target the cellular pathologies underlying Parkinson's Disease. Drug Discov Today Technol 10, e121-e128.Parkinson's Disease (PD) is a devastating neurodegenerative disease that affects over one million patients in the US. Yet, no disease modifying drugs exist, only those that temporarily alleviate symptoms. Because of its poorly defined and highly complex disease etiology, it is essential to embrace unbiased and innovative approaches for identifying new chemical entities that target the underlying toxicities associated with PD. Traditional target-based drug discovery paradigm can suffer from a bias towards a small number of potential targets. Phenotypic screening of both genetic and pharmacological PD models offers an alternative approach to discover compounds that target the initiating causes and effectors of cellular toxicity. The relative paucity of reported phenotypic screens illustrates the intrinsic difficulty in establishing model systems that are both biologically meaningful and adaptable to high-throughput screening. Parallel advances in PD models and in vivo screening technologies will help create opportunities for identifying new therapeutic leads with unanticipated, breakthrough mechanisms of action. Full Text
Thai, T.H., Patterson, H.C., Pham, D.H., Kis-Toth, K., Kaminski, D.A., and Tsokos, G.C. (2013). Deletion of microRNA-155 reduces autoantibody responses and alleviates lupus-like disease in the Fas(lpr) mouse. Proceedings of the National Academy of Sciences of the United States of America 110, 20194-20199.MicroRNA-155 (miR-155) regulates antibody responses and subsequent B-cell effector functions to exogenous antigens. However, the role of miR-155 in systemic autoimmunity is not known. Using the death receptor deficient (Fas(lpr)) lupus-prone mouse, we show here that ablation of miR-155 reduced autoantibody responses accompanied by a decrease in serum IgG but not IgM anti-dsDNA antibodies and a reduction of kidney inflammation. MiR-155 deletion in Fas(lpr) B cells restored the reduced SH2 domain-containing inositol 5'-phosphatase 1 to normal levels. In addition, coaggregation of the Fc gamma receptor IIB with the B-cell receptor in miR-155(-/-)-Fas(lpr) B cells resulted in decreased ERK activation, proliferation, and production of switched antibodies compared with miR-155 sufficient Fas(lpr) B cells. Thus, by controlling the levels of SH2 domain-containing inositol 5'-phosphatase 1, miR-155 in part maintains an activation threshold that allows B cells to respond to antigens. PDF
Theile, C.S., Witte, M.D., Blom, A.E., Kundrat, L., Ploegh, H.L., and Guimaraes, C.P. (2013). Site-specific N-terminal labeling of proteins using sortase-mediated reactions. Nature Protocols 8, 1800-1807.This protocol describes the use of sortase-mediated reactions to label the N terminus of any given protein of interest. The sortase recognition sequence, LPXTG (for Streptococcus aureus sortase A) or LPXTA (for Staphylococcus pyogenes sortase A), can be appended to a variety of probes such as fluorophores, biotin or even to other proteins. The protein to be labeled acts as a nucleophile by attacking the intermediate formed between the probe containing the LPXTG/A motif and the sortase enzyme. If sortase, the protein of interest and a suitably functionalized label are available, the reactions usually require less than 3 h. Full Text
Torikai, H., Reik, A., Soldner, F., Warren, E.H., Yuen, C., Zhou, Y.Y., Crossland, D.L., Huls, H., Littman, N., Zhang, Z.Y. , Jaenisch R, et al. (2013). Toward eliminating HLA class I expression to generate universal cells from allogeneic donors. Blood 122, 1341-1349.Long-term engraftment of allogeneic cells necessitates eluding immune-mediated rejection, which is currently achieved by matching for human leukocyte antigen (HLA) expression, immunosuppression, and/or delivery of donor-derived cells to sanctuary sites. Genetic engineering provides an alternative approach to avoid clearance of cells that are recognized as "non-self" by the recipient. To this end, we developed designer zinc finger nucleases and employed a "hit-and-run" approach to genetic editing for selective elimination of HLA expression. Electro-transfer of mRNA species coding for these engineered nucleases completely disrupted expression of HLA-A on human T cells, including CD19-specific T cells. The HLA-A(neg) T-cell pools can be enriched and evade lysis by HLA-restricted cytotoxic T-cell clones. Recognition by natural killer cells of cells that had lost HLA expression was circumvented by enforced expression of nonclassical HLA molecules. Furthermore, we demonstrate that zinc finger nucleases can eliminate HLA-A expression from embryonic stem cells, which broadens the applicability of this strategy beyond infusing HLA-disparate immune cells. These findings establish that clinically appealing cell types derived from donors with disparate HLA expression can be genetically edited to evade an immune response and provide a foundation whereby cells from a single donor can be administered to multiple recipients. Full Text
Trajkovski, M., and Lodish, H. (2013). MicroRNA networks regulate development of brown adipocytes. Trends in Endocrinology and Metabolism 24, 442-450. Brown adipose tissue (BAT) is specialized for heat generation and energy expenditure as a defense against cold and obesity; in both humans and mice increased amounts of BAT are associated with a lean phenotype and resistance to development of the metabolic syndrome and its complications. Here we summarize recent research showing that several BAT-expressed microRNAs (miRNAs) play important roles in regulating differentiation and metabolism of brown and beige adipocytes; we discuss the key mRNA targets downregulated by these miRNAs and show how these miRNAs affect directly or indirectly transcription factors important for BAT development. We suggest that these miRNAs could be part of novel therapeutics to increase BAT in humans.
Tsun, Z.Y., Bar-Peled, L., Chantranupong, L., Zoncu, R., Wang, T., Kim, C., Spooner, E., and Sabatini, D.M. (2013). The Folliculin Tumor Suppressor Is a GAP for the RagC/D GTPases That Signal Amino Acid Levels to mTOCR 1 Molecular Cell Oct 2 l.The mTORC1 kinase is a master growth regulator that senses numerous environmental cues, including amino acids. The Rag GTPases interact with mTORC1 and signal amino acid sufficiency by promoting the translocation of mTORC1 to the lysosomal surface, its site of activation. The Rags are unusual GTPases in that they function as obligate heterodimers, which consist of RagA or B bound to RagC or D. While the loading of RagA/B with GTP initiates amino acid signaling to mTORC1, the role of RagC/D is unknown. Here, we show that RagC/D is a key regulator of the interaction of mTORC1 with the Rag heterodimer and that, unexpectedly, RagC/D must be GDP bound for the interaction to occur. We identify FLCN and its binding partners, FNIP1/2, as Rag-interacting proteins with GAP activity for RagC/D, but not RagA/B. Thus, we reveal a role for RagC/D in mTORC1 activation and a molecular function for the FLCN tumor suppressor. PDF
Ulitsky, I., and Bartel, D.P. (2013). lincRNAs: Genomics, Evolution, and Mechanisms. Cell 154, 26-46.Long intervening noncoding RNAs (lincRNAs) are transcribed from thousands of loci in mammalian genomes and might play widespread roles in gene regulation and other cellular processes. This Review outlines the emerging understanding of lincRNAs in vertebrate animals, with emphases on how they are being identified and current conclusions and questions regarding their genomics, evolution and mechanisms of action. Full Text
Unhavaithaya2, Y., and Orr-Weaver, T.L. (2013). Centromere proteins CENP-C and CAL1 functionally interact in meiosis for centromere clustering, pairing, and chromosome segregation. Proc Natl Acad Sci U S APublished online before print November 18, 2013 .Meiotic chromosome segregation involves pairing and segregation of homologous chromosomes in the first division and segregation of sister chromatids in the second division. Although it is known that the centromere and kinetochore are responsible for chromosome movement in meiosis as in mitosis, potential specialized meiotic functions are being uncovered. Centromere pairing early in meiosis I, even between nonhomologous chromosomes, and clustering of centromeres can promote proper homolog associations in meiosis I in yeast, plants, and Drosophila. It was not known, however, whether centromere proteins are required for this clustering. We exploited Drosophila mutants for the centromere proteins centromere protein-C (CENP-C) and chromosome alignment 1 (CAL1) to demonstrate that a functional centromere is needed for centromere clustering and pairing. The cenp-C and cal1 mutations result in C-terminal truncations, removing the domains through which these two proteins interact. The mutants show striking genetic interactions, failing to complement as double heterozygotes, resulting in disrupted centromere clustering and meiotic nondisjunction. The cluster of meiotic centromeres localizes to the nucleolus, and this association requires centromere function. In Drosophila, synaptonemal complex (SC) formation can initiate from the centromere, and the SC is retained at the centromere after it disassembles from the chromosome arms. Although functional CENP-C and CAL1 are dispensable for assembly of the SC, they are required for subsequent retention of the SC at the centromere. These results show that integral centromere proteins are required for nuclear position and intercentromere associations in meiosis. Full Text
Unhavaithaya, Y., Park, E.A., Royzman, I., and Orr-Weaver, T.L. (2013). Drosophila Embryonic Cell Cycle Mutants. G3 (Bethesda )Early Online, published on August 26.Nearly all cell division mutants in Drosophila were recovered in late larval/pupal lethal screens, with less than 10 embryonic lethal mutants identified. This is because larval development occurs without a requirement for cell division. Solely the imaginal cells that generate the adult body divide during larval stages, with larval tissues growing by increasing ploidy rather than cell number. Thus most mutants perturbing mitosis or the cell cycle do not manifest a phenotype until the adult body differentiates in late larval and pupal stages. To identify cell cycle components whose maternal pools are depleted in embryogenesis or that have specific functions in embryogenesis, we screened for mutants defective in cell division during embryogenesis. Five new alleles of cyclin E were recovered, ranging from a missense mutation that is viable to stop codons causing embryonic lethality. These permitted us to investigate the requirements for Cyclin E function in neuroblast cell fate determination, a role previously shown for a null cyclin E allele. The mutations causing truncation of the protein affect cell fate of the NB6-4 neuroblast, whereas the weak missense mutation has no effect. We identified mutations in the pavarotti (pav) and tumbleweed (tum) genes needed for cytokinesis by a phenotype of large and multinucleate cells in the embryonic epidermis and nervous system. Other mutations affecting the centromere protein CAL1 and the kinetochore protein Spc105R caused mitotic defects in the nervous system. PDF
Victora, G.D. (2013). Science & SciLifeLab Prize. Stop, go, and evolve. Science 342, 1186. Pathogens evolve fast. RNA-based viruses, such as influenza and HIV, incorporate mutations in their genomes at the brisk rate of one change per 105 bases each time they replicate (the error rate in our own genome is one per ∼1010 bases) Full Text
Vincent, B.M., Lancaster, A.K., Scherz-Shouval, R., Whitesell, L., and Lindquist, S. (2013). Fitness Trade-offs Restrict the Evolution of Resistance to Amphotericin B. PLoS Biol 11, e1001692.The evolution of drug resistance in microbial pathogens provides a paradigm for investigating evolutionary dynamics with important consequences for human health. Candida albicans, the leading fungal pathogen of humans, rapidly evolves resistance to two major antifungal classes, the triazoles and echinocandins. In contrast, resistance to the third major antifungal used in the clinic, amphotericin B (AmB), remains extremely rare despite 50 years of use as monotherapy. We sought to understand this long-standing evolutionary puzzle. We used whole genome sequencing of rare AmB-resistant clinical isolates as well as laboratory-evolved strains to identify and investigate mutations that confer AmB resistance in vitro. Resistance to AmB came at a great cost. Mutations that conferred resistance simultaneously created diverse stresses that required high levels of the molecular chaperone Hsp90 for survival, even in the absence of AmB. This requirement stemmed from severe internal stresses caused by the mutations, which drastically diminished tolerance to external stresses from the host. AmB-resistant mutants were hypersensitive to oxidative stress, febrile temperatures, and killing by neutrophils and also had defects in filamentation and tissue invasion. These strains were avirulent in a mouse infection model. Thus, the costs of evolving resistance to AmB limit the emergence of this phenotype in the clinic. Our work provides a vivid example of the ways in which conflicting selective pressures shape evolutionary trajectories and illustrates another mechanism by which the Hsp90 buffer potentiates the emergence of new phenotypes. Developing antibiotics that deliberately create such evolutionary constraints might offer a strategy for limiting the rapid emergence of drug resistance. Full Text
Wang2, H., Hu, Y.C., Markoulaki, S., Welstead, G.G., Cheng, A.W., Shivalila, C.S., Pyntikova, T., Dadon, D.B., Voytas, D.F., Bogdanove, A.J., David C Page & Rudolf Jaenisch (2013). TALEN-mediated editing of the mouse Y chromosome. Nature Biotechnology Published online 12 May 2013. The functional study of Y chromosome genes has been hindered by a lack of mouse models with specific Y chromosome mutations. We used transcription activator-like effector nuclease (TALEN)-mediated gene editing in mouse embryonic stem cells (mESCs) to produce mice with targeted gene disruptions and insertions in two Y-linked genes-Sry and Uty. TALEN-mediated gene editing is a useful tool for dissecting the biology of the Y chromosome. Full Text
Wang, H., Yang, H., Shivalila, C.S., Dawlaty, M.M., Cheng, A.W., Zhang, F., and Jaenisch, R. (2013). One-Step Generation of Mice Carrying Mutations in Multiple Genes by CRISPR/Cas-Mediated Genome Engineering. Cell. May 1. [Epub ahead of print] Mice carrying mutations in multiple genes are traditionally generated by sequential recombination in embryonic stem cells and/or time-consuming intercrossing of mice with a single mutation. The CRISPR/Cas system has been adapted as an efficient gene-targeting technology with the potential for multiplexed genome editing. We demonstrate that CRISPR/Cas-mediated gene editing allows the simultaneous disruption of five genes (Tet1, 2, 3, Sry, Uty - 8 alleles) in mouse embryonic stem (ES) cells with high efficiency. Coinjection of Cas9 mRNA and single-guide RNAs (sgRNAs) targeting Tet1 and Tet2 into zygotes generated mice with biallelic mutations in both genes with an efficiency of 80%. Finally, we show that coinjection of Cas9 mRNA/sgRNAs with mutant oligos generated precise point mutations simultaneously in two target genes. Thus, the CRISPR/Cas system allows the one-step generation of animals carrying mutations in multiple genes, an approach that will greatly accelerate the in vivo study of functionally redundant genes and of epistatic gene interactions. Full Text
Wang3, Y., Li, J., Yuan, Z., Yuan, B., Zhang, T., Cragun, J.M., Kong, B., and Zheng, W. (2013). PAX8: a sensitive and specific marker to identify cancer cells of ovarian origin for patients prior to neoadjuvant chemotherapy. J Hematol Oncol Aug 19;6:60.BACKGROUND: Neoadjuvant chemotherapy followed by cytoreduction surgery has been used where an accurate cytologic or pathologic diagnosis is usually required before the initiation of neoadjuvant chemotherapy. However, it is difficult to make definitive diagnosis of presence of cancer cells, particularly gynecologic versus non-gynecologic origin, from those ascites specimens due to the absence of specific biomarkers of gynecologic cancers. In the present study, we evaluated if, in addition to the routine morphologic diagnosis, the biomarker PAX8 could be useful in recognition of ovarian epithelial cancer cells prior to the neoadjuvant chemotherapy. METHODS: Two hundred and two cytology specimens including 120 pretreatment ovarian cancer samples, 60 benign controls, and 22 malignant non-gynecologic cases were studied. All cytology slides were morphologically reviewed in a blinded fashion without knowing corresponding pathology diagnosis, if present. A total of 168 cytology specimens with a cell block were stained with PAX8 and Calretinin. These included patients with potential for ovarian cancer neoadjuvant chemotherapy (n=96), metastatic cancers (n=22), and benign controls (n=50). RESULTS: Among the 96 ascitic samples prior to neoadjuvant chemotherapy, 76 (79%) showing morphologic features consistent with cancers of ovarian primary were all PAX+/Calretinin-. The remaining 20 (21%) cases were positive for adenocarcinoma, but morphologically unable to be further classified. Among the 22 metastatic cancers into the pelvis, one case with PAX8+/Calretinin- represented a renal cell carcinoma and the remaining 21 PAX8-/Calretinin- metastatic cancers were either breast metastasis (n=4) and the metastasis from gastrointestinal tract (n=17). Among the 50 benign control pelvic washing cases, 5 PAX8+/Calretinin-cases represented endosalpingiosis (n=4) and endometriosis (n=1), 25 PAX8-/Calretinin+cases showed reactive mesothelial cells, and the remaining 20 specimens with PAX8-/Calretinin- phenotype typically contained inflammatory or blood cells without noticeable diagnostic epithelia. CONCLUSIONS: PAX8 identifies all Mullerian derived benign or malignant epithelia. When combining with Calretinin, PAX8 is a sensitive marker to diagnose the carcinomas of ovarian origin, which will be ideal to be used for those patients with a possible advanced ovarian cancer prior to receiving neoadjuvant chemotherapy. Full Text
Waters, A.J., Bilinski, P., Eichten, S.R., Vaughn, M.W., Ross-Ibarra, J., Gehring, M., and Springer, N.M. (2013). Comprehensive analysis of imprinted genes in maize reveals allelic variation for imprinting and limited conservation with other species. Proceedings of the National Academy of Sciences of the United States of America 110, 19639-19644.In plants, a subset of genes exhibit imprinting in endosperm tissue such that expression is primarily from the maternal or paternal allele. Imprinting may arise as a consequence of mechanisms for silencing of transposons during reproduction, and in some cases imprinted expression of particular genes may provide a selective advantage such that it is conserved across species. Separate mechanisms for the origin of imprinted expression patterns and maintenance of these patterns may result in substantial variation in the targets of imprinting in different species. Here we present deep sequencing of RNAs isolated from reciprocal crosses of four diverse maize genotypes, providing a comprehensive analysis that allows evaluation of imprinting at more than 95% of endosperm-expressed genes. We find that over 500 genes exhibit statistically significant parent-of-origin effects in maize endosperm tissue, but focused our analyses on a subset of these genes that had >90% expression from the maternal allele (69 genes) or from the paternal allele (108 genes) in at least one reciprocal cross. Over 10% of imprinted genes show evidence of allelic variation for imprinting. A comparison of imprinting in maize and rice reveals that 13% of genes with syntenic orthologs in both species exhibit conserved imprinting. Genes that exhibit conserved imprinting between maize and rice have elevated nonsynonymous to synonymous substitution ratios compared with other imprinted genes, suggesting a history of more rapid evolution. Together, these data suggest that imprinting only has functional relevance at a subset of loci that currently exhibit imprinting in maize. PDF
Weng2, J.K. (2013). Elegant biochemistry, chaotic origin.New Phytologist 200, 592-594 Plants are remarkable chemists capable of rapidly exploiting their metabolic toolsets to synthesize a staggering array of structurally and functionally diverse chemicals, known as specialized metabolites. Full Text
Weng, J.K. (2013). The evolutionary paths towards complexity: a metabolic perspective. New Phytologist. 2013 Jul 26. . [Epub ahead of print] As sessile organisms, land plants have exploited their metabolic systems to produce a panoply of structurally and functionally diverse natural chemicals and polymers to adapt to challenging ecosystems. Many of these core and specialized metabolites confer chemical shields against a multitude of abiotic stresses, while others play important roles in plants' interactions with their biotic environments. Plant specialized metabolites can be viewed as complex traits in the sense that the biosynthesis of these molecules typically requires multistep metabolic pathways comprising numerous specific enzymes belonging to diverse protein fold families. Resolving the evolutionary trajectories underlying the emergence of these specialized metabolic pathways will impact a fundamental question in biology - how do complex traits evolve in a Darwinian fashion? Here, I discuss several general patterns observed in rapidly evolving specialized metabolic systems in plants, and surmise mechanistic features at enzyme, pathway and organismal levels that rationalize the remarkable malleability of these systems through stepwise evolution. Future studies, focused on fine sampling of metabolic enzymes and pathways in phylogenetically related plant species, or employing directed evolution strategies in synthetic systems, will significantly broaden our perspective on how biological complexity arises at the metabolic level. Full Text
Whyte, W.A., Orlando, D.A., Hnisz, D., Abraham, B.J., Lin, C.Y., Kagey, M.H., Rahl, P.B., Lee, T.I., and Young, R.A. (2013). Master transcription factors and mediator establish super-enhancers at key cell identity genes. Cell 153, 307-319.Master transcription factors Oct4, Sox2, and Nanog bind enhancer elements and recruit Mediator to activate much of the gene expression program of pluripotent embryonic stem cells (ESCs). We report here that the ESC master transcription factors form unusual enhancer domains at most genes that control the pluripotent state. These domains, which we call super-enhancers, consist of clusters of enhancers that are densely occupied by the master regulators and Mediator. Super-enhancers differ from typical enhancers in size, transcription factor density and content, ability to activate transcription, and sensitivity to perturbation. Reduced levels of Oct4 or Mediator cause preferential loss of expression of super-enhancer-associated genes relative to other genes, suggesting how changes in gene expression programs might be accomplished during development. In other more differentiated cells, super-enhancers containing cell-type-specific master transcription factors are also found at genes that define cell identity. Super-enhancers thus play key roles in the control of mammalian cell identity. Full Text
Witchley, J.N., Mayer, M., Wagner, D.E., Owen, J.H., and Reddien, P.W. (2013). Muscle Cells Provide Instructions for Planarian Regeneration. Cell Reports Aug 14 [Epub ahead of print] .Regeneration requires both potential and instructions for tissue replacement. In planarians, pluripotent stem cells have the potential to produce all new tissue. The identities of the cells that provide regeneration instructions are unknown. Here, we report that position control genes (PCGs) that control regeneration and tissue turnover are expressed in a subepidermal layer of nonneoblast cells. These subepidermal cells coexpress many PCGs. We propose that these subepidermal cells provide a system of body coordinates and positional information for regeneration, and identify them to be muscle cells of the planarian body wall. Almost all planarian muscle cells express PCGs, suggesting a dual function: contraction and control of patterning. PCG expression is dynamic in muscle cells after injury, even in the absence of neoblasts, suggesting that muscle is instructive for regeneration. We conclude that planarian regeneration involves two highly flexible systems: pluripotent neoblasts that can generate any new cell type and muscle cells that provide positional instructions for the regeneration of any body region. Full Text
Witte, M.D., Theile, C.S., Wu, T., Guimaraes, C.P., Blom, A.E., and Ploegh, H.L. (2013). Production of unnaturally linked chimeric proteins using a combination of sortase-catalyzed transpeptidation and click chemistry. Nature Protocols 8, 1808-1819.Chimeric proteins, including bispecific antibodies, are biological tools with therapeutic applications. Genetic fusion and ligation methods allow the creation of N-to-C and C-to-N fused recombinant proteins, but not unnaturally linked N-to-N and C-to-C fusion proteins. This protocol describes a simple procedure for the production of such chimeric proteins, starting from correctly folded proteins and readily available peptides. By equipping the N terminus or C terminus of the proteins of interest with a set of click handles using sortase A, followed by a strain-promoted click reaction, unnatural N-to-N and C-to-C linked (hetero) fusion proteins are established. Examples of proteins that have been conjugated via this method include interleukin-2, interferon-alpha, ubiquitin, antibodies and several single-domain antibodies. If the peptides, sortase A and the proteins of interest are in hand, the unnaturally N-to-N and C-to-C fused proteins can be obtained in 3-4 d. Full Text
Yang, H., Wang, H., Shivalila, C.S., Cheng, A.W., Shi, L., and Jaenisch, R. (2013). One-Step Generation of Mice Carrying Reporter and Conditional Alleles by CRISPR/Cas-Mediated Genome Engineering. Cell Aug 27. [Epub ahead of print] .The type II bacterial CRISPR/Cas system is a novel genome-engineering technology with the ease of multiplexed gene targeting. Here, we created reporter and conditional mutant mice by coinjection of zygotes with Cas9 mRNA and different guide RNAs (sgRNAs) as well as DNA vectors of different sizes. Using this one-step procedure we generated mice carrying a tag or a fluorescent reporter construct in the Nanog, the Sox2, and the Oct4 gene as well as Mecp2 conditional mutant mice. In addition, using sgRNAs targeting two separate sites in the Mecp2 gene, we produced mice harboring the predicted deletions of about 700 bps. Finally, we analyzed potential off-targets of five sgRNAs in gene-modified mice and ESC lines and identified off-target mutations in only rare instances. Full Text
Ye, L., Widlund, A.L., Sims, C.A., Lamming, D.W., Guan, Y.X., Davis, J.G., Sabatini, D.M., Harrison, D.E., Vang, O., and Baur, J.A. (2013). Rapamycin doses sufficient to extend lifespan do not compromise muscle mitochondrial content or endurance. Aging-Us 5, 539-550.Rapamycin extends lifespan in mice, but can have a number of undesirable effects that may ultimately limit its utility in humans. The canonical target of rapamycin, and the one thought to account for its effects on lifespan, is the mammalian/mechanistic target of rapamycin, complex 1 (mTORC1). We have previously shown that at least some of the detrimental side effects of rapamycin are due to "off target" disruption of mTORC2, suggesting they could be avoided by more specific targeting of mTORC1. However, mTORC1 inhibition per se can reduce the mRNA expression of mitochondrial genes and compromise the function of mitochondria in cultured muscle cells, implying that defects in bioenergetics might be an unavoidable consequence of targeting mTORC1 in vivo. Therefore, we tested whether rapamycin, at the same doses used to extend lifespan, affects mitochondrial function in skeletal muscle. While mitochondrial transcripts were decreased, particularly in the highly oxidative soleus muscle, we found no consistent change in mitochondrial DNA or protein levels. In agreement with the lack of change in mitochondrial components, rapamycin-treated mice had endurance equivalent to that of untreated controls, and isolated, permeabilized muscle fibers displayed similar rates of oxygen consumption. We conclude that the doses of rapamycin required to extend life do not cause overt mitochondrial dysfunction in skeletal muscle. Full Text
Youngsaye, W., Hartland, C.L., Morgan, B.J., Ting, A., Nag, P.P., Vincent, B., Mosher, C.A., Bittker, J.A., Dandapani, S., Palmer, M,.Whitesell L, Lindquist S, Schreiber SL, Munoz B.l. (2013). ML212: A small-molecule probe for investigating fluconazole resistance mechanisms in Candida albicans. Beilstein Journal of Organic Chemistry 9, 1501-1507.The National Institutes of Health Molecular Libraries and Probe Production Centers Network (NIH-MLPCN) screened >300,000 compounds to evaluate their ability to restore fluconazole susceptibility in resistant Candida albicans isolates. Additional counter screens were incorporated to remove substances inherently toxic to either mammalian or fungal cells. A substituted indazole possessing the desired bioactivity profile was selected for further development, and initial investigation of structure-activity relationships led to the discovery of ML212. Full Text
Zhang, L., Prak, L., Rayon-Estrada, V., Thiru, P., Flygare, J., Lim, B., and Lodish, H.F. (2013). ZFP36L2 is required for self-renewal of early burst-forming unit erythroid progenitors. Nature Jun 9. [Epub ahead of print].Stem cells and progenitors in many lineages undergo self-renewing divisions, but the extracellular and intracellular proteins that regulate this process are largely unknown. Glucocorticoids stimulate red blood cell formation by promoting self-renewal of early burst-forming unit-erythroid (BFU-E) progenitors. Here we show that the RNA-binding protein ZFP36L2 is a transcriptional target of the glucocorticoid receptor (GR) in BFU-Es and is required for BFU-E self-renewal. ZFP36L2 is normally downregulated during erythroid differentiation from the BFU-E stage, but its expression is maintained by all tested GR agonists that stimulate BFU-E self-renewal, and the GR binds to several potential enhancer regions of ZFP36L2. Knockdown of ZFP36L2 in cultured BFU-E cells did not affect the rate of cell division but disrupted glucocorticoid-induced BFU-E self-renewal, and knockdown of ZFP36L2 in transplanted erythroid progenitors prevented expansion of erythroid lineage progenitors normally seen following induction of anaemia by phenylhydrazine treatment. ZFP36L2 preferentially binds to messenger RNAs that are induced or maintained at high expression levels during terminal erythroid differentiation and negatively regulates their expression levels. ZFP36L2 therefore functions as part of a molecular switch promoting BFU-E self-renewal and a subsequent increase in the total numbers of colony-forming unit-erythroid (CFU-E) progenitors and erythroid cells that are generated. Full Text
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