The following alphabetical list represents papers published in 2016 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.

2016 Titles :

-Absolute Quantification of Matrix Metabolites Reveals the Dynamics of Mitochondrial Metabolism. Chen
-Activation of PKA leads to mesenchymal-to-epithelial transition and loss of tumor-initiating ability. Pattabiraman
-Activation of proto-oncogenes by disruption of chromosome neighborhoods. Hnisz
-All You Need Is Mentorship. Weinberg2
-AMPK promotes tolerance to Ras pathway inhibition by activating autophagy. Sanduja
-Analyzing Single-Molecule Protein Transportation Experiments via Hierarchical Hidden Markov Models. Chen3
-Annexin A2 antibodies but not inhibitors of the annexin A2 heterotetramer impair productive HIV-1 infection of macrophages in vitro.Woodham
-The Antiviral Mechanism of an Influenza A Virus Nucleoprotein-Specific Single-Domain Antibody Fragment. Hanke
-The apo-structure of the leucine sensor Sestrin2 is still elusive. Saxton2
-Asymmetrically localized proteins stabilize basal bodies against ciliary beating forces. Bayless
-Autophagy, Lipophagy and lysosomal lipid storage disorders. Ward
-Back to basics: Refined nuclear reprogramming techniques yield higher-quality stem cells. Buganim
-Bacterial lipids activate, synergize, and inhibit a developmental switch in choanoflagellates. Woznica
-Biomedical Journals and Preprint Services: Friends or Foes? Annesley
-BRCA1/FANCD2/BRG1-Driven DNA Repair Stabilizes the Differentiation State of Human Mammary Epithelial Cells. Wang5
-The Caenorhabditis elegans Protein FIC-1 Is an AMPylase That Covalently Modifies Heat-Shock 70 Family Proteins, Translation Elongation Factors and Histones. Truttmann
-The CASTOR Proteins Are Arginine Sensors for the mTORC1Pathway.Chantranupong
-Cell-Type-Specific Alternative Splicing Governs Cell Fate in the Developing Cerebral Cortex. Zhang
-ChIP-seq Analysis of Human Chronic Myeloid Leukemia Cells. Anders
-Cholesterol-Independent SREBP-1 Maturation Is Linked to ARF1 Inactivation. Smulan
-Cisplatin Resistant Spheroids Model Clinically Relevant Survival Mechanisms in Ovarian Tumors. Chowanadisai
-Citrobacter rodentium NleB inhibits tumor necrosis factor (TNF) receptor-associated factor 3 (TRAF3) ubiquitination to reduce host type I interferon production. Gao
-Clinical heterogeneity associated with KCNA1 mutations include cataplexy and nonataxic presentations. Brownstein
-CNS disease models with human pluripotent stem cells in the CRISPR age. Muffat2
-Co-evolution of Hormone Metabolism and Signaling Networks Expands Plant Adaptive Plasticity .Weng
-The Combined Deficiency of Immunoproteasome Subunits Affects Both the Magnitude and Quality of Pathogen- and Genetic Vaccination-Induced CD8(+) T Cell Responses to the Human Protozoan Parasite Trypanosoma cruzi. Ersching2
-Comparative transcriptomics across the prokaryotic tree of life. Cohen2
-Condensin and Hmo1 Mediate a Starvation-Induced Transcriptional Position Effect within the Ribosomal DNA Array. Wang
-Conserved imprinting associated with unique epigenetic signatures in the Arabidopsis genus. Klosinska
-Conserved Tetramer Junction in the Kinetochore Ndc80 Complex. Valverde
-Control of TSC2-Rheb signaling axis by arginine regulates mTORC1 activity. Carroll
-A Conversation with Rudolf Jaenisch. Neill
-Covalent targeting of remote cysteine residues to develop CDK12 and CDK13 inhibitors. Zhang2
-Crystal Structure and Conformational Change Mechanism of a Bacterial Nramp-Family Divalent Metal Transporter. Bozzi
-Crystal structure of a substrate-engaged SecY protein-translocation channel. Li
-Defining the Essential Function of Yeast Hsf1 Reveals a Compact Transcriptional Program for Maintaining Eukaryotic Proteostasis. Solis
-Dendrimer-RNA nanoparticles generate protective immunity against lethal Ebola, H1N1 influenza, and Toxoplasma gondii challenges with a single dose. Chahal
-DIGIT Is a Conserved Long Noncoding RNA that Regulates GSC Expression to Control Definitive Endoderm Differentiation of Embryonic Stem Cells. Daneshvar
-Directional cerebrospinal fluid movement between brain ventricles in larval zebrafish. Fame
-DMS-MaPseq for genome-wide or targeted RNA structure probing in vivo. Zubradt
-Durable antitumor responses to CD47 blockade require adaptive immune stimulation. Sockolosky
-Dynamic Conformational States Dictate Selectivity toward the Native Substrate in a Substrate-Permissive Acyltransferase. Levsh
-Dynamic control of Hsf1 during heat shock by a chaperone switch and phosphorylation. Zheng
-Editing DNA Methylation in the Mammalian Genome. Liu
-Editorial overview: Cell reprogramming, regeneration and repair. Reddien
-Efficient CRISPR-Cas9 mediated gene disruption in primary erythroid progenitor cells. Li2
-Efficient derivation of microglia-like cells from human pluripotent stem cells.Muffat
-EMT, cell plasticity and metastasis. Chaffer
-Endosperm and imprinting, inextricably linked. Gehring3
-Engineering of Taxadiene Synthase for Improved Selectivity and Yield of a Key Taxol Biosynthetic Intermediate. Edgar
-Environment Dictates Dependence on Mitochondrial Complex I for NAD+ and Aspartate Production and Determines Cancer Cell Sensitivity to Metformin. Gui
-Epigenetics: Cell-type methylomes in the root. Gehring
-Epigenomic analysis detects aberrant super-enhancer DNA methylation in human cancer. Heyn
-ERK and p38 MAPK Activities Determine Sensitivity to PI3K/mTOR Inhibition via Regulation of MYC and YAP.. Muranen
-Evolutionary Inference across Eukaryotes Identifies Specific Pressures Favoring Mitochondrial Gene Retention. Johnston
-Formation of a "Pre-mouth Array" from the Extreme Anterior Domain Is Directed by Neural Crest and Wnt/PCP Signaling. Jacox
-A Fungal-Selective Cytochrome bc1 Inhibitor Impairs Virulence and Prevents the Evolution of Drug Resistance. Vincent
-Generation of Immunity against Pathogens via Single-Domain Antibody-Antigen Constructs. Duarte
-A genome-wide CRISPR screen identifies a restricted set of HIV host dependency factors. Park
-A Genome-wide CRISPR Screen in Toxoplasma Identifies Essential Apicomplexan Genes. Sidik2
-Genome-wide Trans-ethnic Meta-analysis Identifies Seven Genetic Loci Influencing Erythrocyte Traits and a Role for RBPMS in Erythropoiesis. vanRooij
-Germinal Center B Cell Dynamics. Mesin
-Growth of human breast tissues from patient cells in 3D hydrogel scaffolds. Sokol
-Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition). Klionsky
-Hedgehog signaling regulates gene expression in planarian glia. Wang7
-High-fat diet enhances stemness and tumorigenicity of intestinal progenitors. Beyaz
-The history of the Y chromosome in man. Hughes
-Hsp90 in Cancer: Beyond the Usual Suspects. Isaacs
-Human Naive Pluripotent Stem Cells Model X Chromosome Dampening and X Inactivation. Sahakyan
-Human neural crest cells contribute to coat pigmentation in interspecies chimeras after in utero injection into mouse embryos. Cohen
-A Human Trypanosome Suppresses CD8(+) T Cell Priming by Dendritic Cells through the Induction of Immune Regulatory CD4(+) Foxp3(+) T Cells. Ersching
-Identification of a cancer stem cell-specific function for the histone deacetylases, HDAC1 and HDAC7, in breast and ovarian cancer. Witt
-Impact of MicroRNA Levels, Target-Site Complementarity, and Cooperativity on Competing Endogenous RNA-Regulated Gene Expression. Denzler
-Improved Ribosome-Footprint and mRNA Measurements Provide Insights into Dynamics and Regulation of Yeast Translation. Weinberg
-Induced Pluripotent Stem Cells Meet Genome Editing. Hockemeyer
-Induction of Expansion and Folding in Human Cerebral Organoids. Li3
-Inflammation triggers Zeb1-dependent escape from tumor latency. DeCock
-Influence of Mass Resolving Power in Orbital Ion-Trap Mass Spectrometry-Based Metabolomics. Najdekr
-Inhibition of hypothalamic MCT1 expression increases food intake and alters orexigenic and anorexigenic neuropeptide expression. ElizondoVega
-Insulated Neighborhoods: Structural and Functional Units of Mammalian Gene Control. Hnisz2
-An inter-species protein-protein interaction network across vast evolutionary distance. Zhong
-Intrinsically Disordered Proteins Drive Emergence and Inheritance of Biological Traits. Chakrabortee2
-JAK2 V617F stimulates proliferation of erythropoietin- dependent erythroid progenitors and delays their differentiation by activating Stat1 and other non-erythroid signaling pathways. Shi
-Jointly reduced inhibition and excitation underlies circuit-wide changes in cortical processing in Rett syndrome. Banerjee
-Large-Scale Single Guide RNA Library Construction and Use for CRISPR-Cas9-Based Genetic Screens.Wang4
-A Long Noncoding RNA lincRNA-EPS Acts as a Transcriptional Brake to Restrain Inflammation. Atianand
-Longer lifespan in male mice treated with a weakly estrogenic agonist, an antioxidant, an alpha-glucosidase inhibitor or a Nrf2-inducer. Strong
-Longitudinal multiparameter assay of lymphocyte interactions from onset by microfluidic cell pairing and culture. Dura
-Luminidependens (LD) is an Arabidopsis protein with prion behavior. Chakrabortee
-Mechanism of arginine sensing by CASTOR1 upstream of mTORC1. Saxton
-Mediobasal hypothalamic overexpression of DEPTOR protects against high-fat diet-induced obesity. Caron
-Members of the Zinc Cluster Factor Family Alters Virulence in Candida albicans. Issi
-Metabolic Reprogramming of Pancreatic Cancer Mediated by CDK4/6 Inhibition Elicits Unique Vulnerabilities. Franco
-MicroRNA-203 inversely correlates with differentiation grade, targets c-MYC and functions as a tumor suppressor in cSCC. Lohcharoenkal
-A mitotic SKAP isoform regulates spindle positioning at astral microtubule plus end. Kern
-Modeling disease risk through analysis of physical interactions between genetic variants within chromatin regulatory circuitry. Corradin
-Models of human core transcriptional regulatory circuitries. SaintAndre
-Molecular basis of caspase-1 polymerization and its inhibition by a new capping mechanism. Lu
-Molecular characterization of firefly nuptial gifts: a multi-omics approach sheds light on postcopulatory sexual selection. AlWathiqui
-Molecular Criteria for Defining the Naive Human Pluripotent State. Theunissen
-mRNA Poly(A)-tail Changes Specified by Deadenylation Broadly Reshape Translation in Drosophila Oocytes and Early Embryos. Eichhorn
-mTORC1 is Required for Brown Adipose Tissue Recruitment and Metabolic Adaptation to Cold. Labbe
-Mule Regulates the Intestinal Stem Cell Niche via the Wnt Pathway and Targets EphB3 for Proteasomal and Lysosomal Degradation. DominguezBrauer
-Multiple mechanisms contribute to double-strand break repair at rereplication forks in Drosophila follicle cells. Alexander
-Mutations in the substrate binding glycine-rich loop of the mitochondrial processing peptidase-alpha protein (PMPCA) cause a severe mitochondrial disease. Joshi
-Neutrophils suppress intraluminal NK-mediated tumor cell clearance and enhance extravasation of disseminated carcinoma cells. Spiegel
-New insights into the thermal reduction of graphene oxide: Impact of oxygen clustering. Kumar
-O-fucosylated glycoproteins form assemblies in close proximity to the nuclear pore complexes of Toxoplasma gondii. Bandini
-Parallel evolution of male germline epigenetic poising and somatic development in animals. Lesch
-Parent-of-Origin DNA Methylation Dynamics during Mouse Development. Stelzer
-Parkinson-associated risk variant in distal enhancer of alpha-synuclein modulates target gene expression. Soldner
-Peripheral self-reactivity regulates antigen-specific CD8 T-cell responses and cell division under physiological conditions. Swee
-PERK regulates Gq protein-coupled intracellular Ca2+ dynamics in primary cortical neurons. Zhu
-Phenotypic lentivirus screens to identify functional single domain antibodies. Schmidt2
-A PHGDH inhibitor reveals coordination of serine synthesis and one-carbon unit fate. Pacold
-Plasma Membrane Association but Not Midzone Recruitment of RhoGEF ECT2 Is Essential for Cytokinesis. Kotynkova
-Polyspermic fertilization resulting in multipolarity in a sea star zygote. Swartz
-Population-Scale Sequencing Data Enable Precise Estimates of Y-STR Mutation Rates. Willems
-Posttranscriptional Regulation of Glycoprotein Quality Control in the Endoplasmic Reticulum Is Controlled by the E2 Ub-Conjugating Enzyme UBC6e. Hagiwara
-Prodigious plant methylomes. Gehring2
-Rapid capture and labeling of cells on single domain antibodies-functionalized flow cell. Chen2
-The rate of protein synthesis in hematopoietic stem cells is limited partly by 4E-BPs. Signer
-Recent advances in sortase-catalyzed ligation methodology. Antos
-Recurrent somatic mutations in POLR2A define a distinct subset of meningiomas. Clark
-A Regulatory Switch Alters Chromosome Motions at the Metaphase-to-Anaphase Transition.Su
-Replication fork instability and the consequences of fork collisions from rereplication . Alexander2
-Response and resistance to BET bromodomain inhibitors in triple-negative breast cancer.Shu
-RNA G-quadruplexes are globally unfolded in eukaryotic cells and depleted in bacteria. Guo
-Scarless Gene Tagging with One-Step Transformation and Two-Step Selection in Saccharomyces cerevisiae and Schizosaccharomyces pomb. Landgraf
-Selective Y centromere inactivation triggers chromosome shattering in micronuclei and repair by non-homologous end joining. Ly
-Serum Albumin Stimulates Protein Kinase G-dependent Microneme Secretion in Toxoplasma gondii. Brown
-The Shrinking Mitochondrion. Johnston2
-The Simons Genome Diversity Project: 300 genomes from 142 diverse populations. Mallick
-A single domain antibody fragment that recognizes the adaptor ASC defines the role of ASC domains in inflammasome assembly.Schmidt
-Single Guide RNA Library Design and Construction. Wang2
-Size doesn't matter in the heat shock response. Pincus
-A specialized flavone biosynthetic pathway has evolved in the medicinal plant, Scutellaria baicalensis. Zhao
-SPLASH, a hashed identifier for mass spectra. Wohlgemuth
-Stem cells and interspecies chimaeras. Wu
-Stress from Nucleotide Depletion Activates the Transcriptional Regulator HEXIM1 to Suppress Melanoma. Tan
-Structural comparison of the C. elegans and human Ndc80 complexes bound to microtubules reveals distinct binding behavior. WilsonKubalek
-Structurally Defined alphaMHC-II Nanobody-Drug Conjugates: A Therapeutic and Imaging System for B-Cell Lymphoma.Fang
-Structures of TorsinA and its disease-mutant complexed with an activator reveal the molecular basis for primary dystonia. Demircioglu
-Sulfoluciferin is Biosynthesized by a Specialized Luciferin Sulfotransferase in Fireflies. Fallon
-Suppression of 19S proteasome subunits marks emergence of an altered cell state in diverse cancers.Tsvetkov
-Susan Lee Lindquist. Whitesell
-Susan Lee Lindquist (1949-2016). Shorter
-Susan Lindquist: Visionary scientist and peerless mentor. Bevis
-Synthesis of Aza-Rocaglates via ESIPT-Mediated (3+2) Photocycloaddition. Wang6
-System-Wide Adaptations of Desulfovibrio alaskensis G20 to Phosphate-Limited Conditions. Bosak
-Tet1 and Tet2 Protect DNA Methylation Canyons against Hypermethylation. Wiehle
-TGF-beta inhibitors stimulate red blood cell production by enhancing self-renewal of BFU-E erythroid progenitors. Gao2
-The TORC1 pathway to protein destruction. Chantranupong2
-Thymic CD4 T cell selection requires attenuation of March8-mediated MHCII turnover in cortical epithelial cells through CD83. vonRohrscheidt
-Tissue of origin dictates branched-chain amino acid metabolism in mutant Kras-driven cancers. Mayers
-Transnuclear CD8 T cells specific for the immunodominant epitope Gra6 lower acute phase Toxoplasma gondii burden. Sanecka
-TRIB2 reinforces the oncogenic transcriptional program controlled by the TAL1 complex in T-cell acute lymphoblastic leukemia. Tan2
-Two FGFRL-Wnt circuits organize the planarian anteroposterior axis. Scimone
-Ubiquitin-mediated fluctuations in MHC class II facilitate efficient germinal center B cell response. Bannard
-Unique metabolic features of pancreatic cancer stroma: relevance to the tumor compartment, prognosis, and invasive potential. Knudsen
-Unrestrained AMPylation targets cytosolic chaperones and activates the heat shock response. Truttmann2
-Using a Genetically Encoded Sensor to Identify Inhibitors of Toxoplasma gondii Ca2+ Signalling. Sidik
-Usp12 stabilizes the T-cell receptor complex at the cell surface during signaling.Jahan
-Validation of Protein Knockout in Mutant Zebrafish Lines Using In Vitro Translation Assays. Carter
-Viral Packaging and Cell Culture for CRISPR-Based Screens. Wang3
-Visualizing antibody affinity maturation in germinal centers. Tas
-A widely employed germ cell marker is an ancient disordered protein with reproductive functions in diverse eukaryotes. Carmell
-A Workflow for Studying Specialized Metabolism in Nonmodel Eukaryotic Organisms. TorrensSpence
-A zebrafish melanoma model reveals emergence of neural crest identity during melanoma initiation..Kaufman

Alexander2, J.L., and Orr-Weaver, T.L. (2016). Replication fork instability and the consequences of fork collisions from rereplication. Genes & development 30, 2241-2252.Replication forks encounter obstacles that must be repaired or bypassed to complete chromosome duplication before cell division. Proteomic analysis of replication forks suggests that the checkpoint and repair machinery travels with unperturbed forks, implying that they are poised to respond to stalling and collapse. However, impaired fork progression still generates aberrations, including repeat copy number instability and chromosome rearrangements. Deregulated origin firing also causes fork instability if a newer fork collides with an older one, generating double-strand breaks (DSBs) and partially rereplicated DNA. Current evidence suggests that multiple mechanisms are used to repair rereplication damage, yet these can have deleterious consequences for genome integrity. Full Text

Alexander, J.L., Beagan, K., Orr-Weaver, T.L., and McVey, M. (2016). Multiple mechanisms contribute to double-strand break repair at rereplication forks in Drosophila follicle cells. Proceedings of the National Academy of Sciences of the United States of America[Epub ahead of print] Rereplication generates double-strand breaks (DSBs) at sites of fork collisions and causes genomic damage, including repeat instability and chromosomal aberrations. However, the primary mechanism used to repair rereplication DSBs varies across different experimental systems. In Drosophila follicle cells, developmentally regulated rereplication is used to amplify six genomic regions, two of which contain genes encoding eggshell proteins. We have exploited this system to test the roles of several DSB repair pathways during rereplication, using fork progression as a readout for DSB repair efficiency. Here we show that a null mutation in the microhomology-mediated end-joining (MMEJ) component, polymerase theta/mutagen-sensitive 308 (mus308), exhibits a sporadic thin eggshell phenotype and reduced chorion gene expression. Unlike other thin eggshell mutants, mus308 displays normal origin firing but reduced fork progression at two regions of rereplication. We also find that MMEJ compensates for loss of nonhomologous end joining to repair rereplication DSBs in a site-specific manner. Conversely, we show that fork progression is enhanced in the absence of both Drosophila Rad51 homologs, spindle-A and spindle-B, revealing homologous recombination is active and actually impairs fork movement during follicle cell rereplication. These results demonstrate that several DSB repair pathways are used during rereplication in the follicle cells and their contribution to productive fork progression is influenced by genomic position and repair pathway competition. Furthermore, our findings illustrate that specific rereplication DSB repair pathways can have major effects on cellular physiology, dependent upon genomic context. Full Text

AlWathiqui, N., Fallon, T.R., South, A., Weng, J.K., and Lewis, S.M. (2016). Molecular characterization of firefly nuptial gifts: a multi-omics approach sheds light on postcopulatory sexual selection. Sci Rep 6, 38556. Postcopulatory sexual selection is recognized as a key driver of reproductive trait evolution, including the machinery required to produce endogenous nuptial gifts. Despite the importance of such gifts, the molecular composition of the non-gametic components of male ejaculates and their interactions with female reproductive tracts remain poorly understood. During mating, male Photinus fireflies transfer to females a spermatophore gift manufactured by multiple reproductive glands. Here we combined transcriptomics of both male and female reproductive glands with proteomics and metabolomics to better understand the synthesis, composition and fate of the spermatophore in the common Eastern firefly, Photinus pyralis. Our transcriptome of male glands revealed up-regulation of proteases that may enhance male fertilization success and activate female immune response. Using bottom-up proteomics we identified 208 functionally annotated proteins that males transfer to the female in their spermatophore. Targeted metabolomic analysis also provided the first evidence that Photinus nuptial gifts contain lucibufagin, a firefly defensive toxin. The reproductive tracts of female fireflies showed increased gene expression for several proteases that may be involved in egg production. This study offers new insights into the molecular composition of male spermatophores, and extends our understanding of how nuptial gifts may mediate postcopulatory interactions between the sexes. Full Text

Anders, L., and Li, Z. (2016). ChIP-seq Analysis of Human Chronic Myeloid Leukemia Cells. Methods in molecular biology 1465, 129-138.Many transcription factors, chromatin-associated proteins and regulatory DNA elements are genetically and/or epigenetically altered in cancer, including Chronic Myeloid Leukemia (CML). This leads to deregulation of transcription that is often causally linked to the tumorigenic state. Chromatin-immunoprecipitation coupled with massively parallel DNA sequencing (ChIP-seq) is the key technology to study transcription as it allows in vivo whole-genome mapping of epigenetic modifications and interactions of proteins with DNA or chromatin. However, numerous DNA/chromatin-binding proteins, including EZH2, remain difficult to "ChIP," thus yielding genome-wide binding maps of only suboptimal quality. Here, we describe a ChIP-seq protocol optimized for high-quality protein-genome binding maps that have proven especially useful for studying difficult to 'ChIP' transcription regulatory factors in Chronic Myeloid Leukemia (CML) and related malignancies. Full Text

Annesley, T., Scott, M., Bastian, H., Fonseca, V., Ioannidis, J.P., Keller, M.A., and Polka, J. (2016). Biomedical Journals and Preprint Services: Friends or Foes? Clinical chemistry Papers in Press. Here we discuss the concept of preprints with a group of panelists representing various segments of the research and publishing community. Full Text

Antos, J.M., Truttmann, M.C., and Ploegh, H.L. (2016). Recent advances in sortase-catalyzed ligation methodology. Current opinion in structural biology 38, 111-118.The transpeptidation reaction catalyzed by bacterial sortases continues to see increasing use in the construction of novel protein derivatives. In addition to growth in the number of applications that rely on sortase, this field has also seen methodology improvements that enhance reaction performance and scope. In this opinion, we present an overview of key developments in the practice and implementation of sortase-based strategies, including applications relevant to structural biology. Topics include the use of engineered sortases to increase reaction rates, the use of redesigned acyl donors and acceptors to mitigate reaction reversibility, and strategies for expanding the range of substrates that are compatible with a sortase-based approach. Full Text

Atianand, M.K., Hu, W., Satpathy, A.T., Shen, Y., Ricci, E.P., Alvarez-Dominguez, J.R., Bhatta, A., Schattgen, S.A., McGowan, J.D., Blin, J,.Lodish HF, et al. (2016). A Long Noncoding RNA lincRNA-EPS Acts as a Transcriptional Brake to Restrain Inflammation. Cell 165, 1672-1685.Long intergenic noncoding RNAs (lincRNAs) are important regulators of gene expression. Although lincRNAs are expressed in immune cells, their functions in immunity are largely unexplored. Here, we identify an immunoregulatory lincRNA, lincRNA-EPS, that is precisely regulated in macrophages to control the expression of immune response genes (IRGs). Transcriptome analysis of macrophages from lincRNA-EPS-deficient mice, combined with gain-of-function and rescue experiments, revealed a specific role for this lincRNA in restraining IRG expression. Consistently, lincRNA-EPS-deficient mice manifest enhanced inflammation and lethality following endotoxin challenge in vivo. lincRNA-EPS localizes at regulatory regions of IRGs to control nucleosome positioning and repress transcription. Further, lincRNA-EPS mediates these effects by interacting with heterogeneous nuclear ribonucleoprotein L via a CANACA motif located in its 3' end. Together, these findings identify lincRNA-EPS as a repressor of inflammatory responses, highlighting the importance of lincRNAs in the immune system. Full Text

Bandini, G., Haserick, J.R., Motari, E., Ouologuem, D.T., Lourido, S., Roos, D.S., Costello, C.E., Robbins, P.W., and Samuelson, J. (2016). O-fucosylated glycoproteins form assemblies in close proximity to the nuclear pore complexes of Toxoplasma gondii. Proceedings of the National Academy of Sciences of the United States of America (early edition). Toxoplasma gondii is an intracellular parasite that causes disseminated infections in fetuses and immunocompromised individuals. Although gene regulation is important for parasite differentiation and pathogenesis, little is known about protein organization in the nucleus. Here we show that the fucose-binding Aleuria aurantia lectin (AAL) binds to numerous punctate structures in the nuclei of tachyzoites, bradyzoites, and sporozoites but not oocysts. AAL also binds to Hammondia and Neospora nuclei but not to more distantly related apicomplexans. Analyses of the AAL-enriched fraction indicate that AAL binds O-linked fucose added to Ser/Thr residues present in or adjacent to Ser-rich domains (SRDs). Sixty-nine Ser-rich proteins were reproducibly enriched with AAL, including nucleoporins, mRNA-processing enzymes, and cell-signaling proteins. Two endogenous SRDs-containing proteins and an SRD-YFP fusion localize with AAL to the nuclear membrane. Superresolution microscopy showed that the majority of the AAL signal localizes in proximity to nuclear pore complexes. Host cells modify secreted proteins with O-fucose; here we describe the O-fucosylation pathway in the nucleocytosol of a eukaryote. Furthermore, these results suggest O-fucosylation is a mechanism by which proteins involved in gene expression accumulate near the NPC. Full Text

Banerjee, A., Rikhye, R.V., Breton-Provencher, V., Tang, X., Li, C., Li, K., Runyan, C.A., Fu, Z., Jaenisch, R., and Sur, M. (2016). Jointly reduced inhibition and excitation underlies circuit-wide changes in cortical processing in Rett syndrome. PNAS [Epub ahead of print]. Rett syndrome (RTT) arises from loss-of-function mutations in methyl-CpG binding protein 2 gene (Mecp2), but fundamental aspects of its physiological mechanisms are unresolved. Here, by whole-cell recording of synaptic responses in MeCP2 mutant mice in vivo, we show that visually driven excitatory and inhibitory conductances are both reduced in cortical pyramidal neurons. The excitation-to-inhibition (E/I) ratio is increased in amplitude and prolonged in time course. These changes predict circuit-wide reductions in response reliability and selectivity of pyramidal neurons to visual stimuli, as confirmed by two-photon imaging. Targeted recordings reveal that parvalbumin-expressing (PV+) interneurons in mutant mice have reduced responses. PV-specific MeCP2 deletion alone recapitulates effects of global MeCP2 deletion on cortical circuits, including reduced pyramidal neuron responses and reduced response reliability and selectivity. Furthermore, MeCP2 mutant mice show reduced expression of the cation-chloride cotransporter KCC2 (K+/Cl- exporter) and a reduced KCC2/NKCC1 (Na+/K+/Cl- importer) ratio. Perforated patch recordings demonstrate that the reversal potential for GABA is more depolarized in mutant mice, but is restored by application of the NKCC1 inhibitor bumetanide. Treatment with recombinant human insulin-like growth factor-1 restores responses of PV+ and pyramidal neurons and increases KCC2 expression to normalize the KCC2/NKCC1 ratio. Thus, loss of MeCP2 in the brain alters both excitation and inhibition in brain circuits via multiple mechanisms. Loss of MeCP2 from a specific interneuron subtype contributes crucially to the cell-specific and circuit-wide deficits of RTT. The joint restoration of inhibition and excitation in cortical circuits is pivotal for functionally correcting the disorder. Full Text

Bannard, O., McGowan, S.J., Ersching, J., Ishido, S., Victora, G.D., Shin, J.S., and Cyster, J.G. (2016). Ubiquitin-mediated fluctuations in MHC class II facilitate efficient germinal center B cell responses. The Journal of experimental medicine [Epub ahead of print] Antibody affinity maturation occurs in germinal centers (GCs) through iterative rounds of somatic hypermutation and selection. Selection involves B cells competing for T cell help based on the amount of antigen they capture and present on their MHC class II (MHCII) proteins. How GC B cells are able to rapidly and repeatedly transition between mutating their B cell receptor genes and then being selected shortly after is not known. We report that MHCII surface levels and degradation are dynamically regulated in GC B cells. Through ectopic expression of a photoconvertible MHCII-mKikGR chimeric gene, we found that individual GC B cells differed in the rates of MHCII protein turnover. Fluctuations in surface MHCII levels were dependent on ubiquitination and the E3 ligase March1. Increases in March1 expression in centroblasts correlated with decreases in surface MHCII levels, whereas CD83 expression in centrocytes helped to stabilize MHCII at that stage. Defects in MHCII ubiquitination caused GC B cells to accumulate greater amounts of a specific peptide-MHCII (pMHCII), suggesting that MHCII turnover facilitates the replacement of old complexes. We propose that pMHCII complexes are periodically targeted for degradation in centroblasts to favor the presentation of recently acquired antigens, thereby promoting the fidelity and efficiency of selection. Full Text

Bayless, B.A., Galati, D.F., Junker, A.D., Backer, C.B., Gaertig, J., and Pearson, C.G. (2016). Asymmetrically localized proteins stabilize basal bodies against ciliary beating forces. The Journal of cell biology[Epub ahead of print] Basal bodies are radially symmetric, microtubule-rich structures that nucleate and anchor motile cilia. Ciliary beating produces asymmetric mechanical forces that are resisted by basal bodies. To resist these forces, distinct regions within the basal body ultrastructure and the microtubules themselves must be stable. However, the molecular components that stabilize basal bodies remain poorly defined. Here, we determine that Fop1 functionally interacts with the established basal body stability components Bld10 and Poc1. We find that Fop1 and microtubule glutamylation incorporate into basal bodies at distinct stages of assembly, culminating in their asymmetric enrichment at specific triplet microtubule regions that are predicted to experience the greatest mechanical force from ciliary beating. Both Fop1 and microtubule glutamylation are required to stabilize basal bodies against ciliary beating forces. Our studies reveal that microtubule glutamylation and Bld10, Poc1, and Fop1 stabilize basal bodies against the forces produced by ciliary beating via distinct yet interdependent mechanisms. Full Text

Bevis, B.J. (2016). Susan Lindquist: Visionary scientist and peerless mentor. The Journal of cell biology Epub The science universe is dimmer after one of our brightest stars, Susan Lee Lindquist, was taken by cancer on October 27, 2016. Sue was an innovative, creative, out-of-the-box scientific thinker. She had unique biological intuition-an instinct for both the way things worked and the right questions to ask to uncover new research insights. Her wide-ranging career began with the study of protein folding and molecular chaperones, and she went on to show that protein folding can have profound and unexpected biological effects on such diverse processes as cancer, evolution, and neurodegenerative disease. As Sue's laboratory manager, I would like to offer a ground-floor perspective on what made her an exceptional scientist, mentor, and leader. She created a harmonious, collegial environment where collaborative synergy fueled meaningful progress that will impact science for decades to come. Full Text

Beyaz, S., Mana, M.D., Roper, J., Kedrin, D., Saadatpour, A., Hong, S.J., Bauer-Rowe, K.E., Xifaras, M.E., Akkad, A., Arias, E,Luca Pinello,Yarden Katz,Shweta Shinagare,Monther Abu-Remaileh,Maria M. Mihaylova,Dudley W.Lamming,Rizkullah Dogum,Guoji Guo,George W. Bell,Martin Selig,G. Petur Nielsen,Nitin Gupta,Cristina R. Ferrone,Vikram Deshpande,Guo-Cheng Yuan,Stuart H. Orkin, David M. Sabatini & Ömer H. Yilmaz (2016). High-fat diet enhances stemness and tumorigenicity of intestinal progenitors. Nature 531, 53-58.Little is known about how pro-obesity diets regulate tissue stem and progenitor cell function. Here we show that high-fat diet (HFD)-induced obesity augments the numbers and function of Lgr5(+) intestinal stem cells of the mammalian intestine. Mechanistically, a HFD induces a robust peroxisome proliferator-activated receptor delta (PPAR-delta) signature in intestinal stem cells and progenitor cells (non-intestinal stem cells), and pharmacological activation of PPAR-delta recapitulates the effects of a HFD on these cells. Like a HFD, ex vivo treatment of intestinal organoid cultures with fatty acid constituents of the HFD enhances the self-renewal potential of these organoid bodies in a PPAR-delta-dependent manner. Notably, HFD- and agonist-activated PPAR-delta signalling endow organoid-initiating capacity to progenitors, and enforced PPAR-delta signalling permits these progenitors to form in vivo tumours after loss of the tumour suppressor Apc. These findings highlight how diet-modulated PPAR-delta activation alters not only the function of intestinal stem and progenitor cells, but also their capacity to initiate tumours. Full Text

Bosak, T., Schubotz, F., de Santiago-Torio, A., Kuehl, J.V., Carlson, H.K., Watson, N., Daye, M., Summons, R.E., Arkin, A.P., and Deutschbauer, A.M. (2016). System-Wide Adaptations of Desulfovibrio alaskensis G20 to Phosphate-Limited Conditions. PloS one 11, e0168719.The prevalence of lipids devoid of phosphorus suggests that the availability of phosphorus limits microbial growth and activity in many anoxic, stratified environments. To better understand the response of anaerobic bacteria to phosphate limitation and starvation, this study combines microscopic and lipid analyses with the measurements of fitness of pooled barcoded transposon mutants of the model sulfate reducing bacterium Desulfovibrio alaskensis G20. Phosphate-limited G20 has lower growth rates and replaces more than 90% of its membrane phospholipids by a mixture of monoglycosyl diacylglycerol (MGDG), glycuronic acid diacylglycerol (GADG) and ornithine lipids, lacks polyphosphate granules, and synthesizes other cellular inclusions. Analyses of pooled and individual mutants reveal the importance of the high-affinity phosphate transport system (the Pst system), PhoR, and glycolipid and ornithine lipid synthases during phosphate limitation. The phosphate-dependent synthesis of MGDG in G20 and the widespread occurrence of the MGDG/GADG synthase among sulfate reducing partial differential-Proteobacteria implicate these microbes in the production of abundant MGDG in anaerobic environments where the concentrations of phosphate are lower than 10 muM. Numerous predicted changes in the composition of the cell envelope and systems involved in transport, maintenance of cytoplasmic redox potential, central metabolism and regulatory pathways also suggest an impact of phosphate limitation on the susceptibility of sulfate reducing bacteria to other anthropogenic or environmental stresses. Full Text

Bozzi, A.T., Bane, L.B., Weihofen, W.A., Singharoy, A., Guillen, E.R., Ploegh, H.L., Schulten, K., and Gaudet, R. (2016). Crystal Structure and Conformational Change Mechanism of a Bacterial Nramp-Family Divalent Metal Transporter. Structure [Epub ahead of print] The widely conserved natural resistance-associated macrophage protein (Nramp) family of divalent metal transporters enables manganese import in bacteria and dietary iron uptake in mammals. We determined the crystal structure of the Deinococcus radiodurans Nramp homolog (DraNramp) in an inward-facing apo state, including the complete transmembrane (TM) segment 1a (absent from a previous Nramp structure). Mapping our cysteine accessibility scanning results onto this structure, we identified the metal-permeation pathway in the alternate outward-open conformation. We investigated the functional impact of two natural anemia-causing glycine-to-arginine mutations that impaired transition metal transport in both human Nramp2 and DraNramp. The TM4 G153R mutation perturbs the closing of the outward metal-permeation pathway and alters the selectivity of the conserved metal-binding site. In contrast, the TM1a G45R mutation prevents conformational change by sterically blocking the essential movement of that helix, thus locking the transporter in an inward-facing state. Full Text

Brown, K.M., Lourido, S., and Sibley, L.D. (2016). Serum Albumin Stimulates Protein Kinase G-dependent Microneme Secretion in Toxoplasma gondii. Journal of Biological Chemistry 291, 9554-9565.Microneme secretion is essential for motility, invasion, and egress in apicomplexan parasites. Although previous studies indicate that Ca2+ and cGMP control microneme secretion, little is known about how these pathways are naturally activated. Here we have developed genetically encoded indicators for Ca2+ and microneme secretion to better define the signaling pathways that regulate these processes in Toxoplasma gondii. We found that microneme secretion was triggered in vitro by exposure to a single host protein, serum albumin. The natural agonist serum albumin induced microneme secretion in a protein kinase G-dependent manner that correlated with increased cGMP levels. Surprisingly, serum albumin acted independently of elevated Ca2+ and yet it was augmented by artificial agonists that raise Ca2+, such as ethanol. Furthermore, although ethanol elevated intracellular Ca2+, it alone was unable to trigger secretion without the presence of serum or serum albumin. This dichotomy was recapitulated by zaprinast, a phosphodiesterase inhibitor that elevated cGMP and separately increased Ca2+ in a protein kinase G-independent manner leading to microneme secretion. Taken together, these findings reveal that microneme secretion is centrally controlled by protein kinase G and that this pathway is further augmented by elevation of intracellular Ca2+. Full Text

Brownstein, C.A., Beggs, A.H., Rodan, L., Shi, J.H., Towne, M.C., Pelletier, R., Cao, S.Q., Rosenberg, P.A., Urion, D.K., Picker, J., et al. (2016). Clinical heterogeneity associated with KCNA1 mutations include cataplexy and nonataxic presentations. Neurogenetics 17, 11-16.Mutations in the KCNA1 gene are known to cause episodic ataxia/myokymia syndrome type 1 (EA1). Here, we describe two families with unique presentations who were enrolled in an IRB-approved study, extensively phenotyped, and whole exome sequencing (WES) performed. Family 1 had a diagnosis of isolated cataplexy triggered by sudden physical exertion in multiple affected individuals with heterogeneous neurological findings. All enrolled affected members carried a KCNA1 c.941T > C (p.I314T) mutation. Family 2 had an 8-year-old patient with muscle spasms with rigidity for whom WES revealed a previously reported heterozygous missense mutation in KCNA1 c.677C > G (p.T226R), confirming the diagnosis of EA1 without ataxia. WES identified variants in KCNA1 that explain both phenotypes expanding the phenotypic spectrum of diseases associated with mutations of this gene. KCNA1 mutations should be considered in patients of all ages with episodic neurological phenotypes, even when ataxia is not present. This is an example of the power of genomic approaches to identify pathogenic mutations in unsuspected genes responsible for heterogeneous diseases. Full Text

Buganim, Y. (2016). Back to basics: Refined nuclear reprogramming techniques yield higher-quality stem cells. Science 352, 1401. The “hyperdynamic” chromatin state, which characterizes embryonic stem cell (ESC) epigenetic state, facilitates a rapid and efficient reaction to external and internal cues. These cues lead to the activation of key master regulators that drive the cells into their developmental fate. Thus, ESCs hold great promise as a source of diverse differentiated cell types for cell therapy and regenerative medicine. .Full Text

Carmell, M.A., Dokshin, G.A., Skaletsky, H., Hu, Y.C., von Wolfswinkel, J.C., Igarashi, K.J., Bellott, D.W., Nefedov, M., Reddien, P.W., Enders, G.C., Vladimir N Uversky, Craig C Mello & David C Page (2016). A widely employed germ cell marker is an ancient disordered protein with reproductive functions in diverse eukaryotes. eLife 10.7554/eLife.19993. The advent of sexual reproduction and the evolution of a dedicated germline in multicellular organisms are critical landmarks in eukaryotic evolution. We report an ancient family of GCNA (germ cell nuclear antigen) proteins that arose in the earliest eukaryotes, and feature a rapidly evolving intrinsically disordered region (IDR). Phylogenetic analysis reveals that GCNA proteins emerged before the major eukaryotic lineages diverged; GCNA predates the origin of a dedicated germline by a billion years. Gcna gene expression is enriched in reproductive cells across eukarya - either just prior to or during meiosis in single-celled eukaryotes, and in stem cells and germ cells of diverse multicellular animals. Studies of Gcna-mutant C. elegans and mice indicate that GCNA has functioned in reproduction for at least 600 million years. Homology to IDR-containing proteins implicated in DNA damage repair suggests that GCNA proteins may protect the genomic integrity of cells carrying a heritable genome. Full Text

Caron, A., Labbe, S.M., Lanfray, D., Blanchard, P.G., Villot, R., Roy, C., Sabatini, D.M., Richard, D., and Laplante, M. (2016). Mediobasal hypothalamic overexpression of DEPTOR protects against high-fat diet-induced obesity. Molecular metabolism 5, 102-112. The mechanistic target of rapamycin (mTOR) is a serine-threonine kinase that functions into distinct protein complexes (mTORC1 and mTORC2) that regulate energy homeostasis. DEP-domain containing mTOR-interacting protein (DEPTOR) is part of these complexes and is known to dampen mTORC1 function, consequently reducing mTORC1 negative feedbacks and promoting insulin signaling and Akt/PKB activation in several models. Recently, we observed that DEPTOR is expressed in several structures of the brain including the mediobasal hypothalamus (MBH), a region that regulates energy balance. Whether DEPTOR in the MBH plays a functional role in regulating energy balance and hypothalamic insulin signaling has never been tested. METHODS: We have generated a novel conditional transgenic mouse model based on the Cre-LoxP system allowing targeted overexpression of DEPTOR. Mice overexpressing DEPTOR in the MBH were subjected to a metabolic phenotyping and MBH insulin signaling was evaluated. RESULTS: We first report that systemic (brain and periphery) overexpression of DEPTOR prevents high-fat diet-induced obesity, improves glucose metabolism and protects against hepatic steatosis. These phenotypes were associated with a reduction in food intake and feed efficiency and an elevation in oxygen consumption. Strikingly, specific overexpression of DEPTOR in the MBH completely recapitulated these phenotypes. DEPTOR overexpression was associated with an increase in hypothalamic insulin signaling, as illustrated by elevated Akt/PKB activation. CONCLUSION: Altogether, these results support a role for MBH DEPTOR in the regulation of energy balance and metabolism. Full Text

Carroll, B., Maetzel, D., Maddocks, O.D., Otten, G., Ratcliff, M., Smith, G.R., Dunlop, E.A., Passos, J.F., Davies, O.R., Jaenisch, R., et al. (2016). Control of TSC2-Rheb signaling axis by arginine regulates mTORC1 activity. eLife 2016;10.7554/eLife.11058 The mammalian target of rapamycin complex 1 (mTORC1) is the key signalling hub that regulates cellular protein homeostasis, growth and proliferation. Herein, we demonstrate that amino acid arginine acts independent of its metabolism to allow maximal activation of mTORC1 by growth factors, via a mechanism that does not involve regulation of mTORC1 localization to lysosomes. Instead, arginine specifically suppresses lysosomal localization of the TSC complex and interaction with its target small GTPase protein, Rheb. By interfering with TSC-Rheb complex, arginine relieves allosteric inhibition of Rheb by TSC. Arginine is the main amino acid sensed by the mTORC1 pathway in several cell types including human embryonic stem cells (hESCs). Together, our data provide evidence that different growth promoting cues cooperate to a greater extent than previously recognized to achieve tight spatial and temporal regulation of mTORC1 signalling. Full Text

Carter, B.S., Cortes-Campos, C., Chen, X., McCammon, J.M., and Sive, H.L. (2016). Validation of Protein Knockout in Mutant Zebrafish Lines Using In Vitro Translation Assays. Zebrafish.[Epub ahead of print] Advances in genome-editing technology have made creation of zebrafish mutant lines accessible to the community. Experimental validation of protein knockout is a critical step in verifying null mutants, but this can be a difficult task. Absence of protein can be confirmed by Western blotting; however, this approach requires target-specific antibodies that are generally not available for zebrafish proteins. We address this issue using in vitro translation assays, a fast and standard procedure that can be easily implemented. Full Text

Chaffer, C.L., San Juan, B.P., Lim, E., and Weinberg, R.A. (2016). EMT, cell plasticity and metastasis. Cancer metastasis reviews [Epub ahead of print] Carcinoma cells that are induced to suppress their epithelial features and upregulate mesenchymal gene expression programs acquire traits that promote an invasive and metastatic phenotype. This is achieved through the expression of a program termed the epithelial-to-mesenchymal transition (EMT)-a fundamental cell-biological process that plays key roles in embryogenesis and wound healing. Re-activation of the EMT during cancer promotes disease progression and enhances the metastatic phenotype by bestowing upon previously benign carcinoma cell traits such as migration, invasion, resistance to anoikis, chemoresistance and tumour-initiating potential. Herein, we discuss recent insights into the function of the EMT and cancer cell plasticity during cancer progression, with a focus on their role in promoting successful completion of the later stages of the metastatic cascade. Full Text

Chahal, J.S., Khan, O.F., Cooper, C.L., McPartlan, J.S., Tsosie, J.K., Tilley, L.D., Sidik, S.M., Lourido, S., Langer, R., Bavari, S., Ploegh HL, et al. (2016). Dendrimer-RNA nanoparticles generate protective immunity against lethal Ebola, H1N1 influenza, and Toxoplasma gondii challenges with a single dose. Proceedings of the National Academy of Sciences of the United States of America [Epub ahead of print]. Vaccines have had broad medical impact, but existing vaccine technologies and production methods are limited in their ability to respond rapidly to evolving and emerging pathogens, or sudden outbreaks. Here, we develop a rapid-response, fully synthetic, single-dose, adjuvant-free dendrimer nanoparticle vaccine platform wherein antigens are encoded by encapsulated mRNA replicons. To our knowledge, this system is the first capable of generating protective immunity against a broad spectrum of lethal pathogen challenges, including H1N1 influenza, Toxoplasma gondii, and Ebola virus. The vaccine can be formed with multiple antigen-expressing replicons, and is capable of eliciting both CD8+ T-cell and antibody responses. The ability to generate viable, contaminant-free vaccines within days, to single or multiple antigens, may have broad utility for a range of diseases. Full Text

Chakrabortee2, S., Byers, J.S., Jones, S., Garcia, D.M., Bhullar, B., Chang, A., She, R., Lee, L., Fremin, B., Lindquist, S., et al. (2016). Intrinsically Disordered Proteins Drive Emergence and Inheritance of Biological Traits. Cell [Epub ahead of print] Prions are a paradigm-shifting mechanism of inheritance in which phenotypes are encoded by self-templating protein conformations rather than nucleic acids. Here, we examine the breadth of protein-based inheritance across the yeast proteome by assessing the ability of nearly every open reading frame (ORF; approximately 5,300 ORFs) to induce heritable traits. Transient overexpression of nearly 50 proteins created traits that remained heritable long after their expression returned to normal. These traits were beneficial, had prion-like patterns of inheritance, were common in wild yeasts, and could be transmitted to naive cells with protein alone. Most inducing proteins were not known prions and did not form amyloid. Instead, they are highly enriched in nucleic acid binding proteins with large intrinsically disordered domains that have been widely conserved across evolution. Thus, our data establish a common type of protein-based inheritance through which intrinsically disordered proteins can drive the emergence of new traits and adaptive opportunities. Full Text

Chakrabortee, S., Kayatekin, C., Newby, G.A., Mendillo, M.L., Lancaster, A., and Lindquist, S. (2016). Luminidependens (LD) is an Arabidopsis protein with prion behavior. Proceedings of the National Academy of Sciences of the United States of America[Epub ahead of print] Prion proteins provide a unique mode of biochemical memory through self-perpetuating changes in protein conformation and function. They have been studied in fungi and mammals, but not yet identified in plants. Using a computational model, we identified candidate prion domains (PrDs) in nearly 500 plant proteins. Plant flowering is of particular interest with respect to biological memory, because its regulation involves remembering and integrating previously experienced environmental conditions. We investigated the prion-forming capacity of three prion candidates involved in flowering using a yeast model, where prion attributes are well defined and readily tested. In yeast, prions heritably change protein functions by templating monomers into higher-order assemblies. For most yeast prions, the capacity to convert into a prion resides in a distinct prion domain. Thus, new prion-forming domains can be identified by functional complementation of a known prion domain. The prion-like domains (PrDs) of all three of the tested proteins formed higher-order oligomers. Uniquely, the Luminidependens PrD (LDPrD) fully replaced the prion-domain functions of a well-characterized yeast prion, Sup35. Our results suggest that prion-like conformational switches are evolutionarily conserved and might function in a wide variety of normal biological processes. Full Text

Chantranupong2, L., and Sabatini, D.M. The TORC1 pathway to protein destruction. Nature Published online. A study of the proteasome — a protein-degradation complex — reveals an evolutionarily conserved pathway that acts through the protein kinase TORC1 to adjust proteasome levels in response to cellular needs. Full Text

Chantranupong, L., Scaria, S.M., Saxton, R.A., Gygi, M.P., Shen, K., Wyant, G.A., Wang, T., Harper, J.W., Gygi, S.P., and Sabatini, D.M. (2016). The CASTOR Proteins Are Arginine Sensors for the mTORC1 Pathway. Cell [Epub ahead of print]. Amino acids signal to the mTOR complex I (mTORC1) growth pathway through the Rag GTPases. Multiple distinct complexes regulate the Rags, including GATOR1, a GTPase activating protein (GAP), and GATOR2, a positive regulator of unknown molecular function. Arginine stimulation of cells activates mTORC1, but how it is sensed is not well understood. Recently, SLC38A9 was identified as a putative lysosomal arginine sensor required for arginine to activate mTORC1 but how arginine deprivation represses mTORC1 is unknown. Here, we show that CASTOR1, a previously uncharacterized protein, interacts with GATOR2 and is required for arginine deprivation to inhibit mTORC1. CASTOR1 homodimerizes and can also heterodimerize with the related protein, CASTOR2. Arginine disrupts the CASTOR1-GATOR2 complex by binding to CASTOR1 with a dissociation constant of approximately 30 muM, and its arginine-binding capacity is required for arginine to activate mTORC1 in cells. Collectively, these results establish CASTOR1 as an arginine sensor for the mTORC1 pathway. Full Text

Chen2, G.Y., Li, Z., Duarte, J.N., Esteban, A., Cheloha, R.W., Theile, C.S., Fink, G.R., and Ploegh, H.L. (2016). Rapid capture and labeling of cells on single domain antibodies-functionalized flow cell. Biosensors & bioelectronics , in Press Current techniques to characterize leukocyte subgroups in blood require long sample preparation times and sizable sample volumes. A simplified method for leukocyte characterization using smaller blood volumes would thus be useful in diagnostic settings. Here we describe a flow system comprised of two functionalized graphene oxide (GO) surfaces that allow the capture of distinct leukocyte populations from small volumes blood using camelid single-domain antibodyfragments (VHHs) as capture agents. We used site-specifically labeled leukocytes to detect and identify cells exposed to fungal challenge. Combining the chemical and optical properties of GO with the versatility of the VHH scaffold in the context of a flow system provides a quick and efficient method for the capture and characterization of functional leukocytes. Full Text

Chen, W.W., Freinkman, E., Wang, T., Birsoy, K., and Sabatini, D.M. (2016). Absolute Quantification of Matrix Metabolites Reveals the Dynamics of Mitochondrial Metabolism. Cell 166, 1324-1337.e1311.Mitochondria house metabolic pathways that impact most aspects of cellular physiology. While metabolite profiling by mass spectrometry is widely applied at the whole-cell level, it is not routinely possible to measure the concentrations of small molecules in mammalian organelles. We describe a method for the rapid and specific isolation of mitochondria and use it in tandem with a database of predicted mitochondrial metabolites ("MITObolome") to measure the matrix concentrations of more than 100 metabolites across various states of respiratory chain (RC) function. Disruption of the RC reveals extensive compartmentalization of mitochondrial metabolism and signatures unique to the inhibition of each RC complex. Pyruvate enables the proliferation of RC-deficient cells but has surprisingly limited effects on matrix contents. Interestingly, despite failing to restore matrix NADH/NAD balance, pyruvate does increase aspartate, likely through the exchange of matrix glutamate for cytosolic aspartate. We demonstrate the value of mitochondrial metabolite profiling and describe a strategy applicable to other organelles. Full Text

Chen3, Y., Shen, K., Shan, S.O., and Kou, S.C. (2016). Analyzing Single-Molecule Protein Transportation Experiments via Hierarchical Hidden Markov Models. Journal of the American Statistical Association 111, 951-966.To maintain proper cellular functions, over 50% of proteins encoded in the genome need to be transported to cellular membranes. The molecular mechanism behind such a process, often referred to as protein targeting, is not well understood. Single-molecule experiments are designed to unveil the detailed mechanisms and reveal the functions of different molecular machineries involved in the process. The experimental data consist of hundreds of stochastic time traces from the fluorescence recordings of the experimental system. We introduce a Bayesian hierarchical model on top of hidden Markov models (HMMs) to analyze these data and use the statistical results to answer the biological questions. In addition to resolving the biological puzzles and delineating the regulating roles of different molecular complexes, our statistical results enable us to propose a more detailed mechanism for the late stages of the protein targeting process. Full Text

Chowanadisai, W., Messerli, S.M., Miller, D.H., Medina, J.E., Hamilton, J.W., Messerli, M.A., and Brodsky, A.S. (2016). Cisplatin Resistant Spheroids Model Clinically Relevant Survival Mechanisms in Ovarian Tumors. PloS one 11, e0151089.The majority of ovarian tumors eventually recur in a drug resistant form. Using cisplatin sensitive and resistant cell lines assembled into 3D spheroids we profiled gene expression and identified candidate mechanisms and biological pathways associated with cisplatin resistance. OVCAR-8 human ovarian carcinoma cells were exposed to sub-lethal concentrations of cisplatin to create a matched cisplatin-resistant cell line, OVCAR-8R. Genome-wide gene expression profiling of sensitive and resistant ovarian cancer spheroids identified 3,331 significantly differentially expressed probesets coding for 3,139 distinct protein-coding genes (Fc >2, FDR < 0.05) (S2 Table). Despite significant expression changes in some transporters including MDR1, cisplatin resistance was not associated with differences in intracellular cisplatin concentration. Cisplatin resistant cells were significantly enriched for a mesenchymal gene expression signature. OVCAR-8R resistance derived gene sets were significantly more biased to patients with shorter survival. From the most differentially expressed genes, we derived a 17-gene expression signature that identifies ovarian cancer patients with shorter overall survival in three independent datasets. We propose that the use of cisplatin resistant cell lines in 3D spheroid models is a viable approach to gain insight into resistance mechanisms relevant to ovarian tumors in patients. Our data support the emerging concept that ovarian cancers can acquire drug resistance through an epithelial-to-mesenchymal transition. Full Text

Clark, V.E., Harmanci, A.S., Bai, H., Youngblood, M.W., Lee, T.I., Baranoski, J.F., Ercan-Sencicek, A.G., Abraham, B.J., Weintraub, A.S., Hnisz, D, .Matthias Simon, Boris Krischek, E Zeynep Erson-Omay, Octavian Henegariu, Geneive Carrión-Grant, Ketu Mishra-Gorur, Daniel Durán, Johanna E Goldmann, Johannes Schramm, Roland Goldbrunner ,Joseph M Piepmeier, Alexander O Vortmeyer, Jennifer Moliterno Günel, Kaya Bilgüvar, Katsuhito Yasuno, Richard A Young & Murat Günell. (2016). Recurrent somatic mutations in POLR2A define a distinct subset of meningiomas. Nature genetics Published online RNA polymerase II mediates the transcription of all protein-coding genes in eukaryotic cells, a process that is fundamental to life. Genomic mutations altering this enzyme have not previously been linked to any pathology in humans, which is a testament to its indispensable role in cell biology. On the basis of a combination of next-generation genomic analyses of 775 meningiomas, we report that recurrent somatic p.Gln403Lys or p.Leu438_His439del mutations in POLR2A, which encodes the catalytic subunit of RNA polymerase II (ref. 1), hijack this essential enzyme and drive neoplasia. POLR2A mutant tumors show dysregulation of key meningeal identity genes, including WNT6 and ZIC1/ZIC4. In addition to mutations in POLR2A, NF2, SMARCB1, TRAF7, KLF4, AKT1, PIK3CA, and SMO, we also report somatic mutations in AKT3, PIK3R1, PRKAR1A, and SUFU in meningiomas. Our results identify a role for essential transcriptional machinery in driving tumorigenesis and define mutually exclusive meningioma subgroups with distinct clinical and pathological features. Full Text

Cohen2, O., Doron, S., Wurtzel, O., Dar, D., Edelheit, S., Karunker, I., Mick, E., and Sorek, R. (2016). Comparative transcriptomics across the prokaryotic tree of life. Nucleic acids research [Epub ahead of print] Whole-transcriptome sequencing studies from recent years revealed an unexpected complexity in transcriptomes of bacteria and archaea, including abundant non-coding RNAs, cis-antisense transcription and regulatory untranslated regions (UTRs). Understanding the functional relevance of the plethora of non-coding RNAs in a given organism is challenging, especially since some of these RNAs were attributed to 'transcriptional noise'. To allow the search for conserved transcriptomic elements we produced comparative transcriptome maps for multiple species across the microbial tree of life. These transcriptome maps are detailed in annotations, comparable by gene families, and BLAST-searchable by user provided sequences. Our transcriptome collection includes 18 model organisms spanning 10 phyla/subphyla of bacteria and archaea that were sequenced using standardized RNA-seq methods. The utility of the comparative approach, as implemented in our web server, is demonstrated by highlighting genes with exceptionally long 5'UTRs across species, which correspond to many known riboswitches and further suggest novel putative regulatory elements. Our study provides a standardized reference transcriptome to major clinically and environmentally important microbial phyla. The viewer is available at http://exploration.weizmann.ac.il/TCOL, setting a framework for comparative studies of the microbial non-coding genome. Full Text

Cohen, M.A., Wert, K.J., Goldmann, J., Markoulaki, S., Buganim, Y., Fu, D., and Jaenisch, R. (2016). Human neural crest cells contribute to coat pigmentation in interspecies chimeras after in utero injection into mouse embryos. Proceedings of the National Academy of Sciences of the United States of America Early Edition The neural crest (NC) represents multipotent cells that arise at the interphase between ectoderm and prospective epidermis of the neurulating embryo. The NC has major clinical relevance because it is involved in both inherited and acquired developmental abnormalities. The aim of this study was to establish an experimental platform that would allow for the integration of human NC cells (hNCCs) into the gastrulating mouse embryo. NCCs were derived from pluripotent mouse, rat, and human cells and microinjected into embryonic-day-8.5 embryos. To facilitate integration of the NCCs, we used recipient embryos that carried a c-Kit mutation (Wsh/Wsh), which leads to a loss of melanoblasts and thus eliminates competition from the endogenous host cells. The donor NCCs migrated along the dorsolateral migration routes in the recipient embryos. Postnatal mice derived from injected embryos displayed pigmented hair, demonstrating differentiation of the NCCs into functional melanocytes. Although the contribution of human cells to pigmentation in the host was lower than that of mouse or rat donor cells, our results indicate that hNCCs, injected in utero, can integrate into the embryo and form mature functional cells in the animal. This mouse-human chimeric platform allows for a new approach to study NC development and diseases. Full Text

Corradin, O., Cohen, A.J., Luppino, J.M., Bayles, I.M., Schumacher, F.R., and Scacheri, P.C. (2016). Modeling disease risk through analysis of physical interactions between genetic variants within chromatin regulatory circuitry. Nature Published online. geneticsSNPs associated with disease susceptibility often reside in enhancer clusters, or super-enhancers. Constituents of these enhancer clusters cooperate to regulate target genes and often extend beyond the linkage disequilibrium (LD) blocks containing risk SNPs identified in genome-wide association studies (GWAS). We identified 'outside variants', defined as SNPs in weak LD with GWAS risk SNPs that physically interact with risk SNPs as part of a target gene's regulatory circuitry. These outside variants further explain variation in target gene expression beyond that explained by GWAS-associated SNPs. Additionally, the clinical risk associated with GWAS SNPs is considerably modified by the genotype of outside variants. Collectively, these findings suggest a potential model in which outside variants and GWAS SNPs that physically interact in 3D chromatin collude to influence target transcript levels as well as clinical risk. This model offers an additional hypothesis for the source of missing heritability for complex traits. Full Text

Daneshvar, K., Pondick, J.V., Kim, B.M., Zhou, C., York, S.R., Macklin, J.A., Abualteen, A., Tan, B., Sigova, A.A., Marcho, C., et al. (2016). DIGIT Is a Conserved Long Noncoding RNA that Regulates GSC Expression to Control Definitive Endoderm Differentiation of Embryonic Stem Cells. Cell reports 17, 353-365.Long noncoding RNAs (lncRNAs) exhibit diverse functions, including regulation of development. Here, we combine genome-wide mapping of SMAD3 occupancy with expression analysis to identify lncRNAs induced by activin signaling during endoderm differentiation of human embryonic stem cells (hESCs). We find that DIGIT is divergent to Goosecoid (GSC) and expressed during endoderm differentiation. Deletion of the SMAD3-occupied enhancer proximal to DIGIT inhibits DIGIT and GSC expression and definitive endoderm differentiation. Disruption of the gene encoding DIGIT and depletion of the DIGIT transcript reveal that DIGIT is required for definitive endoderm differentiation. In addition, we identify the mouse ortholog of DIGIT and show that it is expressed during development and promotes definitive endoderm differentiation of mouse ESCs. DIGIT regulates GSC in trans, and activation of endogenous GSC expression is sufficient to rescue definitive endoderm differentiation in DIGIT-deficient hESCs. Our study defines DIGIT as a conserved noncoding developmental regulator of definitive endoderm. Full Text

DeCock, J.M., Shibue, T., Dongre, A., Keckesova, Z., Reinhardt, F., and Weinberg, R.A. (2016). Inflammation triggers Zeb1-dependent escape from tumor latency. Cancer research [Epub ahead of print] The emergence of metastatic disease in cancer patients many years or decades after initial successful treatment of primary tumors is well documented but poorly understood at the molecular level. Recent studies have begun exploring the cell-intrinsic programs causing disseminated tumor cells to enter latency and the cellular signals in the surrounding non-permissive tissue microenvironment that maintain the latent state. However, relatively little is known about the mechanisms that enable disseminated tumor cells to escape cancer dormancy or tumor latency. We describe here an in vivo model of solitary metastatic latency in the lung parenchyma. The induction of a localized inflammation in the lungs, initiated by lipopolysaccharide (LPS) treatment, triggers the awakening of these cells, which develop into macroscopic metastases. The escape from latency is dependent on the expression of Zeb1, a key regulator of the epithelial-to-mesenchymal transition (EMT). Furthermore, activation of the EMT program on its own, as orchestrated by Zeb1, is sufficient to incite metastatic outgrowth by causing carcinoma cells to enter stably into a metastasis-initiating cell state. Full Text

Demircioglu, F.E., Sosa, B.A., Ingram, J., Ploegh, H.L., and Schwartz, T.U. (2016). Structures of TorsinA and its disease-mutant complexed with an activator reveal the molecular basis for primary dystonia. eLife 5/e17983. The most common cause of early onset primary dystonia, a neuromuscular disease, is a glutamate deletion (DeltaE) at position 302/303 of TorsinA, a AAA+ ATPase that resides in the endoplasmic reticulum. While the function of TorsinA remains elusive, the DeltaE mutation is known to diminish binding of two TorsinA ATPase activators: lamina-associated protein 1 (LAP1) and its paralog, luminal domain like LAP1 (LULL1). Using a nanobody as a crystallization chaperone, we obtained a 1.4 A crystal structure of human TorsinA in complex with LULL1. This nanobody likewise stabilized the weakened TorsinAE-LULL1 interaction, which enabled us to solve its structure at 1.4 A also. A comparison of these structures shows, in atomic detail, the subtle differences in activator interactions that separate the healthy from the diseased state. This information may provide a structural platform for drug development, as a small molecule that rescues TorsinADeltaE could serve as a cure for primary dystonia. Full Text

Denzler, R., McGeary, S.E., Title, A.C., Agarwal, V., Bartel, D.P., and Stoffel, M. (2016). Impact of MicroRNA Levels, Target-Site Complementarity, and Cooperativity on Competing Endogenous RNA-Regulated Gene Expression. Molecular Cell 64, 565-579.Expression changes of competing endogenous RNAs (ceRNAs) have been proposed to influence microRNA (miRNA) activity and thereby regulate other transcripts containing miRNA-binding sites. Here, we find that although miRNA levels define the extent of repression, they have little effect on the magnitude of the ceRNA expression change required to observe derepression. Canonical 6-nt sites, which typically mediate modest repression, can nonetheless compete for miRNA binding, with potency approximately 20% of that observed for canonical 8-nt sites. In aggregate, low-affinity/background sites also contribute to competition. Sites with extensive additional complementarity can appear as more potent, but only because they induce miRNA degradation. Cooperative binding of proximal sites for the same or different miRNAs does increase potency. These results provide quantitative insights into the stoichiometric relationship between miRNAs and target abundance, target-site spacing, and affinity requirements for ceRNA-mediated gene regulation, and the unusual circumstances in which ceRNA-mediated gene regulation might be observed. Full Text

DominguezBrauer, C., Hao, Z., Elia, A.J., Fortin, J.M., Nechanitzky, R., Brauer, P.M., Sheng, Y., Mana, M.D., Chio, II, Haight, J.,David M. Sabatini, et al. (2016). Mule Regulates the Intestinal Stem Cell Niche via the Wnt Pathway and Targets EphB3 for Proteasomal and Lysosomal Degradation. Cell stem cell. In Press The E3 ubiquitin ligase Mule is often overexpressed in human colorectal cancers, but its role in gut tumorigenesis is unknown. Here, we show in vivo that Mule controls murine intestinal stem and progenitor cell proliferation by modulating Wnt signaling via c-Myc. Mule also regulates protein levels of the receptor tyrosine kinase EphB3 by targeting it for proteasomal and lysosomal degradation. In the intestine, EphB/ephrinB interactions position cells along the crypt-villus axis and compartmentalize incipient colorectal tumors. Our study thus unveils an important new avenue by which Mule acts as an intestinal tumor suppressor by regulation of the intestinal stem cell niche. Full Text

Duarte, J.N., Cragnolini, J.J., Swee, L.K., Bilate, A.M., Bader, J., Ingram, J.R., Rashidfarrokhi, A., Fang, T., Schiepers, A., Hanke, L. ,and Hidde L. Ploegh. (2016). Generation of Immunity against Pathogens via Single-Domain Antibody-Antigen Constructs. Journal of immunology [Epub ahead of print]. mAbs specific for surface proteins on APCs can serve as Ag-delivery vehicles that enhance immunogenicity. The practical use of such constructs is limited by the challenge of expressing and modifying full-sized mAbs. We generated single-domain Ab fragments (VHHs) specific for class II MHC (MHCII), CD11b, and CD36. VHH sequences were modified by inclusion of a C-terminal sortase motif to allow site-specific conjugation with various Ag payloads. We tested T cell activation using VHHs that target distinct APC populations; anti-MHCII adducts elicited strong activation of CD4+ T cells, whereas anti-CD11b showed CD8+ T cell activation superior to targeting via MHCII and CD36. Differences in Ag presentation among constructs were unrelated to dendritic cell subtype or routing to acidic compartments. When coupled to antigenic payloads, anti-MHCII VHH primed Ab responses against GFP, ubiquitin, an OVA peptide, and the alpha-helix of influenza hemagglutinin's stem; the last afforded protection against influenza infection. The versatility of the VHH scaffold and sortase-mediated covalent attachment of Ags suggests their broader application to generate desirable immune responses. Full Text

Dura, B., Servos, M.M., Barry, R.M., Ploegh, H.L., Dougan, S.K., and Voldman, J. (2016). Longitudinal multiparameter assay of lymphocyte interactions from onset by microfluidic cell pairing and culture. Proceedings of the National Academy of Sciences of the United States of America [Epub ahead of print] Resolving how the early signaling events initiated by cell-cell interactions are transduced into diverse functional outcomes necessitates correlated measurements at various stages. Typical approaches that rely on bulk cocultures and population-wide correlations, however, only reveal these relationships broadly at the population level, not within each individual cell. Here, we present a microfluidics-based cell-cell interaction assay that enables longitudinal investigation of lymphocyte interactions at the single-cell level through microfluidic cell pairing, on-chip culture, and multiparameter assays, and allows recovery of desired cell pairs by micromanipulation for off-chip culture and analyses. Well-defined initiation of interactions enables probing cellular responses from the very onset, permitting single-cell correlation analyses between early signaling dynamics and later-stage functional outcomes within same cells. We demonstrate the utility of this microfluidic assay with natural killer cells interacting with tumor cells, and our findings suggest a possible role for the strength of early calcium signaling in selective coordination of subsequent cytotoxicity and IFN-gamma production. Collectively, our experiments demonstrate that this new approach is well-suited for resolving the relationships between complex immune responses within each individual cell. Full Text

Edgar, S., Li, F.S., Qiao, K., Weng, J.K., and Stephanopoulos, G. (2016). Engineering of Taxadiene Synthase for Improved Selectivity and Yield of a Key Taxol Biosynthetic Intermediate. ACS synthetic biology [Epub ahead of print] Attempts at microbial production of the chemotherapeutic agent Taxol (paclitaxel) have met with limited success, due largely to a pathway bottleneck resulting from poor product selectivity of the first hydroxylation step, catalyzed by taxadien-5a-hydroxylase (CYP725A4). Here, we systematically investigate three methodologies, terpene cyclase engineering, P450 engineering, and hydrolase-enzyme screening to overcome this early pathway selectivity bottleneck. We demonstrate that engineering of Taxadiene Synthase, upstream of the promiscuous oxidation step, acts as a practical method for selectivity improvement. Through mutagenesis we achieve a 2.4-fold improvement in yield and selectivity for an alternative cyclization product, taxa-4(20)-11(12)-diene; and for the Taxol precursor taxadien-5alpha-ol, when coexpressed with CYP725A4. This works lays the foundation for the elucidation, engineering, and improved production of Taxol and early Taxol precursors. Full Text

Eichhorn, S.W., Subtelny, A.O., Kronja, I., Kwasnieski, J.C., Orr-Weaver, T.L., and Bartel, D.P. (2016). mRNA Poly(A)-tail Changes Specified by Deadenylation Broadly Reshape Translation in Drosophila Oocytes and Early Embryos. eLife 10.7554/eLife.16955 Because maturing oocytes and early embryos lack appreciable transcription, posttranscriptional regulatory processes control their development. To better understand this control, we profiled translational efficiencies and poly(A)-tail lengths throughout Drosophila oocyte maturation and early embryonic development. The correspondence between translational-efficiency changes and tail-length changes indicated that tail-length changes broadly regulate translation until gastrulation, when this coupling disappears. During egg activation, relative changes in poly(A)-tail length, and thus translational efficiency, were largely retained in the absence of cytoplasmic polyadenylation, which indicated that selective poly(A)-tail shortening primarily specifies these changes. Many translational changes depended on PAN GU and Smaug, and both acted primarily through tail-length changes. Our results also revealed the presence of tail-length-independent mechanisms that maintained translation despite tail-length shortening during oocyte maturation, and prevented essentially all translation of bicoid and several other mRNAs before egg activation. In addition to these fundamental insights, our results provide valuable resources for future studies. Full Text

ElizondoVega, R., Cortes-Campos, C., Barahona, M.J., Carril, C., Ordenes, P., Salgado, M., Oyarce, K., and Garcia-Robles, M.L. (2016). Inhibition of hypothalamic MCT1 expression increases food intake and alters orexigenic and anorexigenic neuropeptide expression. Sci Rep 6, 33606. Hypothalamic glucosensing, which involves the detection of glucose concentration changes by brain cells and subsequent release of orexigenic or anorexigenic neuropeptides, is a crucial process that regulates feeding behavior. Arcuate nucleus (AN) neurons are classically thought to be responsible for hypothalamic glucosensing through a direct sensing mechanism; however, recent data has shown a metabolic interaction between tanycytes and AN neurons through lactate that may also be contributing to this process. Monocarboxylate transporter 1 (MCT1) is the main isoform expressed by tanycytes, which could facilitate lactate release to hypothalamic AN neurons. We hypothesize that MCT1 inhibition could alter the metabolic coupling between tanycytes and AN neurons, altering feeding behavior. To test this, we inhibited MCT1 expression using adenovirus-mediated transfection of a shRNA into the third ventricle, transducing ependymal wall cells and tanycytes. Neuropeptide expression and feeding behavior were measured in MCT1-inhibited animals after intracerebroventricular glucose administration following a fasting period. Results showed a loss in glucose regulation of orexigenic neuropeptides and an abnormal expression of anorexigenic neuropeptides in response to fasting. This was accompanied by an increase in food intake and in body weight gain. Taken together, these results indicate that MCT1 expression in tanycytes plays a role in feeding behavior regulation. Full Text

Ersching, J., Basso, A.S., Kalich, V.L.G., Bortoluci, K.R., and Rodrigues, M.M. (2016). A Human Trypanosome Suppresses CD8(+) T Cell Priming by Dendritic Cells through the Induction of Immune Regulatory CD4(+) Foxp3(+) T Cells. Plos Pathogens 12(6): e1005698. Although CD4(+) Foxp3(+) T cells are largely described in the regulation of CD4(+) T cell responses, their role in the suppression of CD8(+) T cell priming is much less clear. Because the induction of CD8(+) T cells during experimental infection with Trypanosoma cruzi is remarkably delayed and suboptimal, we raised the hypothesis that this protozoan parasite actively induces the regulation of CD8(+) T cell priming. Using an in vivo assay that eliminated multiple variables associated with antigen processing and dendritic cell activation, we found that injection of bone marrow-derived dendritic cells exposed to T. cruzi induced regulatory CD4(+) Foxp3(+) T cells that suppressed the priming of transgenic CD8(+) T cells by peptide-loaded BMDC. This newly described suppressive effect on CD8(+) T cell priming was independent of IL-10, but partially dependent on CTLA-4 and TGF-beta. Accordingly, depletion of Foxp3(+) cells in mice infected with T. cruzi enhanced the response of epitope-specific CD8(+) T cells. Altogether, our data uncover a mechanism by which T. cruzi suppresses CD8(+) T cell responses, an event related to the establishment of chronic infections. Full Text

Ersching2, J., Vasconcelos, J.R., Ferreira, C.P., Caetano, B.C., Machado, A.V., Bruna-Romero, O., Baron, M.A., Ferreira, L.R.P., Cunha-Neto, E., Rock, K.L., et al. (2016). The Combined Deficiency of Immunoproteasome Subunits Affects Both the Magnitude and Quality of Pathogen- and Genetic Vaccination-Induced CD8(+) T Cell Responses to the Human Protozoan Parasite Trypanosoma cruzi. Plos Pathogens 12(4): e1005593. The beta 1i, beta 2i and beta 5i immunoproteasome subunits have an important role in defining the repertoire of MHC class I-restricted epitopes. However, the impact of combined deficiency of the three immunoproteasome subunits in the development of protective immunity to intracellular pathogens has not been investigated. Here, we demonstrate that immunoproteasomes play a key role in host resistance and genetic vaccination-induced protection against the human pathogen Trypanosoma cruzi (the causative agent of Chagas disease), immunity to which is dependent on CD8(+) T cells and IFN-gamma (the classical immunoproteasome inducer). We observed that infection with T. cruzi triggers the transcription of immunoproteasome genes, both in mice and humans. Importantly, genetically vaccinated or T. cruziinfected beta 1i, beta 2i and beta 5i triple knockout (TKO) mice presented significantly lower frequencies and numbers of splenic CD8(+) effector T cells (CD8(+) CD44(high)CD62L(low)) specific for the previously characterized immunodominant (VNHRFTLV) H-2K(b)-restricted T. cruzi epitope. Not only the quantity, but also the quality of parasite-specific CD8(+) T cell responses was altered in TKO mice. Hence, the frequency of double-positive (IFN-gamma(+)/TNF+) or single-positive (IFN-gamma(+)) cells specific for the H-2K(b)-restricted immunodominant as well as subdominant T. cruzi epitopes were higher inWT mice, whereas TNF single-positive cells prevailed among CD8(+) T cells from TKO mice. Contrasting with their WT counterparts, TKO animals were also lethally susceptible to T. cruzi challenge, even after an otherwise protective vaccination with DNA and adenoviral vectors. We conclude that the immunoproteasome subunits are key determinants in host resistance to T. cruzi infection by influencing both the magnitude and quality of CD8(+) T cell responses. Full Text

Fallon, T.R., Li, F.S., Vicent, M.A., and Weng, J.K. (2016). Sulfoluciferin is Biosynthesized by a Specialized Luciferin Sulfotransferase in Fireflies. Biochemistry 55 (24), pp 3341–3344 Firefly luciferin is a specialized metabolite restricted to fireflies (family Lampyridae) and other select families of beetles (order Coleoptera). Firefly luciferin undergoes luciferase-catalyzed oxidation to produce light, thereby enabling the luminous mating signals essential for reproductive success in most bioluminescent beetles. Although firefly luciferin and luciferase have become widely used biotechnological tools, questions remain regarding the physiology and biochemistry of firefly bioluminescence. Here we report sulfoluciferin to be an in vivo derivative of firefly luciferin in fireflies and report the cloning of luciferin sulfotransferase (LST) from the North American firefly Photinus pyralis. LST catalyzes the production of sulfoluciferin from firefly luciferin and the sulfo-donor PAPS. Sulfoluciferin is abundant in several surveyed firefly genera as well as in the bioluminescent elaterid beetle Pyrophorus luminosus at a low level. We propose that sulfoluciferin could serve as a luciferin storage molecule in fireflies and that LST may find use as a new tool to modulate existing biotechnological applications of the firefly bioluminescent system. Full Text

Fame, R.M., Chang, J.T., Hong, A., Aponte-Santiago, N.A., and Sive, H. (2016). Directional cerebrospinal fluid movement between brain ventricles in larval zebrafish. Fluids and barriers of the CNS 13(1) :11. Cerebrospinal fluid (CSF) contained within the brain ventricles contacts neuroepithelial progenitor cells during brain development. Dynamic properties of CSF movement may limit locally produced factors to specific regions of the developing brain. However, there is no study of in vivo CSF dynamics between ventricles in the embryonic brain. We address CSF movement using the zebrafish larva, during the major period of developmental neurogenesis. METHODS: CSF movement was monitored at two stages of zebrafish development: early larva [pharyngula stage; 27-30 h post-fertilization (hpf)] and late larva (hatching period; 51-54 hpf) using photoactivatable Kaede protein to calculate average maximum CSF velocity between ventricles. Potential roles for heartbeat in early CSF movement were investigated using tnnt2a mutant fish (tnnt2a (-/-)) and chemical [2,3 butanedione monoxime (BDM)] treatment. Cilia motility was monitored at these stages using the Tg(betaact:Arl13b-GFP) transgenic fish line. RESULTS: In wild-type early larva there is net CSF movement from the telencephalon to the combined diencephalic/mesencephalic superventricle. This movement directionality reverses at late larval stage. CSF moves directionally from diencephalic to rhombencephalic ventricles at both stages examined, with minimal movement from rhombencephalon to diencephalon. Directional movement is partially dependent on heartbeat, as indicated in assays of tnnt2a (-/-) fish and after BDM treatment. Brain cilia are immotile at the early larval stage. CONCLUSION: These data demonstrate directional movement of the embryonic CSF in the zebrafish model during the major period of developmental neurogenesis. A key conclusion is that CSF moves preferentially from the diencephalic into the rhombencephalic ventricle. In addition, the direction of CSF movement between telencephalic and diencephalic ventricles reverses between the early and late larval stages. CSF movement is partially dependent on heartbeat. At early larval stage, the absence of motile cilia indicates that cilia likely do not direct CSF movement. These data suggest that CSF components may be compartmentalized and could contribute to specialization of the early brain. In addition, CSF movement may also provide directional mechanical signaling. Full Text

Fang, T., Duarte, J.N., Ling, J., Li, Z., Guzman, J.S., and Ploegh, H.L. (2016). Structurally Defined alphaMHC-II Nanobody-Drug Conjugates: A Therapeutic and Imaging System for B-Cell Lymphoma. Angewandte Chemie (International ed in English) 55, 2416-2420.Antibody-drug conjugates (ADCs) of defined structure hold great promise for cancer therapies, but further advances are constrained by the complex structures of full-sized antibodies. Camelid-derived single-domain antibody fragments (VHHs or nanobodies) offer a possible solution to this challenge by providing expedited target screening and validation through switching between imaging and therapeutic activities. We used a nanobody (VHH7) specific for murine MHC-II and rendered "sortase-ready" for the introduction of oligoglycine-modified cytotoxic payloads or NIR fluorophores. The VHH7 conjugates outcompeted commercial monoclonal antibodies (mAbs) for internalization and exhibited high specificity and cytotoxicity against A20 murine B-cell lymphoma. Non-invasive NIR imaging with a VHH7-fluorophore conjugate showed rapid tumor targeting on both localized and metastatic lymphoma models. Subsequent treatment with the nanobody-drug conjugate efficiently controlled tumor growth and metastasis without obvious systemic toxicity. Full Text

Franco, J., Balaji, U., Freinkman, E., Witkiewicz, A.K., and Knudsen, E.S. (2016). Metabolic Reprogramming of Pancreatic Cancer Mediated by CDK4/6 Inhibition Elicits Unique Vulnerabilities. Cell reports[Epub ahead of print] Due to loss of p16ink4a in pancreatic ductal adenocarcinoma (PDA), pharmacological suppression of CDK4/6 could represent a potent target for treatment. In PDA models, CDK4/6 inhibition had a variable effect on cell cycle but yielded accumulation of ATP and mitochondria. Pharmacological CDK4/6 inhibitors induce cyclin D1 protein levels; however, RB activation was required and sufficient for mitochondrial accumulation. CDK4/6 inhibition stimulated glycolytic and oxidative metabolism and was associated with an increase in mTORC1 activity. MTOR and MEK inhibitors potently cooperate with CDK4/6 inhibition in eliciting cell-cycle exit. However, MTOR inhibition fully suppressed metabolism and yielded apoptosis and suppression of tumor growth in xenograft models. The metabolic state mediated by CDK4/6 inhibition increases mitochondrial number and reactive oxygen species (ROS). Concordantly, the suppression of ROS scavenging or BCL2 antagonists cooperated with CDK4/6 inhibition. Together, these data define the impact of therapeutics on PDA metabolism and provide strategies for converting cytostatic response to tumor cell killing. Full Text

Gao2, X., Lee, H.Y., da Rocha, E.L., Zhang, C., Lu, Y.F., Li, D., Feng, Y., Ezike, J., Elmes, R.R., Barrasa, M.I.,Cahan P, Li H, Daley GQ, and Lodish HF (2016). TGF-beta inhibitors stimulate red blood cell production by enhancing self-renewal of BFU-E erythroid progenitors. Blood [Epub ahead of print] Burst-forming unit erythroid progenitors (BFU-Es) are so-named based on their ability to generate in methylcellulose culture large colonies of erythroid cells that consist of "bursts" of smaller erythroid colonies derived from the later CFU-E Epo- dependent progenitors. "Early" BFU-E cells forming large BFU-E colonies presumably have higher capacities for self-renewal than do "late" BFU-E forming small colonies but the mechanism underlying this heterogeneity remains unknown. We show that the type III transforming growth factor (TGF)-beta receptor (TbetaRIII) is a marker that distinguishes "early" and "late" BFU-Es. Transient elevation of TbetaRIII expression promotes TGF-beta signaling during the early BFU-E to late BFU-E transition. Blocking TGF-beta signaling by receptor kinase inhibitor increases early BFU-E cell self-renewal and total erythroblast production, suggesting the use of this type of drug in treating Epo unresponsive anemias. Full Text

Gao, X., Pham, T.H., Feuerbacher, L.A., Chen, K., Hays, M.P., Singh, G., Rueter, C., Guerrero, R.H., and Hardwidge, P.R. (2016). Citrobacter rodentium NleB inhibits tumor necrosis factor (TNF) receptor-associated factor 3 (TRAF3) ubiquitination to reduce host type I interferon production. The Journal of biological chemistry Papers in Press. Interferon signaling plays important roles in both intestinal homeostasis and in the host response to pathogen infection. The extent to which bacterial pathogens inhibit this host pathway is an understudied area of investigation. We characterized Citrobacter rodentium strains bearing deletions in individual type III secretion system effector genes to determine both whether this pathogen inhibits the host type I IFN response and which effector is responsible. The NleB effector limited host IFN-beta production by inhibiting K63-linked ubiquitination of the TNF receptor-associated factor 3 (TRAF3). Inhibition was dependent upon the glycosyltransferase activity of NleB. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a target of NleB during infection, bound to TRAF3 and was required for maximal TRAF3 ubiquitination. NleB glycosyltransferase activity inhibited GAPDH-TRAF3 binding, resulting in reduced TRAF3 ubiquitination. Collectively, our data reveal important interplay between GAPDH and TRAF3 and suggest a mechanism by which the NleB effector inhibits type I IFN signaling. Full Text

Gehring3, M., and Satyaki, P.R. (2016). Endosperm and imprinting, inextricably linked. Plant physiology [Epub ahead of print] This Update focuses on progress over the last several years in understanding imprinted gene expression, from molecular control to physiological significance, within the context of endosperm formation and function. Full Text

Gehring2, M., and Satyaki, P.R. (2016). Prodigious plant methylomes. Genome biology 17, 197.Schmitz and colleagues recently investigated DNA methylation patterns in diverse flowering plant species, finding substantial variation in the extent and distribution of methylation in angiosperms.. Full Text

Gehring, M. (2016). Epigenetics: Cell-type methylomes in the root. Nature plants 2(5):16061.A new study combines cell sorting with DNA methylation and RNA profiling to identify novel epigenomic features in plant roots. Full Text

Gui, D.Y., Sullivan, L.B., Luengo, A., Hosios, A.M., Bush, L.N., Gitego, N., Davidson, S.M., Freinkman, E., Thomas, C.J., and Vander Heiden, M.G. (2016). Environment Dictates Dependence on Mitochondrial Complex I for NAD+ and Aspartate Production and Determines Cancer Cell Sensitivity to Metformin. Cell metabolism [Epub ahead of print] Metformin use is associated with reduced cancer mortality, but how metformin impacts cancer outcomes is controversial. Although metformin can act on cells autonomously to inhibit tumor growth, the doses of metformin that inhibit proliferation in tissue culture are much higher than what has been described in vivo. Here, we show that the environment drastically alters sensitivity to metformin and other complex I inhibitors. We find that complex I supports proliferation by regenerating nicotinamide adenine dinucleotide (NAD)+, and metformin's anti-proliferative effect is due to loss of NAD+/NADH homeostasis and inhibition of aspartate biosynthesis. However, complex I is only one of many inputs that determines the cellular NAD+/NADH ratio, and dependency on complex I is dictated by the activity of other pathways that affect NAD+ regeneration and aspartate levels. This suggests that cancer drug sensitivity and resistance are not intrinsic properties of cancer cells, and demonstrates that the environment can dictate sensitivity to therapies that impact cell metabolism. Full Text

Guo, J.U., and Bartel, D.P. (2016). RNA G-quadruplexes are globally unfolded in eukaryotic cells and depleted in bacteria. Science Vol. 353, Issue 6306, DOI: 10.1126/science.aaf5371. In vitro, some RNAs can form stable four-stranded structures known as G-quadruplexes. Although RNA G-quadruplexes have been implicated in posttranscriptional gene regulation and diseases, direct evidence for their formation in cells has been lacking. Here, we identified thousands of mammalian RNA regions that can fold into G-quadruplexes in vitro, but in contrast to previous assumptions, these regions were overwhelmingly unfolded in cells. Model RNA G-quadruplexes that were unfolded in eukaryotic cells were folded when ectopically expressed in Escherichia coli; however, they impaired translation and growth, which helps explain why we detected few G-quadruplex-forming regions in bacterial transcriptomes. Our results suggest that eukaryotes have a robust machinery that globally unfolds RNA G-quadruplexes, whereas some bacteria have instead undergone evolutionary depletion of G-quadruplex-forming sequences. Full Text

Hagiwara, M., Ling, J., Koenig, P.A., and Ploegh, H.L. (2016). Posttranscriptional Regulation of Glycoprotein Quality Control in the Endoplasmic Reticulum Is Controlled by the E2 Ub-Conjugating Enzyme UBC6e. Molecular cell Volume 63, Issue 5, , Pages 753–767 ER-associated degradation (ERAD) is essential for protein quality control in the ER, not only when the ER is stressed, but also at steady state. We report a new layer of homeostatic control, in which ERAD activity itself is regulated posttranscriptionally and independently of the unfolded protein response by adjusting the endogenous levels of EDEM1, OS-9, and SEL1L (ERAD enhancers). Functional UBC6e requires its precise location in the ER to form a supramolecular complex with Derlin2. This complex targets ERAD enhancers for degradation, a function that depends on UBC6e's enzymatic activity. Ablation of UBC6e causes upregulation of active ERAD enhancers and so increases clearance not only of terminally misfolded substrates, but also of wild-type glycoproteins that fold comparatively slowly in vitro and in vivo. The levels of proteins that comprise the ERAD machinery are thus carefully tuned and adjusted to prevailing needs. Full Text

Hanke, L., Knockenhauer, K.E., Brewer, R.C., van Diest, E., Schmidt, F.I., Schwartz, T.U., and Ploegh, H.L. (2016). The Antiviral Mechanism of an Influenza A Virus Nucleoprotein-Specific Single-Domain Antibody Fragment. MBio. 2016 7(6). pii: e01569-16. Alpaca-derived single-domain antibody fragments (VHHs) that target the influenza A virus nucleoprotein (NP) can protect cells from infection when expressed in the cytosol. We found that one such VHH, alphaNP-VHH1, exhibits antiviral activity similar to that of Mx proteins by blocking nuclear import of incoming viral ribonucleoproteins (vRNPs) and viral transcription and replication in the nucleus. We determined a 3.2-A crystal structure of alphaNP-VHH1 in complex with influenza A virus NP. The VHH binds to a nonconserved region on the body domain of NP, which has been associated with binding to host factors and serves as a determinant of host range. Several of the NP/VHH interface residues determine sensitivity of NP to antiviral Mx GTPases. The structure of the NP/alphaNP-VHH1 complex affords a plausible explanation for the inhibitory properties of the VHH and suggests a rationale for the antiviral properties of Mx proteins. Such knowledge can be leveraged for much-needed novel antiviral strategies. IMPORTANCE: Influenza virus strains can rapidly escape from protection afforded by seasonal vaccines or acquire resistance to available drugs. Additional ways to interfere with the virus life cycle are therefore urgently needed. The influenza virus nucleoprotein is one promising target for antiviral interventions. We have previously isolated alpaca-derived single-domain antibody fragments (VHHs) that protect cells from influenza virus infection if expressed intracellularly. We show here that one such VHH exhibits antiviral activities similar to those of proteins of the cellular antiviral defense (Mx proteins). We determined the three-dimensional structure of this VHH in complex with the influenza virus nucleoprotein and identified the interaction site, which overlaps regions that determine sensitivity of the virus to Mx proteins. Our data define a new vulnerability of influenza virus, help us to better understand the cellular antiviral mechanisms, and provide a well-characterized tool to further study them. Full Text

Heyn, H., Vidal, E., Ferreira, H.J., Vizoso, M., Sayols, S., Gomez, A., Moran, S., Boque-Sastre, R., Guil, S., Martinez-Cardus, A., Charles Y. Lin, Richard A. Young, et al. (2016). Epigenomic analysis detects aberrant super-enhancer DNA methylation in human cancer. Genome biology 17, 11. One of the hallmarks of cancer is the disruption of gene expression patterns. Many molecular lesions contribute to this phenotype, and the importance of aberrant DNA methylation profiles is increasingly recognized. Much of the research effort in this area has examined proximal promoter regions and epigenetic alterations at other loci are not well characterized. RESULTS: Using whole genome bisulfite sequencing to examine uncharted regions of the epigenome, we identify a type of far-reaching DNA methylation alteration in cancer cells of the distal regulatory sequences described as super-enhancers. Human tumors undergo a shift in super-enhancer DNA methylation profiles that is associated with the transcriptional silencing or the overactivation of the corresponding target genes. Intriguingly, we observe locally active fractions of super-enhancers detectable through hypomethylated regions that suggest spatial variability within the large enhancer clusters. Functionally, the DNA methylomes obtained suggest that transcription factors contribute to this local activity of super-enhancers and that trans-acting factors modulate DNA methylation profiles with impact on transforming processes during carcinogenesis. CONCLUSIONS: We develop an extensive catalogue of human DNA methylomes at base resolution to better understand the regulatory functions of DNA methylation beyond those of proximal promoter gene regions. CpG methylation status in normal cells points to locally active regulatory sites at super-enhancers, which are targeted by specific aberrant DNA methylation events in cancer, with putative effects on the expression of downstream genes. Full Text

Hnisz2, D., Day, D.S., and Young, R.A. (2016). Insulated Neighborhoods: Structural and Functional Units of Mammalian Gene Control. Cell 167, 1188-1200.Understanding how transcriptional enhancers control over 20,000 protein-coding genes to maintain cell-type-specific gene expression programs in all human cells is a fundamental challenge in regulatory biology. Recent studies suggest that gene regulatory elements and their target genes generally occur within insulated neighborhoods, which are chromosomal loop structures formed by the interaction of two DNA sites bound by the CTCF protein and occupied by the cohesin complex. Here, we review evidence that insulated neighborhoods provide for specific enhancer-gene interactions, are essential for both normal gene activation and repression, form a chromosome scaffold that is largely preserved throughout development, and are perturbed by genetic and epigenetic factors in disease. Insulated neighborhoods are a powerful paradigm for gene control that provides new insights into development and disease. Full Text

Hnisz, D., Weintraub, A.S., Day, D.S., Valton, A.L., Bak, R.O., Li, C.H., Goldmann, J., Lajoie, B.R., Fan, Z.P., Sigova, A.A,.Reddy J, Borges-Rivera D, Lee TI, Jaenisch R, Porteus MH, Dekker J, Young RA. (2016). Activation of proto-oncogenes by disruption of chromosome neighborhoods. Science[Epub ahead of print] Oncogenes are activated through well-known chromosomal alterations, including gene fusion, translocation and focal amplification. Recent evidence that the control of key genes depends on chromosome structures called insulated neighborhoods led us to investigate whether proto-oncogenes occur within these structures and if oncogene activation can occur via disruption of insulated neighborhood boundaries in cancer cells. We mapped insulated neighborhoods in T-cell acute lymphoblastic leukemia (T-ALL), and found that tumor cell genomes contain recurrent microdeletions that eliminate the boundary sites of insulated neighborhoods containing prominent T-ALL proto-oncogenes. Perturbation of such boundaries in non-malignant cells was sufficient to activate proto-oncogenes. Mutations affecting chromosome neighborhood boundaries were found in many types of cancer. Thus, oncogene activation can occur via genetic alterations that disrupt insulated neighborhoods in malignant cells. Full Text

Hockemeyer, D., and Jaenisch, R. (2016). Induced Pluripotent Stem Cells Meet Genome Editing. Cell stem cell 18, 573-586.It is extremely rare for a single experiment to be so impactful and timely that it shapes and forecasts the experiments of the next decade. Here, we review how two such experiments-the generation of human induced pluripotent stem cells (iPSCs) and the development of CRISPR/Cas9 technology-have fundamentally reshaped our approach to biomedical research, stem cell biology, and human genetics. We will also highlight the previous knowledge that iPSC and CRISPR/Cas9 technologies were built on as this groundwork demonstrated the need for solutions and the benefits that these technologies provided and set the stage for their success. Full Text

Hughes, J.F., and Page, D.C. (2016). The history of the Y chromosome in man. Nature genetics 48, 588-589.Studies of the Y chromosome over the past few decades have opened a window into the history of our species, through the reconstruction and exploitation of a patrilineal (Y-genealogical) tree based on several hundred single-nucleotide variants (SNVs). A new study validates, refines and extends this tree by incorporating >65,000 Y-linked variants identified in 1,244 men representing worldwide diversity. Full Text

Isaacs, J., and Whitesell, L., Editors (2016). ADVANCES IN CANCER RESEARCH Vol. 129 Hsp90 in Cancer: Beyond the Usual Suspects, pp. XIII-XV.

Issi, L., Farrer, R.A., Pastor, K., Landry, B., Delorey, T., Bell, G.W., Thompson, D.A., Cuomo, C.A., and Rao, R.P. (2016). Members of the Zinc Cluster Factor Family Alters Virulence in Candida albicans. Genetics Early Online Virtually all humans are colonized with Candida albicans However, in immunocompromised individuals this benign commensal organism becomes a serious, life-threatening pathogen. Here, we describe and analyze the regulatory networks that modulate innate responses in the host niches. We identified Zcf15 and Zcf29, two Zinc Cluster transcription Factors (ZCF) that are required for C. albicans virulence. Previous sequence analysis of clinical C. albicans isolates from immunocompromised patients indicates that both ZCF genes diverged during clonal evolution. Using in vivo animal models, ex vivo cell culture methods, and in vitro sensitivity assays, we demonstrate that knockout mutants of both ZCF15 and ZCF29 are hypersensitive to reactive oxygen species (ROS), suggesting they help neutralize the host derived ROS produced by phagocytes, as well as establish a sustained infection in vivo Transcriptomic analysis of mutants under resting conditions where cells were not experiencing oxidative stress revealed a large network that control macro and micronutrient homeostasis, which likely contributes to overall pathogen fitness in host niches. Under oxidative stress both transcription factors regulate a separate set of genes involved detoxification of ROS and down regulate ribosome biogenesis. These results are further confirmed by whole genome location analysis, which reveals vastly different binding partners for each TF before and after oxidative stress. Furthermore the absence of a dominant binding motif likely facilitates their mobility and supports the notion they represent a recent expansion of the ZCF family in the pathogenic Candida species. Our analyses provide a framework for understanding new aspects of the interface between C. albicans and host defense response, and extends our understanding of how complex cell behaviors are linked to the evolution of transcription factors. Full Text

Jacox, L., Chen, J., Rothman, A., Lathrop-Marshall, H., and Sive, H. (2016). Formation of a "Pre-mouth Array" from the Extreme Anterior Domain Is Directed by Neural Crest and Wnt/PCP Signaling. Cell reports[Epub ahead of print] The mouth arises from the extreme anterior domain (EAD), a region where the ectoderm and endoderm are directly juxtaposed. Here, we identify a "pre-mouth array" in Xenopus that forms soon after the cranial neural crest has migrated to lie on either side of the EAD. Initially, EAD ectoderm comprises a wide and short epithelial mass that becomes narrow and tall with cells and nuclei changing shape, a characteristic of convergent extension. The resulting two rows of cells-the pre-mouth array-later split down the midline to surround the mouth opening. Neural crest is essential for convergent extension and likely signals to the EAD through the Wnt/planar cell polarity (PCP) pathway. Fzl7 receptor is locally required in EAD ectoderm, while Wnt11 ligand is required more globally. Indeed, heterologous cells expressing Wnt11 can elicit EAD convergent extension. The study reveals a precise cellular mechanism that positions and contributes to the future mouth. Full Text

Jahan, A.S., Lestra, M., Swee, L.K., Fan, Y., Lamers, M.M., Tafesse, F.G., Theile, C.S., Spooner, E., Bruzzone, R., Ploegh, H.L., and Sumana Sanyal (2016). Usp12 stabilizes the T-cell receptor complex at the cell surface during signaling. Proceedings of the National Academy of Sciences of the United States of America [Epub ahead of print]. Posttranslational modifications are central to the spatial and temporal regulation of protein function. Among others, phosphorylation and ubiquitylation are known to regulate proximal T-cell receptor (TCR) signaling. Here we used a systematic and unbiased approach to uncover deubiquitylating enzymes (DUBs) that participate during TCR signaling in primary mouse T lymphocytes. Using a C-terminally modified vinyl methyl ester variant of ubiquitin (HA-Ub-VME), we captured DUBs that are differentially recruited to the cytosol on TCR activation. We identified ubiquitin-specific peptidase (Usp) 12 and Usp46, which had not been previously described in this pathway. Stimulation with anti-CD3 resulted in phosphorylation and time-dependent translocation of Usp12 from the nucleus to the cytosol. Usp12-/- Jurkat cells displayed defective NFkappaB, NFAT, and MAPK activities owing to attenuated surface expression of TCR, which were rescued on reconstitution of wild type Usp12. Proximity-based labeling with BirA-Usp12 revealed several TCR adaptor proteins acting as interactors in stimulated cells, of which LAT and Trat1 displayed reduced expression in Usp12-/- cells. We demonstrate that Usp12 deubiquitylates and prevents lysosomal degradation of LAT and Trat1 to maintain the proximal TCR complex for the duration of signaling. Our approach benefits from the use of activity-based probes in primary cells without any previous genome modification, and underscores the importance of ubiquitin-mediated regulation to refine signaling cascades. Full Text

Johnston2, I., and Williams, B. (2016). The Shrinking Mitochondrion. Scientist 30, 22-23.Full Text

Johnston, I.G., and Williams, B.P. (2016). Evolutionary Inference across Eukaryotes Identifies Specific Pressures Favoring Mitochondrial Gene Retention. Cell systems 2, 101-111 .Since their endosymbiotic origin, mitochondria have lost most of their genes. Although many selective mechanisms underlying the evolution of mitochondrial genomes have been proposed, a data-driven exploration of these hypotheses is lacking, and a quantitatively supported consensus remains absent. We developed HyperTraPS, a methodology coupling stochastic modeling with Bayesian inference, to identify the ordering of evolutionary events and suggest their causes. Using 2015 complete mitochondrial genomes, we inferred evolutionary trajectories of mtDNA gene loss across the eukaryotic tree of life. We find that proteins comprising the structural cores of the electron transport chain are preferentially encoded within mitochondrial genomes across eukaryotes. A combination of high GC content and high protein hydrophobicity is required to explain patterns of mtDNA gene retention; a model that accounts for these selective pressures can also predict the success of artificial gene transfer experiments in vivo. This work provides a general method for data-driven inference of the ordering of evolutionary and progressive events, here identifying the distinct features shaping mitochondrial genomes of present-day species. Full Text

Joshi, M., Anselm, I., Shi, J., Bale, T.A., Towne, M., Schmitz-Abe, K., Crowley, L., Giani, F.C., Kazerounian, S., Markianos, K., et al. (2016). Mutations in the substrate binding glycine-rich loop of the mitochondrial processing peptidase-alpha protein (PMPCA) cause a severe mitochondrial disease. Cold Spring Harbor molecular case studies 2, a000786.We describe a large Lebanese family with two affected members, a young female proband and her male cousin, who had multisystem involvement including profound global developmental delay, severe hypotonia and weakness, respiratory insufficiency, blindness, and lactic acidemia-findings consistent with an underlying mitochondrial disorder. Whole-exome sequencing was performed on DNA from the proband and both parents. The proband and her cousin carried compound heterozygous mutations in the PMPCA gene that encodes for alpha-mitochondrial processing peptidase (alpha-MPP), a protein likely involved in the processing of mitochondrial proteins. The variants were located close to and postulated to affect the substrate binding glycine-rich loop of the alpha-MPP protein. Functional assays including immunofluorescence and western blot analysis on patient's fibroblasts revealed that these variants reduced alpha-MPP levels and impaired frataxin production and processing. We further determined that those defects could be rescued through the expression of exogenous wild-type PMPCA cDNA. Our findings link defective alpha-MPP protein to a severe mitochondrial disease. Full Text

Kaufman, C.K., Mosimann, C., Fan, Z.P., Yang, S., Thomas, A.J., Ablain, J., Tan, J.L., Fogley, R.D., van Rooijen, E., Hagedorn, E.J.,et al, Richard A Young.,and Leonard I. Zonl. (2016). A zebrafish melanoma model reveals emergence of neural crest identity during melanoma initiation. Science 351, aad2197.The "cancerized field" concept posits that cancer-prone cells in a given tissue share an oncogenic mutation, but only discreet clones within the field initiate tumors. Most benign nevi carry oncogenic BRAF(V600E) mutations but rarely become melanoma. The zebrafish crestin gene is expressed embryonically in neural crest progenitors (NCPs) and specifically reexpressed in melanoma. Live imaging of transgenic zebrafish crestin reporters shows that within a cancerized field (BRAF(V600E)-mutant; p53-deficient), a single melanocyte reactivates the NCP state, revealing a fate change at melanoma initiation in this model. NCP transcription factors, including sox10, regulate crestin expression. Forced sox10 overexpression in melanocytes accelerated melanoma formation, which is consistent with activation of NCP genes and super-enhancers leading to melanoma. Our work highlights NCP state reemergence as a key event in melanoma initiation. Full Text

Kern, D.M., Nicholls, P.K., Page, D.C., and Cheeseman, I.M. (2016). A mitotic SKAP isoform regulates spindle positioning at astral microtubule plus ends. The Journal of cell biology[Epub ahead of print] The Astrin/SKAP complex plays important roles in mitotic chromosome alignment and centrosome integrity, but previous work found conflicting results for SKAP function. Here, we demonstrate that SKAP is expressed as two distinct isoforms in mammals: a longer, testis-specific isoform that was used for the previous studies in mitotic cells and a novel, shorter mitotic isoform. Unlike the long isoform, short SKAP rescues SKAP depletion in mitosis and displays robust microtubule plus-end tracking, including localization to astral microtubules. Eliminating SKAP microtubule binding results in severe chromosome segregation defects. In contrast, SKAP mutants specifically defective for plus-end tracking facilitate proper chromosome segregation but display spindle positioning defects. Cells lacking SKAP plus-end tracking have reduced Clasp1 localization at microtubule plus ends and display increased lateral microtubule contacts with the cell cortex, which we propose results in unbalanced dynein-dependent cortical pulling forces. Our work reveals an unappreciated role for the Astrin/SKAP complex as an astral microtubule mediator of mitotic spindle positioning. Full Text

Klionsky, D.J., Sabatini, DM., et al. (2016). Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition). Autophagy 12, 1-222. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. Full Text

Klosinska, M., Picard, C.L., and Gehring, M. (2016). Conserved imprinting associated with unique epigenetic signatures in the Arabidopsis genus. Nature plants 2, 16145. In plants, imprinted gene expression occurs in endosperm seed tissue and is sometimes associated with differential DNA methylation between maternal and paternal alleles1. Imprinting is theorized to have been selected for because of conflict between parental genomes in offspring2, but most studies of imprinting have been conducted in Arabidopsis thaliana, an inbred primarily self-fertilizing species that should have limited parental conflict. We examined embryo and endosperm allele-specific expression and DNA methylation genome-wide in the wild outcrossing species Arabidopsis lyrata. Here we show that the majority of A. lyrata imprinted genes also exhibit parentally biased expression in A. thaliana, suggesting that there is evolutionary conservation in gene imprinting. Surprisingly, we discovered substantial interspecies differences in methylation features associated with paternally expressed imprinted genes (PEGs). Unlike in A. thaliana, the maternal allele of many A. lyrata PEGs was hypermethylated in the CHG context. Increased maternal allele CHG methylation was associated with increased expression bias in favour of the paternal allele. We propose that CHG methylation maintains or reinforces repression of maternal alleles of PEGs. These data suggest that the genes subject to imprinting are largely conserved, but there is flexibility in the epigenetic mechanisms employed between closely related species to maintain monoallelic expression. This supports the idea that imprinting of specific genes is a functional phenomenon, and not simply a byproduct of seed epigenomic reprogramming. Full Text

Knudsen, E.S., Balaji, U., Freinkman, E., McCue, P., and Witkiewicz, A.K. (2016). Unique metabolic features of pancreatic cancer stroma: relevance to the tumor compartment, prognosis, and invasive potential. Oncotarget 10.18632/oncotarget.11893 Pancreatic ductal adenocarcinoma (PDAC) has a dismal prognosis. The aggressiveness and therapeutic recalcitrance of this malignancy has been attributed to multiple factors including the influence of an active desmoplastic stroma. How the stromal microenvironment of PDAC contributes to the fatal nature of this disease is not well defined. In the analysis of clinical specimens, we observed diverse expression of the hypoxic marker carbonic anhydrase IX and the lactate transporter MCT4 in the stromal compartment. These stromal features were associated with the epithelial to mesenchymal phenotype in PDAC tumor cells, and with shorter patient survival. Cultured cancer-associated fibroblasts (CAFs) derived from primary PDAC exhibited a high basal level of hypoxia inducible factor 1a (HIF1alpha) that was both required and sufficient to modulate the expression of MCT4. This event was associated with increased transcription and protein synthesis of HIF1alpha in CAFs relative to PDAC cell lines, while surprisingly the protein turnover rate was equivalent. CAFs utilized glucose predominantly for glycolytic intermediates, whereas glutamine was the preferred metabolite for the TCA cycle. Unlike PDAC cell lines, CAFs were resistant to glucose withdrawal but sensitive to glutamine depletion. Consistent with the lack of reliance on glucose, CAFs could survive the acute depletion of MCT4. In co-culture and xenograft studies CAFs stimulated the invasive potential and metastatic spread of PDAC cell lines through a mechanism dependent on HIF1alpha and MCT4. Together, these data indicate that stromal metabolic features influence PDAC tumor cells to promote invasiveness and metastatic potential and associate with poor outcome in patients with PDAC. Full Text

Kotynkova, K., Su, K.C., West, S.C., and Petronczki, M. (2016). Plasma Membrane Association but Not Midzone Recruitment of RhoGEF ECT2 Is Essential for Cytokinesis. Cell reports 17, 2672-2686.Cytokinesis, the final step of cell division, begins with the formation of a cleavage furrow. How the mitotic spindle specifies the furrow at the equator in animal cells remains unknown. Current models propose that the concentration of the RhoGEF ECT2 at the spindle midzone and the equatorial plasma membrane directs furrow formation. Using chemical genetic and optogenetic tools, we demonstrate that the association of ECT2 with the plasma membrane during anaphase is required and sufficient for cytokinesis. Local membrane targeting of ECT2 leads to unilateral furrowing, highlighting the importance of local ECT2 activity. ECT2 mutations that prevent centralspindlin binding compromise concentration of ECT2 at the midzone and equatorial membrane but sustain cytokinesis. While the association of ECT2 with the plasma membrane is essential for cytokinesis, our data suggest that ECT2 recruitment to the spindle midzone is insufficient to account for equatorial furrowing and may act redundantly with yet-uncharacterized signals. Full Text

Kumar, P.V., Bardhan, N.M., Chen, G.Y., Li, Z.Y., Belcher, A.M., and Grossman, J.C. (2016). New insights into the thermal reduction of graphene oxide: Impact of oxygen clustering. Carbon 100, 90-98.Graphene has attracted interest for a number of applications ranging from electronics, optoelectronics to membrane-based technologies. The thermal reduction of chemically exfoliated graphene oxide (GO) sheets represents an important step for large-scale, solution-based graphene synthesis. Therefore, understanding the reduction process and being able to provide new handles to control the resulting sheet properties is highly desirable. Using atomistic calculations combined with experiments, we study and demonstrate the impact of one such new handle - oxygen clustering on the graphene basal plane - on the structural and electrical properties of reduced GO (rGO) structures. Our calculations reveal that the number of oxygen and carbon atoms removed from the graphene plane during reduction can be tuned depending on the degree of oxygen clustering, without altering the reduction temperature. Further, we demonstrate that rGO thin films with improved sheet resistance (up to 2-fold smaller) can be obtained by facilitating oxygen clustering prior to reduction. Overall, our results highlight that oxygen clustering serves as a useful handle in controlling the structural and electrical properties of the resulting rGO structures, and could be potentially useful toward the synthesis of electrodes, graphene quantum dots and for different graphene-based thin film applications. (C) 2015 Elsevier Ltd. All rights reserved. Full Text

Labbe, S.M., Mouchiroud, M., Caron, A., Secco, B., Freinkman, E., Lamoureux, G., Gelinas, Y., Lecomte, R., Bosse, Y., Chimin, P., et al. (2016). mTORC1 is Required for Brown Adipose Tissue Recruitment and Metabolic Adaptation to Cold. Sci Rep 6, 37223.In response to cold, brown adipose tissue (BAT) increases its metabolic rate and expands its mass to produce heat required for survival, a process known as BAT recruitment. The mechanistic target of rapamycin complex 1 (mTORC1) controls metabolism, cell growth and proliferation, but its role in regulating BAT recruitment in response to chronic cold stimulation is unknown. Here, we show that cold activates mTORC1 in BAT, an effect that depends on the sympathetic nervous system. Adipocyte-specific mTORC1 loss in mice completely blocks cold-induced BAT expansion and severely impairs mitochondrial biogenesis. Accordingly, mTORC1 loss reduces oxygen consumption and causes a severe defect in BAT oxidative metabolism upon cold exposure. Using in vivo metabolic imaging, metabolomics and transcriptomics, we show that mTORC1 deletion impairs glucose and lipid oxidation, an effect linked to a defect in tricarboxylic acid (TCA) cycle activity. These analyses also reveal a severe defect in nucleotide synthesis in the absence of mTORC1. Overall, these findings demonstrate an essential role for mTORC1 in the regulation of BAT recruitment and metabolism in response to cold. Full Text

Landgraf, D., Huh, D., Hallacli, E., and Lindquist, S. (2016). Scarless Gene Tagging with One-Step Transformation and Two-Step Selection in Saccharomyces cerevisiae and Schizosaccharomyces pombe. PloS one 11, e0163950.Gene tagging with fluorescent proteins is commonly applied to investigate the localization and dynamics of proteins in their cellular environment. Ideally, a fluorescent tag is genetically inserted at the endogenous locus at the N- or C- terminus of the gene of interest without disrupting regulatory sequences including the 5' and 3' untranslated region (UTR) and without introducing any extraneous unwanted "scar" sequences, which may create unpredictable transcriptional or translational effects. We present a reliable, low-cost, and highly efficient method for the construction of such scarless C-terminal and N-terminal fusions with fluorescent proteins in yeast. The method relies on sequential positive and negative selection and uses an integration cassette with long flanking regions, which is assembled by two-step PCR, to increase the homologous recombination frequency. The method also enables scarless tagging of essential genes with no need for a complementing plasmid. To further ease high-throughput strain construction, we have computationally automated design of the primers, applied the primer design code to all open reading frames (ORFs) of the budding yeast Saccharomyces cerevisiae (S. cerevisiae) and the fission yeast Schizosaccharomyces pombe (S. pombe), and provide here the computed sequences. To illustrate the scarless N- and C-terminal gene tagging methods in S. cerevisiae, we tagged various genes including the E3 ubiquitin ligase RSP5, the proteasome subunit PRE1, and the eleven Rab GTPases with yeast codon-optimized mNeonGreen or mCherry; several of these represent essential genes. We also implemented the scarless C-terminal gene tagging method in the distantly related organism S. pombe using kanMX6 and HSV1tk as positive and negative selection markers, respectively, as well as ura4. The scarless gene tagging methods presented here are widely applicable to visualize and investigate the functional roles of proteins in living cells. Full Text

Lesch, B.J., Silber, S.J., McCarrey, J.R., and Page, D.C. (2016). Parallel evolution of male germline epigenetic poising and somatic development in animals. Nature genetics [Epub ahead of print] Changes in gene regulation frequently underlie changes in morphology during evolution, and differences in chromatin state have been linked with changes in anatomical structure and gene expression across evolutionary time. Here we assess the relationship between evolution of chromatin state in germ cells and evolution of gene regulatory programs governing somatic development. We examined the poised (H3K4me3/H3K27me3 bivalent) epigenetic state in male germ cells from five mammalian and one avian species. We find that core genes poised in germ cells from multiple amniote species are ancient regulators of morphogenesis that sit at the top of transcriptional hierarchies controlling somatic tissue development, whereas genes that gain poising in germ cells from individual species act downstream of core poised genes during development in a species-specific fashion. We propose that critical regulators of animal development gained an epigenetically privileged state in germ cells, manifested in amniotes by H3K4me3/H3K27me3 poising, early in metazoan evolution. Full Text

Levsh, O., Chiang, Y.C., Tung, C.F., Noel, J.P., Wang, Y., and Weng, J.K. (2016). Dynamic Conformational States Dictate Selectivity toward the Native Substrate in a Substrate-Permissive Acyltransferase. Biochemistry Article ASAP. Hydroxycinnamoyl-CoA:shikimate hydroxycinnamoyltransferase (HCT) is an essential acyltransferase that mediates flux through plant phenylpropanoid metabolism by catalyzing a reaction between p-coumaroyl-CoA and shikimate, yet it also exhibits broad substrate permissiveness in vitro. How do enzymes like HCT avoid functional derailment by cellular metabolites that qualify as non-native substrates? Here, we combine X-ray crystallography and molecular dynamics to reveal distinct dynamic modes of HCT under native and non-native catalysis. We find that essential electrostatic and hydrogen-bonding interactions between the ligand and active site residues, permitted by active site plasticity, are elicited more effectively by shikimate than by other non-native substrates. This work provides a structural basis for how dynamic conformational states of HCT favor native over non-native catalysis by reducing the number of futile encounters between the enzyme and shikimate. Full Text.

Li2, H.J., Shi, J.H., Huang, N.J., Pishesha, N., Natarajan, A., Eng, J.C., and Lodish, H.F. (2016). Efficient CRISPR-Cas9 mediated gene disruption in primary erythroid progenitor cells. Haematologica 101, E216-E219.Here we report a high-efficiency method for CRISPR-Cas9 mediated gene disruption in isolated primary erythroid progenitor cells. We use this method to generate the novel result that Lmna is required in terminal erythroid differentiation. Full Text

Li, L., Park, E., Ling, J., Ingram, J., Ploegh, H., and Rapoport, T.A. (2016). Crystal structure of a substrate-engaged SecY protein-translocation channel. Nature [Epub ahead of print] Hydrophobic signal sequences target secretory polypeptides to a protein-conducting channel formed by a heterotrimeric membrane protein complex, the prokaryotic SecY or eukaryotic Sec61 complex. How signal sequences are recognized is poorly understood, particularly because they are diverse in sequence and length. Structures of the inactive channel show that the largest subunit, SecY or Sec61alpha, consists of two halves that form an hourglass-shaped pore with a constriction in the middle of the membrane and a lateral gate that faces lipid. The cytoplasmic funnel is empty, while the extracellular funnel is filled with a plug domain. In bacteria, the SecY channel associates with the translating ribosome in co-translational translocation, and with the SecA ATPase in post-translational translocation. How a translocating polypeptide inserts into the channel is uncertain, as cryo-electron microscopy structures of the active channel have a relatively low resolution (~10 A) or are of insufficient quality. Here we report a crystal structure of the active channel, assembled from SecY complex, the SecA ATPase, and a segment of a secretory protein fused into SecA. The translocating protein segment inserts into the channel as a loop, displacing the plug domain. The hydrophobic core of the signal sequence forms a helix that sits in a groove outside the lateral gate, while the following polypeptide segment intercalates into the gate. The carboxy (C)-terminal section of the polypeptide loop is located in the channel, surrounded by residues of the pore ring. Thus, during translocation, the hydrophobic segments of signal sequences, and probably bilayer-spanning domains of nascent membrane proteins, exit the lateral gate and dock at a specific site that faces the lipid phase. Full Text

Li3, Y., Muffat, J., Omer, A., Bosch, I., Lancaster, M.A., Sur, M., Gehrke, L., Knoblich, J.A., and Jaenisch, R. (2016). Induction of Expansion and Folding in Human Cerebral Organoids. Cell stem cell [Epub ahead of print] An expansion of the cerebral neocortex is thought to be the foundation for the unique intellectual abilities of humans. It has been suggested that an increase in the proliferative potential of neural progenitors (NPs) underlies the expansion of the cortex and its convoluted appearance. Here we show that increasing NP proliferation induces expansion and folding in an in vitro model of human corticogenesis. Deletion of PTEN stimulates proliferation and generates significantly larger and substantially folded cerebral organoids. This genetic modification allows sustained cell cycle re-entry, expansion of the progenitor population, and delayed neuronal differentiation, all key features of the developing human cortex. In contrast, Pten deletion in mouse organoids does not lead to folding. Finally, we utilized the expanded cerebral organoids to show that infection with Zika virus impairs cortical growth and folding. Our study provides new insights into the mechanisms regulating the structure and organization of the human cortex. Full Text

Liu, X.S., Wu, H., Ji, X., Stelzer, Y., Wu, X., Czauderna, S., Shu, J., Dadon, D., Young, R.A., and Jaenisch, R. (2016). Editing DNA Methylation in the Mammalian Genome. Cell 167, 233-247.e217.Mammalian DNA methylation is a critical epigenetic mechanism orchestrating gene expression networks in many biological processes. However, investigation of the functions of specific methylation events remains challenging. Here, we demonstrate that fusion of Tet1 or Dnmt3a with a catalytically inactive Cas9 (dCas9) enables targeted DNA methylation editing. Targeting of the dCas9-Tet1 or -Dnmt3a fusion protein to methylated or unmethylated promoter sequences caused activation or silencing, respectively, of an endogenous reporter. Targeted demethylation of the BDNF promoter IV or the MyoD distal enhancer by dCas9-Tet1 induced BDNF expression in post-mitotic neurons or activated MyoD facilitating reprogramming of fibroblasts into myoblasts, respectively. Targeted de novo methylation of a CTCF loop anchor site by dCas9-Dnmt3a blocked CTCF binding and interfered with DNA looping, causing altered gene expression in the neighboring loop. Finally, we show that these tools can edit DNA methylation in mice, demonstrating their wide utility for functional studies of epigenetic regulation. Full Text

Lohcharoenkal, W., Harada, M., Loven, J., Meisgen, F., Landen, N.X., Zhang, L., Lapins, J., Mahapatra, K.D., Shi, H., Nissinen, L., et al. (2016). MicroRNA-203 inversely correlates with differentiation grade, targets c-MYC and functions as a tumor suppressor in cSCC. The Journal of investigative dermatology.[Epub ahead of print] Cutaneous squamous cell carcinoma (cSCC) is the second most common cancer and a leading cause of cancer mortality among solid organ transplant (SOT) recipients. MicroRNAs are short RNAs that regulate gene expression and cellular functions. Here we demonstrate a negative correlation between miR-203 expression and the differentiation grade of cSCC. Functionally, miR-203 suppressed cell proliferation, cell motility and angiogenesis-inducing capacity of cSCC cells in vitro and reduced xenograft tumor volume and angiogenesis in vivo. Transcriptomic analysis of cSCC cells with ectopic overexpression of miR-203 revealed dramatic changes in gene networks related to cell cycle and proliferation. Transcription factor enrichment analysis identified c-MYC, as a hub of miR-203-induced transcriptomic changes in SCC. We identified c-MYC as a direct target of miR-203. Highlighting the importance of c-MYC within the miR-203-regulated gene network, overexpression of c-MYC in rescue experiments reversed miR-203-induced growth arrest in cSCC. Together, miR-203 acts as a tumor suppressor in cSCC and its low expression can be a marker for poorly differentiated tumors. Restoration of miR-203 expression may provide therapeutic benefit particularly in poorly differentiated cSCC. Full Text

Lu, A., Li, Y., Schmidt, F.I., Yin, Q., Chen, S., Fu, T.M., Tong, A.B., Ploegh, H.L., Mao, Y., and Wu, H. (2016). Molecular basis of caspase-1 polymerization and its inhibition by a new capping mechanism. Nature structural & molecular biology [Epub ahead of print]. Inflammasomes are cytosolic caspase-1-activation complexes that sense intrinsic and extrinsic danger signals, and trigger inflammatory responses and pyroptotic cell death. Homotypic interactions among Pyrin domains and caspase recruitment domains (CARDs) in inflammasome-complex components mediate oligomerization into filamentous assemblies. Several cytosolic proteins consisting of only interaction domains exert inhibitory effects on inflammasome assembly. In this study, we determined the structure of the human caspase-1 CARD domain (caspase-1CARD) filament by cryo-electron microscopy and investigated the biophysical properties of two caspase-1-like CARD-only proteins: human inhibitor of CARD (INCA or CARD17) and ICEBERG (CARD18). Our results reveal that INCA caps caspase-1 filaments, thereby exerting potent inhibition with low-nanomolar Ki on caspase-1CARD polymerization in vitro and inflammasome activation in cells. Whereas caspase-1CARD uses six complementary surfaces of three types for filament assembly, INCA is defective in two of the six interfaces and thus terminates the caspase-1 filament. Full Text

Ly, P., Teitz, L.S., Kim, D.H., Shoshani, O., Skaletsky, H., Fachinetti, D., Page, D.C., and Cleveland, D.W. (2016). Selective Y centromere inactivation triggers chromosome shattering in micronuclei and repair by non-homologous end joining. Nature cell biology[Epub ahead of print] Chromosome missegregation into a micronucleus can cause complex and localized genomic rearrangements known as chromothripsis, but the underlying mechanisms remain unresolved. Here we developed an inducible Y centromere-selective inactivation strategy by exploiting a CENP-A/histone H3 chimaera to directly examine the fate of missegregated chromosomes in otherwise diploid human cells. Using this approach, we identified a temporal cascade of events that are initiated following centromere inactivation involving chromosome missegregation, fragmentation, and re-ligation that span three consecutive cell cycles. Following centromere inactivation, a micronucleus harbouring the Y chromosome is formed in the first cell cycle. Chromosome shattering, producing up to 53 dispersed fragments from a single chromosome, is triggered by premature micronuclear condensation prior to or during mitotic entry of the second cycle. Lastly, canonical non-homologous end joining (NHEJ), but not homology-dependent repair, is shown to facilitate re-ligation of chromosomal fragments in the third cycle. Thus, initial errors in cell division can provoke further genomic instability through fragmentation of micronuclear DNAs coupled to NHEJ-mediated reassembly in the subsequent interphase. Full Text

Mallick, S., Li, H., Lipson, M., Mathieson, I., Gymrek, M., Racimo, F., Zhao, M., Chennagiri, N., Nordenfelt, S., Tandon, A., et al. (2016). The Simons Genome Diversity Project: 300 genomes from 142 diverse populations. Nature [Epub ahead of print]. Here we report the Simons Genome Diversity Project data set: high quality genomes from 300 individuals from 142 diverse populations. These genomes include at least 5.8 million base pairs that are not present in the human reference genome. Our analysis reveals key features of the landscape of human genome variation, including that the rate of accumulation of mutations has accelerated by about 5% in non-Africans compared to Africans since divergence. We show that the ancestors of some pairs of present-day human populations were substantially separated by 100,000 years ago, well before the archaeologically attested onset of behavioural modernity. We also demonstrate that indigenous Australians, New Guineans and Andamanese do not derive substantial ancestry from an early dispersal of modern humans; instead, their modern human ancestry is consistent with coming from the same source as that of other non-Africans. Full Text

Mayers, J.R., Torrence, M.E., Danai, L.V., Papagiannakopoulos, T., Davidson, S.M., Bauer, M.R., Lau, A.N., Ji, B.W., Dixit, P.D., Hosios, A.M., Elizaveta Freinkman, et al. (2016). Tissue of origin dictates branched-chain amino acid metabolism in mutant Kras-driven cancers. Science 353, 1161-1165.Tumor genetics guides patient selection for many new therapies, and cell culture studies have demonstrated that specific mutations can promote metabolic phenotypes. However, whether tissue context defines cancer dependence on specific metabolic pathways is unknown. Kras activation and Trp53 deletion in the pancreas or the lung result in pancreatic ductal adenocarinoma (PDAC) or non-small cell lung carcinoma (NSCLC), respectively, but despite the same initiating events, these tumors use branched-chain amino acids (BCAAs) differently. NSCLC tumors incorporate free BCAAs into tissue protein and use BCAAs as a nitrogen source, whereas PDAC tumors have decreased BCAA uptake. These differences are reflected in expression levels of BCAA catabolic enzymes in both mice and humans. Loss of Bcat1 and Bcat2, the enzymes responsible for BCAA use, impairs NSCLC tumor formation, but these enzymes are not required for PDAC tumor formation, arguing that tissue of origin is an important determinant of how cancers satisfy their metabolic requirements. Full Text

Mesin, L., Ersching, J., and Victora, G.D. (2016). Germinal Center B Cell Dynamics. Immunity 45, 471-482.Germinal centers (GCs) are the site of antibody diversification and affinity maturation and as such are vitally important for humoral immunity. The study of GC biology has undergone a renaissance in the past 10 years, with a succession of findings that have transformed our understanding of the cellular dynamics of affinity maturation. In this review, we discuss recent developments in the field, with special emphasis on how GC cellular and clonal dynamics shape antibody affinity and diversity during the immune response. Full Text

Muffat2, J., Li, Y., and Jaenisch, R. (2016). CNS disease models with human pluripotent stem cells in the CRISPR age. Current opinion in cell biology 43, 96-103.In vitro differentiation of human pluripotent stem cells provides a systematic platform to investigate the physiological development and function of the human nervous system, as well as the etiology and consequence when these processes go awry. Recent development in three-dimensional (3D) organotypic culture systems allows modeling of the complex structure formation of the human CNS, and the intricate interactions between various resident neuronal and glial cell types. Combined with an ever-expanding genome editing and regulation toolkit such as CRISPR/Cas9, it is now a possibility to study human neurological disease in the relevant molecular, cellular and anatomical context. In this article, we review recent progress in 3D neural culture and the implications for disease modeling. Full Text

Muffat, J., Li, Y., Yuan, B., Mitalipova, M., Omer, A., Corcoran, S., Bakiasi, G., Tsai, L.H., Aubourg, P., Ransohoff, R.M & Rudolph Jaenisch. (2016). Efficient derivation of microglia-like cells from human pluripotent stem cells. Nature Medicine Advance online publication. Microglia, the only lifelong resident immune cells of the central nervous system (CNS), are highly specialized macrophages that have been recognized to have a crucial role in neurodegenerative diseases such as Alzheimer's, Parkinson's and adrenoleukodystrophy (ALD). However, in contrast to other cell types of the human CNS, bona fide microglia have not yet been derived from cultured human pluripotent stem cells. Here we establish a robust and efficient protocol for the rapid production of microglia-like cells from human (h) embryonic stem (ES) and induced pluripotent stem (iPS) cells that uses defined serum-free culture conditions. These in vitro pluripotent stem cell-derived microglia-like cells (termed pMGLs) faithfully recapitulate the expected ontogeny and characteristics of their in vivo counterparts, and they resemble primary fetal human and mouse microglia. We generated these cells from multiple disease-specific cell lines and find that pMGLs derived from an hES model of Rett syndrome are smaller than their isogenic controls. We further describe a platform to study the integration and live behavior of pMGLs in organotypic 3D cultures. This modular differentiation system allows for the study of microglia in highly defined conditions as they mature in response to developmentally relevant cues, and it provides a framework in which to study the long-term interactions of microglia residing in a tissue-like environment. Full Text

Muranen, T., Selfors, L.M., Hwang, J., Gallegos, L.L., Coloff, J.L., Thoreen, C.C., Kang, S.A., Sabatini, D.M., Mills, G.B., and Brugge, J.S. (2016). ERK and p38 MAPK Activities Determine Sensitivity to PI3K/mTOR Inhibition via Regulation of MYC and YAP. Cancer research [Epub ahead of print] Aberrant activation of the PI3K/mTOR pathway is a common feature of many cancers and an attractive target for therapy, but resistance inevitably evolves as is the case for any cancer cell-targeted therapy. In animal tumor models, chronic inhibition of PI3K/mTOR initially inhibits tumor growth, but over time, tumor cells escape inhibition. In this study, we identified a context-dependent mechanism of escape whereby tumor cells upregulated the proto-oncogene transcriptional regulators c-MYC and YAP1. This mechanism was dependent on both constitutive ERK activity as well as inhibition of the stress kinase p38. Inhibition of p38 relieved proliferation arrest and allowed upregulation of MYC and YAP through stabilization of CREB. These data provide new insights into cellular signaling mechanisms that influence resistance to PI3K/mTOR inhibitors. Furthermore, they suggest that therapies that inactivate YAP or MYC or augment p38 activity could enhance the efficacy of PI3K/mTOR inhibitors.. Full Text

Najdekr, L., Friedecky, D., Tautenhahn, R., Pluskal, T., Wang, J., Huang, Y., and Adam, T. (2016). Influence of Mass Resolving Power in Orbital Ion-Trap Mass Spectrometry-Based Metabolomics. Analytical Chemistry 88, 11429-11435.Modern separation methods in conjunction with high-resolution accurate mass (HRAM) spectrometry can provide an enormous number of features characterized by exact mass and chromatographic behavior. Higher mass resolving power usually requires longer scanning times, and thus fewer data points are acquired across the target peak. This could cause difficulties for quantification, feature detection and deconvolution. The aim of this work was to describe the influence of mass spectrometry resolving power on profiling metabolomics experiments. From metabolic databases (HMDB, LipidMaps, KEGG), a list of compounds (41 474) was compiled and potential adducts and isotopes were calculated (622 110 features). The number of distinguishable masses was calculated for up to 3840k resolution. To evaluate these models, human plasma samples were analyzed by LC-HRMS on an Orbitrap Elite hybrid mass spectrometer (Thermo Fisher Scientific, CA, USA) at resolving power settings of 15k (7.8 Hz) up to a maximum of 480k (1.2 Hz). Software XCMS 1.44, MZmine 2.13.1, and Compound Discoverer 2.0.0.303 were used for evaluation. In plasma samples, the number of detected features increased sharply up to 60k in both positive and negative mode. However, beyond these values, it either flattened out or decreased owing to technical limitations. In conclusion, the most effective mass resolving powers for profiling analyses of metabolite rich biofluids on the Orbitrap Elite were around 60 000120 000 fwhm to retrieve the highest amount of information. The region between 400-800 m/z was influenced the most by resolution. Full Text

Neill, U.S.., and Jaenisch, R. (2016). A Conversation with Rudolf Jaenisch. Developmental Dynamics 245, 698-701 Rudolf Jaenisch of the Whitehead Institute at MIT is a remarkable scientist at the center of the study of epigenetics. Jaenisch created the very first transgenic mice and did the first experiment showing that therapeutic cloning could correct a genetic defect. He also conducted the first proof of principle experiments with induced pluripotent stem (iPS) cells to correct sickle cell anemia and Parkinson disease in rodents.Full Text

Pacold, M.E., Brimacombe, K.R., Chan, S.H., Rohde, J.M., Lewis, C.A., Swier, L.J., Possemato, R., Chen, W.W., Sullivan, L.B., Fiske, B.P.,Cho S, Freinkman E, Abu-Remaileh M , Liu CM , Zhou M, Koh MJ, Chung H, et al, and Sabatini DM (2016). A PHGDH inhibitor reveals coordination of serine synthesis and one-carbon unit fate. Nature chemical biology [Epub ahead of print] Serine is both a proteinogenic amino acid and the source of one-carbon units essential for de novo purine and deoxythymidine synthesis. In the canonical pathway of glucose-derived serine synthesis, Homo sapiens phosphoglycerate dehydrogenase (PHGDH) catalyzes the first, rate-limiting step. Genetic loss of PHGDH is toxic toward PHGDH-overexpressing breast cancer cell lines even in the presence of exogenous serine. Here, we used a quantitative high-throughput screen to identify small-molecule PHGDH inhibitors. These compounds reduce the production of glucose-derived serine in cells and suppress the growth of PHGDH-dependent cancer cells in culture and in orthotopic xenograft tumors. Surprisingly, PHGDH inhibition reduced the incorporation into nucleotides of one-carbon units from glucose-derived and exogenous serine. We conclude that glycolytic serine synthesis coordinates the use of one-carbon units from endogenous and exogenous serine in nucleotide synthesis, and we suggest that one-carbon unit wasting thus may contribute to the efficacy of PHGDH inhibitors in vitro and in vivo. Full Text

Park, R.J., Wang, T., Koundakjian, D., Hultquist, J.F., Lamothe-Molina, P., Monel, B., Schumann, K., Yu, H., Krupzcak, K.M., Garcia-Beltran, W,.David M. Sabatini, et al. (2016). A genome-wide CRISPR screen identifies a restricted set of HIV host dependency factors. Nature genetics advance online publication Host proteins are essential for HIV entry and replication and can be important nonviral therapeutic targets. Large-scale RNA interference (RNAi)-based screens have identified nearly a thousand candidate host factors, but there is little agreement among studies and few factors have been validated. Here we demonstrate that a genome-wide CRISPR-based screen identifies host factors in a physiologically relevant cell system. We identify five factors, including the HIV co-receptors CD4 and CCR5, that are required for HIV infection yet are dispensable for cellular proliferation and viability. Tyrosylprotein sulfotransferase 2 (TPST2) and solute carrier family 35 member B2 (SLC35B2) function in a common pathway to sulfate CCR5 on extracellular tyrosine residues, facilitating CCR5 recognition by the HIV envelope. Activated leukocyte cell adhesion molecule (ALCAM) mediates cell aggregation, which is required for cell-to-cell HIV transmission. We validated these pathways in primary human CD4+ T cells through Cas9-mediated knockout and antibody blockade. Our findings indicate that HIV infection and replication rely on a limited set of host-dispensable genes and suggest that these pathways can be studied for therapeutic intervention. Full Text

Pattabiraman, D.R., Bierie, B., Kober, K.I., Thiru, P., Krall, J.A., Zill, C., Reinhardt, F., Tam, W.L., and Weinberg, R.A. (2016). Activation of PKA leads to mesenchymal-to-epithelial transition and loss of tumor-initiating ability. Science 351, aad3680.The epithelial-to-mesenchymal transition enables carcinoma cells to acquire malignancy-associated traits and the properties of tumor-initiating cells (TICs). TICs have emerged in recent years as important targets for cancer therapy, owing to their ability to drive clinical relapse and enable metastasis. Here, we propose a strategy to eliminate mesenchymal TICs by inducing their conversion to more epithelial counterparts that have lost tumor-initiating ability. We report that increases in intracellular levels of the second messenger, adenosine 3',5'-monophosphate, and the subsequent activation of protein kinase A (PKA) induce a mesenchymal-to-epithelial transition (MET) in mesenchymal human mammary epithelial cells. PKA activation triggers epigenetic reprogramming of TICs by the histone demethylase PHF2, which promotes their differentiation and loss of tumor-initiating ability. This study provides proof-of-principle for inducing an MET as differentiation therapy for TICs and uncovers a role for PKA in enforcing and maintaining the epithelial state. Full Text

Pincus, D. (2016). Size doesn't matter in the heat shock response. Current genetics [Epub ahead of print] Heat shock factor 1 (Hsf1) is a transcription factor that is often described as the master regulator of the heat shock response in all eukaryotes. However, due to its essentiality in yeast, Hsf1's contribution to the transcriptome under basal and heat shock conditions has never been directly determined. Using a chemical genetics approach that allowed rapid Hsf1 inactivation, my colleagues and I have recently shown that the bulk of the heat shock response is Hsf1 independent. Rather than inducing genes responsible for carrying out the various cellular processes required for adaptation to thermal stress, Hsf1 controls a dedicated set of chaperone protein genes devoted to restoring protein-folding homeostasis. The limited scope of the Hsf1 regulon belies its outsize importance in cellular fitness. Full Text

Reddien, P.W., and Tanaka, E.M. (2016). Editorial overview: Cell reprogramming, regeneration and repair. Current Opinion in Genetics & Development 40, IV-VI.Full Text

Sahakyan, A., Kim, R., Chronis, C., Sabri, S., Bonora, G., Theunissen, T.W., Kuoy, E., Langerman, J., Clark, A.T., Jaenisch, R., et al. (2016). Human Naive Pluripotent Stem Cells Model X Chromosome Dampening and X Inactivation. Cell stem cell [Epub ahead of print] Naive human embryonic stem cells (hESCs) can be derived from primed hESCs or directly from blastocysts, but their X chromosome state has remained unresolved. Here, we show that the inactive X chromosome (Xi) of primed hESCs was reactivated in naive culture conditions. Like cells of the blastocyst, the resulting naive cells contained two active X chromosomes with XIST expression and chromosome-wide transcriptional dampening and initiated XIST-mediated X inactivation upon differentiation. Both establishment of and exit from the naive state (differentiation) happened via an XIST-negative XaXa intermediate. Together, these findings identify a cell culture system for functionally exploring the two X chromosome dosage compensation processes in early human development: X dampening and X inactivation. However, remaining differences between naive hESCs and embryonic cells related to mono-allelic XIST expression and non-random X inactivation highlight the need for further culture improvement. As the naive state resets Xi abnormalities seen in primed hESCs, it may provide cells better suited for downstream applications. Full Text

SaintAndre, V., Federation, A.J., Lin, C.Y., Abraham, B.J., Reddy, J., Lee, T.I., Bradner, J.E., and Young, R.A. (2016). Models of human core transcriptional regulatory circuitries. Genome research [Epub ahead of print] A small set of core transcription factors (TFs) dominates control of the gene expression program in embryonic stem cells and other well-studied cellular models. These core TFs collectively regulate their own gene expression, thus forming an interconnected auto-regulatory loop that can be considered the core transcriptional regulatory circuitry (CRC) for that cell type. There is limited knowledge of core TFs, and thus models of core regulatory circuitry, for most cell types. We recently discovered that genes encoding known core TFs forming CRCs are driven by super-enhancers, which provides an opportunity to systematically predict CRCs in poorly studied cell types through super-enhancer mapping. Here, we use super-enhancer maps to generate CRC models for 75 human cell and tissue types. These core circuitry models should prove valuable for further investigating cell-type-specific transcriptional regulation in healthy and diseased cells. Full Text

Sanduja, S., Feng, Y., Mathis, R.A., Sokol, E.S., Reinhardt, F., Halaban, R., and Gupta, P.B. (2016). AMPK promotes tolerance to Ras pathway inhibition by activating autophagy. Oncogene advance online publication. Targeted inhibitors of oncogenic Ras (rat sarcoma viral oncogene)-Raf signaling have shown great promise in the clinic, but resistance remains a major challenge: 30% of tumors with pathway mutations do not respond to targeted inhibitors, and of the 70% that do respond, all eventually develop resistance. Before cancer cells acquire resistance, they respond to initial drug treatment either by undergoing apoptosis ('addiction') or by surviving treatment albeit with reduced growth ('tolerance'). As these drug-tolerant cells serve as a reservoir from which resistant cells eventually emerge, inhibiting the pathways that confer tolerance could potentially delay or even prevent recurrence. Here, we show that melanomas and other cancers acquire tolerance to Ras-Raf pathway inhibitors by activating autophagy, which is mediated by the cellular energy sensor AMP-activated protein kinase (AMPK). Blocking this AMPK-mediated autophagy sensitizes drug-tolerant melanomas to Ras-Raf pathway inhibitors. Conversely, activating AMPK signaling and autophagy enables melanomas that would otherwise be addicted to the Ras-Raf pathway to instead tolerate pathway inhibition. These findings identify a key mechanism of tolerance to Ras-Raf pathway inhibitors and suggest that blocking either AMPK or autophagy in combination with these targeted inhibitors could increase tumor regression and decrease the likelihood of eventual recurrence.Oncogene advance online publication, 4 April 2016; doi:10.1038/onc.2016.70. Full Text

Sanecka, A., Yoshida, N., Dougan, S.K., Jackson, J., Shastri, N., Ploegh, H., Blanchard, N., and Frickel, E.M. (2016). Transnuclear CD8 T cells specific for the immunodominant epitope Gra6 lower acute phase Toxoplasma gondii burden. Immunology[Epub ahead of print] We generated a CD8 T cell receptor transnuclear (TN) mouse specific to the Ld -restricted immunodominant epitope of GRA6 from Toxoplasma gondii as a source of cells to facilitate further investigation into the CD8 T cell mediated response against this pathogen. The TN T cells bound Ld -Gra6 tetramer and proliferated upon unspecific and peptide-specific stimulation. The TCR beta sequence of the Gra6-specific TN CD8 T cells is identical in its V- and J-region to the TCR beta harboured by a hybridoma line generated in response to Gra6 peptide. Adoptively transferred Gra6 TN CD8 T cells proliferated upon Toxoplasma infection in vivo and exhibited an activated phenotype similar to host CD8 T cells specific to Gra6. The brain of Toxoplasma-infected mice carried Gra6 TN cells already at day 8 post-infection. Both Gra6 TN mice as well as adoptively transferred Gra6 TN cells were able to significantly reduce the parasite burden in the acute phase of Toxoplasma infection. Overall, the Gra6 TN mouse represents a functional tool to study the protective and immunodominant specific CD8 T cell response to Toxoplasma both in the acute as well as the chronic phase of infection. This article is protected by copyright. All rights reserved. Full Text

Saxton2, R.A., Knockenhauer, K.E., Schwartz, T.U., and Sabatini, D.M. (2016). The apo-structure of the leucine sensor Sestrin2 is still elusive. Science signaling 9(446):ra92. Sestrin2 is a GATOR2-interacting protein that directly binds leucine and is required for the inhibition of mTORC1 under leucine deprivation, indicating that it is a leucine sensor for the mTORC1 pathway. We recently reported the structure of Sestrin2 in complex with leucine [Protein Data Bank (PDB) ID, 5DJ4] and demonstrated that mutations in the leucine-binding pocket that alter the affinity of Sestrin2 for leucine result in a corresponding change in the leucine sensitivity of mTORC1 in cells. A lower resolution structure of human Sestrin2 (PDB ID, 5CUF), which was crystallized in the absence of exogenous leucine, showed Sestrin2 to be in a nearly identical conformation as the leucine-bound structure. On the basis of this observation, it has been argued that leucine binding does not affect the conformation of Sestrin2 and that Sestrin2 may not be a sensor for leucine. We show that simple analysis of the reported "apo"-Sestrin2 structure reveals the clear presence of prominent, unmodeled electron density in the leucine-binding pocket that exactly accommodates the leucine observed in the higher resolution structure. Refining the reported apo-structure with leucine eliminated the large Fobs-Fcalc difference density at this position and improved the working and free R factors of the model. Consistent with this result, our own structure of Sestrin2 crystallized in the absence of exogenous leucine also contained electron density that is best explained by leucine. Thus, the structure of apo-Sestrin2 remains elusive. Full Text

Saxton, R.A., Chantranupong, L., Knockenhauer, K.E., Schwartz, T.U., and Sabatini, D.M. (2016). Mechanism of arginine sensing by CASTOR1 upstream of mTORC1. Nature 536, 229–233. The mechanistic Target of Rapamycin Complex 1 (mTORC1) is a major regulator of eukaryotic growth that coordinates anabolic and catabolic cellular processes with inputs such as growth factors and nutrients, including amino acids. In mammals arginine is particularly important, promoting diverse physiological effects such as immune cell activation, insulin secretion, and muscle growth, largely mediated through activation of mTORC1 (refs 4, 5, 6, 7).Arginine activates mTORC1 upstream of the Rag family of GTPases, through either the lysosomal amino acid transporter SLC38A9 or the GATOR2-interacting Cellular Arginine Sensor for mTORC1 (CASTOR1). However, the mechanism by which the mTORC1 pathway detects and transmits this arginine signal has been elusive. Here, we present the 1.8 A crystal structure of arginine-bound CASTOR1. Homodimeric CASTOR1 binds arginine at the interface of two Aspartate kinase, Chorismate mutase, TyrA (ACT) domains, enabling allosteric control of the adjacent GATOR2-binding site to trigger dissociation from GATOR2 and downstream activation of mTORC1. Our data reveal that CASTOR1 shares substantial structural homology with the lysine-binding regulatory domain of prokaryotic aspartate kinases, suggesting that the mTORC1 pathway exploited an ancient, amino-acid-dependent allosteric mechanism to acquire arginine sensitivity. Together, these results establish a structural basis for arginine sensing by the mTORC1 pathway and provide insights into the evolution of a mammalian nutrient sensor. Full Text

Schmidt2, F.I., Hanke, L., Morin, B., Brewer, R., Brusic, V., Whelan, S.P., and Ploegh, H.L. (2016). Phenotypic lentivirus screens to identify functional single domain antibodies. Nature microbiology 1, 16080.Manipulation of proteins is key in assessing their in vivo function. Although genetic ablation is straightforward, reversible and specific perturbation of protein function remains a challenge. Single domain antibody fragments, such as camelid-derived VHHs, can serve as inhibitors or activators of intracellular protein function, but functional testing of identified VHHs is laborious. To address this challenge, we have developed a lentiviral screening approach to identify VHHs that elicit a phenotype when expressed intracellularly. We identified 19 antiviral VHHs that protect human A549 cells from lethal infection with influenza A virus (IAV) or vesicular stomatitis virus (VSV), respectively. Both negative-sense RNA viruses are vulnerable to VHHs uniquely specific for their respective nucleoproteins. Antiviral VHHs prevented nuclear import of viral ribonucleoproteins or mRNA transcription, respectively, and may provide clues for novel antiviral reagents. In principle, the screening approach described here should be applicable to identify inhibitors of any pathogen or biological pathway. Full Text

Schmidt, F.I., Lu, A., Chen, J.W., Ruan, J., Tang, C., Wu, H., and Ploegh, H.L. (2016). A single domain antibody fragment that recognizes the adaptor ASC defines the role of ASC domains in inflammasome assembly. The Journal of experimental medicine [Epub ahead of print] Myeloid cells assemble inflammasomes in response to infection or cell damage; cytosolic sensors activate pro-caspase-1, indirectly for the most part, via the adaptors ASC and NLRC4. This leads to secretion of proinflammatory cytokines and pyroptosis. To explore complex formation under physiological conditions, we generated an alpaca single domain antibody, VHHASC, which specifically recognizes the CARD of human ASC via its type II interface. VHHASCnot only impairs ASCCARDinteractions in vitro,but also inhibits inflammasome activation in response to NLRP3, AIM2, and NAIP triggers when expressed in living cells, highlighting a role of ASC in all three types of inflammasomes. VHHASCleaves the Pyrin domain of ASC functional and stabilizes a filamentous intermediate of inflammasome activation. Incorporation of VHHASC-EGFP into these structures allowed the visualization of endogenous ASCPYDfilaments for the first time. These data revealed that cross-linking of ASCPYDfilaments via ASCCARDmediates the assembly of ASC foci. Full Text

Scimone, M.L., Cote, L.E., Rogers, T., and Reddien, P.W. (2016). Two FGFRL-Wnt circuits organize the planarian anteroposterior axis.eLife 2016;10.7554/eLife.12845 How positional information instructs adult tissue maintenance is poorly understood. Planarians undergo whole-body regeneration and tissue turnover, providing a model for adult positional information studies. Genes encoding secreted and transmembrane components of multiple developmental pathways are predominantly expressed in planarian muscle cells. Several of these genes regulate regional identity, consistent with muscle harboring positional information. Here, single-cell RNA-sequencing of 115 muscle cells from distinct anterior-posterior regions identified 44 regionally expressed genes, including multiple Wnt and ndk/FGF receptor-like (ndl/FGFRL) genes. Two distinct FGFRL-Wnt circuits, involving juxtaposed anterior FGFRL and posterior Wnt expression domains, controlled planarian head and trunk patterning. ndl-3 and wntP-2 inhibition expanded the trunk, forming ectopic mouths and secondary pharynges, which independently extended and ingested food. fz5/8-4 inhibition, like that of ndk and wntA, caused posterior brain expansion and ectopic eye formation. Our results suggest that FGFRL-Wnt circuits operate within a body-wide coordinate system to control adult axial positioning. Full Text

Shi, J., Yuan, B., Hu, W., and Lodish, H. JAK2 V617F stimulates proliferation of erythropoietin- dependent erythroid progenitors and delays their differentiation by activating Stat1 and other non-erythroid signaling pathways. Experimental hematology [Epub ahead of print]Our study focuses on JAK2 V617F function at the terminal erythropoiesis stage.However, activation of abnormal signaling pathways in the HSCs and early progenitorsalso disturbs early hematopoietic development. Over-expression of hyperactive EpoRR129C in early hematopoietic progenitors, inducing strong Epo signaling, indeed leads to severe leukemia . JAK2 V617F alters the expression of several genes, includingNF-E2, in early hematopoietic progenitors. Transgenic mouse models with elevated ormutated NF-E2 levels develop MPN-like phenotypes and leads to leukemictransformation . Moreover, JAK2 V617F has been shown to affect the selfrenewaland differentiation of HSCs. Therefore, JAK2 V617F ET patients orJAK2 V617F positive chronic granulocytic patients may have other defects in earlyhematopoiesis that results in erythroid progenitor cell defects. Full Text

Shorter, J., and Gitler, A.D. (2016). Susan Lee Lindquist (1949-2016). Nature 540, 40 Susan Lee Lindquist revealed the profound ramifications of a process that most scientists had overlooked: protein folding. Through elegant experiments in yeast, plants, flies and human cells, Lindquist demonstrated how this process by which proteins adopt their proper shapes fuels evolution. It can buffer the effects of genetic variation, allow new traits to emerge and enable the rapid evolution of new adaptations. Her insights have paved the way for innovative strategies to treat diseases including Alzheimer's, Parkinson's and cancer. Full Text

Shu, S., Lin, C.Y., He, H.H., Witwicki, R.M., Tabassum, D.P., Roberts, J.M., Janiszewska, M., Jin Huh, S., Liang, Y., Ryan, J., Anders L, Young RA, et al. (2016). Response and resistance to BET bromodomain inhibitors in triple-negative breast cancer. Nature . [Epub ahead of print] Triple-negative breast cancer (TNBC) is a heterogeneous and clinically aggressive disease for which there is no targeted therapy. BET bromodomain inhibitors, which have shown efficacy in several models of cancer, have not been evaluated in TNBC. These inhibitors displace BET bromodomain proteins such as BRD4 from chromatin by competing with their acetyl-lysine recognition modules, leading to inhibition of oncogenic transcriptional programs. Here we report the preferential sensitivity of TNBCs to BET bromodomain inhibition in vitro and in vivo, establishing a rationale for clinical investigation and further motivation to understand mechanisms of resistance. In paired cell lines selected for acquired resistance to BET inhibition from previously sensitive TNBCs, we failed to identify gatekeeper mutations, new driver events or drug pump activation. BET-resistant TNBC cells remain dependent on wild-type BRD4, which supports transcription and cell proliferation in a bromodomain-independent manner. Proteomic studies of resistant TNBC identify strong association with MED1 and hyper-phosphorylation of BRD4 attributable to decreased activity of PP2A, identified here as a principal BRD4 serine phosphatase. Together, these studies provide a rationale for BET inhibition in TNBC and present mechanism-based combination strategies to anticipate clinical drug resistance. Full Text

Sidik2, S.M., Huet, D., Ganesan, S.M., Huynh, M.H., Wang, T., Nasamu, A.S., Thiru, P., Saeij, J.P., Carruthers, V.B., Niles, J.C.,and Sebastian Lourido (2016). A Genome-wide CRISPR Screen in Toxoplasma Identifies Essential Apicomplexan Genes. Cell 166, 1423-1435.e1412.Apicomplexan parasites are leading causes of human and livestock diseases such as malaria and toxoplasmosis, yet most of their genes remain uncharacterized. Here, we present the first genome-wide genetic screen of an apicomplexan. We adapted CRISPR/Cas9 to assess the contribution of each gene from the parasite Toxoplasma gondii during infection of human fibroblasts. Our analysis defines approximately 200 previously uncharacterized, fitness-conferring genes unique to the phylum, from which 16 were investigated, revealing essential functions during infection of human cells. Secondary screens identify as an invasion factor the claudin-like apicomplexan microneme protein (CLAMP), which resembles mammalian tight-junction proteins and localizes to secretory organelles, making it critical to the initiation of infection. CLAMP is present throughout sequenced apicomplexan genomes and is essential during the asexual stages of the malaria parasite Plasmodium falciparum. These results provide broad-based functional information on T. gondii genes and will facilitate future approaches to expand the horizon of antiparasitic interventions. Full Text

Sidik, S.M., Hortua Triana, M.A., Paul, A.S., El Bakkouri, M., Hackett, C.G., Tran, F., Westwood, N.J., Hui, R., Zuercher, W.J., Duraisingh, M.T.,Silvia N.J. Moreno,and Sebastian Lourido. (2016). Using a Genetically Encoded Sensor to Identify Inhibitors of Toxoplasma gondii Ca2+ Signalling. The Journal of biological chemistry Papers in Press. The lifecycles of apicomplexan parasites progress in accordance with fluxes in cytosolic Ca2+. Such fluxes are necessary for events like motility and egress from host cells. We used genetically-encoded Ca2+ indicators (GCaMPs) to develop a cell-based phenotypic screen for compounds that modulate Ca2+ signalling in the model apicomplexan Toxoplasma gondii. In doing so, we took advantage of the phosphodiesterase inhibitor zaprinast, which we show acts in part through cGMP-dependent protein kinase (PKG) to raise levels of cytosolic Ca2+. We define the pool of Ca2+ regulated by PKG to be a neutral store distinct from the endoplasmic reticulum. Screening a library of 823 ATP mimetics, we identify both inhibitors and enhancers of Ca2+ signalling. Two such compounds constitute novel PKG inhibitors, and prevent zaprinast from increasing cytosolic Ca2+. The enhancers identified are capable of releasing intracellular Ca2+ stores independently of zaprinast or PKG. One of these enhancers blocks parasite egress and invasion, and shows strong antiparasitic activity against T. gondii. The same compound inhibits invasion of the most lethal malaria parasite, Plasmodium falciparum. Inhibition of Ca2+-related phenotypes in these two apicomplexan parasites suggests that depletion of intracellular Ca2+ stores by the enhancer may be an effective antiparasitic strategy. These results establish a powerful new strategy for identifying compounds that modulate the essential parasite signalling pathways regulated by Ca2+, underscoring the importance of these pathways and the therapeutic potential of their inhibition. Full Text

Signer, R.A., Qi, L., Zhao, Z., Thompson, D., Sigova, A.A., Fan, Z.P., DeMartino, G.N., Young, R.A., Sonenberg, N., and Morrison, S.J. (2016). The rate of protein synthesis in hematopoietic stem cells is limited partly by 4E-BPs. Genes & development [Epub ahead of print] Adult stem cells must limit their rate of protein synthesis, but the underlying mechanisms remain largely unexplored. Differences in protein synthesis among hematopoietic stem cells (HSCs) and progenitor cells did not correlate with differences in proteasome activity, total RNA content, mRNA content, or cell division rate. However, adult HSCs had more hypophosphorylated eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) and 4E-BP2 as compared with most other hematopoietic progenitors. Deficiency for 4E-BP1 and 4E-BP2 significantly increased global protein synthesis in HSCs, but not in other hematopoietic progenitors, and impaired their reconstituting activity, identifying a mechanism that promotes HSC maintenance by attenuating protein synthesis. Full Text

Smulan, L.J., Ding, W., Freinkman, E., Gujja, S., Edwards, Y.J., and Walker, A.K. (2016). Cholesterol-Independent SREBP-1 Maturation Is Linked to ARF1 Inactivation. Cell report 16, 1–10. Lipogenesis requires coordinated expression of genes for fatty acid, phospholipid, and triglyceride synthesis. Transcription factors, such as SREBP-1 (Sterol regulatory element binding protein), may be activated in response to feedback mechanisms linking gene activation to levels of metabolites in the pathways. SREBPs can be regulated in response to membrane cholesterol and we also found that low levels of phosphatidylcholine (a methylated phospholipid) led to SBP-1/SREBP-1 maturation in C. elegans or mammalian models. To identify additional regulatory components, we performed a targeted RNAi screen in C. elegans, finding that both lpin-1/Lipin 1 (which converts phosphatidic acid to diacylglycerol) and arf-1.2/ARF1 (a GTPase regulating Golgi function) were important for low-PC activation of SBP-1/SREBP-1. Mechanistically linking the major hits of our screen, we find that limiting PC synthesis or LPIN1 knockdown in mammalian cells reduces the levels of active GTP-bound ARF1. Thus, changes in distinct lipid ratios may converge on ARF1 to increase SBP-1/SREBP-1 activity. Full Text

Sockolosky, J.T., Dougan, M., Ingram, J.R., Ho, C.C., Kauke, M.J., Almo, S.C., Ploegh, H.L., and Garcia, K.C. (2016). Durable antitumor responses to CD47 blockade require adaptive immune stimulation. Proceedings of the National Academy of Sciences of the United States of America [Epub ahead of print] Therapeutic antitumor antibodies treat cancer by mobilizing both innate and adaptive immunity. CD47 is an antiphagocytic ligand exploited by tumor cells to blunt antibody effector functions by transmitting an inhibitory signal through its receptor signal regulatory protein alpha (SIRPalpha). Interference with the CD47-SIRPalpha interaction synergizes with tumor-specific monoclonal antibodies to eliminate human tumor xenografts by enhancing macrophage-mediated antibody-dependent cellular phagocytosis (ADCP), but synergy between CD47 blockade and ADCP has yet to be demonstrated in immunocompetent hosts. Here, we show that CD47 blockade alone or in combination with a tumor-specific antibody fails to generate antitumor immunity against syngeneic B16F10 tumors in mice. Durable tumor immunity required programmed death-ligand 1 (PD-L1) blockade in combination with an antitumor antibody, with incorporation of CD47 antagonism substantially improving response rates. Our results highlight an underappreciated contribution of the adaptive immune system to anti-CD47 adjuvant therapy and suggest that targeting both innate and adaptive immune checkpoints can potentiate the vaccinal effect of antitumor antibody therapy. Full Text

Sokol, E.S., Miller, D.H., Breggia, A., Spencer, K.C., Arendt, L.M., and Gupta, P.B. (2016). Growth of human breast tissues from patient cells in 3D hydrogel scaffolds. Breast cancer research 18 : 19. Three-dimensional (3D) cultures have proven invaluable for expanding human tissues for basic research and clinical applications. In both contexts, 3D cultures are most useful when they (1) support the outgrowth of tissues from primary human cells that have not been immortalized through extensive culture or viral infection and (2) include defined, physiologically relevant components. Here we describe a 3D culture system with both of these properties that stimulates the outgrowth of morphologically complex and hormone-responsive mammary tissues from primary human breast epithelial cells. METHODS: Primary human breast epithelial cells isolated from patient reduction mammoplasty tissues were seeded into 3D hydrogels. The hydrogel scaffolds were composed of extracellular proteins and carbohydrates present in human breast tissue and were cultured in serum-free medium containing only defined components. The physical properties of these hydrogels were determined using atomic force microscopy. Tissue growth was monitored over time using bright-field and fluorescence microscopy, and maturation was assessed using morphological metrics and by immunostaining for markers of stem cells and differentiated cell types. The hydrogel tissues were also studied by fabricating physical models from confocal images using a 3D printer. RESULTS: When seeded into these 3D hydrogels, primary human breast epithelial cells rapidly self-organized in the absence of stromal cells and within 2 weeks expanded to form mature mammary tissues. The mature tissues contained luminal, basal, and stem cells in the correct topological orientation and also exhibited the complex ductal and lobular morphologies observed in the human breast. The expanded tissues became hollow when treated with estrogen and progesterone, and with the further addition of prolactin produced lipid droplets, indicating that they were responding to hormones. Ductal branching was initiated by clusters of cells expressing putative mammary stem cell markers, which subsequently localized to the leading edges of the tissue outgrowths. Ductal elongation was preceded by leader cells that protruded from the tips of ducts and engaged with the extracellular matrix. CONCLUSIONS: These 3D hydrogel scaffolds support the growth of complex mammary tissues from primary patient-derived cells. We anticipate that this culture system will empower future studies of human mammary gland development and biology. Full Text

Soldner, F., Stelzer, Y., Shivalila, C.S., Abraham, B.J., Latourelle, J.C., Barrasa, M.I., Goldmann, J., Myers, R.H., Young, R.A., and Jaenisch, R. (2016). Parkinson-associated risk variant in distal enhancer of alpha-synuclein modulates target gene expression. Nature[Epub ahead of print] Genome-wide association studies (GWAS) have identified numerous genetic variants associated with complex diseases, but mechanistic insights are impeded by a lack of understanding of how specific risk variants functionally contribute to the underlying pathogenesis. It has been proposed that cis-acting effects of non-coding risk variants on gene expression are a major factor for phenotypic variation of complex traits and disease susceptibility. Recent genome-scale epigenetic studies have highlighted the enrichment of GWAS-identified variants in regulatory DNA elements of disease-relevant cell types. Furthermore, single nucleotide polymorphism (SNP)-specific changes in transcription factor binding are correlated with heritable alterations in chromatin state and considered a major mediator of sequence-dependent regulation of gene expression. Here we describe a novel strategy to functionally dissect the cis-acting effect of genetic risk variants in regulatory elements on gene expression by combining genome-wide epigenetic information with clustered regularly-interspaced short palindromic repeats (CRISPR)/Cas9 genome editing in human pluripotent stem cells. By generating a genetically precisely controlled experimental system, we identify a common Parkinson's disease associated risk variant in a non-coding distal enhancer element that regulates the expression of alpha-synuclein (SNCA), a key gene implicated in the pathogenesis of Parkinson's disease. Our data suggest that the transcriptional deregulation of SNCA is associated with sequence-dependent binding of the brain-specific transcription factors EMX2 and NKX6-1. This work establishes an experimental paradigm to functionally connect genetic variation with disease-relevant phenotypes. Full Text

Solis, E.J., Pandey, J.P., Zheng, X., Jin, D.X., Gupta, P.B., Airoldi, E.M., Pincus, D., and Denic, V. (2016). Defining the Essential Function of Yeast Hsf1 Reveals a Compact Transcriptional Program for Maintaining Eukaryotic Proteostasis. Molecular cell In Press, Corrected Proof Despite its eponymous association with the heat shock response, yeast heat shock factor 1 (Hsf1) is essential even at low temperatures. Here we show that engineered nuclear export of Hsf1 results in cytotoxicity associated with massive protein aggregation. Genome-wide analysis revealed that Hsf1 nuclear export immediately decreased basal transcription and mRNA expression of 18 genes, which predominately encode chaperones. Strikingly, rescuing basal expression of Hsp70 and Hsp90 chaperones enabled robust cell growth in the complete absence of Hsf1. With the exception of chaperone gene induction, the vast majority of the heat shock response was Hsf1 independent. By comparative analysis of mammalian cell lines, we found that only heat shock-induced but not basal expression of chaperones is dependent on the mammalian Hsf1 homolog (HSF1). Our work reveals that yeast chaperone gene expression is an essential housekeeping mechanism and provides a roadmap for defining the function of HSF1 as a driver of oncogenesis. Full Text

Spiegel, A., Brooks, M.W., Houshyar, S., Reinhardt, F., Ardolino, M., Fessler, E., Chen, M.B., Krall, J.A., DeCock, J., Zervantonakis, I.K., Weinberg RA, et al. (2016). Neutrophils suppress intraluminal NK-mediated tumor cell clearance and enhance extravasation of disseminated carcinoma cells. Cancer discovery [Epub ahead of print] Immune cells promote the initial metastatic dissemination of carcinoma cells from primary tumors. In contrast to their well-studied functions in the initial stages of metastasis, the specific roles of immunocytes in facilitating progression through the critical later steps of the invasion-metastasis cascade remain poorly understood. Here we define novel functions of neutrophils in promoting intraluminal survival and extravasation at sites of metastatic dissemination. We show that CD11b+/Ly6G+ neutrophils enhance metastasis formation via two distinct mechanisms. First, neutrophils inhibit natural killer cell function, which leads to a significant increase in the intraluminal survival time of tumor cells. Thereafter, neutrophils operate to facilitate extravasation of tumor cells through the secretion of IL-1beta and matrix metalloproteinases. These results identify neutrophils as key regulators of intraluminal survival and extravasation through their crosstalk with host cells and disseminating carcinoma cells. Full Text

Stelzer, Y., Wu, H., Song, Y., Shivalila, C.S., Markoulaki, S., and Jaenisch, R. (2016). Parent-of-Origin DNA Methylation Dynamics during Mouse Development. Cell reports 16, 3167-3180.Parent-specific differentially methylated regions (DMRs) are established during gametogenesis and regulate parent-specific expression of imprinted genes. Monoallelic expression of imprinted genes is essential for development, suggesting that imprints are faithfully maintained in embryos and adults. To test this hypothesis, we targeted a reporter for genomic methylation to the imprinted Dlk1-Dio3 intergenic DMR (IG-DMR) to assess the methylation of both parental alleles at single-cell resolution. Biallelic gain or loss of IG-DMR methylation occurred in a small fraction of mouse embryonic stem cells, significantly affecting developmental potency. Mice carrying the reporter in either parental allele showed striking parent-specific changes in IG-DMR methylation, causing substantial and consistent tissue- and cell-type-dependent signatures in embryos and postnatal animals. Furthermore, dynamics in DNA methylation persisted during adult neurogenesis, resulting in inter-individual diversity. This substantial cell-cell DNA methylation heterogeneity implies that dynamic DNA methylation variations in the adult may be of functional importance. Full Text

Strong, R., Miller, R.A., Antebi, A., Astle, C.M., Bogue, M., Denzel, M.S., Fernandez, E., Flurkey, K., Hamilton, K.L., Lamming, D.W, David M.Sabatini, et al. (2016). Longer lifespan in male mice treated with a weakly estrogenic agonist, an antioxidant, an alpha-glucosidase inhibitor or a Nrf2-inducer. Aging cell[Epub ahead of print] The National Institute on Aging Interventions Testing Program (ITP) evaluates agents hypothesized to increase healthy lifespan in genetically heterogeneous mice. Each compound is tested in parallel at three sites, and all results are published. We report the effects of lifelong treatment of mice with four agents not previously tested: Protandim, fish oil, ursodeoxycholic acid (UDCA) and metformin - the latter with and without rapamycin, and two drugs previously examined: 17-alpha-estradiol and nordihydroguaiaretic acid (NDGA), at doses greater and less than used previously. 17-alpha-estradiol at a threefold higher dose robustly extended both median and maximal lifespan, but still only in males. The male-specific extension of median lifespan by NDGA was replicated at the original dose, and using doses threefold lower and higher. The effects of NDGA were dose dependent and male specific but without an effect on maximal lifespan. Protandim, a mixture of botanical extracts that activate Nrf2, extended median lifespan in males only. Metformin alone, at a dose of 0.1% in the diet, did not significantly extend lifespan. Metformin (0.1%) combined with rapamycin (14 ppm) robustly extended lifespan, suggestive of an added benefit, based on historical comparison with earlier studies of rapamycin given alone. The alpha-glucosidase inhibitor, acarbose, at a concentration previously tested (1000 ppm), significantly increased median longevity in males and 90th percentile lifespan in both sexes, even when treatment was started at 16 months. Neither fish oil nor UDCA extended lifespan. These results underscore the reproducibility of ITP longevity studies and illustrate the importance of identifying optimal doses in lifespan studies. Full Text

Su, K.C., Barry, Z., Schweizer, N., Maiato, H., Bathe, M., and Cheeseman, I.M. (2016). A Regulatory Switch Alters Chromosome Motions at the Metaphase-to-Anaphase Transition. Cell reports 17, 1728-1738.To achieve chromosome segregation during mitosis, sister chromatids must undergo a dramatic change in their behavior to switch from balanced oscillations at the metaphase plate to directed poleward motion during anaphase. However, the factors that alter chromosome behavior at the metaphase-to-anaphase transition remain incompletely understood. Here, we perform time-lapse imaging to analyze anaphase chromosome dynamics in human cells. Using multiple directed biochemical, genetic, and physical perturbations, our results demonstrate that differences in the global phosphorylation states between metaphase and anaphase are the major determinant of chromosome motion dynamics. Indeed, causing a mitotic phosphorylation state to persist into anaphase produces dramatic metaphase-like oscillations. These induced oscillations depend on both kinetochore-derived and polar ejection forces that oppose poleward motion. Thus, our analysis of anaphase chromosome motion reveals that dephosphorylation of multiple mitotic substrates is required to suppress metaphase chromosome oscillatory motions and achieve directed poleward motion for successful chromosome segregation. Full Text

Swartz, S.Z. (2016). Polyspermic fertilization resulting in multipolarity in a sea star zygote. Molecular reproduction and development 83, 375.

Swee, L.K., Tan, Z.W., Sanecka, A., Yoshida, N., Patel, H., Grotenbreg, G., Frickel, E.M., and Ploegh, H.L. (2016). Peripheral self-reactivity regulates antigen-specific CD8 T-cell responses and cell division under physiological conditions. Open biology 6(11). pii: 160293. 6T-cell identity is established by the expression of a clonotypic T-cell receptor (TCR), generated by somatic rearrangement of TCRalpha and beta genes. The properties of the TCR determine both the degree of self-reactivity and the repertoire of antigens that can be recognized. For CD8 T cells, the relationship between TCR identity-hence reactivity to self-and effector function(s) remains to be fully understood and has rarely been explored outside of the H-2b haplotype. We measured the affinity of three structurally distinct CD8 T-cell-derived TCRs that recognize the identical H-2 Ld-restricted epitope, derived from the Rop7 protein of Toxoplasma gondii We used CD8 T cells obtained from mice generated by somatic cell nuclear transfer as the closest approximation of primary T cells with physiological TCR rearrangements and TCR expression levels. First, we demonstrate the common occurrence of secondary rearrangements in endogenously rearranged loci. Furthermore, we characterized and compared the response of Rop7-specific CD8 T-cell clones upon Toxoplasma gondii infection as well as effector function and TCR signalling upon antigenic stimulation in vitro Antigen-independent TCR cross-linking in vitro uncovered profound intrinsic differences in the effector functions between T-cell clones. Finally, by assessing the degree of self-reactivity and comparing the transcriptomes of naive Rop7 CD8 T cells, we show that lower self-reactivity correlates with lower effector capacity, whereas higher self-reactivity is associated with enhanced effector function as well as cell cycle entry under physiological conditions. Altogether, our data show that potential effector functions and basal proliferation of CD8 T cells are set by self-reactivity thresholds. Full Text

Tan, J.L., Fogley, R.D., Flynn, R.A., Ablain, J., Yang, S., Saint-Andre, V., Fan, Z.P., Do, B.T., Laga, A.C., Fujinaga, K.,Young RA, et al. (2016). Stress from Nucleotide Depletion Activates the Transcriptional Regulator HEXIM1 to Suppress Melanoma. Molecular Cell 62, 34-46.Studying cancer metabolism gives insight into tumorigenic survival mechanisms and susceptibilities. In melanoma, we identify HEXIM1, a transcription elongation regulator, as a melanoma tumor suppressor that responds to nucleotide stress. HEXIM1 expression is low in melanoma. Its overexpression in a zebrafish melanoma model suppresses cancer formation, while its inactivation accelerates tumor onset in vivo. Knockdown of HEXIM1 rescues zebrafish neural crest defects and human melanoma proliferation defects that arise from nucleotide depletion. Under nucleotide stress, HEXIM1 is induced to form an inhibitory complex with P-TEFb, the kinase that initiates transcription elongation, to inhibit elongation at tumorigenic genes. The resulting alteration in gene expression also causes anti-tumorigenic RNAs to bind to and be stabilized by HEXIM1. HEXIM1 plays an important role in inhibiting cancer cell-specific gene transcription while also facilitating anti-cancer gene expression. Our study reveals an important role for HEXIM1 in coupling nucleotide metabolism with transcriptional regulation in melanoma. Full Text

Tan2, S.H., Yam, A.W.Y., Lawton, L.N., Wong, R.W.J., Young, R.A., Look, A.T., and Sanda, T. (2016). TRIB2 reinforces the oncogenic transcriptional program controlled by the TAL1 complex in T-cell acute lymphoblastic leukemia. Leukemia 30, 959-962 In this study, we identified molecular pathways regulated by TRIB2 in T-ALL cells. .Full Text

Tas, J.M., Mesin, L., Pasqual, G., Targ, S., Jacobsen, J.T., Mano, Y.M., Chen, C.S., Weill, J.C., Reynaud, C.A., Browne, E.P., Michael Meyer-Hermann, and Gabriel D. Victora.(2016). Visualizing antibody affinity maturation in germinal centers. Science [Epub ahead of print] Antibodies somatically mutate to attain high affinity in germinal centers (GCs). There, competition between B cell clones and among somatic mutants of each clone drives an increase in average affinity across the population. The extent to which higher-affinity cells eliminating competitors restricts clonal diversity is unknown. By combining multiphoton microscopy and sequencing, we show that tens to hundreds of distinct B cell clones seed each GC and that GCs lose clonal diversity at widely disparate rates. Furthermore, efficient affinity maturation can occur in the absence of homogenizing selection, ensuring that many clones can mature in parallel within the same GC. Our findings have implications for development of vaccines in which antibodies with non-immunodominant specificities must be elicited, as is the case for HIV-1 and influenza. Full Text

Theunissen, T.W., Friedli, M., He, Y., Planet, E., O'Neil, R.C., Markoulaki, S., Pontis, J., Wang, H., Iouranova, A., Imbeault, M.,Julien Duc,Malkiel A. Cohen,Katherine J. Wert,Rosa Castanon,Zhuzhu Zhang,Yanmei Huang,Joseph R. Nery,Jesse Drotar,Tenzin Lungjangwa,Didier Trono,Joseph R. Ecker, and Rudolf Jaenisch (2016). Molecular Criteria for Defining the Naive Human Pluripotent State. Cell stem cell In Press Recent studies have aimed to convert cultured human pluripotent cells to a naive state, but it remains unclear to what extent the resulting cells recapitulate in vivo naive pluripotency. Here we propose a set of molecular criteria for evaluating the naive human pluripotent state by comparing it to the human embryo. We show that transcription of transposable elements provides a sensitive measure of the concordance between pluripotent stem cells and early human development. We also show that induction of the naive state is accompanied by genome-wide DNA hypomethylation, which is reversible except at imprinted genes, and that the X chromosome status resembles that of the human preimplantation embryo. However, we did not see efficient incorporation of naive human cells into mouse embryos. Overall, the different naive conditions we tested showed varied relationships to human embryonic states based on molecular criteria, providing a backdrop for future analysis of naive human pluripotency. Full Text

TorrensSpence, M.P., Fallon, T.R., and Weng, J.K. (2016). A Workflow for Studying Specialized Metabolism in Nonmodel Eukaryotic Organisms. Methods in enzymology 576, 69-97.Eukaryotes contain a diverse tapestry of specialized metabolites, many of which are of significant pharmaceutical and industrial importance to humans. Nevertheless, exploration of specialized metabolic pathways underlying specific chemical traits in nonmodel eukaryotic organisms has been technically challenging and historically lagged behind that of the bacterial systems. Recent advances in genomics, metabolomics, phylogenomics, and synthetic biology now enable a new workflow for interrogating unknown specialized metabolic systems in nonmodel eukaryotic hosts with greater efficiency and mechanistic depth. This chapter delineates such workflow by providing a collection of state-of-the-art approaches and tools, ranging from multiomics-guided candidate gene identification to in vitro and in vivo functional and structural characterization of specialized metabolic enzymes. As already demonstrated by several recent studies, this new workflow opens up a gateway into the largely untapped world of natural product biochemistry in eukaryotes. Full Text

Truttmann2, M.C., Zheng, X., Hanke, L., Damon, J.R., Grootveld, M., Krakowiak, J., Pincus, D., and Ploegh, H.L. (2016). Unrestrained AMPylation targets cytosolic chaperones and activates the heat shock response. Proceedings of the National Academy of Sciences of the United States of America[Epub ahead of print][Epub ahead of print] Protein AMPylation is a conserved posttranslational modification with emerging roles in endoplasmic reticulum homeostasis. However, the range of substrates and cell biological consequences of AMPylation remain poorly defined. We expressed human and Caenorhabditis elegans AMPylation enzymes-huntingtin yeast-interacting protein E (HYPE) and filamentation-induced by cyclic AMP (FIC)-1, respectively-in Saccharomyces cerevisiae, a eukaryote that lacks endogenous protein AMPylation. Expression of HYPE and FIC-1 in yeast induced a strong cytoplasmic Hsf1-mediated heat shock response, accompanied by attenuation of protein translation, massive protein aggregation, growth arrest, and lethality. Overexpression of Ssa2, a cytosolic heat shock protein (Hsp)70, was sufficient to partially rescue growth. In human cell lines, overexpression of active HYPE similarly induced protein aggregation and the HSF1-dependent heat shock response. Excessive AMPylation also abolished HSP70-dependent influenza virus replication. Our findings suggest a mode of Hsp70 inactivation by AMPylation and point toward a role for protein AMPylation in the regulation of cellular protein homeostasis beyond the endoplasmic reticulum. Full Text

Truttmann, M.C., Cruz, V.E., Guo, X., Engert, C., Schwartz, T.U., and Ploegh, H.L. (2016). The Caenorhabditis elegans Protein FIC-1 Is an AMPylase That Covalently Modifies Heat-Shock 70 Family Proteins, Translation Elongation Factors and Histones. PLoS genetics 12, e1006023.Protein AMPylation by Fic domain-containing proteins (Fic proteins) is an ancient and conserved post-translational modification of mostly unexplored significance. Here we characterize the Caenorhabditis elegans Fic protein FIC-1 in vitro and in vivo. FIC-1 is an AMPylase that localizes to the nuclear surface and modifies core histones H2 and H3 as well as heat shock protein 70 family members and translation elongation factors. The three-dimensional structure of FIC-1 is similar to that of its human ortholog, HYPE, with 38% sequence identity. We identify a link between FIC-1-mediated AMPylation and susceptibility to the pathogen Pseudomonas aeruginosa, establishing a connection between AMPylation and innate immunity in C. elegans. Full Text

Tsvetkov, P., Sokol, E., Jin, D., Brune, Z., Thiru, P., Ghandi, M., Garraway, L.A., Gupta, P.B., Santagata, S., Whitesell, L.,and Lindquist S. (2016). Suppression of 19S proteasome subunits marks emergence of an altered cell state in diverse cancers. Proceedings of the National Academy of Sciences of the United States of America[Epub ahead of print] The use of proteasome inhibitors to target cancer's dependence on altered protein homeostasis has been greatly limited by intrinsic and acquired resistance. Analyzing data from thousands of cancer lines and tumors, we find that those with suppressed expression of one or more 19S proteasome subunits show intrinsic proteasome inhibitor resistance. Moreover, such proteasome subunit suppression is associated with poor outcome in myeloma patients, where proteasome inhibitors are a mainstay of treatment. Beyond conferring resistance to proteasome inhibitors, proteasome subunit suppression also serves as a sentinel of a more global remodeling of the transcriptome. This remodeling produces a distinct gene signature and new vulnerabilities to the proapoptotic drug, ABT-263. This frequent, naturally arising imbalance in 19S regulatory complex composition is achieved through a variety of mechanisms, including DNA methylation, and marks the emergence of a heritably altered and therapeutically relevant state in diverse cancers. Full Text

Valverde, R., Ingram, J., and Harrison, S.C. (2016). Conserved Tetramer Junction in the Kinetochore Ndc80 Complex. Cell reports 17, 1915-1922.The heterotetrameric Ndc80 complex establishes connectivity along the principal longitudinal axis of a kinetochore. Its two heterodimeric subcomplexes, each with a globular end and a coiled-coil shaft, connect end-to-end to create a approximately 600 A long rod spanning the gap from centromere-proximal structures to spindle microtubules. Neither subcomplex has a known function on its own, but the heterotetrameric organization and the characteristics of the junction are conserved from yeast to man. We have determined crystal structures of two shortened ("dwarf") Ndc80 complexes that contain the full tetramer junction and both globular ends. The junction connects two alpha-helical coiled coils through regions of four-chain and three-chain overlap. The complexity of its structure depends on interactions among conserved amino-acid residues, suggesting a binding site for additional cellular factor(s) not yet identified. Full Text

vanRooij, F.J., Qayyum, R., Smith, A.V., Zhou, Y., Trompet, S., Tanaka, T., Keller, M.F., Chang, L.C., Schmidt, H., Yang, M.L, .Brian J. Abraham, et al. (2016). Genome-wide Trans-ethnic Meta-analysis Identifies Seven Genetic Loci Influencing Erythrocyte Traits and a Role for RBPMS in Erythropoiesis. American journal of human genetics[Epub ahead of print]. Genome-wide association studies (GWASs) have identified loci for erythrocyte traits in primarily European ancestry populations. We conducted GWAS meta-analyses of six erythrocyte traits in 71,638 individuals from European, East Asian, and African ancestries using a Bayesian approach to account for heterogeneity in allelic effects and variation in the structure of linkage disequilibrium between ethnicities. We identified seven loci for erythrocyte traits including a locus (RBPMS/GTF2E2) associated with mean corpuscular hemoglobin and mean corpuscular volume. Statistical fine-mapping at this locus pointed to RBPMS at this locus and excluded nearby GTF2E2. Using zebrafish morpholino to evaluate loss of function, we observed a strong in vivo erythropoietic effect for RBPMS but not for GTF2E2, supporting the statistical fine-mapping at this locus and demonstrating that RBPMS is a regulator of erythropoiesis. Our findings show the utility of trans-ethnic GWASs for discovery and characterization of genetic loci influencing hematologic traits. Full Text

Vincent, B.M., Langlois, J.B., Srinivas, R., Lancaster, A.K., Scherz-Shouval, R., Whitesell, L., Tidor, B., Buchwald, S.L., and Lindquist, S. (2016). A Fungal-Selective Cytochrome bc1 Inhibitor Impairs Virulence and Prevents the Evolution of Drug Resistance. Cell chemical biology [Epub ahead of print] To cause disease, a microbial pathogen must adapt to the challenges of its host environment. The leading fungal pathogen Candida albicans colonizes nutrient-poor bodily niches, withstands attack from the immune system, and tolerates treatment with azole antifungals, often evolving resistance. To discover agents that block these adaptive strategies, we screened 300,000 compounds for inhibition of azole tolerance in a drug-resistant Candida isolate. We identified a novel indazole derivative that converts azoles from fungistatic to fungicidal drugs by selective inhibition of mitochondrial cytochrome bc1. We synthesized 103 analogs to optimize potency (half maximal inhibitory concentration 0.4 muM) and fungal selectivity (28-fold over human). In addition to reducing azole resistance, targeting cytochrome bc1 prevents C. albicans from adapting to the nutrient-deprived macrophage phagosome and greatly curtails its virulence in mice. Inhibiting mitochondrial respiration and restricting metabolic flexibility with this synthetically tractable chemotype provides an attractive therapeutic strategy to limit both fungal virulence and drug resistance. Full Text

vonRohrscheidt, J., Petrozziello, E., Nedjic, J., Federle, C., Krzyzak, L., Ploegh, H.L., Ishido, S., Steinkasserer, A., and Klein, L. (2016). Thymic CD4 T cell selection requires attenuation of March8-mediated MHCII turnover in cortical epithelial cells through CD83. The Journal of experimental medicine [Epub ahead of print] Deficiency of CD83 in thymic epithelial cells (TECs) dramatically impairs thymic CD4 T cell selection. CD83 can exert cell-intrinsic and -extrinsic functions through discrete protein domains, but it remains unclear how CD83's capacity to operate through these alternative functional modules relates to its crucial role in TECs. In this study, using viral reconstitution of gene function in TECs, we found that CD83's transmembrane domain is necessary and sufficient for thymic CD4 T cell selection. Moreover, a ubiquitination-resistant MHCII variant restored CD4 T cell selection in Cd83-/- mice. Although during dendritic cell maturation CD83 is known to stabilize MHCII through opposing the ubiquitin ligase March1, regulation of March1 did not account for CD83's TEC-intrinsic role. Instead, we provide evidence that MHCII in cortical TECs (cTECs) is targeted by March8, an E3 ligase of as yet unknown physiological substrate specificity. Ablating March8 in Cd83-/- mice restored CD4 T cell development. Our results identify CD83-mediated MHCII stabilization through antagonism of March8 as a novel functional adaptation of cTECs for T cell selection. Furthermore, these findings suggest an intriguing division of labor between March1 and March8 in controlling inducible versus constitutive MHCII expression in hematopoietic antigen-presenting cells versus TECs. Full Text

Wang, D., Mansisidor, A., Prabhakar, G., and Hochwagen, A. (2016). Condensin and Hmo1 Mediate a Starvation-Induced Transcriptional Position Effect within the Ribosomal DNA Array. Cell reports Volume 14(5)1010–1017. Repetitive DNA arrays are important structural features of eukaryotic genomes that are often heterochromatinized to suppress repeat instability. It is unclear, however, whether all repeats within an array are equally subject to heterochromatin formation and gene silencing. Here, we show that in starving Saccharomyces cerevisiae, silencing of reporter genes within the ribosomal DNA (rDNA) array is less pronounced in outer repeats compared with inner repeats. This position effect is linked to the starvation-induced contraction of the nucleolus. We show that the chromatin regulators condensin and Hmo1 redistribute within the rDNA upon starvation; that Hmo1, like condensin, is required for nucleolar contraction; and that the position effect partially depends on both proteins. Starvation-induced nucleolar contraction and differential desilencing of the outer rDNA repeats may provide a mechanism to activate rDNA-encoded RNAPII transcription units without causing general rDNA instability.. Full Text

Wang5, H., Bierie, B., Li, A.G., Pathania, S., Toomire, K., Dimitrov, S.D., Liu, B., Gelman, R., Giobbie-Hurder, A., Feunteun, J., et al. (2016). BRCA1/FANCD2/BRG1-Driven DNA Repair Stabilizes the Differentiation State of Human Mammary Epithelial Cells. Molecular cell [Epub ahead of print] An abnormal differentiation state is common in BRCA1-deficient mammary epithelial cells, but the underlying mechanism is unclear. Here, we report a convergence between DNA repair and normal, cultured human mammary epithelial (HME) cell differentiation. Surprisingly, depleting BRCA1 or FANCD2 (Fanconi anemia [FA] proteins) or BRG1, a mSWI/SNF subunit, caused HME cells to undergo spontaneous epithelial-to-mesenchymal transition (EMT) and aberrant differentiation. This also occurred when wild-type HMEs were exposed to chemicals that generate DNA interstrand crosslinks (repaired by FA proteins), but not in response to double-strand breaks. Suppressed expression of DeltaNP63 also occurred in each of these settings, an effect that links DNA damage to the aberrant differentiation outcome. Taken together with somatic breast cancer genome data, these results point to a breakdown in a BRCA/FA-mSWI/SNF-DeltaNP63-mediated DNA repair and differentiation maintenance process in mammary epithelial cells that may contribute to sporadic breast cancer development. Full Text

Wang7, I.E., Lapan, S.W., Scimone, M.L., Clandinin, T.R., and Reddien, P.W. (2016). Hedgehog signaling regulates gene expression in planarian glia. eLife 10.7554/eLife.16996 Hedgehog signaling is critical for vertebrate central nervous system (CNS) development, but its role in CNS biology in other organisms is poorly characterized. In the planarian Schmidtea mediterranea, hedgehog (hh) is expressed in medial cephalic ganglia neurons, suggesting a possible role in CNS maintenance or regeneration. We performed RNA sequencing of planarian brain tissue following RNAi of hh and patched (ptc), which encodes the Hh receptor. Two misregulated genes, intermediate filament-1 (if-1) and calamari (cali), were expressed in a previously unidentified non-neural CNS cell type. These cells expressed orthologs of astrocyte-associated genes involved in neurotransmitter uptake and metabolism, and extended processes enveloping regions of high synapse concentration. We propose that these cells are planarian glia. Planarian glia were distributed broadly, but only expressed if-1 and cali in the neuropil near hh+ neurons. Planarian glia and their regulation by Hedgehog signaling present a novel tractable system for dissection of glia biology. Full Text

Wang2, T., Lander, E.S., and Sabatini, D.M. (2016). Single Guide RNA Library Design and Construction. Cold Spring Harbor protocols 2016, pdb.prot090803.This protocol describes how to generate a single guide RNA (sgRNA) library for use in genetic screens. There are many online tools available for predicting sgRNA sequences with high target specificity and/or cleavage activity. Here, we refer the user to genome-wide sgRNA sequence predictions that we have developed for both the human and mouse and that are available from the Broad Institute website. Once a set of target genes and corresponding sgRNA sequences has been identified, customized oligonucleotide pools can be rapidly synthesized by a number of commercial vendors. Thereafter, as described here, the oligonucleotides can be efficiently cloned into an appropriate lentiviral expression vector backbone. The resulting plasmid pool can then be packaged into lentiviral particles and used to generate knockouts in any cell line of choice. Full Text

Wang3, T., Lander, E.S., and Sabatini, D.M. (2016). Viral Packaging and Cell Culture for CRISPR-Based Screens. Cold Spring Harbor protocols 2016, pdb.prot090811.This protocol describes how to perform the tissue culture and high-throughput sequencing library preparation for a CRISPR-based screen. First, pantropic lentivirus is prepared from a single guide RNA (sgRNA) plasmid pool and applied to the target cells. Following antibiotic selection and a harvest of the initial population, cells are then cultured under the desired screening condition(s) for 14 population doublings. The sgRNA barcode sequences integrated in the genomic DNA of each cell population are amplified and subject to high-throughput sequencing. Guidelines for downstream analysis of the sequencing data are also provided. Full Text

Wang4, T., Lander, E.S., and Sabatini, D.M. (2016). Large-Scale Single Guide RNA Library Construction and Use for CRISPR-Cas9-Based Genetic Screens. Cold Spring Harbor protocols 2016, pdb.top086892.The ability to systematically disrupt genes serves as a powerful tool for understanding their function. The programmable CRISPR-Cas9 system enables efficient targeting of large numbers of genes through the use of single guide RNA (sgRNA) libraries. In cultured mammalian cells, collections of knockout mutants can be readily generated by means of transduction of Cas9-sgRNA lentiviral pools, screened for a phenotype of interest, and counted using high-throughput DNA sequencing. This technique represents the first general method for undertaking systematic loss-of-function genetic screens in mammalian cells. Here, we introduce the methodology and rationale for conducting CRISPR-based screens, focusing on distinguishing positive and negative selection strategies. Full Text

Wang6, W., Cencic, R., Whitesell, L., Pelletier, J., and Porco, J.A., Jr. (2016). Synthesis of Aza-Rocaglates via ESIPT-Mediated (3+2) Photocycloaddition. ChemistryA European Journal[Epub ahead of print] Synthesis of aza-rocaglates, nitrogen-containing analogues of the rocaglate natural products, is reported. The route features ESIPT-mediated (3+2) photocycloaddition of 1-alkyl-2-aryl-3-hydroxyquinolinones with the dipolarophile methyl cinnamate. A continuous photoflow reactor was utilized for photocycloadditions. An array of compounds bearing the hexahydrocyclopenta[b]indole core structure was synthesized and evaluated in translation inhibition assays. Full Text

Ward, C., Martinez-Lopez, N., Otten, E.G., Carroll, B., Maetzel, D., Singh, R., Sarkar, S., and Korolchuk, V.I. (2016). Autophagy, Lipophagy and lysosomal lipid storage disorders. Biochimica et biophysica acta [Epub ahead of print] Autophagy is a catabolic process with an essential function in the maintenance of cellular and tissue homeostasis. It is primarily recognised for its role in the degradation of dysfunctional proteins and unwanted organelles, however in recent years the range of autophagy substrates has also been extended to lipids. Degradation of lipids via autophagy is termed lipophagy. The ability of autophagy to contribute to the maintenance of lipo-homeostasis becomes particularly relevant in the context of genetic lysosomal storage disorders where perturbations of autophagic flux have been suggested to contribute to the disease aetiology. Here we review recent discoveries of the molecular mechanisms mediating lipid turnover by the autophagy pathways. We further focus on the relevance of autophagy, and specifically lipophagy, to the disease mechanisms. Moreover, autophagy is also discussed as a potential therapeutic target in several key lysosomal storage disorders. Full Text

Weinberg, D.E., Shah, P., Eichhorn, S.W., Hussmann, J.A., Plotkin, J.B., and Bartel, D.P. (2016). Improved Ribosome-Footprint and mRNA Measurements Provide Insights into Dynamics and Regulation of Yeast Translation. Cell reports [Epub ahead of print] Ribosome-footprint profiling provides genome-wide snapshots of translation, but technical challenges can confound its analysis. Here, we use improved methods to obtain ribosome-footprint profiles and mRNA abundances that more faithfully reflect gene expression in Saccharomyces cerevisiae. Our results support proposals that both the beginning of coding regions and codons matching rare tRNAs are more slowly translated. They also indicate that emergent polypeptides with as few as three basic residues within a ten-residue window tend to slow translation. With the improved mRNA measurements, the variation attributable to translational control in exponentially growing yeast was less than previously reported, and most of this variation could be predicted with a simple model that considered mRNA abundance, upstream open reading frames, cap-proximal structure and nucleotide composition, and lengths of the coding and 5' UTRs. Collectively, our results provide a framework for executing and interpreting ribosome-profiling studies and reveal key features of translational control in yeast. Full Text

Weinberg2, R.A., Schuldiner, M., Wu, H., Stevens, B., Nielsen, J., Hiesinger, P.R., and Hassan, B.A. (2016). All You Need Is Mentorship. Cell 164, 1092-1093. If I, as a mentor, can imbue my trainees with this last skill—a taste for important problems—I view their experience with me as a major success! Full Text

Weng, J.K., Ye, M.L., Li, B., and Noel, J.P. (2016). Co-evolution of Hormone Metabolism and Signaling Networks Expands Plant Adaptive Plasticity. Cell 166, 881-893.Classically, hormones elicit specific cellular responses by activating dedicated receptors. Nevertheless, the biosynthesis and turnover of many of these hormone molecules also produce chemically related metabolites. These molecules may also possess hormonal activities; therefore, one or more may contribute to the adaptive plasticity of signaling outcomes in host organisms. Here, we show that a catabolite of the plant hormone abscisic acid (ABA), namely phaseic acid (PA), likely emerged in seed plants as a signaling molecule that fine-tunes plant physiology, environmental adaptation, and development. This trait was facilitated by both the emergence- selection of a PA reductase that modulates PA concentrations and by the functional diversification of the ABA receptor family to perceive and respond to PA. Our results suggest that PA serves as a hormone in seed plants through activation of a subset of ABA receptors. This study demonstrates that the co-evolution of hormone metabolism and signaling networks can expand organismal resilience. Full Text

Whitesell, L., and Santagata, S. (2016). Susan Lindquist (1949-2016). Science (New York, NY) 354, 974 On 27 October 2016, Susan Lee Lindquist, professor at the Massachusetts Institute of Technology (MIT), died of cancer at the age of 67. She was a formidable academic leader, dedicated mentor, beloved friend, and devoted wife and mother who will be deeply missed. .Full Text

Wiehle, L., Raddatz, G., Musch, T., Dawlaty, M.M., Jaenisch, R., Lyko, F., and Breiling, A. (2016). Tet1 and Tet2 Protect DNA Methylation Canyons against Hypermethylation. Molecular and Cellular Biology 36, 452-461.DNA methylation is a dynamic epigenetic modification with an important role in cell fate specification and reprogramming. The Ten eleven translocation (Tet) family of enzymes converts 5-methylcytosine to 5-hydroxymethylcytosine, which promotes passive DNA demethylation and functions as an intermediate in an active DNA demethylation process. Tet1/Tet2 double-knockout mice are characterized by developmental defects and epigenetic instability, suggesting a requirement for Tet-mediated DNA demethylation for the proper regulation of gene expression during differentiation. Here, we used whole-genome bisulfite and transcriptome sequencing to characterize the underlying mechanisms. Our results uncover the hypermethylation of DNA methylation canyons as the genomic key feature of Tet1/Tet2 double-knockout mouse embryonic fibroblasts. Canyon hypermethylation coincided with disturbed regulation of associated genes, suggesting a mechanistic explanation for the observed Tet-dependent differentiation defects. Based on these results, we propose an important regulatory role of Tet-dependent DNA demethylation for the maintenance of DNA methylation canyons, which prevents invasive DNA methylation and allows functional regulation of canyon-associated genes. Full Text

Willems, T., Gymrek, M., Poznik, G.D., Tyler-Smith, C., and Erlich, Y. (2016). Population-Scale Sequencing Data Enable Precise Estimates of Y-STR Mutation Rates. American journal of human genetics[Epub ahead of print] Short tandem repeats (STRs) are mutation-prone loci that span nearly 1% of the human genome. Previous studies have estimated the mutation rates of highly polymorphic STRs by using capillary electrophoresis and pedigree-based designs. Although this work has provided insights into the mutational dynamics of highly mutable STRs, the mutation rates of most others remain unknown. Here, we harnessed whole-genome sequencing data to estimate the mutation rates of Y chromosome STRs (Y-STRs) with 2-6 bp repeat units that are accessible to Illumina sequencing. We genotyped 4,500 Y-STRs by using data from the 1000 Genomes Project and the Simons Genome Diversity Project. Next, we developed MUTEA, an algorithm that infers STR mutation rates from population-scale data by using a high-resolution SNP-based phylogeny. After extensive intrinsic and extrinsic validations, we harnessed MUTEA to derive mutation-rate estimates for 702 polymorphic STRs by tracing each locus over 222,000 meioses, resulting in the largest collection of Y-STR mutation rates to date. Using our estimates, we identified determinants of STR mutation rates and built a model to predict rates for STRs across the genome. These predictions indicate that the load of de novo STR mutations is at least 75 mutations per generation, rivaling the load of all other known variant types. Finally, we identified Y-STRs with potential applications in forensics and genetic genealogy, assessed the ability to differentiate between the Y chromosomes of father-son pairs, and imputed Y-STR genotypes. Full Text

WilsonKubalek, E.M., Cheeseman, I.M., and Milligan, R.A. (2016). Structural comparison of the C. elegans and human Ndc80 complexes bound to microtubules reveals distinct binding behavior. Molecular biology of the cell . [Epub ahead of print] During cell division, kinetochores must remain tethered to the plus ends of dynamic microtubule polymers. However, the molecular basis for robust kinetochore-microtubule interactions remains poorly understood. The conserved 4-subunit Ndc80 complex plays an essential and direct role in generating dynamic kinetochore-microtubule attachments. Here, we compare the binding of the C. elegans and human Ndc80 complexes to microtubules at high resolution using cryo-electron microscopy reconstructions. Despite the conserved roles of the Ndc80 complex in diverse organisms, we find that the attachment mode of these complexes for microtubules is distinct. The human Ndc80 complex binds every tubulin monomer along the microtubule protofilament, whereas the C. elegans Ndc80 complex binds more tightly to beta-tubulin. In addition, the C. elegans Ndc80 complex tilts more toward the adjacent protofilament. These structural differences in the Ndc80 complex between different species may play significant roles in the nature of kinetochore-microtubule interactions. Full Text

Witt, A.E., Lee, C.W., Lee, T.I., Azzam, D.J., Wang, B., Caslini, C., Petrocca, F., Grosso, J., Jones, M., Cohick, E.B.,A B Gropper, R A Young, et al(2016). Identification of a cancer stem cell-specific function for the histone deacetylases, HDAC1 and HDAC7, in breast and ovarian cancer. Oncogene advance online publication Tumours are comprised of a highly heterogeneous population of cells, of which only a small subset of stem-like cells possess the ability to regenerate tumours in vivo. These cancer stem cells (CSCs) represent a significant clinical challenge as they are resistant to conventional cancer therapies and play essential roles in metastasis and tumour relapse. Despite this realization and great interest in CSCs, it has been difficult to develop CSC-targeted treatments due to our limited understanding of CSC biology. Here, we present evidence that specific histone deacetylases (HDACs) play essential roles in the CSC phenotype. Utilizing a novel CSC model, we discovered that the HDACs, HDAC1 and HDAC7, are specifically over-expressed in CSCs when compared to non-stem-tumour-cells (nsTCs). Furthermore, we determine that HDAC1 and HDAC7 are necessary to maintain CSCs, and that over-expression of HDAC7 is sufficient to augment the CSC phenotype. We also demonstrate that clinically available HDAC inhibitors (HDACi) targeting HDAC1 and HDAC7 can be used to preferentially target CSCs. These results provide actionable insights that can be rapidly translated into CSC-specific therapies.Oncogene advance online publication, 3 October 2016; doi:10.1038/onc.2016.337. Full Text

Wohlgemuth, G., Mehta, S.S., Mejia, R.F., Neumann, S., Pedrosa, D., Pluskal, T., Schymanski, E.L., Willighagen, E.L., Wilson, M., Wishart, D.S., et al. (2016). SPLASH, a hashed identifier for mass spectra. Nature biotechnology 34, 1099-1101 Over the past few years, as the use of mass spectrometry (MS) has increased, multiple spectral libraries, databases and software frameworks have been created to enable sharing and searching of MS data. However, finding all the spectra that correspond to a specific compound across different databases continues to be a challenge. A spectral identifier that improves the exchange of mass spectra, as well as provenance and duplicate detection, would address these issues and enhance searchability..Full Text

Woodham, A.W., Sanna, A.M., Taylor, J.R., Skeate, J.G., Da Silva, D.M., Dekker, L.V., and Kast, W.M. (2016). Annexin A2 antibodies but not inhibitors of the annexin A2 heterotetramer impair productive HIV-1 infection of macrophages in vitro. Virology journal 13, 187.During sexual transmission of human immunodeficiency virus (HIV), macrophages are initial targets for HIV infection. Secretory leukocyte protease inhibitor (SLPI) has been shown to protect against HIV infection of macrophages through interactions with annexin A2 (A2), which is found on the macrophage cell surface as a heterotetramer (A2t) consisting of A2 and S100A10. Therefore, we investigated potential protein-protein interactions between A2 and HIV-1 gp120 through a series of co-immunoprecipitation assays and a single molecule pulldown (SiMPull) technique. Additionally, inhibitors of A2t (A2ti) that target the interaction between A2 and S100A10 were tested for their ability to impair productive HIV-1 infection of macrophages. Our data suggest that interactions between HIV-1 gp120 and A2 exist, though this interaction may be indirect. Furthermore, an anti-A2 antibody impaired HIV-1 particle production in macrophages in vitro, whereas A2ti did not indicating that annexin A2 may promote HIV-1 infection of macrophages in its monomeric rather than tetrameric form. Full Text

Woznica, A., Cantley, A.M., Beemelmanns, C., Freinkman, E., Clardy, J., and King, N. (2016). Bacterial lipids activate, synergize, and inhibit a developmental switch in choanoflagellates. Proceedings of the National Academy of Sciences of the United States of America[Epub ahead of print] In choanoflagellates, the closest living relatives of animals, multicellular rosette development is regulated by environmental bacteria. The simplicity of this evolutionarily relevant interaction provides an opportunity to identify the molecules and regulatory logic underpinning bacterial regulation of development. We find that the rosette-inducing bacterium Algoriphagus machipongonensis produces three structurally divergent classes of bioactive lipids that, together, activate, enhance, and inhibit rosette development in the choanoflagellate Salpingoeca rosetta. One class of molecules, the lysophosphatidylethanolamines (LPEs), elicits no response on its own but synergizes with activating sulfonolipid rosette-inducing factors (RIFs) to recapitulate the full bioactivity of live Algoriphagus. LPEs, although ubiquitous in bacteria and eukaryotes, have not previously been implicated in the regulation of a host-microbe interaction. This study reveals that multiple bacterially produced lipids converge to activate, enhance, and inhibit multicellular development in a choanoflagellate. Full Text

Wu, J., Greely, H.T., Jaenisch, R., Nakauchi, H., Rossant, J., and Belmonte, J.C. (2016). Stem cells and interspecies chimaeras. Nature 540, 51-59.Chimaeras are both monsters of the ancient imagination and a long-established research tool. Recent advances, particularly those dealing with the identification and generation of various kinds of stem cells, have broadened the repertoire and utility of mammalian interspecies chimaeras and carved out new paths towards understanding fundamental biology as well as potential clinical applications. Full Text

Zhang2, T., Kwiatkowski, N., Olson, C.M., Dixon-Clarke, S.E., Abraham, B.J., Greifenberg, A.K., Ficarro, S.B., Elkins, J.M., Liang, Y., Hannett, N.M., Richard A Young, et al. (2016). Covalent targeting of remote cysteine residues to develop CDK12 and CDK13 inhibitors. Nature chemical biology doi:10.1038/nchembio.2166 Cyclin-dependent kinases 12 and 13 (CDK12 and CDK13) play critical roles in the regulation of gene transcription. However, the absence of CDK12 and CDK13 inhibitors has hindered the ability to investigate the consequences of their inhibition in healthy cells and cancer cells. Here we describe the rational design of a first-in-class CDK12 and CDK13 covalent inhibitor, THZ531. Co-crystallization of THZ531 with CDK12-cyclin K indicates that THZ531 irreversibly targets a cysteine located outside the kinase domain. THZ531 causes a loss of gene expression with concurrent loss of elongating and hyperphosphorylated RNA polymerase II. In particular, THZ531 substantially decreases the expression of DNA damage response genes and key super-enhancer-associated transcription factor genes. Coincident with transcriptional perturbation, THZ531 dramatically induced apoptotic cell death. Small molecules capable of specifically targeting CDK12 and CDK13 may thus help identify cancer subtypes that are particularly dependent on their kinase activities. Full Text

Zhang, X., Chen, M.H., Wu, X., Kodani, A., Fan, J., Doan, R., Ozawa, M., Ma, J., Yoshida, N., Reiter, J.F., et al. (2016). Cell-Type-Specific Alternative Splicing Governs Cell Fate in the Developing Cerebral Cortex. Cell 166, 1147-1162.e1115.Alternative splicing is prevalent in the mammalian brain. To interrogate the functional role of alternative splicing in neural development, we analyzed purified neural progenitor cells (NPCs) and neurons from developing cerebral cortices, revealing hundreds of differentially spliced exons that preferentially alter key protein domains-especially in cytoskeletal proteins-and can harbor disease-causing mutations. We show that Ptbp1 and Rbfox proteins antagonistically govern the NPC-to-neuron transition by regulating neuron-specific exons. Whereas Ptbp1 maintains apical progenitors partly through suppressing a poison exon of Flna in NPCs, Rbfox proteins promote neuronal differentiation by switching Ninein from a centrosomal splice form in NPCs to a non-centrosomal isoform in neurons. We further uncover an intronic human mutation within a PTBP1-binding site that disrupts normal skipping of the FLNA poison exon in NPCs and causes a brain-specific malformation. Our study indicates that dynamic control of alternative splicing governs cell fate in cerebral cortical development. Full Text

Zhao, Q., Zhang, Y., Wang, G., Hill, L., Weng, J.K., Chen, X.Y., Xue, H., and Martin, C. (2016). A specialized flavone biosynthetic pathway has evolved in the medicinal plant, Scutellaria baicalensis. Science advances 2, e1501780.Wogonin and baicalein are bioactive flavones in the popular Chinese herbal remedy Huang-Qin (Scutellaria baicalensis Georgi). These specialized flavones lack a 4'-hydroxyl group on the B ring (4'-deoxyflavones) and induce apoptosis in a wide spectrum of human tumor cells in vitro and inhibit tumor growth in vivo in different mouse tumor models. Root-specific flavones (RSFs) from Scutellaria have a variety of reported additional beneficial effects including antioxidant and antiviral properties. We describe the characterization of a new pathway for the synthesis of these compounds, in which pinocembrin (a 4'-deoxyflavanone) serves as a key intermediate. Although two genes encoding flavone synthase II (FNSII) are expressed in the roots of S. baicalensis, FNSII-1 has broad specificity for flavanones as substrates, whereas FNSII-2 is specific for pinocembrin. FNSII-2 is responsible for the synthesis of 4'-deoxyRSFs, such as chrysin and wogonin, wogonoside, baicalein, and baicalin, which are synthesized from chrysin. A gene encoding a cinnamic acid-specific coenzyme A ligase (SbCLL-7), which is highly expressed in roots, is required for the synthesis of RSFs by FNSII-2, as demonstrated by gene silencing. A specific isoform of chalcone synthase (SbCHS-2) that is highly expressed in roots producing RSFs is also required for the synthesis of chrysin. Our studies reveal a recently evolved pathway for biosynthesis of specific, bioactive 4'-deoxyflavones in the roots of S. baicalensis. Full Text

Zheng, X., Krakowiak, J., Patel, N., Beyzavi, A., Ezike, J., Khalil, A.S., and Pincus, D. (2016). Dynamic control of Hsf1 during heat shock by a chaperone switch and phosphorylation. eLife 5. pii: e18638. Heat shock factor (Hsf1) regulates the expression of molecular chaperones to maintain protein homeostasis. Despite its central role in stress resistance, disease and aging, the mechanisms that control Hsf1 activity remain unresolved. Here we show that in budding yeast, Hsf1 basally associates with the chaperone Hsp70 and this association is transiently disrupted by heat shock, providing the first evidence that a chaperone repressor directly regulates Hsf1 activity. We develop and experimentally validate a mathematical model of Hsf1 activation by heat shock in which unfolded proteins compete with Hsf1 for binding to Hsp70. Surprisingly, we find that Hsf1 phosphorylation, previously thought to be required for activation, in fact only positively tunes Hsf1 and does so without affecting Hsp70 binding. Our work reveals two uncoupled forms of regulation - an ON/OFF chaperone switch and a tunable phosphorylation gain - that allow Hsf1 to flexibly integrate signals from the proteostasis network and cell signaling pathways. Full Text

Zhong, Q., Pevzner, S.J., Hao, T., Wang, Y., Mosca, R., Menche, J., Taipale, M., Tasan, M., Fan, C., Yang, X. ,Lindquist, S. et al. (2016). An inter-species protein-protein interaction network across vast evolutionary distance. Molecular systems biology 12, 865.In cellular systems, biophysical interactions between macromolecules underlie a complex web of functional interactions. How biophysical and functional networks are coordinated, whether all biophysical interactions correspond to functional interactions, and how such biophysical-versus-functional network coordination is shaped by evolutionary forces are all largely unanswered questions. Here, we investigate these questions using an "inter-interactome" approach. We systematically probed the yeast and human proteomes for interactions between proteins from these two species and functionally characterized the resulting inter-interactome network. After a billion years of evolutionary divergence, the yeast and human proteomes are still capable of forming a biophysical network with properties that resemble those of intra-species networks. Although substantially reduced relative to intra-species networks, the levels of functional overlap in the yeast-human inter-interactome network uncover significant remnants of co-functionality widely preserved in the two proteomes beyond human-yeast homologs. Our data support evolutionary selection against biophysical interactions between proteins with little or no co-functionality. Such non-functional interactions, however, represent a reservoir from which nascent functional interactions may arise. Full Text

Zhu, S., McGrath, B.C., Bai, Y., Tang, X., and Cavener, D.R. (2016). PERK regulates Gq protein-coupled intracellular Ca2+ dynamics in primary cortical neurons. Molecular brain 9, 87. PERK (EIF2AK3) is an ER-resident eIF2alpha kinase required for behavioral flexibility and metabotropic glutamate receptor-dependent long-term depression via its translational control. Motivated by the recent discoveries that PERK regulates Ca2+ dynamics in insulin-secreting beta-cells underlying glucose-stimulated insulin secretion, and modulates Ca2+ signals-dependent working memory, we explored the role of PERK in regulating Gq protein-coupled Ca2+ dynamics in pyramidal neurons. We found that acute PERK inhibition by the use of a highly specific PERK inhibitor reduced the intracellular Ca2+ rise stimulated by the activation of acetylcholine, metabotropic glutamate and bradykinin-2 receptors in primary cortical neurons. More specifically, acute PERK inhibition increased IP3 receptor mediated ER Ca2+ release, but decreased receptor-operated extracellular Ca2+ influx. Impaired Gq protein-coupled intracellular Ca2+ rise was also observed in genetic Perk knockout neurons. Taken together, our findings reveal a novel role of PERK in neurons, which is eIF2alpha-independent, and suggest that the impaired working memory in forebrain-specific Perk knockout mice may stem from altered Gq protein-coupled intracellular Ca2+ dynamics in cortical pyramidal neurons. Full Text

Zubradt, M., Gupta, P., Persad, S., Lambowitz, A.M., Weissman, J.S., and Rouskin, S. (2016). DMS-MaPseq for genome-wide or targeted RNA structure probing in vivo.Nature methods[Epub ahead of print]. Coupling of structure-specific in vivo chemical modification to next-generation sequencing is transforming RNA secondary structure studies in living cells. The dominant strategy for detecting in vivo chemical modifications uses reverse transcriptase truncation products, which introduce biases and necessitate population-average assessments of RNA structure. Here we present dimethyl sulfate (DMS) mutational profiling with sequencing (DMS-MaPseq), which encodes DMS modifications as mismatches using a thermostable group II intron reverse transcriptase. DMS-MaPseq yields a high signal-to-noise ratio, can report multiple structural features per molecule, and allows both genome-wide studies and focused in vivo investigations of even low-abundance RNAs. We apply DMS-MaPseq for the first analysis of RNA structure within an animal tissue and to identify a functional structure involved in noncanonical translation initiation. Additionally, we use DMS-MaPseq to compare the in vivo structure of pre-mRNAs with their mature isoforms. These applications illustrate DMS-MaPseq's capacity to dramatically expand in vivo analysis of RNA structure. Full Text

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