The following alphabetical list represents papers published in 2015 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.

2015 Titles :

-Abundant contribution of short tandem repeats to gene expression variation in humans.Gymrek
-Accommodating family life: mentoring future female faculty members. Lodish
-Activator-inhibitor coupling between Rho signalling and actin assembly makes the cell cortex an excitable medium. Bement
-Adaptive Regulation of Testis Gene Expression and Control of Male Fertility by the Drosophila Harpin RNA Pathway. Wen
-Addressing the Genetics of Human Mental Health Disorders in Model Organisms. McCammon
-Affinity Inequality among Serum Antibodies That Originate in Lymphoid Germinal Center. Kang
-Allosteric activation of apicomplexan calcium-dependent protein kinases. Ingram
-Amino Acid Management in Cancer. Tsun
-Asymmetric apportioning of aged mitochondria between daughter cells is required for stemness. Katajisto
-The Biology and Evolution of Mammalian Y Chromosomes. Hughes2
-The calcium signaling toolkit of the Apicomplexan parasites Toxoplasma gondii and Plasmodium spp. Lourido
-CASC15-S Is a Tumor Suppressor lncRNA at the 6p22 Neuroblastoma Susceptibility Locus. Russell
-CDK7-Dependent Transcriptional Addiction in Triple-Negative Breast Cancer.Wang2
-CEACAM1 regulates TIM-3-mediated tolerance and exhaustion. Huang
-The CENP-L-N Complex Forms a Critical Node in an Integrated Meshwork of Interactions at the Centromere-Kinetochore Interface. McKinley2
-Challenges in understanding psychiatric disorders and developing therapeutics: a role for zebrafish. McCammon2
-Chemical Genomics-Based Antifungal Drug Discovery: Targeting Glycosylphosphatidylinositol (GPI) Precursor Biosynthesis. Mann
-Chromatin proteomic profiling reveals novel proteins associated with histone-marked genomic regions. Ji
-Chromosome Segregation: A Spatial Code to Correct Kinetochore-Microtubule Attachments. Monda
-Clinical heterogeneity associated with KCNA1 mutations include cataplexy and nonataxic presentations. Brownstein
-Clonal Deletion Prunes but Does Not Eliminate Self-Specific alphabeta CD8 T Lymphocytes. Yu
-Combined Loss of Tet1 and Tet2 Promotes B Cell, but Not Myeloid Malignancies, in Mice. Zhao
-A Comparative Genomics Perspective on the Origin of Multicellularity and Early Animal Evolution. Srivastava
-Compromising the 19S proteasome complex protects cells from reduced flux through the proteasome. Tsvetkov
-Convergence of Developmental and Oncogenic Signaling Pathways at Transcriptional Super-Enhancers.Hnisz
-Cooperative nutrient accumulation sustains growth of mammalian cells. Son
-Creating Patient-Specific Neural Cells for the In Vitro Study of Brain Disorders. Brennand
-A CRISPR system permits genetic engineering of essential genes and gene families. Vyas
-The cytoplasmic prolyl-tRNA synthetase of the malaria parasite is a dual-stage target of febrifugine and its analogs. Herman
-De Novo Reconstruction of Adipose Tissue Transcriptomes Reveals Long Non-coding RNA Regulators of Brown Adipocyte Development.AlvarezDominguez
-Differentiation of Human Parthenogenetic Pluripotent Stem Cells Reveals Multiple Tissue- and Isoform-Specific Imprinted Transcripts. Stelzer
-Discovery of an Unconventional Centromere in Budding Yeast Redefines Evolution of Point Centromeres. Kobayashi
-Disruption of Sphingolipid Biosynthesis Blocks Phagocytosis of Candida albicans. Tafesse
-Disruption of the Rag-Ragulator Complex by c17orf59 Inhibits mTORC1. Schweitzer
-Dissecting risk haplotypes in sporadic Alzheimer's disease. Soldner
-Distinct EMT programs control normal mammary stem cells and tumour-initiating cells. Ye
-Distinct Organization and Regulation of the Outer Kinetochore KMN Network Downstream of CENP-C and CENP-T .Rago
-Efficient reprogramming of human fibroblasts and blood-derived endothelial progenitor cells using non-modified RNA for reprogramming and immune evasion. Poleganov
-Endothelial thermotolerance impairs nanoparticle transport in tumors. Bagley
-Engineering lipid overproduction in the oleaginous yeast Yarrowia lipolytica. Qiao
-Enzyme-Mediated Modification of Single-Domain Antibodies for Imaging Modalities with Different Characteristics.Rashidian2
-Epithelial-Mesenchymal Plasticity: A Central Regulator of Cancer Progression. Ye2
-An Essential Role of the Mitochondrial Electron Transport Chain in Cell Proliferation Is to Enable Aspartate Synthesis. Birsoy
-Evasion of Innate Cytosolic DNA Sensing by a Gammaherpesvirus Facilitates Establishment of Latent Infection.Sun
-Failure to replicate the STAP cell phenomenon. DeLosAngeles1
-5-Hydroxymethylcytosine Is Not Present in Appreciable Quantities in Arabidopsis DNA. Erdmann
-Fluorophore-Conjugated Holliday Junctions for Generating Super-Bright Antibodies and Antibody Fragments. Li
-A Gene Regulatory Program for Meiotic Prophase in the Fetal Ovary. Soh
-A Generic and Cell-Type-Specific Wound Response Precedes Regeneration in Planarians.Wurtzel
-Genetic predisposition to neuroblastoma mediated by a LMO1 super-enhancer polymorphism. Oldridge
-The genome of the vervet (Chlorocebus aethiops sabaeus) . Warren
-A genome-scale in vivo loss-of-function screen identifies Phf6 as a lineage-specific regulator of leukemia cell growth. Meacham
-Genome-wide association study follow-up identifies cyclin A2 as a regulator of the transition through cytokinesis during terminal erythropoiesis. Ludwig
-Germinal Center Selection and the Antibody Response to Influenza. Victora
-The GOBLET training portal: a global repository of bioinformatics training materials, courses and trainers. Corpas
-Graphene Oxide Nanosheets Modified with Single-Domain Antibodies for Rapid and Efficient Capture of Cells.Chen
-GSK-3beta phosphorylation of cytoplasmic dynein reduces Ndel1 binding to intermediate chains and alters dynein motility. Gao
-Hallmarks of pluripotency, DeLosAngeles2
-How does multistep tumorigenesis really proceed? Chaffer
-HypE-specific Nanobodies as Tools to modulate HypE-mediated Target AMPylation. Truttmann
-Identification and characterization of essential genes in the human genome. Wang3
-Identification of an oncogenic RAB protein. Wheeler
-Identification of Direct Kinase Substrates Using Analogue-Sensitive Alleles.Rothenberg
-Identification of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase as a novel autophagy regulator by high content shRNA screening.Strohecker
-Increasing the efficiency of precise genome editing with CRISPR-Cas9 by inhibition of nonhomologous end joining. Maruyama
-Independent regulation of vertebral number and vertebral identity by microRNA-196 paralogs. Wong
-Induced Pluripotency and Epigenetic Reprogramming. Hochedlinger
-Inferring transient particle transport dynamics in live cells. Monnier
-Intracellular Expression of Camelid Single-Domain Antibodies Specific for Influenza Virus Nucleoprotein Uncovers Distinct Features of Its Nuclear Localization. Ashour
-Introducing the Plastic Surgery Research Network: A Comprehensive Online Platform to Facilitate Interdisciplinary Plastic Surgery Research. Jalali
-Licensing of Primordial Germ Cells for Gametogenesis Depends on Genital Ridge Signaling. Hu
-Long non-coding RNAs as regulators of the endocrine system. Knoll
-Lysosomal amino acid transporter SLC38A9 signals arginine sufficiency to mTORC1.Wang
-The Menu of Features that Define Primary MicroRNAs and Enable De Novo Design of MicroRNA Genes. Fang
-MERAV: a tool for comparing gene expression across human tissues and cell types. Shaul
-Methylation-sensitive expression of a DNA demethylase gene serves as an epigenetic rheostat. Williams
-The molecular basis for centromere identity and function. McKinley
-Monitoring Dynamics of DNA Methylation at Single-Cell Resolution during Development and Disease. Stelzer3
-The mTORC1/4E-BP pathway coordinates hemoglobin production with L-leucine availability. Chung
-A niche role for cancer exosomes in metastasis. Zhang
-Noninvasive imaging of immune responses. Rashidian
-Nontoxic antimicrobials that evade drug resistance. Davis
-Nutrient-Sensing Mechanisms across Evolution. Chantranupong
-Nutrient-sensing mechanisms and pathways. Efeyan
-One-Step Enzymatic Modification of the Cell Surface Redirects Cellular Cytotoxicity and Parasite Tropism. Swee
-Parkinson-causing alpha-synuclein missense mutations shift native tetramers to monomers as a mechanism for disease initiation. Dettmer
-Periodic retinoic acid-STRA8 signaling intersects with periodic germ-cell competencies to regulate spermatogenesis. Endo
-Perturbation-Expression Analysis Identifies RUNX1 as a Regulator of Human Mammary Stem Cell Differentiation. Sokol
-Polyploidy .Frawley
-PPAR-alpha and glucocorticoid receptor synergize to promote erythroid progenitor self-renewal. Lee
-Predicting effective microRNA target sites in mammalian mRN. Agarwal
-Principles of Long Noncoding RNA Evolution Derived from Direct Comparison of Transcriptomes in 17 Species. Hezroni
-Profiling lymphocyte interactions at the single-cell level by microfluidic cell pairing .Dura
-Recurrent mTORC1-activating RRAGC mutations in follicular lymphoma. Okosun
-Replication Fork Progression during Re-replication Requires the DNA Damage Checkpoint and Double-Strand Break Repair. Alexander
-A respiratory chain controlled signal transduction cascade in the mitochondrial intermembrane space mediates hydrogen peroxide signaling. Patterson
-RNA: An Expanding View of Function and Evolution. Han
-The role of tanycytes in hypothalamic glucosensing. ElizondoVega
-SCNT-Derived ESCs with Mismatched Mitochondria Trigger an Immune Response in Allogeneic Hosts Deuse
-Sensitivity-Enhanced NMR Reveals Alterations in Protein Structure by Cellular Milieu. Frederick
-Sequencing the cap-snatching repertoire of H1N1 influenza provides insight into the mechanism of viral transcription initiation. Koppstein
-Sex chromosome-to-autosome transposition events counter Y-chromosome gene loss in mammals.Hughes
-SHMT2 drives glioma cell survival in ischaemia but imposes a dependence on glycine clearance. Kim
-Simultaneous Site-Specific Dual Protein Labeling Using Protein Prenyltransferases. Zhang2
-Site-specific protein modification using immobilized sortase in batch and continuous-flow systems. Witte
-Small-molecule enhancers of autophagy modulate cellular disease phenotypes suggested by human genetics.Kuo
-Solid-immersion fluorescence microscopy with increased emission and super resolution. Liau
-Structural basis for leucine sensing by the Sestrin2-mTORC1 pathway. Saxton
-Structural biology. Structural basis for chemokine recognition and activation of a viral G protein-coupled receptor. Burg
-Supporting Aspartate Biosynthesis Is an Essential Function of Respiration in Proliferating Cell. Sullivan
-A Systematic Approach to Identify Candidate Transcription Factors that Control Cell Identity. DAlessio
-teashirt is required for head-versus-tail regeneration polarity in planarians. Owen
-Tet1 and Tet2 protect DNA methylation canyons against hypermethylation. Wiehle
-TET1 is a tumor suppressor of hematopoietic malignancy . Cimmino
-TEX11 is mutated in infertile men with azoospermia and regulates genome-wide recombination rates in mouse. Yang
-3D Chromosome Regulatory Landscape of Human Pluripotent Cells.Ji2
-Thrombospondin-1 repression is mediated via distinct mechanisms in fibroblasts and epithelial cells. Watnick
-Toward stem cell-based phenotypic screens for neurodegenerative diseases. Khurana
-The Toxoplasma Dense Granule Proteins GRA17 and GRA23 Mediate the Movement of Small Molecules between the Host and the Parasitophorous Vacuole. Gold
-Toxoplasma gondii Superinfection and Virulence during Secondary Infection Correlate with the Exact ROP5/ROP18 Allelic Combination.Jensen
-Tracing Dynamic Changes of DNA Methylation at Single-Cell Resolution. Stelzer2
-Transcription dynamically patterns the meiotic chromosome-axis interface.Sun2
-Transcription factor trapping by RNA in gene regulatory elements.Sigova
-TRIB2 reinforces the oncogenic transcriptional program controlled by the TAL1 complex in T-cell acute lymphoblastic leukemia. Tan
-Triggering Positive Selection of Germinal Center B Cells by Antigen Targeting to DEC-205. Pasqual
-Tumor Cell-Derived Periostin Regulates Cytokines That Maintain Breast Cancer Stem Cells.Lambert
-Understanding replication fork progression, stability, and chromosome fragility by exploiting the Suppressor of Underreplication protein. Nordman
-Use of F-18-2-Fluorodeoxyglucose to Label Antibody Fragments for Immuno-Positron Emission Tomography of Pancreatic Cancer .Rashidian2
-When bigger is better: the role of polyploidy in organogenesis. OrrWeaver
-Widespread Macromolecular Interaction Perturbations in Human Genetic Disorders. Sahni
-X-linked macrocytic dyserythropoietic anemia in females with an ALAS2 mutation. Sankaran
-Zebrafish adult-derived hypothalamic neurospheres generate gonadotropin-releasing hormone (GnRH) neurons. CortesCampos
-Zebrafish cerebrospinal fluid mediates cell survival through a retinoid signaling pathway . Chang

Agarwal, V., Bell, G.W., Nam, J.W., and Bartel, D.P. (2015). Predicting effective microRNA target sites in mammalian mRNA. eLife 4: e05005. MicroRNA targets are often recognized through pairing between the miRNA seed region and complementary sites within target mRNAs, but not all of these canonical sites are equally effective, and both computational and in vivo UV-crosslinking approaches suggest that many mRNAs are targeted through non-canonical interactions. Here, we show that recently reported non-canonical sites do not mediate repression despite binding the miRNA, which indicates that the vast majority of functional sites are canonical. Accordingly, we developed an improved quantitative model of canonical targeting, using a compendium of experimental datasets that we pre-processed to minimize confounding biases. This model, which considers site type and another 14 features to predict the most effectively targeted mRNAs, performed significantly better than existing models and was as informative as the best high-throughput in vivo crosslinking approaches. It drives the latest version of TargetScan (v7.0; targetscan.org), thereby providing a valuable resource for placing miRNAs into gene-regulatory networks. Full Text

Alexander, J.L., Barrasa, M.I., and Orr-Weaver, T.L. (2015). Replication Fork Progression during Re-replication Requires the DNA Damage Checkpoint and Double-Strand Break Repair. Current biology : CBAvailable online 4 June 2015. Replication origins are under tight regulation to ensure activation occurs only once per cell cycle [1, 2]. Origin re-firing in a single S phase leads to the generation of DNA double-strand breaks (DSBs) and activation of the DNA damage checkpoint [2-7]. If the checkpoint is blocked, cells enter mitosis with partially re-replicated DNA that generates chromosome breaks and fusions [5]. These types of chromosomal aberrations are common in numerous human cancers, suggesting that re-replication events contribute to cancer progression. It was proposed that fork instability and DSBs formed during re-replication are the result of head-to-tail collisions and collapse of adjacent replication forks [3]. However, previously studied systems lack the resolution to determine whether the observed DSBs are generated at sites of fork collisions. Here, we utilize the Drosophila ovarian follicle cells, which exhibit re-replication under precise developmental control [8-10], to model the consequences of re-replication at actively elongating forks. Re-replication occurs from specific replication origins at six genomic loci, termed Drosophila amplicons in follicle cells (DAFCs) [10-12]. Precise developmental timing of DAFC origin firing permits identification of forks at defined points after origin initiation [13, 14]. Here, we show that DAFC re-replication causes fork instability and generates DSBs at sites of potential fork collisions. Immunofluorescence and ChIP-seq demonstrate the DSB marker gammaH2Av is enriched at elongating forks. Fork progression is reduced in the absence of DNA damage checkpoint components and nonhomologous end-joining (NHEJ), but not homologous recombination. NHEJ appears to continually repair forks during re-replication to maintain elongation. Full Text

AlvarezDominguez, J.R., Bai, Z., Xu, D., Yuan, B., Lo, K.A., Yoon, M.J., Lim, Y.C., Knoll, M., Slavov, N., Chen, S.,Harvey F. Lodish et al. (2015). De Novo Reconstruction of Adipose Tissue Transcriptomes Reveals Long Non-coding RNA Regulators of Brown Adipocyte Development. Cell metabolism Volume 21, Issue 5, p764–776 Brown adipose tissue (BAT) protects against obesity by promoting energy expenditure via uncoupled respiration. To uncover BAT-specific long non-coding RNAs (lncRNAs), we used RNA-seq to reconstruct de novo transcriptomes of mouse brown, inguinal white, and epididymal white fat and identified approximately 1,500 lncRNAs, including 127 BAT-restricted loci induced during differentiation and often targeted by key regulators PPARgamma, C/EBPalpha, and C/EBPbeta. One of them, lnc-BATE1, is required for establishment and maintenance of BAT identity and thermogenic capacity. lnc-BATE1 inhibition impairs concurrent activation of brown fat and repression of white fat genes and is partially rescued by exogenous lnc-BATE1 with mutated siRNA-targeting sites, demonstrating a function in trans. We show that lnc-BATE1 binds heterogeneous nuclear ribonucleoprotein U and that both are required for brown adipogenesis. Our work provides an annotated catalog for the study of fat depot-selective lncRNAs and establishes lnc-BATE1 as a regulator of BAT development and physiology. Full Text

Ashour, J., Schmidt, F.I., Hanke, L., Cragnolini, J., Cavallari, M., Altenburg, A., Brewer, R., Ingram, J., Shoemaker, C., and Ploegh, H.L. (2015). Intracellular Expression of Camelid Single-Domain Antibodies Specific for Influenza Virus Nucleoprotein Uncovers Distinct Features of Its Nuclear Localization. Journal of Virology 89, 2792-2800 Perturbation of protein-protein interactions relies mostly on genetic approaches or on chemical inhibition. Small RNA viruses, such as influenza A virus, do not easily lend themselves to the former approach, while chemical inhibition requires that the target protein be druggable. A lack of tools thus constrains the functional analysis of flu-encoded proteins. We generated a panel of camelid-derived single domain antibody fragments (VHHs) against influenza nucleoprotein (NP), a viral protein essential for nuclear trafficking and packaging of the influenza genome. We show that these VHHs can target NP in living cells and perturb NP's function during infection. Cytosolic expression of NP-specific VHHs (αNP-VHHs) disrupts virus replication at an early stage of the life cycle. Based on their specificity, these VHHs fall into two distinct groups. Both prevent nuclear import of the vRNP complex without disrupting nuclear import of NP alone. Different stages of the virus life cycle thus rely on distinct nuclear localization motifs of NP. Their molecular characterization may afford new means of intervention in the virus life cycle. Full Text.

Bagley, A.F., Scherz-Shouval, R., Galie, P.A., Zhang, A.Q., Wyckoff, J., Whitesell, L., Chen, C.S., Lindquist, S., and Bhatia, S.N. (2015). Endothelial thermotolerance impairs nanoparticle transport in tumors. Cancer research. [Epub ahead of print]. The delivery of diagnostic and therapeutic agents to solid tumors is limited by physical transport barriers within tumors, and such restrictions directly contribute to decreased therapeutic efficacy and the emergence of drug resistance. Nanomaterials designed to perturb the local tumor environment with precise spatiotemporal control have demonstrated potential to enhance drug delivery in preclinical models. Here, we investigated the ability of one class of heat-generating nanomaterials called plasmonic nanoantennae to enhance tumor transport in a xenograft model of ovarian cancer. We observed a temperature-dependent increase in the transport of diagnostic nanoparticles into tumors. However, a transient, reversible reduction in this enhanced transport was seen upon re-exposure to heating, consistent with the development of vascular thermotolerance. Harnessing these observations, we designed an improved treatment protocol combining plasmonic nanoantennae with diffusion-limited chemotherapies. Using a microfluidic endothelial model and genetic tools to inhibit the heat-shock response (HSR), we found that the ability of thermal preconditioning to limit heat-induced cytoskeletal disruption is an important component of vascular thermotolerance. This work therefore highlights the clinical relevance of cellular adaptations to nanomaterials and identifies molecular pathways whose modulation could improve the exposure of tumors to therapeutic agents. Full Text

Bement, W.M., Leda, M., Moe, A.M., Kita, A.M., Larson, M.E., Golding, A.E., Pfeuti, C., Su, K.C., Miller, A.L., Goryachev, A.B., et al. (2015). Activator-inhibitor coupling between Rho signalling and actin assembly makes the cell cortex an excitable medium. Nature cell biology. Published online. Animal cell cytokinesis results from patterned activation of the small GTPase Rho, which directs assembly of actomyosin in the equatorial cortex. Cytokinesis is restricted to a portion of the cell cycle following anaphase onset in which the cortex is responsive to signals from the spindle. We show that shortly after anaphase onset oocytes and embryonic cells of frogs and echinoderms exhibit cortical waves of Rho activity and F-actin polymerization. The waves are modulated by cyclin-dependent kinase 1 (Cdk1) activity and require the Rho GEF (guanine nucleotide exchange factor), Ect2. Surprisingly, during wave propagation, although Rho activity elicits F-actin assembly, F-actin subsequently inactivates Rho. Experimental and modelling results show that waves represent excitable dynamics of a reaction-diffusion system with Rho as the activator and F-actin the inhibitor. We propose that cortical excitability explains fundamental features of cytokinesis including its cell cycle regulation. Full Text

Birsoy, K., Wang, T., Chen, W.W., Freinkman, E., Abu-Remaileh, M., and Sabatini, D.M. (2015). An Essential Role of the Mitochondrial Electron Transport Chain in Cell Proliferation Is to Enable Aspartate Synthesis. Cell 162, 540-551.The mitochondrial electron transport chain (ETC) enables many metabolic processes, but why its inhibition suppresses cell proliferation is unclear. It is also not well understood why pyruvate supplementation allows cells lacking ETC function to proliferate. We used a CRISPR-based genetic screen to identify genes whose loss sensitizes human cells to phenformin, a complex I inhibitor. The screen yielded GOT1, the cytosolic aspartate aminotransferase, loss of which kills cells upon ETC inhibition. GOT1 normally consumes aspartate to transfer electrons into mitochondria, but, upon ETC inhibition, it reverses to generate aspartate in the cytosol, which partially compensates for the loss of mitochondrial aspartate synthesis. Pyruvate stimulates aspartate synthesis in a GOT1-dependent fashion, which is required for pyruvate to rescue proliferation of cells with ETC dysfunction. Aspartate supplementation or overexpression of an aspartate transporter allows cells without ETC activity to proliferate. Thus, enabling aspartate synthesis is an essential role of the ETC in cell proliferation. Full Text

Brennand, K.J., Marchetto, M.C., Benvenisty, N., Brustle, O., Ebert, A., Izpisua Belmonte, J.C., Kaykas, A., Lancaster, M.A., Livesey, F.J., McConnell, M.J., Frank Soldner, and Rudolf Jaenisch. (2015). Creating Patient-Specific Neural Cells for the In Vitro Study of Brain Disorders. Stem cell reports.[Epub ahead of print] As a group, we met to discuss the current challenges for creating meaningful patient-specific in vitro models to study brain disorders. Although the convergence of findings between laboratories and patient cohorts provided us confidence and optimism that hiPSC-based platforms will inform future drug discovery efforts, a number of critical technical challenges remain. This opinion piece outlines our collective views on the current state of hiPSC-based disease modeling and discusses what we see to be the critical objectives that must be addressed collectively as a field. Full Text

Brownstein, C.A., Beggs, A.H., Rodan, L., Shi, J., Towne, M.C., Pelletier, R., Cao, S., Rosenberg, P.A., Urion, D.K., Picker, J., et al. (2015). Clinical heterogeneity associated with KCNA1 mutations include cataplexy and nonataxic presentations. Neurogenetics[Epub ahead of print] 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

Burg, J.S., Ingram, J.R., Venkatakrishnan, A.J., Jude, K.M., Dukkipati, A., Feinberg, E.N., Angelini, A., Waghray, D., Dror, R.O., Ploegh, H.L., et al. (2015). Structural biology. Structural basis for chemokine recognition and activation of a viral G protein-coupled receptor. Science 347, 1113-1117.Chemokines are small proteins that function as immune modulators through activation of chemokine G protein-coupled receptors (GPCRs). Several viruses also encode chemokines and chemokine receptors to subvert the host immune response. How protein ligands activate GPCRs remains unknown. We report the crystal structure at 2.9 angstrom resolution of the human cytomegalovirus GPCR US28 in complex with the chemokine domain of human CX3CL1 (fractalkine). The globular body of CX3CL1 is perched on top of the US28 extracellular vestibule, whereas its amino terminus projects into the central core of US28. The transmembrane helices of US28 adopt an active-state-like conformation. Atomic-level simulations suggest that the agonist-independent activity of US28 may be due to an amino acid network evolved in the viral GPCR to destabilize the receptor's inactive state. Full Text.

Chaffer, C.L., and Weinberg, R.A. (2015). How does multistep tumorigenesis really proceed? Cancer discovery 5, 22-24. Identifying the cancer cells-of-origin is of great interest, as it holds the potential to elucidate biologic mechanisms inherent in the normal cell state that have been co-opted to drive the oncogenic cell state. An emerging concept, proposed here, states that cancer stem cells, key players in cancer initiation and metastasis, arise when transit-amplifying cells with mutant genomes dedifferentiate and enter the stem cell state. This model contrasts with the notion that cancer stem cells are the direct products of neoplastically transformed normal tissue stem cells. Cancer Discov; 5(1); 22-4. (c)2015 AACR. Full Text.

Chang, J.T., Lehtinen, M.K., and Sive, H. (2015). Zebrafish cerebrospinal fluid mediates cell survival through a retinoid signaling pathway. Developmental neurobiology (ahead of print). Cerebrospinal fluid (CSF) includes conserved factors whose function is largely unexplored. To assess the role of CSF during embryonic development, CSF was repeatedly drained from embryonic zebrafish brain ventricles soon after their inflation. Removal of CSF increased cell death in the diencephalon, indicating a survival function. Factors within the CSF are required for neuroepithelial cell survival as injected mouse CSF but not artificial CSF could prevent cell death after CSF depletion. Mass spectrometry analysis of the CSF identified retinol binding protein 4 (Rbp4), which transports retinol, the precursor to retinoic acid (RA). Consistent with a role for Rbp4 in cell survival, inhibition of Rbp4 or RA synthesis increased neuroepithelial cell death. Conversely, ventricle injection of exogenous human RBP4 plus retinol, or RA alone prevented cell death after CSF depletion. Zebrafish rbp4 is highly expressed in the yolk syncytial layer, suggesting Rbp4 protein and retinol/RA precursors can be transported into the CSF from the yolk. In accord with this suggestion, injection of human RBP4 protein into the yolk prevents neuroepithelial cell death in rbp4 loss-of-function embryos. Together, these data support the model that Rbp4 and RA precursors are present within the CSF and used for synthesis of RA, which promotes embryonic neuroepithelial survival. This article is protected by copyright. All rights reserved. PDF

Chantranupong, L., Wolfson, R.L., and Sabatini, D.M. (2015). Nutrient-Sensing Mechanisms across Evolution. Cell 161, 67-83.For organisms to coordinate their growth and development with nutrient availability, they must be able to sense nutrient levels in their environment. Here, we review select nutrient-sensing mechanisms in a few diverse organisms. We discuss how these mechanisms reflect the nutrient requirements of specific species and how they have adapted to the emergence of multicellularity in eukaryotes. Full Text.

Chen, G.Y., Li, Z., Theile, C.S., Bardhan, N.M., Kumar, P.V., Duarte, J.N., Maruyama, T., Rashidfarrokh, A., Belcher, A.M., and Ploegh, H.L. (2015). Graphene Oxide Nanosheets Modified with Single-Domain Antibodies for Rapid and Efficient Capture of Cells. Chemistry a European Journal [Epub ahead of print]. Peripheral blood can provide valuable information on an individual's immune status. Cell-based assays typically target leukocytes and their products. Characterization of leukocytes from whole blood requires their separation from the far more numerous red blood cells.1 Current methods to classify leukocytes, such as recovery on antibody-coated beads or fluorescence-activated cell sorting require long sample preparation times and relatively large sample volumes.2 A simple method that enables the characterization of cells from a small peripheral whole blood sample could overcome limitations of current analytical techniques. We describe the development of a simple graphene oxide surface coated with single-domain antibody fragments. This format allows quick and efficient capture of distinct WBC subpopulations from small samples ( approximately 30 muL) of whole blood in a geometry that does not require any specialized equipment such as cell sorters or microfluidic devices. Full Text

Chung, J., Bauer, D.E., Ghamari, A., Nizzi, C.P., Deck, K.M., Kingsley, P.D., Yien, Y.Y., Huston, N.C., Chen, C., Schultz, IJ, Harvey F. Lodish, et al. (2015). The mTORC1/4E-BP pathway coordinates hemoglobin production with L-leucine availability. Science signaling 8, ra34. In multicellular organisms, the mechanisms by which diverse cell types acquire distinct amino acids and how cellular function adapts to their availability are fundamental questions in biology. We found that increased neutral essential amino acid (NEAA) uptake was a critical component of erythropoiesis. As red blood cells matured, expression of the amino acid transporter gene Lat3 increased, which increased NEAA import. Inadequate NEAA uptake by pharmacologic inhibition or RNAi-mediated knockdown of LAT3 triggered a specific reduction in hemoglobin production in zebrafish embryos and murine erythroid cells through the mTORC1 (mammalian target of rapamycin complex 1)/4E-BP (eukaryotic translation initiation factor 4E–binding protein) pathway. CRISPR-mediated deletion of members of the 4E-BP family in murine erythroid cells rendered them resistant to mTORC1 and LAT3 inhibition and restored hemoglobin production. These results identify a developmental role for LAT3 in red blood cells and demonstrate that mTORC1 serves as a homeostatic sensor that couples hemoglobin production at the translational level to sufficient uptake of NEAAs, particularly l-leucine. Full Text

Cimmino, L., Dawlaty, M.M., Ndiaye-Lobry, D., Yap, Y.S., Bakogianni, S., Yu, Y., Bhattacharyya, S., Shaknovich, R., Geng, H., Lobry, C. ,Rudolph Jaenisch, et al. (2015). TET1 is a tumor suppressor of hematopoietic malignancy. Nature immunologyPublished online. The methylcytosine dioxygenase TET1 (‘ten-eleven translocation 1’) is an important regulator of 5-hydroxymethylcytosine (5hmC) in embryonic stem cells. The diminished expression of TET proteins and loss of 5hmC in many tumors suggests a critical role for the maintenance of this epigenetic modification. Here we found that deletion of Tet1 promoted the development of B cell lymphoma in mice. TET1 was required for maintenance of the normal abundance and distribution of 5hmC, which prevented hypermethylation of DNA, and for regulation of the B cell lineage and of genes encoding molecules involved in chromosome maintenance and DNA repair. Whole-exome sequencing of TET1-deficient tumors revealed mutations frequently found in non-Hodgkin B cell lymphoma (B-NHL), in which TET1 was hypermethylated and transcriptionally silenced. Our findings provide in vivo evidence of a function for TET1 as a tumor suppressor of hematopoietic malignancy. Full Text.

Corpas, M., Jimenez, R.C., Bongcam-Rudloff, E., Budd, A., Brazas, M.D., Fernandes, P.L., Gaeta, B., van Gelder, C., Korpelainen, E., Lewitter, F., et al. (2015). The GOBLET training portal: a global repository of bioinformatics training materials, courses and trainers. Bioinformatics 31, 140-142.A Summary: Rapid technological advances have led to an explosion of biomedical data in recent years. The pace of change has inspired new collaborative approaches for sharing materials and resources to help train life scientists both in the use of cutting-edge bioinformatics tools and databases and in how to analyse and interpret large datasets. A prototype platform for sharing such training resources was recently created by the Bioinformatics Training Network (BTN). Building on this work, we have created a centralized portal for sharing training materials and courses, including a catalogue of trainers and course organizers, and an announcement service for training events. For course organizers, the portal provides opportunities to promote their training events; for trainers, the portal offers an environment for sharing materials, for gaining visibility for their work and promoting their skills; for trainees, it offers a convenient one-stop shop for finding suitable training resources and identifying relevant training events and activities locally and worldwide. Full Text.

CortesCampos, C., Letelier, J., Ceriani, R., and Whitlock, K.E. (2015). Zebrafish adult-derived hypothalamic neurospheres generate gonadotropin-releasing hormone (GnRH) neurons. Biology open [Epub ahead of print] Gonadotropin-releasing hormone (GnRH) is a hypothalamic decapeptide essential for fertility in vertebrates. Human male patients lacking GnRH and treated with hormone therapy can remain fertile after cessation of treatment suggesting that new GnRH neurons can be generated during adult life. We used zebrafish to investigate the neurogenic potential of the adult hypothalamus. Previously we have characterized the development of GnRH cells in the zebrafish linking genetic pathways to the differentiation of neuromodulatory and endocrine GnRH cells in specific regions of the brain. Here, we developed a new method to obtain neural progenitors from the adult hypothalamus in vitro. Using this system, we show that neurospheres derived from the adult hypothalamus can be maintained in culture and subsequently differentiate glia and neurons. Importantly, the adult derived progenitors differentiate into neurons containing GnRH and the number of cells is increased through exposure to either testosterone or GnRH, hormones used in therapeutic treatment in humans. Finally, we show in vivo that a neurogenic niche in the hypothalamus contains GnRH positive neurons. Thus, we demonstrated for the first time that neurospheres can be derived from the hypothalamus of the adult zebrafish and that these neural progenitors are capable of producing GnRH containing neurons. Full Text

DAlessio, A.C., Fan, Z.P., Wert, K.J., Baranov, P., Cohen, M.A., Saini, J.S., Cohick, E., Charniga, C., Dadon, D., Hannett, N.M.,Michael J. Young,Sally Temple,Rudolf Jaenisch,Tong Ihn Lee,and Richard A. Young(2015). A Systematic Approach to Identify Candidate Transcription Factors that Control Cell Identity. Stem cell reports. [Epub ahead of print] Hundreds of transcription factors (TFs) are expressed in each cell type, but cell identity can be induced through the activity of just a small number of core TFs. Systematic identification of these core TFs for a wide variety of cell types is currently lacking and would establish a foundation for understanding the transcriptional control of cell identity in development, disease, and cell-based therapy. Here, we describe a computational approach that generates an atlas of candidate core TFs for a broad spectrum of human cells. The potential impact of the atlas was demonstrated via cellular reprogramming efforts where candidate core TFs proved capable of converting human fibroblasts to retinal pigment epithelial-like cells. These results suggest that candidate core TFs from the atlas will prove a useful starting point for studying transcriptional control of cell identity and reprogramming in many human cell types. Full Text

Davis, S.A., Vincent, B.M., Endo, M.M., Whitesell, L., Marchillo, K., Andes, D.R., Lindquist, S., and Burke, M.D. (2015). Nontoxic antimicrobials that evade drug resistance. Nature chemical biology Published online 01 June 2015 Drugs that act more promiscuously provide fewer routes for the emergence of resistant mutants. This benefit, however, often comes at the cost of serious off-target and dose-limiting toxicities. The classic example is the antifungal amphotericin B (AmB), which has evaded resistance for more than half a century. We report markedly less toxic amphotericins that nevertheless evade resistance. They are scalably accessed in just three steps from the natural product, and they bind their target (the fungal sterol ergosterol) with far greater selectivity than AmB. Hence, they are less toxic and far more effective in a mouse model of systemic candidiasis. To our surprise, exhaustive efforts to select for mutants resistant to these more selective compounds revealed that they are just as impervious to resistance as AmB. Thus, highly selective cytocidal action and the evasion of resistance are not mutually exclusive, suggesting practical routes to the discovery of less toxic, resistance-evasive therapies. Full Text

DeLosAngeles1, A., Ferrari, F., Fujiwara, Y., Mathieu, R., Lee, S., Tu, H.C., Ross, S., Chou, S., Nguyen, M., Wu, Z. ,Horold W. Theunissen ,Benjamin E. Powell, Sumeth Imsoonthornruksa, Rudolf Jaenisch,et al. (2015). Failure to replicate the STAP cell phenomenon. Nature 525, E6-9 In summary, our replication attempts and genetic analysis indicate that existing STAP protocols are neither robust nor reproducible. To substantiate future claims of reprogramming and alternative states of potency, we urge a rigorous application of several independent means for validating functional pluripotency and genomic profiling to confirm cell line provenance. Ultimately, the essential standard of robustness and reproducibility must be met for new claims to exert a positive and lasting influence on the research community. Full Text

DeLosAngeles2, A., Ferrari, F., Xi, R., Fujiwara, Y., Benvenisty, N., Deng, H., Hochedlinger, K., Jaenisch, R., Lee, S., Leitch, H.G., et al. (2015). Hallmarks of pluripotency. Nature 525, 469-478.Stem cells self-renew and generate specialized progeny through differentiation, but vary in the range of cells and tissues they generate, a property called developmental potency. Pluripotent stem cells produce all cells of an organism, while multipotent or unipotent stem cells regenerate only specific lineages or tissues. Defining stem-cell potency relies upon functional assays and diagnostic transcriptional, epigenetic and metabolic states. Here we describe functional and molecular hallmarks of pluripotent stem cells, propose a checklist for their evaluation, and illustrate how forensic genomics can validate their provenance. Full Text

Dettmer, U., Newman, A.J., Soldner, F., Luth, E.S., Kim, N.C., von Saucken, V.E., Sanderson, J.B., Jaenisch, R., Bartels, T., and Selkoe, D. (2015). Parkinson-causing alpha-synuclein missense mutations shift native tetramers to monomers as a mechanism for disease initiation. Nature communications 6, 7314. Beta-Sheet-rich alpha-synuclein (alphaS) aggregates characterize Parkinson's disease (PD). alphaS was long believed to be a natively unfolded monomer, but recent work suggests it also occurs in alpha-helix-rich tetramers. Crosslinking traps principally tetrameric alphaS in intact normal neurons, but not after cell lysis, suggesting a dynamic equilibrium. Here we show that freshly biopsied normal human brain contains abundant alphaS tetramers. The PD-causing mutation A53T decreases tetramers in mouse brain. Neurons derived from an A53T patient have decreased tetramers. Neurons expressing E46K do also, and adding 1-2 E46K-like mutations into the canonical alphaS repeat motifs (KTKEGV) further reduces tetramers, decreases alphaS solubility and induces neurotoxicity and round inclusions. The other three fPD missense mutations likewise decrease tetramer:monomer ratios. The destabilization of physiological tetramers by PD-causing missense mutations and the neurotoxicity and inclusions induced by markedly decreasing tetramers suggest that decreased alpha-helical tetramers and increased unfolded monomers initiate pathogenesis. Tetramer-stabilizing compounds should prevent this. Full Text

Deuse, T., Wang, D., Stubbendorff, M., Itagaki, R., Grabosch, A., Greaves, L.C., Alawi, M., Grunewald, A., Hu, X.M., Hua, X.Q.,Rudolf Jaenisch, et al. (2015). SCNT-Derived ESCs with Mismatched Mitochondria Trigger an Immune Response in Allogeneic Hosts. Cell Stem Cell 16, 33-38.The generation of pluripotent stem cells by somatic cell nuclear transfer (SCNT) has recently been achieved in human cells and sparked new interest in this technology. The authors reporting this methodical breakthrough speculated that SCNT would allow the creation of patient-matched embryonic stem cells, even in patients with hereditary mitochondrial diseases. However, herein we show that mismatched mitochondria in nuclear-transfer-derived embryonic stem cells (NT-ESCs) possess alloantigenicity and are subject to immune rejection. In a murine transplantation setup, we demonstrate that allogeneic mitochondria in NT-ESCs, which are nucleus-identical to the recipient, may trigger an adaptive alloimmune response that impairs the survival of NT-ESC grafts. The immune response is adaptive, directed against mitochondrial content, and amenable for tolerance induction. Mitochondrial alloantigenicity should therefore be considered when developing therapeutic SCNT-based strategies. Full Text.

Dura, B., Dougan, S.K., Barisa, M., Hoehl, M.M., Lo, C.T., Ploegh, H.L., and Voldman, J. (2015). Profiling lymphocyte interactions at the single-cell level by microfluidic cell pairing. Nature communications 6, 5940. Establishing a successful immune response requires cell-cell interactions, where the nature of antigen presentation dictates functional outcomes. Methods to study these interactions, however, suffer from limited throughput and a lack of control over cell pairing. Here we describe a microfluidic platform that achieves high-throughput deterministic pairing of lymphocytes with a defined contact time, thereby allowing accurate assessment of early activation events for each pair in controlled microenvironments. More importantly, the platform allows the capture of dynamic processes and static parameters from both partners simultaneously, thus enabling pairwise-correlated multiparametric profiling of lymphocyte interactions over hundreds of pairs in a single experiment. Using our platform, we characterized early activation dynamics of CD8 T cells (OT-1 and TRP1 transnuclear (TN)) and investigated the extent of heterogeneity in T-cell activation and the correlation of multiple readouts. The results establish our platform as a promising tool for quantitative investigation of lymphocyte interactions. Full Text.

Efeyan, A., Comb, W.C., and Sabatini, D.M. (2015). Nutrient-sensing mechanisms and pathways. Nature 517, 302-310.The ability to sense and respond to fluctuations in environmental nutrient levels is a requisite for life. Nutrient scarcity is a selective pressure that has shaped the evolution of most cellular processes. Different pathways that detect intracellular and extracellular levels of sugars, amino acids, lipids and surrogate metabolites are integrated and coordinated at the organismal level through hormonal signals. During food abundance, nutrient-sensing pathways engage anabolism and storage, whereas scarcity triggers homeostatic mechanisms, such as the mobilization of internal stores through autophagy. Nutrient-sensing pathways are commonly deregulated in human metabolic diseases. Full Text.

ElizondoVega, R., Cortes-Campos, C., Barahona, M.J., Oyarce, K.A., Carril, C.A., and Garcia-Robles, M.A. (2015). The role of tanycytes in hypothalamic glucosensing. Journal of cellular and molecular medicine. Vol XX, No X, 2015 pp. 1-12. Tanycytes are elongated hypothalamic glial cells that cover the basal walls of the third ventricle; their apical regions contact the cerebrospinal fluid (CSF), and their processes reach hypothalamic neuronal nuclei that control the energy status of an organism. These nuclei maintain the balance between energy expenditure and intake, integrating several peripheral signals and triggering cellular responses that modify the feeding behaviour and peripheral glucose homeostasis. One of the most important and well-studied signals that control this process is glucose; however, the mechanism by which this molecule is sensed remains unknown. We along with others have proposed that tanycytes play a key role in this process, transducing changes in CSF glucose concentration to the neurons that control energy status. Recent studies have demonstrated the expression and function of monocarboxylate transporters and canonical pancreatic beta cell glucose sensing molecules, including glucose transporter 2 and glucokinase, in tanycytes. These and other data, which will be discussed in this review, suggest that hypothalamic glucosensing is mediated through a metabolic interaction between tanycytes and neurons through lactate. This article will summarize the recent evidence that supports the importance of tanycytes in hypothalamic glucosensing, and discuss the possible mechanisms involved in this process. Finally, it is important to highlight that a detailed analysis of this mechanism could represent an opportunity to understand the evolution of associated pathologies, including diabetes and obesity, and identify new candidates for therapeutic intervention. Full Text

Endo, T., Romer, K.A., Anderson, E.L., Baltus, A.E., de Rooij, D.G., and Page, D.C. (2015). Periodic retinoic acid-STRA8 signaling intersects with periodic germ-cell competencies to regulate spermatogenesis. Proceedings of the National Academy of Sciences of the United States of America Early Edition. Mammalian spermatogenesis-the transformation of stem cells into millions of haploid spermatozoa-is elaborately organized in time and space. We explored the underlying regulatory mechanisms by genetically and chemically perturbing spermatogenesis in vivo, focusing on spermatogonial differentiation, which begins a series of amplifying divisions, and meiotic initiation, which ends these divisions. We first found that, in mice lacking the retinoic acid (RA) target gene Stimulated by retinoic acid gene 8 (Stra8), undifferentiated spermatogonia accumulated in unusually high numbers as early as 10 d after birth, whereas differentiating spermatogonia were depleted. We thus conclude that Stra8, previously shown to be required for meiotic initiation, also promotes (but is not strictly required for) spermatogonial differentiation. Second, we found that injection of RA into wild-type adult males induced, independently, precocious spermatogonial differentiation and precocious meiotic initiation; thus, RA acts instructively on germ cells at both transitions. Third, the competencies of germ cells to undergo spermatogonial differentiation or meiotic initiation in response to RA were found to be distinct, periodic, and limited to particular seminiferous stages. Competencies for both transitions begin while RA levels are low, so that the germ cells respond as soon as RA levels rise. Together with other findings, our results demonstrate that periodic RA-STRA8 signaling intersects with periodic germ-cell competencies to regulate two distinct, cell-type-specific responses: spermatogonial differentiation and meiotic initiation. This simple mechanism, with one signal both starting and ending the amplifying divisions, contributes to the prodigious output of spermatozoa and to the elaborate organization of spermatogenesis. PDF

Erdmann, R.M., Souza, A.L., Clish, C.B., and Gehring, M. (2015). 5-Hydroxymethylcytosine Is Not Present in Appreciable Quantities in Arabidopsis DNA. G3-Genes Genomes Genetics 5, 1-8.5-Hydroxymethylcytosine (5-hmC) is an intermediate in active demethylation in metazoans, as well as a potentially stable epigenetic mark. Previous reports investigating 5-hydroxymethylcytosine in plants have reached conflicting conclusions. We systematically investigated whether 5-hmC is present in plant DNA using a range of methods. Using the model organism Arabidopsis thaliana, in addition to other plant species, we assayed the amount or distribution of 5-hydroxymethylcytosine by thin-layer chromatography, immunoprecipitation-chip, ELISA, enzymatic radiolabeling, and mass spectrometry. The failure to observe 5-hydroxymethylcytosine by thin-layer chromatography established an upper bound for the possible fraction of the nucleotide in plant DNA. Antibody-based methods suggested that there were low levels of 5-hmC in plant DNA, but these experiments were potentially confounded by cross-reactivity with the abundant base 5-methylcytosine. Enzymatic radiolabeling and mass spectrometry, the most sensitive methods for detection that we used, failed to detect 5-hydroxymethylcytosine in A. thaliana genomic DNA isolated from a number of different tissue types and genetic backgrounds. Taken together, our results led us to conclude that 5-hmC is not present in biologically relevant quantities within plant genomic DNA. Full Text.

Fang, W., and Bartel, D.P. (2015). The Menu of Features that Define Primary MicroRNAs and Enable De Novo Design of MicroRNA Genes. Mol Cell [Epub ahead of print] MicroRNAs (miRNAs) are small regulatory RNAs processed from stem-loop regions of primary transcripts (pri-miRNAs), with the choice of stem loops for initial processing largely determining what becomes a miRNA. To identify sequence and structural features influencing this choice, we determined cleavage efficiencies of >50,000 variants of three human pri-miRNAs, focusing on the regions intractable to previous high-throughput analyses. Our analyses revealed a mismatched motif in the basal stem region, a preference for maintaining or improving base pairing throughout the remainder of the stem, and a narrow stem-length preference of 35 +/- 1 base pairs. Incorporating these features with previously identified features, including three primary-sequence motifs, yielded a unifying model defining mammalian pri-miRNAs in which motifs help orient processing and increase efficiency, with the presence of more motifs compensating for structural defects. This model enables generation of artificial pri-miRNAs, designed de novo, without reference to any natural sequence yet processed more efficiently than natural pri-miRNAs. Full Text

Frawley, L.E., and Orr-Weaver, T.L. (2015). Polyploidy. Current biology : CB 25, R353-358.Polyploidy is defined as an increase in genome DNA content. Throughout the plant and animal kingdoms specific cell types become polyploid as part of their differentiation programs. When this occurs in subsets of tissues within an organism it is termed somatic polyploidy, because it is distinct from the increase in ploidy that is inherited through the germline and present in every cell type of the organism. Germline polyploidy is common in plants and occurs in some animals, such as amphibians, but will not be discussed further here. Somatic polyploid cells can be mononucleate or multinucleate, and the replicated sister chromatids can remain attached and aligned, producing polytene chromosomes, or they can be dispersed (Figure 1). In this Primer, we focus on why somatic polyploidy occurs and how cells become polyploid - the first of these issues being more speculative, given the status of the field. Full Text

Frederick, K.K., Michaelis, V.K., Corzilius, B., Ong, T.C., Jacavone, A.C., Griffin, R.G., and Lindquist, S. (2015). Sensitivity-Enhanced NMR Reveals Alterations in Protein Structure by Cellular Milieus. Cell
163, 620–628 Biological processes occur in complex environments containing a myriad of potential interactors. Unfortunately, limitations on the sensitivity of biophysical techniques normally restrict structural investigations to purified systems, at concentrations that are orders of magnitude above endogenous levels. Dynamic nuclear polarization (DNP) can dramatically enhance the sensitivity of nuclear magnetic resonance (NMR) spectroscopy and enable structural studies in biologically complex environments. Here, we applied DNP NMR to investigate the structure of a protein containing both an environmentally sensitive folding pathway and an intrinsically disordered region, the yeast prion protein Sup35. We added an exogenously prepared isotopically labeled protein to deuterated lysates, rendering the biological environment "invisible" and enabling highly efficient polarization transfer for DNP. In this environment, structural changes occurred in a region known to influence biological activity but intrinsically disordered in purified samples. Thus, DNP makes structural studies of proteins at endogenous levels in biological contexts possible, and such contexts can influence protein structure. Full Text

Gao, F.J., Hebbar, S., Gao, X.A., Alexander, M., Pandey, J.P., Walla, M.D., Cotham, W.E., King, S.J., and Smith, D.S. (2015). GSK-3beta phosphorylation of cytoplasmic dynein reduces Ndel1 binding to intermediate chains and alters dynein motility. Traffic [Epub ahead of print] Glycogen synthase kinase 3 (GSK-3) has been linked to regulation of kinesin-dependent axonal transport in squid and flies, and to indirect regulation of cytoplasmic dynein. We have now found evidence for direct regulation of dynein by mammalian GSK-3beta in both neurons and non-neuronal cells. GSK-3beta coprecipitates with and phosphorylates mammalian dynein. Phosphorylation of dynein intermediate chain (IC) reduces its interaction with Ndel1, a protein that contributes to dynein force generation. Two conserved residues, S87/T88 in IC-1B and S88/T89 in IC-2C, have been identified as GSK-3 targets by both mass spectrometry and site-directed mutagenesis. These sites are within a Ndel1-binding domain, and mutation of both sites alters the interaction of IC's with Ndel1. Dynein motility is stimulated by 1) pharmacological and genetic inhibition of GSK-3beta; 2) an insulin-sensitizing agent (rosiglitazone); 3) manipulating an insulin response pathway that leads to GSK-3beta inactivation. Thus our study connects a well-characterized insulin-signaling pathway directly to dynein stimulation via GSK-3 inhibition. PDF

Gold, D.A., Kaplan, A.D., Lis, A., Bett, G.C., Rosowski, E.E., Cirelli, K.M., Bougdour, A., Sidik, S.M., Beck, J.R., Lourido, S., et al. (2015). The Toxoplasma Dense Granule Proteins GRA17 and GRA23 Mediate the Movement of Small Molecules between the Host and the Parasitophorous Vacuole. Cell Host Microbe 17, 642-652.Toxoplasma gondii is a protozoan pathogen in the phylum Apicomplexa that resides within an intracellular parasitophorous vacuole (PV) that is selectively permeable to small molecules through unidentified mechanisms. We have identified GRA17 as a Toxoplasma-secreted protein that localizes to the parasitophorous vacuole membrane (PVM) and mediates passive transport of small molecules across the PVM. GRA17 is related to the putative Plasmodium translocon protein EXP2 and conserved across PV-residing Apicomplexa. The PVs of GRA17-deficient parasites have aberrant morphology, reduced permeability to small molecules, and structural instability. GRA17-deficient parasites proliferate slowly and are avirulent in mice. These GRA17-deficient phenotypes are rescued by complementation with Plasmodium EXP2. GRA17 functions synergistically with a related protein, GRA23. Exogenous expression of GRA17 or GRA23 alters the membrane conductance properties of Xenopus oocytes in a manner consistent with a large non-selective pore. Thus, GRA17 and GRA23 provide a molecular basis for PVM permeability and nutrient access. Full Text

Gymrek, M., Willems, T., Guilmatre, A., Zeng, H., Markus, B., Georgiev, S., Daly, M.J., Price, A.L., Pritchard, J.K., Sharp, A.J.and Yaniv Erlich. (2015). Abundant contribution of short tandem repeats to gene expression variation in humans.Nature Genetics advance online publication. The contribution of repetitive elements to quantitative human traits is largely unknown. Here we report a genome-wide survey of the contribution of short tandem repeats (STRs), which constitute one of the most polymorphic and abundant repeat classes, to gene expression in humans. Our survey identified 2,060 significant expression STRs (eSTRs). These eSTRs were replicable in orthogonal populations and expression assays. We used variance partitioning to disentangle the contribution of eSTRs from that of linked SNPs and indels and found that eSTRs contribute 10-15% of the cis heritability mediated by all common variants. Further functional genomic analyses showed that eSTRs are enriched in conserved regions, colocalize with regulatory elements and may modulate certain histone modifications. By analyzing known genome-wide association study (GWAS) signals and searching for new associations in 1,685 whole genomes from deeply phenotyped individuals, we found that eSTRs are enriched in various clinically relevant conditions. These results highlight the contribution of STRs to the genetic architecture of quantitative human traits. Full Text

Han, X., Chen, Y., Wang, L., Fang, W., Zhang, N., and Zhu, Q. (2015). RNA: An Expanding View of Function and Evolution. Evolutionary bioinformatics online 11, 77-79.The sequencing capability behind RNA-seq is still progressing at an astonishing pace, with the cost of sequencing the same sample dropping exponentially. The scope of transcriptome characterization is continually expanding, especially fueled by large-scale tissue-specific and developmental-stage-specific studies.12 We expect more such studies will emerge in the next few years to further push the limit. The accumulation of expression data, associated with fast increasing genotype information, will lead to a comprehensive identification of functional regulatory variants and deeper understanding of mechanisms of gene regulation. .Full Text

Herman, J.D., Pepper, L.R., Cortese, J.F., Estiu, G., Galinsky, K., Zuzarte-Luis, V., Derbyshire, E.R., Ribacke, U., Lukens, A.K., Santos, S.A., et al. (2015). The cytoplasmic prolyl-tRNA synthetase of the malaria parasite is a dual-stage target of febrifugine and its analogs. Science translational medicine 7, 288ra277.The emergence of drug resistance is a major limitation of current antimalarials. The discovery of new druggable targets and pathways including those that are critical for multiple life cycle stages of the malaria parasite is a major goal for developing next-generation antimalarial drugs. Using an integrated chemogenomics approach that combined drug resistance selection, whole-genome sequencing, and an orthogonal yeast model, we demonstrate that the cytoplasmic prolyl-tRNA (transfer RNA) synthetase (PfcPRS) of the malaria parasite Plasmodium falciparum is a biochemical and functional target of febrifugine and its synthetic derivative halofuginone. Febrifugine is the active principle of a traditional Chinese herbal remedy for malaria. We show that treatment with febrifugine derivatives activated the amino acid starvation response in both P. falciparum and a transgenic yeast strain expressing PfcPRS. We further demonstrate in the Plasmodium berghei mouse model of malaria that halofuginol, a new halofuginone analog that we developed, is active against both liver and asexual blood stages of the malaria parasite. Halofuginol, unlike halofuginone and febrifugine, is well tolerated at efficacious doses and represents a promising lead for the development of dual-stage next-generation antimalarials. Full Text

Hezroni, H., Koppstein, D., Schwartz, M.G., Avrutin, A., Bartel, D.P., and Ulitsky, I. (2015). Principles of Long Noncoding RNA Evolution Derived from Direct Comparison of Transcriptomes in 17 Species. Cell reports Available online 7 May 2015. The inability to predict long noncoding RNAs from genomic sequence has impeded the use of comparative genomics for studying their biology. Here, we develop methods that use RNA sequencing (RNA-seq) data to annotate the transcriptomes of 16 vertebrates and the echinoid sea urchin, uncovering thousands of previously unannotated genes, most of which produce long intervening noncoding RNAs (lincRNAs). Although in each species, >70% of lincRNAs cannot be traced to homologs in species that diverged >50 million years ago, thousands of human lincRNAs have homologs with similar expression patterns in other species. These homologs share short, 5'-biased patches of sequence conservation nested in exonic architectures that have been extensively rewired, in part by transposable element exonization. Thus, over a thousand human lincRNAs are likely to have conserved functions in mammals, and hundreds beyond mammals, but those functions require only short patches of specific sequences and can tolerate major changes in gene architecture. Full Text

Hnisz, D., Schuijers, J., Lin, C.Y., Weintraub, A.S., Abraham, B.J., Lee, T.I., Bradner, J.E., and Young, R.A. (2015). Convergence of Developmental and Oncogenic Signaling Pathways at Transcriptional Super-Enhancers. Mol Cell [Epub ahead of print] Super-enhancers and stretch enhancers (SEs) drive expression of genes that play prominent roles in normal and disease cells, but the functional importance of these clustered enhancer elements is poorly understood, so it is not clear why genes key to cell identity have evolved regulation by such elements. Here, we show that SEs consist of functional constituent units that concentrate multiple developmental signaling pathways at key pluripotency genes in embryonic stem cells and confer enhanced responsiveness to signaling of their associated genes. Cancer cells frequently acquire SEs at genes that promote tumorigenesis, and we show that these genes are especially sensitive to perturbation of oncogenic signaling pathways. Super-enhancers thus provide a platform for signaling pathways to regulate genes that control cell identity during development and tumorigenesis. Full Text.

Hochedlinger, K., and Jaenisch, R. (2015). Induced Pluripotency and Epigenetic Reprogramming. Cold Spring Harbor perspectives in biology.Dec 1;7(12). pii: a019448. Induced pluripotency defines the process by which somatic cells are converted into induced pluripotent stem cells (iPSCs) upon overexpression of a small set of transcription factors. In this article, we put transcription factor-induced pluripotency into a historical context, review current methods to generate iPSCs, and discuss mechanistic insights that have been gained into the process of reprogramming. In addition, we focus on potential therapeutic applications of induced pluripotency and emerging technologies to efficiently engineer the genomes of human pluripotent cells for scientific and therapeutic purposes. Full Text

Hu, Y.C., Nicholls, P.K., Soh, Y.Q., Daniele, J.R., Junker, J.P., van Oudenaarden, A., and Page, D.C. (2015). Licensing of Primordial Germ Cells for Gametogenesis Depends on Genital Ridge Signaling. PLoS genetics 11, e1005019.In mouse embryos at mid-gestation, primordial germ cells (PGCs) undergo licensing to become gametogenesis-competent cells (GCCs), gaining the capacity for meiotic initiation and sexual differentiation. GCCs then initiate either oogenesis or spermatogenesis in response to gonadal cues. Germ cell licensing has been considered to be a cell-autonomous and gonad-independent event, based on observations that some PGCs, having migrated not to the gonad but to the adrenal gland, nonetheless enter meiosis in a time frame parallel to ovarian germ cells -- and do so regardless of the sex of the embryo. Here we test the hypothesis that germ cell licensing is cell-autonomous by examining the fate of PGCs in Gata4 conditional mutant (Gata4 cKO) mouse embryos. Gata4, which is expressed only in somatic cells, is known to be required for genital ridge initiation. PGCs in Gata4 cKO mutants migrated to the area where the genital ridge, the precursor of the gonad, would ordinarily be formed. However, these germ cells did not undergo licensing and instead retained characteristics of PGCs. Our results indicate that licensing is not purely cell-autonomous but is induced by the somatic genital ridge. Full Text.

Huang, Y.H., Zhu, C., Kondo, Y., Anderson, A.C., Gandhi, A., Russell, A., Dougan, S.K., Petersen, B.S., Melum, E., Pertel, T,.Hidde L. Ploegh, et al. (2015). CEACAM1 regulates TIM-3-mediated tolerance and exhaustion. Nature 517, 386-U566.T-cell immunoglobulin domain and mucin domain-3 (TIM-3, also known as HAVCR2) is an activation-induced inhibitory molecule involved in tolerance and shown to induce T-cell exhaustion in chronic viral infection and cancers'. Under some conditions, TIM-3 expression has also been shown to be stimulatory. Considering that TIM-3, like cytotoxic T lymphocyte antigen 4 (CTLA-4) and programmed death 1 (PD-1), is being targeted for cancer immunotherapy, it is important to identify the circumstances under which TIM-3 can inhibit and activate T-cell responses. Here we show that TIM-3 is co-expressed and forms a heterodimer with carcinoembryonic antigen cell adhesion molecule 1 (CEACAM1), another well-known molecule expressed on activated T cells and involved in T-cell inhibition(6-10). Biochemical, biophysical and X-ray crystallography studies show that the membranedistal immunoglobulin-variable (IgV) -like amino-terminal domain of each is crucial to these interactions. The presence of CEACAM 1 endows TIM-3 with inhibitory function. CEACAM1 facilitates the maturation and cell surface expression of TIM-3 by forming a heterodimeric interaction in cis through the highly related membranedistal N-terminal domains of each molecule. CEACAM 1 and TIM-3 also bind in trans through their N-terminal domains. Both cis and trans interactions between CEACAM1 and TIM-3 determine the tolerance-inducing function of TIM-3. In a mouse adoptive transfer colitis model, CEACAM 1 -deficient T cells are hyper-inflammatory with reduced cell surface expression of TIM-3 and regulatory cytokines, and this is restored by T-cell-specific CEACAM 1 expression. During chronic viral infection and in a tumour environment, CEACAM1 and TIM-3 mark exhausted T cells. Co-blockade of CEACAM1 and TIM-3 leads to enhancement of anti-tumour immune responses with improved elimination of tumours in mouse colorectal cancer models. Thus, CEACAM 1 serves as a heterophilic ligand for TIM-3 that is required for its ability to mediate T-cell inhibition, and this interaction has a crucial role in regulating autoimmunity and anti-tumour immunity. Full Text.

Hughes2and Y chromosomes evolved from ordinary autosomes beginning at least 180 million years ago. Despite their shared ancestry, mammalian Y chromosomes display enormous variation among species in size, gene content, and structural complexity. Several unique features of the Y chromosome-its lack of a homologous partner for crossing over, its functional specialization for spermatogenesis, and its high degree of sequence amplification-contribute to this extreme variation. However, amid this evolutionary turmoil many commonalities have been revealed that have contributed to our understanding of the selective pressures driving the evolution and biology of the Y chromosome. Two biological themes have defined Y-chromosome research over the past six decades: testis determination and spermatogenesis. A third biological theme begins to emerge from recent insights into the Y chromosome's roles beyond the reproductive tract-a theme that promises to broaden the reach of Y-chromosome research by shedding light on fundamental sex differences in human health and disease. Expected final online publication date for the Annual Review of Genetics Volume 49 is November 23, 2015. Please see http://www.annualreviews.org/catalog/pubdates.aspx for revised estimates. Full Text

Hughes, J.F., Skaletsky, H., Koutseva, N., Pyntikova, T., and Page, D.C. (2015). Sex chromosome-to-autosome transposition events counter Y-chromosome gene loss in mammals. Genome biology 16, 104. BACKGROUND: Although the mammalian X and Y chromosomes evolved from a single pair of autosomes, they are highly differentiated: the Y chromosome is dramatically smaller than the X and has lost most of its genes. The surviving genes are a specialized set with extraordinary evolutionary longevity. Most mammalian lineages have experienced delayed, or relatively recent, loss of at least one conserved Y-linked gene. An extreme example of this phenomenon is in the Japanese spiny rat, where the Y chromosome has disappeared altogether. In this species, many Y-linked genes were rescued by transposition to new genomic locations, but until our work presented here, this has been considered an isolated case. RESULTS: We describe eight cases of genes that have relocated to autosomes in mammalian lineages where the corresponding Y-linked gene has been lost. These gene transpositions originated from either the X or Y chromosomes, and are observed in diverse mammalian lineages: occurring at least once in marsupials, apes, and cattle, and at least twice in rodents and marmoset. For two genes - EIF1AX/Y and RPS4X/Y - transposition to autosomes occurred independently in three distinct lineages. CONCLUSIONS: Rescue of Y-linked gene loss through transposition to autosomes has previously been reported for a single isolated rodent species. However, our findings indicate that this compensatory mechanism is widespread among mammalian species. Thus, Y-linked gene loss emerges as an additional driver of gene transposition from the sex chromosomes, a phenomenon thought to be driven primarily by meiotic sex chromosome inactivation. Full Text

Ingram, J.R., Knockenhauer, K.E., Markus, B.M., Mandelbaum, J., Ramek, A., Shan, Y., Shaw, D.E., Schwartz, T.U., Ploegh, H.L., and Lourido, S. (2015). Allosteric activation of apicomplexan calcium-dependent protein kinases. Proceedings of the National Academy of Sciences of the United States of America Vol. 112 no. 36, E4975–E4984. Calcium-dependent protein kinases (CDPKs) comprise the major group of Ca2+-regulated kinases in plants and protists. It has long been assumed that CDPKs are activated, like other Ca2+-regulated kinases, by derepression of the kinase domain (KD). However, we found that removal of the autoinhibitory domain from Toxoplasma gondii CDPK1 is not sufficient for kinase activation. From a library of heavy chain-only antibody fragments (VHHs), we isolated an antibody (1B7) that binds TgCDPK1 in a conformation-dependent manner and potently inhibits it. We uncovered the molecular basis for this inhibition by solving the crystal structure of the complex and simulating, through molecular dynamics, the effects of 1B7-kinase interactions. In contrast to other Ca2+-regulated kinases, the regulatory domain of TgCDPK1 plays a dual role, inhibiting or activating the kinase in response to changes in Ca2+ concentrations. We propose that the regulatory domain of TgCDPK1 acts as a molecular splint to stabilize the otherwise inactive KD. This dependence on allosteric stabilization reveals a novel susceptibility in this important class of parasite enzymes. Full Text

Jalali, M., and Saldanha, F. (2015). Introducing the Plastic Surgery Research Network: A Comprehensive Online Platform to Facilitate Interdisciplinary Plastic Surgery Research. Plastic and Reconstructive Surgery 135, 1083E-1085E. Full text

Jensen, K.D., Camejo, A., Melo, M.B., Cordeiro, C., Julien, L., Grotenbreg, G.M., Frickel, E.M., Ploegh, H.L., Young, L., and Saeij, J.P. (2015). Toxoplasma gondii Superinfection and Virulence during Secondary Infection Correlate with the Exact ROP5/ROP18 Allelic Combination. mBio 6 ( 2) e02280-14. The intracellular parasite Toxoplasma gondii infects a wide variety of vertebrate species globally. Infection in most hosts causes a lifelong chronic infection and generates immunological memory responses that protect the host against new infections. In regions where the organism is endemic, multiple exposures to T. gondii likely occur with great frequency, yet little is known about the interaction between a chronically infected host and the parasite strains from these areas. A widely used model to explore secondary infection entails challenge of chronically infected or vaccinated mice with the highly virulent type I RH strain. Here, we show that although vaccinated or chronically infected C57BL/6 mice are protected against the type I RH strain, they are not protected against challenge with most strains prevalent in South America or another type I strain, GT1. Genetic and genomic analyses implicated the parasite-secreted rhoptry effectors ROP5 and ROP18, which antagonize the host's gamma interferon-induced immunity-regulated GTPases (IRGs), as primary requirements for virulence during secondary infection. ROP5 and ROP18 promoted parasite superinfection in the brains of challenged survivors. We hypothesize that superinfection may be an important mechanism to generate T. gondii strain diversity, simply because two parasite strains would be present in a single meal consumed by the feline definitive host. Superinfection may drive the genetic diversity of Toxoplasma strains in South America, where most isolates are IRG resistant, compared to North America, where most strains are IRG susceptible and are derived from a few clonal lineages. In summary, ROP5 and ROP18 promote Toxoplasma virulence during reinfection. IMPORTANCE: Toxoplasma gondii is a widespread parasite of warm-blooded animals and currently infects one-third of the human population. A long-standing assumption in the field is that prior exposure to this parasite protects the host from subsequent reexposure, due to the generation of protective immunological memory. However, this assumption is based on clinical data and mouse models that analyze infections with strains common to Europe infections with strains common to Europe and North America. In contrast, we found that the majority of strains sampled from around the world, in particular those from South America, were able to kill or reinfect the brains of hosts previously exposed to T. gondii. The T. gondii virulence factors ROP5 and ROP18, which inhibit key host effectors that mediate parasite killing, were required for these phenotypes. We speculate that these results underpin clinical observations that pregnant women previously exposed to Toxoplasma can develop congenital infection upon reexposure to South American strains. Full Text.

Ji2, X., Dadon, D.B., Powell, B.E., Fan, Z.P., Borges-Rivera, D., Shachar, S., Weintraub, A.S., Hnisz, D., Pegoraro, G., Lee, T.I., Tom Misteli, Rudolf Jaenisch, and Richard A. Young (2015). 3D Chromosome Regulatory Landscape of Human Pluripotent Cells. Cell Stem Cell Online Now In this study, we describe the 3D chromosome regulatory landscape of human naive and primed embryonic stem cells. To devise this map, we identified transcriptional enhancers and insulators in these cells and placed them within the context of cohesin-associated CTCF-CTCF loops using cohesin ChIA-PET data. The CTCF-CTCF loops we identified form a chromosomal framework of insulated neighborhoods, which in turn form topologically associating domains (TADs) that are largely preserved during the transition between the naive and primed states. Regulatory changes in enhancer-promoter interactions occur within insulated neighborhoods during cell state transition. The CTCF anchor regions we identified are conserved across species, influence gene expression, and are a frequent site of mutations in cancer cells, underscoring their functional importance in cellular regulation. These 3D regulatory maps of human pluripotent cells therefore provide a foundation for future interrogation of the relationships between chromosome structure and gene control in development and disease. Full Text

Ji, X., Dadon, D.B., Abraham, B.J., Lee, T.I., Jaenisch, R., Bradner, J.E., and Young, R.A. (2015). Chromatin proteomic profiling reveals novel proteins associated with histone-marked genomic regions. Proceedings of the National Academy of Sciences of the United States of America[Epub ahead of print] More than a thousand proteins are thought to contribute to mammalian chromatin and its regulation, but our understanding of the genomic occupancy and function of most of these proteins is limited. Here we describe an approach, which we call "chromatin proteomic profiling," to identify proteins associated with genomic regions marked by specifically modified histones. We used ChIP-MS to identify proteins associated with genomic regions marked by histones modified at specific lysine residues, including H3K27ac, H3K4me3, H3K79me2, H3K36me3, H3K9me3, and H4K20me3, in ES cells. We identified 332 known and 114 novel proteins associated with these histone-marked genomic segments. Many of the novel candidates have been implicated in various diseases, and their chromatin association may provide clues to disease mechanisms. More than 100 histone modifications have been described, so similar chromatin proteomic profiling studies should prove to be valuable for identifying many additional chromatin-associated proteins in a broad spectrum of cell types. Full Text.

Kang, M., Eisen, T.J., Eisen, E.A., Chakraborty, A.K., and Eisen, H.N. (2015). Affinity Inequality among Serum Antibodies That Originate in Lymphoid Germinal Centers. PloS one 10, e0139222.Upon natural infection with pathogens or vaccination, antibodies are produced by a process called affinity maturation. As affinity maturation ensues, average affinity values between an antibody and ligand increase with time. Purified antibodies isolated from serum are invariably heterogeneous with respect to their affinity for the ligands they bind, whether macromolecular antigens or haptens (low molecular weight approximations of epitopes on antigens). However, less is known about how the extent of this heterogeneity evolves with time during affinity maturation. To shed light on this issue, we have taken advantage of previously published data from Eisen and Siskind (1964). Using the ratio of the strongest to the weakest binding subsets as a metric of heterogeneity (or affinity inequality), we analyzed antibodies isolated from individual serum samples. The ratios were initially as high as 50-fold, and decreased over a few weeks after a single injection of small antigen doses to around unity. This decrease in the effective heterogeneity of antibody affinities with time is consistent with Darwinian evolution in the strong selection limit. By contrast, neither the average affinity nor the heterogeneity evolves much with time for high doses of antigen, as competition between clones of the same affinity is minimal. Full Text

Katajisto, P., Dohla, J., Chaffer, C., Pentinmikko, N., Marjanovic, N., Iqbal, S., Zoncu, R., Chen, W., Weinberg, R.A., and Sabatini, D.M. (2015). Asymmetric apportioning of aged mitochondria between daughter cells is required for stemness. Science. Published Online April 2 2015. By dividing asymmetrically, stem cells can generate two daughter cells with distinct fates. However, evidence is limited in mammalian systems for the selective apportioning of subcellular contents between daughters. We followed the fates of old and young organelles during the division of human mammary stemlike cells and found that such cells apportion aged mitochondria asymmetrically between daughter cells. Daughter cells that received fewer old mitochondria maintained stem cell traits. Inhibition of mitochondrial fission disrupted both the age-dependent subcellular localization and segregation of mitochondria and caused loss of stem cell properties in the progeny cells. Hence, mechanisms exist for mammalian stemlike cells to asymmetrically sort aged and young mitochondria, and these are important for maintaining stemness properties. Full Text.

Khurana, V., Tardiff, D.F., Chung, C.Y., and Lindquist, S. (2015). Toward stem cell-based phenotypic screens for neurodegenerative diseases. Nature reviews Neurology. Published online19 May 2015. In the absence of a single preventive or disease-modifying strategy, neurodegenerative diseases are becoming increasingly prevalent in our ageing population. The mechanisms underlying neurodegeneration are poorly understood, making the target-based drug screening strategies that are employed by the pharmaceutical industry fraught with difficulty. However, phenotypic screening in neurons and glia derived from patients is now conceivable through unprecedented developments in reprogramming, transdifferentiation, and genome editing. We outline progress in this nascent field, but also consider the formidable hurdles to identifying robust, disease-relevant and screenable cellular phenotypes in patient-derived cells. We illustrate how analysis in the simple baker's yeast cell Saccharaomyces cerevisiae is driving discovery in patient-derived neurons, and how approaches in this model organism can establish a paradigm to guide the development of stem cell-based phenotypic screens. Full Text

Kim, Dohoon , Kivanc Birsoy, Elizaveta Freinkman, Richard L. Possemato, Yakov Chudnovsky, Michael E. Pacold ,Walter W. Chen, Jason R. Cantor ,Manjae Kwon, Seong Woo Kang, and David M. Sabatini. (2015). SHMT2 drives glioma cell survival in ischaemia but imposes a dependence on glycine clearance. Nature Published online. Cancer cells adapt their metabolic processes to support rapid proliferation, but less is known about how cancer cells alter metabolism to promote cell survival in a poorly vascularized tumour microenvironment. Here we identify a key role for serine and glycine metabolism in the survival of brain cancer cells within the ischaemic zones of gliomas. In human glioblastoma multiforme, mitochondrial serine hydroxymethyltransferase (SHMT2) and glycine decarboxylase (GLDC) are highly expressed in the pseudopalisading cells that surround necrotic foci. We find that SHMT2 activity limits that of pyruvate kinase (PKM2) and reduces oxygen consumption, eliciting a metabolic state that confers a profound survival advantage to cells in poorly vascularized tumour regions. GLDC inhibition impairs cells with high SHMT2 levels as the excess glycine not metabolized by GLDC can be converted to the toxic molecules aminoacetone and methylglyoxal. Thus, SHMT2 is required for cancer cells to adapt to the tumour environment, but also renders these cells sensitive to glycine cleavage system inhibition. Full Text

Knoll, M., Lodish, H.F., and Sun, L. (2015). Long non-coding RNAs as regulators of the endocrine system. Nature Reviews Endocrinology[Epub ahead of print] Long non-coding RNAs (lncRNAs) are a large and diverse group of RNAs that are often lineage-specific and that regulate multiple biological functions. Many are nuclear and are essential parts of ribonucleoprotein complexes that modify chromatin segments and establish active or repressive chromatin states; others are cytosolic and regulate the stability of mRNA or act as microRNA sponges. This Review summarizes the current knowledge of lncRNAs as regulators of the endocrine system, with a focus on the identification and mode of action of several endocrine-important lncRNAs. We highlight lncRNAs that have a role in the development and function of pancreatic beta cells, white and brown adipose tissue, and other endocrine organs, and discuss the involvement of these molecules in endocrine dysfunction (for example, diabetes mellitus). We also address the associations of lncRNAs with nuclear receptors involved in major hormonal signalling pathways, such as estrogen and androgen receptors, and the relevance of these associations in certain endocrine cancers. Full Text.

Kobayashi, N., Suzuki, Y., Schoenfeld, L.W., Muller, C.A., Nieduszynski, C., Wolfe, K.H., and Tanaka, T.U. (2015). Discovery of an Unconventional Centromere in Budding Yeast Redefines Evolution of Point Centromeres. Current biology : CB [Epub ahead of print] Centromeres are the chromosomal regions promoting kinetochore assembly for chromosome segregation. In many eukaryotes, the centromere consists of up to mega base pairs of DNA. On such "regional centromeres," kinetochore assembly is mainly defined by epigenetic regulation [1]. By contrast, a clade of budding yeasts (Saccharomycetaceae) has a "point centromere" of 120-200 base pairs of DNA, on which kinetochore assembly is defined by the consensus DNA sequence [2, 3]. During evolution, budding yeasts acquired point centromeres, which replaced ancestral, regional centromeres [4]. All known point centromeres among different yeast species share common consensus DNA elements (CDEs) [5, 6], implying that they evolved only once and stayed essentially unchanged throughout evolution. Here, we identify a yeast centromere that challenges this view: that of the budding yeast Naumovozyma castellii is the first unconventional point centromere with unique CDEs. The N. castellii centromere CDEs are essential for centromere function but have different DNA sequences from CDEs in other point centromeres. Gene order analyses around N. castellii centromeres indicate their unique, and separate, evolutionary origin. Nevertheless, they are still bound by the ortholog of the CBF3 complex, which recognizes CDEs in other point centromeres. The new type of point centromere originated prior to the divergence between N. castellii and its close relative Naumovozyma dairenensis and disseminated to all N. castellii chromosomes through extensive genome rearrangement. Thus, contrary to the conventional view, point centromeres can undergo rapid evolutionary changes. These findings give new insights into the evolution of point centromeres. Full Text

Koppstein, D., Ashour, J., and Bartel, D.P. (2015). Sequencing the cap-snatching repertoire of H1N1 influenza provides insight into the mechanism of viral transcription initiation. Nucleic acids research Published online: April 21, 2015 The influenza polymerase cleaves host RNAs approximately 10-13 nucleotides downstream of their 5' ends and uses this capped fragment to prime viral mRNA synthesis. To better understand this process of cap snatching, we used high-throughput sequencing to determine the 5' ends of A/WSN/33 (H1N1) influenza mRNAs. The sequences provided clear evidence for nascent-chain realignment during transcription initiation and revealed a strong influence of the viral template on the frequency of realignment. After accounting for the extra nucleotides inserted through realignment, analysis of the capped fragments indicated that the different viral mRNAs were each prepended with a common set of sequences and that the polymerase often cleaved host RNAs after a purine and often primed transcription on a single base pair to either the terminal or penultimate residue of the viral template. We also developed a bioinformatic approach to identify the targeted host transcripts despite limited information content within snatched fragments and found that small nuclear RNAs and small nucleolar RNAs contributed the most abundant capped leaders. These results provide insight into the mechanism of viral transcription initiation and reveal the diversity of the cap-snatched repertoire, showing that noncoding transcripts as well as mRNAs are used to make influenza mRNAs. Full Text

Kuo, S.Y., Castoreno, A.B., Aldrich, L.N., Lassen, K.G., Goel, G., Dancik, V., Kuballa, P., Latorre, I., Conway, K.L., Sarkar, S.,Maetzel D, Jaenisch R, et al. (2015). Small-molecule enhancers of autophagy modulate cellular disease phenotypes suggested by human genetics. Proceedings of the National Academy of Sciences of the United States of America [Epub ahead of print]. Studies of human genetics and pathophysiology have implicated the regulation of autophagy in inflammation, neurodegeneration, infection, and autoimmunity. These findings have motivated the use of small-molecule probes to study how modulation of autophagy affects disease-associated phenotypes. Here, we describe the discovery of the small-molecule probe BRD5631 that is derived from diversity-oriented synthesis and enhances autophagy through an mTOR-independent pathway. We demonstrate that BRD5631 affects several cellular disease phenotypes previously linked to autophagy, including protein aggregation, cell survival, bacterial replication, and inflammatory cytokine production. BRD5631 can serve as a valuable tool for studying the role of autophagy in the context of cellular homeostasis and disease. Full Text

Lambert, A.W., Wong, C.K., Ozturk, S., Papageorgis, P., Raghunathan, R., Alekseyev, Y., Gower, A.C., Reinhard, B.M., Abdolmaleky, H.M., and Thiagalingam, S. (2015). Tumor Cell-Derived Periostin Regulates Cytokines That Maintain Breast Cancer Stem Cells. Mol Cancer Research [Epub ahead of print] Basal-like breast cancer (BLBC) is an aggressive subtype of breast cancer which is often enriched with cancer stem cells (CSCs), but the underlying molecular basis for this connection remains elusive. We hypothesized that BLBC cells are able to establish a niche permissive to the maintenance of CSCs and found that tumor cell-derived periostin (POSTN), a component of the extracellular matrix, as well as a corresponding cognate receptor, integrin alphavbeta3, are highly expressed in a subset of BLBC cell lines as well as in cancer stem cell-enriched populations. Furthermore, we demonstrated that an intact periostin-integrin beta3 signaling axis is required for the maintenance of breast CSCs. POSTN activates the ERK signaling pathway and regulates NF-kappaB-mediated transcription of key cytokines, namely IL6 and IL8, which in turn control downstream activation of STAT3. In summary, these findings suggest that BLBC cells have an innate ability to establish a microenvironmental niche supportive of CSCs. IMPLICATIONS: The findings reported here indicate that POSTN produced by CSCs acts to reinforce the stem cell state through the activation of integrin receptors and the production of key cytokines. Full Text

Lee, H.Y., Gao, X., Barrasa, M.I., Li, H., Elmes, R.R., Peters, L.L., and Lodish, H.F. (2015). PPAR-alpha and glucocorticoid receptor synergize to promote erythroid progenitor self-renewal. Nature Published online
11 May 2015. Many acute and chronic anaemias, including haemolysis, sepsis and genetic bone marrow failure diseases such as Diamond-Blackfan anaemia, are not treatable with erythropoietin (Epo), because the colony-forming unit erythroid progenitors (CFU-Es) that respond to Epo are either too few in number or are not sensitive enough to Epo to maintain sufficient red blood cell production. Treatment of these anaemias requires a drug that acts at an earlier stage of red cell formation and enhances the formation of Epo-sensitive CFU-E progenitors. Recently, we showed that glucocorticoids specifically stimulate self-renewal of an early erythroid progenitor, burst-forming unit erythroid (BFU-E), and increase the production of terminally differentiated erythroid cells. Here we show that activation of the peroxisome proliferator-activated receptor alpha (PPAR-alpha) by the PPAR-alpha agonists GW7647 and fenofibrate synergizes with the glucocorticoid receptor (GR) to promote BFU-E self-renewal. Over time these agonists greatly increase production of mature red blood cells in cultures of both mouse fetal liver BFU-Es and mobilized human adult CD34+ peripheral blood progenitors, with a new and effective culture system being used for the human cells that generates normal enucleated reticulocytes. Although Ppara-/- mice show no haematological difference from wild-type mice in both normal and phenylhydrazine (PHZ)-induced stress erythropoiesis, PPAR-alpha agonists facilitate recovery of wild-type but not Ppara-/- mice from PHZ-induced acute haemolytic anaemia. We also show that PPAR-alpha alleviates anaemia in a mouse model of chronic anaemia. Finally, both in control and corticosteroid-treated BFU-E cells, PPAR-alpha co-occupies many chromatin sites with GR; when activated by PPAR-alpha agonists, additional PPAR-alpha is recruited to GR-adjacent sites and presumably facilitates GR-dependent BFU-E self-renewal. Our discovery of the role of PPAR-alpha agonists in stimulating self-renewal of early erythroid progenitor cells suggests that the clinically tested PPAR-alpha agonists we used may improve the efficacy of corticosteroids in treating Epo-resistant anaemias. Full Text

Li, Z., Theile, C.S., Chen, G.Y., Bilate, A.M., Duarte, J.N., Avalos, A.M., Fang, T., Barberena, R., Sato, S., and Ploegh, H.L. (2015). Fluorophore-Conjugated Holliday Junctions for Generating Super-Bright Antibodies and Antibody Fragments. Angewandte Chemie Early View. The site-specific modification of proteins with fluorophores can render a protein fluorescent without compromising its function. To avoid self-quenching from multiple fluorophores installed in close proximity, we used Holliday junctions to label proteins site-specifically. Holliday junctions enable modification with multiple fluorophores at reasonably precise spacing. We designed a Holliday junction with three of its four arms modified with a fluorophore of choice and the remaining arm equipped with a dibenzocyclooctyne substituent to render it reactive with an azide-modified fluorescent single-domain antibody fragment or an intact immunoglobulin produced in a sortase-catalyzed reaction. These fluorescent Holliday junctions improve fluorescence yields for both single-domain and full-sized antibodies without deleterious effects on antigen binding. Full Text

Liau, Z.L., Liau, A.A., Porter, J.M., Salmon, W.C., Sheu, S.S., and Chen, J.J. (2015). Solid-immersion fluorescence microscopy with increased emission and super resolution. Journal of Applied Physics 117(1) 014502. We investigate solid-immersion fluorescence microscopy suitable for super-resolution nanotechnology and biological imaging, and have observed limit of resolution as small as 15 nm with microspheres, mitochondria, and chromatin fibers. We have further observed that fluorescence efficiency increases with excitation power density, implicating appreciable stimulated emission and increased resolution. We discuss potential advantages of the solid-immersion microscopy, including combined use with previously established super-resolution techniques for reaching deeper beyond the conventional diffraction limit. . Full Text.

Lodish, H.F. (2015). Accommodating family life: mentoring future female faculty members. Trends in cell biology Articles in press.The demands of family life are crucial factors in successfully retaining women in science. Retention efforts should focus on creating a family-friendly environment within the laboratory and the institute. Based on my own experiences, I suggest ways to attract top young scientists and support their development into leading researchers. Full Text.

Lourido, S., and Moreno, S.N.J. (2015). The calcium signaling toolkit of the Apicomplexan parasites Toxoplasma gondii and Plasmodium spp. Cell calcium 57, 186-193.Apicomplexan parasites have complex life cycles, frequently split between different hosts and reliant on rapid responses as the parasites react to changing environmental conditions. Calcium ion (Ca2+) signaling is consequently essential for the cellular and developmental changes that support Apicomplexan parasitism. Apicomplexan genomes reveal a rich repertoire of genes involved in calcium signaling, although many of the genes responsible for observed physiological changes remain unknown. There is evidence, for example, for the presence of a nifedipine-sensitive calcium entry mechanism in Toxoplasma, but the molecular components involved in Ca2+ entry in both Toxoplasma and Plasmodium, have not been identified. The major calcium stores are the endoplasmic reticulum (ER), the acidocalcisomes, and the plant-like vacuole in Toxoplasma, or the food vacuole in Plasmodium spp. Pharmacological evidence suggests that Ca2+ release from intracellular stores may be mediated by inositol 1,4,5-trisphosphate (IP3) or cyclic ADP ribose (cADPR) although there is no molecular evidence for the presence of receptors for these second messengers in the parasites. Several Ca2+-ATPases are present in Apicomplexans and a putative mitochondrial Ca2+/H+ exchanger has been identified. Apicomplexan genomes contain numerous genes encoding Ca2+-binding proteins, with the notable expansion of calcium-dependent protein kinases (CDPKs), whose study has revealed roles in gliding motility, microneme secretion, host cell invasion and egress, and parasite differentiation. Microneme secretion has also been shown to depend on the C2 domain containing protein DOC2 in both Plasmodium spp. and Toxoplasma, providing further evidence for the complex transduction of Ca2+ signals in these organisms. The characterization of these pathways could lead to the discovery of novel drug targets and to a better understanding of the role of Ca2+ in these parasites.

Ludwig, L.S., Cho, H., Wakabayashi, A., Eng, J.C., Ulirsch, J.C., Fleming, M.D., Lodish, H.F., and Sankaran, V.G. (2015). Genome-wide association study follow-up identifies cyclin A2 as a regulator of the transition through cytokinesis during terminal erythropoiesis. American Journal of Hematology[Epub ahead of print]. Genome-wide association studies (GWAS) hold tremendous promise to improve our understanding of human biology. Recent GWAS have revealed over 75 loci associated with erythroid traits, including the 4q27 locus that is associated with red blood cell size (mean corpuscular volume, MCV). The close linkage disequilibrium block at this locus harbors the CCNA2 gene that encodes cyclin A2. CCNA2 mRNA is highly expressed in human and murine erythroid progenitor cells and regulated by the essential erythroid transcription factor GATA1. To understand the role of cyclin A2 in erythropoiesis, we have reduced expression of this gene using short hairpin RNAs in a primary murine erythroid culture system. We demonstrate that cyclin A2 levels affect erythroid cell size by regulating the passage through cytokinesis during the final cell division of terminal erythropoiesis. Our study provides new insight into cell cycle regulation during terminal erythropoiesis and more generally illustrates the value of functional GWAS follow-up to gain mechanistic insight into hematopoiesis.

Mann, P.A., McLellan, C.A., Koseoglu, S., Si, Q., Kuzmin, E., Flattery, A., Harris, G., Sher, X., Murgolo, N., Wang, H., Lindquist S, et al. (2015). Chemical Genomics-Based Antifungal Drug Discovery: Targeting Glycosylphosphatidylinositol (GPI) Precursor Biosynthesis. ACS infectious diseases 1, 59-72.Steadily increasing antifungal drug resistance and persistent high rates of fungal-associated mortality highlight the dire need for the development of novel antifungals. Characterization of inhibitors of one enzyme in the GPI anchor pathway, Gwt1, has generated interest in the exploration of targets in this pathway for further study. Utilizing a chemical genomics-based screening platform referred to as the Candida albicans fitness test (CaFT), we have identified novel inhibitors of Gwt1 and a second enzyme in the glycosylphosphatidylinositol (GPI) cell wall anchor pathway, Mcd4. We further validate these targets using the model fungal organism Saccharomyces cerevisiae and demonstrate the utility of using the facile toolbox that has been compiled in this species to further explore target specific biology. Using these compounds as probes, we demonstrate that inhibition of Mcd4 as well as Gwt1 blocks the growth of a broad spectrum of fungal pathogens and exposes key elicitors of pathogen recognition. Interestingly, a strong chemical synergy is also observed by combining Gwt1 and Mcd4 inhibitors, mirroring the demonstrated synthetic lethality of combining conditional mutants of GWT1 and MCD4. We further demonstrate that the Mcd4 inhibitor M720 is efficacious in a murine infection model of systemic candidiasis. Our results establish Mcd4 as a promising antifungal target and confirm the GPI cell wall anchor synthesis pathway as a promising antifungal target area by demonstrating that effects of inhibiting it are more general than previously recognized. Full Text

Maruyama, T., Dougan, S.K., Truttmann, M.C., Bilate, A.M., Ingram, J.R., and Ploegh, H.L. (2015). Increasing the efficiency of precise genome editing with CRISPR-Cas9 by inhibition of nonhomologous end joining. Nature biotechnology Published online 23 March 2015 Methods to introduce targeted double-strand breaks (DSBs) into DNA enable precise genome editing by increasing the rate at which externally supplied DNA fragments are incorporated into the genome through homologous recombination. The efficiency of these methods is limited by nonhomologous end joining (NHEJ), an alternative DNA repair pathway that competes with homology-directed repair (HDR). To promote HDR at the expense of NHEJ, we targeted DNA ligase IV, a key enzyme in the NHEJ pathway, using the inhibitor Scr7. Scr7 treatment increased the efficiency of HDR-mediated genome editing, using Cas9 in mammalian cell lines and in mice for all four genes examined, up to 19-fold. This approach should be applicable to other customizable endonucleases, such as zinc finger nucleases and transcription activator-like effector nucleases, and to nonmammalian cells with sufficiently conserved mechanisms of NHEJ and HDR. Full Text.

McCammon2, J.M., and Sive, H. (2015). Challenges in understanding psychiatric disorders and developing therapeutics: a role for zebrafish. Disease models & mechanisms 8, 647-656.The treatment of psychiatric disorders presents three major challenges to the research and clinical community: defining a genotype associated with a disorder, characterizing the molecular pathology of each disorder and developing new therapies. This Review addresses how cellular and animal systems can help to meet these challenges, with an emphasis on the role of the zebrafish. Genetic changes account for a large proportion of psychiatric disorders and, as gene variants that predispose to psychiatric disease are beginning to be identified in patients, these are tractable for study in cellular and animal systems. Defining cellular and molecular criteria associated with each disorder will help to uncover causal physiological changes in patients and will lead to more objective diagnostic criteria. These criteria should also define co-morbid pathologies within the nervous system or in other organ systems. The definition of genotypes and of any associated pathophysiology is integral to the development of new therapies. Cell culture-based approaches can address these challenges by identifying cellular pathology and by high-throughput screening of gene variants and potential therapeutics. Whole-animal systems can define the broadest function of disorder-associated gene variants and the organismal impact of candidate medications. Given its evolutionary conservation with humans and its experimental tractability, the zebrafish offers several advantages to psychiatric disorder research. These include assays ranging from molecular to behavioural, and capability for chemical screening. There is optimism that the multiple approaches discussed here will link together effectively to provide new diagnostics and treatments for psychiatric patients. Full Text

McCammon, J.M., and Sive, H. (2015). Addressing the Genetics of Human Mental Health Disorders in Model Organisms. Annual review of genomics and human genetics [Epub ahead of print] Mental health disorders are notoriously difficult to diagnose and treat for a variety of reasons, including genetic heterogeneity, comorbidities, and qualitative diagnostic criteria. Discovery of the molecular pathology underlying these disorders is crucial to the development of quantitative biomarkers and novel therapeutics. In this review, we discuss contributions to the mental health field of different cellular and whole-animal approaches in characterizing psychiatric genetics and molecular pathology. These approaches include mammalian cell and neuronal culture, cerebral organoids, induced pluripotent stem cells, and the whole-animal models of nematodes, flies, mollusks, frogs, mice, and zebrafish, on the last of which we place extra emphasis. Integrative use of these cellular and animal systems in a complementary and informative fashion maximizes the potential contributions to the mental health field as a whole. Expected final online publication date for the Annual Review of Genomics and Human Genetics Volume 16 is August 31, 2015. Please see http://www.annualreviews.org/catalog/pubdates.aspx for revised estimates. Full Text

McKinley2, K.L., Sekulic, N., Guo, L.Y., Tsinman, T., Black, B.E., and Cheeseman, I.M. (2015). The CENP-L-N Complex Forms a Critical Node in an Integrated Meshwork of Interactions at the Centromere-Kinetochore Interface. Mol Cell 60, 886-898.During mitosis, the macromolecular kinetochore complex assembles on the centromere to orchestrate chromosome segregation. The properties and architecture of the 16-subunit Constitutive Centromere-Associated Network (CCAN) that allow it to build a robust platform for kinetochore assembly are poorly understood. Here, we use inducible CRISPR knockouts and biochemical reconstitutions to define the interactions between the human CCAN proteins. We find that the CCAN does not assemble as a linear hierarchy, and instead, each sub-complex requires multiple non-redundant interactions for its localization to centromeres and the structural integrity of the overall assembly. We demonstrate that the CENP-L-N complex plays a crucial role at the core of this assembly through interactions with CENP-C and CENP-H-I-K-M. Finally, we show that the CCAN is remodeled over the cell cycle such that sub-complexes depend on their interactions differentially. Thus, an interdependent meshwork within the CCAN underlies the centromere specificity and stability of the kinetochore. Full Text

McKinley, K.L., and Cheeseman, I.M. (2015). The molecular basis for centromere identity and function. Nature reviews Molecular cell biology Published online The centromere is the region of the chromosome that directs its segregation in mitosis and meiosis. Although the functional importance of the centromere has been appreciated for more than 130 years, elucidating the molecular features and properties that enable centromeres to orchestrate chromosome segregation is an ongoing challenge. Most eukaryotic centromeres are defined epigenetically and require the presence of nucleosomes containing the histone H3 variant centromere protein A (CENP-A; also known as CENH3). Ongoing work is providing important molecular insights into the central requirements for centromere identity and propagation, and the mechanisms by which centromeres recruit kinetochores to connect to spindle microtubules. Full Text

Meacham, C.E., Lawton, L.N., Soto-Feliciano, Y.M., Pritchard, J.R., Joughin, B.A., Ehrenberger, T., Fenouille, N., Zuber, J., Williams, R.T., Young, R.A., et al. (2015). A genome-scale in vivo loss-of-function screen identifies Phf6 as a lineage-specific regulator of leukemia cell growth. Genes & development 29, 483-488.We performed a genome-scale shRNA screen for modulators of B-cell leukemia progression in vivo. Results from this work revealed dramatic distinctions between the relative effects of shRNAs on the growth of tumor cells in culture versus in their native microenvironment. Specifically, we identified many "context-specific" regulators of leukemia development. These included the gene encoding the zinc finger protein Phf6. While inactivating mutations in PHF6 are commonly observed in human myeloid and T-cell malignancies, we found that Phf6 suppression in B-cell malignancies impairs tumor progression. Thus, Phf6 is a "lineage-specific" cancer gene that plays opposing roles in developmentally distinct hematopoietic malignancies. Full Text.

Monda, J.K., and Cheeseman, I.M. (2015). Chromosome Segregation: A Spatial Code to Correct Kinetochore-Microtubule Attachments. Current biology : CB 25, R601-603.Erroneous kinetochore-microtubule interactions must be detected and corrected before a cell enters anaphase to prevent chromosome mis-segregation. Two new studies describe an Aurora A-mediated error correction mechanism based on the spatial position of a chromosome within the mitotic spindle. Full Text

Monnier, N., Barry, Z., Park, H.Y., Su, K.C., Katz, Z., English, B.P., Dey, A., Pan, K., Cheeseman, I.M., Singer, R.H., et al. (2015). Inferring transient particle transport dynamics in live cells. Nature methods[Epub ahead of print] Live-cell imaging and particle tracking provide rich information on mechanisms of intracellular transport. However, trajectory analysis procedures to infer complex transport dynamics involving stochastic switching between active transport and diffusive motion are lacking. We applied Bayesian model selection to hidden Markov modeling to infer transient transport states from trajectories of mRNA-protein complexes in live mouse hippocampal neurons and metaphase kinetochores in dividing human cells. The software is available at http://hmm-bayes.org/. Full Text

Nordman, J.T., and Orr-Weaver, T.L. (2015). Understanding replication fork progression, stability, and chromosome fragility by exploiting the Suppressor of Underreplication protein. BioEssays Early View 9 June 2015 There are many layers of regulation governing DNA replication to ensure that genetic information is accurately transmitted from mother cell to daughter cell. While much of the control occurs at the level of origin selection and firing, less is known about how replication fork progression is controlled throughout the genome. In Drosophila polytene cells, specific regions of the genome become repressed for DNA replication, resulting in underreplication and decreased copy number. Importantly, underreplicated domains share properties with common fragile sites. The Suppressor of Underreplication protein SUUR is essential for this repression. Recent work established that SUUR functions by directly inhibiting replication fork progression, raising several interesting questions as to how replication fork progression and stability can be modulated within targeted regions of the genome. Here we discuss potential mechanisms by which replication fork inhibition can be achieved and the consequences this has on genome stability and copy number control. Full Text

Okosun, J., Wolfson, R.L., Wang, J., Araf, S., Wilkins, L., Castellano, B.M., Escudero-Ibarz, L., Al Seraihi, A.F., Richter, J., Bernhart, S.H.,Alejo Efeyan & David M Sabatini et al. (2015). Recurrent mTORC1-activating RRAGC mutations in follicular lymphoma. Nature Genetics Published online .Follicular lymphoma is an incurable B cell malignancy characterized by the t(14;18) translocation and mutations affecting the epigenome. Although frequent gene mutations in key signaling pathways, including JAK-STAT, NOTCH and NF-kappaB, have also been defined, the spectrum of these mutations typically overlaps with that in the closely related diffuse large B cell lymphoma (DLBCL). Using a combination of discovery exome and extended targeted sequencing, we identified recurrent somatic mutations in RRAGC uniquely enriched in patients with follicular lymphoma (17%). More than half of the mutations preferentially co-occurred with mutations in ATP6V1B2 and ATP6AP1, which encode components of the vacuolar H+-ATP ATPase (V-ATPase) known to be necessary for amino acid-induced activation of mTORC1. The RagC variants increased raptor binding while rendering mTORC1 signaling resistant to amino acid deprivation. The activating nature of the RRAGC mutations, their existence in the dominant clone and their stability during disease progression support their potential as an excellent candidate for therapeutic targeting. Full Text

Oldridge, D.A., Wood, A.C., Weichert-Leahey, N., Crimmins, I., Sussman, R., Winter, C., McDaniel, L.D., Diamond, M., Hart, L.S., Zhu, , .Brian J. Abraham, Lars Anders, ,Richard A Young, et al. (2015). Genetic predisposition to neuroblastoma mediated by a LMO1 super-enhancer polymorphism. Nature [Epub ahead of print] Neuroblastoma is a paediatric malignancy that typically arises in early childhood, and is derived from the developing sympathetic nervous system. Clinical phenotypes range from localized tumours with excellent outcomes to widely metastatic disease in which long-term survival is approximately 40% despite intensive therapy. A previous genome-wide association study identified common polymorphisms at the LMO1 gene locus that are highly associated with neuroblastoma susceptibility and oncogenic addiction to LMO1 in the tumour cells. Here we investigate the causal DNA variant at this locus and the mechanism by which it leads to neuroblastoma tumorigenesis. We first imputed all possible genotypes across the LMO1 locus and then mapped highly associated single nucleotide polymorphism (SNPs) to areas of chromatin accessibility, evolutionary conservation and transcription factor binding sites. We show that SNP rs2168101 G>T is the most highly associated variant (combined P = 7.47 x 10-29, odds ratio 0.65, 95% confidence interval 0.60-0.70), and resides in a super-enhancer defined by extensive acetylation of histone H3 lysine 27 within the first intron of LMO1. The ancestral G allele that is associated with tumour formation resides in a conserved GATA transcription factor binding motif. We show that the newly evolved protective TATA allele is associated with decreased total LMO1 expression (P = 0.028) in neuroblastoma primary tumours, and ablates GATA3 binding (P < 0.0001). We demonstrate allelic imbalance favouring the G-containing strand in tumours heterozygous for this SNP, as demonstrated both by RNA sequencing (P < 0.0001) and reporter assays (P = 0.002). These findings indicate that a recently evolved polymorphism within a super-enhancer element in the first intron of LMO1 influences neuroblastoma susceptibility through differential GATA transcription factor binding and direct modulation of LMO1 expression in cis, and this leads to an oncogenic dependency in tumour cells. Full Text

OrrWeaver, T.L. (2015). When bigger is better: the role of polyploidy in organogenesis. Trends in genetics : TIG Online Now Articles . Defining how organ size is regulated, a process controlled not only by the number of cells but also by the size of the cells, is a frontier in developmental biology. Large cells are produced by increasing DNA content or ploidy, a developmental strategy employed throughout the plant and animal kingdoms. The widespread use of polyploidy during cell differentiation makes it important to define how this hypertrophy contributes to organogenesis. I discuss here examples from a variety of animals and plants in which polyploidy controls organ size, the size and function of specific tissues within an organ, or the differentiated properties of cells. In addition, I highlight how polyploidy functions in wound healing and tissue regeneration. Full Text

Owen, J.H., Wagner, D.E., Chen, C.C., Petersen, C.P., and Reddien, P.W. (2015). teashirt is required for head-versus-tail regeneration polarity in planarians. Development (Cambridge, England) Articles in Press. Regeneration requires that the identities of new cells are properly specified to replace missing tissues. The Wnt signaling pathway serves a central role in specifying posterior cell fates during planarian regeneration. We identified a gene encoding a homolog of the Teashirt family of zinc-finger proteins in the planarian Schmidtea mediterranea to be a target of Wnt signaling in intact animals and at posterior-facing wounds. Inhibition of Smed-teashirt (teashirt) by RNA interference (RNAi) resulted in the regeneration of heads in place of tails, a phenotype previously observed with RNAi of the Wnt pathway genes beta-catenin-1, wnt1, Dvl-1/2 or wntless. teashirt was required for beta-catenin-1-dependent activation of posterior genes during regeneration. These findings identify teashirt as a transcriptional target of Wnt signaling required for Wnt-mediated specification of posterior blastemas. Full Text.

Pasqual, G., Angelini, A., and Victora, G.D. (2015). Triggering Positive Selection of Germinal Center B Cells by Antigen Targeting to DEC-205. Methods in molecular biology) 1291, 125-134.Germinal centers (GCs) are the site of maturation of antibody affinity and are thus of key importance to humoral immunity. The study of B-cell dynamics and selection within the GC has been hampered by the limited number of techniques available to manipulate GC output in vivo. Here, we describe an approach to trigger positive selection of B cells in vivo by targeting antigen specifically to a subpopulation of GC B cells via the surface lectin DEC-205 and forcing their interaction with T follicular helper cells. Targeted GC B cells can then be followed over time as they progress through the stages of positive selection. Full Text.

Patterson H.C., Gerbeth, C., Thiru, P., Vogtle, N.F., Knoll, M., Shahsafaei, A., Samocha, K.E., Huang, C.X., Harden, M.M., Song, R.,Cynthia Chen, Jennifer Kao, Jiahai Shi, Wendy Salmon, Yoav D. Shaul, George W. Bell and Harvey F. Lodish. (2015). A respiratory chain controlled signal transduction cascade in the mitochondrial intermembrane space mediates hydrogen peroxide signaling. Proc Natl Acad Sci U S A. 2015 Oct 20;112(42):E5679-88. Reactive oxygen species (ROS) such as hydrogen peroxide (H2O2) govern cellular homeostasis by inducing signaling. H2O2 modulates the activity of phosphatases and many other signaling molecules through oxidation of critical cysteine residues, which led to the notion that initiation of ROS signaling is broad and nonspecific, and thus fundamentally distinct from other signaling pathways. Here, we report that H2O2 signaling bears hallmarks of a regular signal transduction cascade. It is controlled by hierarchical signaling events resulting in a focused response as the results place the mitochondrial respiratory chain upstream of tyrosine-protein kinase Lyn, Lyn upstream of tyrosine-protein kinase SYK (Syk), and Syk upstream of numerous targets involved in signaling, transcription, translation, metabolism, and cell cycle regulation. The active mediators of H2O2 signaling colocalize as H2O2 induces mitochondria-associated Lyn and Syk phosphorylation, and a pool of Lyn and Syk reside in the mitochondrial intermembrane space. Finally, the same intermediaries control the signaling response in tissues and species responsive to H2O2 as the respiratory chain, Lyn, and Syk were similarly required for H2O2 signaling in mouse B cells, fibroblasts, and chicken DT40 B cells. Consistent with a broad role, the Syk pathway is coexpressed across tissues, is of early metazoan origin, and displays evidence of evolutionary constraint in the human. These results suggest that H2O2 signaling is under control of a signal transduction pathway that links the respiratory chain to the mitochondrial intermembrane space-localized, ubiquitous, and ancient Syk pathway in hematopoietic and nonhematopoietic cells. Full Text

Poleganov, M.A., Eminli, S., Beissert, T., Herz, S., Moon, J.I., Goldmann, J., Beyer, A., Heck, R., Burkhart, I., Barea Roldan, D., et al. (2015). Efficient reprogramming of human fibroblasts and blood-derived endothelial progenitor cells using non-modified RNA for reprogramming and immune evasion. Human gene therapy [Epub ahead of print] mRNA reprogramming results in the generation of genetically stable induced pluripotent stem (iPS) cells while avoiding the risks of genomic integration. Previously published mRNA reprogramming protocols have proven to be inconsistent, time consuming, and mainly restricted to fibroblasts thereby demonstrating the need for a simple, but reproducible protocol applicable to various cell types. So far there have been no published reports using mRNA to reprogram any cell type derived from human blood. Non-modified synthetic mRNAs are immunogenic and activate cellular defense mechanisms, which can lead to cell death and inhibit mRNA translation upon repetitive transfection. Hence to overcome RNA related toxicity we combined non-modified reprogramming mRNAs [OCT4, SOX2, KLF4, cMYC, NANOG and LIN28 (OSKMNL)] with immune evasion mRNAs [E3, K3 and B18R (EKB)] from Vaccinia virus (VACV). Additionally we included mature, double stranded microRNAs (miRNAs) from the 302/367 cluster, which are known to enhance the reprogramming process, to develop a robust reprogramming protocol for the generation of stable iPS cell lines from both human fibroblasts and human blood-outgrowth endothelial progenitor cells (EPCs). Our novel combination of RNAs enables the cell to tolerate repetitive transfections for the generation of stable iPS cell colonies from human fibroblasts within 11 days while requiring only four transfections. Moreover, our method resulted in the first known mRNA-vectored reprogramming of human blood-derived EPCs within 10 days while requiring only eight daily transfections. Full Text

Qiao, K., Imam Abidi, S.H., Liu, H., Zhang, H., Chakraborty, S., Watson, N., Kumaran Ajikumar, P., and Stephanopoulos, G. (2015). Engineering lipid overproduction in the oleaginous yeast Yarrowia lipolytica. Metabolic engineering [Epub ahead of print] Conversion of carbohydrates to lipids at high yield and productivity is essential for cost-effective production of renewable biodiesel. Although some microorganisms can convert sugars to oils, conversion yields and rates are typically low due primarily to allosteric inhibition of the lipid biosynthetic pathway by saturated fatty acids. By reverse engineering the mammalian cellular obese phenotypes, we identified the delta-9 stearoyl-CoA desaturase (SCD) as a rate limiting step and target for the metabolic engineering of the lipid synthesis pathway in Yarrowia lipolytica. Simultaneous overexpression of SCD, Acetyl-CoA carboxylase (ACC1), and Diacylglyceride acyl-transferase (DGA1) in Y. lipolytica yielded an engineered strain exhibiting highly desirable phenotypes of fast cell growth and lipid overproduction including high carbon to lipid conversion yield (84.7% of theoretical maximal yield), high lipid titers ( approximately 55g/L), and enhanced tolerance to glucose and cellulose-derived sugars. Moreover, the engineered strain shows a three-fold growth advantage over the wild type strain. As a result, a maximal lipid productivity of approximately 1g/L/h is obtained during the stationary phase. Furthermore, we show that the engineered yeast required cytoskeleton remodeling in eliciting the obesity phenotype. Altogether, our work describes the development of a microbial catalyst with the highest reported lipid yield, titer and productivity to date. This is an important step towards the development of an efficient and cost-effective process for biodiesel production from renewable resources. Full Text.

Rago, F., Gascoigne, K.E., and Cheeseman, I.M. Distinct Organization and Regulation of the Outer Kinetochore KMN Network Downstream of CENP-C and CENP-T. Current biology : CB Feb 4.[Epub ahead of print] The kinetochore provides a vital connection between chromosomes and spindle microtubules [1, 2]. Defining the molecular architecture of the core kinetochore components is critical for understanding the mechanisms by which the kinetochore directs chromosome segregation. The KNL1/Mis12 complex/Ndc80 complex (KMN) network acts as the primary microtubule-binding interface at kinetochores [3] and provides a platform to recruit regulatory proteins [4]. Recent work found that the inner kinetochore components CENP-C and CENP-T act in parallel to recruit the KMN network to kinetochores [5-8]. However, due to the presence of these dual pathways, it has not been possible to distinguish differences in the nature of kinetochore assembly downstream of CENP-C or CENP-T. Here, we separated these pathways by targeting CENP-C and CENP-T independently to an ectopic chromosomal locus in human cells. Our work reveals that the organization of the KMN network components downstream of CENP-C and CENP-T is distinct. CENP-C recruits the Ndc80 complex through its interactions with KNL1 and the Mis12 complex. In contrast, CENP-T directly interacts with Ndc80, which in turn promotes KNL1/Mis12 complex recruitment through a separate region on CENP-T, resulting in functional relationships for KMN network localization that are inverted relative to the CENP-C pathway. We also find that distinct regulatory paradigms control the assembly of these pathways, with Aurora B kinase promoting KMN network recruitment to CENP-C and cyclin-dependent kinase (CDK) regulating KMN network recruitment to CENP-T. This work reveals unexpected complexity for the architecture and regulation of the core components of the kinetochore-microtubule interface. Full Text.

Rashidian2, M., Keliher, E.J., Dougan, M., Juras, P.K., Cavallari, M., Wojtkiewicz, G.R., Jacobsen, J.T., Edens, J.G., Tas, J.M.J., Victora, G, Ralph Weissleder and Hidde Ploegh (2015). Use of F-18-2-Fluorodeoxyglucose to Label Antibody Fragments for Immuno-Positron Emission Tomography of Pancreatic Cancer. ACS Central Science 1, 142-147.We generated F-18-labeled antibody fragments for positron emission tomography (PET) imaging using a sortase-mediated reaction to install a trans-cyclooctene-functionalized short peptide onto proteins of interest, followed by reaction with a tetrazine-labeled-F-18-2-deoxyfluoroglucose (FDG). The method is rapid, robust, and site-specific (radiochemical yields > 25%, not decay corrected). The availability of F-18-2-deoxyfluoroglucose avoids the need for more complicated chemistries used to generate carbon-fluorine bonds. We demonstrate the utility of the method by detecting heterotopic pancreatic tumors in mice by PET, using anti-Class II MHC single domain antibodies. We correlate macroscopic PET images with microscopic two-photon visualization of the tumor. Our approach provides easy access to F-18-labeled antibodies and their fragments at a level of molecular specificity that complements conventional F-18-FDG imaging. Full Text . Full Text

Rashidian, M., Keliher, E.J., Bilate, A.M., Duarte, J.N., Wojtkiewicz, G.R., Jacobsen, J.T., Cragnolini, J., Swee, L.K., Victora, G.D., Weissleder, R., and Hidde L. Ploegh (2015). Noninvasive imaging of immune responses. Proceedings of the National Academy of Sciences of the United States of America Early Edition.. At their margins, tumors often contain neutrophils, dendritic cells, and activated macrophages, which express class II MHC and CD11b products. The interplay between stromal cells, tumor cells, and migratory cells such as lymphocytes creates opportunities for noninvasive imaging of immune responses. We developed alpaca-derived antibody fragments specific for mouse class II MHC and CD11b products, expressed on the surface of a variety of myeloid cells. We validated these reagents by flow cytometry and two-photon microscopy to obtain images at cellular resolution. To enable noninvasive imaging of the targeted cell populations, we developed a method to site-specifically label VHHs [the variable domain (VH) of a camelid heavy-chain only antibody] with 18F or 64Cu. Radiolabeled VHHs rapidly cleared the circulation (t1/2 approximately 20 min) and clearly visualized lymphoid organs. We used VHHs to explore the possibility of imaging inflammation in both xenogeneic and syngeneic tumor models, which resulted in detection of tumors with remarkable specificity. We also imaged the infiltration of myeloid cells upon injection of complete Freund's adjuvant. Both anti-class II MHC and anti-CD11b VHHs detected inflammation with excellent specificity. Given the ease of manufacture and labeling of VHHs, we believe that this method could transform the manner in which antitumor responses and/or infectious events may be tracked. Full Text

Rothenberg, D.A., Gordon, E.A., White, F.M., and Lourido, S. (2016). Identification of Direct Kinase Substrates Using Analogue-Sensitive Alleles. Methods in molecular biology 1355, 71-84. Identifying the substrates of protein kinases remains a major obstacle in the elucidation of eukaryotic signaling pathways. Promiscuity among kinases and their substrates coupled with the extraordinary plasticity of phosphorylation networks renders traditional genetic approaches or small-molecule inhibitors problematic when trying to determine the direct substrates of an individual kinase. Here we describe methods to label, enrich, and identify the direct substrates of analogue-sensitive kinases by exploiting their steric complementarity to artificial ATP analogues. Using calcium-dependent protein kinases of Toxoplasma gondii as a model for these approaches, this protocol brings together numerous advances that enable labeling of kinase targets in semi-permeabilized cells, quantification of direct labeling over background, and highly specific enrichment of targeted phosphopeptides. Full Text

Russell, M.R., Penikis, A., Oldridge, D.A., Alvarez-Dominguez, J.R., McDaniel, L., Diamond, M., Padovan, O., Raman, P., Li, Y., Wei, J.S., et al. (2015). CASC15-S Is a Tumor Suppressor lncRNA at the 6p22 Neuroblastoma Susceptibility Locus. Cancer research 75(15); 1-12. Chromosome 6p22 was identified recently as a neuroblastoma susceptibility locus, but its mechanistic contributions to tumorigenesis are as yet undefined. Here we report that the most highly significant single-nucleotide polymorphism (SNP) associations reside within CASC15, a long noncoding RNA that we define as a tumor suppressor at 6p22. Low-level expression of a short CASC15 isoform (CASC15-S) associated highly with advanced neuroblastoma and poor patient survival. In human neuroblastoma cells, attenuating CASC15-S increased cellular growth and migratory capacity. Gene expression analysis revealed downregulation of neuroblastoma-specific markers in cells with attenuated CASC15-S, with concomitant increases in cell adhesion and extracellular matrix transcripts. Altogether, our results point to CASC15-S as a mediator of neural growth and differentiation, which impacts neuroblastoma initiation and progression.. Full Text

Sahni, N., Yi, S., Taipale, M., Fuxman Bass, J.I., Coulombe-Huntington, J., Yang, F., Peng, J., Weile, J., Karras, G.I., Wang, Y. ,Irina Krykbaeva, Susan Lindquist and Marc Vidal. (2015). Widespread Macromolecular Interaction Perturbations in Human Genetic Disorders. Cell 161, 647-660.How disease-associated mutations impair protein activities in the context of biological networks remains mostly undetermined. Although a few renowned alleles are well characterized, functional information is missing for over 100,000 disease-associated variants. Here we functionally profile several thousand missense mutations across a spectrum of Mendelian disorders using various interaction assays. The majority of disease-associated alleles exhibit wild-type chaperone binding profiles, suggesting they preserve protein folding or stability. While common variants from healthy individuals rarely affect interactions, two-thirds of disease-associated alleles perturb protein-protein interactions, with half corresponding to "edgetic" alleles affecting only a subset of interactions while leaving most other interactions unperturbed. With transcription factors, many alleles that leave protein-protein interactions intact affect DNA binding. Different mutations in the same gene leading to different interaction profiles often result in distinct disease phenotypes. Thus disease-associated alleles that perturb distinct protein activities rather than grossly affecting folding and stability are relatively widespread. Full Text

Sankaran, V.G., Ulirsch, J.C., Tchaikovskii, V., Ludwig, L.S., Wakabayashi, A., Kadirvel, S., Lindsley, R.C., Bejar, R., Shi, J.H., Lovitch, S.B., et al. (2015). X-linked macrocytic dyserythropoietic anemia in females with an ALAS2 mutation. Journal of Clinical Investigation 125, 1665-1669.Macrocytic anemia with abnormal erythropoiesis is a common feature of megaloblastic anemias, congenital dyserythropoietic anemias, and myelodysplastic syndromes. Here, we characterized a family with multiple female individuals who have macrocytic anemia. The proband was noted to have dyserythropoiesis and iron overload. After an extensive diagnostic evaluation that did not provide insight into the cause of the disease, whole-exome sequencing of multiple family members revealed the presence of a mutation in the X chromosomal gene ALAS2, which encodes 5'-aminolevulinate synthase 2, in the affected females. We determined that this mutation (Y365C) impairs binding of the essential cofactor pyridoxal 5'-phosphate to ALAS2, resulting in destabilization of the enzyme and consequent loss of function. X inactivation was not highly skewed in wbc from the affected individuals. In contrast, and consistent with the severity of the ALAS2 mutation, there was a complete skewing toward expression of the WT allele in mRNA from reticulocytes that could be recapitulated in primary erythroid cultures. Together, the results of the X inactivation and mRNA studies illustrate how this X-linked dominant mutation in ALAS2 can perturb normal erythropoiesis through cell-nonautonomous effects. Moreover, our findings highlight the value of whole-exome sequencing in diagnostically challenging cases for the identification of disease etiology and extension of the known phenotypic spectrum of disease. Full Text

Saxton, R.A., Knockenhauer, K.E., Wolfson, R.L., Chantranupong, L., Pacold, M.E., Wang, T., Schwartz, T.U., and Sabatini, D.M. (2015). Structural basis for leucine sensing by the Sestrin2-mTORC1 pathway. Science Published Online. Eukaryotic cells coordinate growth with the availability of nutrients through mTOR complex 1 (mTORC1), a master growth regulator. Leucine is of particular importance and activates mTORC1 via the Rag GTPases and their regulators GATOR1 and GATOR2. Sestrin2 interacts with GATOR2 and is a leucine sensor. We present the 2.7-A crystal structure of Sestrin2 in complex with leucine. Leucine binds through a single pocket that coordinates its charged functional groups and confers specificity for the hydrophobic side chain. A loop encloses leucine and forms a lid-latch mechanism required for binding. A structure-guided mutation in Sestrin2 that decreases its affinity for leucine leads to a concomitant increase in the leucine concentration required for mTORC1 activation in cells. These results provide a structural mechanism of amino acid sensing by the mTORC1 pathway. Full Text

Schweitzer, L.D., Comb, W.C., Bar-Peled, L., and Sabatini, D.M. (2015). Disruption of the Rag-Ragulator Complex by c17orf59 Inhibits mTORC1. Cell reports 12, 1445–1455. mTORC1 controls key processes that regulate cell growth, including mRNA translation, ribosome biogenesis, and autophagy. Environmental amino acids activate mTORC1 by promoting its recruitment to the cytosolic surface of the lysosome, where its kinase is activated downstream of growth factor signaling. mTORC1 is brought to the lysosome by the Rag GTPases, which are tethered to the lysosomal membrane by Ragulator, a lysosome-bound scaffold. Here, we identify c17orf59 as a Ragulator-interacting protein that regulates mTORC1 activity through its interaction with Ragulator at the lysosome. The binding of c17orf59 to Ragulator prevents Ragulator interaction with the Rag GTPases, both in cells and in vitro, and decreases Rag GTPase lysosomal localization. Disruption of the Rag-Ragulator interaction by c17orf59 impairs mTORC1 activation by amino acids by preventing mTOR from reaching the lysosome. By disrupting the Rag-Ragulator interaction to inhibit mTORC1, c17orf59 expression may represent another mechanism to modulate nutrient sensing by mTORC1. Full Text

Shaul, Y.D., Yuan, B., Thiru, P., Nutter-Upham, A., McCallum, S., Lanzkron, C., Bell, G.W., and Sabatini, D.M. (2015) MERAV: a tool for comparing gene expression across human tissues and cell types. Nucleic Acids ResearchsFirst published online: November 30, 2015 The oncogenic transformation of normal cells into malignant, rapidly proliferating cells requires major alterations in cell physiology. For example, the transformed cells remodel their metabolic processes to supply the additional demand for cellular building blocks. We have recently demonstrated essential metabolic processes in tumor progression through the development of a methodological analysis of gene expression. Here, we present the Metabolic gEne RApid Visualizer (MERAV, http://merav.wi.mit.edu), a web-based tool that can query a database comprising approximately 4300 microarrays, representing human gene expression in normal tissues, cancer cell lines and primary tumors. MERAV has been designed as a powerful tool for whole genome analysis which offers multiple advantages: one can search many genes in parallel; compare gene expression among different tissue types as well as between normal and cancer cells; download raw data; and generate heatmaps; and finally, use its internal statistical tool. Most importantly, MERAV has been designed as a unique tool for analyzing metabolic processes as it includes matrixes specifically focused on metabolic genes and is linked to the Kyoto Encyclopedia of Genes and Genomes pathway search. Full Text

Sigova, A.A., Abraham, B.J., Ji, X., Molinie, B., Hannett, N.M., Guo, Y.E., Jangi, M., Giallourakis, C.C., Sharp, P.A., and Young, R.A. (2015). Transcription factor trapping by RNA in gene regulatory elements. Science Published Online Transcription factors (TFs) bind specific sequences in promoter-proximal and distal DNA elements in order to regulate gene transcription. RNA is transcribed from both of these DNA elements, and some DNA-binding TFs bind RNA. Hence, RNA transcribed from regulatory elements may contribute to stable TF occupancy at these sites. We show that the ubiquitously expressed TF YY1 binds to both gene regulatory elements and also to their associated RNA species genome-wide. Reduced transcription of regulatory elements diminishes YY1 occupancy whereas artificial tethering of RNA enhances YY1 occupancy at these elements. We propose that RNA makes a modest but important contribution to the maintenance of certain TFs at gene regulatory elements and suggest that transcription of regulatory elements produces a positive feedback loop that contributes to the stability of gene expression programs. Full Text

Soh, Y.Q., Junker, J.P., Gill, M.E., Mueller, J.L., van Oudenaarden, A., and Page, D.C. (2015). A Gene Regulatory Program for Meiotic Prophase in the Fetal Ovary. PLoS genetics 11, e1005531.The chromosomal program of meiotic prophase, comprising events such as laying down of meiotic cohesins, synapsis between homologs, and homologous recombination, must be preceded and enabled by the regulated induction of meiotic prophase genes. This gene regulatory program is poorly understood, particularly in organisms with a segregated germline. We characterized the gene regulatory program of meiotic prophase as it occurs in the mouse fetal ovary. By profiling gene expression in the mouse fetal ovary in mutants with whole tissue and single-cell techniques, we identified 104 genes expressed specifically in pre-meiotic to pachytene germ cells. We characterized the regulation of these genes by 1) retinoic acid (RA), which induces meiosis, 2) Dazl, which is required for germ cell competence to respond to RA, and 3) Stra8, a downstream target of RA required for the chromosomal program of meiotic prophase. Initial induction of practically all identified meiotic prophase genes requires Dazl. In the presence of Dazl, RA induces at least two pathways: one Stra8-independent, and one Stra8-dependent. Genes vary in their induction by Stra8, spanning fully Stra8-independent, partially Stra8-independent, and fully Stra8-dependent. Thus, Stra8 regulates the entirety of the chromosomal program but plays a more nuanced role in governing the gene expression program. We propose that Stra8-independent gene expression enables the stockpiling of selected meiotic structural proteins prior to the commencement of the chromosomal program. Unexpectedly, we discovered that Stra8 is required for prompt down-regulation of itself and Rec8. Germ cells that have expressed and down-regulated Stra8 are refractory to further Stra8 expression. Negative feedback of Stra8, and subsequent resistance to further Stra8 expression, may ensure a single, restricted pulse of Stra8 expression. Collectively, our findings reveal a gene regulatory logic by which germ cells prepare for the chromosomal program of meiotic prophase, and ensure that it is induced only once. Full Text

Son, S., Stevens, M.M., Chao, H.X., Thoreen, C., Hosios, A.M., Schweitzer, L.D., Weng, Y., Wood, K., Sabatini, D., Vander Heiden, M.G., et al. (2015). Cooperative nutrient accumulation sustains growth of mammalian cells. Scientific reports 5, 17401.The coordination of metabolic processes to allow increased nutrient uptake and utilization for macromolecular synthesis is central for cell growth. Although studies of bulk cell populations have revealed important metabolic and signaling requirements that impact cell growth on long time scales, whether the same regulation influences short-term cell growth remains an open question. Here we investigate cell growth by monitoring mass accumulation of mammalian cells while rapidly depleting particular nutrients. Within minutes following the depletion of glucose or glutamine, we observe a growth reduction that is larger than the mass accumulation rate of the nutrient. This indicates that if one particular nutrient is depleted, the cell rapidly adjusts the amount that other nutrients are accumulated, which is consistent with cooperative nutrient accumulation. Population measurements of nutrient sensing pathways involving mTOR, AKT, ERK, PKA, MST1, or AMPK, or pro-survival pathways involving autophagy suggest that they do not mediate this growth reduction. Furthermore, the protein synthesis rate does not change proportionally to the mass accumulation rate over these time scales, suggesting that intracellular metabolic pools buffer the growth response. Our findings demonstrate that cell growth can be regulated over much shorter time scales than previously appreciated. Full Text

Sokol, E.S., Sanduja, S., Jin, D.X., Miller, D.H., Mathis, R.A., and Gupta, P.B. (2015). Perturbation-Expression Analysis Identifies RUNX1 as a Regulator of Human Mammary Stem Cell Differentiation. PLoS computational biology 11, e1004161.The search for genes that regulate stem cell self-renewal and differentiation has been hindered by a paucity of markers that uniquely label stem cells and early progenitors. To circumvent this difficulty we have developed a method that identifies cell-state regulators without requiring any markers of differentiation, termed Perturbation-Expression Analysis of Cell States (PEACS). We have applied this marker-free approach to screen for transcription factors that regulate mammary stem cell differentiation in a 3D model of tissue morphogenesis and identified RUNX1 as a stem cell regulator. Inhibition of RUNX1 expanded bipotent stem cells and blocked their differentiation into ductal and lobular tissue rudiments. Reactivation of RUNX1 allowed exit from the bipotent state and subsequent differentiation and mammary morphogenesis. Collectively, our findings show that RUNX1 is required for mammary stem cells to exit a bipotent state, and provide a new method for discovering cell-state regulators when markers are not available. Full Text

Soldner, F., and Jaenisch, R. (2015). Dissecting risk haplotypes in sporadic Alzheimer's disease. Cell Stem Cell 16, 341-342.Understanding how genetic risk variants contribute to complex diseases is crucial for predicting disease susceptibility and developing patient-tailored therapies. In this issue of Cell Stem Cell, Young et al. (2015) dissect the function of common non-coding risk haplotypes in the SORL1 locus in the pathogenesis of sporadic Alzheimer's disease using patient-derived induced pluripotent stem cells. Full Text.

Srivastava, M. (2015). A Comparative Genomics Perspective on the Origin of Multicellularity and Early Animal Evolution. In Evolutionary Transitions to Multicellular Life: Principles and Mechanisms, pp. 269-299. Full Text

Stelzer3, Y., and Jaenisch, R. (2015). Monitoring Dynamics of DNA Methylation at Single-Cell Resolution during Development and Disease. Cold Spring Harbor symposia on quantitative biology [Epub ahead of print] DNA methylation is a broadly studied epigenetic modification that is essential for normal mammalian development. Over the years, numerous methodologies were developed trying to cope with the intrinsic challenge of reading the "second dimension" epigenetic code. The recent rapid expansion of sequencing technologies has made it possible to fully chart the methylation landscape of different cell types at single-base resolution. Surprisingly, accumulating data suggest that, in addition to the massive epigenome remodeling during early development, cell type and tissue specification is associated with high levels of DNA methylation dynamics at distal regulatory elements. However, current methods provide only a static "snapshot" of DNA methylation, thus precluding the study of real-time methylation dynamics during cell fate changes. Here we review the principles of a new approach that enables monitoring loci-specific DNA methylation dynamics at single-cell resolution. We also discuss potential applications and promises for implementing this methodology to study DNA methylation changes during development and disease. Full Text

Stelzer2, Y., Shivalila, C.S., Soldner, F., Markoulaki, S., and Jaenisch, R. (2015). Tracing Dynamic Changes of DNA Methylation at Single-Cell Resolution. Cell 163, 218-229.Mammalian DNA methylation plays an essential role in development. To date, only snapshots of different mouse and human cell types have been generated, providing a static view on DNA methylation. To enable monitoring of methylation status as it changes over time, we establish a reporter of genomic methylation (RGM) that relies on a minimal imprinted gene promoter driving a fluorescent protein. We show that insertion of RGM proximal to promoter-associated CpG islands reports the gain or loss of DNA methylation. We further utilized RGM to report endogenous methylation dynamics of non-coding regulatory elements, such as the pluripotency-specific super enhancers of Sox2 and miR290. Loci-specific DNA methylation changes and their correlation with transcription were visualized during cell-state transition following differentiation of mouse embryonic stem cells and during reprogramming of somatic cells to pluripotency. RGM will allow the investigation of dynamic methylation changes during development and disease at single-cell resolution. Full Text

Stelzer, Y., Bar, S., Bartok, O., Afik, S., Ronen, D., Kadener, S., and Benvenisty, N. (2015). Differentiation of Human Parthenogenetic Pluripotent Stem Cells Reveals Multiple Tissue- and Isoform-Specific Imprinted Transcripts. Cell reports 31 March Ahead of Print. Parental imprinting results in monoallelic parent-of-origin-dependent gene expression. However, many imprinted genes identified by differential methylation do not exhibit complete monoallelic expression. Previous studies demonstrated complex tissue-dependent expression patterns for some imprinted genes. Still, the complete magnitude of this phenomenon remains largely unknown. By differentiating human parthenogenetic induced pluripotent stem cells into different cell types and combining DNA methylation with a 5' RNA sequencing methodology, we were able to identify tissue- and isoform-dependent imprinted genes in a genome-wide manner. We demonstrate that nearly half of all imprinted genes express both biallelic and monoallelic isoforms that are controlled by tissue-specific alternative promoters. This study provides a global analysis of tissue-specific imprinting in humans and suggests that alternative promoters are central in the regulation of imprinted genes. Full Text.

Strohecker, A.M., Joshi, S., Possemato, R., Abraham, R.T., Sabatini, D.M., and White, E. (2015). Identification of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase as a novel autophagy regulator by high content shRNA screening. Oncogene ADVANCE ONLINE PUBLICATION. Deregulation of autophagy has been linked to multiple degenerative diseases and cancer, thus the identification of novel autophagy regulators for potential therapeutic intervention is important. To meet this need, we developed a high content image-based short hairpin RNA screen monitoring levels of the autophagy substrate p62/SQSTM1. We identified 186 genes whose loss caused p62 accumulation indicative of autophagy blockade, and 67 genes whose loss enhanced p62 elimination indicative of autophagy stimulation. One putative autophagy stimulator, 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 4 (PFKFB4), drives flux through pentose phosphate pathway. Knockdown of PFKFB4 in prostate cancer cells increased p62 and reactive oxygen species (ROS), but surprisingly increased autophagic flux. Addition of the ROS scavenger N-acetyl cysteine prevented p62 accumulation in PFKFB4-depleted cells, suggesting that the upregulation of p62 and autophagy was a response to oxidative stress caused by PFKFB4 elimination. Thus, PFKFB4 suppresses oxidative stress and p62 accumulation, without which autophagy is stimulated likely as a ROS detoxification response.Oncogene advance online publication. Full Text.

Sullivan, L.B., Gui, D.Y., Hosios, A.M., Bush, L.N., Freinkman, E., and Vander Heiden, M.G. (2015). Supporting Aspartate Biosynthesis Is an Essential Function of Respiration in Proliferating Cells. Cell 162, 552-563.Mitochondrial respiration is important for cell proliferation; however, the specific metabolic requirements fulfilled by respiration to support proliferation have not been defined. Here, we show that a major role of respiration in proliferating cells is to provide electron acceptors for aspartate synthesis. This finding is consistent with the observation that cells lacking a functional respiratory chain are auxotrophic for pyruvate, which serves as an exogenous electron acceptor. Further, the pyruvate requirement can be fulfilled with an alternative electron acceptor, alpha-ketobutyrate, which provides cells neither carbon nor ATP. Alpha-ketobutyrate restores proliferation when respiration is inhibited, suggesting that an alternative electron acceptor can substitute for respiration to support proliferation. We find that electron acceptors are limiting for producing aspartate, and supplying aspartate enables proliferation of respiration deficient cells in the absence of exogenous electron acceptors. Together, these data argue a major function of respiration in proliferating cells is to support aspartate synthesis. Full Text

Sun, C., Schattgen, S.A., Pisitkun, P., Jorgensen, J.P., Hilterbrand, A.T., Wang, L.J., West, J.A., Hansen, K., Horan, K.A., Jakobsen, M.,.Hidde L. Ploegh, et al. (2015). Evasion of Innate Cytosolic DNA Sensing by a Gammaherpesvirus Facilitates Establishment of Latent Infection. Journal of Immunology Published January 16, 2015. Herpesviruses are DNA viruses harboring the capacity to establish lifelong latent-recurrent infections. There is limited knowledge about viruses targeting the innate DNA-sensing pathway, as well as how the innate system impacts on the latent reservoir of herpesvirus infections. In this article, we report that murine gammaherpesvirus 68 (MHV68), in contrast to alpha- and beta-herpesviruses, induces very limited innate immune responses through DNA-stimulated pathways, which correspondingly played only a minor role in the control of MHV68 infections in vivo. Similarly, Kaposi's sarcoma-associated herpesvirus also did not stimulate immune signaling through the DNA-sensing pathways. Interestingly, an MHV68 mutant lacking deubiquitinase (DUB) activity, embedded within the large tegument protein open reading frame (ORF)64, gained the capacity to stimulate the DNA-activated stimulator of IFN genes (STING) pathway. We found that ORF64 targeted a step in the DNA-activated pathways upstream of the bifurcation into the STING and absent in melanoma 2 pathways, and lack of the ORF64 DUB was associated with impaired delivery of viral DNA to the nucleus, which, instead, localized to the cytoplasm. Correspondingly, the ORF64 DUB active site mutant virus exhibited impaired ability to establish latent infection in wild-type, but not STING-deficient, mice. Thus, gammaherpesviruses evade immune activation by the cytosolic DNA-sensing pathway, which, in the MHV68 model, facilitates establishment of infections. Full Text.

Sun2, X., Huang, L., Markowitz, T.E., Blitzblau, H.G., Chen, D., Klein, F., and Hochwagen, A. (2015). Transcription dynamically patterns the meiotic chromosome-axis interface. eLife 4 : e074244. Meiotic chromosomes are highly compacted yet remain transcriptionally active. To understand how chromosome folding accommodates transcription, we investigated the assembly of the axial element, the proteinaceous structure that compacts meiotic chromosomes and promotes recombination and fertility. We found that the axial element proteins of budding yeast are flexibly anchored to chromatin by the ring-like cohesin complex. The ubiquitous presence of cohesin at sites of convergent transcription provides well-dispersed points for axis attachment and thus chromosome compaction. Axis protein enrichment at these sites directly correlates with the propensity for recombination initiation nearby. A separate modulating mechanism that requires the conserved axial-element component Hop1 biases axis protein binding towards small chromosomes. Importantly, axis anchoring by cohesin is adjustable and readily displaced in the direction of transcription by the transcriptional machinery. We propose that such robust but flexible tethering allows the axial element to promote recombination while easily adapting to changes in chromosome activity. Full Text

Swee, L.K., Lourido, S., Bell, G.W., Ingram, J.R., and Ploegh, H.L. (2015). One-Step Enzymatic Modification of the Cell Surface Redirects Cellular Cytotoxicity and Parasite Tropism. ACS Chemical Biology 10, 460-465.Surface display of engineered proteins has many useful applications. The expression of a synthetic chimeric antigen receptor composed of an extracellular tumor-specific antibody fragment linked to a cytosolic activating motif in engineered T cells is now considered a viable approach for the treatment of leukemias. The risk of de novo tumor development, inherent in the transfer of genetically engineered cells, calls for alternative approaches for the functionalization of the lymphocyte plasma membrane. We demonstrate the conjugation of LPXTG-tagged probes and LPXTG-bearing proteins to endogenous acceptors at the plasma membrane in a single step using sortase A. We successfully conjugated biotin probes not only to mouse hematopoietic cells but also to yeast cells, 293T cells, and Toxoplasma gondii. Installation of single domain antibodies on activated CD8 T cell redirects cell-specific cytotoxicity to cells that bear the relevant antigen. Likewise, conjugation of Toxoplasma gondii with single domain antibodies targets the pathogen to cells that express the antigen recognized by these single domain antibodies. This simple and robust enzymatic approach enables engineering of the plasma membrane for research or therapy under physiological reaction conditions that ensure the viability of the modified cells. Full Text.

Tan, S.H., Yam, A.W., Lawton, L.N., Wong, R.W., Young, R.A., Look, T.A., and Sanda, T. (2015). TRIB2 reinforces the oncogenic transcriptional program controlled by the TAL1 complex in T-cell acute lymphoblastic leukemia. Leukemia article preview 23 July 2015.

Tafesse, F.G., Rashidfarrokhi, A., Schmidt, F.I., Freinkman, E., Dougan, S., Dougan, M., Esteban, A., Maruyama, T., Strijbis, K., and Ploegh, H.L. (2015). Disruption of Sphingolipid Biosynthesis Blocks Phagocytosis of Candida albicans. PLoS pathogens 11, e1005188.The ability of phagocytes to clear pathogens is an essential attribute of the innate immune response. The role of signaling lipid molecules such as phosphoinositides is well established, but the role of membrane sphingolipids in phagocytosis is largely unknown. Using a genetic approach and small molecule inhibitors, we show that phagocytosis of Candida albicans requires an intact sphingolipid biosynthetic pathway. Blockade of serine-palmitoyltransferase (SPT) and ceramide synthase-enzymes involved in sphingolipid biosynthesis- by myriocin and fumonisin B1, respectively, impaired phagocytosis by phagocytes. We used CRISPR/Cas9-mediated genome editing to generate Sptlc2-deficient DC2.4 dendritic cells, which lack serine palmitoyl transferase activity. Sptlc2-/- DC2.4 cells exhibited a stark defect in phagocytosis, were unable to bind fungal particles and failed to form a normal phagocytic cup to engulf C. albicans. Supplementing the growth media with GM1, the major ganglioside present at the cell surface, restored phagocytic activity of Sptlc2-/- DC2.4 cells. While overall membrane trafficking and endocytic pathways remained functional, Sptlc2-/- DC2.4 cells express reduced levels of the pattern recognition receptors Dectin-1 and TLR2 at the cell surface. Consistent with the in vitro data, compromised sphingolipid biosynthesis in mice sensitizes the animal to C. albicans infection. Sphingolipid biosynthesis is therefore critical for phagocytosis and in vivo clearance of C. albicans. Full Text

Truttmann, M.C., Wu, Q., Stiegeler, S., Duarte, J.N., Ingram, J., and Ploegh, H.L. (2015). HypE-specific Nanobodies as Tools to modulate HypE-mediated Target AMPylation. The Journal of biological chemistry [Epub ahead of print] The covalent addition of mono-AMP to target proteins (AMPylation) by Fic domain containing proteins is a poorly understood yet highly conserved post-translational modification. Here we describe the generation, evaluation and application of four HypE specific nanobodies, three of which inhibit HypE mediated target AMPylation in vitro, and a fourth that acts as an activator. All VHHs bind HypE when expressed as GFP-fusions in intact cells. We observe localization of HypE at the nuclear envelope and further identify histones H2 to H4, but not H1, as novel in vitro targets of the human Fic protein. Its role in histone modification provides a possible link between AMPylation and regulation of gene expression. Full Text.

Tsun, Z.Y., and Possemato, R. (2015). Amino Acid Management in Cancer. Seminars in cell & developmental biology[Epub ahead of print] Amino acids have a dual role in cellular metabolism, as they are both the building blocks for protein synthesis and intermediate metabolites which fuel other biosynthetic reactions. Recent work has demonstrated that deregulation of both arms of amino acid management are common alterations seen in cancer. Among the most highly consumed nutrients by cancer cells are the amino acids glutamine and serine, and the biosynthetic pathways that metabolize them are required in various cancer subtypes and the object of current efforts to target cancer metabolism. Also altered in cancer are components of the machinery which sense amino acid sufficiency, nucleated by the mechanistic target of rapamycin (mTOR), a key regulator of cell growth via modulation of key processes including protein synthesis and autophagy. The precise ways in which altered amino acid management supports cellular transformation remains mostly elusive, and a fuller mechanistic understanding of these processes will be important for efforts to exploit such alterations for cancer therapy. Full Text

Tsvetkov, P., Mendillo, M.L., Zhao, J., Carette, J.E., Merrill, P.H., Cikes, D., Varadarajan, M., van Diemen, F.R., Penninger, J.M., Goldberg, A.L., Sandro Santagata, Susan Lindquist, et al. (2015). Compromising the 19S proteasome complex protects cells from reduced flux through the proteasome. eLife 4 : e08467. Proteasomes are central regulators of protein homeostasis in eukaryotes. Proteasome function is vulnerable to environmental insults, cellular protein imbalance and targeted pharmaceuticals. Yet, mechanisms that cells deploy to counteract inhibition of this central regulator are little understood. To find such mechanisms, we reduced flux through the proteasome to the point of toxicity with specific inhibitors and performed genome-wide screens for mutations that allowed cells to survive. Counter to expectation, reducing expression of individual subunits of the proteasome's 19S regulatory complex increased survival. Strong 19S reduction was cytotoxic but modest reduction protected cells from inhibitors. Protection was accompanied by an increased ratio of 20S to 26S proteasomes, preservation of protein degradation capacity and reduced proteotoxic stress. While compromise of 19S function can have a fitness cost under basal conditions, it provided a powerful survival advantage when proteasome function was impaired. This means of rebalancing proteostasis is conserved from yeast to humans. Full Text

Victora, G.D., and Wilson, P.C. (2015). Germinal Center Selection and the Antibody Response to Influenza. Cell 163, 545-548.In this Minireview, we discuss basic aspects of germinal center biology in the context of immunity to influenza infection and speculate on how the simultaneous evolutionary races of virus and antibody may impact our efforts to design a universal influenza vaccine. Full Text

Vyas, V.K., Barrasa, M.I., and Fink, G.R. (2015). A CRISPR system permits genetic engineering of essential genes and gene families. Science advances 1, e1500248.Candida albicans is a pathogenic yeast that causes mucosal and systematic infections with high mortality. The absence of facile molecular genetics has been a major impediment to analysis of pathogenesis. The lack of meiosis coupled with the absence of plasmids makes genetic engineering cumbersome, especially for essential functions and gene families. We describe a C. albicans CRISPR system that overcomes many of the obstacles to genetic engineering in this organism. The high frequency with which CRISPR-induced mutations can be directed to target genes enables easy isolation of homozygous gene knockouts, even without selection. Moreover, the system permits the creation of strains with mutations in multiple genes, gene families, and genes that encode essential functions. This CRISPR system is also effective in a fresh clinical isolate of undetermined ploidy. Our method transforms the ability to manipulate the genome of Candida and provides a new window into the biology of this pathogen. Full Text

Wang3, T., Birsoy, K., Hughes, N.W., Krupczak, K.M., Post, Y., Wei, J.J., Lander, E.S., and Sabatini, D.M. (2015). Identification and characterization of essential genes in the human genome. Science [Epub ahead of print]. Large-scale genetic analysis of lethal phenotypes has elucidated the molecular underpinnings of many biological processes. Using the bacterial clustered regularly interspaced short palindromic repeats (CRISPR) system, we constructed a genome-wide single-guide RNA (sgRNA) library to screen for genes required for proliferation and survival in a human cancer cell line. Our screen revealed the set of cell-essential genes, which was validated by an orthogonal gene-trap-based screen and comparison with yeast gene knockouts. This set is enriched for genes that encode components of fundamental pathways, are expressed at high levels, and contain few inactivating polymorphisms in the human population. We also uncovered a large group of uncharacterized genes involved in RNA processing, a number of whose products localize to the nucleolus. Lastly, screens in additional cell lines showed a high degree of overlap in gene essentiality, but also revealed differences specific to each cell line and cancer type that reflect the developmental origin, oncogenic drivers, paralogous gene expression pattern, and chromosomal structure of each line. These results demonstrate the power of CRISPR-based screens and suggest a general strategy for identifying liabilities in cancer cells. Full Text.

Wang, S., Tsun, Z.Y., Wolfson, R.L., Shen, K., Wyant, G.A., Plovanich, M.E., Yuan, E.D., Jones, T.D., Chantranupong, L., Comb, W, Wang T, Bar-Peled L, Zoncu R, Straub C, Kim C, Park J ,Sabatini BL, Sabatini DM, et al. (2015). Metabolism. Lysosomal amino acid transporter SLC38A9 signals arginine sufficiency to mTORC1. Science 347, 188-194.The mechanistic target of rapamycin complex 1 (mTORC1) protein kinase is a master growth regulator that responds to multiple environmental cues. Amino acids stimulate, in a Rag-, Ragulator-, and vacuolar adenosine triphosphatase-dependent fashion, the translocation of mTORC1 to the lysosomal surface, where it interacts with its activator Rheb. Here, we identify SLC38A9, an uncharacterized protein with sequence similarity to amino acid transporters, as a lysosomal transmembrane protein that interacts with the Rag guanosine triphosphatases (GTPases) and Ragulator in an amino acid-sensitive fashion. SLC38A9 transports arginine with a high Michaelis constant, and loss of SLC38A9 represses mTORC1 activation by amino acids, particularly arginine. Overexpression of SLC38A9 or just its Ragulator-binding domain makes mTORC1 signaling insensitive to amino acid starvation but not to Rag activity. Thus, SLC38A9 functions upstream of the Rag GTPases and is an excellent candidate for being an arginine sensor for the mTORC1 pathway. Full Text.

Wang2, Y., Zhang, T., Kwiatkowski, N., Abraham, B.J., Lee, T.I., Xie, S., Yuzugullu, H., Von, T., Li, H., Lin, Z, Richard A. Young, et al. (2015). CDK7-Dependent Transcriptional Addiction in Triple-Negative Breast Cancer. Cell 163, 174-186.Triple-negative breast cancer (TNBC) is a highly aggressive form of breast cancer that exhibits extremely high levels of genetic complexity and yet a relatively uniform transcriptional program. We postulate that TNBC might be highly dependent on uninterrupted transcription of a key set of genes within this gene expression program and might therefore be exceptionally sensitive to inhibitors of transcription. Utilizing kinase inhibitors and CRISPR/Cas9-mediated gene editing, we show here that triple-negative but not hormone receptor-positive breast cancer cells are exceptionally dependent on CDK7, a transcriptional cyclin-dependent kinase. TNBC cells are unique in their dependence on this transcriptional CDK and suffer apoptotic cell death upon CDK7 inhibition. An "Achilles cluster" of TNBC-specific genes is especially sensitive to CDK7 inhibition and frequently associated with super-enhancers. We conclude that CDK7 mediates transcriptional addiction to a vital cluster of genes in TNBC and CDK7 inhibition may be a useful therapy for this challenging cancer. Full Text

Warren, W.C., Jasinska, A.J., Garcia-Perez, R., Svardal, H., Tomlinson, C., Rocchi, M., Archidiacono, N., Capozzi, O., Minx, P., Montague, M.J.,Jennifer Hughes, et al. (2015). The genome of the vervet (Chlorocebus aethiops sabaeus). Genome Research [Epub ahead of print] We describe a genome reference of the African green monkey or vervet (Chlorocebus aethiops). This member of the Old World monkey (OWM) superfamily is uniquely valuable for genetic investigations of simian immunodeficiency virus (SIV), of which it is the most abundant natural host species, and of a wide range of health-related phenotypes assessed in Caribbean vervets (C. a. sabaeus), whose numbers have expanded dramatically since Europeans introduced small numbers of their ancestors from West Africa during the colonial era. We use the reference to characterize the genomic relationship between vervets and other primates, the intrageneric phylogeny of vervet subspecies, and genome-wide structural variations of a pedigreed C. a. sabaeus population. Through comparative analyses with human and rhesus macaque, we characterize at high resolution the unique chromosomal fission events that differentiate the vervets and their close relatives from most other catarrhine primates, in whom karyotype is highly conserved. We also provide a summary of transposable elements and contrast these with the rhesus macaque and human. Analysis of sequenced genomes representing each of the main vervet subspecies supports previously hypothesized relationships between these populations, which range across most of sub-Saharan Africa, while uncovering high levels of genetic diversity within each. Sequence-based analyses of major histocompatibility complex (MHC) polymorphisms reveal extremely low diversity in Caribbean C. a. sabaeus vervets, as compared to vervets from putatively ancestral West African regions. In the C. a. sabaeus research population we discover the first structural variations that are in some cases predicted to have a deleterious effect; future studies will determine the phenotypic impact of these variations. Full Text

Watnick, R.S., Rodriguez, R.K., Wang, S., Blois, A.L., Rangarajan, A., Ince, T., and Weinberg, R.A. (2015). Thrombospondin-1 repression is mediated via distinct mechanisms in fibroblasts and epithelial cells. Oncogene 34, 2823-2835.Tumor-associated angiogenesis is postulated to be regulated by the balance between pro- and anti-angiogenic factors. We demonstrate here that the critical step in establishing the angiogenic capability of human tumor cells is the repression of a key secreted anti-angiogenic factor, thrombospondin-1 (Tsp-1). This repression is essential for tumor formation by mammary epithelial cells and kidney cells engineered to express SV40 early region proteins, hTERT, and H-RasV12. In transformed epithelial cells, a signaling pathway leading from Ras to Tsp-1 repression induces the sequential activation of PI3 kinase, Rho and ROCK, leading to activation of Myc through phosphorylation, thereby enabling Myc to repress Tsp-1 transcription. In transformed fibroblasts, however, the repression of Tsp-1 can be achieved by an alternative mechanism involving inactivation of both p53 and pRb. We thus describe novel mechanisms by which the activation of oncogenes in epithelial cells and the inactivation of tumor suppressors in fibroblasts permits angiogenesis and, in turn, tumor formation. Full Text

Wen, J.Y., Duan, H., Bejarano, F., Okamura, K., Fabian, L., Brill, J.A., Bortolamiol-Becet, D., Martin, R., Ruby, J.G., and Lai, E.C. (2015). Adaptive Regulation of Testis Gene Expression and Control of Male Fertility by the Drosophila Harpin RNA Pathway. Molecular Cell 57, 165-178.Although endogenous siRNAs (endo-siRNAs) have been described in many species, still little is known about their endogenous utility. Here, we show that Drosophila hairpin RNAs (hpRNAs) generate an endo-siRNA class with predominant expression in testes. Although hpRNAs are universally recently evolved, we identify highly complementary protein-coding targets for all hpRNAs. Importantly, we find broad evidence for evolutionary divergences that preferentially maintain compensatory pairing between hpRNAs and targets, serving as first evidence for adaptive selection for siRNA-mediated target regulation in metazoans. We demonstrate organismal impact of hpRNA activity, since knockout of hpRNA1 derepresses its target ATP synthase-beta in testes and compromises spermatogenesis and male fertility. Moreover, we reveal surprising male-specific impact of RNAi factors on germ cell development and fertility, consistent with testis-directed function of the hpRNA pathway. Finally, the collected hpRNA loci chronicle an evolutionary timeline that reflects their origins from prospective target genes, mirroring a strategy described for plant miRNAs. Full Text.

Wheeler, D.B., Zoncu, R., Root, D.E., Sabatini, D.M., and Sawyers, C.L. (2015). Identification of an oncogenic RAB protein. Science Published Online. In an shRNA screen for genes that affect AKT phosphorylation, we identified the RAB35 small GTPase-a protein previously implicated in endomembrane trafficking-as a regulator of the PI3K pathway. Depletion of RAB35 suppresses AKT phosphorylation in response to growth factors, whereas expression of a dominant active GTPase-deficient mutant of RAB35 constitutively activates the PI3K/AKT pathway. RAB35 functions downstream of growth factor receptors and upstream of PDK1 and mTORC2 and co-purifies with PI3K in immunoprecipitation assays. Two somatic RAB35 mutations found in human tumors generate alleles that constitutively activate PI3K/AKT signaling, suppress apoptosis, and transform cells in a PI3K-dependent manner. Furthermore, oncogenic RAB35 is sufficient to drive PDGFRalpha to LAMP2-positive endomembranes in the absence of ligand, suggesting there may be latent oncogenic potential in dysregulated endomembrane trafficking. Full Text

Wiehle, L., Raddatz, G., Musch, T., Dawlaty, M.M., Jaenisch, R., Lyko, F., and Breiling, A. (2015). Tet1 and Tet2 protect DNA methylation canyons against hypermethylation. Molecular and cellular biology.. [Epub ahead of print] DNA methylation is a dynamic epigenetic modification with an important role in cell fate specification and reprogramming. The Ten eleven translocation family of enzymes converts 5-methylcytosine to 5-hydroxymethylcytosine which promotes passive DNA demethylation and functions as 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

Williams, B.P., Pignatta, D., Henikoff, S., and Gehring, M. (2015). Methylation-sensitive expression of a DNA demethylase gene serves as an epigenetic rheostat. PLoS genetics 11, e1005142.Genomes must balance active suppression of transposable elements (TEs) with the need to maintain gene expression. In Arabidopsis, euchromatic TEs are targeted by RNA-directed DNA methylation (RdDM). Conversely, active DNA demethylation prevents accumulation of methylation at genes proximal to these TEs. It is unknown how a cellular balance between methylation and demethylation activities is achieved. Here we show that both RdDM and DNA demethylation are highly active at a TE proximal to the major DNA demethylase gene ROS1. Unexpectedly, and in contrast to most other genomic targets, expression of ROS1 is promoted by DNA methylation and antagonized by DNA demethylation. We demonstrate that inducing methylation in the ROS1 proximal region is sufficient to restore ROS1 expression in an RdDM mutant. Additionally, methylation-sensitive expression of ROS1 is conserved in other species, suggesting it is adaptive. We propose that the ROS1 locus functions as an epigenetic rheostat, tuning the level of demethylase activity in response to methylation alterations, thus ensuring epigenomic stability. Full Text.

Witte, M.D., Wu, T., Guimaraes, C.P., Theile, C.S., Blom, A.E., Ingram, J.R., Li, Z., Kundrat, L., Goldberg, S.D., and Ploegh, H.L. (2015). Site-specific protein modification using immobilized sortase in batch and continuous-flow systems. Nature protocols 10, 508-516.Transpeptidation catalyzed by sortase A allows the preparation of proteins that are site-specifically and homogeneously modified with a wide variety of functional groups, such as fluorophores, PEG moieties, lipids, glycans, bio-orthogonal reactive groups and affinity handles. This protocol describes immobilization of sortase A on a solid support (Sepharose beads). Immobilization of sortase A simplifies downstream purification of a protein of interest after labeling of its N or C terminus. Smaller batch and larger-scale continuous-flow reactions require only a limited amount of enzyme. The immobilized enzyme can be reused for multiple cycles of protein modification reactions. The described protocol also works with a Ca(2+)-independent variant of sortase A with increased catalytic activity. This heptamutant variant of sortase A (7M) was generated by combining previously published mutations, and this immobilized enzyme can be used for the modification of calcium-senstive substrates or in instances in which low temperatures are needed. Preparation of immobilized sortase A takes 1-2 d. Batch reactions take 3-12 h and flow reactions proceed at 0.5 ml h(-1), depending on the geometry of the reactor used. Full Text.

Wolfson, R.L., Chantranupong, L., Saxton, R.A., Shen, K., Scaria, S.M., Cantor, J.R., and Sabatini, D.M. (2015). Sestrin2 is a leucine sensor for the mTORC1 pathway. Science Epub Leucine is a proteogenic amino acid that also regulates many aspects of mammalian physiology, in large part by activating the mTOR complex 1 (mTORC1) protein kinase, a master growth controller. Amino acids signal to mTORC1 through the Rag guanosine triphosphatases (GTPases). Several factors regulate the Rags, including GATOR1, a GTPase-activating protein (GAP); GATOR2, a positive regulator of unknown function; and Sestrin2, a GATOR2-interacting protein that inhibits mTORC1 signaling. We find that leucine, but not arginine, disrupts the Sestrin2-GATOR2 interaction by binding to Sestrin2 with a dissociation constant of 20 muM, which is the leucine concentration that half-maximally activates mTORC1. The leucine-binding capacity of Sestrin2 is required for leucine to activate mTORC1 in cells. These results indicate that Sestrin2 is a leucine sensor for the mTORC1 pathway. Full Text

Wong, S.F., Agarwal, V., Mansfield, J.H., Denans, N., Schwartz, M.G., Prosser, H.M., Pourquie, O., Bartel, D.P., Tabin, C.J., and McGlinn, E. (2015). Independent regulation of vertebral number and vertebral identity by microRNA-196 paralogs. Proceedings of the National Academy of Sciences of the United States of America Early Edition.The Hox genes play a central role in patterning the embryonic anterior-to-posterior axis. An important function of Hox activity in vertebrates is the specification of different vertebral morphologies, with an additional role in axis elongation emerging. The miR-196 family of microRNAs (miRNAs) are predicted to extensively target Hox 3' UTRs, although the full extent to which miR-196 regulates Hox expression dynamics and influences mammalian development remains to be elucidated. Here we used an extensive allelic series of mouse knockouts to show that the miR-196 family of miRNAs is essential both for properly patterning vertebral identity at different axial levels and for modulating the total number of vertebrae. All three miR-196 paralogs, 196a1, 196a2, and 196b, act redundantly to pattern the midthoracic region, whereas 196a2 and 196b have an additive role in controlling the number of rib-bearing vertebra and positioning of the sacrum. Independent of this, 196a1, 196a2, and 196b act redundantly to constrain total vertebral number. Loss of miR-196 leads to a collective up-regulation of numerous trunk Hox target genes with a concomitant delay in activation of caudal Hox genes, which are proposed to signal the end of axis extension. Additionally, we identified altered molecular signatures associated with the Wnt, Fgf, and Notch/segmentation pathways and demonstrate that miR-196 has the potential to regulate Wnt activity by multiple mechanisms. By feeding into, and thereby integrating, multiple genetic networks controlling vertebral number and identity, miR-196 is a critical player defining axial formulae. Full Text

Wurtzel, O., Cote, L.E., Poirier, A., Satija, R., Regev, A., and Reddien, P.W. (2015). A Generic and Cell-Type-Specific Wound Response Precedes Regeneration in Planarians. Developmental cell 35, 632-645.Regeneration starts with injury. Yet how injuries affect gene expression in different cell types and how distinct injuries differ in gene expression remain unclear. We defined the transcriptomes of major cell types of planarians-flatworms that regenerate from nearly any injury-and identified 1,214 tissue-specific markers across 13 cell types. RNA sequencing on 619 single cells revealed that wound-induced genes were expressed either in nearly all cell types or specifically in one of three cell types (stem cells, muscle, or epidermis). Time course experiments following different injuries indicated that a generic wound response is activated with any injury regardless of the regenerative outcome. Only one gene, notum, was differentially expressed early between anterior- and posterior-facing wounds. Injury-specific transcriptional responses emerged 30 hr after injury, involving context-dependent patterning and stem-cell-specialization genes. The regenerative requirement of every injury is different; however, our work demonstrates that all injuries start with a common transcriptional response. Full Text

Yang, F., Silber, S., Leu, N.A., Oates, R.D., Marszalek, J.D., Skaletsky, H., Brown, L.G., Rozen, S., Page, D.C., and Wang, P.J. (2015). TEX11 is mutated in infertile men with azoospermia and regulates genome-wide recombination rates in mouse. EMBO molecular medicine Early View. Genome-wide recombination is essential for genome stability, evolution, and speciation. Mouse Tex11, an X-linked meiosis-specific gene, promotes meiotic recombination and chromosomal synapsis. Here, we report that TEX11 is mutated in infertile men with non-obstructive azoospermia and that an analogous mutation in the mouse impairs meiosis. Genetic screening of a large cohort of idiopathic infertile men reveals that TEX11 mutations, including frameshift and splicing acceptor site mutations, cause infertility in 1% of azoospermic men. Functional evaluation of three analogous human TEX11 missense mutations in transgenic mouse models identified one mutation (V748A) as a potential infertility allele and found two mutations non-causative. In the mouse model, an intronless autosomal Tex11 transgene functionally substitutes for the X-linked Tex11 gene, providing genetic evidence for the X-to-autosomal retrotransposition evolution phenomenon. Furthermore, we find that TEX11 protein levels modulate genome-wide recombination rates in both sexes. These studies indicate that TEX11 alleles affecting expression level or substituting single amino acids may contribute to variations in recombination rates between sexes and among individuals in humans. Full Text

Ye2, X., and Weinberg, R.A. (2015). Epithelial-Mesenchymal Plasticity: A Central Regulator of Cancer Progression. Trends in Cell Biology [Epub ahead of print] The epithelial-mesenchymal transition (EMT) program has emerged as a central driver of tumor malignancy. Moreover, the recently uncovered link between passage through an EMT and acquisition of stem-like properties indicates that activation of the EMT programs serves as a major mechanism for generating cancer stem cells (CSCs); that is, a subpopulation of cancer cells that are responsible for initiating and propagating the disease. In this review, we summarize the evidence supporting the widespread involvement of the EMT program in tumor pathogenesis and attempt to rationalize the connection between the EMT program and acquisition of stem cell traits. We propose that epithelial-mesenchymal plasticity is likely controlled by multiple varients of the core EMT program, and foresee the need to resolve the various programs and the molecular mechanisms that underlie them. Full Text

Ye, X., Tam, W.L., Shibue, T., Kaygusuz, Y., Reinhardt, F., Ng Eaton, E., and Weinberg, R.A. (2015). Distinct EMT programs control normal mammary stem cells and tumour-initiating cells .Nature 525,
256–260. Tumour-initiating cells (TICs) are responsible for metastatic dissemination and clinical relapse in a variety of cancers. Analogies between TICs and normal tissue stem cells have led to the proposal that activation of the normal stem-cell program within a tissue serves as the major mechanism for generating TICs. Supporting this notion, we and others previously established that the Slug epithelial-to-mesenchymal transition-inducing transcription factor (EMT-TF), a member of the Snail family, serves as a master regulator of the gland-reconstituting activity of normal mammary stem cells, and that forced expression of Slug in collaboration with Sox9 in breast cancer cells can efficiently induce entrance into the TIC state. However, these earlier studies focused on xenograft models with cultured cell lines and involved ectopic expression of EMT-TFs, often at non-physiological levels. Using genetically engineered knock-in reporter mouse lines, here we show that normal gland-reconstituting mammary stem cells residing in the basal layer of the mammary epithelium and breast TICs originating in the luminal layer exploit the paralogous EMT-TFs Slug and Snail, respectively, which induce distinct EMT programs. Broadly, our findings suggest that the seemingly similar stem-cell programs operating in TICs and normal stem cells of the corresponding normal tissue are likely to differ significantly in their details. Full Text

Yu, W., Jiang, N., Ebert, P.J., Kidd, B.A., Muller, S., Lund, P.J., Juang, J., Adachi, K., Tse, T., Birnbaum, M.E,.Gijsbert M. Grotenbreg, et al. (2015). Clonal Deletion Prunes but Does Not Eliminate Self-Specific alphabeta CD8 T Lymphocytes. Immunity 42, 929-941.It has long been thought that clonal deletion efficiently removes almost all self-specific T cells from the peripheral repertoire. We found that self-peptide MHC-specific CD8+ T cells in the blood of healthy humans were present in frequencies similar to those specific for non-self antigens. For the Y chromosome-encoded SMCY antigen, self-specific T cells exhibited only a 3-fold lower average frequency in males versus females and were anergic with respect to peptide activation, although this inhibition could be overcome by a stronger stimulus. We conclude that clonal deletion prunes but does not eliminate self-specific T cells and suggest that to do so would create holes in the repertoire that pathogens could readily exploit. In support of this hypothesis, we detected T cells specific for all 20 amino acid variants at the p5 position of a hepatitis C virus epitope in a random group of blood donors. Full Text

Zhang2, Y., Blanden, M.J., Sudheer, C., Gangopadhyay, S.A., Rashidian, M., Hougland, J.L., and Distefano, M.D. (2015). Simultaneous Site-Specific Dual Protein Labeling Using Protein Prenyltransferases. Bioconjugate chemistry 26, 2542-2553.Site-specific protein labeling is an important technique in protein chemistry and is used for diverse applications ranging from creating protein conjugates to protein immobilization. Enzymatic reactions, including protein prenylation, have been widely exploited as methods to accomplish site-specific labeling. Enzymatic prenylation is catalyzed by prenyltransferases, including protein farnesyltransferase (PFTase) and geranylgeranyltransferase type I (GGTase-I), both of which recognize C-terminal CaaX motifs with different specificities and transfer prenyl groups from isoprenoid diphosphates to their respective target proteins. A number of isoprenoid analogues containing bioorthogonal functional groups have been used to label proteins of interest via PFTase-catalyzed reaction. In this study, we sought to expand the scope of prenyltransferase-mediated protein labeling by exploring the utility of rat GGTase-I (rGGTase-I). First, the isoprenoid specificity of rGGTase-I was evaluated by screening eight different analogues and it was found that those with bulky moieties and longer backbone length were recognized by rGGTase-I more efficiently. Taking advantage of the different substrate specificities of rat PFTase (rPFTase) and rGGTase-I, we then developed a simultaneous dual labeling method to selectively label two different proteins by using isoprenoid analogue and CaaX substrate pairs that were specific to only one of the prenyltransferases. Using two model proteins, green fluorescent protein with a C-terminal CVLL sequence (GFP-CVLL) and red fluorescent protein with a C-terminal CVIA sequence (RFP-CVIA), we demonstrated that when incubated together with both prenyltransferases and the selected isoprenoid analogues, GFP-CVLL was specifically modified with a ketone-functionalized analogue by rGGTase-I and RFP-CVIA was selectively labeled with an alkyne-containing analogue by rPFTase. By switching the ketone-containing analogue to an azide-containing analogue, it was possible to create protein tail-to-tail dimers in a one-pot procedure through the copper(I)-catalyzed alkyne-azide cycloaddition (CuAAC) reaction. Overall, with the flexibility of using different isoprenoid analogues, this system greatly extends the utility of protein labeling using prenyltransferases. Full Text

Zhang, Y., and Wang, X.F. (2015). A niche role for cancer exosomes in metastasis. Nature cell biology 17, 709-711 .Cancer cells are known to secrete exosomes with pro-metastatic effects. Pancreatic-cancer-derived exosomes are now shown to promote liver metastasis by eliciting pre-metastatic niche formation through a multi-step process. This involves uptake of exosome-derived factors by liver Kupffer cells and hepatic stellate cell activation to generate a fibrotic microenvironment with immune cell infiltrates that favours metastasis. Full Text

Zhao, Z., Chen, L., Dawlaty, M.M., Pan, F., Weeks, O., Zhou, Y., Cao, Z., Shi, H., Wang, J., Lin, L., Rudolph Jaenisch, et al. (2015). Combined Loss of Tet1 and Tet2 Promotes B Cell, but Not Myeloid Malignancies, in Mice. Cell reports.Volume 13, Issue 8, 1692–1704. TET1/2/3 are methylcytosine dioxygenases that regulate cytosine hydroxymethylation. Tet1/2 are abundantly expressed in HSC/HPCs and are implicated in hematological malignancies. Tet2 deletion in mice causes myeloid malignancies, while Tet1-null mice develop B cell lymphoma after an extended period of latency. Interestingly, TET1/2 are often concomitantly downregulated in acute B-lymphocytic leukemia. Here, we investigated the overlapping and non-redundant functions of Tet1/2 using Tet1/2 double-knockout (DKO) mice. DKO and Tet2-/- HSC/HPCs show overlapping and unique 5hmC and 5mC profiles. DKO mice exhibit strikingly decreased incidence and delayed onset of myeloid malignancies in comparison to Tet2-/- mice and in contrast develop lethal B cell malignancies. Transcriptome analysis of DKO tumors reveals expression changes in many genes dysregulated in human B cell malignancies, including LMO2, BCL6, and MYC. These results highlight the critical roles of TET1/2 individually and together in the pathogenesis of hematological malignancies. Full Text

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