The following alphabetical list represents papers published in 2010 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.

 

-Activation of Amp-Activated Protein Kinase Signaling Pathway by Adiponectin and Insulin in Mouse Adipocytes: Requirement of Acyl-Coa Synthetases Fatp1 and Acsl1 and Association with an Elevation in Amp/Atp Ratio. Liu
-Adiponectin Deficiency Promotes Tumor Growth in Mice by Reducing Macrophage Infiltration. Sun
-Alpha-Synuclein: Membrane Interactions and Toxicity in Parkinson's Disease. Auluck
-Alteration of Processing Induced by a Single Nucleotide Polymorphism in Pri-Mir-126. Harnprasopwat
-The Angelman Syndrome Protein Ube3a Regulates Synapse Development by Ubiquitinating Arc. Greer
-Association of Tat with Promoters of Pten and Pp2a Subunits Is Key to Transcriptional Activation of Apoptotic Pathways in Hiv-Infected Cd4+T Cells Kim
-Aurora B Kinase Controls the Targeting of the Astrin-Skap Complex to Bioriented Kinetochores Schmidt
-Aurora B Phosphorylates Spatially Distinct Targets to Differentially Regulate the Kinetochore-Microtubule Interface Welburn
-Autocrine Tgf-Beta and Stromal Cell-Derived Factor-1 (Sdf-1) Signaling Drives the Evolution of Tumor-Promoting Mammary Stromal Myofibroblasts. Kojima
-Biochemical, Cell Biological, and Genetic Assays to Analyze Amyloid and Prion Aggregation in Yeast. Alberti
-c-Myc Regulates Transcriptional Pause Release. Rahl
-Cancer Cell of Origin: Spotlight on Luminal Progenitors.Chaffer
-Characterization and Structural Studies of the Plasmodium Falciparum Ubiquitin and Nedd8 Hydrolase Uchl3 ArtavanisTsakonas
-Chimpanzee and Human Y Chromosomes Are Remarkably Divergent in Structure and Gene Content. Hughes
-Chromatin Structure and Gene Expression Programs of Human Embryonic and Induced Pluripotent Stem Cells Guenther
-Chromosome Segregation: Taking the Passenger Seat Vader
-Combinatorial Development of Biomaterials for Clonal Growth of Human Pluripotent Stem Cells. Mei
-Comparison of Contractile Behavior of Native Murine Ventricular Tissue and Cardiomyocytes Derived from Embryonic or Induced Pluripotent Stem Cells Xi
-Compounds from an Unbiased Chemical Screen Reverse Both Er-to-Golgi Trafficking Defects and Mitochondrial Dysfunction in Parkinson's Disease Models. Su
-Concurrent Suppression of Integrin {Alpha}5, Radixin, and Rhoa Phenocopies the Effects of Mir-31 on Metastasis Valastyan
-Convergent Evolution of Chicken Z and Human X Chromosomes by Expansion and Gene Acquisition. Bellott
-Core Epithelial-to-Mesenchymal Transition Interactome Gene-Expression Signature Is Associated with Claudin-Low and Metaplastic Breast Cancer Subtypes. Taube
-A Crucial Overseer of Tumor Metastasis and Other Emerging Roles Valastyan
-Deficiency of Antigen-Presenting Cell Invariant Chain Reduces Atherosclerosis in Mice. Sun
-Derivation of Pre-X Inactivation Human Embryonic Stem Cells under Physiological Oxygen Concentrations. Lengner
-Detecting Separate Time Scales in Genetic Expression Data. Orlando
-Developmental Control of the DNA Replication and Transcription Programs. Nordman
-Differential Regulation of Effector- and Central-Memory Responses to Toxoplasma Gondii Infection by Il-12 Revealed by Tracking of Tgd057-Specific Cd8+T Cells. Wilson
-Differential Requirements for Clathrin-Dependent Endocytosis at Sites of Cell-Substrate Adhesion. Batchelder
-Differentiated Parkinson Patient-Derived Induced Pluripotent Stem Cells Grow in the Adult Rodent Brain and Reduce Motor Asymmetry in Parkinsonian Rats. Hargus
-Discovering Homotypic Binding Events at High Spatial Resolution. Guo
-Distinct Roles for Mir-1 and Mir-133a in the Proliferation and Differentiation of Rhambomyosarcoma Cells. Rao
-Down-Regulation of Myc Is Essential for Terminal Erythroid Maturation. Jayapal
-A Drag Correlation for a Nonporous Sphere Steadily Approaching an Impermeable Plane at Finite Reynolds Numbers. Ryu
-Enhancing Stress Resistance and Production Phenotypes through Transcriptome Engineering. Lam
-Epigenetic Memory in Induced Pluripotent Stem Cells. Kim
-Epigenetics in the Extreme: Prions and the Inheritance of Environmentally Acquired Traits Halfmann
-Epithelial Mesenchymal Transition Traits in Human Breast Cancer Cell Lines Parallel the Cd44(Hi/)Cd24(Lo/-) Stem Cell Phenotype in Human Breast Cancer. Blick
-Epithelial Relaxation Mediated by the Myosin Phosphatase Regulator Mypt1 Is Required for Brain Ventricle Lumen Expansion and Hindbrain Morphogenesis. Gutzman
-Expanding the Microrna Targeting Code: Functional Sites with Centered Pairing. Shin
-Expansion of Human Cord Blood Hematopoietic Stem Cells for Transplantation. Chou
-Fatp2 Is a Hepatic Fatty Acid Transporter and Peroxisomal Very Long-Chain Acyl-Coa Synthetase Falcon
-Feed-Forward Regulation of a Cell Fate Determinant by an Rna-Binding Protein Generates Asymmetry in Yeast Wolf
-Fetal Liver Hepatic Progenitors Are Supportive Stromal Cells for Hematopoietic Stem Cells. Chou
-Formation of Antiviral Cytoplasmic Granules During Orthopoxvirus Infection. SimpsonHolley
-Formation, Regulation and Evolution of Caenorhabditis Elegans 3'utrs. Jan
-From Stem Cell to Erythroblast: Regulation of Red Cell Production at Multiple Levels by Multiple Hormones. Lodish
-Functional Genomics, Proteomics, and Regulatory DNA Analysis in Isogenic Settings Using Zinc Finger Nuclease-Driven Transgenesis into a Safe Harbor Locus in the Human Genome. Dekelver
-Gene Expression Profiling - Clusters of Possibilities Bergkvist
-Generation of Ipscs from Cultured Human Malignant Cells. Carette
-Genotype to Phenotype: A Complex Problem. Dowell
-Global Control of Motor Neuron Topography Mediated by the Repressive Actions of a Single Hox Gene. Jung
-The Hcmv Membrane Glycoprotein Us10 Selectively Targets Hla-G for Degradation. Park
-Hif-1 {Alpha} Synergizes with Glucocorticoids to Promote Bfu-E Progenitor Self-Renewal. Flygare
-Histone H3k27ac Separates Active from Poised Enhancers and Predicts Developmental State Creyghton
-Homeodomain-Interacting Protein Kinase 2 Plays an Important Role in Normal Terminal Erythroid Differentiation Hattangadi
-Hsp90 and Environmental Stress Transform the Adaptive Value of Natural Genetic Variation Jarosz
-Hsp90 at the Hub of Protein Homeostasis: Emerging Mechanistic Insights.Taipale
-Human Embryonic Stem Cells with Biological and Epigenetic Characteristics Similar to Those of Mouse Escs. Hanna
-Identification of Functional Elements and Regulatory Circuits by Drosophila Modencode. Roy
-Immunity and Immunopathology to Viruses: What Decides the Outcome? Rouse
-Injectable in Situ Cross-Linking Hydrogels for Local Antifungal Therapy. Hudson
-Integrating Multiple Evidence Sources to Predict Transcription Factor Binding in the Human Genome Ernst
-Ips Cell Technology in Regenerative Medicine. Lengner
-Kinetochore Assembly: If You Build It, They Will Come. Gascoigne
-Lysosomal Dysfunction Promotes Cleavage and Neurotoxicity of Tau in Vivo. Khurana
-Mammalian Micrornas: Experimental Evaluation of Novel and Previously Annotated Genes Chiang
-Mammalian Micrornas Predominantly Act to Decrease Target Mrna Levels. Guo
-A Mec1- and Pp4-Dependent Checkpoint Couples Centromere Pairing to Meiotic Recombination Falk
-Mediator and Cohesin Connect Gene Expression and Chromatin Architecture. Kagey
-Meiosis: A Prdm9 Guide to the Hotspots of Recombination. Hochwagen
-Metastasis Suppression: A Role of the Dice(R) Valastyan
-Metastatic Colonization: Settlement, Adaptation and Propagation of Tumor Cells in a Foreign Tissue Environment. Shibue
-The Mi-2-Like Smed-Chd4 Gene Is Required for Stem Cell Differentiation in the Planarian Schmidtea Mediterranea. Scimone
-Microrna Mir-125b Causes Leukemia Bousquet
-Micrornas Prevent Precocious Gene Expression and Enable Pattern Formation During Plant Embryogenesis Nodine
-Mir-191 Regulates Mouse Erythroblast Enucleation by Down-Regulating Riok3 and Mxi1. Zhang
-Mir-9, a Myc/Mycn-Activated Microrna, Regulates E-Cadherin and Cancer Metastasis. Ma
-The Mir-17-92 Microrna Polycistron Regulates Mll Leukemia Stem Cell Potential by Modulating P21 Expression Wong
-A Mitotic Phosphorylation Feedback Network Connects Cdk1, Plk1, 53bp1, and Chk2 to Inactivate the G2/M DNA Damage Checkpoint. vanVugt
-Modelling Neurodegeneration in Saccharomyces Cerevisiae: Why Cook with Baker's Yeast? Khurana
-Mtor: From Growth Signal Integration to Cancer, Diabetes and Ageing Zoncu
-Mtorc1 Activates Srebp-1c and Uncouples Lipogenesis from Gluconeogenesis Laplante
-Mtorc1 Controls Fasting-Induced Ketogenesis and Its Modulation by Ageing. Sengupta
-Musashi-2 Regulates Normal Hematopoiesis and Promotes Aggressive Myeloid Leukemia. Kharas
-Native Capillary Isoelectric Focusing for the Separation of Protein Complex Isoforms and Subcomplexes. Fonslow
-A Novel Mutation in the Mir-128b Gene Reduces Mirna Processing and Leads to Glucocorticoid Resistance of Mll-Af4 Acute Lymphocytic Leukemia Cells. Kotani
-Of Mice and (Wo)Men: Mouse Models of Breast Cancer Metastasis to Bone. Goldstein
-Optical Trapping with High Forces Reveals Unexpected Behaviors of Prion Fibrils Dong
-Planarian Regeneration Involves Distinct Stem Cell Responses to Wounds and Tissue Absence. Wenemoser
-Pluripotency and Cellular Reprogramming: Facts, Hypotheses, Unresolved Issues. Hanna
-Pluripotent Stem Cells Are Highly Susceptible Targets for Syngeneic, Allogeneic, and Xenogeneic Natural Killer Cells. Dressel
-Point: Hypotheses First. Weinberg
-Polyamine Pathway Contributes to the Pathogenesis of Parkinson Disease. Lewandowski
-Position-Dependent Silencing of Germline V Beta Segments on Tcr Beta Alleles Containing Preassembled V Beta Dj Beta C Beta 1 Genes Brady
-Prb and E2f4 Play Distinct Cell-Intrinsic Roles in Fetal Erythropoiesis. Zhang
-Prion Induction Involves an Ancient System for the Sequestration of Aggregated Proteins and Heritable Changes in Prion Fragmentation Tyedmers
-Prions, Protein Homeostasis, and Phenotypic Diversity. Halfmann
-Protein Folding Sculpting Evolutionary Change. Lindquist
-Protein Homeostasis and the Phenotypic Manifestation of Genetic Diversity: Principles and Mechanisms. Jarosz
-Quality and Quantity Control at the Endoplasmic Reticulum. Hegde
-Rac Gtpases Play Multiple Roles in Erythropoiesis. Ji
-Ragulator-Rag Complex Targets Mtorc1 to the Lysosomal Surface and Is Necessary for Its Activation by Amino Acids. Sancak
-Reconstructing Sex Chromosome Evolution. Page
-Reconstructing the Evolution of Vertebrate Sex Chromosomes. Bellott
-Regulated Targeting of Protein Phosphatase 1 to the Outer Kinetochore by Knl1 Opposes Aurora B Kinase. Liu
-Regulation of the Mtor Complex 1 Pathway by Nutrients, Growth Factors, and Stress Sengupta
-Reprogramming of Human Peripheral Blood Cells to Induced Pluripotent Stem Cells Staerk
-Rictor Phosphorylation on the Thr-1135 Site Does Not Require Mammalian Target of Rapamycin Complex 2. Boulbes
-Ronin/Hcf-1 Binds to a Hyperconserved Enhancer Element and Regulates Genes Involved in the Growth of Embryonic Stem Cells. Dejosez
-Short Rnas Are Transcribed from Repressed Polycomb Target Genes and Interact with Polycomb Repressive Complex-2 Kanhere
-Spatial Expression Profiles in the Xenopus Laevis Oocytes Measured with Qpcr-Tomography. Sindelka
-The Stats on Naive Ipsc Reprogramming. Hanna
-Strong Intranucleoid Interactions Organize the Escherichia Coli Chromosome into a Nucleoid Filament. Wiggins
-Structure of the Human Mtor Complex I and Its Implications for Rapamycin Inhibition. Yip
-A Structural Element within the Huwe1 Hect Domain Modulates Self-Ubiquitination and Substrate Ubiquitination Activities. Pandya
-T-Cell Receptor-Driven Lymphomagenesis in Mice Derived from a Reprogrammed T Cell. Serwold
-Tcr Beta Feedback Signals Inhibit the Coupling of Recombinationally Accessible V Beta 14 Segments with Dj Beta Complexes. YangIott
-Ten Years of Genetics and Genomics: What Have We Achieved and Where Are We Heading? Heard
-Tension at Embo's Aneuploidy Workshop. DeWulf
-The Tetraspanin Cd82 Is Specifically Recruited to Fungal and Bacterial Phagosomes Prior to Acidification. ArtavanisTsakonas
-Thalassemia: An Overview of 50 Years of Clinical Research. Sankaran
-Therapeutic Silencing of Mir-10b Inhibits Metastasis in a Mouse Mammary Tumor Model. Ma
-Three Quite Different Things That Matter to Me. Lindquist
-Transcriptional Role of Cyclin D1 in Development Revealed by a Genetic-Proteomic Screen Bienvenu
-The Transmembrane Segment of a Tail-Anchored Protein Determines Its Degradative Fate through Dislocation from the Endoplasmic Reticulum. Claessen
-Transnuclear Mice with Predefined T Cell Receptor Specificities against Toxoplasma Gondii Obtained Via Scnt. Kirak
-Tumor-Host Interactions: A Far-Reaching Relationship. McAllister
-Two-Step Imprinted X-Inactivation: Repeat Vs Genic Silencing in the Mouse. Namekawa
-The Use of Surface Modified Poly(Glycerol-Co-Sebacic Acid) in Retinal Transplantation. Pritchard

Alberti, S., Halfmann, R., and Lindquist, S. Biochemical, Cell Biological, and Genetic Assays to Analyze Amyloid and Prion Aggregation in Yeast. In Methods in Enzymology, Vol 470: Guide to Yeast Genetics: , pp. 709-734.
Protein aggregates are associated with a variety of debilitating human diseases, but they can have functional roles as well. Both pathological and nonpathological protein aggregates display tremendous diversity, with substantial differences in aggregate size, morphology, and structure. Among the different aggregation types, amyloids are particularly remarkable, because of their high degree of order and their ability to form self-perpetuating conformational states. Amyloids form the structural basis for a group of proteins called prions, which have the ability to generate new phenotypes by a simple switch in protein conformation that does not involve changes in the sequence of the DNA. Although protein aggregates are notoriously difficult to study, recent technological developments and, in particular, the use of yeast prions as model systems, have been very instrumental in understanding fundamental aspects of aggregation. Here, we provide a range of biochemical, cell biological and yeast genetic methods that are currently used in our laboratory to study protein aggregation and the formation of amyloids and prions. PDF

ArtavanisTsakonas, K., Kasperkovitz, P.V., Papa, E., Cardenas, M.L., Khan, N.S., Van der Veen, A.G., Ploegh, H.L., and Vyas, J.M. (2010). The Tetraspanin Cd82 Is Specifically Recruited to Fungal and Bacterial Phagosomes Prior to Acidification. Infection & Immunity .Dec 13. [Epub ahead of print] CD82 is a member of the tetraspanin superfamily whose physiological role is best described in the context of cancer metastasis. However, CD82 also associates with components of the Class II MHC antigen presentation pathway including Class II MHC molecules, the peptide loading machinery, as well as CD63, another tetraspanin, suggesting a role for CD82 in antigen presentation. Here we observe the dynamic rearrangement of CD82 after pathogen uptake by imaging CD82-mRFP1 expressed in primary living dendritic cells. CD82 showed rapid and specific recruitment to Cryptococcus neoformans-containing phagosomes when compared to polystyrene-containing phagosomes, similar to CD63. CD82 was also actively recruited to phagosomes containing other pathogenic fungi including Candida albicans and Aspergillus fumigatus. Recruitment of CD82 to fungal phagosomes occurred independently of TLR signaling. Recruitment was not limited to fungi as bacterial organisms including E. coli and S. aureus also induced CD82 recruitment to the phagosome. CD82 intersected the endocytic pathway used by LPS, implicating CD82 in trafficking of small, pathogen associated molecules. Despite its partial overlap with lysosomal compartments, CD82 recruitment to C. neoformans-containing phagosomes occurred independently of phagosome acidification. Kinetic analysis of fluorescence imaging reveals that CD82 and Class II MHC simultaneously appear in the phagosome, indicating that these two proteins may be associated. Together, these data indicate that the CD82 tetraspanin is specifically recruited to pathogen-containing phagosomes prior to fusion with lysosomes. PDF

ArtavanisTsakonas, K., Weihofen, W.A., Antos, J.M., Coleman, B.I., Comeaux, C.A., Duraisingh, M.T., Gaudet, R., and Ploegh, H.L. Characterization and Structural Studies of the Plasmodium Falciparum Ubiquitin and Nedd8 Hydrolase Uchl3. Journal of Biological Chemistry 285, 6857-6866. Like their human hosts, Plasmodium falciparum parasites rely on the ubiquitin-proteasome system for survival. We previously identified PfUCHL3, a deubiquitinating enzyme, and here we characterize its activity and changes in active site architecture upon binding to ubiquitin. We find strong evidence that PfUCHL3 is essential to parasite survival. The crystal structures of both PfUCHL3 alone and in complex with the ubiquitin-based suicide substrate UbVME suggest a rather rigid active site crossover loop that likely plays a role in restricting the size of ubiquitin adduct substrates. Molecular dynamics simulations of the structures and a model of the PfUCHL3-PfNedd8 complex allowed the identification of shared key interactions of ubiquitin and PfNedd8 with PfUCHL3, explaining the dual specificity of this enzyme. Distinct differences observed in ubiquitin binding between PfUCHL3 and its human counterpart make it likely that the parasitic DUB can be selectively targeted while leaving the human enzyme unaffected. Full Text.

Auluck, P.K., Caraveo, G., and Lindquist, S. Alpha-Synuclein: Membrane Interactions and Toxicity in Parkinson's Disease. Annu Rev Cell Dev Biol Vol. 26 2010. In the late 1990s, mutations in the synaptic protein alpha-synuclein (alpha-syn) were identified in families with hereditary Parkinson's disease (PD). Rapidly, alpha-syn became the target of numerous investigations that have transformed our understanding of the pathogenesis underlying this disorder. alpha-Syn is the major component of Lewy bodies (LBs), cytoplasmic protein aggregates that form in the neurons of PD patients. alpha-Syn interacts with lipid membranes and adopts amyloid conformations that deposit within LBs. Work in yeast and other model systems has revealed that alpha-syn-associated toxicity might be the consequence of abnormal membrane interactions and alterations in vesicle trafficking. Here we review evidence regarding alpha-syn's normal interactions with membranes and regulation of synaptic vesicles as well as how overexpression of alpha-syn yields global cellular dysfunction. Finally, we present a model linking vesicle dynamics to toxicity with the sincere hope that understanding these disease mechanisms will lead to the development of novel, potent therapeutics. Expected final online publication date for the Annual Review of Cell and Developmental Biology Volume 26 is October 06, 2010. Please see http://www.annualreviews.org/catalog/pubdates.aspx for revised estimates. Full Text.

Batchelder, E.M., and Yarar, D. Differential Requirements for Clathrin-Dependent Endocytosis at Sites of Cell-Substrate Adhesion. Molecular Biology of the Cell.Jul 14. [Epub ahead of print] Monitoring Editor: Sandra Lemmon Clathrin-dependent endocytosis is a major route for the cellular import of macromolecules and occurs at the interface between the cell and its surroundings. However, little is known about the influences of cell-substrate attachment in clathrin-coated vesicle formation. Using biochemical and imaging-based methods, we find that cell-substrate adhesion reduces the rate of endocytosis. Clathrin-coated pits (CCPs) in proximity to substrate contacts exhibit slower dynamics in comparison to CCPs found more distant from adhesions. Direct manipulation of the extra-cellular matrix (ECM) to modulate adhesion demonstrates that tight adhesion dramatically reduces clathrin-dependent endocytosis and extends the lifetimes of clathrin structures. This reduction is in part mediated by integrin-matrix engagement. In addition, we demonstrate that actin cytoskeletal dynamics are differentially required for efficient endocytosis, with a stronger requirement for actin polymerization in areas of adhesion. Together, these results reveal that cell-substrate adhesion regulates clathrin-dependent endocytosis, and suggests that actin assembly facilitates vesicle formation at sites of adhesion. PDF

Bellott, D.W., Skaletsky, H., Pyntikova, T., Mardis, E.R., Graves, T., Kremitzki, C., Brown, L.G., Rozen, S., Warren, W.C., Wilson, R.K., and David C Page. Convergent Evolution of Chicken Z and Human X Chromosomes by Expansion and Gene Acquisition. Nature. Jul 11. [Epub ahead of print] In birds, as in mammals, one pair of chromosomes differs between the sexes. In birds, males are ZZ and females ZW. In mammals, males are XY and females XX. Like the mammalian XY pair, the avian ZW pair is believed to have evolved from autosomes, with most change occurring in the chromosomes found in only one sex-the W and Y chromosomes. By contrast, the sex chromosomes found in both sexes-the Z and X chromosomes-are assumed to have diverged little from their autosomal progenitors. Here we report findings that challenge this assumption for both the chicken Z chromosome and the human X chromosome. The chicken Z chromosome, which we sequenced essentially to completion, is less gene-dense than chicken autosomes but contains a massive tandem array containing hundreds of duplicated genes expressed in testes. A comprehensive comparison of the chicken Z chromosome with the finished sequence of the human X chromosome demonstrates that each evolved independently from different portions of the ancestral genome. Despite this independence, the chicken Z and human X chromosomes share features that distinguish them from autosomes: the acquisition and amplification of testis-expressed genes, and a low gene density resulting from an expansion of intergenic regions. These features were not present on the autosomes from which the Z and X chromosomes originated but were instead acquired during the evolution of Z and X as sex chromosomes. We conclude that the avian Z and mammalian X chromosomes followed convergent evolutionary trajectories, despite their evolving with opposite (female versus male) systems of heterogamety. More broadly, in birds and mammals, sex chromosome evolution involved not only gene loss in sex-specific chromosomes, but also marked expansion and gene acquisition in sex chromosomes common to males and females. Full Text.

Bellott, D.W., and Page, D.C. Reconstructing the Evolution of Vertebrate Sex Chromosomes. Cold Spring Harbor Symposia on Quantitative Biology 2010 May 27. [Epub ahead of print]. Sex chromosomes and their evolution have captivated researchers since their discovery. For more than 100 years, the dominant model of sex chromosome evolution has held that differentiated sex chromosomes, such as the X and Y chromosomes of mammals or the Z and W chromosomes of birds, evolved from ordinary autosomes, primarily through the degeneration of the sex-specific Y or W chromosome. At the same time, the sex chromosomes shared between sexes, the X and Z chromosomes, are expected to remain essentially untouched. This model was based on limited cytogenetic and genetic data. Only in the last decade, with the advent of genomics, has the complete sequence of any sex chromosome pair become available. High-quality finished sequences of the human and chimpanzee Y chromosomes, as well as the human X chromosome, have revealed sequence features unanticipated by the traditional model of sex chromosome evolution. Large, highly identical, tandem and inverted arrays of testis-expressed genes are major sources of innovation in gene content on sex-specific chromosomes as well as sex-shared chromosomes. Accounting for the emergence of these ampliconic structures presents a challenge for future studies of sex chromosome evolution. Full Text.

Bergkvist, A., Rusnakova, V., Sindelka, R., Garda, J.M.A., Sjogreen, B., Lindh, D., Forootan, A., and Kubista, M. Gene Expression Profiling - Clusters of Possibilities. Methods 50, 323-335. Advances in qPCR technology allow studies of increasingly large systems comprising many genes and samples. The increasing data sizes allow expression profiling both in the gene and the samples dimension while also putting higher demands on sound statistical analysis and expertise to handle and interpret its results. We distinguish between exploratory and confirmatory statistical studies. In this paper we demonstrate several techniques available for exploratory studies on a system of Xenopus laevis development from egg to tadpole. Techniques include hierarchical clustering, heatmap, principal component analysis and self-organizing maps. We stress that even though exploratory studies are excellent for generating hypotheses, results have not been proven statistically significant until an independent confirmatory study has been performed. An exploratory study may certainly be valuable in its own right, and there are often not enough resources to report both an exploratory and a confirmatory study at the same time. However, exploratory and confirmatory studies are intimately connected and we would like to raise that awareness among qPCR practitioners. We suggest that scientific reports should always have a hypothesis focus. Reports are either hypothesis generating, from an exploratory study, or hypothesis validating, from a confirmatory study, or both. In either case, we suggest the generated or validated hypotheses be specifically stated. Full Text.

Bienvenu, F., Jirawatnotai, S., Elias, J.E., Meyer, C.A., Mizeracka, K., Marson, A., Frampton, G.M., Cole, M.F., Odom, D.T., Odajima, J., Yan Geng, Agnieszka Zagozdzon, Marie Jecrois, Richard A. Young, X. Shirley Liu, Constance L. Cepko Steven P. Gygi & Piotr Sicinski. Transcriptional Role of Cyclin D1 in Development Revealed by a Genetic-Proteomic Screen. Nature 463, 374-378. Cyclin D1 belongs to the core cell cycle machinery, and it is frequently overexpressed in human cancers(1,2). The full repertoire of cyclin D1 functions in normal development and oncogenesis is unclear at present. Here we developed Flag-and haemagglutinin-tagged cyclin D1 knock-in mouse strains that allowed a high-throughput mass spectrometry approach to search for cyclin D1-binding proteins in different mouse organs. In addition to cell cycle partners, we observed several proteins involved in transcription. Genome-wide location analyses (chromatin immunoprecipitation coupled to DNA microarray; ChIP-chip) showed that during mouse development cyclin D1 occupies promoters of abundantly expressed genes. In particular, we found that in developing mouse retinas-an organ that critically requires cyclin D1 function(3,4)-cyclin D1 binds the upstream regulatory region of the Notch1 gene, where it serves to recruit CREB binding protein (CBP) histone acetyltransferase. Genetic ablation of cyclin D1 resulted in decreased CBP recruitment, decreased histone acetylation of the Notch1 promoter region, and led to decreased levels of the Notch1 transcript and protein in cyclin D1-null (Ccnd1(-/-)) retinas. Transduction of an activated allele of Notch1 into Ccnd1(-/-) retinas increased proliferation of retinal progenitor cells, indicating that upregulation of Notch1 signalling alleviates the phenotype of cyclin D1-deficiency. These studies show that in addition to its well-established cell cycle roles, cyclin D1 has an in vivo transcriptional function in mouse development. Our approach, which we term 'genetic-proteomic', can be used to study the in vivo function of essentially any protein. Full Text

Blick, T., Hugo, H., Widodo, E., Waltham, M., Pinto, C., Mani, S.A., Weinberg, R.A., Neve, R.M., Lenburg, M.E., and Thompson, E.W. Epithelial Mesenchymal Transition Traits in Human Breast Cancer Cell Lines Parallel the Cd44(Hi/)Cd24(Lo/-) Stem Cell Phenotype in Human Breast Cancer. Journal of Mammary Gland Biology and Neoplasia 15, 235-252. We review here the recently emerging relationship between epithelial-mesenchymal transition (EMT) and breast cancer stem cells (BCSC), and provide analyses of published data on human breast cancer cell lines, supporting their utility as a model for the EMT/BCSC state. Genome-wide transcriptional profiling of these cell lines has confirmed the existence of a subgroup with mesenchymal tendencies and enhanced invasive properties ('Basal B'/Mesenchymal), distinct from subgroups with either predominantly luminal ('Luminal') or mixed basal/luminal ('Basal A') features (Neve et al. Cancer Cell, 2006). A literature-derived EMT gene signature has shown specific enrichment within the Basal B subgroup of cell lines, consistent with their over-expression of various EMT transcriptional drivers. Basal B cell lines are found to resemble BCSC, being CD44(high)CD24(low). Moreover, gene products that distinguish Basal B from Basal A and Luminal cell lines (Basal B Discriminators) showed close concordance with those that define BCSC isolated from clinical material, as reported by Shipitsin et al. (Cancer Cell, 2007). CD24 mRNA levels varied across Basal B cell lines, correlating with other Basal B Discriminators. Many gene products correlating with CD24 status in Basal B cell lines were also differentially expressed in isolated BCSC. These findings confirm and extend the importance of the cellular product of the EMT with Basal B cell lines, and illustrate the value of analysing these cell lines for new leads that may improve breast cancer outcomes. Gene products specific to Basal B cell lines may serve as tools for the detection, quantification, and analysis of BCSC/EMT attributes. Full Text.

Bousquet, M., Harris, M.H., Zhou, B., and Lodish, H.F. (2010). Microrna Mir-125b Causes Leukemia. Proc Natl Acad Sci U S A. Nov 30. [Epub ahead of print] MicroRNA miR-125b has been implicated in several kinds of leukemia. The chromosomal translocation t(2;11)(p21;q23) found in patients with myelodysplasia and acute myeloid leukemia leads to an overexpression of miR-125b of up to 90-fold normal. Moreover, miR-125b is also up-regulated in patients with B-cell acute lymphoblastic leukemia carrying the t(11;14)(q24;q32) translocation. To decipher the presumed oncogenic mechanism of miR-125b, we used transplantation experiments in mice. All mice transplanted with fetal liver cells ectopically expressing miR-125b showed an increase in white blood cell count, in particular in neutrophils and monocytes, associated with a macrocytic anemia. Among these mice, half died of B-cell acute lymphoblastic leukemia, T-cell acute lymphoblastic leukemia, or a myeloproliferative neoplasm, suggesting an important role for miR-125b in early hematopoiesis. Furthermore, coexpression of miR-125b and the BCR-ABL fusion gene in transplanted cells accelerated the development of leukemia in mice, compared with control mice expressing only BCR-ABL, suggesting that miR-125b confers a proliferative advantage to the leukemic cells. Thus, we show that overexpression of miR-125b is sufficient both to shorten the latency of BCR-ABL-induced leukemia and to independently induce leukemia in a mouse model.PDF.

Boulbes, D., Chen, C.H., Shaikenov, T., Agarwal, N.K., Peterson, T.R., Addona, T.A., Keshishian, H., Carr, S.A., Magnuson, M.A., Sabatini, D.M., et al. Rictor Phosphorylation on the Thr-1135 Site Does Not Require Mammalian Target of Rapamycin Complex 2. Molecular Cancer Research 8, 896-906. In animal cells, growth factors coordinate cell proliferation and survival by regulating the phosphoinositide 3-kinase/Akt signaling pathway. Deregulation of this signaling pathway is common in a variety of human cancers. The PI3K-dependent signaling kinase complex defined as mammalian target of rapamycin complex 2 (mTORC2) functions as a regulatory Ser-473 kinase of Akt. We find that activation of mTORC2 by growth factor signaling is linked to the specific phosphorylation of its component rictor on Thr-1135. The phosphorylation of this site is induced by the growth factor stimulation and expression of the oncogenic forms of ras or PI3K. Rictor phosphorylation is sensitive to the inhibition of PI3K, mTOR, or expression of integrin-linked kinase. The substitution of wild-type rictor with its specific phospho-mutants in rictor null mouse embryonic fibroblasts did not alter the growth factor-dependent phosphorylation of Akt, indicating that the rictor Thr-1135 phosphorylation is not critical in the regulation of the mTORC2 kinase activity. We found that this rictor phosphorylation takes place in the mTORC2-deficient cells, suggesting that this modification might play a role in the regulation of not only mTORC2 but also the mTORC2-independent function of rictor. Full Text.

Brady, B.L., Oropallo, M.A., Yang-Iott, K.S., Serwold, T., Hochedlinger, K., Jaenisch, R., Weissman, I.L., and Bassing, C.H. Position-Dependent Silencing of Germline V Beta Segments on Tcr Beta Alleles Containing Preassembled V Beta Dj Beta C Beta 1 Genes. Journal of Immunology 185, 3564-3573. The genomic organization of TCR beta loci enables V beta-to-DJ beta 2 rearrangements on alleles with assembled V beta DJ beta C beta 1 genes, which could have deleterious physiologic consequences. To determine whether such V beta rearrangements occur and, if so, how they might be regulated, we analyzed mice with TCR beta alleles containing preassembled functional V beta DJ beta C beta 1 genes. V beta 10 segments were transcribed, rearranged, and expressed in thymocytes when located immediately upstream of a V beta 1DJ beta C beta 1 gene, but not on alleles with a V beta 14DJ beta C beta 1 gene. Germline V beta 10 transcription was silenced in mature alpha beta T cells. This allele-dependent and developmental stage-specific silencing of V beta 10 correlated with increased CpG methylation and decreased histone acetylation over the V beta 10 promoter and coding region. Transcription, rearrangement, and expression of the V beta 4 and V beta 16 segments located upstream of V beta 10 were silenced on alleles containing either V beta DJ beta C beta 1 gene; sequences within V beta 4, V beta 16, and the V beta 4/V beta 16-V beta 10 intergenic region exhibited constitutive high CpG methylation and low histone acetylation. Collectively, our data indicate that the position of V beta segments relative to assembled V beta DJ beta C beta 1 genes influences their rearrangement and suggest that DNA sequences between V beta segments may form boundaries between active and inactive V beta chromatin domains upstream of V beta DJ beta C beta genes. The Journal of Immunology, 2010, 185: 3564-3573. Full Text.

Carette, J.E., Pruszak, J., Varadarajan, M., Blomen, V.A., Gokhale, S., Camargo, F.D., Wernig, M., Jaenisch, R., and Brummelkamp, T.R. Generation of Ipscs from Cultured Human Malignant Cells. Blood. Mar 16. [Epub ahead of print] Induced pluripotent stem cells (iPSCs) can be generated from various differentiated cell types by the expression of a set of defined transcription factors. So far, iPSCs have been generated from primary cells but it is unclear if human cancer cell lines can be reprogrammed. Here we describe the generation and characterization of iPS cells derived from human chronic myeloid leukemia (CML) cells. We show that despite the presence of oncogenic mutations these cells acquired pluripotency by the expression of four transcription factors and underwent differentiation into cell types derived of all three germ layers during teratoma formation. Interestingly, although the parental cell line was strictly dependent on continuous signaling of the BCR-ABL oncogene -also termed oncogene addiction- reprogrammed cells lost this dependency and became resistant to the BCR-ABL inhibitor imatinib. This finding indicates that the therapeutic agent imatinib targets cells in a specific epigenetic differentiated cell state and this may contribute to its inability to fully eradicate disease in CML patients. PDF

Chaffer, C.L., and Weinberg, R.A. Cancer Cell of Origin: Spotlight on Luminal Progenitors. Cell Stem Cell 7, 271-272. Does basal type breast cancer arise from oncogenic transformation of a basal cell type? In this issue of Cell Stem Cell, Molyneux et al. (2010) investigate the provenance of the basal-type BRCA1 breast carcinoma and come up with unanticipated results. Full Text.

Chiang, H.R., Schoenfeld, L.W., Ruby, J.G., Auyeung, V.C., Spies, N., Baek, D., Johnston, W.K., Russ, C., Luo, S., Babiarz, J.E., Blelloch R, Schroth GP, Nusbaum C, and Bartel DP. Mammalian Micrornas: Experimental Evaluation of Novel and Previously Annotated Genes. Genes and Development.Apr 22. [Epub ahead of print] MicroRNAs (miRNAs) are small regulatory RNAs that derive from distinctive hairpin transcripts. To learn more about the miRNAs of mammals, we sequenced 60 million small RNAs from mouse brain, ovary, testes, embryonic stem cells, three embryonic stages, and whole newborns. Analysis of these sequences confirmed 398 annotated miRNA genes and identified 108 novel miRNA genes. More than 150 previously annotated miRNAs and hundreds of candidates failed to yield sequenced RNAs with miRNA-like features. Ectopically expressing these previously proposed miRNA hairpins also did not yield small RNAs, whereas ectopically expressing the confirmed and newly identified hairpins usually did yield small RNAs with the classical miRNA features, including dependence on the Drosha endonuclease for processing. These experiments, which suggest that previous estimates of conserved mammalian miRNAs were inflated, provide a substantially revised list of confidently identified murine miRNAs from which to infer the general features of mammalian miRNAs. Our analyses also revealed new aspects of miRNA biogenesis and modification, including tissue-specific strand preferences, sequential Dicer cleavage of a metazoan precursor miRNA (pre-miRNA), consequential 5' heterogeneity, newly identified instances of miRNA editing, and evidence for widespread pre-miRNA uridylation reminiscent of miRNA regulation by Lin28. PDF

Chou, S., Chu, P., Hwang, W., and Lodish, H. Expansion of Human Cord Blood Hematopoietic Stem Cells for Transplantation. Cell Stem Cell 7 (4) : 427-428. A recent Science paper reported a purine derivative that expands human cord blood hematopoietic stem cells in culture (Boitano et al., 2010) by antagonizing the aryl hydrocarbon receptor. Major problems need to be overcome before ex vivo HSC expansion can be used clinically. Full Text.

Chou, S., and Lodish, H.F. Fetal Liver Hepatic Progenitors Are Supportive Stromal Cells for Hematopoietic Stem Cells. Proc Natl Acad Sci U S A.Apr 12. [Epub ahead of print] Previously we showed that the approximately 2% of fetal liver cells reactive with an anti-CD3epsilon monoclonal antibody support ex vivo expansion of both fetal liver and bone marrow hematopoietic stem cells (HSCs); these cells express two proteins important for HSC ex vivo expansion, IGF2, and angiopoietin-like 3. Here we show that these cells do not express any CD3 protein and are not T cells; rather, we purified these HSC-supportive stromal cells based on the surface phenotype of SCF(+)DLK(+). Competitive repopulating experiments show that SCF(+)DLK(+) cells support the maintenance of HSCs in ex vivo culture. These are the principal fetal liver cells that express not only angiopoietin-like 3 and IGF2, but also SCF and thrombopoietin, two other growth factors important for HSC expansion. They are also the principal fetal liver cells that express CXCL12, a factor required for HSC homing, and also alpha-fetoprotein (AFP), indicating that they are fetal hepatic stem or progenitor cells. Immunocytochemistry shows that >93% of the SCF(+) cells express DLK and Angptl3, and a portion of SCF(+) cells also expresses CXCL12. Thus SCF(+)DLK(+) cells are a highly homogenous population that express a complete set of factors for HSC expansion and are likely the primary stromal cells that support HSC expansion in the fetal liver. PDF

Claessen, J.H., Mueller, B., Spooner, E., Pivorunas, V.L., and Ploegh, H.L. The Transmembrane Segment of a Tail-Anchored Protein Determines Its Degradative Fate through Dislocation from the Endoplasmic Reticulum. J Biol Chem. Apr 30. [Epub ahead of print]Terminally misfolded proteins that accumulate in the endoplasmic reticulum (ER) are dislocated and targeted for ubiquitin-dependent destruction by the proteasome. UBC6e is a tail-anchored E2 ubiquitin-conjugating enzyme that is part of a dislocation complex nucleated by the ER-resident protein SEL1L. Little is known about the turnover of tail-anchored ER proteins. We constructed a set of UBC6e transmembrane domain replacement mutants and found that the tail-anchor of UBC6e is vital for its function, its stability and its mode of membrane integration, the latter step dependent on the ASNA1/TRC40 chaperone. We constructed a tail-anchored UBC6e variant that requires for its removal from the ER membrane not only YOD1 and p97, two cytosolic proteins involved in the extraction of ER transmembrane or luminal proteins, but also UBXD8, AUP1 and members of the Derlin family. Degradation of tail-anchored proteins thus relies on components that are also used in other aspects of protein quality control in the ER. PDF

Creyghton, M.P., Cheng, A.W., Welstead, G.G., Kooistra, T., Carey, B.W., Steine, E.J., Hanna, J., Lodato, M.A., Frampton, G.M., Sharp, P.A., Laurie A. Boyer, Richard A. Young, and Rudolf Jaenisch (2010). Histone H3k27ac Separates Active from Poised Enhancers and Predicts Developmental State. Proc Natl Acad Sci U S A. Nov 24. [Epub ahead of print] Developmental programs are controlled by transcription factors and chromatin regulators, which maintain specific gene expression programs through epigenetic modification of the genome. These regulatory events at enhancers contribute to the specific gene expression programs that determine cell state and the potential for differentiation into new cell types. Although enhancer elements are known to be associated with certain histone modifications and transcription factors, the relationship of these modifications to gene expression and developmental state has not been clearly defined. Here we interrogate the epigenetic landscape of enhancer elements in embryonic stem cells and several adult tissues in the mouse. We find that histone H3K27ac distinguishes active enhancers from inactive/poised enhancer elements containing H3K4me1 alone. This indicates that the amount of actively used enhancers is lower than previously anticipated. Furthermore, poised enhancer networks provide clues to unrealized developmental programs. Finally, we show that enhancers are reset during nuclear reprogramming. PDF

DeWulf, P., and Cheeseman, I.M. Tension at Embo's Aneuploidy Workshop. Embo Reports 11, 727-729.The EMBO 2010 Workshop on Chromosome Segregation and Aneuploidy, held between 19 and 23 June at Edinburgh University's Royal College of Surgeons, highlighted basic mitotic mechanisms, chromosome-based defects linked to human diseases and the processes that connect them. Full Text.

Dejosez, M., Levine, S.S., Frampton, G.M., Whyte, W.A., Stratton, S.A., Barton, M.C., Gunaratne, P.H., Young, R.A., and Zwaka, T.P. Ronin/Hcf-1 Binds to a Hyperconserved Enhancer Element and Regulates Genes Involved in the Growth of Embryonic Stem Cells. Genes & Development 24, 1479-1484. Self-renewing embryonic stem (ES) cells have an exceptional need for timely biomass production, yet the transcriptional control mechanisms responsible for meeting this requirement are largely unknown. We report here that Ronin (Thap11), which is essential for the self-renewal of ES cells, binds with its transcriptional coregulator, Hcf-1, to a highly conserved enhancer element that previously lacked a recognized binding factor. The subset of genes bound by Ronin/Hcf-1 function primarily in transcription initiation, mRNA splicing, and cell metabolism; genes involved in cell signaling and cell development are conspicuously underrepresented in this target gene repertoire. Although Ronin/Hcf-1 represses the expression of some target genes, its activity at promoter sites more often leads to the up-regulation of genes essential to protein biosynthesis and energy production. We propose that Ronin/Hcf-1 controls a genetic program that contributes to the unimpeded growth of ES cells. Full Text.

DeKelver, R.C., Choi, V.M., Moehle, E.A., Paschon, D.E., Hockemeyer, D., Meijsing, S.H., Sancak, Y., Cui, X., Steine, E.J., Miller, J.C., Tam P, Bartsevich VV, Meng X, Rupniewski I, Gopalan SM, Sun HC, Pitz KJ, Rock JM, Zhang L, Davis GD,Rebar EJ, Cheeseman IM, Yamamoto KR, Sabatini DM, Jaenisch R, Gregory PD, Urnov FD. Functional Genomics, Proteomics, and Regulatory DNA Analysis in Isogenic Settings Using Zinc Finger Nuclease-Driven Transgenesis into a Safe Harbor Locus in the Human Genome. Genome Research Aug;20(8):1133-42. Isogenic settings are routine in model organisms, yet remain elusive for genetic experiments on human cells. We describe the use of designed zinc finger nucleases (ZFNs) for efficient transgenesis without drug selection into the PPP1R12C gene, a "safe harbor" locus known as AAVS1. ZFNs enable targeted transgenesis at a frequency of up to 15% following transient transfection of both transformed and primary human cells, including fibroblasts and hES cells. When added to this locus, transgenes such as expression cassettes for shRNAs, small-molecule-responsive cDNA expression cassettes, and reporter constructs, exhibit consistent expression and sustained function over 50 cell generations. By avoiding random integration and drug selection, this method allows bona fide isogenic settings for high-throughput functional genomics, proteomics, and regulatory DNA analysis in essentially any transformed human cell type and in primary cells. Full Text.

Dong J,, Castro, C.E., Boyce, M.C., Lang, M.J., and Lindquist, S. (2010). Optical Trapping with High Forces Reveals Unexpected Behaviors of Prion Fibrils. Nature Struct Mol Biol. Published online28 November 2010 Amyloid fibrils are important in diverse cellular functions, feature in many human diseases and have potential applications in nanotechnology. Here we describe methods that combine optical trapping and fluorescent imaging to characterize the forces that govern the integrity of amyloid fibrils formed by a yeast prion protein. A crucial advance was to use the self-templating properties of amyloidogenic proteins to tether prion fibrils, enabling their manipulation in the optical trap. At normal pulling forces the fibrils were impervious to disruption. At much higher forces (up to 250 pN), discontinuities occurred in force-extension traces before fibril rupture. Experiments with selective amyloid-disrupting agents and mutations demonstrated that such discontinuities were caused by the unfolding of individual subdomains. Thus, our results reveal unusually strong noncovalent intermolecular contacts that maintain fibril integrity even when individual monomers partially unfold and extend fibril length. Full Text.

Dowell, R.D., Ryan, O., Jansen, A., Cheung, D., Agarwala, S., Danford, T., Bernstein, D.A., Rolfe, P.A., Heisler, L.E., Chin, B., Nislow C, Giaever G, Phillips PC, Fink GR, Gifford DK, and Boone C. Genotype to Phenotype: A Complex Problem. Science 328, 469-469.We generated a high-resolution whole-genome sequence and individually deleted 5100 genes in Sigma1278b, a Saccharomyces cerevisiae strain closely related to reference strain S288c. Similar to the variation between human individuals, Sigma1278b and S288c average 3.2 single-nucleotide polymorphisms per kilobase. A genome-wide comparison of deletion mutant phenotypes identified a subset of genes that were conditionally essential by strain, including 44 essential genes unique to Sigma1278b and 13 unique to S288c. Genetic analysis indicates the conditional phenotype was most often governed by complex genetic interactions, depending on multiple background-specific modifiers. Our comprehensive analysis suggests that the presence of a complex set of modifiers will often underlie the phenotypic differences between individuals Full Text.

Dressel, R., Nolte, J., Elsner, L., Novota, P., Guan, K., Streckfuss-Bomeke, K., Hasenfuss, G., Jaenisch, R., and Engel, W. Pluripotent Stem Cells Are Highly Susceptible Targets for Syngeneic, Allogeneic, and Xenogeneic Natural Killer Cells. FASEB J.Feb 9. [Epub ahead of print] Multipotent adult germ-line stem cells (maGSCs) and induced pluripotent stem cells (iPSCs) could be used to generate autologous cells for therapeutic purposes, which are expected to be tolerated by the recipient. However, effects of the immune system on these cells have not been investigated. We have compared the susceptibility of maGSC lines to IL-2-activated natural killer (NK) cells with embryonic stem cell (ESC) lines, iPSCs, and F9 teratocarcinoma cells. The killing of pluripotent cell lines by syngeneic, allogeneic, and xenogeneic killer cells ranged between 48 and 265% in chromium release assays when compared to YAC-1 cells, which served as highly susceptible reference cells. With the exception of 2 maGSC lines, they expressed ligands for the activating NK receptor NKG2D that belong to the RAE-1 family, and killing could be inhibited by soluble NKG2D, demonstrating a functional role of these molecules. Furthermore, ligands of the activating receptor DNAM-1 were frequently expressed. The susceptibility to NK cells might constitute a common feature of pluripotent cells. It could result in rejection after transplantation, as suggested by a reduced teratoma growth after NK cell activation in vivo, but it might also offer a strategy to deplete contaminating pluripotent cells before grafting of differentiated cells.-Dressel, R., Nolte, J., Elsner, L., Novota, P., Guan, K., Streckfuss-Bomeke, K., Hasenfuss, G., Jaenisch, R., Engel, W. Pluripotent stem cells are highly susceptible targets for syngeneic, allogeneic, and xenogeneic natural killer cells. PDF.

Ernst, J., Plasterer, H.L., Simon, I., and Bar-Joseph, Z. Integrating Multiple Evidence Sources to Predict Transcription Factor Binding in the Human Genome. Genome Research 20, 526-536. Information about the binding preferences of many transcription factors is known and characterized by a sequence binding motif. However, determining regions of the genome in which a transcription factor binds based on its motif is a challenging problem, particularly in species with large genomes, since there are often many sequences containing matches to the motif but are not bound. Several rules based on sequence conservation or location, relative to a transcription start site, have been proposed to help differentiate true binding sites from random ones. Other evidence sources may also be informative for this task. We developed a method for integrating multiple evidence sources using logistic regression classifiers. Our method works in two steps. First, we infer a score quantifying the general binding preferences of transcription factor binding at all locations based on a large set of evidence features, without using any motif specific information. Then, we combined this general binding preference score with motif information for specific transcription factors to improve prediction of regions bound by the factor. Using cross-validation and new experimental data we show that, surprisingly, the general binding preference can be highly predictive of true locations of transcription factor binding even when no binding motif is used. When combined with motif information our method outperforms previous methods for predicting locations of true binding. Full Text.

Falcon, A., Doege, H., Fluitt, A., Tsang, B., Watson, N., Kay, M.A., and Stahl, A. Fatp2 Is a Hepatic Fatty Acid Transporter and Peroxisomal Very Long-Chain Acyl-Coa Synthetase. American Journal of Physiology-Endocrinology and Metabolism 299, E384-E393. Falcon A, Doege H, Fluitt A, Tsang B, Watson N, Kay MA, Stahl A. FATP2 is a hepatic fatty acid transporter and peroxisomal very long-chain acyl-CoA synthetase. Am J Physiol Endocrinol Metab 299: E384-E393, 2010. First published June 8, 2010; doi:10.1152/ajpendo.00226.2010.-Fatty acid transport protein (FATP) 2, a member of the FATP family of fatty acid uptake mediators, has independently been identified as a hepatic peroxisomal very long-chain acyl-CoA synthetase (VLACS). Here we address whether FATP2 is 1) a peroxisomal enzyme, 2) a plasma membrane-associated long-chain fatty acid (LCFA) transporter, or 3) a multifunctional protein. We found that, in mouse livers, only a minor fraction of FATP2 localizes to peroxisomes, where it contributes to approximately half of the peroxisomal VLACS activity. However, total hepatic (V)LACS activity was not significantly affected by loss of FATP2, while LCFA uptake was reduced by 40%, indicating a more prominent role in hepatic LCFA uptake. This suggests FATP2 as a potential target for a therapeutic intervention of hepatosteatosis. Adeno-associated virus 8-based short hairpin RNA expression vectors were used to achieve liver-specific FATP2 knockdown, which significantly reduced hepatosteatosis in the face of continued high-fat feeding, concomitant with improvements in liver physiology, fasting glucose, and insulin levels. Based on our findings, we propose a model in which FATP2 is a multifunctional protein that shows subcellular localization-dependent activity and is a major contributor to peroxisomal (V) LACS activity and hepatic fatty acid uptake, suggesting FATP2 as a potential novel target for the treatment of nonalcoholic fatty liver disease. Full Text.

Falk, J.E., Chan, A.C., Hoffmann, E., and Hochwagen, A. A Mec1- and Pp4-Dependent Checkpoint Couples Centromere Pairing to Meiotic Recombination. Dev Cell 19, 599-611. The faithful alignment of homologous chromosomes during meiotic prophase requires the coordination of DNA double-strand break (DSB) repair with large-scale chromosome reorganization. Here we identify the phosphatase PP4 (Pph3/Psy2) as a mediator of this process in Saccharomyces cerevisiae. In pp4 mutants, early stages of crossover repair and homology-independent pairing of centromeres are coordinately blocked. We traced the loss of centromere pairing to the persistent phosphorylation of the chromosomal protein Zip1 on serine 75. Zip1-S75 is a consensus site for the ATR-like checkpoint kinase Mec1, and centromere pairing is restored in mec1 mutants. Importantly, Zip1-S75 phosphorylation does not alter chromosome synapsis or DSB repair, indicating that Mec1 separates centromere pairing from the other functions of Zip1. The centromeric localization and persistent activity of PP4 during meiotic prophase suggest a model whereby Zip1-S75 phosphorylation dynamically destabilizes homology-independent centromere pairing in response to recombination initiation, thereby coupling meiotic chromosome dynamics to DSB repair. PDF.

Flygare, J., Rayon Estrada, V., Shin, C., Gupta, S., and Lodish, H.F. (2010). Hif-1 {Alpha} Synergizes with Glucocorticoids to Promote Bfu-E Progenitor Self-Renewal. Blood.Dec 21. [Epub ahead of print] With the aim of finding small molecules that stimulate erythropoiesis earlier than erythropoietin and that enhance CFU-E production, we studied the mechanism by which glucocorticoids increase CFU-E formation. Using BFU-E and CFU-E progenitors purified by a new technique, we demonstrate that glucocorticoids stimulate the earliest (BFU-E) progenitors to undergo limited self-renewal, which increases formation of CFU-E cells > 20-fold. Interestingly, glucocorticoids induce expression of genes in BFU-E cells that contain promoter regions highly enriched for hypoxia-induced factor 1 alpha (HIF1alpha) binding sites. This suggests activation of HIF1alpha may enhance or replace the effect of glucocorticoids on BFU-E self-renewal. Indeed, HIF1alpha activation by a prolyl hydroxylase inhibitor (PHI) synergizes with glucocorticoids and enhances production of CFU-Es 170-fold. Since PHIs are able to increase erythroblast production at very low concentrations of glucocorticoids, PHI-induced stimulation of BFU-E progenitors thus represents a conceptually new therapeutic window for treating Epo-resistant anemia. PDF

Fonslow, B.R., Kang, S.A., Gestaut, D.R., Graczyk, B., Davis, T.N., Sabatini, D.M., and Yates Iii, J.R. Native Capillary Isoelectric Focusing for the Separation of Protein Complex Isoforms and Subcomplexes. Analytical Chemistry Jul 8. [Epub ahead of print]. Here we report the use of capillary isoelectric focusing under native conditions for the separation of protein complex isoforms and subcomplexes. Using biologically relevant HIS-tag and FLAG-tag purified protein complexes, we demonstrate the separations of protein complex isoforms of the mammalian target of rapamycin complex (mTORC1 and 2) and the subcomplexes and different phosphorylation states of the Dam1 complex. The high efficiency capillary isoelectric focusing separation allowed for resolution of protein complexes and subcomplexes similar in size and biochemical composition. By performing separations with native buffers and reduced temperature (15 degrees C) we were able to maintain the complex integrity of the more thermolabile mTORC2 during isoelectric focusing and detection (<45 min). Increasing the separation temperature allowed us to monitor dissociation of the Dam1 complex into its subcomplexes (25 degrees C) and eventually its individual protein components (30 degrees C). The separation of two different phosphorylation states of the Dam1 complex, generated from an in vitro kinase assay with Mps1 kinase, was straightforward due to the large pI shift upon multiple phosphorylation events. The separation of the protein complex isoforms of mTORC, on the other hand, required the addition of a small pI range (4-6.5) of ampholytes to improve resolution and stability of the complexes. We show that native capillary isoelectric focusing is a powerful method for the difficult separations of large, similar, unstable protein complexes. This method shows potential for differentiation of protein complex isoform and subcomplex compositions, post-translational modifications, architectures, stabilities, equilibria, and relative abundances under biologically relevant conditions. Full Text.

Gascoigne, K.E., and Cheeseman, I.M. Kinetochore Assembly: If You Build It, They Will Come. Curr Opin Cell Biol.Aug 9. [Epub ahead of print] Accurate chromosome segregation requires the interaction of chromosomes with the microtubules from the mitotic spindle. This interaction is mediated by the macro-molecular kinetochore complex, which assembles only at the centromeric region of each chromosome. However, how this site is specified and how assembly of the kinetochore structure is regulated in coordination with cell cycle progression remains unclear. Recent studies have begun to shed light on the mechanisms underlying assembly of this complex structure. PDF

Goldstein, R.H., Weinberg, R.A., and Rosenblatt, M. Of Mice and (Wo)Men: Mouse Models of Breast Cancer Metastasis to Bone. Journal of Bone and Mineral Research 25, 431-436.While no animal model can fully recapitulate the progression of breast cancer tumorigenesis and metastasis from initial epithelial cell transformation to extravasation and final arrest in the skeleton, advances in genetic and surgical techniques have combined to move the field progressively closer to using models of human osteotropic metastasis that more precisely mirror disease pathogenesis in humans. The importance of these advanced models is clear, namely, identification of new molecular targets to block metastasis, testing of metastasis-specific therapies in a preclinical setting, and a greater understanding of the interactions of carcinoma cells with the stromal microenvironments that they encounter in the primary tumor and in sites of metastatic dissemination, including the bone. It is our hope that these discoveries and the greater understanding of cancer metastasis that will emerge from these models will begin to address the urgent medical need of the thousands of women who are diagnosed with metastatic breast cancer each year. Full Text.

Greer, P.L., Hanayama, R., Bloodgood, B.L., Mardinly, A.R., Lipton, D.M., Flavell, S.W., Kim, T.K., Griffith, E.C., Waldon, Z., Maehr, R, Ploegh HL, Chowdhury S, Worley PF, Steen J, Greenberg ME. The Angelman Syndrome Protein Ube3a Regulates Synapse Development by Ubiquitinating Arc. Cell 140, 704-716. Angelman Syndrome is a debilitating neurological disorder caused by mutation of the E3 ubiquitin ligase Ube3A, a gene whose mutation has also recently been associated with autism spectrum disorders (ASDs). The function of Ube3A during nervous system development and how Ube3A mutations give rise to cognitive impairment in individuals with Angleman Syndrome and ASDs are not clear. We report here that experience-driven neuronal activity induces Ube3A transcription and that Ube3A then regulates excitatory synapse development by controlling the degradation of Arc, a synaptic protein that promotes the internalization of the AMPA subtype of glutamate receptors. We find that disruption of Ube3A function in neurons leads to an increase in Arc expression and a concomitant decrease in the number of AMPA receptors at excitatory synapses. We propose that this deregulation of AMPA receptor expression at synapses may contribute to the cognitive dysfunction that occurs in Angelman Syndrome and possibly other ASDs. Full Text.

Guenther, M.G., Frampton, G.M., Soldner, F., Hockemeyer, D., Mitalipova, M., Jaenisch, R., and Young, R.A. Chromatin Structure and Gene Expression Programs of Human Embryonic and Induced Pluripotent Stem Cells. Cell Stem Cell 7, 249-257. Knowledge of both the global chromatin structure and the gene expression programs of human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) should provide a robust means to assess whether the genomes of these cells have similar pluripotent states. Recent studies have suggested that ESCs and iPSCs represent different pluripotent states with substantially different gene expression profiles. We describe here a comparison of global chromatin structure and gene expression data for a panel of human ESCs and iPSCs. Genome-wide maps of nucleosomes with histone H3K4me3 and H3K27me3 modifications indicate that there is little difference between ESCs and iPSCs with respect to these marks. Gene expression profiles confirm that the transcriptional programs of ESCs and iPSCs show very few consistent differences. Although some variation in chromatin structure and gene expression was observed in these cell lines, these variations did not serve to distinguish ESCs from iPSCs. PDF

Guenther, M.G., and Young, R.A. Transcription. Repressive Transcription. Science 329, 150-151. How are active and repressed portions of the genome established and maintained during development? In vertebrates, about 2 m of DNA is packaged into chromatin in a manner that allows for active transcription of some loci and repression of others. Most chromatin regulators do not recognize specific DNA sequences, so how are they recruited to specific sites throughout the genome? For actively transcribed genes, transcription factors or the transcription initiation apparatus recruit regulators associated with active chromatin (1). For genes that are repressed, recent studies suggest a counterintuitive model: Transcription initiates the formation of repressive chromatin Full Text.

Guo, Y., Papachristoudis, G., Altshuler, R.C., Gerber, G.K., Jaakkola, T.S., Gifford, D.K., and Mahony, S. Discovering Homotypic Binding Events at High Spatial Resolution. Bioinformatics.Oct 21. [Epub ahead of print] MOTIVATION: Clusters of protein-DNA interaction events involving the same transcription factor are known to act as key components of invertebrate and mammalian promoters and enhancers. However, detecting closely spaced homotypic events from ChIP-Seq data is challenging because random variation in the ChIP fragmentation process obscures event locations. RESULTS: The Genome Positioning System (GPS) can predict protein-DNA interaction events at high spatial resolution from ChIP-Seq data while retaining the ability to resolve closely spaced events that appear as a single cluster of reads. GPS models observed reads using a complexity penalized mixture model and efficiently predicts event locations with a segmented EM algorithm. An optional mode permits GPS to align common events across distinct experiments. GPS detects more joint events in synthetic and actual ChIP-Seq data and has superior spatial resolution when compared with other methods. In addition, the specificity and sensitivity of GPS are superior to or comparable with other methods. AVAILABILITY: http://cgs.csail.mit.edu/gps CONTACT: gifford@mit.edu, mahony@MIT.EDU SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online. PDF

Guo, H., Ingolia, N.T., Weissman, J.S., and Bartel, D.P. Mammalian Micrornas Predominantly Act to Decrease Target Mrna Levels. Nature 466, 835-840. MicroRNAs (miRNAs) are endogenous approximately 22-nucleotide RNAs that mediate important gene-regulatory events by pairing to the mRNAs of protein-coding genes to direct their repression. Repression of these regulatory targets leads to decreased translational efficiency and/or decreased mRNA levels, but the relative contributions of these two outcomes have been largely unknown, particularly for endogenous targets expressed at low-to-moderate levels. Here, we use ribosome profiling to measure the overall effects on protein production and compare these to simultaneously measured effects on mRNA levels. For both ectopic and endogenous miRNA regulatory interactions, lowered mRNA levels account for most (>/=84%) of the decreased protein production. These results show that changes in mRNA levels closely reflect the impact of miRNAs on gene expression and indicate that destabilization of target mRNAs is the predominant reason for reduced protein output. Full Text.

Gutzman, J.H., and Sive, H. Epithelial Relaxation Mediated by the Myosin Phosphatase Regulator Mypt1 Is Required for Brain Ventricle Lumen Expansion and Hindbrain Morphogenesis. Development 137, 795-804. We demonstrate that in the zebrafish hindbrain, cell shape, rhombomere morphogenesis and, unexpectedly, brain ventricle lumen expansion depend on the contractile state of the neuroepithelium. The hindbrain neural tube opens in a specific sequence, with initial separation along the midline at rhombomere boundaries, subsequent openings within rhombomeres and eventual coalescence of openings into the hindbrain ventricle lumen. A mutation in the myosin phosphatase regulator mypt1 results in a small ventricle due to impaired stretching of the surrounding neuroepithelium. Although initial hindbrain opening remains normal, mypt1 mutant rhombomeres do not undergo normal morphological progression. Three-dimensional reconstruction demonstrates cell shapes within rhombomeres and at rhombomere boundaries are abnormal in mypt1 mutants. Wild-type cell shape requires that surrounding cells are also wild type, whereas mutant cell shape is autonomously regulated. Supporting the requirement for regulation of myosin function during hindbrain morphogenesis, wild-type embryos show dynamic levels of phosphorylated myosin regulatory light chain (pMRLC). By contrast, mutants show continuously high pMRLC levels, with concentration of pMRLC and myosin II at the apical side of the epithelium, and myosin II and actin concentration at rhombomere boundaries. Brain ventricle lumen expansion, rhombomere morphology and cell shape are rescued by inhibition of myosin II function, indicating that each defect is a consequence of overactive myosin. We suggest that the epithelium must ;relax', via activity of myosin phosphatase, to allow for normal hindbrain morphogenesis and expansion of the brain ventricular lumen. Epithelial relaxation might be a widespread strategy to facilitate tube inflation in many organs. Full Text.

Halfmann, R., and Lindquist, S. Epigenetics in the Extreme: Prions and the Inheritance of Environmentally Acquired Traits. Science 330, 629-632. Prions are an unusual form of epigenetics: Their stable inheritance and complex phenotypes come about through protein folding rather than nucleic acid-associated changes. With intimate ties to protein homeostasis and a remarkable sensitivity to stress, prions are a robust mechanism that links environmental extremes with the acquisition and inheritance of new traits. Full Text.

Halfmann, R., Alberti, S., and Lindquist, S. Prions, Protein Homeostasis, and Phenotypic Diversity. Trends in Cell Biology.Jan 11. [Epub ahead of print] Prions are fascinating but often misunderstood protein aggregation phenomena. The traditional association of the mammalian prion protein with disease has overshadowed a potentially more interesting attribute of prions: their ability to create protein-based molecular memories. In fungi, prions alter the relationship between genotype and phenotype in a heritable way that diversifies clonal populations. Recent findings in yeast indicate that prions might be much more common than previously realized. Moreover, prion-driven phenotypic diversity increases under stress, and can be amplified by the dynamic maturation of prion-initiating states. In this report, we suggest that these qualities allow prions to act as 'bet-hedging' devices that facilitate the adaptation of yeasts to stressful environments, and might speed the evolution of new traits. Full Text

Hanna, J.H., Saha, K., and Jaenisch, R. (2010). Pluripotency and Cellular Reprogramming: Facts, Hypotheses, Unresolved Issues. Cell 143, 508-525. Direct reprogramming of somatic cells to induced pluripotent stem cells by ectopic expression of defined transcription factors has raised fundamental questions regarding the epigenetic stability of the differentiated cell state. In addition, evidence has accumulated that distinct states of pluripotency can interconvert through the modulation of both cell-intrinsic and exogenous factors. To fully realize the potential of in vitro reprogrammed cells, we need to understand the molecular and epigenetic determinants that convert one cell type into another. Here we review recent advances in this rapidly moving field and emphasize unresolved and controversial questions. Full Text.

Hanna J.H. The Stats on Naive Ipsc Reprogramming. Cell Stem Cell 7, 274-276. Pluripotency can be induced in somatic cells via ectopic expression of defined transcription factors. In this issue of Cell Stem Cell, Yang et al. (2010) demonstrate that Lif/Stat3 signaling directly contributes to the in vitro induction of murine naive pluripotency. Full Text.

Hanna, J., Cheng, A.W., Saha, K., Kim, J., Lengner, C.J., Soldner, F., Cassady, J.P., Muffat, J., Carey, B.W., and Jaenisch, R. Human Embryonic Stem Cells with Biological and Epigenetic Characteristics Similar to Those of Mouse Escs. Proc Natl Acad Sci U S A.May 4. [Epub ahead of print] Human and mouse embryonic stem cells (ESCs) are derived from blastocyst-stage embryos but have very different biological properties, and molecular analyses suggest that the pluripotent state of human ESCs isolated so far corresponds to that of mouse-derived epiblast stem cells (EpiSCs). Here we rewire the identity of conventional human ESCs into a more immature state that extensively shares defining features with pluripotent mouse ESCs. This was achieved by ectopic induction of Oct4, Klf4, and Klf2 factors combined with LIF and inhibitors of glycogen synthase kinase 3beta (GSK3beta) and mitogen-activated protein kinase (ERK1/2) pathway. Forskolin, a protein kinase A pathway agonist which can induce Klf4 and Klf2 expression, transiently substitutes for the requirement for ectopic transgene expression. In contrast to conventional human ESCs, these epigenetically converted cells have growth properties, an X-chromosome activation state (XaXa), a gene expression profile, and a signaling pathway dependence that are highly similar to those of mouse ESCs. Finally, the same growth conditions allow the derivation of human induced pluripotent stem (iPS) cells with similar properties as mouse iPS cells. The generation of validated "naive" human ESCs will allow the molecular dissection of a previously undefined pluripotent state in humans and may open up new opportunities for patient-specific, disease-relevant research. PDF.

Hargus, G., Cooper, O., Deleidi, M., Levy, A., Lee, K., Marlow, E., Yow, A., Soldner, F., Hockemeyer, D., Hallett, P.J.,Osborn T, Jaenisch R, Isacson O.. Differentiated Parkinson Patient-Derived Induced Pluripotent Stem Cells Grow in the Adult Rodent Brain and Reduce Motor Asymmetry in Parkinsonian Rats. Proceedings of the National Academy of Sciences of the United States of America 107, 15921-15926. Recent advances in deriving induced pluripotent stem(iPS) cells from patients offer new possibilities for biomedical research and clinical applications, as these cells could be used for autologous transplantation. We differentiated iPS cells from patients with Parkinson's disease (PD) into dopaminergic (DA) neurons and show that these DA neurons can be transplanted without signs of neurodegeneration into the adult rodent striatum. The PD patient iPS (PDiPS) cell-derived DA neurons survived at high numbers, showed arborization, and mediated functional effects in an animal model of PD as determined by reduction of amphetamine-and apomorphine-induced rotational asymmetry, but only a few DA neurons projected into the host striatum at 16 wk after transplantation. We next applied FACS for the neural cell adhesion molecule NCAM on differentiated PDiPS cells before transplantation, which resulted in surviving DA neurons with functional effects on amphetamine-induced rotational asymmetry in a 6-OHDA animal model of PD. Morphologically, we found that PDiPS cell-derived non-DA neurons send axons along white matter tracts into specific close and remote gray matter target areas in the adult brain. Such findings establish the transplantation of human PDiPS cell-derived neurons as a long-term in vivo method to analyze potential disease-related changes in a physiological context. Our data also demonstrate proof of principle of survival and functional effects of PDiPS cell-derived DA neurons in an animal model of PD and encourage further development of differentiation protocols to enhance growth and function of implanted PDiPS cell-derived DA neurons in regard to potential therapeutic applications. Full Text.

Harnprasopwat, R., Ha, D., Toyoshima, T., Lodish, H., Tojo, A., and Kotani, A. Alteration of Processing Induced by a Single Nucleotide Polymorphism in Pri-Mir-126. Biochem Biophys Res Commun.Jul 16. [Epub ahead of print] MicroRNAs (miRNAs) are small non-coding RNAs that inhibit expression of specific target genes at the post-transcriptional level. Sequence variations in miRNA genes, including pri-miRNAs, pre-miRNAs and mature miRNAs, could potentially influence the processing and/or target selection of miRNAs. In this study, we have found the single nucleotide polymorphism (SNP) at the twenty-fourth nucleotide (+24) of the mature miR-126 in the genome of RS4;11 cells, derived from a MLL-AF4 ALL patient. Through a series of in vivo analyzes, we found that this miR-126 SNP significantly blocks the processing of pri-miRNA to mature miRNA, as well as reduces miRNA-mediated translational suppression. Moreover, its frequency is different among races. Thus, our study emphasizes the importance of identifying new miRNA SNP and its contribution to miRNA biogenesis which is possible link to human genetic disease. Full Text.

Hattangadi, S.M., Burke, K.A., and Lodish, H.F. Homeodomain-Interacting Protein Kinase 2 Plays an Important Role in Normal Terminal Erythroid Differentiation. Blood.Mar 15. [Epub ahead of print] Gene-targeting experiments report that the homeodomain-interacting protein kinases 1 and 2, Hipk1 and Hipk2, are essential but redundant in hematopoietic development-because Hipk1/Hipk2 double-deficient animals exhibit severe defects in hematopoiesis and vasculogenesis while the single knockouts do not. These serine-threonine kinases phosphorylate, and consequently modify the functions of, several important hematopoietic transcription factors and cofactors. Here we show that Hipk2 knockdown alone plays a significant role in terminal fetal liver erythroid differentiation. Hipk1 and Hipk2 are highly induced during primary mouse fetal liver erythropoiesis. Specific knockdown of Hipk2 inhibits terminal erythroid cell proliferation-explained in part by impaired cell cycle progression as well as increased apoptosis-and terminal enucleation as well as the accumulation of hemoglobin. Hipk2 knockdown also reduces the transcription of many genes involved in proliferation and apoptosis as well as important, erythroid-specific genes involved in hemoglobin biosynthesis-such as alpha-globin and mitoferrin 1-demonstrating that Hipk2 plays an important role in some but not all aspects of normal terminal erythroid differentiation. PDF.

Heard, E., Tishkoff, S., Todd, J.A., Vidal, M., Wagner, G.P., Wang, J., Weigel, D., and Young, R. Ten Years of Genetics and Genomics: What Have We Achieved and Where Are We Heading? Nature Reviews Genetics 11, 723-733. To celebrate the first 10 years of Nature Reviews Genetics, we asked eight leading researchers for their views on the key developments in genetics and genomics in the past decade and the prospects for the future. Their responses highlight the incredible changes that the field has seen, from the explosion of genomic data and the many possibilities it has opened up to the ability to reprogramme adult cells to pluripotency. The way ahead looks similarly exciting as we address questions such as how cells function as systems and how complex interactions among genetics, epigenetics and the environment combine to shape phenotypes. Full Text

Hegde, R.S., and Ploegh, H.L. Quality and Quantity Control at the Endoplasmic Reticulum. Current Opinion in Cell Biology 22, 437-446. The endoplasmic reticulum (ER) is the site of maturation for secretory and membrane proteins that together make up about one third of the cellular proteome. Cells carefully control the synthetic output of this organelle to regulate both quality and quantity of proteins that emerge. Here, we synthesize current concepts underlying the pathways that mediate protein degradation from the ER and their deployment under physiologic and pathologic conditions. PDF.

Hochwagen, A., and Marais, G.A. Meiosis: A Prdm9 Guide to the Hotspots of Recombination. Curr Biol 20, R271-R274. During meiosis, homologous recombination occurs preferentially at defined hotspots. In mammals, the fast-evolving DNA-binding domain of PRDM9 has now been identified as a major hotspot determinant that may explain the rapid rates of hotspot redistribution during evolution. Full Text.

Hudson, S.P., Langer, R., Fink, G.R., and Kohane, D.S. Injectable in Situ Cross-Linking Hydrogels for Local Antifungal Therapy. Biomaterials 31, 1444-1452. Invasive fungal infections can be devastating, particularly in immunocompromised patients, and difficult to treat with systemic drugs. Furthermore, systemic administration of those medications can have severe side effects. We have developed an injectable local antifungal treatment for direct administration into existing or potential sites of fungal infection. Amphotericin B (AmB), a hydrophobic, potent, and broad-spectrum antifungal agent, was rendered water-soluble by conjugation to a dextran-aldehyde polymer. The dextranaldehyde-AmB conjugate retained antifungal efficacy against Candida albicans. Mixing carboxymethylcellulose-hydrazide with dextran-aldehyde formed a gel that cross-linked in situ by formation of hydrazone bonds. The gel provided in vitro release of antifungal activity for 11 days. and contact with the gel killed Candida for three weeks. There was no apparent tissue toxicity in the murine peritoneum and the gel caused no adhesions. Gels produced by entrapment of a suspension of AmB in CMC-dextran without conjugation of drug to polymers did not release fungicidal activity, but did kill on contact Injectable systems of these types, containing soluble or insoluble drug formulations, could be useful for treatment of local antifungal infections, with or without concurrent systemic therapy. . Full Text

Hughes, J.F., Skaletsky, H., Pyntikova, T., Graves, T.A., van Daalen, S.K., Minx, P.J., Fulton, R.S., McGrath, S.D., Locke, D.P., Friedman, C.,Trask BJ, Mardis ER, Warren WC, Repping S, Rozen S, Wilson RK, and Page DC Chimpanzee and Human Y Chromosomes Are Remarkably Divergent in Structure and Gene Content. Nature. Jan 13. [Epub ahead of print] The human Y chromosome began to evolve from an autosome hundreds of millions of years ago, acquiring a sex-determining function and undergoing a series of inversions that suppressed crossing over with the X chromosome. Little is known about the recent evolution of the Y chromosome because only the human Y chromosome has been fully sequenced. Prevailing theories hold that Y chromosomes evolve by gene loss, the pace of which slows over time, eventually leading to a paucity of genes, and stasis. These theories have been buttressed by partial sequence data from newly emergent plant and animal Y chromosomes, but they have not been tested in older, highly evolved Y chromosomes such as that of humans. Here we finished sequencing of the male-specific region of the Y chromosome (MSY) in our closest living relative, the chimpanzee, achieving levels of accuracy and completion previously reached for the human MSY. By comparing the MSYs of the two species we show that they differ radically in sequence structure and gene content, indicating rapid evolution during the past 6 million years. The chimpanzee MSY contains twice as many massive palindromes as the human MSY, yet it has lost large fractions of the MSY protein-coding genes and gene families present in the last common ancestor. We suggest that the extraordinary divergence of the chimpanzee and human MSYs was driven by four synergistic factors: the prominent role of the MSY in sperm production, 'genetic hitchhiking' effects in the absence of meiotic crossing over, frequent ectopic recombination within the MSY, and species differences in mating behaviour. Although genetic decay may be the principal dynamic in the evolution of newly emergent Y chromosomes, wholesale renovation is the paramount theme in the continuing evolution of chimpanzee, human and perhaps other older MSYs. Full Text.

Jan, C.H., Friedman, R.C., Ruby, J.G., and Bartel, D.P. (2010). Formation, Regulation and Evolution of Caenorhabditis Elegans 3'utrs. Nature. 2010 Nov 17. [Epub ahead of print] Post-transcriptional gene regulation frequently occurs through elements in mRNA 3' untranslated regions (UTRs). Although crucial roles for 3'UTR-mediated gene regulation have been found in Caenorhabditis elegans, most C. elegans genes have lacked annotated 3'UTRs. Here we describe a high-throughput method for reliable identification of polyadenylated RNA termini, and we apply this method, called poly(A)-position profiling by sequencing (3P-Seq), to determine C. elegans 3'UTRs. Compared to standard methods also recently applied to C. elegans UTRs, 3P-Seq identified 8,580 additional UTRs while excluding thousands of shorter UTR isoforms that do not seem to be authentic. Analysis of this expanded and corrected data set suggested that the high A/U content of C. elegans 3'UTRs facilitated genome compaction, because the elements specifying cleavage and polyadenylation, which are A/U rich, can more readily emerge in A/U-rich regions. Indeed, 30% of the protein-coding genes have mRNAs with alternative, partially overlapping end regions that generate another 10,480 cleavage and polyadenylation sites that had gone largely unnoticed and represent potential evolutionary intermediates of progressive UTR shortening. Moreover, a third of the convergently transcribed genes use palindromic arrangements of bidirectional elements to specify UTRs with convergent overlap, which also contributes to genome compaction by eliminating regions between genes. Although nematode 3'UTRs have median length only one-sixth that of mammalian 3'UTRs, they have twice the density of conserved microRNA sites, in part because additional types of seed-complementary sites are preferentially conserved. These findings reveal the influence of cleavage and polyadenylation on the evolution of genome architecture and provide resources for studying post-transcriptional gene regulation. Full Text.

Jarosz, D.F., and Lindquist, S. (2010). Hsp90 and Environmental Stress Transform the Adaptive Value of Natural Genetic Variation. Science 330, 1820-1824. How can species remain unaltered for long periods yet also undergo rapid diversification? By linking genetic variation to phenotypic variation via environmental stress, the Hsp90 protein-folding reservoir might promote both stasis and change. However, the nature and adaptive value of Hsp90-contingent traits remain uncertain. In ecologically and genetically diverse yeasts, we find such traits to be both common and frequently adaptive. Most are based on preexisting variation, with causative polymorphisms occurring in coding and regulatory sequences alike. A common temperature stress alters phenotypes similarly. Both selective inhibition of Hsp90 and temperature stress increase correlations between genotype and phenotype. This system broadly determines the adaptive value of standing genetic variation and, in so doing, has influenced the evolution of current genomes. Full Text.

Jarosz, D.F., Taipale, M., and Lindquist, S. Protein Homeostasis and the Phenotypic Manifestation of Genetic Diversity: Principles and Mechanisms. Annu Rev Genet 44, 189-216. Changing a single nucleotide in a genome can have profound consequences under some conditions, but the same change can have no consequences under others. Indeed, organisms can be surprisingly robust to environmental and genetic perturbations. Yet, the mechanisms underlying such robustness are controversial. Moreover, how they might affect evolutionary change remains enigmatic. Here, we review the recently appreciated central role of protein homeostasis in buffering and potentiating genetic variation and discuss how these processes mediate the critical influence of the environment on the relationship between genotype and phenotype. Deciphering how robustness emerges from biological organization and the mechanisms by which it is overcome in changing environments will lead to a more complete understanding of both fundamental evolutionary processes and diverse human diseases. Full Text.

Jayapal, S., Lee, K.L., Ji, P., Kaldis, P., Lim, B., and Lodish, H.F. (2010). Down-Regulation of Myc Is Essential for Terminal Erythroid Maturation. Journal of Biological Chemistry 285, 40252-40265. Terminal differentiation of mammalian erythroid progenitors involves 4-5 cell divisions and induction of many erythroid important genes followed by chromatin and nuclear condensation and enucleation. The protein levels of c-Myc (Myc) are reduced dramatically during late stage erythroid maturation, coinciding with cell cycle arrest in G(1) phase and enucleation, suggesting possible roles for c-Myc in either or both of these processes. Here we demonstrate that ectopic Myc expression affects terminal erythroid maturation in a dose-dependent manner. Expression of Myc at physiological levels did not affect erythroid differentiation or cell cycle shutdown but specifically blocked erythroid nuclear condensation and enucleation. Continued Myc expression prevented deacetylation of several lysine residues in histones H3 and H4 that are normally deacetylated during erythroid maturation. The histone acetyltransferase Gcn5 was up-regulated by Myc, and ectopic Gcn5 expression partially blocked enucleation and inhibited the late stage erythroid nuclear condensation and histone deacetylation. When overexpressed at levels higher than the physiological range, Myc blocked erythroid differentiation, and the cells continued to proliferate in cytokine-free, serum-containing culture medium with an early erythroblast morphology. Gene expression analysis demonstrated the dysregulation of erythropoietin signaling pathway and the up-regulation of several positive regulators of G(1)-S cell cycle checkpoint by supraphysiological levels of Myc. These results reveal an important dose-dependent function of Myc in regulating terminal maturation in mammalian erythroid cells. Full Text.

Ji, P., and Lodish, H.F. Rac Gtpases Play Multiple Roles in Erythropoiesis. Haematologica-the Hematology Journal 95 (1) :2-4. Full Text.

Jung, H., Lacombe, J., Mazzoni, E.O., Liem, K.F., Grinstein, J., Mahony, S., Mukhopadhyay, D., Gifford, D.K., Young, R.A., Anderson, K.V., Hynek Wichterle, Jeremy S. Dasen. Global Control of Motor Neuron Topography Mediated by the Repressive Actions of a Single Hox Gene. Neuron 67, 781-796. In the developing spinal cord, regional and combinatorial activities of Hox transcription factors are critical in controlling motor neuron fates along the rostrocaudal axis, exemplified by the precise pattern of limb innervation by more than fifty Hox-dependent motor pools. The mechanisms by which motor neuron diversity is constrained to limb levels are, however, not well understood. We show that a single Hox gene, Hoxc9, has an essential role in organizing the motor system through global repressive activities. Hoxc9 is required for the generation of thoracic motor columns, and in its absence, neurons acquire the fates of limb-innervating populations. Unexpectedly, multiple Hox genes are derepressed in Hoxc9 mutants, leading to motor pool disorganization and alterations in the connections by thoracic and forelimb-level subtypes. Genome-wide analysis of Hoxc9 binding suggests that this mode of repression is mediated by direct interactions with Hox regulatory elements, independent of chromatin marks typically associated with repressed Hox genes. PDF

Kagey, M.H., Newman, J.J., Bilodeau, S., Zhan, Y., Orlando, D.A., van Berkum, N.L., Ebmeier, C.C., Goossens, J., Rahl, P.B., Levine, S.S., Taatjes DJ, Dekker J, and Young RA. Mediator and Cohesin Connect Gene Expression and Chromatin Architecture. Nature. Aug 18. [Epub ahead of print] Transcription factors control cell-specific gene expression programs through interactions with diverse coactivators and the transcription apparatus. Gene activation may involve DNA loop formation between enhancer-bound transcription factors and the transcription apparatus at the core promoter, but this process is not well understood. Here we report that mediator and cohesin physically and functionally connect the enhancers and core promoters of active genes in murine embryonic stem cells. Mediator, a transcriptional coactivator, forms a complex with cohesin, which can form rings that connect two DNA segments. The cohesin-loading factor Nipbl is associated with mediator-cohesin complexes, providing a means to load cohesin at promoters. DNA looping is observed between the enhancers and promoters occupied by mediator and cohesin. Mediator and cohesin co-occupy different promoters in different cells, thus generating cell-type-specific DNA loops linked to the gene expression program of each cell. Full Text.

Kanhere, A., Viiri, K., Araujo, C.C., Rasaiyaah, J., Bouwman, R.D., Whyte, W.A., Pereira, C.F., Brookes, E., Walker, K., Bell, G.W., Richard A. Young et al. Short Rnas Are Transcribed from Repressed Polycomb Target Genes and Interact with Polycomb Repressive Complex-2. Molecular Cell 38, 675-688. Polycomb proteins maintain cell identity by repressing the expression of developmental regulators specific for other cell types. Polycomb repressive complex-2 (PRC2) catalyzes trimethylation of histone H3 lysine-27 (H3K27me3). Although repressed, PRC2 targets are generally associated with the transcriptional initiation marker H3K4me3, but the significance of this remains unclear. Here, we identify a class of short RNAs, similar to 50-200 nucleotides in length, transcribed from the 5' end of polycomb target genes in primary T cells and embryonic stem cells. Short RNA transcription is associated with RNA polymerase II and H3K4me3, occurs in the absence of mRNA transcription, and is independent of polycomb activity. Short RNAs form stem-loop structures resembling PRC2 binding sites in Xist, interact with PRC2 through SUZ12, cause gene repression in cis, and are depleted from polycomb target genes activated during cell differentiation. We propose that short RNAs play a role in the association of PRC2 with its target genes. Full Text.

Kharas, M.G., Lengner, C.J., Al-Shahrour, F., Bullinger, L., Ball, B., Zaidi, S., Morgan, K., Tam, W., Paktinat, M., Okabe, R., Gozo M, Einhorn W, Lane SW, Scholl C, Fröhling S, Fleming M, Ebert BL, Gilliland DG, Jaenisch R, Daley GQ. Musashi-2 Regulates Normal Hematopoiesis and Promotes Aggressive Myeloid Leukemia. Nature Medicine.Volume 16: 903–908 RNA-binding proteins of the Musashi (Msi) family are expressed in stem cell compartments and in aggressive tumors, but they have not yet been widely explored in the blood. Here we demonstrate that Msi2 is the predominant form expressed in hematopoietic stem cells (HSCs), and its knockdown leads to reduced engraftment and depletion of HSCs in vivo. Overexpression of human MSI2 in a mouse model increases HSC cell cycle progression and cooperates with the chronic myeloid leukemia-associated BCR-ABL1 oncoprotein to induce an aggressive leukemia. MSI2 is overexpressed in human myeloid leukemia cell lines, and its depletion leads to decreased proliferation and increased apoptosis. Expression levels in human myeloid leukemia directly correlate with decreased survival in patients with the disease, thereby defining MSI2 expression as a new prognostic marker and as a new target for therapy in acute myeloid leukemia (AML). Full Text.

Khurana, V., Elson-Schwab, I., Fulga, T.A., Sharp, K.A., Loewen, C.A., Mulkearns, E., Tyynela, J., Scherzer, C.R., and Feany, M.B. Lysosomal Dysfunction Promotes Cleavage and Neurotoxicity of Tau in Vivo. Plos Genetics 2010 Jul 15;6(7):e1001026. Expansion of the lysosomal system, including cathepsin D upregulation, is an early and prominent finding in Alzheimer's disease brain. Cell culture studies, however, have provided differing perspectives on the lysosomal connection to Alzheimer's disease, including both protective and detrimental influences. We sought to clarify and molecularly define the connection in vivo in a genetically tractable model organism. Cathepsin D is upregulated with age in a Drosophila model of Alzheimer's disease and related tauopathies. Genetic analysis reveals that cathepsin D plays a neuroprotective role because genetic ablation of cathepsin D markedly potentiates tau-induced neurotoxicity. Further, generation of a C-terminally truncated form of tau found in Alzheimer's disease patients is significantly increased in the absence of cathepsin D. We show that truncated tau has markedly increased neurotoxicity, while solubility of truncated tau is decreased. Importantly, the toxicity of truncated tau is not affected by removal of cathepsin D, providing genetic evidence that modulation of neurotoxicity by cathepsin D is mediated through C-terminal cleavage of tau. We demonstrate that removing cathepsin D in adult postmitotic neurons leads to aberrant lysosomal expansion and caspase activation in vivo, suggesting a mechanism for C-terminal truncation of tau. We also demonstrate that both cathepsin D knockout mice and cathepsin D-deficient sheep show abnormal C-terminal truncation of tau and accompanying caspase activation. Thus, caspase cleavage of tau may be a molecular mechanism through which lysosomal dysfunction and neurodegeneration are causally linked in Alzheimer's disease. Full Text.

Khurana, V., and Lindquist, S. Modelling Neurodegeneration in Saccharomyces Cerevisiae: Why Cook with Baker's Yeast? Nature Reviews Neuroscience Apr 28. [Epub ahead of print]. In ageing populations, neurodegenerative diseases increase in prevalence, exacting an enormous toll on individuals and their communities. Multiple complementary experimental approaches are needed to elucidate the mechanisms underlying these complex diseases and to develop novel therapeutics. Here, we describe why the budding yeast Saccharomyces cerevisiae has a unique role in the neurodegeneration armamentarium. As the best-understood and most readily analysed eukaryotic organism, S. cerevisiae is delivering mechanistic insights into cell-autonomous mechanisms of neurodegeneration at an interactome-wide scale. Full Text.

Kim, N., Kukkonen, S., Gupta, S., and Aldovini, A. (2010). Association of Tat with Promoters of Pten and Pp2a Subunits Is Key to Transcriptional Activation of Apoptotic Pathways in Hiv-Infected Cd4+T Cells. Plos Pathogens 6(9). pii: e1001103.. Apoptosis in HIV-1-infected CD4+ primary T cells is triggered by the alteration of the PI3K and p53 pathways, which converge on the FOXO3a transcriptional activator. Tat alone can cause activation of FOXO3a and of its proapoptotic target genes. To understand how Tat affects this pathway, we carried out ChIP-Chip experiments with Tat. Tat associates with the promoters of PTEN and two PP2A subunit genes, but not with the FOXO3a promoter. PTEN and PP2A encode phosphatases, whose levels and activity are increased when Tat is expressed. They counteract phosphorylation of Akt1 and FOXO3a, and so activate transcriptional activity of FOXO3a. FOXO3a promotes increased transcription of Egr-1, which can further stimulate the transcription of PTEN, thereby reinforcing the pathway that leads to FOXO3a transcriptional activation. RNAi experiments support the role of PTEN and PP2A in the initiation of the Tat-mediated cascade, which is critical to apoptosis. The increased accumulation of PTEN and PP2A subunit mRNAs during Tat expression is more likely to be the result of increased transcription initiation and not relief of promoter-proximal pausing of RNAPII. The Tat-PTEN and -PP2A promoter interactions provide a mechanistic explanation of Tat-mediated apoptosis in CD4+ T cells. Full Text.

Kim, K., Doi, A., Wen, B., Ng, K., Zhao, R., Cahan, P., Kim, J., Aryee, M.J., Ji, H., Ehrlich, L.I., Yabuuchi A, Takeuchi A, Cunniff KC, Hongguang H, McKinney-Freeman S, Naveiras O, Yoon TJ, Irizarry RA, Jung N, Seita J, Hanna J, Murakami P, Jaenisch R, Weissleder R, Orkin SH, Weissman IL, Feinberg AP, Daley GQ.. Epigenetic Memory in Induced Pluripotent Stem Cells. Nature. Published online19 July 2010 Somatic cell nuclear transfer and transcription-factor-based reprogramming revert adult cells to an embryonic state, and yield pluripotent stem cells that can generate all tissues. Through different mechanisms and kinetics, these two reprogramming methods reset genomic methylation, an epigenetic modification of DNA that influences gene expression, leading us to hypothesize that the resulting pluripotent stem cells might have different properties. Here we observe that low-passage induced pluripotent stem cells (iPSCs) derived by factor-based reprogramming of adult murine tissues harbour residual DNA methylation signatures characteristic of their somatic tissue of origin, which favours their differentiation along lineages related to the donor cell, while restricting alternative cell fates. Such an 'epigenetic memory' of the donor tissue could be reset by differentiation and serial reprogramming, or by treatment of iPSCs with chromatin-modifying drugs. In contrast, the differentiation and methylation of nuclear-transfer-derived pluripotent stem cells were more similar to classical embryonic stem cells than were iPSCs. Our data indicate that nuclear transfer is more effective at establishing the ground state of pluripotency than factor-based reprogramming, which can leave an epigenetic memory of the tissue of origin that may influence efforts at directed differentiation for applications in disease modelling or treatment. Full Text.

Kirak, O., Frickel, E.M., Grotenbreg, G.M., Suh, H., Jaenisch, R., and Ploegh, H.L. Transnuclear Mice with Predefined T Cell Receptor Specificities against Toxoplasma Gondii Obtained Via Scnt. Science 328, 243-248. Mice that are transgenic for rearranged antigen-specific T cell receptors (TCRs) are essential tools to study T cell development and function. Such TCRs are usually isolated from the relevant T cells after long-term culture, often after repeated antigen stimulation, which unavoidably skews the T cell population used. Random genomic integration of the TCR alpha and beta chain and expression from nonendogenous promoters represent additional drawbacks of transgenics. Using epigenetic reprogramming via somatic cell nuclear transfer, we demonstrated that T cells with predefined specificities against Toxoplasma gondii can be used to generate mouse models that express the TCR from their endogenous loci, without experimentally introduced genetic modification. The relative ease and speed with which such transnuclear models can be obtained holds promise for the construction of other disease models. Full Text.

Kojima, Y., Acar, A., Eaton, E.N., Mellody, K.T., Scheel, C., Ben-Porath, I., Onder, T.T., Wang, Z.C., Richardson, A.L., Weinberg, R.A., and Akira Orimo (2010). Autocrine Tgf-Beta and Stromal Cell-Derived Factor-1 (Sdf-1) Signaling Drives the Evolution of Tumor-Promoting Mammary Stromal Myofibroblasts. Proceedings of the National Academy of Sciences of the United States of America 107, 20009-20014. Much interest is currently focused on the emerging role of tumor-stroma interactions essential for supporting tumor progression. Carcinoma-associated fibroblasts (CAFs), frequently present in the stroma of human breast carcinomas, include a large number of myofibroblasts, a hallmark of activated fibroblasts. These fibroblasts have an ability to substantially promote tumorigenesis. However, the precise cellular origins of CAFs and the molecular mechanisms by which these cells evolve into tumor-promoting myofibroblasts remain unclear. Using a coimplantation breast tumor xenograft model, we show that resident human mammary fibroblasts progressively convert into CAF myofibroblasts during the course of tumor progression. These cells increasingly acquire two autocrine signaling loops, mediated by TGF-beta and SDF-1 cytokines, which both act in autostimulatory and cross-communicating fashions. These autocrine-signaling loops initiate and maintain the differentiation of fibroblasts into myofibroblasts and the concurrent tumor-promoting phenotype. Collectively, these findings indicate that the establishment of the self-sustaining TGF-beta and SDF-1 autocrine signaling gives rise to tumor-promoting CAF myofibroblasts during tumor progression. This autocrine-signaling mechanism may prove to be an attractive therapeutic target to block the evolution of tumor-promoting CAFs. Full Text.

Kotani, A., Ha, D., Schotte, D., den Boer, M.L., Armstrong, S.A., and Lodish, H.F. A Novel Mutation in the Mir-128b Gene Reduces Mirna Processing and Leads to Glucocorticoid Resistance of Mll-Af4 Acute Lymphocytic Leukemia Cells. Cell Cycle 9, 1037-1042. MLL-AF4 Acute Lymphocytic Leukemia has a poor prognosis, and the mechanisms by which these leukemias develop are not understood despite intensive research based on well-known concepts and methods. MicroRNAs ( miRNAs) are a new class of small noncoding RNAs that post-transcriptionally regulate expression of target mRNA transcripts. We recently reported that ectopic expression of miR-128b together with miR-221, two of the miRNAs downregulated in MLL-AF4 ALL, restores glucocorticoid resistance through downregulation of the MLL-AF4 chimeric fusion proteins MLL-AF4 and AF4-MLL that are generated by chromosomal translocation t(4; 11). Here we report the identification of new mutations in miR-128b in RS4; 11 cells, derived from MLL-AF4 ALL patient. One novel mutation significantly reduces the processing of miR-128b. Finally, this base change occurs in a primary MLL-AF4 ALL sample as an acquired mutation. These results demonstrate that the novel mutation in miR-128b in MLL-AF4 ALL alters the processing of miR-128b and that the resultant downregulation of mature miR-128b contributes to glucocorticoid resistance through the failure to downregulate the fusion oncogenes. PDF

Lam, F.H., Hartner, F.S., Fink, G.R., and Stephanopoulos, G. Enhancing Stress Resistance and Production Phenotypes through Transcriptome Engineering. In Methods in Enzymology, Vol 470: Guide to Yeast Genetics: , pp. 509-532.As Saccharomyces cerevisiae is engineered further as a microbial factory for industrially relevant but potentially cytotoxic molecules such as ethanol, issues of cell viability arise that threaten to place a biological limit on output capacity and/or the use of less refined production conditions. Evidence suggests that one naturally evolved mode of survival in deleterious environments involves the complex, multigenic interplay between disparate stress response and homeostasis mechanisms. Rational engineering of such resistance would require a systems-level understanding of cellular behavior that is, in general, not yet available. To circumvent this limitation, we have developed a phenotype discovery approach termed global transcription machinery engineering (gTME) that allows for the generation and selection of nonphysiological traits. We alter gene expression on a genome-wide scale by selecting for dominant mutations in a randomly mutagenized general transcription factor. The gene encoding the mutated transcription factor resides on a plasmid in a strain carrying the unaltered chromosomal allele. Thus, although the dominant mutations may destroy the essential function of the plasmid-borne variant, alteration of the transcriptome with minimal perturbation to normal cellular processes is possible via the presence of the native genomic allele. Achieving a phenotype of interest involves the construction and diversity evaluation of yeast libraries harboring random sequence variants of a chosen transcription factor and the subsequent selection and validation of mutant strains. We describe the rationale and procedures associated with each step in the context of generating strains possessing enhanced ethanol tolerance. PDF

Laplante, M., and Sabatini, D.M. Mtorc1 Activates Srebp-1c and Uncouples Lipogenesis from Gluconeogenesis.Proc Natl Acad Sci U S A. 107(8):3281-2. Epub 2010 Feb 18 The identification of mTORC1 as an insulin-regulated component controlling lipogenesis, but not gluconeogenesis, provides a basis for understanding the selective nature of hepatic insulin resistance. . Full Text.

Lengner, C.J., Gimelbrant, A.A., Erwin, J.A., Cheng, A.W., Guenther, M.G., Welstead, G.G., Alagappan, R., Frampton, G.M., Xu, P., Muffat, J., Sandro Santagata, Doug Powers, C. Brent Barrett, Richard A. Young, Jeannie T. Lee, Rudolf Jaenisch, and Maisam Mitalipova . Derivation of Pre-X Inactivation Human Embryonic Stem Cells under Physiological Oxygen Concentrations. Cell. May 12. [Epub ahead of print] The presence of two active X chromosomes (XaXa) is a hallmark of the ground state of pluripotency specific to murine embryonic stem cells (ESCs). Human ESCs (hESCs) invariably exhibit signs of X chromosome inactivation (XCI) and are considered developmentally more advanced than their murine counterparts. We describe the establishment of XaXa hESCs derived under physiological oxygen concentrations. Using these cell lines, we demonstrate that (1) differentiation of hESCs induces random XCI in a manner similar to murine ESCs, (2) chronic exposure to atmospheric oxygen is sufficient to induce irreversible XCI with minor changes of the transcriptome, (3) the Xa exhibits heavy methylation of the XIST promoter region, and (4) XCI is associated with demethylation and transcriptional activation of XIST along with H3K27-me3 deposition across the Xi. These findings indicate that the human blastocyst contains pre-X-inactivation cells and that this state is preserved in vitro through culture under physiological oxygen. Full Text.

Lengner, C.J. Ips Cell Technology in Regenerative Medicine. Ann N Y Acad Sci 1192, 38-44. The promise of treating human genetic and degenerative diseases through the application of pluripotent cell-based tissue engineering and regenerative medicine has come significantly closer to realization since the isolation of human embryonic stem (ES) cells. While the study of ES cells has greatly increased our fundamental understanding of pluripotency, technical and ethical limitations have been seemingly insurmountable impediments to the application of these cells in the clinic. The recent discovery that somatic mammalian cells can be epigenetically reprogrammed to a pluripotent state through the exogenous expression of the transcription factors OCT4, SOX2, KLF4, and c-MYC has yielded a new cell type for potential application in regenerative medicine, the induced pluripotent stem (iPS) cell. Here we discuss how advances in iPS cell technology have led to the generation of patient-specific cell lines that can potentially be used to model human diseases and ultimately act as therapeutic agents. PDF.

Lewandowski, N.M., Ju, S.L., Verbitsky, M., Ross, B., Geddie, M.L., Rockenstein, E., Adame, A., Muhammad, A., Vonsattel, J.P., Ringe, D.,Cote L, Lindquist S, Masliah E, Petsko GA, Marder K, Clark LN, Small SA. Polyamine Pathway Contributes to the Pathogenesis of Parkinson Disease. Proceedings of the National Academy of Sciences of the United States of America 107, 16970-16975. The full complement of molecular pathways contributing to the pathogenesis of Parkinson disease (PD) remains unknown. Here we address this issue by taking a broad approach, beginning by using functional MRI to identify brainstem regions differentially affected and resistant to the disease. Relying on these imaging findings, we then profiled gene expression levels from postmortem brainstem regions, identifying a disease-related decrease in the expression of the catabolic polyamine enzyme spermidine/spermine N1-acetyl-transferase 1 (SAT1). Next, a range of studies were completed to support the pathogenicity of this finding. First, to test for a causal link between polyamines and alpha-synuclein toxicity, we investigated a yeast model expressing alpha-synuclein. Polyamines were found to enhance the toxicity of alpha-synuclein, and an unbiased genome-wide screen for modifiers of alpha-synuclein toxicity identified Tpo4, a member of a family of proteins responsible for polyamine transport. Second, to test for a causal link between SAT1 activity and PD histopathology, we investigated a mouse model expressing alpha-synuclein. DENSPM(N1, N11-diethylnorspermine), a polyamine analog that increases SAT1 activity, was found to reduce PD histopathology, whereas Berenil (diminazene aceturate), a pharmacological agent that reduces SAT1 activity, worsened the histopathology. Third, to test for a genetic link, we sequenced the SAT1 gene and a rare but unique disease-associated variant was identified. Taken together, the findings from human patients, yeast, and a mouse model implicate the polyamine pathway in PD pathogenesis. Full Text.

Lindquist, S. (2010). Three Quite Different Things That Matter to Me. Mol Biol Cell 21, 3804. I'm grateful to be asked to comment on cell biology and the next 50 years. There is much to say. But let me focus on just three quite different things that particularly matter to me. Full Text.

Lindquist, S. Protein Folding Sculpting Evolutionary Change. Cold Spring Harbor Symposia on Quantitative Biology. 2009 74:103-8. Epub 2010 Our work suggests that the forces that govern protein folding exert a profound effect on how genotypes are translated into phenotypes and that this in turn has strong effects on evolutionary processes. Molecular chaperones, also known as "heat-shock proteins" (Hsps), promote the correct folding and maturation of many other proteins in the cell. Hsp90 is an abundant and highly specialized chaperone that works on a particularly interesting group of client proteins: metastable signal transducers that are key regulators of a broad spectrum of biological processes. Such proteins often have evolved to finish folding only when they have received a specific signal, such as the binding of a ligand or a posttranslational modification. Importantly, the folding of Hsp90 clients is particularly sensitive to changes in the external and internal environment of the cell. Therefore, Hsp90 is uniquely positioned to couple environmental contingencies to the evolution of new traits. Our work has helped to define two mechanisms by which Hsp90 might influence the acquisition of new phenotypes. First, by robustly maintaining signaling pathways, Hsp90 can buffer the effects of mutations in those pathways, allowing the storage of cryptic genetic variation that is released by stress. In this case, when the Hsp90 buffer is compromised by environmental stress, new traits appear. These traits can also be assimilated, so that they become manifest even in the absence of stress, when genetic recombination and selection enrich causative variants in subsequent generations. Second, Hsp90 can potentiate the effects of genetic variation, allowing new mutations to produce immediate phenotypes. In this case, when Hsp90 function is compromised, new traits are lost. These traits can also be assimilated, so that they are maintained under environmental stress, but this is achieved through new mutations. We have discovered these powerful evolutionary mechanisms in fruit flies, mustard plants, and fungi, but expect them to operate in all eukaryotes. Another line of work relating protein folding to the evolution of new traits involves protein-based hereditary elements known as prions. These produce changes in phenotype through heritable, self-perpetuating changes in protein conformation. Because changes in protein homeostasis occur with environmental stress, prions can be cured or induced by stress, creating heritable new phenotypes that depend on the genetic variation present in the organism. Both prions and Hsp90 provide plausible mechanisms for allowing genetic diversity and fluctuating environments to fuel the pace of evolutionary change. The multiple mechanisms by which protein folding can influence the evolution of new traits provide both a new paradigm for understanding rapid, stepwise evolution and a framework for targeted therapeutic interventions. PDF

Liu, D., Vleugel, M., Backer, C.B., Hori, T., Fukagawa, T., Cheeseman, I.M., and Lampson, M.A. Regulated Targeting of Protein Phosphatase 1 to the Outer Kinetochore by Knl1 Opposes Aurora B Kinase. Journal of Cell Biology 188, 809-820. Regulated interactions between kinetochores and spindle microtubules are essential to maintain genomic stability during chromosome segregation. The Aurora B kinase phosphorylates kinetochore substrates to destabilize kinetochore-microtubule interactions and eliminate incorrect attachments. These substrates must be dephosphorylated to stabilize correct attachments, but how opposing kinase and phosphatase activities are coordinated at the kinetochore is unknown. Here, we demonstrate that a conserved motif in the kinetochore protein KNL1 directly interacts with and targets protein phosphatase 1 (PP1) to the outer kinetochore. PP1 recruitment by KNL1 is required to dephosphorylate Aurora B substrates at kinetochores and stabilize microtubule attachments. PP1 levels at kinetochores are regulated and inversely proportional to local Aurora B activity. Indeed, we demonstrate that phosphorylation of KNL1 by Aurora B disrupts the KNL1-PP1 interaction. In total, our results support a positive feedback mechanism by which Aurora B activity at kinetochores not only targets substrates directly, but also prevents localization of the opposing phosphatase. Full Text.

Liu, Q., Gauthier, M.S., Sun, L., Ruderman, N., and Lodish, H. Activation of Amp-Activated Protein Kinase Signaling Pathway by Adiponectin and Insulin in Mouse Adipocytes: Requirement of Acyl-Coa Synthetases Fatp1 and Acsl1 and Association with an Elevation in Amp/Atp Ratio. FASEB J.Jul 28. [Epub ahead of print] Adiponectin activates AMP-activated protein kinase (AMPK) in adipocytes, but the underlying mechanism remains unclear. Here we tested the hypothesis that AMP, generated in activating fatty acids to their CoA derivatives, catalyzed by acyl-CoA synthetases, is involved in AMPK activation by adiponectin. Moreover, in adipocytes, insulin affects the subcellular localization of acyl-CoA synthetase FATP1. Thus, we also tested whether insulin activates AMPK in these cells and, if so, whether it activates through a similar mechanism. We examined these hypotheses by measuring the AMP/ATP ratio and AMPK activation on adiponectin and insulin stimulation and after knocking down acyl-CoA synthetases in adipocytes. We show that adiponectin activation of AMPK is accompanied by an approximately 2-fold increase in the cellular AMP/ATP ratio. Moreover, FATP1 and Acsl1, the 2 major acyl-CoA synthetase isoforms in adipocytes, are essential for AMPK activation by adiponectin. We also show that after 40 min. insulin activated AMPK in adipocytes, which was coupled with a 5-fold increase in the cellular AMP/ATP ratio. Knockdown studies show that FATP1 and Acsl1 are required for these processes, as well as for stimulation of long-chain fatty acid uptake by adiponection and insulin. These studies demonstrate that a change in cellular energy state is associated with AMPK activation by both adiponectin and insulin, which requires the activity of FATP1 and Acsl1.-Liu, Q., Gauthier, M.-L., Sun, L., Ruderman, N., Lodish, H. Activation of AMP-activated protein kinase signaling pathway by adiponectin and insulin in mouse adipocytes: requirement of acyl-CoA synthetases FATP1 and Acsl1 and association with an elevation in AMP/ATP ratio. PDF.

Lodish, H., Flygare, J., and Chou, S. From Stem Cell to Erythroblast: Regulation of Red Cell Production at Multiple Levels by Multiple Hormones. IUBMB Life. Mar 19. [Epub ahead of print] This article reviews the regulation of production of red blood cells at several levels: (1) the ability of erythropoietin and adhesion to a fibronectin matrix to stimulate the rapid production of red cells by inducing terminal proliferation and differentiation of committed erythroid CFU-E progenitors; (2) the regulated expansion of the pool of earlier BFU-E erythroid progenitors by glucocorticoids and other factors that occurs during chronic anemia or inflammation; and (3) the expansion of thehematopoietic cell pool to produce more progenitors of all hematopoietic lineages. . Full Text.

Ma, L., Reinhardt, F., Pan, E., Soutschek, J., Bhat, B., Marcusson, E.G., Teruya-Feldstein, J., Bell, G.W., and Weinberg, R.A. Therapeutic Silencing of Mir-10b Inhibits Metastasis in a Mouse Mammary Tumor Model. Nature Biotechnology Published Online 28 March. MicroRNAs (miRNAs) are increasingly implicated in the regulation of metastasis. Despite their potential as targets for anti-metastatic therapy, miRNAs have only been silenced in normal tissues of rodents and nonhuman primates. Therefore, the development of effective approaches for sequence-specific inhibition of miRNAs in tumors remains a scientific and clinical challenge. Here we show that systemic treatment of tumor-bearing mice with miR-10b antagomirs-a class of chemically modified anti-miRNA oligonucleotide-suppresses breast cancer metastasis. Both in vitro and in vivo, silencing of miR-10b with antagomirs significantly decreases miR-10b levels and increases the levels of a functionally important miR-10b target, Hoxd10. Administration of miR-10b antagomirs to mice bearing highly metastatic cells does not reduce primary mammary tumor growth but markedly suppresses formation of lung metastases in a sequence-specific manner. The miR-10b antagomir, which is well tolerated by normal animals, appears to be a promising candidate for the development of new anti-metastasis agents. Full Text.

Ma, L., Young, J., Prabhala, H., Pan, E., Mestdagh, P., Muth, D., Teruya-Feldstein, J., Reinhardt, F., Onder, T.T., Valastyan, S., Westermann F, Speleman F, Vandesompele J, & Weinberg RA. . Mir-9, a Myc/Mycn-Activated Microrna, Regulates E-Cadherin and Cancer Metastasis. Nature Cell Biology Published Online: 21 February . MicroRNAs (miRNAs) are increasingly implicated in regulating the malignant progression of cancer. Here we show that miR-9, which is upregulated in breast cancer cells, directly targets CDH1, the E-cadherin-encoding messenger RNA, leading to increased cell motility and invasiveness. miR-9-mediated E-cadherin downregulation results in the activation of beta-catenin signalling, which contributes to upregulated expression of the gene encoding vascular endothelial growth factor (VEGF); this leads, in turn, to increased tumour angiogenesis. Overexpression of miR-9 in otherwise non-metastatic breast tumour cells enables these cells to form pulmonary micrometastases in mice. Conversely, inhibiting miR-9 by using a 'miRNA sponge' in highly malignant cells inhibits metastasis formation. Expression of miR-9 is activated by MYC and MYCN, both of which directly bind to the mir-9-3 locus. Significantly, in human cancers, miR-9 levels correlate with MYCN amplification, tumour grade and metastatic status. These findings uncover a regulatory and signalling pathway involving a metastasis-promoting miRNA that is predicted to directly target expression of the key metastasis-suppressing protein E-cadherin. PDF.

McAllister, S.S., and Weinberg, R.A. Tumor-Host Interactions: A Far-Reaching Relationship. Journal of Clinical Oncology.Jul 19. [Epub ahead of print] Carcinomas are composed of neoplastic epithelial cells, which form the heart of the tumor, as well as a variety of mesenchymal cell types and extracellular matrix components that comprise the tumor stroma, often termed its microenvironment. The normal counterparts of some stromal cells are thought to limit tumor growth, while tumor-associated stromal cells have been convincingly shown to actively promote tumor progression via complex heterotypic interactions with the nearby carcinoma cells. More recent advances have revealed that tumor-host interactions extend well beyond the local tissue microenvironment (ie, interactions between the neoplastic cells and the nearby stroma) and that tumors not only respond to, but actively perturb host organs at distant anatomic sites. This indicates that many aspects of tumor biology can only be explained by a detailed understanding of both local and systemic interactions, yet we currently have only a fragmentary understanding of both processes. In this review, we address the recent advances in our understanding of the contributions of local and systemic environments to cancer progression, the ability of tumors to actively perturb the host environment, and current therapeutic approaches that are designed to disrupt tumor-host relationships.

Mei, Y., Saha, K., Bogatyrev, S.R., Yang, J., Hook, A.L., Kalcioglu, Z.I., Cho, S.W., Mitalipova, M., Pyzocha, N., Rojas, F., Jaenisch, R. et al.Combinatorial Development of Biomaterials for Clonal Growth of Human Pluripotent Stem Cells. Nature Materials 9, 768-778. Both human embryonic stem cells and induced pluripotent stem cells can self-renew indefinitely in culture; however, present methods to clonally grow them are inefficient and poorly defined for genetic manipulation and therapeutic purposes. Here we develop the first chemically defined, xeno-free, feeder-free synthetic substrates to support robust self-renewal of fully dissociated human embryonic stem and induced pluripotent stem cells. Material properties including wettability, surface topography, surface chemistry and indentation elastic modulus of all polymeric substrates were quantified using high-throughput methods to develop structure-function relationships between material properties and biological performance. These analyses show that optimal human embryonic stem cell substrates are generated from monomers with high acrylate content, have a moderate wettability and employ integrin alpha(v)beta(3) and alpha(v)beta(5) engagement with adsorbed vitronectin to promote colony formation. The structure-function methodology employed herein provides a general framework for the combinatorial development of synthetic substrates for stem cell culture. Full Text.

Namekawa, S.H., Payer, B., Huynh, K.D., Jaenisch, R., and Lee, J.T. Two-Step Imprinted X-Inactivation: Repeat Vs Genic Silencing in the Mouse. Molecular Cell Biology. 19 April . [Epub ahead of print] Mammals compensate for unequal X-linked gene dosages between the sexes by inactivating one X-chromosome in the female. In marsupials and in the early mouse embryo, X-chromosome inactivation (XCI) is imprinted to occur selectively on the paternal X-chromosome (X(P)). The mechanisms and events underlying X(P) imprinting remain unclear. Here, we find that the imprinted X(P) can be functionally divided into two domains, one comprising traditional coding genes (genic) and the other comprising intergenic repetitive elements. X(P) repetitive elements are silenced by the 2-cell stage, does not require Xist, and occurs several divisions prior to genic silencing. By contrast, genic silencing initiates at the morula-to-blastocyst stage and absolutely requires Xist. Genes translocate into the pre-silenced repeat region as they are inactivated, whereas active genes remain outside. Thus, during the gamete-embryo transition, imprinted XCI occurs in two steps, with repeat silencing preceding genic inactivation. Nucleolar association may underlie epigenetic asymmetry of X(P) and X(M). We hypothesize that transgenerational information (the imprint) may be carried by repeats from the paternal germline or that, alternatively, repetitive elements may be silenced at the 2-cell stage in a parent-of-origin-specific manner. Our model incorporates aspects of the 'classical', 'de novo', and 'preinactivation' hypotheses and suggests that Xist RNA functions relatively late during preimplantation mouse development. Full Text.

Nodine, M.D., and Bartel, D.P. (2010). Micrornas Prevent Precocious Gene Expression and Enable Pattern Formation During Plant Embryogenesis. Genes Dev 24, 2678-2692. Arabidopsis embryos lacking DICER-LIKE1 (DCL1), which is required for microRNA (miRNA) biogenesis, arrest early in development. To assess the functions of embryonic miRNAs, we determined the developmental and molecular consequences of DCL1 loss. We found that DCL1 is required for cell differentiation events as early as the eight-cell stage and soon thereafter for proper division of the hypophysis and subprotoderm cells. By the early globular ( approximately 32-cell) stage, dcl1-null mutant embryos overexpress approximately 50 miRNA targets. In dcl1 eight-cell embryos, the two most up-regulated targets are those of miR156 and encode SPL10 and SPL11 transcription factors. SPL10 and SPL11 are derepressed >150-fold in dcl1 embryos and are redundantly required for the dcl1 early patterning defects. Moreover, as early as the eight-cell stage, miR156-mediated repression of zygotic SPL transcripts prevents premature accumulation of transcripts from genes normally induced during the embryonic maturation phase. Thus, the first perceptible molecular function of plant embryonic miRNAs is the opposite of that in vertebrates; in vertebrates, miRNAs sharpen the first developmental transition, whereas in plants, they forestall developmental transitions by repressing mRNAs that act later. We propose that, by preventing precocious expression of differentiation-promoting transcription factors, miRNAs enable proper embryonic patterning. Full Text.

Nordman, J., Li, S., Eng, T., Macalpine, D., and Orr-Weaver, T.L. (2010). Developmental Control of the DNA Replication and Transcription Programs. Genome Research Dec 22. [Epub ahead of print] Polyploid or polytene cells, which have more than 2C DNA content, are widespread throughout nature and present in most differentiated Drosophila tissues. These cells also can display differential replication, that is, genomic regions of increased or decreased DNA copy number relative to overall genomic ploidy. How frequently differential replication is used as a developmental strategy remains unclear. Here, we use genome-wide array-based comparative genomic hybridization (aCGH) to profile differential DNA replication in isolated and purified larval fat body and midgut tissues of Drosophila, and we compare them with recent aCGH profiles of the larval salivary gland. We identify sites of euchromatic underreplication that are common to all three tissues and others that are tissue specific. We demonstrate that both common and tissue-specific underreplicated sites are dependent on the Suppressor of Underreplication protein, SUUR. mRNA-seq profiling shows that whereas underreplicated regions are generally transcriptionally silent in the larval midgut and salivary gland, transcriptional silencing and underreplication have been uncoupled in the larval fat body. In addition to revealing the prevalence of differential replication, our results show that transcriptional silencing and underreplication can be mechanistically uncoupled. PDF.

Orlando, D.A., Brady, S.M., Fink, T.M.A., Benfey, P.N., and Ahnert, S.E. Detecting Separate Time Scales in Genetic Expression Data. BMC Genomics Jun 16;11:381. Background: Biological processes occur on a vast range of time scales, and many of them occur concurrently. As a result, system-wide measurements of gene expression have the potential to capture many of these processes simultaneously. The challenge however, is to separate these processes and time scales in the data. In many cases the number of processes and their time scales is unknown. This issue is particularly relevant to developmental biologists, who are interested in processes such as growth, segmentation and differentiation, which can all take place simultaneously, but on different time scales. Results: We introduce a flexible and statistically rigorous method for detecting different time scales in time-series gene expression data, by identifying expression patterns that are temporally shifted between replicate datasets. We apply our approach to a Saccharomyces cerevisiae cell-cycle dataset and an Arabidopsis thaliana root developmental dataset. In both datasets our method successfully detects processes operating on several different time scales. Furthermore we show that many of these time scales can be associated with particular biological functions. Conclusions: The spatiotemporal modules identified by our method suggest the presence of multiple biological processes, acting at distinct time scales in both the Arabidopsis root and yeast. Using similar large-scale expression datasets, the identification of biological processes acting at multiple time scales in many organisms is now possible. Full Text.

Page, D.C., Hughes, J.F., Bellott, D.W., Mueller, J.L., Gill, M.E., Larracuente, A., Graves, T., Muzny, D., Warren, W.C., Gibbs, R.A., Richard K Wilson and Helen Skaletsky (2010). Reconstructing Sex Chromosome Evolution. Genome Biology 11(Suppl 1):I21 Full Text.

Pandya, R.K., Partridge, J.R., Love, K.R., Schwartz, T.U., and Ploegh, H.L. A Structural Element within the Huwe1 Hect Domain Modulates Self-Ubiquitination and Substrate Ubiquitination Activities. Journal of Biological Chemistry 285, 5664-5673. E3 ubiquitin ligases catalyze the final step of ubiquitin conjugation and regulate numerous cellular processes. The HECT class of E3 ubiquitin (Ub) ligases directly transfers Ub from bound E2 enzyme to a myriad of substrates. The catalytic domain of HECT Ub ligases has a bilobal architecture that separates the E2 binding region and catalytic site. An important question regarding HECT domain function is the control of ligase activity and specificity. Here we present a functional analysis of the HECT domain of the E3 ligase HUWE1 based on crystal structures and show that a single N-terminal helix significantly stabilizes the HECT domain. We observe that this element modulates HECT domain activity, as measured by self-ubiquitination induced in the absence of this helix, as distinct from its effects on Ub conjugation of substrate Mcl-1. Such subtle changes to the protein may be at the heart of the vast spectrum of substrate specificities displayed by HECT domain E3 ligases. Full Text.

Park, B., Spooner, E., Houser, B.L., Strominger, J.L., and Ploegh, H.L. The Hcmv Membrane Glycoprotein Us10 Selectively Targets Hla-G for Degradation. J Exp Med.2010 Aug 16. [Epub ahead of print] Human cytomegalovirus (HCMV) encodes an endoplasmic reticulum (ER)-resident transmembrane glycoprotein, US10, expressed early in the replicative cycle of HCMV as part of the same cluster that encodes the known immunoevasins US2, US3, US6, and US11. We show that US10 down-regulates cell surface expression of HLA-G, but not that of classical class I MHC molecules. The unique and short cytoplasmic tail of HLA-G (RKKSSD) is essential in its role as a US10 substrate, and a tri-leucine motif in the cytoplasmic tail of US10 is responsible for down-regulation of HLA-G. Both the kinetics of HLA-G degradation and the mechanisms responsible appear to be distinct from those used by the US2 and US11 pathways, suggesting the existence of a third route of protein dislocation from the ER. We show that US10-mediated degradation of HLA-G interferes with HLA-G-mediated NK cell inhibition. Given the role of HLA-G in protecting the fetus from attack by the maternal immune system and in directing the differentiation of human dendritic cells to promote the evolution of regulatory T cells, HCMV likely targets the HLA-G-dependent axis of immune recognition no less efficiently than it interferes with classical class I MHC-restricted antigen presentation. Full Text.

Pritchard, C.D., Arner, K.M., Neal, R.A., Neeley, W.L., Bojo, P., Bachelder, E., Holz, J., Watson, N., Botchwey, E.A., Langer, R.S., Ghosh FK. The Use of Surface Modified Poly(Glycerol-Co-Sebacic Acid) in Retinal Transplantation. Biomaterials 31, 2153-2162. Retinal transplantation experiments have advanced considerably during recent years, but remaining diseased photoreceptor cells in the host retina and inner retinal cells in the transplant physically obstruct the development of graft-host neuronal contacts which are required for vision. Recently, we developed methods for the isolation of donor photoreceptor layers in vitro, and the selective removal of host photoreceptors in vivo using biodegradable elastomeric membranes composed of poly(glycerol-co-sebacic acid) (PGS). Here, we report the surface modification of PGS membranes to promote the attachment of photoreceptor layers, allowing the resulting composite to be handled surgically as a single entity. PGS membranes were chemically modified with peptides containing an arginine-glycine-aspartic acid (RGD) extracellular matrix ligand sequence. PGS membranes were also coated with electrospun nanofiber meshes, containing laminin and poly(epsilon-caprolactone) (PCL). Following in vitro co-culture of biomaterial membranes with isolated embryonic retinal tissue, composites were tested for surgical handling and examined with hematoxylin and eosin staining and immunohistochemical markers. Electrospun nanofibers composed of laminin and PCL promoted sufficient cell adhesion for simultaneous transplantation of isolated photoreceptor layers and PGS membranes. Composites developed large populations of recoverin and rhodopsin labeled photoreceptors. Furthermore, ganglion cells, rod bipolar cells and All amacrine cells were absent in co-cultured retinas as observed by neurofilament, PKC and parvalbumin labeling respectively. These results facilitate retinal transplantation experiments in which a composite graft composed of a biodegradable membrane adhered to an immature retina dominated by photoreceptor cells may be delivered in a single surgery, with the possibility of improving graft-host neuronal connections. Full Text.

Rahl, Peter B, Charles Y. Lin, Amy C. Seila, Ryan A. Flynn, Scott McCuine, Christopher B. Burge, Phillip A. Sharp, Richard A. Young .c-Myc Regulates Transcriptional Pause Release Cell, Volume 141, Issue 3, 432-445 Recruitment of the RNA polymerase II (Pol II) transcription initiation apparatus to promoters by specific DNA-binding transcription factors is well recognized as a key regulatory step in gene expression. We report here that promoter-proximal pausing is a general feature of transcription by Pol II in mammalian cells and thus an additional step where regulation of gene expression occurs. This suggests that some transcription factors recruit the transcription apparatus to promoters, whereas others effect promoter-proximal pause release. Indeed, we find that the transcription factor c-Myc, a key regulator of cellular proliferation, plays a major role in Pol II pause release rather than Pol II recruitment at its target genes. We discuss the implications of these results for the role of c-Myc amplification in human cancer .Full Text

Rao, P.K., Missiaglia, E., Shields, L., Hyde, G., Yuan, B., Shepherd, C.J., Shipley, J., and Lodish, H.F. Distinct Roles for Mir-1 and Mir-133a in the Proliferation and Differentiation of Rhambomyosarcoma Cells. FASEB J.May 13. [Epub ahead of print] Rhabdomyosarcoma is the most common soft tissue sarcoma in the pediatric population. As this tumor has an undifferentiated myogenic phenotype, agents that promote differentiation hold particular promise as part of a novel therapeutic approach to combat this type of cancer. In this report, we focus on the contribution of two microRNAs (miRNAs) in rhabdomyosarcomas. Levels of miR-1 and miR-133a are drastically reduced in representative cell lines from each major rhabdomyosarcoma subtype (embryonal and alveolar). Introduction of miR-1 and miR-133a into an embryonal rhabdomyosarcoma-derived cell line is cytostatic, thereby suggesting a tumor suppressor-like role for these myogenic miRNAs. Transcriptional profiling of cells after miR-1 and miR-133a expression reveals that miR-1 (but not miR-133a) exerts a strong promyogenic influence on these poorly differentiated tumor cells. We identify mRNAs that are down-regulated by these miRNAs and propose roles for miR-1 and miR-133a in repressing isoforms of genes that are normally not expressed in muscle. Finally, we show that mRNA targets of miR-1 and miR-133a are up-regulated in rhabdomyosarcomas, suggesting a causative role for these miRNAs in the development of rhabdomyosarcomas. More important, these results point to the promise of enhancing rhabdomyosarcoma therapy using miRNAs as agents that mediate cytostasis and promote muscle differentiation.-Rao, P. K., Missiaglia, E., Shields, L., Hyde, G., Yuan, B., Shepherd, C. J., Shipley, J., Lodish, H. F. Distinct roles for Mir-1 and Mir-133a in the proliferation and differentiation of rhambomyosarcoma cells. PDF

Rouse, B.T., and Sehrawat, S. Immunity and Immunopathology to Viruses: What Decides the Outcome? Nature Reviews Immunology 10, 514-526. Many viruses infect humans and most are controlled satisfactorily by the immune system with limited damage to host tissues. Some viruses, however, do cause overt damage to the host, either in isolated cases or as a reaction that commonly occurs after infection. The outcome is influenced by properties of the infecting virus, the circumstances of infection and several factors controlled by the host. In this Review, we focus on host factors that influence the outcome of viral infection, including genetic susceptibility, the age of the host when infected, the dose and route of infection, the induction of anti-inflammatory cells and proteins, as well as the presence of concurrent infections and past exposure to cross-reactive agents. Full Text.

Roy, S., Ernst, J., Kharchenko, P.V., Kheradpour, P., Negre, N., Eaton, M.L., Landolin, J.M., Bristow, C.A., Ma, L.J., Lin, M.F., Nordman, J, Orr-Weaver, T, et al (2010). Identification of Functional Elements and Regulatory Circuits by Drosophila Modencode. Science 330, 1787-1797. To gain insight into how genomic information is translated into cellular and developmental programs, the Drosophila model organism Encyclopedia of DNA Elements (modENCODE) project is comprehensively mapping transcripts, histone modifications, chromosomal proteins, transcription factors, replication proteins and intermediates, and nucleosome properties across a developmental time course and in multiple cell lines. We have generated more than 700 data sets and discovered protein-coding, noncoding, RNA regulatory, replication, and chromatin elements, more than tripling the annotated portion of the Drosophila genome. Correlated activity patterns of these elements reveal a functional regulatory network, which predicts putative new functions for genes, reveals stage-and tissue-specific regulators, and enables gene-expression prediction. Our results provide a foundation for directed experimental and computational studies in Drosophila and related species and also a model for systematic data integration toward comprehensive genomic and functional annotation. Full Text.

Ryu, S., and Matsudaira, P. A Drag Correlation for a Nonporous Sphere Steadily Approaching an Impermeable Plane at Finite Reynolds Numbers. Chemical Engineering Science 65, 4913-4915. A particle experiences resistance while moving in a viscous fluid and this drag force increases in the vicinity of no-slip surfaces due to the wall effect. For a nonporous solid spherical particle steadily approaching an impermeable plane, Brenner analytically obtained the wall effect correction factor for the small Reynolds number (Brenner, 1961). For finite Reynolds numbers Wu and Lee calculated the correction factor from their numerical simulation (Wu, 1998). Unifying the results of these previous studies, we propose a compact form of the wall effect correction factor that is valid for Reynolds numbers less than 40. PDF

Sancak, Y., Bar-Peled, L., Zoncu, R., Markhard, A.L., Nada, S., and Sabatini, D.M. Ragulator-Rag Complex Targets Mtorc1 to the Lysosomal Surface and Is Necessary for Its Activation by Amino Acids. Cell.Apr 7. [Epub ahead of print] The mTORC1 kinase promotes growth in response to growth factors, energy levels, and amino acids, and its activity is often deregulated in disease. The Rag GTPases interact with mTORC1 and are proposed to activate it in response to amino acids by promoting mTORC1 translocation to a membrane-bound compartment that contains the mTORC1 activator, Rheb. We show that amino acids induce the movement of mTORC1 to lysosomal membranes, where the Rag proteins reside. A complex encoded by the MAPKSP1, ROBLD3, and c11orf59 genes, which we term Ragulator, interacts with the Rag GTPases, recruits them to lysosomes, and is essential for mTORC1 activation. Constitutive targeting of mTORC1 to the lysosomal surface is sufficient to render the mTORC1 pathway amino acid insensitive and independent of Rag and Ragulator, but not Rheb, function. Thus, Rag-Ragulator-mediated translocation of mTORC1 to lysosomal membranes is the key event in amino acid signaling to mTORC1. Full Text.

Sankaran, V.G., and Nathan, D.G. (2010). Thalassemia: An Overview of 50 Years of Clinical Research. Hematol Oncol Clin North Am 24, 1005-1020. The thalassemias are attributable to the defective production of the alpha- and beta-globin polypeptides of hemoglobin. Significant discoveries have illuminated the pathophysiology and enhanced the prevention and treatment of the thalassemias, and this article reviews many of the advances that have occurred in the past 50 years. However, the application of new approaches to the treatment of these disorders has been slow, particularly in the developing world where the diseases are common, but there is definite progress. This article emphasizes how the increasing knowledge of cellular and molecular biology are facilitating the development of more effective therapies for these patients. Full Text.

Schmidt, J.C., Kiyomitsu, T., Hori, T., Backer, C.B., Fukagawa, T., and Cheeseman, I.M. Aurora B Kinase Controls the Targeting of the Astrin-Skap Complex to Bioriented Kinetochores. J Cell Biol.Oct 11. [Epub ahead of print]. During mitosis, kinetochores play multiple roles to generate interactions with microtubules, and direct chromosome congression, biorientation, error correction, and anaphase segregation. However, it is unclear what changes at the kinetochore facilitate these distinct activities. Here, we describe a complex of the spindle- and kinetochore-associated protein Astrin, the small kinetochore-associated protein (SKAP), and the dynein light chain LC8. Although most dynein-associated proteins localize to unaligned kinetochores in an Aurora B-dependent manner, Astrin, SKAP, and LC8 localization is antagonized by Aurora B such that they target exclusively to bioriented kinetochores. Astrin-SKAP-depleted cells fail to maintain proper chromosome alignment, resulting in a spindle assembly checkpoint-dependent mitotic delay. Consistent with a role in stabilizing bioriented attachments, Astrin and SKAP bind directly to microtubules and are required for CLASP localization to kinetochores. In total, our results suggest that tension-dependent Aurora B phosphorylation can act to control outer kinetochore composition to provide distinct activities to prometaphase and metaphase kinetochores. Full Text.

Scimone, M.L., Meisel, J., and Reddien, P.W. The Mi-2-Like Smed-Chd4 Gene Is Required for Stem Cell Differentiation in the Planarian Schmidtea Mediterranea. Development 137, 1231-1241. Freshwater planarians are able to regenerate any missing part of their body and have extensive tissue turnover because of the action of dividing cells called neoblasts. Neoblasts provide an excellent system for in vivo study of adult stem cell biology. We identified the Smed-CHD4 gene, which is predicted to encode a chromatin-remodeling protein similar to CHD4/Mi-2 proteins, as required for planarian regeneration and tissue homeostasis. Following inhibition of Smed-CHD4 with RNA interference (RNAi), neoblast numbers were initially normal, despite an inability of the animals to regenerate. However, the proliferative response of neoblasts to amputation or growth stimulation in Smed-CHD4(RNAi) animals was diminished. Smed-CHD4(RNAi) animals displayed a dramatic reduction in the numbers of certain neoblast progeny cells. Smed-CHD4 was required for the formation of these neoblast progeny cells. Together, these results indicate that Smed-CHD4 is required for neoblasts to produce progeny cells committed to differentiation in order to control tissue turnover and regeneration and suggest a crucial role for CHD4 proteins in stem cell differentiation. Full Text.

Sengupta, S., Peterson, T.R., Laplante, M., Oh, S., and Sabatini, D.M. (2010). Mtorc1 Controls Fasting-Induced Ketogenesis and Its Modulation by Ageing. Nature 468, 1100-1104. The multi-component mechanistic target of rapamycin complex 1 (mTORC1) kinase is the central node of a mammalian pathway that coordinates cell growth with the availability of nutrients, energy and growth factors. Progress has been made in the identification of mTORC1 pathway components and in understanding their functions in cells, but there is relatively little known about the role of the pathway in vivo. Specifically, we have little knowledge regarding the role mTOCR1 has in liver physiology. In fasted animals, the liver performs numerous functions that maintain whole-body homeostasis, including the production of ketone bodies for peripheral tissues to use as energy sources. Here we show that mTORC1 controls ketogenesis in mice in response to fasting. We find that liver-specific loss of TSC1 (tuberous sclerosis 1), an mTORC1 inhibitor, leads to a fasting-resistant increase in liver size, and to a pronounced defect in ketone body production and ketogenic gene expression on fasting. The loss of raptor (regulatory associated protein of mTOR, complex 1) an essential mTORC1 component, has the opposite effects. In addition, we find that the inhibition of mTORC1 is required for the fasting-induced activation of PPARalpha (peroxisome proliferator activated receptor alpha), the master transcriptional activator of ketogenic genes, and that suppression of NCoR1 (nuclear receptor co-repressor 1), a co-repressor of PPARalpha, reactivates ketogenesis in cells and livers with hyperactive mTORC1 signalling. Like livers with activated mTORC1, livers from aged mice have a defect in ketogenesis, which correlates with an increase in mTORC1 signalling. Moreover, we show that the suppressive effects of mTORC1 activation and ageing on PPARalpha activity and ketone production are not additive, and that mTORC1 inhibition is sufficient to prevent the ageing-induced defect in ketogenesis. Thus, our findings reveal that mTORC1 is a key regulator of PPARalpha function and hepatic ketogenesis and suggest a role for mTORC1 activity in promoting the ageing of the liver. Full Text.

Sengupta, S., Peterson, T.R., and Sabatini, D.M. Regulation of the Mtor Complex 1 Pathway by Nutrients, Growth Factors, and Stress. Mol Cell 40, 310-322. The large serine/threonine protein kinase mTOR regulates cellular and organismal homeostasis by coordinating anabolic and catabolic processes with nutrient, energy, and oxygen availability and growth factor signaling. Cells and organisms experience a wide variety of insults that perturb the homeostatic systems governed by mTOR and therefore require appropriate stress responses to allow cells to continue to function. Stress can manifest from an excess or lack of upstream signals or as a result of genetic perturbations in upstream effectors of the pathway. mTOR nucleates two large protein complexes that are important nodes in the pathways that help buffer cells from stresses, and are implicated in the progression of stress-associated phenotypes and diseases, such as aging, tumorigenesis, and diabetes. This review focuses on the key components of the mTOR complex 1 pathway and on how various stresses impinge upon them. Full Text.

Serwold, T., Hochedlinger, K., Swindle, J., Hedgpeth, J., Jaenisch, R., and Weissman, I.L. (2010). T-Cell Receptor-Driven Lymphomagenesis in Mice Derived from a Reprogrammed T Cell. Proceedings of the National Academy of Sciences of the United States of America 107, 18939-18943. The conversion of mature somatic cells into pluripotent stem cells, both by nuclear transfer and transduction with specific "reprogramming" genes, represents a major advance in regenerative medicine. Pluripotent stem cell lines can now be generated from an individual's own cells, facilitating the generation of immunologically acceptable stem cell-based therapeutics. Many cell types can undergo nuclear reprogramming, leading to the question of whether the identity of the reprogrammed cell of origin has a biological consequence. Peripheral blood, containing a mixture of T, B, NK, and myeloid cell types, represents one potential source of reprogrammable cells. In this study, we describe the unique case of mice derived from a reprogrammed T cell. These mice have prerearranged T-cell receptor (TCR) genes in all cells. Surprisingly, approximate to 50% of mice with prerearranged TCR genes develop spontaneous T cell lymphomas, which originate in the thymus. The lymphomas arise from developing T cells, and contain activated Notch1, similar to most human and mouse T-cell acute lymphoblastic lymphomas. Furthermore, lymphomagenesis requires the expression of both prerearranged TCR alpha and TCR beta genes, indicating a critical role for TCR signaling. Furthermore, inhibitors of multiple branches of TCR signaling suppress lymphoma growth, implicating TCR signaling as an essential component in lymphoma proliferation. The lymphomagenesis in mice derived from a reprogrammed T cell demonstrates the deleterious consequences of misregulation of the TCR rearrangement and signaling pathways and illustrates one case of cellular reprogramming where the identity of the cell of origin has profound consequences. Full Text.

Shibue, T., and Weinberg, R.A. . Metastatic Colonization: Settlement, Adaptation and Propagation of Tumor Cells in a Foreign Tissue Environment. Seminars in Cancer Biology .Dec 8. [Epub ahead of print] Disseminated tumor cells must negotiate multiple situations that challenge their viability and/or proliferative capacity before they can successfully colonize distant organ sites. Thus, the shear stress caused by the blood flow may physically damage tumor cells during their translocation from primary tumors to distant organs via the circulation. In addition, the tissue microenvironment of distant organs is generally unfamiliar to tumor cells, limiting their proliferation within the parenchyma of these organs. Each of these situations involves various types of interactions between tumor cells and host components, which either support or inhibit the establishment and subsequent progression of metastases. The initial formation of micrometastases, as well as their subsequent growth-often termed colonization-therefore require complex adaptations by tumor cells to various host components, most of which are never encountered by these cells during their growth within primary tumor sites. These difficulties explain why the colonization of distant organs by disseminated tumor cells is an extraordinarily demanding task and thus inefficient, and suggests a number of potential targets that might be used in the future to interfere therapeutically with this process. Studying the details of tumor-host interactions at each of the steps leading up to successful metastatic colonization may therefore pave the way for designing therapeutic strategies to counteract the metastatic spread of malignant tumors. Full Text

Shin, C., Nam, J.W., Farh, K.K., Chiang, H.R., Shkumatava, A., and Bartel, D.P. Expanding the Microrna Targeting Code: Functional Sites with Centered Pairing. Mol Cell 38, 789-802. Most metazoan microRNA (miRNA) target sites have perfect pairing to the seed region, located near the miRNA 5' end. Although pairing to the 3' region sometimes supplements seed matches or compensates for mismatches, pairing to the central region has been known to function only at rare sites that impart Argonaute-catalyzed mRNA cleavage. Here, we present "centered sites," a class of miRNA target sites that lack both perfect seed pairing and 3'-compensatory pairing and instead have 11-12 contiguous Watson-Crick pairs to the center of the miRNA. Although centered sites can impart mRNA cleavage in vitro (in elevated Mg(2+)), in cells they repress protein output without consequential Argonaute-catalyzed cleavage. Our study also identified extensively paired sites that are cleavage substrates in cultured cells and human brain. This expanded repertoire of cleavage targets and the identification of the centered site type help explain why central regions of many miRNAs are evolutionarily conserved. Full Text.

SimpsonHolley, M., Kedersha, N., Dower, K., Rubins, K.H., Anderson, P., Hensley, L.E., and Connor, J.H. Formation of Antiviral Cytoplasmic Granules During Orthopoxvirus Infection. J Virology Dec 8. [Epub ahead of print] Vaccinia virus mutants lacking the dsRNA-binding E3L protein (DeltaE3L VV) show restricted replication in most cell types, as dsRNA produced by vaccinia activates PKR, leading to eIF2alpha phosphorylation and impaired translation initiation. Here we show that cells infected with DeltaE3L VV assemble cytoplasmic granular structures which surround the vaccinia replication factories at an early stage of the nonproductive infection. These structures contain the stress granule-associated proteins G3BP, TIA-1, and USP10 as well as PolyA-containing RNA. These structures lack large ribosomal subunit proteins, suggesting that they are translationally inactive. Formation of these punctuate structures correlates with restricted replication, as they occur in >80% of cells infected with DeltaE3L VV, but in only 10% of cells infected with wild-type VV. We therefore refer to these structures as antiviral granules (AVGs). Formation of AVGs requires PKR and phosphorylated eIF2alpha, as MEFs lacking PKR displayed reduced granule formation and MEFs lacking phosphorylatable eIF2alpha showed no granule formation. In both cases, these decreased levels of AVG formation correlated with increased DeltaE3L VV replication. Surprisingly, MEFs lacking the AVG component protein TIA-1 supported increased replication of DeltaE3L VV, despite increased eIF2alpha phosphorylation and the assembly of AVGs that lacked TIA-1. These data indicate that the effective PKR mediated restriction of DeltaE3L VV replication requires AVG formation subsequent to eIF2alpha phosphorylation. This is a novel finding that supports the hypothesis that the formation of subcellular protein aggregates is an intentional and important component of the successful cellular antiviral response. PDF

Sindelka, R., Sidova, M., Svec, D., and Kubista, M. Spatial Expression Profiles in the Xenopus Laevis Oocytes Measured with Qpcr-Tomography. Methods Jan 5 [Epub ahead of print] qPCR tomography was developed to study mRNA localization in complex biological samples that are embedded and cryo-sectioned. After total RNA extraction and reverse transcription, the spatial profiles of mRNAs and other functional RNAs were determined by qPCR. The Xenopus laevis oocyte was selected as model, because of its large size (more than 1 mm) and large amount of total RNA ( approximately 5 ug). Fifteen sections along the animal-vegetal axis were cut and prepared for quantification of 31 RNA targets using the high-throughput real-time RT-PCR (qPCR) BioMark platform. mRNAs were found to have two localization patterns, animal/central or vegetal. Because of the high resolution in sectioning, it was possible to distinguish two subgroups of the vegetal gene patterns: germ plasm determinant pattern and profile of other vegetal genes. Full Text

Staerk, J., Dawlaty, M.M., Gao, Q., Maetzel, D., Hanna, J., Sommer, C.A., Mostoslavsky, G., and Jaenisch, R. Reprogramming of Human Peripheral Blood Cells to Induced Pluripotent Stem Cells. Cell Stem Cell 7, 20-24.Our study demonstrates that peripheral blood can be utilized as an easily accessible source of patient tissue for reprogramming without the need to extensively maintain cell cultures prior to reprogramming experiments. PDF

Su, L.H.J., Auluck, P.K., Outeiro, T.F., Yeger-Lotem, E., Kritzer, J.A., Tardiff, D.F., Strathearn, K.E., Liu, F., Cao, S.S., Hamamichi, S., Bell GW and Susan Lindquist. Compounds from an Unbiased Chemical Screen Reverse Both Er-to-Golgi Trafficking Defects and Mitochondrial Dysfunction in Parkinson's Disease Models. Disease Models & Mechanisms 3, 194-208. alpha-Synuclein (alpha-syn) is a small lipid-binding protein involved in vesicle trafficking whose function is poorly characterized. It is of great interest to human biology and medicine because alpha-syn dysfunction is associated with several neurodegenerative disorders, including Parkinson's disease (PD). We previously created a yeast model of alpha-syn pathobiology, which established vesicle trafficking as a process that is particularly sensitive to alpha-syn expression. We also uncovered a core group of proteins with diverse activities related to alpha-syn toxicity that is conserved from yeast to mammalian neurons. Here, we report that a yeast strain expressing a somewhat higher level of alpha-syn also exhibits strong defects in mitochondria! function. Unlike our previous strain, genetic suppression of endoplasmic reticulum (ER)-to-Golgi trafficking alone does not suppress alpha-syn toxicity in this strain. In an effort to identify individual compounds that could simultaneously rescue these apparently disparate pathological effects of alpha-syn, we screened a library of 115,000 compounds. We identified a class of small molecules that reduced alpha-syn toxicity at micromolar concentrations in this higher toxicity strain. These compounds reduced the formation of alpha-syn foci, re-established ER-to-Golgi trafficking and ameliorated alpha-syn-mediated damage to mitochondria. They also corrected the toxicity of alpha-syn in nematode neurons and in primary rat neuronal midbrain cultures. Remarkably, the compounds also protected neurons against rotenone-induced toxicity, which has been used to model the mitochondrial defects associated with PD in humans. That single compounds are capable of rescuing the diverse toxicities of alpha-syn in yeast and neurons suggests that they are acting on deeply rooted biological processes that connect these toxicities and have been conserved for a billion years of eukaryotic evolution. Thus, it seems possible to develop novel therapeutic strategies to simultaneously target the multiple pathological features of PD. Full Text.

Sun, J.S., Hartvigsen, K., Chou, M.Y., Zhang, Y.D., Sukhova, G.K., Zhang, J., Lopez-Ilasaca, M., Diehl, C.J., Yakov, N., Harats, D., Jacob George, Joseph L. Witztum, Peter Libby, Hidde Ploegh, and Guo-Ping Shi. Deficiency of Antigen-Presenting Cell Invariant Chain Reduces Atherosclerosis in Mice. Circulation 122, 808-U121. Background-Adaptive immunity and innate immunity play important roles in atherogenesis. Invariant chain (CD74) mediates antigen-presenting cell antigen presentation and T-cell activation. This study tested the hypothesis that CD74-deficient mice have reduced numbers of active T cells and resist atherogenesis. Methods and Results-In low-density lipoprotein receptor-deficient (Ldlr(-/-)) mice, CD74 deficiency (Ldlr(-/-)Cd74(-/-)) significantly reduced atherosclerosis and CD25(+)-activated T cells in the atheromata. Although Ldlr(-/-)Cd74(-/-) mice had decreased levels of plasma immunoglobulin (Ig) G1, IgG2b, and IgG2c against malondialdehyde-modified LDL (MDA-LDL), presumably as a result of impaired antigen-presenting cell function, Ldlr(-/-)Cd74(-/-) mice showed higher levels of anti-MDA-LDL IgM and IgG3. After immunization with MDA-LDL, Ldlr(-/-)Cd74(-/-) mice had lower levels of all anti-MDA-LDL Ig isotypes compared with Ldlr(-/-) mice. As anticipated, only Ldlr(-/-) splenocytes responded to in vitro stimulation with MDA-LDL, producing Th1/Th2 cytokines. Heat shock protein-65 immunization enhanced atherogenesis in Ldlr(-/-) mice, but Ldlr(-/-) Cd74(-/-) mice remained protected. Compared with Ldlr(-/-) mice, Ldlr(-/-)Cd74(-/-) mice had higher anti-MDALDL autoantibody titers, fewer lesion CD25(+)-activated T cells, impaired release of Th1/Th2 cytokines from antigen-presenting cells after heat shock protein-65 stimulation, and reduced levels of all plasma anti-heat shock protein-65 Ig isotypes. Cytofluorimetry of splenocytes and peritoneal cavity cells of MDA-LDL- or heat shock protein-65-immunized mice showed increased percentages of autoantibody-producing marginal zone B and B-1 cells in Ldlr(-/-)Cd74(-/-) mice compared with Ldlr(-/-) mice. Conclusions-Invariant chain deficiency in Ldlr(-/-) mice reduced atherosclerosis. This finding was associated with an impaired adaptive immune response to disease-specific antigens. Concomitantly, an unexpected increase in the number of innate-like peripheral B-1 cell populations occurred, resulting in increased IgM/IgG3 titers to the oxidation-specific epitopes. Full Text.

Sun, Y., and Lodish, H.F. Adiponectin Deficiency Promotes Tumor Growth in Mice by Reducing Macrophage Infiltration. PLoS One 2010 Aug 5;5(8). pii: e11987. Adiponectin is an adipocyte-derived plasma protein that has been implicated in regulating angiogenesis, but the role of adiponectin in regulating this process is still controversial. In this study, in order to determine whether adiponectin affects tumor growth and tumor induced vascularization, we implanted B16F10 melanoma and Lewis Lung Carcinoma cells subcutaneously into adiponectin knockout and wild-type control mice, and found that adiponectin deficiency markedly promoted the growth of both tumors. Immunohistochemical analyses indicated that adiponectin deficiency reduced macrophage recruitment to the tumor, but did not affect cancer cell mitosis, apoptosis, or tumor-associated angiogenesis. In addition, treatment with recombinant adiponectin did not affect the proliferation of cultured B16F10 tumor cells. Importantly, the restoration of microphage infiltration at an early stage of tumorigenesis by means of co-injection of B16F10 cells and macrophages reversed the increased tumor growth in adiponectin knockout mice. Thus, we conclude that the enhanced tumor growth observed in adiponectin deficient mice is likely due to the reduction of macrophage infiltration rather than enhanced angiogenesis. Full Text.

Taipale, M., Jarosz, D.F., and Lindquist, S. Hsp90 at the Hub of Protein Homeostasis: Emerging Mechanistic Insights. Nature Reviews Molecular Cell Biology .Jun 9. [Epub ahead of print] Heat shock protein 90 (HSP90) is a highly conserved molecular chaperone that facilitates the maturation of a wide range of proteins (known as clients). Clients are enriched in signal transducers, including kinases and transcription factors. Therefore, HSP90 regulates diverse cellular functions and exerts marked effects on normal biology, disease and evolutionary processes. Recent structural and functional analyses have provided new insights on the transcriptional and biochemical regulation of HSP90 and the structural dynamics it uses to act on a diverse client repertoire. Comprehensive understanding of how HSP90 functions promises not only to provide new avenues for therapeutic intervention, but to shed light on fundamental biological questions. Full Text.

Taube, J.H., Herschkowitz, J.I., Komurov, K., Zhou, A.Y., Gupta, S., Yang, J., Hartwell, K., Onder, T.T., Gupta, P.B., Evans, K.W., Hollier BG, Ram PT, Lander ES, Rosen JM, Weinberg RA, Mani SA Core Epithelial-to-Mesenchymal Transition Interactome Gene-Expression Signature Is Associated with Claudin-Low and Metaplastic Breast Cancer Subtypes. Proceedings of the National Academy of Sciences of the United States of America 107, 15449-15454. The epithelial-to-mesenchymal transition (EMT) produces cancer cells that are invasive, migratory, and exhibit stem cell characteristics, hallmarks of cells that have the potential to generate metastases. Inducers of the EMT include several transcription factors (TFs), such as Goosecoid, Snail, and Twist, as well as the secreted TGF-beta 1. Each of these factors is capable, on its own, of inducing an EMT in the human mammary epithelial (HMLE) cell line. However, the interactions between these regulators are poorly understood. Overexpression of each of the above EMT inducers up-regulates a subset of other EMT-inducing TFs, with Twist, Zeb1, Zeb2, TGF-beta 1, and FOXC2 being commonly induced. Up-regulation of Slug and FOXC2 by either Snail or Twist does not depend on TGF-beta 1 signaling. Gene expression signatures (GESs) derived by overexpressing EMT-inducing TFs reveal that the Twist GES and Snail GES are the most similar, although the Goosecoid GES is the least similar to the others. An EMT core signature was derived from the changes in gene expression shared by up-regulation of Gsc, Snail, Twist, and TGF-beta 1 and by down-regulation of E-cadherin, loss of which can also trigger an EMT in certain cell types. The EMT core signature associates closely with the claudin-low and metaplastic breast cancer subtypes and correlates negatively with pathological complete response. Additionally, the expression level of FOXC1, another EMT inducer, correlates strongly with poor survival of breast cancer patients. Full Text.

Tyedmers, J., Treusch, S., Dong, J., McCaffery, J.M., Bevis, B., and Lindquist, S. Prion Induction Involves an Ancient System for the Sequestration of Aggregated Proteins and Heritable Changes in Prion Fragmentation. Proc Natl Acad Sci U S A. Published online before print April 26, 2010 When the translation termination factor Sup35 adopts the prion state, [PSI (+)], the read-through of stop codons increases, uncovering hidden genetic variation and giving rise to new, often beneficial, phenotypes. Evidence suggests that prion induction involves a process of maturation, but this has never been studied in detail. To do so, we used a visually tractable prion model consisting of the Sup35 prion domain fused to GFP (PrD-GFP) and overexpressed it to achieve induction in many cells simultaneously. PrD-GFP first assembled into Rings as previously described. Rings propagated for many generations before the protein transitioned into a Dot structure. Dots transmitted the [PSI (+)] phenotype through mating and meiosis, but Rings did not. Surprisingly, the underlying amyloid conformation of PrD-GFP was identical in Rings and Dots. However, by electron microscopy, Rings consisted of very long uninterrupted bundles of fibers, whereas Dot fibers were highly fragmented. Both forms were deposited at the IPOD, a biologically ancient compartment for the deposition of irreversibly aggregated proteins that we propose is the site of de novo prion induction. We find that oxidatively damaged proteins are also localized there, helping to explain how proteotoxic stresses increase the rate of prion induction. Curing PrD-GFP prions, by inhibiting Hsp104's fragmentation activity, reversed the induction process: Dot cells produced Rings before PrD-GFP reverted to the soluble state. Thus, formation of the genetically transmissible prion state is a two-step process that involves an ancient system for the asymmetric inheritance of damaged proteins and heritable changes in the extent of prion fragmentation. PDF

Vader, G., and Lens, S.M. Chromosome Segregation: Taking the Passenger Seat. Curr Biol 20, R879-881. The chromosomal passenger complex (CPC) is a major regulator of mitotic and meiotic chromosome segregation. Three recent papers now elucidate the mechanisms that determine the localization of the CPC to the inner centromere. Full Text.

Valastyan, S., and Weinberg, R.A. (2010). Metastasis Suppression: A Role of the Dice(R). Genome Biol 11, 141. Recent studies have implicated the microRNA biogenesis enzyme Dicer as a suppressor of breast carcinoma metastasis and elucidated upstream signaling pathways that control Dicer levels. Full Text.

Valastyan, S., Chang, A., Benaich, N., Reinhardt, F., and Weinberg, R.A. Concurrent Suppression of Integrin {Alpha}5, Radixin, and Rhoa Phenocopies the Effects of Mir-31 on Metastasis. Cancer Research Jun 8. [Epub ahead of print]. miR-31 inhibits breast cancer metastasis via the pleiotropic suppression of a cohort of prometastatic target genes that include integrin alpha(5) (ITGA5), radixin (RDX), and RhoA. We previously showed that the concomitant overexpression of ITGA5, RDX, and RhoA was capable of overriding the antimetastatic effects of ectopically expressed miR-31 in vivo. However, these prior studies failed to investigate whether the combined suppression of the endogenous mRNAs encoding these three proteins recapitulated the in vivo consequences of miR-31 expression on metastasis. We show here that short hairpin RNA-mediated concurrent downregulation of ITGA5, RDX, and RhoA is sufficient to phenocopy the full spectrum of described influences of miR-31 on metastasis in vivo, including the effects of this microRNA (miRNA) on local invasion, early post-intravasation events, and metastatic colonization. These findings provide mechanistic insights into the metastatic process and have implications about the importance of pleiotropy for the biological actions of miRNAs. Full Text.

Valastyan, S., and Weinberg, R.A. Mir-31: A Crucial Overseer of Tumor Metastasis and Other Emerging Roles. Cell Cycle Jun 17;9(11). [Epub ahead of print]. MicroRNAs constitute a family of pleiotropically acting short regulatory RNAs. Increasingly, specific microRNAs have been implicated as key modulators of a variety of normal physiologic processes; moreover, the aberrant activity of certain microRNAs has been linked to the pathogenesis of multiple diseases. The microRNA miR-31 has been identified as a crucial overseer of several normal and diseased phenotypes. Here, we describe current knowledge regarding the functions of miR-31, with an emphasis placed upon the role of this microRNA in neoplastic development and tumor metastasis. Additionally, we highlight a number of recent reports concerning the contributions of miR-31 to other pathological states, the role of this microRNA in normal physiology, and the upstream mechanisms by which miR-31 expression levels are regulated. Assessed collectively, existing evidence suggests that miR-31 concomitantly regulates a number of essential signaling pathways in mammalian cells. For these reasons, further elucidation of the biological actions of miR-31 may prove significant for the prognosis and remediation of various pathological states. Full Text.

vanVugt, M., Gardino, A.K., Linding, R., Ostheimer, G.J., Reinhardt, H.C., Ong, S.E., Tan, C.S., Miao, H., Keezer, S.M., Li, J.J., Pawson T, Lewis TA, Carr SA, Smerdon SJ, Brummelkamp TR, Yaffe MB. A Mitotic Phosphorylation Feedback Network Connects Cdk1, Plk1, 53bp1, and Chk2 to Inactivate the G2/M DNA Damage Checkpoint. PLoS Biol 8(1): e1000287. DNA damage checkpoints arrest cell cycle progression to facilitate DNA repair. The ability to survive genotoxic insults depends not only on the initiation of cell cycle checkpoints but also on checkpoint maintenance. While activation of DNA damage checkpoints has been studied extensively, molecular mechanisms involved in sustaining and ultimately inactivating cell cycle checkpoints are largely unknown. Here, we explored feedback mechanisms that control the maintenance and termination of checkpoint function by computationally identifying an evolutionary conserved mitotic phosphorylation network within the DNA damage response. We demonstrate that the non-enzymatic checkpoint adaptor protein 53BP1 is an in vivo target of the cell cycle kinases Cyclin-dependent kinase-1 and Polo-like kinase-1 (Plk1). We show that Plk1 binds 53BP1 during mitosis and that this interaction is required for proper inactivation of the DNA damage checkpoint. 53BP1 mutants that are unable to bind Plk1 fail to restart the cell cycle after ionizing radiation-mediated cell cycle arrest. Importantly, we show that Plk1 also phosphorylates the 53BP1-binding checkpoint kinase Chk2 to inactivate its FHA domain and inhibit its kinase activity in mammalian cells. Thus, a mitotic kinase-mediated negative feedback loop regulates the ATM-Chk2 branch of the DNA damage signaling network by phosphorylating conserved sites in 53BP1 and Chk2 to inactivate checkpoint signaling and control checkpoint duration. Full Text.

Weinberg, R. Point: Hypotheses First. Nature 464, 678. There is little to show for all the time and money invested in genomic studies of cancer and the approach is undermining tried-and-tested ways of doing, and of building, science. Full Text.

Welburn, J.P., Vleugel, M., Liu, D., Yates, J.R., 3rd, Lampson, M.A., Fukagawa, T., and Cheeseman, I.M. Aurora B Phosphorylates Spatially Distinct Targets to Differentially Regulate the Kinetochore-Microtubule Interface. Mol Cell 38, 383-392. Accurate chromosome segregation requires carefully regulated interactions between kinetochores and microtubules, but how plasticity is achieved to correct diverse attachment defects remains unclear. Here we demonstrate that Aurora B kinase phosphorylates three spatially distinct targets within the conserved outer kinetochore KNL1/Mis12 complex/Ndc80 complex (KMN) network, the key player in kinetochore-microtubule attachments. The combinatorial phosphorylation of the KMN network generates graded levels of microtubule-binding activity, with full phosphorylation severely compromising microtubule binding. Altering the phosphorylation state of each protein causes corresponding chromosome segregation defects. Importantly, the spatial distribution of these targets along the kinetochore axis leads to their differential phosphorylation in response to changes in tension and attachment state. In total, rather than generating exclusively binary changes in microtubule binding, our results suggest a mechanism for the tension-dependent fine-tuning of kinetochore-microtubule interactions. Full Text.

Wenemoser, D., and Reddien, P.W. Planarian Regeneration Involves Distinct Stem Cell Responses to Wounds and Tissue Absence. Developmental Biologyl. Jun 19. [Epub ahead of print] Regeneration requires signaling from a wound site for detection of the wound, and a mechanism that determines the nature of the injury to specify the appropriate regenerative response. Wound signals and tissue responses to wounds that elicit regeneration remain poorly understood. Planarians are able to regenerate from essentially any type of injury and present a novel system for the study of wound responses in regeneration initiation. Newly developed molecular and cellular tools now enable study of regeneration initiation using the planarian Schmidtea mediterranea. Planarian regeneration requires adult stem cells called neoblasts and amputation triggers two peaks in neoblast mitoses early in regeneration. We demonstrate that the first mitotic peak is a body-wide response to any injury and that a second, local, neoblast response is induced only when injury results in missing tissue. This second response was characterized by recruitment of neoblasts to wounds, even in areas that lack neoblasts in the intact animal. Subsequently, these neoblasts were induced to divide and differentiate near the wound, leading to formation of new tissue. We conclude that there exist two functionally distinct signaling phases of the stem cell wound response that distinguish between simple injury and situations that require the regeneration of missing tissue. Full Text.

Wiggins, P.A., Cheveralls, K.C., Martin, J.S., Lintner, R., and Kondev, J. Strong Intranucleoid Interactions Organize the Escherichia Coli Chromosome into a Nucleoid Filament. Proc Natl Acad Sci U S A.Mar 1. [Epub ahead of print] The stochasticity of chromosome organization was investigated by fluorescently labeling genetic loci in live Escherichia coli cells. In spite of the common assumption that the chromosome is well modeled by an unstructured polymer, measurements of the locus distributions reveal that the E. coli chromosome is precisely organized into a nucleoid filament with a linear order. Loci in the body of the nucleoid show a precision of positioning within the cell of better than 10% of the cell length. The precision of interlocus distance of genomically-proximate loci was better than 4% of the cell length. The measured dependence of the precision of interlocus distance on genomic distance singles out intranucleoid interactions as the mechanism responsible for chromosome organization. From the magnitude of the variance, we infer the existence of an as-yet uncharacterized higher-order DNA organization in bacteria. We demonstrate that both the stochastic and average structure of the nucleoid is captured by a fluctuating elastic filament model. PDF

Wilson, D.C., Grotenbreg, G.M., Liu, K.N., Zhao, Y.L., Frickel, E.M., Gubbels, M.J., Ploegh, H.L., and Yap, G.S. Differential Regulation of Effector- and Central-Memory Responses to Toxoplasma Gondii Infection by Il-12 Revealed by Tracking of Tgd057-Specific Cd8+T Cells. Plos Pathogens 6(3): e1000815 Production of the pro-inflammatory cytokine IL-12 by innate phagocytes drives the differentiation of IFN-gamma-producing effector T cells during Toxoplasma gondii infection. However, the role of IL-12 in the regulation of memory CD8+ T cell differentiation and function during murine toxoplasmosis is unclear. To track memory CTL development, we identified a novel H-2K(b)-restricted CTL population specific for the Toxoplasma antigen tgd057. Tgd057-specific CTLs were induced by both vaccination and natural peroral infection, and were representative of the polyclonal CTL population. Tgd057-specific primary effector cells required IL-12 for the differentiation of KLRG1+ effector subpopulations and IFN-gamma production in response to restimulation with parasite-infected cells, but not to restimulation with cognate peptide. The effect of IL-12 deficiency during the primary response was profoundly imprinted on memory CTLs, which continued to show defects in cell numbers, KLRG1+ effector memory subpopulation differentiation, and IFN-gamma recall responses. Importantly, isolated CD62L hi KLRG1- CD8+ T cells differentiated in the absence of IL-12 were enhanced in their ability to generate IFN-gamma-producing secondary tgd057-specific effector cells. Our data, for the first time, demonstrate the negative impact of IL-12 signaling on the quality of the central memory CTL compartment. Thus, despite the beneficial role of IL-12 in promoting effector differentiation, excessive exposure to IL-12 during CTL priming may limit the development of long-term protective immunity through the decreased fitness of central memory CTL responses. Full Text.

Wolf, J.J., Dowell, R.D., Mahony, S., Rabani, M., Gifford, D.K., and Fink, G.R. Feed-Forward Regulation of a Cell Fate Determinant by an Rna-Binding Protein Generates Asymmetry in Yeast. Genetics.Apr 9. [Epub ahead of print] Saccharomyces cerevisiae can divide asymmetrically so that the mother and daughter cells have different fates. We show that the RNA-binding protein Khd1 regulates asymmetric expression of FLO11 to determine daughter cell fate during filamentous growth. Khd1 represses transcription of FLO11 indirectly through its regulation of ASH1 mRNA. Khd1 also represses FLO11 through a post-transcriptional mechanism independent of ASH1. Cross-linking immunoprecipitation (CLIP) coupled with high-throughput sequencing shows that Khd1 directly binds repetitive sequences in FLO11 mRNA. Khd1 inhibits translation through this interaction, establishing feed-forward repression of FLO11. This regulation enables changes in FLO11 expression between mother and daughter cells, which establishes the asymmetry required for the developmental transition between yeast form and filamentous growth.PDF

Wong, P., Iwasaki, M., Somervaille, T.C., Ficara, F., Carico, C., Arnold, C., Chen, C.Z., and Cleary, M.L. The Mir-17-92 Microrna Polycistron Regulates Mll Leukemia Stem Cell Potential by Modulating P21 Expression. Cancer Research Apr 20. [Epub ahead of print]. Despite advances in defining the critical molecular determinants for leukemia stem cell (LSC) generation and maintenance, little is known about the roles of microRNAs in LSC biology. Here, we identify microRNAs that are differentially expressed in LSC-enriched cell fractions (c-kit(+)) in a mouse model of MLL leukemia. Members of the miR-17 family were notably more abundant in LSCs compared with their normal counterpart granulocyte-macrophage progenitors and myeloblast precursors. Expression of miR-17 family microRNAs was substantially reduced concomitant with leukemia cell differentiation and loss of self-renewal, whereas forced expression of a polycistron construct encoding miR-17-19b miRNAs significantly shortened the latency for MLL leukemia development. Leukemias expressing increased levels of the miR-17-19b construct displayed a higher frequency of LSCs, more stringent block of differentiation, and enhanced proliferation associated with reduced expression of p21, a cyclin-dependent kinase inhibitor previously implicated as a direct target of miR-17 microRNAs. Knockdown of p21 in MLL-transformed cells phenocopied the overexpression of the miR-17 polycistron, including a significant decrease in leukemia latency, validating p21 as a biologically relevant and direct in vivo target of the miR-17 polycistron in MLL leukemia. Expression of c-myc, a crucial upstream regulator of the miR-17 polycistron, correlated with miR-17-92 levels, enhanced self-renewal, and LSC potential. Thus, microRNAs quantitatively regulate LSC self-renewal in MLL-associated leukemia in part by modulating the expression of p21, a known regulator of normal stem cell function. Full Text.

Xi, J., Khalil, M., Shishechian, N., Hannes, T., Pfannkuche, K., Liang, H., Fatima, A., Haustein, M., Suhr, F., Bloch, W., Reppel M, Saric T, Wernig M, Jaenisch R, Brockmeier K, Hescheler J, and Pillekamp F. Comparison of Contractile Behavior of Native Murine Ventricular Tissue and Cardiomyocytes Derived from Embryonic or Induced Pluripotent Stem Cells. FASEB Journal (published ahead of print 6 April). Cardiomyocytes generated from embryonic stem cells (ESCs) and induced pluripotent stem (iPS) cells are suggested for repopulation of destroyed myocardium. Because contractile properties are crucial for functional regeneration, we compared cardiomyocytes differentiated from ES cells (ESC-CMs) and iPS cells (iPS-CMs). Native myocardium served as control. Murine ESCs or iPS cells were differentiated 11 d in vitro and cocultured 5-7 d with irreversibly injured myocardial tissue slices. Vital embryonic ventricular tissue slices of similar age served for comparison. Force-frequency relationship (FFR), effects of Ca(2+), Ni(2+), nifedipine, ryanodine, beta-adrenergic, and muscarinic modulation were studied during loaded contractions. FFR was negative for ESC-CMs and iPS-CMs. FFR was positive for embryonic tissue and turned negative after treatment with ryanodine. In all groups, force of contraction and relaxation time increased with the concentration of Ca(2+) and decreased with nifedipine. Force was reduced by Ni(2+). Isoproterenol (1 muM) increased the force most pronounced in embryonic tissue (207+/-31%, n=7; ESC-CMs: 123+/-5%, n=4; iPS-CMs: 120+/-4%, n=8). EC50 values were similar. Contractile properties of iPS-CMs and ESC-CMs were similar, but they were significantly different from ventricular tissue of comparable age. The results indicate immaturity of the sarcoplasmic reticulum and the beta-adrenergic response of iPS-CMs and ESC-CMs.-Xi, J., Khalil, M., Shishechian, N., Hannes, T., Pfannkuche, K., Liang, H., Fatima, A., Haustein, M., Suhr, F., Bloch, W., Reppel, M., Saric, T., Wernig, M., Jaenisch, R., Brockmeier, K., Hescheler, J., Pillekamp, F. Comparison of contractile behavior of native murine ventricular tissue and cardiomyocytes derived from embryonic or induced pluripotent stem cells. PDF.

YangIott, K.S., Carpenter, A.C., Rowh, M.A.W., Steinel, N., Brady, B.L., Hochedlinger, K., Jaenisch, R., and Bassing, C.H. Tcr Beta Feedback Signals Inhibit the Coupling of Recombinationally Accessible V Beta 14 Segments with Dj Beta Complexes. Journal of Immunology 184, 1369-1378. Ag receptor allelic exclusion is thought to occur through monoallelic initiation and subsequent feedback inhibition of recombinational accessibility. However, our previous analysis of mice containing a V(D)J recombination reporter inserted into V beta 14 (V beta 14(Rep)) indicated that V beta 14 chromatin accessibility is biallelic. To determine whether V beta 14 recombinational accessibility is subject to feedback inhibition, we analyzed TCR beta rearrangements in V beta 14(Rep) mice containing a preassembled in-frame transgenic V beta 8.2D beta 1J beta 1.1 or an endogenous V beta 14D beta 1J beta 1.4 rearrangement on the homologous chromosome. Expression of either preassembled V beta DJ beta C beta-chain accelerated thymocyte development because of enhanced cellular selection, demonstrating that the rate-limiting step in early alpha beta T cell development is the assembly of an in-frame V beta DJ beta rearrangement. Expression of these preassembled V beta DJ beta rearrangements inhibited endogenous V beta 14-to-DJ beta rearrangements as expected. However, in contrast to results predicted by the accepted model of TCR beta feedback inhibition, we found that expression of these preassembled TCR beta-chains did not down-regulate recombinational accessibility of V beta 14 chromatin. Our findings suggest that TCR beta-mediated feedback inhibition of V beta 14 rearrangements depends on inherent properties of V beta 14, D beta, and J beta recombination signal sequences. Full Text.

Yip, C.K., Murata, K., Walz, T., Sabatini, D.M., and Kang, S.A. Structure of the Human Mtor Complex I and Its Implications for Rapamycin Inhibition. Molecular Cell 38, 768-774. The mammalian target of rapamycin complex 1 (mTORC1) regulates cell growth in response to the nutrient and energy status of the cell, and its deregulation is common in human cancers. Little is known about the overall architecture and subunit organization of this essential signaling complex. We have determined the three-dimensional (3D) structure of the fully assembled human mTORC1 by cryo-electron microscopy (cryo-EM). Our analyses reveal that mTORC1 is an obligate dimer with an overall rhomboid shape and a central cavity. The dimeric interfaces are formed by interlocking interactions between the mTOR and raptor subunits. Extended incubation with FKBP12-rapamycin compromises the structural integrity of mTORC1 in a stepwise manner, leading us to propose a model in which rapamycin inhibits mTORC1-mediated phosphorylation of 4E-BP1 and S6K1 through different mechanisms. Full Text.

Zhang, J., Lee, E.Y., Liu, Y., Berman, S.D., Lodish, H.F., and Lees, J.A. Prb and E2f4 Play Distinct Cell-Intrinsic Roles in Fetal Erythropoiesis. Cell Cycle 9(2). [Epub ahead of print]. The retinoblastoma tumor suppressor protein pRB functions, at least in part, by directly binding to and modulating the activity of the E2F transcription factors. Previous studies have shown that both E2F4 and pRB play important roles in fetal erythropoiesis. Given that these two proteins interact directly we investigated the overlap of E2F4 and pRB function in this process by analyzing E2f4(-/-), conditional Rb knockout (Rb(1lox/1lox)), and compound E2f4(-/-);Rb(1lox/1lox) embryos. At E15.5 E2f4(-/-) and Rb(1lox/1lox) fetal erythroid cells display distinct abnormalities in their differentiation profiles. When cultured in vitro, both E2f4(-/-) and Rb(1lox/1lox) erythroid cells show defects in cell cycle progression. Surprisingly, analysis of cell cycle profiling suggests that E2F4 and pRB control cell cycle exit through different mechanisms. Moreover, only pRB, but not E2F4, promotes cell survival in erythroid cells. We observed an additive rather than a synergistic impact upon the erythroid defects in the compound E2f4(/);Rb(1lox/1lox) embryos. We further found that fetal liver macrophage development is largely normal regardless of genotype. Taken together, our results show that E2F4 and pRB play independent cell-intrinsic roles in fetal erythropoiesis. PDF.

Zhang, L., Flygare, J., Wong, P., Lim, B., and Lodish, H.F. (2010). Mir-191 Regulates Mouse Erythroblast Enucleation by Down-Regulating Riok3 and Mxi1. Genes and Development.Dec 31. [Epub ahead of print] Using RNA-seq technology, we found that the majority of microRNAs (miRNAs) present in CFU-E erythroid progenitors are down-regulated during terminal erythroid differentiation. Of the developmentally down-regulated miRNAs, ectopic overexpression of miR-191 blocks erythroid enucleation but has minor effects on proliferation and differentiation. We identified two erythroid-enriched and developmentally up-regulated genes, Riok3 and Mxi1, as direct targets of miR-191. Knockdown of either Riok3 or Mxi1 blocks enucleation, and either physiological overexpression of miR-191 or knockdown of Riok3 or Mxi1 blocks chromatin condensation. Thus, down-regulation of miR-191 is essential for erythroid chromatin condensation and enucleation by allowing up-regulation of Riok3 and Mxi1. PDF.

Zoncu, R., Efeyan, A., and Sabatini, D.M. (2010). Mtor: From Growth Signal Integration to Cancer, Diabetes and Ageing. Nat Rev Mol Cell Biol.Dec 15. [Epub ahead of print] In all eukaryotes, the target of rapamycin (TOR) signalling pathway couples energy and nutrient abundance to the execution of cell growth and division, owing to the ability of TOR protein kinase to simultaneously sense energy, nutrients and stress and, in metazoans, growth factors. Mammalian TOR complex 1 (mTORC1) and mTORC2 exert their actions by regulating other important kinases, such as S6 kinase (S6K) and Akt. In the past few years, a significant advance in our understanding of the regulation and functions of mTOR has revealed the crucial involvement of this signalling pathway in the onset and progression of diabetes, cancer and ageing. Full Text.

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