The following alphabetical list represents papers published in 2008 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.

-Aberrant Chromatin at Genes Encoding Stem Cell Regulators in Human Mixed-Lineage Leukemia.Guenther
-Activation and Transposition of Endogenous Retroviral Elements in Hypomethylation Induced Tumors in Mice. Howard
-Alternative Isoform Regulation in Human Tissue Transcriptomes. Wang
-Analysis of the Mouse Embryonic Stem Cell Regulatory Networks Obtained by Chip-Chip and Chip-Pet. Mathur
-Angiopoietin-Like 5 and Igfbp2 Stimulate Ex Vivo Expansion of Human Cord Blood Hematopoietic Stem Cells as Assayed by Nod/Scid Transplantation. .Zhang
-Cancer Cell Metabolism: Warburg and Beyond. Hsu
-The Cd8 T Cell Response against Murine Gamma Herpes Virus (Mhv-68) Is Directed Towards a Broad Repertoire of Epitopes from Both Early and Late Antigens. GredmarkRuss
-Cell Type-Specific DNA Methylation Patterns in the Human Breast. BloushtainQimron
-Chaperone-Dependent Amyloid Assembly Protects Cells from Prion Toxicity. Douglas
-Characterization and Classification of Zebrafish Brain Morphology Mutants. Lowery
-Cleavage of the Star Strand Facilitates Assembly of Some Micrornas into Ago2-Containing Silencing Complexes in Mammals Shin
-C-Myc Is Dispensable for Direct Reprogramming of Mouse Fibroblasts.Wernig
-Coevolution in the Tumor Microenvironment. Weinberg
-Concurrent Detection of Secreted Products from Human Lymphocytes by Microengraving: Cytokines and Antigen-Reactive Antibodies. Bradshaw
-Connecting Microrna Genes to the Core Transcriptional Regulatory Circuitry of Embryonic Stem Cells. Marson
-Continuous Elimination of Oxidized Nucleotides Is Necessary to Prevent Rapid Onset of Cellular Senescence Rai
-Crystallographic Conformers of Actin in a Biologically Active Bundle of Filaments.Cong
-Derivation and Manipulation of Murine Embryonic Stem Cells. Meissner
-Detection of Compounds That Rescue Rab1-Synuclein Toxicity. Fleming
-Differential Effects of Usp14 and Uch-L1 on the Ubiquitin Proteasome System and Synaptic Activity. Walters
-Dimerization by a Cytokine Receptor Is Necessary for Constitutive Activation of Jak2v617f.Lu
-Direct and Selective Elimination of Specific Prions and Amyloids by 4,5-Dianilinophthalimide and Analogs. Wang
-Direct Reprogramming of Terminally Differentiated Mature B Lymphocytes to Pluripotency. Hanna
-Discovery of Cd8+ T Cell Epitopes in Chlamydia Trachomatis Infection through Use of Caged Class I Mhc Tetramers. Grotenbreg
-Discovery of Quinolinediones Exhibiting a Heat Shock Response and Angiogenesis Inhibition. Hargreaves
-Divergent Transcription from Active Promoters. Seila
-The Drosophila Hairpin Rna Pathway Generates Endogenous Short Interfering Rnas Okamura
-A Drug-Inducible System for Direct Reprogramming of Human Somatic Cells to Pluripotency Hockemeyer
-A Drug-Inducible Transgenic System for Direct Reprogramming of Multiple Somatic Cell Types. Wernig
-Dynamic, Morphotype-Specific Candida Albicans Beta-Glucan Exposure During Infection and Drug Treatment. Wheeler
-Early Origins and Evolution of Micrornas and Piwi-Interacting Rnas in Animals Grimson
-Ectodomain Cleavage of the Egf Ligands Hb-Egf, Neuregulin1-{Beta}, and Tgf-{Alpha} Is Specifically Triggered by Different Stimuli and Involves Different Pkc Isoenzymes Herrlich
-Electron Microscopy of Hydrated Samples. Timp
-An Embryonic Stem Cell-Like Gene Expression Signature in Poorly Differentiated Aggressive Human Tumors.BenPorath
-Endogenous Sirna and Mirna Targets Identified by Sequencing of the Arabidopsis Degradome AddoQuaye
-Enucleation of Cultured Mouse Fetal Erythroblasts Requires Rac Gtpases and Mdia2. Ji
-Epithelial-Mesenchymal Transition: At the Crossroads of Development and Tumor Metastasis. Yang
-The Epithelial-Mesenchymal Transition Generates Cells with Properties of Stem Cells. Mani
-Filling the Mosaic of P53 Actions: P53 Represses Rhamm Expression. Godar
-Flo1 Is a Variable Green Beard Gene That Drives Biofilm-Like Cooperation in Budding Yeast. Smukalla
-Folate Deficiency Induces Genomic Uracil Misincorporation and Hypomethylation but Does Not Increase DNA Point Mutations Linhart
-Formation of the Zebrafish Midbrain-Hindbrain Boundary Constriction Requires Laminin-Dependent Basal Constriction. Gutzman
-A Functional Proteomics Approach Links the Ubiquitin-Related Modifier Urm1 to a Trna Modification Pathway. Schlieker
-Gene Nomenclature Guidelines for the Planarian Schmidtea Mediterranea Reddien
-Gene Therapy of Diamond Blackfan Anemia Cd34(+) Cells Leads to Improved Erythroid Development and Engraftment Following Transplantation Flygare
-Genetic Substitution of Cdk1 by Cdk2 Leads to Embryonic Lethality and Loss of Meiotic Function of Cdk2. Satyanarayana
-Genome-Scale DNA Methylation Maps of Pluripotent and Differentiated Cells. Meissner
-Genome-Wide Analysis of the H3k4 Histone Demethylase Rbp2 Reveals a Transcriptional Program Controlling Differentiation. LopezBigas
-Germ Cell-Intrinsic and -Extrinsic Factors Govern Meiotic Initiation in Mouse Embryos Lin
-Global Analysis of the Meiotic Crossover Landscape Chen
-Growth-Inhibitory and Tumor- Suppressive Functions of P53 Depend on Its Repression of Cd44 Expression. Godar
-Heat Shock Factor 1 Regulates Lifespan as Distinct from Disease Onset in Prion Disease Steele
-Highly Parallel Identification of Essential Genes in Cancer Cells. Luo
-Hsp90 Affects the Expression of Genetic Variation and Developmental Stability in Quantitative Traits. Sangster
-Hsp90-Buffered Genetic Variation Is Common in Arabidopsis Thaliana. Sangster
-Hsp104 Antagonizes Alpha-Synuclein Aggregation and Reduces Dopaminergic Degeneration in a Rat Model of Parkinson Disease LoBianco
-Hsp104, Hsp70 and Hsp40 Interplay Regulates Formation, Growth and Elimination of Sup35 Prions. Shorter
-H2az Is Enriched at Polycomb Complex Target Genes in Es Cells and Is Necessary for Lineage Commitment. Creyghton
-Identification and Characterization of Ctrp9, a Novel Secreted Glycoprotein, from Adipose Tissue That Reduces Serum Glucose in Mice and Forms Heterotrimers with Adiponectin. Wong
-The Impact of Micrornas on Protein Output. Baek
-Impaired Erad and Er Stress Are Early and Specific Events in Polyglutamine Toxicity. Duennwald
-Improved Polymerase Ribozyme Efficiency on Hydrophobic Assemblies. Muller-In Retrospect: The Chromosome Trail. Weinberg
-Increased Mtorc1 Signaling Upregulates Stress. Reiling
-Induction of Pluripotent Stem Cells from Primary Human Fibroblasts with Only Oct4 and Sox2. Huangfu
-Isolation and Functional Characterization of Side Population Stem Cells. Johnnidis
-Isolation of a Drosophila Amplification Origin Developmentally Activated by Transcription. Xie
-The Known Unknowns of Antigen Processing and Presentation Vyas
-Leaving Home Early: Reexamination of the Canonical Models of Tumor Progression Weinberg
-Loss of E-Cadherin Promotes Metastasis Via Multiple Downstream Transcriptional Pathways. Onder
-Lymphotoxin-Dependent Prion Replication in Inflammatory Stromal Cells of Granulomas. Heikenwalder
-Lysosomal Cysteine and Aspartic Proteases Are Heterogeneously Expressed and Act Redundantly to Initiate Human Invariant Chain Degradation Constantino
-The Many Faces of Tumor Dormancy Weinberg
-Mapping Key Features of Transcriptional Regulatory Circuitry in Embryonic Stem Cells. Cole
-Mat Formation in Saccharomyces Cerevisiae Requires Nutrient and Ph Gradients Reynolds
-Mechanisms of Malignant Progression Weinberg
-Meiosis. Hochwagen
-Meiotic Failure in Male Mice Lacking an X-Linked Factor. Yang
-Micromanagement of the Immune System by Micrornas. Lodish
-Micromanagers of Malignancy: Role of Micrornas in Regulating Metastasis Ma
-Micrornas in the Hox Network: An Apparent Link to Posterior Prevalence Yekta
-Molecular architecture of the kinetochore-microtubule interface.Cheeseman
-Molecular, Biochemical and Functional Characterizations of C1q/Tnf Family Members: Adipose-Tissue-Selective Expression Patterns, Regulation by Ppar-Gamma Agonist, Cysteine-Mediated Oligomerizations, Combinatorial Associations and Metabolic Functions.Wong
-Mouse Maelstrom, a Component of Nuage, Is Essential for Spermatogenesis and Transposon Repression in Meiosis Soper
-The Mouse X Chromosome Is Enriched for Multicopy Testis Genes Showing Postmeiotic Expression Mueller
-Msy Breakpoint Mapper, a Database of Sequence-Tagged Sites Useful in Defining Naturally Occurring Deletions in the Human Y Chromosome. Lange
-Mutations in the Chromosomal Passenger Complex and the Condensin Complex Differentially Affect Synaptonemal Complex Disassembly and Metaphase I Configuration in Drosophila Female Meiosis. Resnick
-N-Linked Glycosylation Does Not Impair Proteasomal Degradation but Affects Class I Major Histocompatibility Complex Presentation Kario
-Neurons Derived from Reprogrammed Fibroblasts Functionally Integrate into the Fetal Brain and Improve Symptoms of Rats with Parkinson's Disease. Wernig
-Nucleo-cytoplasmic Shuttling of the Golgi Phosphatidylinositol 4-Kinase Pik1 Is Regulated by 14-3-3 Proteins and Coordinates Golgi Function with Cell Growth. Demmel
-Oncogenic Kras-Induced Leukemogeneis: Hematopoietic Stem Cells as the Initial Target and Lineage-Specific Progenitors as the Potential Targets for Final Leukemic Transformation. Zhang
-Optimal Optical Trap for Bacterial Viability. Mirsaidov
-Optimization of the Surfaces Used to Capture Antibodies from Single Hybridomas Reduces the Time Required for Microengraving Ronan
-Parasite Stage-Specific Recognition of Endogenous Toxoplasma Gondii-Derived Cd8(+) T Cell Epitopes. Frickel
-The Parkinson's disease protein alpha-synuclein disrupts cellular Rab homeostasis Gitler
-The Pluripotency Regulator Oct4: A Role in Somatic Stem Cells? Lengner
-Power-Limited Contraction Dynamics of Vorticella Convallaria: An Ultrafast Biological Spring.Upadhyaya
-Prion Switching in Response to Environmental Stress Tyedmers
-Profiling Antibody Responses by Multiparametric Analysis of Primary B Cells. Story
-Promoter Cpg Methylation Contributes to Es Cell Gene Regulation in Parallel with Oct4/Nanog, Pcg Complex, and Histone H3k4/K27 Trimethylation. Fouse
-Proteolytic Cleavage in an Endolysosomal Compartment Is Required for Activation of Toll-Like Receptor 9 Park
-The Rag Gtpases Bind Raptor and Mediate Amino Acid Signaling to Mtorc1 Sancak
-Ras Oncogenes: Split Personalities. Karnoub
-Reaction-Mapped Quantitative Multiplexed Polymerase Chain Reaction on a Microfluidic Device Ueberfeld
-Regulation of Apc/C Activators in Mitosis and Meiosis. Pesin
-Regulation of Progenitor Cell Proliferation and Granulocyte Function by Microrna-223 Johnnidis
-Reply Ma
-The Reprogramming Language of Pluripotency. Welstead
-Reprogramming of Murine and Human Somatic Cells Using a Single Polycistronic Vector. Carey
-Reprogramming of Neural Progenitor Cells into Induced Pluripotent Stem Cells in the Absence of Exogenous Sox2 Expression Eminli
-Reprogramming of Somatic Cell Identity. Hanna
-Retrospective: Judah Folkman (1933-2008) Hanahan
-The Role of Calorie Restriction and Sirt1 in Prion-Mediated Neurodegeneration Chen
-Sel1l Nucleates a Protein Complex Required for Dislocation of Misfolded Glycoproteins. Mueller
-Sequential Expression of Pluripotency Markers During Direct Reprogramming of Mouse Somatic Cells. Brambrink
-A Single Hox Locus in Drosophila Produces Functional Micrornas from Opposite DNA Strands. Stark
-Sister Chromatid Cohesion Independent Roles of Cohesin in Meiotic Chromosome Morphogenesis and Pairing. Brar
-Smed-Beta-Catenin-1 Is Required for Anteroposterior Blastema Polarity in Planarian Regeneration. Petersen
-Stem Cells, the Molecular Circuitry of Pluripotency and Nuclear Reprogramming. Jaenisch
-Somatic Cell Nuclear Transfer and Derivation of Embryonic Stem Cells in the Mouse Markoulaki
-Stra8 and Its Inducer, Retinoic Acid, Regulate Meiotic Initiation in Both Spermatogenesis and Oogenesis in Mice. Anderson
-Substrate Filtering by the Active-Site Crossover Loop in Uchl3 Revealed by Sortagging and Gain-of-Function Mutations Popp
-Systemic Endocrine Instigation of Indolent Tumor Growth Requires Osteopontin McAllister
-Systematic Evaluation of Variability in Chip-Chip Experiments Using Predefined DNA Targets. Johnson
-Tcf3 Is an Integral Component of the Core Regulatory Circuitry of Embryonic Stem Cells. Cole
-384-Channel Parallel Microfluidic Cytometer for Rare-Cell Screening. McKenna
-Toward a Molecular Structure of the Eukaryotic Kinetochore Welburn
-Twisted Epithelial-Mesenchymal Transition Blocks Senescence Weinberg
-Unc93b1 Delivers Nucleotide-Sensing Toll-Like Receptors to Endolysosomes. Kim
-Uncovering the Gene Knockout Landscape for Improved Lycopene Production in E. Coli .Alper
-Wnt Signaling Promotes Reprogramming of Somatic Cells to Pluripotency. Marson
-A Yeast Tdp-43 Proteinopathy Model: Exploring the Molecular Determinants of Tdr-43 Aggregation and Cellular Toxicity. Johnson

AddoQuaye C., Eshoo, T.W., Bartel, D.P., and Axtell, M.J. (2008). Endogenous Sirna and Mirna Targets Identified by Sequencing of the Arabidopsis Degradome. Current Biology 18, 758-762. MicroRNAs (miRNAs) regulate the expression of target mRNAs in plants and animals [1]. Plant miRNA targets have been predicted on the basis of their extensive and often conserved complementarity to the miRNAs [2-4], as well as on miRNA overexpression experiments [5]; many of these target predictions have been confirmed by isolation of the products of miRNA-directed cleavage. Here, we present a transcriptome-wide experimental method, called "degradome sequencing," to directly detect cleaved miRNA targets without relying on predictions or overexpression. The 5' ends of polyadenylated, uncapped mRNAs from Arabidopsis were directly sampled, resulting in an empirical snapshot of the degradome. miRNA-mediated-cleavage products were easily discerned from an extensive background of degraded mRNAs, which collectively covered the majority of the annotated transcriptome. Many previously known Arabidopsis miRNA targets were confirmed, and several novel targets were also discovered. Quantification of cleavage fragments revealed that those derived from TAS transcripts, which are unusual in their production of abundant secondary small interfering RNAs (siRNAs), accumulated to very high levels. A subset of secondary siRNAs are also known to direct cleavage of targets in trans [6]; degradome sequencing revealed many cleaved targets of these trans-acting siRNAs (ta-siRNAs). This empirical method is broadly applicable to the discovery and quantification of cleaved targets of small RNAs without a priori predictions. Full Text.

Alper, H., and Stephanopoulos, G. (2008). Uncovering the Gene Knockout Landscape for Improved Lycopene Production in E. Coli. Applied Microbiology and Biotechnology 78, 801-810. Systematic and combinatorial genetic approaches for the identification of gene knockout and overexpression targets have been effectively employed in the improvement of cellular phenotypes. Previously, we demonstrated how two of these tools, metabolic modeling and transposon mutagenesis, can be combined to identify strains of interest spanning the metabolic landscape of recombinant lycopene production in Escherichia coli. However, it is unknown how to best select multiple-gene knockout targets. Hence, this study seeks to understand how the overall order of gene selection, or search trajectory, biases the exploration and topology of the metabolic landscape. In particular, transposon mutagenesis and selection were employed in the background of eight different knockout genotypes. Collectively, 800,000 mutants were analyzed in hopes of exhaustively identifying all advantageous gene knockout targets. Several interesting observations, including clusters of gene functions, recurrence, and divergent genotypes, demonstrate the complexity of mapping only one genotype to one phenotype. One particularly interesting mutant, the ?hnr?yliE genotype, exhibited a drastically improved lycopene production capacity in basic minimal medium in comparison to the best strains identified in previous studies. Full Text.

Anderson, E.L., Baltus, A.E., Roepers-Gajadien, H.L., Hassold, T.J., de Rooij, D.G., van Pelt, A.M., and Page, D.C. (2008). Stra8 and Its Inducer, Retinoic Acid, Regulate Meiotic Initiation in Both Spermatogenesis and Oogenesis in Mice. Proc Natl Acad Sci U S A.Sep 17. [Epub ahead of print] In eukaryotes, diploid cells give rise to haploid cells via meiosis, a program of two cell divisions preceded by one round of DNA replication. Although key molecular components of the meiotic apparatus are highly conserved among eukaryotes, the mechanisms responsible for initiating the meiotic program have diverged substantially among eukaryotes. This raises a related question in animals with two distinct sexes: Within a given species, are similar or different mechanisms of meiotic initiation used in the male and female germ lines? In mammals, this question is underscored by dramatic differences in the timing of meiotic initiation in males and females. Stra8 is a vertebrate-specific, cytoplasmic factor expressed by germ cells in response to retinoic acid. We previously demonstrated that Stra8 gene function is required for meiotic initiation in mouse embryonic ovaries. Here we report that, on an inbred C57BL/6 genetic background, the same factor is also required for meiotic initiation in germ cells of juvenile mouse testes. In juvenile C57BL/6 males lacking Stra8 gene function, the early mitotic development of germ cells appears to be undisturbed. However, these cells then fail to undergo the morphological changes that define meiotic prophase, and they do not display the molecular hallmarks of meiotic chromosome cohesion, synapsis and recombination. We conclude that, in mice, Stra8 regulates meiotic initiation in both spermatogenesis and oogenesis. Taken together with previous observations, our present findings indicate that, in both the male and female germ lines, meiosis is initiated through retinoic acid induction of Stra8. PDF.

Baek, D., Villen, J., Shin, C., Camargo, F.D., Gygi, S.P., and Bartel, D.P. (2008). The Impact of Micrornas on Protein Output. Nature Jul 30. [Epub ahead of print]. MicroRNAs are endogenous approximately 23-nucleotide RNAs that can pair to sites in the messenger RNAs of protein-coding genes to downregulate the expression from these messages. MicroRNAs are known to influence the evolution and stability of many mRNAs, but their global impact on protein output had not been examined. Here we use quantitative mass spectrometry to measure the response of thousands of proteins after introducing microRNAs into cultured cells and after deleting mir-223 in mouse neutrophils. The identities of the responsive proteins indicate that targeting is primarily through seed-matched sites located within favourable predicted contexts in 3' untranslated regions. Hundreds of genes were directly repressed, albeit each to a modest degree, by individual microRNAs. Although some targets were repressed without detectable changes in mRNA levels, those translationally repressed by more than a third also displayed detectable mRNA destabilization, and, for the more highly repressed targets, mRNA destabilization usually comprised the major component of repression. The impact of microRNAs on the proteome indicated that for most interactions microRNAs act as rheostats to make fine-scale adjustments to protein output. Full Text.

BenPorath, I., Thomson, M.W., Carey, V.J., Ge, R., Bell, G.W., Regev, A., and Weinberg, R.A. (2008). An Embryonic Stem Cell-Like Gene Expression Signature in Poorly Differentiated Aggressive Human Tumors. Nat Genet 40, 499-507. Cancer cells possess traits reminiscent of those ascribed to normal stem cells. It is unclear, however, whether these phenotypic similarities reflect the activity of common molecular pathways. Here, we analyze the enrichment patterns of gene sets associated with embryonic stem (ES) cell identity in the expression profiles of various human tumor types. We find that histologically poorly differentiated tumors show preferential overexpression of genes normally enriched in ES cells, combined with preferential repression of Polycomb-regulated genes. Moreover, activation targets of Nanog, Oct4, Sox2 and c-Myc are more frequently overexpressed in poorly differentiated tumors than in well-differentiated tumors. In breast cancers, this ES-like signature is associated with high-grade estrogen receptor (ER)-negative tumors, often of the basal-like subtype, and with poor clinical outcome. The ES signature is also present in poorly differentiated glioblastomas and bladder carcinomas. We identify a subset of ES cell-associated transcription regulators that are highly expressed in poorly differentiated tumors. Our results reveal a previously unknown link between genes associated with ES cell identity and the histopathological traits of tumors and support the possibility that these genes contribute to stem cell-like phenotypes shown by many tumors. Full Text.

BloushtainQimron, N., Yao, J., Snyder, E.L., Shipitsin, M., Campbell, L.L., Mani, S.A., Hua, M., Chen, H.Y., Ustyansky, V., Antosiewicz, J.E., et al. (2008). Cell Type-Specific DNA Methylation Patterns in the Human Breast. Proceedings of the National Academy of Sciences of the United States of America 105, 14076-14081. Cellular identity and differentiation are determined by epigenetic programs. The characteristics of these programs in normal human mammary epithelium and their similarity to those in stem cells are unknown. To begin investigating these issues, we analyzed the DNA methylation and gene expression profiles of distinct subpopulations of mammary epithelial cells by using MSDK (methylation-specific digital karyotyping) and SAGE (serial analysis of gene expression). We identified discrete cell-type and differentiation state-specific DNA methylation and gene expression patterns that were maintained in a subset of breast carcinomas and correlated with clinically relevant tumor subtypes. CD44+ cells were the most hypomethylated and highly expressed several transcription factors with known stem cell function including HOXA10 and TCF3. Many of these genes were also hypomethylated in BMP4-treated compared with undifferentiated human embryonic stem (ES) cells that we analyzed by MSDK for comparison. Further highlighting the similarity of epigenetic programs of embryonic and mammary epithelial cells, genes highly expressed in CD44+ relative to more differentiated CD24+ cells were significantly enriched for Suz12 targets in ES cells. The expression of FOXC1, one of the transcription factors hypomethylated and highly expressed in CD44+ cells, induced a progenitor-like phenotype in differentiated mammary epithelial cells. These data suggest that epigenetically controlled transcription factors play a key role in regulating mammary epithelial cell phenotypes and imply similarities among epigenetic programs that define progenitor cell characteristics. PDF

Bradshaw, E.M., Kent, S.C., Tripuraneni, V., Orban, T., Ploegh, H.L., Hafler, D.A., and Love, J.C. (2008). Concurrent Detection of Secreted Products from Human Lymphocytes by Microengraving: Cytokines and Antigen-Reactive Antibodies. Clinical Immunology 129, 10-18. Cell surface determinants, cytokines and antibodies secreted by hematopoietic cells are used to classify their lineage and function. Currently available techniques are unable to elucidate multiple secreted proteins white also assigning phenotypic surface-displayed markers to the individual living cells. Here, a soft lithographic method, microengraving, was adapted for the multiplexed interrogation of populations of individual human peripheral blood mononuclear cells for secreted cytokines (IFN-gamma and IL-6), antigen-specific antibodies, and lineage-specific surface-expressed markers. Application of the method to a clinical sample from a recent-onset Type 1 diabetic subject with a positive titer of anti-insulin antibodies showed that similar to 0.58% of circulating CD19(+) B cells secreted proinsulin-reactive antibodies of the IgG isotype and 2-3% of circulating cells secreted IL-6. These data demonstrate the utility of microengraving for interrogating multiple phenotypes of single human cells concurrently and for detecting rare populations of cells by their secreted products.

Brambrink, T., Foreman, R., Welstead, G.G., Lengner, C.J., Wernig, M., Suh, H., and Jaenisch, R. (2008). Sequential Expression of Pluripotency Markers During Direct Reprogramming of Mouse Somatic Cells. Cell Stem Cell 2, 151-159. Pluripotency can be induced in differentiated murine and human cells by retroviral transduction of Oct4, Sox2, Klf4, and c-Myc. We have devised a reprogramming strategy in which these four transcription factors are expressed from doxycycline (dox)-inducible lentiviral vectors. Using these inducible constructs, we derived induced pluripotent stem (PS) cells from mouse embryonic fibroblasts (MEFs) and found that transgene silencing is a prerequisite for normal cell differentiation. We have analyzed the timing of known pluripotency marker activation during mouse PS cell derivation and observed that alkaline phosphatase (AP) was activated first, followed by stage-specific embryonic antigen 1 (SSEA1). Expression of Nanog and the endogenous Oct4 gene, marking fully reprogrammed cells, was only observed late in the process. Importantly, the virally transduced cDNAs needed to be expressed for at least 12 days in order to generate PS cells. Our results are a step toward understanding some of the molecular events governing epigenetic reprogramming. Full Text .

Brar, G.A., Hochwagen, A., Ee, L., and Amon, A. (2008). Sister Chromatid Cohesion Independent Roles of Cohesin in Meiotic Chromosome Morphogenesis and Pairing. Molecular Biology of the Cell.Dec 10. [Epub ahead of print] Sister chromatid cohesion, mediated by cohesin complexes, is laid down during DNA replication and is essential for the accurate segregation of chromosomes. Previous studies indicated that, in addition to its cohesion function, cohesins are essential for completion of recombination, pairing, meiotic chromosome axis formation and assembly of the Synaptonemal Complex (SC). Using mutants in the cohesin subunit Rec8, in which phosphorylated residues were mutated to alanines, we show that cohesin phosphorylation is not only important for cohesin removal, but that cohesin's meiotic prophase functions are distinct from each other. We find pairing and SC formation to be dependent on Rec8, but independent of the presence of a sister chromatid and hence sister chromatid cohesion. We identified mutants in REC8 that differentially affect Rec8's cohesion, pairing, recombination, chromosome axis and SC assembly function. These findings define Rec8 as a key determinant of meiotic chromosome morphogenesis and a central player in multiple meiotic events. PDF

Carey, B.W., Markoulaki, S., Hanna, J., Saha, K., Gao, Q., Mitalipova, M., and Jaenisch, R. (2008). Reprogramming of Murine and Human Somatic Cells Using a Single Polycistronic Vector. Proc Natl Acad Sci U S A. Directed reprogramming of somatic cells by defined factors provides a novel method for the generation of patient-specific stem cells with the potential to bypass both the practical and ethical concerns associated with somatic cell nuclear transfer (SCNT) and human embryonic stem (hES) cells. Although the generation of induced pluripotent stem (iPS) cells has proven a robust technology in mouse and human, a major impediment to the use of iPS cells for therapeutic purposes has been the viral-based delivery of the reprogramming factors because multiple proviral integrations pose the danger of insertional mutagenesis. Here we report a novel approach to reduce the number of viruses necessary to reprogram somatic cells by delivering reprogramming factors in a single virus using 2A "self-cleaving" peptides, which support efficient polycistronic expression from a single promoter. We find that up to four reprogramming factors (Oct4, Sox2, Klf4, and c-Myc) can be expressed from a single virus to generate iPS cells in both embryonic and adult somatic mouse cells and we show that a single proviral copy is sufficient to generate iPS cells from mouse embryonic fibroblasts. In addition we have generated human induced pluripotent stem (hiPS) cell lines from human keratinocytes, demonstrating that a single polycistronic virus can reprogram human somatic cells. Full Text.

Cheeseman, I.M., and Desai, A. (2008). Molecular architecture of the kinetochore-microtubule interface. Nat Rev Mol Cell Biol 9, 33-46. Segregation of the replicated genome during cell division in eukaryotes requires the kinetochore to link centromeric DNA to spindle microtubules. The kinetochore is composed of a number of conserved protein complexes that direct its specification and assembly, bind to spindle microtubules and regulate chromosome segregation. Recent studies have identified more than 80 kinetochore components, and are revealing how these proteins are organized into the higher order kinetochore structure, as well as how they function to achieve proper chromosome segregation. Full Text.

Chen, D., Steele, A.D., Hutter, G., Bruno, J., Govindarajan, A., Easlon, E., Lin, S.J., Aguzzi, A., Lindquist, S., and Guarente, L. (2008). The Role of Calorie Restriction and Sirt1 in Prion-Mediated Neurodegeneration. Experimental Gerontology 43, 1086-1093. A central focus of aging research is to determine how calorie restriction (CR) extends lifespan and delays diseases of aging. SIRT1, the mammalian ortholog of Sir2 in yeast, is a longevity factor which mediates dietary restriction in diverse species. In addition, SIRT1 plays a protective role in several models of neurodegenerative disease. We tested the role of SIRT1 in mediating the effects of CR in a mouse model of prion disease. Prion diseases are protein misfolding disorders of the central nervous system with many similarities to other neurodegenerative diseases, including deposition of aggregated protein, gliosis, and loss of synapses and neurons. We report that the onset of prion disease is delayed by CR and in the SIRT1 KO mice fed ad libitum. CR exerts no further effect on the SIRT1 KO strain, suggesting the effects of CR and SIRT1 deletion are mechanistically coupled. In conjunction, SIRT1 is downregulated in certain brain regions of CR mice. The expression of PrP mRNA and protein is reduced in the brains of CR mice and in SIRT1 knockout mice, suggesting a possible mechanism for the delayed onset of disease, as PrP levels are a critical determinant of how quickly mice succumb to prion disease. Surprisingly, CR greatly shortens the duration of clinical symptoms of prion disease and ultimately shortens lifespan of prion-inoculated mice in a manner that is independent of SIRT1. Taken together, our results suggest a more complex interplay between CR, SIRT1, and neurodegenerative diseases than previously appreciated..

Chen, S.Y., Tsubouchi, T., Rockmill, B., Sandler, J.S., Richards, D.R., Vader, G., Hochwagen, A., Roeder, G.S., and Fung, J.C. (2008). Global Analysis of the Meiotic Crossover Landscape. Developmental Cell 15, 401-415. Tight control of the number and distribution of crossovers is of great importance for meiosis. Crossovers establish chiasmata, which are physical connections between homologous chromosomes that provide the tension necessary to align chromosomes on the meiotic spindle. Understanding the mechanisms underlying crossover control has been hampered by the difficulty in determining crossover distributions. Here, we present a microarray-based method to analyze multiple aspects of crossover control simultaneously and rapidly, at high resolution, genome-wide, and on a cell-by-cell basis. Using this approach, we show that loss of interference in zip2 and zip4/spo22 mutants is accompanied by a reduction in crossover homeostasis, thus connecting these two levels of crossover control. We also provide evidence to suggest that repression of crossing over at telomeres and centromeres arises from different mechanisms. Lastly, we uncover a surprising role for the synaptonemal complex component Zip1 in repressing crossing over at the centromere. Full Text.

Cole, M.F., and Young, R.A. (2008). Mapping Key Features of Transcriptional Regulatory Circuitry in Embryonic Stem Cells. Cold Spring Harbor Symposia on Quantitative Biology Vol. 73 The process by which a single fertilized egg develops into a human being with more than 200 cell types-each with a distinct gene expression pattern controlling its cellular state -is poorly understood. Knowledge of the transcriptional regulatory circuitry h t at establishes and maintains gene expression programs in mammalian cells is fundamental to understanding development and should provide the foundation for improved diagnosis and treatment of disease. Although it is not yet feasible to map the entirety of this circuitry in vertebrate cells, recent work in embryonic stem (ES) cells has demonstrated that core features of the circuitry can be discovered through studies involving selected regulators. Here, we highlight the fundamental insights that have emerged from studies that examined the role of transcription factors, chromatin regulators, signaling pathways, and noncoding RNAs in the regulatory circuitry of ES cells. Maps of regulatory circuitry and the insights that have emerged from these studies have improved our understanding of global gene expression and are facilitating efforts to reprogram cells for disease therapeutics and regenerative medicine..

Cole, M.F., Johnstone, S.E., Newman, J.J., Kagey, M.H., and Young, R.A. (2008). Tcf3 Is an Integral Component of the Core Regulatory Circuitry of Embryonic Stem Cells. Genes Dev 22, 746-755. Embryonic stem (ES) cells have a unique regulatory circuitry, largely controlled by the transcription factors Oct4, Sox2, and Nanog, which generates a gene expression program necessary for pluripotency and self-renewal. How external signals connect to this regulatory circuitry to influence ES cell fate is not known. We report here that a terminal component of the canonical Wnt pathway in ES cells, the transcription factor T-cell factor-3 (Tcf3), co-occupies promoters throughout the genome in association with the pluripotency regulators Oct4 and Nanog. Thus, Tcf3 is an integral component of the core regulatory circuitry of ES cells, which includes an autoregulatory loop involving the pluripotency regulators. Both Tcf3 depletion and Wnt pathway activation cause increased expression of Oct4, Nanog, and other pluripotency factors and produce ES cells that are refractory to differentiation. Our results suggest that the Wnt pathway, through Tcf3, brings developmental signals directly to the core regulatory circuitry of ES cells to influence the balance between pluripotency and differentiation. Full Text.

Cong, Y., Topf, M., Sali, A., Matsudaira, P., Dougherty, M., Chiu, W., and Schmid, M.F. (2008). Crystallographic Conformers of Actin in a Biologically Active Bundle of Filaments. Journal of Molecular Biology 375, 331-336. Actin carries out many of its cellular functions through its filamentous form; thus, understanding the detailed structure of actin filaments is an essential step in achieving a mechanistic understanding of actin function. The acrosomal bundle in the Limulus sperm has been shown to be a quasi-crystalline array with an asymmetric unit composed of a filament with 14 actin-scruin pairs. The bundle in its true discharge state penetrates the jelly coat of the egg. Our previous electron crystallographic reconstruction demonstrated that the actin filament cross-linked by scruin in this acrosomal bundle state deviates significantly from a perfect F-actin helix. In that study, the tertiary structure of each of the 14 actin protomers in the asymmetric unit of the bundle filament was assumed to be constant. In the current study, an actin filament atomic model in the acrosomal bundle has been refined by combining rigid-body docking with multiple actin crystal structures from the Protein Data Bank and constrained energy minimization. Our observation demonstrates that actin protomers adopt different tertiary conformations when they form an actin filament in the bundle. The scruin and bundle packing forces appear to influence the tertiary and quaternary conformations of actin in the filament of this biologically active bundle. . Full Text.

Costantino, C.M., Hang, H.C., Kent, S.C., Hafler, D.A., and Ploegh, H.L. (2008). Lysosomal Cysteine and Aspartic Proteases Are Heterogeneously Expressed and Act Redundantly to Initiate Human Invariant Chain Degradation. Journal of Immunology 180, 2876-2885. Presentation of Ag by class II MHC is regulated by lysosomal proteases that not only destroy the class II invariant chain (Ii) chaperone but also generate the peptide Ag that is loaded onto the class II MHC dimer. We sought to determine the extent to which asparagine endopeptidase (AEP) influences human Ag and Ii processing. Our data confirm the constructive function of AEP in tetanus toxoid processing, but they are discordant with findings that suggest a destructive role for AEP in processing of the immunodominant myelin basic protein epitope. Furthermore, we observed no effect on invariant chain processing following AEP inhibition for several distinct allelic variants of human class II MHC products. We find that cysteine and aspartic proteases, as well as AEP, can act redundantly to initiate Ii processing. We detected considerable variation in lysosomal activity between different EBV-transformed B cell lines, but these differences do not result in altered regulation of invariant chain catabolism. We propose that, as for bound peptide Ag, the identity of the lysosomal enzyme that initiates invariant chain cleavage is dependent on the class II MHC allelic variants expressed.PDF

Creyghton, M.P., Markoulaki, S., Levine, S.S., Hanna, J., Lodato, M.A., Sha, K., Young, R.A., Jaenisch, R., and Boyer, L.A. (2008). H2az Is Enriched at Polycomb Complex Target Genes in Es Cells and Is Necessary for Lineage Commitment. Cell. 5 Nov. Article in Press, Corrected Proof Elucidating how chromatin influences gene expression patterns and ultimately cell fate is fundamental to understanding development and disease. The histone variant H2AZ has emerged as a key regulator of chromatin function and plays an essential but unknown role during mammalian development. Here, genome-wide analysis reveals that H2AZ occupies the promoters of developmentally important genes in a manner that is remarkably similar to that of the Polycomb group (PcG) protein Suz12. By using RNAi, we demonstrate a role for H2AZ in regulating target gene expression, find that H2AZ and PcG protein occupancy is interdependent at promoters, and further show that H2AZ is necessary for ES cell differentiation. Notably, H2AZ occupies a different subset of genes in lineage-committed cells, suggesting that its dynamic redistribution is necessary for cell fate transitions. Thus, H2AZ, together with PcG proteins, may establish specialized chromatin states in ES cells necessary for the proper execution of developmental gene expression programs. Full Text.

Demmel, L., Beck, M., Klose, C., Schlaitz, A.L., Gloor, Y., Hsu, P.P., Havlis, J., Shevchenko, A., Krause, E., Kalaidzidis, Y., et al. (2008). Nucleo-cytoplasmic Shuttling of the Golgi Phosphatidylinositol 4-Kinase Pik1 Is Regulated by 14-3-3 Proteins and Coordinates Golgi Function with Cell Growth. Mol Biol Cell. Jan 2 [Epub ahead of print] Monitoring Editor: Benjamin Glick The yeast phosphatidylinositol 4-kinase Pik1p is essential for proliferation and controls Golgi homeostasis and transport of newly synthesized proteins from this compartment. At the Golgi, phosphatidylinositol 4-phosphate recruits multiple cytosolic effectors involved in formation of post-Golgi transport vesicles. A second pool of catalytically active Pik1p localizes to the nucleus. The physiological significance and regulation of this dual localization of the lipid kinase remains unknown. Here we show that Pik1p binds to the redundant 14-3-3 proteins Bmh1p and Bmh2p. We provide evidence that nucleo-cytoplasmic shuttling of Pik1p involves phosphorylation and that 14-3-3 proteins bind Pik1p in the cytoplasm. Nutrient deprivation results in relocation of Pik1p from the Golgi to the nucleus and increases the amount of Pik1p - 14-3-3 complex, a process reversed upon restored nutrient supply. These data suggest a role of Pik1p nucleo-cytoplasmic shuttling in coordination of biosynthetic transport from the Golgi with nutrient signaling. PDF

Douglas, P.M., Treusch, S., Ren, H.Y., Halfmann, R., Duennwald, M.L., Lindquist, S., and Cyr, D.M. (2008). Chaperone-Dependent Amyloid Assembly Protects Cells from Prion Toxicity. Proceedings of the National Academy of Sciences of the United States of America 105, 7206-7211. Protein conformational diseases are associated with the aberrant accumulation of amyloid protein aggregates, but whether amyloid formation is cytotoxic or protective is unclear. To address this issue, we investigated a normally benign amyloid formed by the yeast prion [RNQ(+)]. Surprisingly, modest overexpression of Rnq1 protein was deadly, but only when preexisting Rnq1 was in the [RNQ(+)] prion conformation. Molecular chaperones protect against protein aggregation diseases and are generally believed to do so by solubilizing their substrates. The Hsp40 chaperone, Sis1, suppressed Rnq1 proteotoxicity, but instead of blocking Rnq1 protein aggregation, it stimulated conversion of soluble Rnq1 to [RNQ(+)] amyloid. Furthermore, interference with Sis1-mediated [RNQ(+)] amyloid formation exacerbated Rnq1 toxicity. These and other data establish that even subtle changes in the folding homeostasis of an amyloidogenic protein can create a severe proteotoxic gain-of-function phenotype and that chaperone-mediated amyloid assembly can be cytoprotective. The possible relevance of these findings to other phenomena, including prion-driven neurodegenerative diseases and heterokaryon incompatibility in fungi, is discussed. Full Text.

Duennwald, M.L., and Lindquist, S. (2008). Impaired Erad and Er Stress Are Early and Specific Events in Polyglutamine Toxicity. Genes and Development .Nov 17. [Epub ahead of print] Protein misfolding, whether caused by aging, environmental factors, or genetic mutations, is a common basis for neurodegenerative diseases. The misfolding of proteins with abnormally long polyglutamine (polyQ) expansions causes several neurodegenerative disorders, such as Huntington's disease (HD). Although many cellular pathways have been documented to be impaired in HD, the primary triggers of polyQ toxicity remain elusive. We report that yeast cells and neuron-like PC12 cells expressing polyQ-expanded huntingtin (htt) fragments display a surprisingly specific, immediate, and drastic defect in endoplasmic reticulum (ER)-associated degradation (ERAD). We further decipher the mechanistic basis for this defect in ERAD: the entrapment of the essential ERAD proteins Npl4, Ufd1, and p97 by polyQ-expanded htt fragments. In both yeast and mammalian neuron-like cells, overexpression of Npl4 and Ufd1 ameliorates polyQ toxicity. Our results establish that impaired ER protein homeostasis is a broad and highly conserved contributor to polyQ toxicity in yeast, in PC12 cells, and, importantly, in striatal cells expressing full-length polyQ-expanded huntingtin. PDF

Eminli, S., Utikal, J., Arnold, K., Jaenisch, R., and Hochedlinger, K. (2008). Reprogramming of Neural Progenitor Cells into Induced Pluripotent Stem Cells in the Absence of Exogenous Sox2 Expression. Stem Cells 26, 2467-2474. Expression of the transcription factors Oct4, Sox2, Klf4, and c-Myc in mesodermal and endodermal derivatives, including fibroblasts, lymphocytes, liver, stomach, and beta cells, generates induced pluripotent stem (iPS) cells. It remains unknown, however, whether cell types of the ectodermal lineage are equally amenable to reprogramming into iPS cells by the same combination of factors. To test this, we have isolated genetically marked neural progenitor cells (NPCs) from neonatal mouse brains and infected them with viral vectors expressing Oct4, Sox2, Klf4, and c-Myc. Infected NPCs gave rise to iPS cells that expressed markers of embryonic stem cells, showed demethylation of pluripotency genes, formed teratomas, and contributed to viable chimeras. In contrast to other somatic cell types, NPCs expressed high levels of endogenous Sox2 and thus did not require viral Sox2 expression for reprogramming into iPS cells. Our data show that in addition to mesoderm- and endoderm-derived cell types, neural progenitor cells of the ectodermal lineage can be reprogrammed into iPS cells, suggesting that in vitro reprogramming is a universal process. These results also imply that the combination of factors necessary for reprogramming is dependent on cellular context.. Full Text.

Fleming, J., Outeiro, T.F., Slack, M., Lindquist, S.L., and Bulawa, C.E. (2008). Detection of Compounds That Rescue Rab1-Synuclein Toxicity. In Small Gtpases in Disease, Pt B, pp. 339-351..

Frickel, E.M., Sahoo, N., Hopp, J., Gubbels, M.J., Craver, M.P.J., Knoll, L.J., Ploegh, H.L., and Grotenbreg, G.M. (2008). Parasite Stage-Specific Recognition of Endogenous Toxoplasma Gondii-Derived Cd8(+) T Cell Epitopes. Journal of Infectious Diseases 198, 1625-1633. Background. BALB/c mice control infection with the obligate intracellular parasite Toxoplasma gondii and develop a latent chronic infection in the brain, as do immunocompetent humans. Interferon-gamma-producing CD8(+) T cells provide essential protection against T. gondii infection, but the epitopes recognized have so far remained elusive. Methods. We employed caged major histocompatibility complex molecules to generate similar to 250 H-2L(d) tetramers and to distinguish T. gondii-specific CD8(+) T cells in BALB/c mice. Results. We identified 2 T. gondii-specific H-2L(d)-restricted T cell epitopes, one from dense granule protein GRA4 and the other from rhoptry protein ROP7. H-2L(d)/GRA4 reactive T cells from multiple organ sources predominated 2 weeks after infection, while the reactivity of the H-2L(d)/ROP7 T cells peaked 6-8 weeks after infection. BALB/c animals infected with T. gondii mutants defective in establishing a chronic infection showed altered levels of antigen-specific T cells, depending on the T. gondii mutant used. Conclusions. Our results shed light on the identity and the parasite stage-specificity of 2 CD8(+) T cell epitopes recognized in the acute and chronic phase of infection with T. gondii .Full Text

Flygare, J., Olsson, K., Richter, J., and Karlsson, S. (2008). Gene Therapy of Diamond Blackfan Anemia Cd34(+) Cells Leads to Improved Erythroid Development and Engraftment Following Transplantation. Experimntal Hematology.Article in Press OBJECTIVE: Diamond-Blackfan anemia (DBA) is a rare congenital hypoplastic anemia caused by mutations in ribosomal protein (RP) genes. Our aim is to develop gene therapy for DBA patients with mutations in RPS19. We previously demonstrated that RPS19 gene transfer partially corrects erythroid development in vitro. In this study, we asked if RPS19 gene transfer corrects erythroid development in unsorted cells transplanted to immunodeficient mice and if the RPS19-corrected fraction has a proliferative advantage after transplantation. We further determined if high level of RPS19 expression is required for correction. MATERIAL AND METHODS: Mobilized peripheral blood CD34(+) cells were transduced by oncoretroviral vector particles pseudotyped with the feline endogenous retrovirus envelope. Vectors containing two different promoters with different RPS19 transgene expression levels were compared. Transduced cells were transplanted to immunocompromised nonobese diabetic/severe combined immunodeficient-beta2 microglobulin null mice in order to assess therapeutic effects of RPS19 gene transfer in vivo. RESULTS: We show that correction of erythroid development requires high RPS19 expression. The corrected fraction of unselected DBA cells have a survival advantage in vivo, suggesting that successful gene therapy may only require correction of a fraction of the patient cells. CONCLUSION: Our findings are fundamental for development of clinical gene therapy for DBA because they demonstrate increased engraftment of RPS19-transduced cells without selection of gene-corrected cells prior to transplantation, an essential prelude to studying long-term therapeutic effects in emerging animal models for DBA. Full Text.

Fouse, S.D., Shen, Y., Pellegrini, M., Cole, S., Meissner, A., Van Neste, L., Jaenisch, R., and Fan, G.P. (2008). Promoter Cpg Methylation Contributes to Es Cell Gene Regulation in Parallel with Oct4/Nanog, Pcg Complex, and Histone H3k4/K27 Trimethylation. Cell Stem Cell 2, 160-169. We report here genome-wide mapping of DNA methylation patterns at proximal promoter regions in mouse embryonic stem (mES) cells. Most methylated genes are differentiation associated and repressed in mES cells. By contrast, the unmethylated gene set includes many housekeeping and pluripotency genes. By crossreferencing methylation patterns to genome-wide mapping of histone H3 lysine (K) 4/27 trimethylation and binding of Oct4, Nanog, and Polycomb proteins on gene promoters, we found that promoter DNA methylation is the only marker of this group present on similar to 30% of genes, many of which are silenced in mES cells. In demethylated mutant mES cells, we saw upregulation of a subset of X-linked genes and developmental genes that are methylated in wild-type mES cells, but lack either H3K4 and H3K27 trimethylation or association with Polycomb, Oct4, or Nanog. Our data suggest that in mES cells promoter methylation represents a unique epigenetic program that complements other regulatory mechanisms to ensure appropriate gene expression. Full Text.

Gitler, A.D., Bevis, B.J., Shorter, J., Strathearn, K.E., Hamamichi, S., Su, L.J., Caldwell, K.A., Caldwell, G.A., Rochet, J.C., McCaffery, J.M.,Charles Barlowe, and Susan Lindquist (2008). The Parkinson's disease protein alpha-synuclein disrupts cellular Rab homeostasis. Proc Natl Acad Sci U S A 105, 145-150. alpha-Synuclein (alpha-syn), a protein of unknown function, is the most abundant protein in Lewy bodies, the histological hallmark of Parkinson's disease (PD). In yeast alpha-syn inhibits endoplasmic reticulum (ER)-to-Golgi (ER-->Golgi) vesicle trafficking, which is rescued by overexpression of a Rab GTPase that regulates ER-->Golgi trafficking. The homologous Rab1 rescues alpha-syn toxicity in dopaminergic neuronal models of PD. Here we investigate this conserved feature of alpha-syn pathobiology. In a cell-free system with purified transport factors alpha-syn inhibited ER-->Golgi trafficking in an alpha-syn dose-dependent manner. Vesicles budded efficiently from the ER, but their docking or fusion to Golgi membranes was inhibited. Thus, the in vivo trafficking problem is due to a direct effect of alpha-syn on the transport machinery. By ultrastructural analysis the earliest in vivo defect was an accumulation of morphologically undocked vesicles, starting near the plasma membrane and growing into massive intracellular vesicular clusters in a dose-dependent manner. By immunofluorescence/immunoelectron microscopy, these clusters were associated both with alpha-syn and with diverse vesicle markers, suggesting that alpha-syn can impair multiple trafficking steps. Other Rabs did not ameliorate alpha-syn toxicity in yeast, but RAB3A, which is highly expressed in neurons and localized to presynaptic termini, and RAB8A, which is localized to post-Golgi vesicles, suppressed toxicity in neuronal models of PD. Thus, alpha-syn causes general defects in vesicle trafficking, to which dopaminergic neurons are especially sensitive. Full Text.

Godar, S., and Weinberg, R.A. (2008). Filling the Mosaic of P53 Actions: P53 Represses Rhamm Expression. Cell Cycle Nov 15;7(22) : 3479-80. [Epub ahead of print]. Full Text.

Godar, S., Ince, T.A., Bell, G.W., Feldser, D., Donaher, J.L., Bergh, J., Liu, A., Miu, K., Watnick, R.S., Reinhardt, F., McAllister SS, Jacks T, and Weinberg RA. (2008). Growth-Inhibitory and Tumor- Suppressive Functions of P53 Depend on Its Repression of Cd44 Expression. Cell 134, 62-73. The p53 tumor suppressor is a key mediator of cellular responses to various stresses. Here, we show that under conditions of basal physiologic and cell-culture stress, p53 inhibits expression of the CD44 cell-surface molecule via binding to a noncanonical p53-binding sequence in the CD44 promoter. This interaction enables an untransformed cell to respond to stress-induced, p53-dependent cytostatic and apoptotic signals that would otherwise be blocked by the actions of CD44. In the absence of p53 function, the resulting derepressed CD44 expression is essential for the growth and tumor-initiating ability of highly tumorigenic mammary epithelial cells. In both tumorigenic and nontumorigenic cells, CD44's expression is positively regulated by p63, a paralogue of p53. Our data indicate that CD44 is a key tumor-promoting agent in transformed tumor cells lacking p53 function. They also suggest that the derepression of CD44 resulting from inactivation of p53 can potentially aid the survival of immortalized, premalignant cells. Full Text.

GredmarkRuss, S., Cheung, E.J., Isaacson, M.K., Ploegh, H.L., and Grotenbreg, G.M. (2008). The Cd8 T Cell Response against Murine Gamma Herpes Virus (Mhv-68) Is Directed Towards a Broad Repertoire of Epitopes from Both Early and Late Antigens. Journal of Virolology Oct 15. [Epub ahead of print] Infection of mice with murine gamma herpes virus (MHV-68) robustly activates CD8 T cells, but only six Class I MHC restricted epitopes have been described to date for the widely used H-2(b) haplotype mice. To explore the specificity and kinetics of the CTL response in MHV-68 infected C57BL/6 mice, we screened for H-2K(b) and H-2D(b) restricted epitopes using a set of 384 candidate epitopes in a MHC tetramer based approach, and identified 19 new epitopes in 16 different ORFs. Of the six known H-2K(b)- and H-2D(b)-restricted epitopes, we confirmed a response against three and did not detect CD8 T cell specific responses for the remaining three. The peak of the CD8 T cell response to most peptides occurs between 6 and 10 days post infection (dpi). The respective MHC tetramer-positive CD8 T cells display an activated/effector phenotype (CD62(lo) and CD44(hi)) and produce IFN-gamma upon peptide stimulation ex vivo. MHV-68 infection in vivo elicits a response to multiple viral epitopes, derived from both early and late viral antigens, illustrating a far broader T cell repertoire and more rapid activation than previously recorded. PDF

Grimson, A., Srivastava, M., Fahey, B., Woodcroft, B.J., Chiang, H.R., King, N., Degnan, B.M., Rokhsar, D.S., and Bartel, D.P. (2008). Early Origins and Evolution of Micrornas and Piwi-Interacting Rnas in Animals. Nature.Oct 1. [Epub ahead of print] In bilaterian animals, such as humans, flies and worms, hundreds of microRNAs (miRNAs), some conserved throughout bilaterian evolution, collectively regulate a substantial fraction of the transcriptome. In addition to miRNAs, other bilaterian small RNAs, known as Piwi-interacting RNAs (piRNAs), protect the genome from transposons. Here we identify small RNAs from animal phyla that diverged before the emergence of the Bilateria. The cnidarian Nematostella vectensis (starlet sea anemone), a close relative to the Bilateria, possesses an extensive repertoire of miRNA genes, two classes of piRNAs and a complement of proteins specific to small-RNA biology comparable to that of humans. The poriferan Amphimedon queenslandica (sponge), one of the simplest animals and a distant relative of the Bilateria, also possesses miRNAs, both classes of piRNAs and a full complement of the small-RNA machinery. Animal miRNA evolution seems to have been relatively dynamic, with precursor sizes and mature miRNA sequences differing greatly between poriferans, cnidarians and bilaterians. Nonetheless, miRNAs and piRNAs have been available as classes of riboregulators to shape gene expression throughout the evolution and radiation of animal phyla. Full Text.

Grotenbreg, G.M., Roan, N.R., Guillen, E., Meijers, R., Wang, J.H., Bell, G.W., Starnbach, M.N., and Ploegh, H.L. (2008). Discovery of Cd8+ T Cell Epitopes in Chlamydia Trachomatis Infection through Use of Caged Class I Mhc Tetramers. Proc Natl Acad Sci U S A. 2008 Feb 1 [Epub ahead of print]. Class I MHC tetramers allow direct phenotypic identification of CD8(+) T cell populations, but their production remains laborious. A peptide exchange strategy that employs class I MHC products loaded with conditional ligands (caged MHC molecules) provides a fast and straightforward method to obtain diverse arrays of class I MHC tetramers and facilitates CD8(+) T cell epitope discovery. Here, we describe the development of photocleavable analogs of the FAPGNYPAL (SV9) epitope that bind H-2K(b) and H-2D(b) with full retention of their structural and functional integrity. We ranked all possible H-2K(b) octameric and H-2D(b) nonameric epitopes that span the genome of Chlamydia trachomatis and prepared MHC tetramers from approximately 2,000 of the highest scoring peptides by replacement of the SV9 analog with the peptide of choice. The resulting 2,000-member class I MHC tetramer array allowed the discovery of two variants of an epitope derived from polymorphic membrane protein I (PmpI) and an assessment of the kinetics of emergence and the effector function of the corresponding CD8(+) T cells. PDF

Guenther, M.G., Lawton, L.N., Rozovskaia, T., Frampton, G.M., Levine, S.S., Volkert, T.L., Croce, C.M., Nakamura, T., Canaani, E., and Young, R.A. (2008). Aberrant Chromatin at Genes Encoding Stem Cell Regulators in Human Mixed-Lineage Leukemia. Genes Dev 22, 3403-3408. Mixed-lineage leukemia (MLL) fusion proteins are potent inducers of leukemia, but how these proteins generate aberrant gene expression programs is poorly understood. Here we show that the MLL-AF4 fusion protein occupies developmental regulatory genes important for hematopoietic stem cell identity and self-renewal in human leukemia cells. These MLL-AF4-bound regions have grossly altered chromatin structure, with histone modifications catalyzed by trithorax group proteins and DOT1 extending across large domains. Our results define direct targets of the MLL fusion protein, reveal the global role of epigenetic misregulation in leukemia, and identify new targets for therapeutic intervention in cancer. Full Text.

Gutzman, J.H., Graeden, E.G., Lowery, L.A., Holley, H.S., and Sive, H. (2008). Formation of the Zebrafish Midbrain-Hindbrain Boundary Constriction Requires Laminin-Dependent Basal Constriction. Mechanisms of Development.Jul 18. [Epub ahead of print]The midbrain-hindbrain boundary (MHB) is a highly conserved fold in the vertebrate embryonic brain. We have termed the deepest point of this fold the MHB constriction (MHBC) and have begun to define the mechanisms by which it develops. In the zebrafish, the MHBC is formed soon after neural tube closure, concomitant with inflation of the brain ventricles. The MHBC is unusual, as it forms by bending the basal side of the neuroepithelium. At single cell resolution, we show that zebrafish MHBC formation involves two steps. The first is a shortening of MHB cells to approximately 75% of the length of surrounding cells. The second is basal constriction, and apical expansion, of a small group of cells that contribute to the MHBC. In the absence of inflated brain ventricles, basal constriction still occurs, indicating that the MHBC is not formed as a passive consequence of ventricle inflation. In laminin mutants, basal constriction does not occur, indicating an active role for the basement membrane in this process. Apical expansion also fails to occur in laminin mutants, suggesting that apical expansion may be dependent on basal constriction. This study demonstrates laminin-dependent basal constriction as a previously undescribed molecular mechanism for brain morphogenesis. PDF.

Hanahan, D., and Weinberg, R.A. (2008). Retrospective: Judah Folkman (1933-2008). Science 319, 1055. Full Text.

Hanna, J., Carey, B.W., and Jaenisch, R. (2008). Reprogramming of Somatic Cell Identity. Cold Spring Harbor Symposia on Quantitative Biology Vol. 73 All mammalian somatic cells originate from a single fertilized cell, the zygote, and share identical genetic information despite the dramatic changes in cell structure and function that accompany organismal development.The genome is subjected to a wide array of epigenetic modifications during lineage specification, a process that contributes to the implementation and maintenance of specific gene expression programs in somatic cells. Nuclear transfer and cell-fusion experiments demonstrate that the epigenetic signature directing a cell identity can be erased and modified into that of another cell type. Furthermore, in the case of cloning, differentiated cells can be reprogrammed back to pluripotency to support the reexpression of all developmental programs. Recent breakthroughs highlight the importance of transcription factors as well as epigenetic modifiers in the establishment, maintenance, and rewiring of cell identity. By focusing on reprogramming of terminally differentiated lymphocytes, we review and highlight recent insights into the molecular mechanisms and cellular events potentially underlying programming and reprogramming of somatic cell identity in mammals.

Hanna, J., Markoulaki, S., Schorderet, P., Carey, B.W., Beard, C., Wernig, M., Creyghton, M.P., Steine, E.J., Cassady, J.P., Foreman, R., Rudolph Jaenisch et al. (2008). Direct Reprogramming of Terminally Differentiated Mature B Lymphocytes to Pluripotency. Cell 133, 250-264. Pluripotent cells can be derived from fibroblasts by ectopic expression of defined transcription factors. A fundamental unresolved question is whether terminally differentiated cells can be reprogrammed to pluripotency. We utilized transgenic and inducible expression of four transcription factors (Oct4, Sox2, Klf4, and c-Myc) to reprogram mouse B lymphocytes. These factors were sufficient to convert nonterminally differentiated B cells to a pluripotent state. However, reprogramming of mature B cells required additional interruption with the transcriptional state maintaining B cell identity by either ectopic expression of the myeloid transcription factor CCAAT/enhancer-binding-protein-alpha (C/EBPalpha) or specific knockdown of the B cell transcription factor Pax5. Multiple iPS lines were clonally derived from both nonfully and fully differentiated B lymphocytes, which gave rise to adult chimeras with germline contribution, and to late-term embryos when injected into tetraploid blastocysts. Our study provides definite proof for the direct nuclear reprogramming of terminally differentiated adult cells to pluripotency. Full Text.

Hargreaves, R.H.J., David, C.L., Whitesell, L.J., LaBarbera, D.V., Jamil, A., Chapuis, J.C., and Skibo, E.B. (2008). Discovery of Quinolinediones Exhibiting a Heat Shock Response and Angiogenesis Inhibition. Journal of Medicinal Chemistry 51, 2492-2501. A series of substituted quinoline-5,8-diones were synthesized and evaluated as inhibitors of the chaperone protein Hsp90 using two assays: competition for binding to C-terminal ATP-binding site and competition for binding to N-terminal ATP-binding site. In addition, the ability of the compounds to induce the heat shock response was determined using a reporter fibroblast cell line. Of all the compounds assayed, only 6-aziridinyl-2-biphenylquinoline-5,8-dione induced a heat shock response and did so without interacting at the ATP binding sites of Hsp90. COMPARE analysis was carried out on quinoline-5,8-diones active in the National Cancer Institute's 60-cell line screen with the goal of discovering quinoline-5,8-dione structures that interact with other cellular targets (molecular targets) important for cancer chemotherapy. COMPARE analysis led to the discovery of a combretastatin- like quinoline-5,8-dione structure that, in fact, inhibited angiogenesis. Full Text.

Heikenwalder, M., Kurrer, M.O., Margalith, I., Kranich, J., Zeller, N., Haybaeck, J., Polymenidou, M., Matter, M., Bremer, J., Jackson, W.S.,Lindquist S., et al. (2008). Lymphotoxin-Dependent Prion Replication in Inflammatory Stromal Cells of Granulomas. Immunity 29, 998-1008. Prior to invading the nervous system, prions frequently colonize lymphoid organs and sites of inflammatory lymphoneogenesis, where they colocalize with Mfge8(+) follicular dendritic cells (FDCs). Here, we report that soft-tissue granulomas, a frequent feature of chronic inflammation, expressed the cellular prion protein (PrPC, encoded by Prnp) and the lymphotoxin receptor (LT beta R), even though they lacked FDCs and did not display lymphoneogenesis. After intraperitoneal prion inoculation, granulomas of Prnp(+/+) mice, but not Prnp(-/-) granulomas or unaffected Prnp(+/+) skin, accumulated prion infectivity and disease-associated prion protein. Bone-marrow transfers between Prnp(+/+) and Prnp(-/-) mice and administration of lymphotoxin signaling antagonists indicated that prion replication required radioresistant PrPC-expressing cells and LT beta R signaling. Granulomatous PrPC was mainly expressed by stromal LT beta R+ mesenchymal cells that were absent from unaffected subcutis. Hence, granulomas can act as clinically silent reservoirs of prion infectivity. Furthermore, lymphotoxin-dependent prion replication can occur in inflammatory stromal cells that are distinct from FDCs. Full Text.

Herrlich, A., Klinman, E., Fu, J., Sadegh, C., and Lodish, H. (2008). Ectodomain Cleavage of the Egf Ligands Hb-Egf, Neuregulin1-{Beta}, and Tgf-{Alpha} Is Specifically Triggered by Different Stimuli and Involves Different Pkc Isoenzymes. FASEB J. Aug 29. [Epub ahead of print] Metalloproteinase cleavage of transmembrane proteins (ectodomain cleavage), including the epidermal growth factor (EGF) ligands heparin-binding EGF-like growth factor (HB-EGF), neuregulin (NRG), and transforming growth factor-alpha (TGF-alpha), is important in many cellular signaling pathways and is disregulated in many diseases. It is largely unknown how physiological stimuli of ectodomain cleavage-hypertonic stress, phorbol ester, or activation of G-protein-coupled receptors [e.g., by lysophosphatidic acid (LPA)]-are molecularly connected to metalloproteinase activation. To study this question, we developed a fluorescence-activated cell sorting (FACS) -based assay that measures cleavage of EGF ligands in single living cells. EGF ligands expressed in mouse lung epithelial cells are differentially and specifically cleaved depending on the stimulus. Inhibition of protein kinase C (PKC) isoenzymes or metalloproteinase inhibition by batimastat (BB94) showed that different regulatory signals are used by different stimuli and EGF substrates, suggesting differential effects that act on the substrate, the metalloproteinase, or both. For example, hypertonic stress led to strong cleavage of HB-EGF and NRG but only moderate cleavage of TGF-alpha. HB-EGF, NRG, and TGF-alpha cleavage was not dependent on PKC, and only HB-EGF and NRG cleavage were inhibited by BB94. In contrast, phorbol 12-myristate-13-acetate (TPA) -induced cleavage of HB-EGF, NRG, and TGF-alpha was dependent on PKC and sensitive to BB94 inhibition. LPA led to significant cleavage of only NRG and TGF-alpha and was inhibited by BB94; only LPA-induced NRG cleavage required PKC. Surprisingly, specific inhibition of atypical PKCs zeta and iota [not activated by diacylglycerol (DAG) and calcium] significantly enhanced TPA-induced NRG cleavage. Employed in a high-throughput cloning strategy, our cleavage assay should allow the identification of candidate proteins involved in signal transduction of different extracellular stimuli into ectodomain cleavage.. PDF

Hockemeyer, D., Soldner, F., Cook, E.G., Gao, Q., Mitalipova, M., and Jaenisch, R. (2008). A Drug-Inducible System for Direct Reprogramming of Human Somatic Cells to Pluripotency. Cell Stem Cell 3, 346-353. Current approaches to reprogram human somatic cells to pluripotent iPSCs utilize viral transduction of different combinations of transcription factors. These protocols are highly inefficient because only a small fraction of cells carry the appropriate number and stoichiometry of proviral insertions to initiate the reprogramming process. Here we have generated genetically homogeneous "secondary" somatic cells, which carry the reprogramming factors as defined doxycycline (DOX)-inducible transgenes. These cells were obtained by infecting fibroblasts with DOX-inducible lentiviruses, isolating "primary" iPSCs in the presence of the drug, and finally differentiating to "secondary" fibroblasts. When "secondary" fibroblast lines were cultured in the presence of DOX without further viral infection, up to 2% of the cells were reprogrammed to pluripotent "secondary" human iPSCs. This system will facilitate the characterization of the reprogramming process and provides a unique platform for genetic or chemical screens to enhance reprogramming or replace individual factors. PDF

Hochwagen, A. (2008). Meiosis. Current Biology 18, R641-645. Full Text.

Howard, G., Eiges, R., Gaudet, F., Jaenisch, R., and Eden, A. (2008). Activation and Transposition of Endogenous Retroviral Elements in Hypomethylation Induced Tumors in Mice. Oncogene 27, 404-408. Genomewide DNA hypomethylation is a consistent finding in human tumors, but the importance of this change for human tumorigenesis remains an open question. We have previously reported that mice carrying a hypomorphic allele for the maintenance DNA methyltransferase (Dnmt1(chip/-)) are hypomethylated and develop thymic lymphomas, demonstrating that genomewide DNA hypomethylation can induce tumors. Hypomethylated cells exhibit inherent chromosomal instability, which is revealed in the lymphomas as a consistent trisomy of chromosome 15. We now report another aspect of the molecular basis for tumor development upon DNA hypomethylation. Seven out of 16 hypomethylation-induced lymphomas were found to contain an intracisternal A particle (IAP) somatic insertion in the middle of the Notch1 genomic locus, leading to generation of an oncogenic form of Notch1 in the tumors. This finding suggests that the molecular basis for hypomethylation-induced tumors in this model involves chromosomal instability events accompanied by activation of endogenous retroviral elements. Our findings validate the proposed role of DNA methylation in suppression of transposable elements in mammalian cells and demonstrate the importance of DNA methylation for normal cell function as well as the potential consequences of spontaneously occurring or chemically induced DNA hypomethylation. Full Text.

Hsu, P.P., and Sabatini, D.M. (2008). Cancer Cell Metabolism: Warburg and Beyond. Cell 134, 703-707. Described decades ago, the Warburg effect of aerobic glycolysis is a key metabolic hallmark of cancer, yet its significance remains unclear. In this Essay, we re-examine the Warburg effect and establish a framework for understanding its contribution to the altered metabolism of cancer cells. Full Text.

Huangfu, D.W., Osafune, K., Maehr, R., Guo, W., Eijkelenboom, A., Chen, S., Muhlestein, W., and Melton, D.A. (2008). Induction of Pluripotent Stem Cells from Primary Human Fibroblasts with Only Oct4 and Sox2. Nature Biotechnology 26, 1269-1275. Ectopic expression of defined sets of genetic factors can reprogram somatic cells to induced pluripotent stem (iPS) cells that closely resemble embryonic stem (ES) cells. The low efficiency with which iPS cells are derived hinders studies on the molecular mechanism of reprogramming, and integration of viral transgenes, in particular the oncogenes c-Myc and Klf4, may handicap this method for human therapeutic applications. Here we report that valproic acid (VPA), a histone deacetylase inhibitor, enables reprogramming of primary human fibroblasts with only two factors, Oct4 and Sox2, without the need for the oncogenes c-Myc or Klf4. The two factor-induced human iPS cells resemble human ES cells in pluripotency, global gene expression profiles and epigenetic states. These results support the possibility of reprogramming through purely chemical means, which would make therapeutic use of reprogrammed cells safer and more practical. Full Text.

Jaenisch R., and Young, R. (2008). Stem Cells, the Molecular Circuitry of Pluripotency and Nuclear Reprogramming. Cell 132, 567-582. Reprogramming of somatic cells to a pluripotent embryonic stem cell-like state has been achieved by nuclear transplantation of a somatic nucleus into an enucleated egg and most recently by introducing defined transcription factors into somatic cells. Nuclear reprogramming is of great medical interest, as it has the potential to generate a source of patient-specific cells. Here, we review strategies to reprogram somatic cells to a pluripotent embryonic state and discuss our understanding of the molecular mechanisms of reprogramming based on recent insights into the regulatory circuitry of the pluripotent state. Full Text.

Ji, P., Jayapal, S.R., and Lodish, H.F. (2008). Enucleation of Cultured Mouse Fetal Erythroblasts Requires Rac Gtpases and Mdia2. Nature Cell Biology. Published online: 10 February 2008 Mammalian erythroid cells undergo enucleation, an asymmetric cell division involving extrusion of a pycnotic nucleus enveloped by the plasma membrane. The mechanisms that power and regulate the enucleation process have remained obscure. Here, we show that deregulation of Rac GTPase during a late stage of erythropoiesis completely blocks enucleation of cultured mouse fetal erythroblasts without affecting their proliferation or differentiation. Formation of the contractile actin ring (CAR) on the plasma membrane of enucleating erythroblasts was disrupted by inhibition of Rac GTPases. Furthermore, we demonstrate that mDia2, a downstream effector of Rho GTPases and a formin protein required for nucleation of unbranched actin filaments, is also required for enucleation of mouse fetal erythroblasts. We show that Rac1 and Rac2 bind to mDia2 in a GTP-dependent manner and that downregulation of mDia2, but not mDia1, by small interfering RNA (siRNA) during the late stages of erythropoiesis blocked both CAR formation and erythroblast enucleation. Additionally, overexpression of a constitutively active mutant of mDia2 rescued the enucleation defects induced by the inhibition of Rac GTPases. These results reveal important roles for Rac GTPases and their effector mDia2 in enucleation of mammalian erythroblasts. Full Text.

Johnnidis, J.B., and Camargo, F.D. (2008). Isolation and Functional Characterization of Side Population Stem Cells. Methods Mol Biol 430, 183-193. The "side population" (SP) phenotype is a manifestation of primitive cells' ability to efficiently efflux the fluorescent DNA-staining dye Hoechst 33342 and can be used as the basis by which to isolate these cells using flow cytometry. In the bone marrow (BM), the SP defines a cell subset with a highly homogeneous content of hematopoietic stem cells (HSCs). In this chapter, we describe a protocol to reproducibly isolate murine BM SP cells, as well as analytic measures, such as single cell transplantation, that can be used to assess the functionality of SP-derived stem cells.

Johnnidis, J.B., Harris, M.H., Wheeler, R.T., Stehling-Sun, S., Lam, M.H., Kirak, O., Brummelkamp, T.R., Fleming, M.D., and Camargo, F.D. (2008). Regulation of Progenitor Cell Proliferation and Granulocyte Function by Microrna-223. Nature Feb 17 [Epub ahead of print]. MicroRNAs are abundant in animal genomes and have been predicted to have important roles in a broad range of gene expression programmes. Despite this prominence, there is a dearth of functional knowledge regarding individual mammalian microRNAs. Using a loss-of-function allele in mice, we report here that the myeloid-specific microRNA-223 (miR-223) negatively regulates progenitor proliferation and granulocyte differentiation and activation. miR-223 (also called Mirn223) mutant mice have an expanded granulocytic compartment resulting from a cell-autonomous increase in the number of granulocyte progenitors. We show that Mef2c, a transcription factor that promotes myeloid progenitor proliferation, is a target of miR-223, and that genetic ablation of Mef2c suppresses progenitor expansion and corrects the neutrophilic phenotype in miR-223 null mice. In addition, granulocytes lacking miR-223 are hypermature, hypersensitive to activating stimuli and display increased fungicidal activity. As a consequence of this neutrophil hyperactivity, miR-223 mutant mice spontaneously develop inflammatory lung pathology and exhibit exaggerated tissue destruction after endotoxin challenge. Our data support a model in which miR-223 acts as a fine-tuner of granulocyte production and the inflammatory response. Full Text.

Johnson, B.S., McCaffery, J.M., Lindquist, S., and Gitler, A.D. (2008). A Yeast Tdp-43 Proteinopathy Model: Exploring the Molecular Determinants of Tdr-43 Aggregation and Cellular Toxicity. Proceedings of the National Academy of Sciences of the United States of America 105, 6439-6444. Protein misfolding is intimately associated with devastating human neurodegenerative diseases, including Alzheimer's, Huntington's, and Parkinson's. Although disparate in their pathophysiology, many of these disorders share a common theme, manifested in the accumulation of insoluble protein aggregates in the brain. Recently, the major disease protein found in the pathological inclusions of two of these diseases, amyotrophic lateral sclerosis (ALS) and frontal temporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U), was identified as the 43-kDa TAR-DNA-binding protein (TDP-43), providing a molecular link between them. TDP-43 is a ubiquitously expressed nuclear protein that undergoes a pathological conversion to an aggregated cytoplasmic localization in affected regions of the nervous system. Whether TDP-43 itself can convey toxicity and whether its abnormal aggregation is a cause or consequence of pathogenesis remain unknown. We report a yeast model to define mechanisms governing TDP-43 subcellular localization and aggregation. Remarkably, this simple model recapitulates several salient features of human TDP-43 proteinopathies, including conversion from nuclear localization to cytoplasmic aggregation. We establish a connection between this aggregation and toxicity. The pathological features of TDP-43 are distinct from those of yeast models of other protein-misfolding diseases, such as polyglutamine. This suggests that the yeast model reveals specific aspects of the underlying biology of the disease protein rather than general cellular stresses associated with accumulating misfolded proteins. This work provides a mechanistic framework for investigating the toxicity of TDP-43 aggregation relevant to human disease and establishes a manipulable, high-throughput model for discovering potential therapeutic strategies. Full Text

Johnson, D.S., Li, W., Gordon, D.B., Bhattacharjee, A., Curry, B., Ghosh, J., Brizuela, L., Carroll, J.S., Brown, M., Flicek, P., Scott A. McCuine, et al. (2008). Systematic Evaluation of Variability in Chip-Chip Experiments Using Predefined DNA Targets. Genome Research 18, 393-403. The most widely used method for detecting genome-wide protein-DNA interactions is chromatin immunoprecipitation on tiling microarrays, commonly known as ChIP-chip. Here, we conducted the first objective analysis of tiling array platforms, amplification procedures, and signal detection algorithms in a simulated ChIP-chip experiment. Mixtures of human genomic DNA and "spike-ins" comprised of nearly 100 human sequences at various concentrations were hybridized to four tiling array platforms by eight independent groups. Blind to the number of spike-ins, their locations, and the range of concentrations, each group made predictions of the spike-in locations. We found that microarray platform choice is not the primary determinant of overall performance. In fact, variation in performance between labs, protocols, and algorithms within the same array platform was greater than the variation in performance between array platforms. However, each array platform had unique performance characteristics that varied with tiling resolution and the number of replicates, which have implications for cost versus detection power. Long oligonucleotide arrays were slightly more sensitive at detecting very low enrichment. On all platforms, simple sequence repeats and genome redundancy tended to result in false positives. LM-PCR and WGA, the most popular sample amplification techniques, reproduced relative enrichment levels with high fidelity. Performance among signal detection algorithms was heavily dependent on array platform. The spike-in DNA samples and the data presented here provide a stable benchmark against which future ChIP platforms, protocol improvements, and analysis methods can be evaluated. Full Text.

Kario, E., Tirosh, B., Ploegh, H.L., and Navon, A. (2008). N-Linked Glycosylation Does Not Impair Proteasomal Degradation but Affects Class I Major Histocompatibility Complex Presentation. Journal of Biological Chemistry 283, 244-254. The addition of N-linked glycans to nascent polypeptides occurs cotranslationally in the endoplasmic reticulum (ER). For many proteins the state of the glycans serves as an indicator, which allows the ER quality control system to monitor the conformation of polypeptides upon folding. Proteins that fail to fold in the ER are often dislocated to the cytoplasm, where they are subjected to proteasomal degradation. Although the addition of N-linked glycans occurs within the ER, non-lysosomal removal of the glycans occurs in the cytosol by the action of peptide N-glycanase (PNGase). In this study, we investigated the interplay between PNGase action and proteasomal degradation of ER misfolded proteins (i.e. whether PNGase acts prior to or following proteasomal degradation). Interestingly, we found that glycan removal from N-terminally extended peptides modulates the presentation of class I major histocompatibility complex-restricted epitopes. Our findings provide direct evidence that the proteasome is capable of degrading glycoproteins without prior removal of their glycans. This degradation is independent of either the identity of the glycosylated protein or the type and number of N-linked glycans it harbors. We also captured and characterized glycopeptides generated following proteasomal degradation of RNaseB. Although the carbohydrate moiety reduced the variability of the degradation products that include the glycosylated residue ( local effect), the overall global digestion pattern of RNaseB was unaffected. Together with earlier findings by others, our data support a model in which PNGase may act both upstream and downstream to proteasomal degradation and demonstrates its important role in class I major histocompatibility complex antigen presentation. Full Text.

Karnoub, A.E., and Weinberg, R.A. (2008). Ras Oncogenes: Split Personalities. Nat Rev Mol Cell Biol 9, 517-531. Extensive research on the Ras proteins and their functions in cell physiology over the past 30 years has led to numerous insights that have revealed the involvement of Ras not only in tumorigenesis but also in many developmental disorders. Despite great strides in our understanding of the molecular and cellular mechanisms of action of the Ras proteins, the expanding roster of their downstream effectors and the complexity of the signalling cascades that they regulate indicate that much remains to be learnt. Full Text.

Kim, Y.M., Brinkmann, M.M., Paquet, M.E., and Ploegh, H.L. (2008). Unc93b1 Delivers Nucleotide-Sensing Toll-Like Receptors to Endolysosomes. Nature Published online 27 February 2008. Signalling by means of toll-like receptors (TLRs) is essential for the development of innate and adaptive immune responses. UNC93B1, essential for signalling of TLR3, TLR7 and TLR9 in both humans and mice, physically interacts with these TLRs in the endoplasmic reticulum (ER). Here we show that the function of the polytopic membrane protein UNC93B1 is to deliver the nucleotide-sensing receptors TLR7 and TLR9 from the ER to endolysosomes. In dendritic cells of 3d mice, which express an UNC93B1 missense mutant (H412R) incapable of TLR binding, neither TLR7 nor TLR9 exits the ER. Furthermore, the trafficking and signalling defects of the nucleotide-sensing TLRs in 3d dendritic cells are corrected by expression of wild-type UNC93B1. However, UNC93B1 is dispensable for ligand recognition and signal initiation by TLRs. To our knowledge, UNC93B1 is the first protein to be identified as a molecule specifically involved in trafficking of nucleotide-sensing TLRs. By inhibiting the interaction between UNC93B1 and TLRs it should be possible to achieve specific regulation of the nucleotide-sensing TLRs without compromising signalling via the cell-surface-disposed TLRs. Full Text..

Lange, J., Skaletsky, H., Bell, G.W., and Page, D.C. (2008). Msy Breakpoint Mapper, a Database of Sequence-Tagged Sites Useful in Defining Naturally Occurring Deletions in the Human Y Chromosome. Nucleic Acids Res 36, D809-814. Y chromosome deletions arise frequently in human populations, where they cause sex reversal and Turner syndrome and predispose individuals to infertility and germ cell cancer. Knowledge of the nucleotide sequence of the male-specific region of the Y chromosome (MSY) makes it possible to precisely demarcate such deletions and the repertoires of genes lost, offering insights into mechanisms of deletion and the molecular etiologies of associated phenotypes. Such deletion mapping is usually conducted using polymerase chain reaction (PCR) assays for the presence or absence of a series of Y-chromosomal DNA markers, or sequence-tagged sites (STSs). In the course of mapping intact and aberrant Y chromosomes during the past two decades, we and our colleagues have developed robust PCR assays for 1287 Y-specific STSs. These PCR assays amplify 1698 loci at an average spacing of <14 kb across the MSY euchromatin. To facilitate mapping of deletions, we have compiled a database of these STSs, MSY Breakpoint Mapper (http://breakpointmapper.wi.mit.edu/). When queried, this online database provides regionally targeted catalogs of STSs and nearby genes. MSY Breakpoint Mapper is useful for efficiently and systematically defining the breakpoint(s) of virtually any naturally occurring Y chromosome deletion. Full Text.

Lengner, C.J., Welstead, G.G., and Jaenisch, R. (2008). The Pluripotency Regulator Oct4: A Role in Somatic Stem Cells? Cell Cycle 7(6) 15 March 2008 . Since its discovery as a critical regulator of pluripotency in embryonic stem (ES) cells and the inner cells mass of the developing blastocyst, the Pou domaincontaining transcription factor Oct4 has become a proxy for "stemness" in numerous studies of somatic stem cells as it's presence is often taken as evidence of pluripotency in these cells. Recent studies, however, have demonstrated that not only is Oct4 dispensable for maintaining potency in somatic stem cell compartments, but also that the methods applied to detect Oct4 and the interpretation of the resulting data may be flawed. Here we contrast pathways known to govern pluripotency in embryonic stem cells with those in adult stem cells and critically discuss the concept of pluripotency in adult stem cells of the mammalian soma.

Lin, Y., Gill, M.E., Koubova, J., and Page, D.C. (2008). Germ Cell-Intrinsic and -Extrinsic Factors Govern Meiotic Initiation in Mouse Embryos. Science 322, 1685-1687. Retinoic acid (RA) is an essential extrinsic inducer of meiotic initiation in mammalian germ cells. However, RA acts too widely in mammalian development to account, by itself, for the cell-type and temporal specificity of meiotic initiation. We considered parallels to yeast, in which extrinsic and intrinsic factors combine to restrict meiotic initiation. We demonstrate that, in mouse embryos, extrinsic and intrinsic factors together regulate meiotic initiation. The mouse RNA-binding protein DAZL, which is expressed by postmigratory germ cells, is a key intrinsic factor, enabling those cells to initiate meiosis in response to RA. Within a brief developmental window, Dazl-expressing germ cells in both XX and XY embryos actively acquire the ability to interpret RA as a meiosis-inducing signal. Full Text.

Linhart, H.G., Troen, A., Bell, G.W., Cantu, E., Chao, W.H., Moran, E., Steine, E., He, T., and Jaenisch, R. (2008). Folate Deficiency Induces Genomic Uracil Misincorporation and Hypomethylation but Does Not Increase DNA Point Mutations. Gastroenterology.Oct 9. [Epub ahead of print] BACKGROUND AND AIMS: Epidemiologic studies have linked nutritional folate deficiency to an increased risk of cancer, but recent trials suggest that folate supplementation does not protect against tumor formation. Our aim was to analyze the genetic and epigenetic consequences of folate deficiency and to investigate whether impairment of the uracil base excision repair pathway can enhance its effects. METHODS: Wild-type mice and those deficient in uracil DNA glycosylase (Ung(-/-)) were placed on a folate-deficient diet for 8 months. We measured tumor incidence in major organs, DNA mutation rates, DNA mutation spectra, local DNA methylation, and global DNA methylation in colon epithelial cells. RESULTS: The experimental diet increased plasma homocysteine (60%, P < .001) and DNA uracil content (24%, P < .05) but not tumor formation. Global DNA methylation was slightly decreased in splenocytes (9.1%) and small intestinal epithelial cells (4.2%), and significantly reduced in colon epithelial cells (7.2%, P < .04). No gene-specific changes in methylation were detected at the mouse B1 element, the H19 DMR, or the Oct4 gene. By lambdaCII assay and sequencing analysis of 730 mutants, we found that Ung(-/-) mice had a higher frequency of point mutations and increased C:G to T:A transitions at non-CpG sites. However, folate deficiency had no additional effect on the DNA mutation frequency or spectrum in Ung(-/-) or wild-type mice. CONCLUSIONS: Contradicting current concepts, these findings indicate that the effects of a low-folate diet on DNA methylation and point mutations are insufficient to promote tumor development, even in the presence of Ung deficiency. Full Text.

LoBianco, C., Shorter, J., Regulier, E., Lashuel, H., Iwatsubo, T., Lindquist, S., and Aebischer, P. (2008). Hsp104 Antagonizes Alpha-Synuclein Aggregation and Reduces Dopaminergic Degeneration in a Rat Model of Parkinson Disease. Journal of Clinical Investigation(New Online) Parkinson disease (PD) is characterized by dopaminergic neurodegeneration and intracellular inclusions of alpha-synuclein amyloid fibers, which are stable and difficult to dissolve. Whether inclusions are neuroprotective or pathological remains controversial, because prefibrillar oligomers may be more toxic than amyloid inclusions. Thus, whether therapies should target inclusions, preamyloid oligomers, or both is a critically important issue. In yeast, the protein-remodeling factor Hsp104 cooperates with Hsp70 and Hsp40 to dissolve and reactivate aggregated proteins. Metazoans, however, have no Hsp104 ortholog. Here we introduced Hsp104 into a rat PD model. Remarkably, Hsp104 reduced formation of phosphorylated alpha-synuclein inclusions and prevented nigrostriatal dopaminergic neurodegeneration induced by PD-linked alpha-synuclein (A30P). An in vitro assay employing pure proteins revealed that Hsp104 prevented fibrillization of alpha-synuclein and PD-linked variants (A30P, A53T, E46K). Hsp104 coupled ATP hydrolysis to the disassembly of preamyloid oligomers and amyloid fibers composed of alpha-synuclein. Furthermore, the mammalian Hsp70 and Hsp40 chaperones, Hsc70 and Hdj2, enhanced alpha-synuclein fiber disassembly by Hsp104. Hsp104 likely protects dopaminergic neurons by antagonizing toxic alpha-synuclein assemblies and might have therapeutic potential for PD and other neurodegenerative amyloidoses. Full Text.

Lodish, H.F., Zhou, B., Liu, G., and Chen, C.Z. (2008). Micromanagement of the Immune System by Micrornas. Nat Rev Immunol.advance online publication, 21 January 2008 MicroRNAs (miRNAs) are an abundant class of evolutionarily conserved small non-coding RNAs that are thought to control gene expression by targeting mRNAs for degradation or translational repression. Emerging evidence suggests that miRNA-mediated gene regulation represents a fundamental layer of genetic programmes at the post-transcriptional level and has diverse functional roles in animals. Here, we provide an overview of the mechanisms by which miRNAs regulate gene expression, with specific focus on the role of miRNAs in regulating the development of immune cells and in modulating innate and adaptive immune responses. Full Text.

Lopez-Bigas, N., Kisiel, T.A., DeWaal, D.C., Holmes, K.B., Volkert, T.L., Gupta, S., Love, J., Murray, H.L., Young, R.A., and Benevolenskaya, E.V. (2008). Genome-Wide Analysis of the H3k4 Histone Demethylase Rbp2 Reveals a Transcriptional Program Controlling Differentiation. Molecular Cell 31, 520-530. Retinoblastoma protein (pRB) mediates cell-cycle withdrawal and differentiation by interacting with a variety of proteins. RB-Binding Protein 2 (RBP2) has been shown to be a key effector. We sought to determine transcriptional regulation by RBP2 genome-wide by using location analysis and gene expression profiling experiments. We describe that RBP2 shows high correlation with the presence of H3K4me3 and its target genes are separated into two functionally distinct classes: differentiation-independent and differentiation-dependent genes. The former class is enriched by genes that encode mitochondrial proteins, while the latter is represented by cell-cycle genes. We demonstrate the role of RBP2 in mitochondrial biogenesis, which involves regulation of H3K4me3-modified nucleosomes. Analysis of expression changes upon RBP2 depletion depicted genes with a signature of differentiation control, analogous to the changes seen upon reintroduction of pRB. We conclude that, during differentiation, RBP2 exerts inhibitory effects on multiple genes through direct interaction with their promoters. Full Text.

Lowery, L.A., De Rienzo, G., Gutzman, J.H., and Sive, H. (2008). Characterization and Classification of Zebrafish Brain Morphology Mutants.Anatatomical Record (Hoboken). 2008 Dec 2. [Epub ahead of print]. The mechanisms by which the vertebrate brain achieves its three-dimensional structure are clearly complex, requiring the functions of many genes. Using the zebrafish as a model, we have begun to define genes required for brain morphogenesis, including brain ventricle formation, by studying 16 mutants previously identified as having embryonic brain morphology defects. We report the phenotypic characterization of these mutants at several timepoints, using brain ventricle dye injection, imaging, and immunohistochemistry with neuronal markers. Most of these mutants display early phenotypes, affecting initial brain shaping, whereas others show later phenotypes, affecting brain ventricle expansion. In the early phenotype group, we further define four phenotypic classes and corresponding functions required for brain morphogenesis. Although we did not use known genotypes for this classification, basing it solely on phenotypes, many mutants with defects in functionally related genes clustered in a single class. In particular, Class 1 mutants show midline separation defects, corresponding to epithelial junction defects; Class 2 mutants show reduced brain ventricle size; Class 3 mutants show midbrain-hindbrain abnormalities, corresponding to basement membrane defects; and Class 4 mutants show absence of ventricle lumen inflation, corresponding to defective ion pumping. Later brain ventricle expansion requires the extracellular matrix, cardiovascular circulation, and transcription/splicing-dependent events. We suggest that these mutants define processes likely to be used during brain morphogenesis throughout the vertebrates.

Lu, X.H., Huang, L.J.S., and Lodish, H.F. (2008). Dimerization by a Cytokine Receptor Is Necessary for Constitutive Activation of Jak2v617f. Journal of Biological Chemistry 283, 5258-5266. The majority of the BCR-ABL-negative myeloproliferative disorders express the mutant JAK2, JAK2V617F. Previously we showed that constitutive activation of this oncogenic JAK2 mutant in Ba/F3 or 32D cells requires coexpression of a cognate homodimeric cytokine receptor, such as the EpoR. However, overexpression of JAK2V617F in Ba/F3 cells renders them cytokine-independent for growth in the absence of an exogenous cytokine receptor. Here, we demonstrated that JAK2V617F domains required for receptor association are essential for cytokine-independent growth by overexpressed JAK2V617F, suggesting JAK2V617F is binding to an unknown endogenous cytokine receptor(s) for its activation. We further showed that disruption of EpoR dimerization by coexpressing a truncated EpoR disrupted JAK2V617F-mediated transformation, indicating that EpoR dimerization plays an essential role in the activation of JAK2V617F. Interestingly, coexpression of JAK2V617F with EpoR mutants that retain JAK2 binding but are defective in mediating Epo-dependent JAK2 activation due to mutations in a conserved juxtamembrane motif does lead to cytokine-independent activation of JAK2V617F. Overall, these findings confirm that JAK2V617F requires binding to a dimerized cytokine receptor for its activation, and that the key EpoR juxtamembrane regulatory motif essential for Epo-dependent JAK2 activation is not essential for the activation of JAK2V617F. The structure of the activated JAK2V617F is thus likely to be different from that of the activated wild-type JAK2, raising the possibility of developing a specifically targeted therapy for myeloproliferative disorders. Full Text.

Luo, B., Cheung, H.W., Subramanian, A., Sharifnia, T., Okamoto, M., Yang, X.P., Hinkle, G., Boehm, J.S., Beroukhim, R., Weir, B.A., Sabatini DM, et al. (2008). Highly Parallel Identification of Essential Genes in Cancer Cells. Proceedings of the National Academy of Sciences of the United States of America 105, 20380-20385. More complete knowledge of the molecular mechanisms underlying cancer will improve prevention, diagnosis and treatment. Efforts such as The Cancer Genome Atlas are systematically characterizing the structural basis of cancer, by identifying the genomic mutations associated with each cancer type. A powerful complementary approach is to systematically characterize the functional basis of cancer, by identifying the genes essential for growth and related phenotypes in different cancer cells. Such information would be particularly valuable for identifying potential drug targets. Here, we report the development of an efficient, robust approach to perform genome-scale pooled shRNA screens for both positive and negative selection and its application to systematically identify cell essential genes in 12 cancer cell lines. By integrating these functional data with comprehensive genetic analyses of primary human tumors, we identified known and putative oncogenes such as EGFR, KRAS, MYC, BCR-ABL, MYB, CRKL, and CDK4 that are essential for cancer cell proliferation and also altered in human cancers. We further used this approach to identify genes involved in the response of cancer cells to tumoricidal agents and found 4 genes required for the response of CML cells to imatinib treatment: PTPN1, NF1, SMARCB1, and SMARCE1, and 5 regulators of the response to FAS activation, FAS, FADD, CASP8, ARID1A and CBX1. Broad application of this highly parallel genetic screening strategy will not only facilitate the rapid identification of genes that drive the malignant state and its response to therapeutics but will also enable the discovery of genes that participate in any biological process. Full Text.

Ma, L., Teruya-Feldstein, J., and Weinberg, R.A. (2008). Ma Et Al. Reply. Nature 455, E9. Replying to: H. E. Gee, et al. 455, 10.1038/nature07362 (2008)Gee et al. contest that microRNA-10b is not a prognostic marker for metastasis risk in breast cancer. However, their observations do not bear on the pro-metastatic roles of microRNA-10b (miR-10b) as we described them, nor do they undermine our conclusions. Full Text.

Ma, L., and Weinberg, R.A. (2008). Micromanagers of Malignancy: Role of Micrornas in Regulating Metastasis. Trends in Genetics Jul 30. [Epub ahead of print] It has become evident that cancer pathogenesis involves, among other macromolecules, a class of small regulatory RNAs named microRNAs, and that microRNA expression profiles are associated with prognosis and therapeutic outcome in several human cancers. Although the oncogenic or tumor-suppressing functions of several microRNAs have been characterized, the mechanistic roles played by microRNAs specifically in mediating metastasis have been addressed only recently. In this review, we focus on our emerging understanding of the contributions of microRNAs to malignant progression, specifically their functions in mediating tumor invasion and metastasis. These findings illuminate the molecular basis of metastasis and begin to connect small-RNA discoveries to the development of novel clinical biomarkers and therapeutic targets in neoplastic diseases. PDF

Mani S.A., Guo, W., Liao, M.J., Eaton, E.N., Ayyanan, A., Zhou, A.Y., Brooks, M., Reinhard, F., Zhang, C.C., Shipitsin, M., Robert A Weinberg et al(2008). The Epithelial-Mesenchymal Transition Generates Cells with Properties of Stem Cells. Cell 133, 704-715. The epithelial-mesenchymal transition (EMT) is a key developmental program that is often activated during cancer invasion and metastasis. We here report that the induction of an EMT in immortalized human mammary epithelial cells (HMLEs) results in the acquisition of mesenchymal traits and in the expression of stem-cell markers. Furthermore, we show that those cells have an increased ability to form mammospheres, a property associated with mammary epithelial stem cells. Independent of this, stem cell-like cells isolated from HMLE cultures form mammospheres and express markers similar to those of HMLEs that have undergone an EMT. Moreover, stem-like cells isolated either from mouse or human mammary glands or mammary carcinomas express EMT markers. Finally, transformed human mammary epithelial cells that have undergone an EMT form mammospheres, soft agar colonies, and tumors more efficiently. These findings illustrate a direct link between the EMT and the gain of epithelial stem cell properties. Full Text.

Markoulaki, S., Meissner, A., and Jaenisch, R. (2008). Somatic Cell Nuclear Transfer and Derivation of Embryonic Stem Cells in the Mouse. Methods 45, 101-114. Addressing the fundamental questions of nuclear equivalence in somatic cells has fascinated scientists for decades and has resulted in the development of somatic cell nuclear transfer (SCNT) or animal cloning. SCNT involves the transfer of the nucleus of a somatic cell into the cytoplasm of an egg whose own chromosomes have been removed. In the mouse, SCNT has not only been successfully used to address the issue of nuclear equivalence, but has been used as a model system to test the hypothesis that embryonic stem cells (ESCs) derived from NT blastocysts have the potential to correct-through genetic manipulations-degenerative diseases. This paper aims to provide a comprehensive description of SCNT in the mouse and the derivation of ESCs from blastocysts generated by this technique. SCNT is a very challenging and inefficient procedure because it is technically complex, it bypasses the normal events of gamete interactions and egg activation, and it depends on adequate reprogramming of the somatic cell nucleus in vivo. Improvements in any or all those aspects may enhance the efficiency and applicability of SCNT. ESC derivation from SCNT blastocysts, on the other hand, requires the survival of only a few successfully reprogrammed cells, which have the capacity to proliferate indefinitely in vitro, maintain correct genetic and epigenetic status, and differentiate into any cell type in the body-characteristics that are essential for transplantation therapy or any other in vivo application. PDF

Marson, A., Foreman, R., Chevalier, B., Bilodeau, S., Kahn, M., Young, R.A., and Jaenisch, R. (2008). Wnt Signaling Promotes Reprogramming of Somatic Cells to Pluripotency. Cell Stem Cell 3, 132-135. Full Text.

Marson, A., Levine, S.S., Cole, M.F., Frampton, G.M., Brambrink, T., Johnstone, S., Guenther, M.G., Johnston, W.K., Wernig, M., Newman, J.,Young RA et al. (2008). Connecting Microrna Genes to the Core Transcriptional Regulatory Circuitry of Embryonic Stem Cells. Cell 134, 521-533. MicroRNAs (miRNAs) are crucial for normal embryonic stem (ES) cell self-renewal and cellular differentiation, but how miRNA gene expression is controlled by the key transcriptional regulators of ES cells has not been established. We describe here the transcriptional regulatory circuitry of ES cells that incorporates protein-coding and miRNA genes based on high-resolution ChIP-seq data, systematic identification of miRNA promoters, and quantitative sequencing of short transcripts in multiple cell types. We find that the key ES cell transcription factors are associated with promoters for miRNAs that are preferentially expressed in ES cells and with promoters for a set of silent miRNA genes. This silent set of miRNA genes is co-occupied by Polycomb group proteins in ES cells and shows tissue-specific expression in differentiated cells. These data reveal how key ES cell transcription factors promote the ES cell miRNA expression program and integrate miRNAs into the regulatory circuitry controlling ES cell identity. Full Text.

Mathur, D., Danford, T.W., Boyer, L.A., Young, R.A., Gifford, D.K., and Jaenisch, R. (2008). Analysis of the Mouse Embryonic Stem Cell Regulatory Networks Obtained by Chip-Chip and Chip-Pet. Genome Biology 9(8):R126. Epub 2008 Aug 13. Background: Genomewide approaches have begun to reveal the transcriptional networks responsible for pluripotency in embryonic stem (ES) cells. Chromatin Immunoprecipitation (ChIP) followed either by hybridization to a microarray platform (ChIP-chip) or by DNA sequencing (ChIP-PET), has identified binding targets of the ES cell transcription factors OCT4 and NANOG in humans and mice, respectively. These studies have provided an outline of the transcriptional framework involved in maintaining pluripotency. Recent evidence with comparing multiple technologies suggests that expanding these datasets using different platforms would be a useful resource for examining the mechanisms underlying pluripotency regulation. Results: We have now identified OCT4 and NANOG genomic targets in mouse ES cells by ChIPchip and provided the means to compare these data with previously reported ChIP-PET results in mouse ES cells. We have mapped the sequences of OCT4 and NANOG binding events from each dataset to genomic coordinates, providing a valuable resource to facilitate a better understanding of the ES cell regulatory circuitry. Interestingly, although considerable differences are observed in OCT4 and NANOG occupancy as identified by each method, a substantial number of targets in both datasets are enriched for genes that have known roles in cell fate specification and that are differentially expressed upon Oct4 or Nanog knockdown. Conclusion: This study suggests that each dataset is a partial representation of the overall ES cell regulatory circuitry, and through integrating binding data obtained by ChIP chip and ChIP-PET, the methods presented here provide a useful means for integrating datasets obtained by different techniques in the future. Full Text.

McAllister, S.S., Gifford, A.M., Greiner, A.L., Kelleher, S.P., Saelzler, M.P., Ince, T.A., Reinhardt, F., Harris, L.N., Hylander, B.L., Repasky, E.A.,and Robert A Weinberg (2008). Systemic Endocrine Instigation of Indolent Tumor Growth Requires Osteopontin. Cell 133, 994-1005. The effects of primary tumors on the host systemic environment and resulting contributions of the host to tumor growth are poorly understood. Here, we find that human breast carcinomas instigate the growth of otherwise-indolent tumor cells, micrometastases, and human tumor surgical specimens located at distant anatomical sites. This systemic instigation is accompanied by incorporation of bone-marrow cells (BMCs) into the stroma of the distant, once-indolent tumors. We find that BMCs of hosts bearing instigating tumors are functionally activated prior to their mobilization; hence, when coinjected with indolent cells, these activated BMCs mimic the systemic effects imparted by instigating tumors. Secretion of osteopontin by instigating tumors is necessary for BMC activation and the subsequent outgrowth of the distant otherwise-indolent tumors. These results reveal that outgrowth of indolent tumors can be governed on a systemic level by endocrine factors released by certain instigating tumors, and hold important experimental and therapeutic implications. Full Text.

McKenna, B.K., Selim, A.A., Richard Bringhurst, F., and Ehrlich, D.J. (2009). 384-Channel Parallel Microfluidic Cytometer for Rare-Cell Screening. Lab Chip 9, 305-310. We have constructed a 384-channel parallel microfluidic cytometer (PMC). The multichannel architecture allows 384 unique samples for a cell-based screen to be read out in approximately 6-10 min, about 30-times the speed of a conventional fluorescence-activated cytometer system (FACS). This architecture also allows the signal integration time to be varied over a larger range than is practical in single-channel FACS and is suitable for detection of rare-cells in a high background of negatives. The signal-to-noise advantages have been confirmed by using the system to count rare clonal osteocytes in the most difficult early stages of an expression-cloning screen for the carboxy-terminal parathyroid hormone receptor (CPTHR). This problem requires finding several dozen positive cells in a background of one million negatives. The system is automated around a scanning laser confocal detector and a 96-tip robotic pipettor and can maintain in vitro cultures on-system in 384-well plates. It is therefore directly practical for biology applications using existing high-throughput culture facilities. The PMC system lends itself to high-sample-number cytometry with an unusual capability for time synchronization and rare-cell sensitivity. A limited ability to handle large sample numbers has restricted applications of single-channel FACS in combinatorial cell assays; therefore the PMC could have a significant application in high-throughput screening. Full Text.

Meissner, A., Eminli, S., and Jaenisch, R. (2009). Derivation and Manipulation of Murine Embryonic Stem Cells. Methods Mol Biol 482, 3-19. Pluripotent embryonic stem (ES) cell lines were first isolated over 25 years ago and remain an essential tool in molecular and developmental biology to this day. In particular, the use of homologous recombination and subsequent generation of ES-derived mice has greatly facilitated research across all fields. Moreover, ES cells represent an extremely attractive model to study events in early development. In this chapter, we will describe the derivation and propagation of murine ES cells. This is followed by a description of targeting ES cells and a protocol for the generation of mice by diploid and tetraploid blastocyst injections. Full Text.

Meissner, A., Mikkelsen, T.S., Gu, H., Wernig, M., Hanna, J., Sivachenko, A., Zhang, X., Bernstein, B.E., Nusbaum, C., Jaffe, D.B., Wernig M, Jaenisch R et al(2008). Genome-Scale DNA Methylation Maps of Pluripotent and Differentiated Cells. Nature. Jul 6. [Epub ahead of print] DNA methylation is essential for normal development and has been implicated in many pathologies including cancer. Our knowledge about the genome-wide distribution of DNA methylation, how it changes during cellular differentiation and how it relates to histone methylation and other chromatin modifications in mammals remains limited. Here we report the generation and analysis of genome-scale DNA methylation profiles at nucleotide resolution in mammalian cells. Using high-throughput reduced representation bisulphite sequencing and single-molecule-based sequencing, we generated DNA methylation maps covering most CpG islands, and a representative sampling of conserved non-coding elements, transposons and other genomic features, for mouse embryonic stem cells, embryonic-stem-cell-derived and primary neural cells, and eight other primary tissues. Several key findings emerge from the data. First, DNA methylation patterns are better correlated with histone methylation patterns than with the underlying genome sequence context. Second, methylation of CpGs are dynamic epigenetic marks that undergo extensive changes during cellular differentiation, particularly in regulatory regions outside of core promoters. Third, analysis of embryonic-stem-cell-derived and primary cells reveals that 'weak' CpG islands associated with a specific set of developmentally regulated genes undergo aberrant hypermethylation during extended proliferation in vitro, in a pattern reminiscent of that reported in some primary tumours. More generally, the results establish reduced representation bisulphite sequencing as a powerful technology for epigenetic profiling of cell populations relevant to developmental biology, cancer and regenerative medicine. Full Text.

Mirsaidov, U., Timp, W., Timp, K., Mir, M., Matsudaira, P., and Timp, G. (2008). Optimal Optical Trap for Bacterial Viability. Physical Review E 78. Optical trapping is a powerful tool for the micromanipulation of living cells-especially bacteria-but photodamage induced by the laser beam can adversely affect viability. We have explored optical trapping conditions in the near infrared (840-930 nm) that preserve the viability of E. coli, as measured by gene expression of green fluorescent protein. We have found that time-sharing the optical traps, i.e., dwelling only 10 mu s-1 ms on the cell, improves viability relative to continuous wave (CW) exposure for the same exposure time. We have also observed that similar to CW traps the photodamage in a time-shared trap depends weakly on wavelength, but linearly on peak power, implying an effect induced by single photon absorption. Taken altogether, integrating the exposure time and peak power, the data indicate that there is a lethal energy dose of about 5 J for E. coli. Thus a single parameter-the energy-can be used to describe the limitation on viability. Full Text.

Mueller, B., Klemm, E.J., Spooner, E., Claessen, J.H., and Ploegh, H.L. (2008). Sel1l Nucleates a Protein Complex Required for Dislocation of Misfolded Glycoproteins. Proc Natl Acad Sci U S A. Aug 26;105(34):12325-30.Membrane and secretory proteins that fail to pass quality control in the endoplasmic reticulum are discharged into the cytosol and degraded by the proteasome. Many of the mammalian components involved in this process remain to be identified. We performed a biochemical search for proteins that interact with SEL1L, a protein that is part of the mammalian HRD1 ligase complex and involved in substrate recognition. SEL1L is crucial for dislocation of Class I major histocompatibility complex heavy chains by the human cytomegalovirus US11 protein. We identified AUP1, UBXD8, UBC6e, and OS9 as functionally important components of this degradation complex in mammalian cells, as confirmed by mutagenesis and dominant negative versions of these proteins. Full Text.

Mueller, J.L., Mahadevaiah, S.K., Park, P.J., Warburton, P.E., Page, D.C., and Turner, J.M. (2008). The Mouse X Chromosome Is Enriched for Multicopy Testis Genes Showing Postmeiotic Expression. Nature Genetics May 4 [Epub ahead of print]. According to the prevailing view, mammalian X chromosomes are enriched in spermatogenesis genes expressed before meiosis and deficient in spermatogenesis genes expressed after meiosis. The paucity of postmeiotic genes on the X chromosome has been interpreted as a consequence of meiotic sex chromosome inactivation (MSCI)-the complete silencing of genes on the XY bivalent at meiotic prophase. Recent studies have concluded that MSCI-initiated silencing persists beyond meiosis and that most genes on the X chromosome remain repressed in round spermatids. Here, we report that 33 multicopy gene families, representing approximately 273 mouse X-linked genes, are expressed in the testis and that this expression is predominantly in postmeiotic cells. RNA FISH and microarray analysis show that the maintenance of X chromosome postmeiotic repression is incomplete. Furthermore, X-linked multicopy genes exhibit a similar degree of expression as autosomal genes. Thus, not only is the mouse X chromosome enriched for spermatogenesis genes functioning before meiosis, but in addition, approximately 18% of mouse X-linked genes are expressed in postmeiotic cells. Full Text.

Muller, U.F., and Bartel, D.P. (2008). Improved Polymerase Ribozyme Efficiency on Hydrophobic Assemblies. RNA Jan 29 [Epub ahead of print]. During an early step in the evolution of life, RNA served both as genome and as catalyst, according to the RNA world hypothesis. For self-replication, the RNA organisms must have contained an RNA that catalyzes RNA polymerization. As a first step toward recapitulating an RNA world in the laboratory, a polymerase ribozyme was generated previously by in vitro evolution and design. However, the efficiency of this ribozyme is about 100-fold too low for self-replication because of a low affinity of the ribozyme to its primer/template substrate. To improve the substrate interactions by colocalizing ribozyme and substrate on micelles, we attached hydrophobic anchors to both RNAs. We show here that the hydrophobic anchors led to aggregates with the expected size of the corresponding micelles. The micelle formation increased the polymerization yield of full-length products by 3- to 20-fold, depending on substrates and reaction conditions. With the best-characterized substrate, the improvement in polymerization efficiency was primarily due to reduced sequence-specific stalling on partially extended substrates. We discuss how, during the origin of life, micellar ribozyme aggregates could have acted as precursors to membrane-encapsulated life forms. PDF. Okamura, K., Chung, W.J., Ruby, J.G., Guo, H.L., Bartel, D.P., and Lai, E.C. (2008). The Drosophila Hairpin Rna Pathway Generates Endogenous Short Interfering Rnas. Nature 453, 803-U808. In contrast to microRNAs and Piwi- associated RNAs, short interfering RNAs ( siRNAs) are seemingly dispensable for host- directed gene regulation in Drosophila. This notion is based on the fact that mutants lacking the core siRNA- generating enzyme Dicer- 2 or the predominant siRNA effector Argonaute 2 are viable, fertile and of relatively normal morphology(1,2). Moreover, endogenous Drosophila siRNAs have not yet been identified. Here we report that siRNAs derived from long hairpin RNA genes ( hpRNAs) programme Slicer complexes that can repress endogenous target transcripts. The Drosophila hpRNA pathway is a hybrid mechanism that combines canonical RNA interference factors ( Dicer- 2, Hen1 ( known as CG12367) and Argonaute 2) with a canonical microRNA factor ( Loquacious) to generate similar to 21- nucleotide siRNAs. These novel regulatory RNAs reveal unexpected complexity in the sorting of small RNAs, and open a window onto the biological usage of endogenous RNA interference in Drosophila. Full Text.

Okamura, K., Chung, W.J., Ruby, J.G., Guo, H.L., Bartel, D.P., and Lai, E.C. (2008). The Drosophila Hairpin Rna Pathway Generates Endogenous Short Interfering Rnas. Nature 453, 803-806 In contrast to microRNAs and Piwi- associated RNAs, short interfering RNAs ( siRNAs) are seemingly dispensable for host- directed gene regulation in Drosophila. This notion is based on the fact that mutants lacking the core siRNA- generating enzyme Dicer- 2 or the predominant siRNA effector Argonaute 2 are viable, fertile and of relatively normal morphology(1,2). Moreover, endogenous Drosophila siRNAs have not yet been identified. Here we report that siRNAs derived from long hairpin RNA genes ( hpRNAs) programme Slicer complexes that can repress endogenous target transcripts. The Drosophila hpRNA pathway is a hybrid mechanism that combines canonical RNA interference factors ( Dicer- 2, Hen1 ( known as CG12367) and Argonaute 2) with a canonical microRNA factor ( Loquacious) to generate similar to 21- nucleotide siRNAs. These novel regulatory RNAs reveal unexpected complexity in the sorting of small RNAs, and open a window onto the biological usage of endogenous RNA interference in Drosophila. Full Text.

Onder, T.T., Gupta, P.B., Mani, S.A., Yang, J., Lander, E.S., and Weinberg, R.A. (2008). Loss of E-Cadherin Promotes Metastasis Via Multiple Downstream Transcriptional Pathways. Cancer Res 68, 3645-3654. Loss of the epithelial adhesion molecule E-cadherin is thought to enable metastasis by disrupting intercellular contacts-an early step in metastatic dissemination. To further investigate the molecular basis of this notion, we use two methods to inhibit E-cadherin function that distinguish between E-cadherin's cell-cell adhesion and intracellular signaling functions. Whereas the disruption of cell-cell contacts alone does not enable metastasis, the loss of E-cadherin protein does, through induction of an epithelial-to-mesenchymal transition, invasiveness, and anoikis resistance. We find the E-cadherin binding partner beta-catenin to be necessary, but not sufficient, for induction of these phenotypes. In addition, gene expression analysis shows that E-cadherin loss results in the induction of multiple transcription factors, at least one of which, Twist, is necessary for E-cadherin loss-induced metastasis. These findings indicate that E-cadherin loss in tumors contributes to metastatic dissemination by inducing wide-ranging transcriptional and functional changes.. Full Text.

Park, B., Brinkmann, M.M., Spooner, E., Lee, C.C., Kim, Y.M., and Ploegh, H.L. (2008). Proteolytic Cleavage in an Endolysosomal Compartment Is Required for Activation of Toll-Like Receptor 9. Nature Immunology Published online: 19 October 2008. Toll-like receptors (TLRs) activate the innate immune system in response to pathogens. Here we show that TLR9 proteolytic cleavage is a prerequisite for TLR9 signaling. Inhibition of lysosomal proteolysis rendered TLR9 inactive. The carboxy-terminal fragment of TLR9 thus generated included a portion of the TLR9 ectodomain, as well as the transmembrane and cytoplasmic domains. This cleavage fragment bound to the TLR9 ligand CpG DNA and, when expressed in Tlr9(-/-) dendritic cells, restored CpG DNA-induced cytokine production. Although cathepsin L generated the requisite TLR9 cleavage products in a cell-free in vitro system, several proteases influenced TLR9 cleavage in intact cells. Lysosomal proteolysis thus contributes to innate immunity by facilitating specific cleavage of TLR9. Full Text.

Pesin, J.A., and Orr-Weaver, T.L. (2008). Regulation of Apc/C Activators in Mitosis and Meiosis. Annual Review of Cell and Developmental Biology Vol. 24. The anaphase-promoting complex/cyclosome (APC/C) is a multisubunit E3 ubiquitin ligase that triggers the degradation of multiple substrates during mitosis. Cdc20/Fizzy and Cdh1/Fizzy-related activate the APC/C and confer substrate specificity through complex interactions with both the core APC/C and substrate proteins. The regulation of Cdc20 and Cdh1 is critical for proper APC/C activity and occurs in multiple ways: targeted protein degradation, phosphorylation, and direct binding of inhibitory proteins. During the specialized divisions of meiosis, the activity of the APC/C must be modified to achieve proper chromosome segregation. Recent studies show that one way in which APC/C activity is modified is through the use of meiosis-specific APC/C activators. Furthermore, regulation of the APC/C during meiosis is carried out by both mitotic regulators of the APC/C as well as meiosisspecific regulators. Here, we review the regulation of APC/C activators during mitosis and the role and regulation of the APC/C during female meiosis. Full Text.

Petersen, C.P., and Reddien, P.W. (2008). Smed-Beta-Catenin-1 Is Required for Anteroposterior Blastema Polarity in Planarian Regeneration. Science 319, 327-330. Planarian flatworms can regenerate heads at anterior- facing wounds and tails at posterior- facing wounds throughout the body. How this regeneration polarity is specified has been a classic problem for more than a century. We identified a planarian gene, Smed-beta catenin-1, that controls regeneration polarity. Posterior- facing blastemas regenerate a head instead of a tail in Smed-beta catenin- 1( RNAi) animals. Smed-beta catenin- 1 is required after wounding and at any posterior- facing wound for polarity. Additionally, intact Smed-beta catenin- 1( RNAi) animals display anteriorization during tissue turnover. Five Wnt genes and a secreted Frizzled- related Wnt antagonist- like gene are expressed in domains along the anteroposterior axis that reset to new positions during regeneration, which suggests that Wnts control polarity through Smed-beta catenin-1. Our data suggest that beta-catenin specifies the posterior character of the anteroposterior axis throughout the Bilateria and specifies regeneration polarity in planarians. Full Text.

Popp, M.W., Artavanis-Tsakonas, K., and Ploegh, H.L. (2008). Substrate Filtering by the Active-Site Crossover Loop in Uchl3 Revealed by Sortagging and Gain-of-Function Mutations. Journal of Biological Chemistry Papers in Press. Published on December 1, 2008 Determining how deubiquitinating enzymes discriminate between ubiquitin-conjugated substrates is critical to understand their function. Through application of a novel protein cleavage and tagging technique, sortagging, we show that human UCHL3 and the Plasmodium falciparum homologue, members of the ubiquitin c-terminal hydrolase family, use a unique active site crossover loop to restrict access of bulky ubiquitin adducts to the active site. Although it provides connectivity for critical active site residues in UCHL3, physical integrity of the crossover loop is dispensable for catalysis. By enlarging the active site crossover loop, we have constructed gain-of-function mutants that can accept substrates that the parent enzyme cannot, including ubiquitin chains of various linkages. PDF

Rai, P., Onder, T.T., Young, J.J., McFaline, J.L., Pang, B., Dedon, P.C., and Weinberg, R.A. (2008). Continuous Elimination of Oxidized Nucleotides Is Necessary to Prevent Rapid Onset of Cellular Senescence. Proc Natl Acad Sci U S A. early edition 31 December Reactive oxygen species (ROS) appear to play a role in limiting both cellular and organismic lifespan. However, because of their pleiotropic effects, it has been difficult to ascribe a specific role to ROS in initiating the process of cellular senescence. We have studied the effects of oxidative DNA damage on cell proliferation, believing that such damage is of central importance to triggering senescence. To do so, we devised a strategy to decouple levels of 8-oxoguanine, a major oxidative DNA lesion, from ROS levels. Suppression of MTH1 expression, which hydrolyzes 8-oxo-dGTP, was accompanied by increased total cellular 8-oxoguanine levels and caused early-passage primary and telomerase-immortalized human skin fibroblasts to rapidly undergo senescence, doing so without altering cellular ROS levels. This senescent phenotype recapitulated several salient features of replicative senescence, notably the presence of senescence-associated beta-galactosidase (SA beta-gal) activity, apparently irreparable genomic DNA breaks, and elevation of p21(Cip1), p53, and p16(INK4A) tumor suppressor protein levels. Culturing cells under low oxygen tension (3%) largely prevented the shMTH1-dependent senescent phenotype. These results indicate that the nucleotide pool is a critical target of intracellular ROS and that oxidized nucleotides, unless continuously eliminated, can rapidly induce cell senescence through signaling pathways very similar to those activated during replicative senescence.PDF

Reddien, P.W., Newmark, P.A., and Alvarado, A.S. (2008). Gene Nomenclature Guidelines for the Planarian Schmidtea Mediterranea. Developmental Dynamics Published Online: 15 Jul 2008. We describe a gene nomenclature system for the freshwater planarian Schmidtea mediterranea. Guidelines are specified for designating names for genes and proteins, as well as for describing RNA-mediated genetic interference (RNAi) experiments. The proposed conventions aim to avoid multiple names being ascribed to single genes and to establish a uniform, simple method for naming genes in S. mediterranea that is readily understood by researchers working on planarians and other organisms . Full Text.

Reiling, J.H., and Sabatini, D.M. (2008). Increased Mtorc1 Signaling Upregulates Stress. Mol Cell 29, 533-535. In this issue of Molecular Cell, Ozcan et al. (2008) show that the loss of the tuberous sclerosis tumor suppressor complex induces endoplasmic reticulum stress, leading to attenuation of insulin receptor signaling activity via the unfolded protein response.Full Text.

Resnick, T.D., Dej, K.J., Xiang, Y., Hawley, R.S., Ahn, C., and Orr-Weaver, T.L. (2008). Mutations in the Chromosomal Passenger Complex and the Condensin Complex Differentially Affect Synaptonemal Complex Disassembly and Metaphase I Configuration in Drosophila Female Meiosis. Genetics. Dec 22. [Epub ahead of print]. Production of haploid gametes relies on the specially regulated meiotic cell cycle. Analyses of the role of essential mitotic regulators in meiosis have been hampered by a shortage of appropriate alleles in metazoans. We characterized female-sterile alleles of the condensin complex component dcap-g and used them to define roles for condensin in Drosophila female meiosis. In mitosis, the condensin complex is required for sister-chromatid resolution and contributes to chromosome condensation. In meiosis, we demonstrate a role for dcap-g in disassembly of the synaptonemal complex and for proper retention of the chromosomes in a metaphase I arrested state. The chromosomal passenger complex also is known to have mitotic roles in chromosome condensation and is required in some systems for localization of the condensin complex. We used the QA26 allele of passenger component incenp to investigate the role of the passenger complex in oocyte meiosis. Strikingly, in incenp(QA26) mutants maintenance of the synaptonemal complex is disrupted. In contrast to the dcap-g mutants, the incenp mutation leads to a failure of paired homologous chromosomes to biorient, such that bivalents frequently orient towards only one pole in prometaphase and metaphase I. We show that incenp interacts genetically with ord, suggesting an important functional relationship between them in meiotic chromosome dynamics. The dcap-g and incenp mutations cause maternal effect lethality, with embryos from mutant mothers arrested in the initial mitotic divisions.PDF

Reynolds, T.B., Jansen, A., Peng, X., and Fink, G.R. (2008). Mat Formation in Saccharomyces Cerevisiae Requires Nutrient and Ph Gradients. Eukaryotic Cell 7, 122-130. The ability of Saccharomyces cerevisiae to form morphologically complex colony-like structures called mats requires expression of the cell surface glycoprotein Flo11p and growth on a semisolid surface. As the mat grows, it forms two visually distinct populations called the rim (edge of the mat) and the hub ( interior of the mat), which can be physically separated from one another based on their agar adherence properties. Here, we show that growth of the mat on a semisolid agar surface creates concentric glucose and pH gradients in the medium that are required for the differentiation of the hub and rim. Disruption of the pathways that respond to changing levels of glucose block mat formation by decreasing FLO11 expression. However, in wild-type cells, Flo11p is expressed in both portions of the structure. The difference in adherence between the rim and hub appears to be a consequence of the reduced adherence of Flo11p at the elevated pH of the rim. Full Text.

Ronan, J.L., Story, C.M., Papa, E., and Love, J.C. (2008). Optimization of the Surfaces Used to Capture Antibodies from Single Hybridomas Reduces the Time Required for Microengraving. Journal of Immunological Methods. 21 November Article in Press The most common method for the generation of monoclonal antibodies involves the identification and isolation of hybridomas from polyclonal populations. The discovery of new antibodies for biochemical and immunohistochemical assays in a rapid and efficient manner, however, remains a challenge. Here, a series of experiments are described that realize significant improvements to an approach for screening large numbers of single cells to identify antigen-specific monoclonal antibodies in a high-throughput manner (10(5)-10(6) cells in less than 12 h). The soft lithographic process called microengraving yields microarrays of monoclonal antibodies that can be correlated to individual hybridomas; the cells can then be retrieved and expanded to establish new cell lines. The factors examined here included the glass slide used for the microarray, the buffer used to deposit capture antibodies onto the glass, the type of polyclonal antibodies used to capture the secreted antibodies, and the time required for microengraving. Compared to earlier reports of this method, these studies resulted in increased signal-to-noise ratios for individual elements in the microarrays produced, and a considerable decrease in the time required to produce one microarray from a set of cells (from 2-4 h to 3-10 min). These technical advances will improve the throughput and reduce the costs for this alternative to traditional screening by limiting serial dilution. Full Text.

Sancak, Y., Peterson, T.R., Shaul, Y.D., Lindquist, R.A., Thoreen, C.C., Bar-Peled, L., and Sabatini, D.M. (2008). The Rag Gtpases Bind Raptor and Mediate Amino Acid Signaling to Mtorc1. Science.Published Online May 22, 2008 The multiprotein mTORC1 protein kinase complex is the central component of a pathway that promotes growth in response to insulin, energy levels, and amino acids, and is deregulated in common cancers. We find that the Rag proteins-a family of four related small guanosine triphosphatases (GTPases)-interact with mTORC1 in an amino acid-sensitive manner and are necessary for the activation of the mTORC1 pathway by amino acids. A Rag mutant that is constitutively bound to GTP interacted strongly with mTORC1, and its expression within cells made the mTORC1 pathway resistant to amino acid deprivation. Conversely, expression of a GDP-bound Rag mutant prevented stimulation of mTORC1 by amino acids. The Rag proteins do not directly stimulate the kinase activity of mTORC1, but, like amino acids, promote the intracellular localization of mTOR to a compartment that also contains its activator Rheb. Full Text.

Sangster, T.A., Salathia, N., Undurraga, S., Milo, R., Schellenberg, K., Lindquist, S., and Queitsch, C. (2008). Hsp90 Affects the Expression of Genetic Variation and Developmental Stability in Quantitative Traits. Proc Natl Acad Sci U S A 105, 2963-2968. Modulation of the activity of the molecular chaperone HSP90 has been extensively discussed as a means to alter phenotype in many traits and organisms. Such changes can be due to the exposure of cryptic genetic variation, which in some instances may also be accomplished by mild environmental alteration. Should such polymorphisms be widespread, natural selection may be more effective at producing phenotypic change in suboptimal environments. However, the frequency and identity of buffered polymorphisms in natural populations are unknown. Here, we employ quantitative genetic dissection of an Arabidopsis thaliana developmental response, hypocotyl elongation in the dark, to detail the underpinnings of genetic variation responsive to HSP90 modulation. We demonstrate that HSP90-dependent alleles occur in continuously distributed, environmentally responsive traits and are amenable to quantitative genetic mapping techniques. Furthermore, such alleles are frequent in natural populations and can have significant effects on natural phenotypic variation. We also find that HSP90 modulation has both general and allele-specific effects on developmental stability; that is, developmental stability is a phenotypic trait that can be affected by natural variation. However, effects of revealed variation on trait means outweigh effects of decreased developmental stability, and the HSP90-dependent trait alterations could be acted on by natural selection. Thus, HSP90 may centrally influence canalization, assimilation, and the rapid evolutionary alteration of phenotype through the concealment and exposure of cryptic genetic variation. Full Text.

Sangster, T.A., Salathia, N., Lee, H.N., Watanabe, E., Schellenberg, K., Morneau, K., Wang, H., Undurraga, S., Queitsch, C., and Lindquist, S. (2008). Hsp90-Buffered Genetic Variation Is Common in Arabidopsis Thaliana. Proc Natl Acad Sci U S A 105, 2969-2974. HSP90 is a protein chaperone particularly important in the maturation of a diverse set of proteins that regulate key steps in a multitude of biological processes. Alterations in HSP90 function produce altered phenotypes at low penetrance in natural populations. Previous work has shown that at least some of these phenotypes are due to genetic variation that remains phenotypically cryptic until it is revealed by the impairment of HSP90 function. Exposure of such "buffered" genetic polymorphisms can also be accomplished by environmental stress, linking the appearance of new phenotypes to defects in protein homeostasis. Should such polymorphisms be widespread, natural selection may be more effective at producing phenotypic change in suboptimal environments. In evaluating this hypothesis, a key unknown factor is the frequency with which HSP90-buffered polymorphisms occur in natural populations. Here, we present Arabidopsis thaliana populations suitable for genetic mapping that have constitutively reduced HSP90 levels. We employ quantitative genetic techniques to examine the HSP90-dependent polymorphisms affecting a host of plastic plant life-history traits. Our results demonstrate that HSP90-dependent natural variation is present at high frequencies in A. thaliana, with an expectation that at least one HSP90-dependent polymorphism will affect nearly every quantitative trait in progeny of two different wild lines. Hence, HSP90 is likely to occupy a central position in the translation of genotypic variation into phenotypic differences. Full Text.

Satyanarayana, A., Berthet, C., Lopez-Molina, J., Coppola, V., Tessarollo, L., and Kaldis, P. (2008). Genetic Substitution of Cdk1 by Cdk2 Leads to Embryonic Lethality and Loss of Meiotic Function of Cdk2. Development 135, 3389-3400. It was believed that Cdk2-cyclin E complexes are essential to drive cells through the G1-S phase transition. However, it was discovered recently that the mitotic kinase Cdk1 (Cdc2a) compensates for the loss of Cdk2. In the present study, we tested whether Cdk2 can compensate for the loss of Cdk1. We generated a knockin mouse in which the Cdk2 cDNA was knocked into the Cdk1 locus (Cdk1(Cdk2KI)). Substitution of both copies of Cdk1 by Cdk2 led to early embryonic lethality, even though Cdk2 was expressed from the Cdk1 locus. In addition, we generated Cdk2(-/-) Cdk1(+/Cdk2KI) mice in which one copy of Cdk2 and one copy of Cdk1 were expressed from the Cdk1 locus and the Cdk2 gene was deleted from the endogenous Cdk2 locus. We found that both male and female Cdk2(-/-) Cdk1(+/Cdk2KI) mice were sterile, similar to Cdk2(-/-) mice, even though they expressed the Cdk2 protein from the Cdk1 locus in testes. The translocational and cell cycle properties of knockin Cdk2 in Cdk2(-/-) Cdk1(+/Cdk2KI) cells were comparable to those of endogenous Cdk2, but we detected premature transcriptional activation of Cdk1 during liver regeneration in the absence of Cdk2. This study provides evidence of the molecular differences between Cdk2 and Cdk1 and highlights that the timing of transcriptional activation and the genetic locus play important roles in determining the function of Cdk proteins in vivo. Full Text.

Schlieker, C.D., Van der Veen, A.G., Damon, J.R., Spooner, E., and Ploegh, H.L. (2008). A Functional Proteomics Approach Links the Ubiquitin-Related Modifier Urm1 to a Trna Modification Pathway. Proc Natl Acad Sci U S A. Nov 18. [Epub ahead of print] Urm1 is a highly conserved ubiquitin-related modifier of unknown function. A reduction of cellular Urm1 levels causes severe cytokinesis defects in HeLa cells, resulting in the accumulation of enlarged multinucleated cells. To understand the underlying mechanism, we applied a functional proteomics approach and discovered an enzymatic activity that links Urm1 to a tRNA modification pathway. Unlike ubiquitin (Ub) and many Ub-like modifiers, which are commonly conjugated to proteinaceous targets, Urm1 is activated by an unusual mechanism to yield a thiocarboxylate intermediate that serves as sulfur donor in tRNA thiolation reactions. This mechanism is reminiscent of that used by prokaryotic sulfur carriers and thus defines the evolutionary link between ancient Ub progenitors and the eukaryotic Ub/Ub-like modification systems. PDF

Seila, A.C., Calabrese, J.M., Levine, S.S., Yeo, G.W., Rahl, P.B., Flynn, R.A., Young, R.A., and Sharp, P.A. (2008). Divergent Transcription from Active Promoters. Science 322, 1849-1851. Transcription initiation by RNA polymerase II ( RNAPII) is thought to occur unidirectionally from most genes. Here, we present evidence of widespread divergent transcription at protein- encoding gene promoters. Transcription start site- associated RNAs ( TSSa- RNAs) nonrandomly flank active promoters, with peaks of antisense and sense short RNAs at 250 nucleotides upstream and 50 nucleotides downstream of TSSs, respectively. Northern analysis shows that TSSa- RNAs are subsets of an RNA population 20 to 90 nucleotides in length. Promoter- associated RNAPII and H3K4- trimethylated histones, transcription initiation hallmarks, colocalize at sense and antisense TSSa- RNA positions; however, H3K79- dimethylated histones, characteristic of elongating RNAPII, are only present downstream of TSSs. These results suggest that divergent transcription over short distances is common for active promoters and may help promoter regions maintain a state poised for subsequent regulation. Full Text

Shin, C. (2008). Cleavage of the Star Strand Facilitates Assembly of Some Micrornas into Ago2-Containing Silencing Complexes in Mammals. Mol Cells Aug 27;26(3). [Epub ahead of print]. In animals, microRNAs (miRNAs) and small interfering RNAs (siRNAs) repress expression of protein coding genes by assembling distinct RNA-induced silencing complexes (RISCs). It has previously been shown that passenger-strand cleavage is a predominant mechanism when siRNA duplexes are loaded into Argonaute2 (Ago2)-containing RISC, while an unwinding bypass mechanism is favored for miRNA duplexes with mismatches. Here I present experimental data indicating that some mammalian miRNAs are assembled into Ago2-containing RISC by cleaving their corresponding miRNA star strands. This phenomenon may depend on the secondary structure near the scissile phosphate of the miRNA duplex. In addition, I show that ATP is not required for star-strand cleavage in this process. Taken together, the data here provide insight into the miRNA-loading mechanisms in mammals. Full Text.

Shorter, J., and Lindquist, S. (2008). Hsp104, Hsp70 and Hsp40 Interplay Regulates Formation, Growth and Elimination of Sup35 Prions. Embo Journal 27, 2712-2724. Self-templating amyloid forms of Sup35 constitute the yeast prion [PSI+]. How the protein-remodelling factor, Hsp104, collaborates with other chaperones to regulate [PSI+] inheritance remains poorly delineated. Here, we report how the Ssa and Ssb components of the Hsp70 chaperone system directly affect Sup35 prionogenesis and cooperate with Hsp104. We identify the ribosome-associated Ssb1:Zuo1:Ssz1 complex as a potent antagonist of Sup35 prionogenesis. The Hsp40 chaperones, Sis1 and Ydj1, preferentially interact with Sup35 oligomers and fibres compared with monomers, and facilitate Ssa1 and Ssb1 binding. Various Hsp70: Hsp40 pairs block prion nucleation by disassembling molten oligomers and binding mature oligomers. By binding fibres, Hsp70: Hsp40 pairs occlude prion recognition elements and inhibit seeded assembly. These inhibitory activities are partially relieved by the nucleotide exchange factor, Fes1. Low levels of Hsp104 stimulate prionogenesis and alleviate inhibition by some Hsp70: Hsp40 pairs. At high concentrations, Hsp104 eliminates Sup35 prions. This activity is reduced when Ssa1, or enhanced when Ssb1, is incorporated into nascent prions. These findings illuminate several facets of the chaperone interplay that underpins [PSI+] inheritance. Full Text.

Smukalla, S., Caldara, M., Pochet, N., Beauvais, A., Guadagnini, S., Yan, C., Vinces, M.D., Jansen, A., Prevost, M.C., Latge, J.P., Jean-Paul Latgé, Gerald R. Fink, Kevin R. Foster and Kevin J. Verstrepen (2008). Flo1 Is a Variable Green Beard Gene That Drives Biofilm-Like Cooperation in Budding Yeast. Cell 135, 726-737. The budding yeast, Saccharomyces cerevisiae, has emerged as an archetype of eukaryotic cell biology. Here we show that S. cerevisiae is also a model for the evolution of cooperative behavior by revisiting flocculation, a self-adherence phenotype lacking in most laboratory strains. Expression of the gene FLO1 in the laboratory strain S288C restores flocculation, an altered physiological state, reminiscent of bacterial biofilms. Flocculation protects the FLO1 expressing cells from multiple stresses, including antimicrobials and ethanol. Furthermore, FLO1(+) cells avoid exploitation by nonexpressing flo1 cells by self/non-self recognition: FLO1(+) cells preferentially stick to one another, regardless of genetic relatedness across the rest of the genome. Flocculation, therefore, is driven by one of a few known "green beard genes,'' which direct cooperation toward other carriers of the same gene. Moreover, FLO1 is highly variable among strains both in expression and in sequence, suggesting that flocculation in S. cerevisiae is a dynamic, rapidly evolving social trait. Full Text.

Soper, S.F.C., van der Heijden, G.W., Hardiman, T.C., Goodheart, M., Martin, S.L., de Boer, P., and Bortvin, A. (2008). Mouse Maelstrom, a Component of Nuage, Is Essential for Spermatogenesis and Transposon Repression in Meiosis. Developmental Cell 15, 285-297. Tight control of transposon activity is essential for the integrity of the germline. Recently, a germ-cell-specific organelle, nuage, was proposed to play a role in transposon repression. To test this hypothesis, we disrupted a murine homolog of a Drosophila nuage protein Maelstrom. Effects on male meiotic chromosome synapsis and derepression of transposable elements (TEs) were observed. In the adult Mael(-/-) testes, LINE-1 (L1) derepression occurred at the onset of meiosis. As a result, Mael(-/-) spermatocytes were flooded with L1 ribonucleoproteins (RNPs) that accumulated in large cytoplasmic enclaves and nuclei. Mael(-/-) spermatocytes with nuclear L1 RNPs exhibited massive DNA damage and severe chromosome asynapsis even in the absence of SP011-generated meiotic double-strand breaks. This study demonstrates that MAEL, a nuage component, is indispensable for the silencing of TEs and identifies the initiation of meiosis as an important step in TE control in the male germline. Full Text.

Stark, A., Bushati, N., Jan, C.H., Kheradpour, P., Hodges, E., Brennecke, J., Bartel, D.P., Cohen, S.M., and Kellis, M. (2008). A Single Hox Locus in Drosophila Produces Functional Micrornas from Opposite DNA Strands. Genes & Development 22, 8-13. MicroRNAs (miRNAs) are similar to 22-nucleotide RNAs that are processed from characteristic precursor hairpins and pair to sites in messages of protein-coding genes to direct post-transcriptional repression. Here, we report that the miRNA iab-4 locus in the Drosophila Hox cluster is transcribed convergently from both DNA strands, giving rise to two distinct functional miRNAs. Both sense and antisense miRNA products target neighboring Hox genes via highly conserved sites, leading to homeotic transformations when ectopically expressed. We also report sense/antisense miRNAs in mouse and find antisense transcripts close to many miRNAs in both flies and mammals, suggesting that additional sense/antisense pairs exist. Full Text.

Steele, A.D., Hutter, G., Jackson, W.S., Heppner, F.L., Borkowski, A.W., King, O.D., Raymond, G.J., Aguzzi, A., and Lindquist, S. (2008). Heat Shock Factor 1 Regulates Lifespan as Distinct from Disease Onset in Prion Disease. Proc Natl Acad Sci U S A.Aug 29. [Epub ahead of print] Prion diseases are fatal, transmissible, neurodegenerative diseases caused by the misfolding of the prion protein (PrP). At present, the molecular pathways underlying prion-mediated neurotoxicity are largely unknown. We hypothesized that the transcriptional regulator of the stress response, heat shock factor 1 (HSF1), would play an important role in prion disease. Uninoculated HSF1 knockout (KO) mice used in our study do not show signs of neurodegeneration as assessed by survival, motor performance, or histopathology. When inoculated with Rocky Mountain Laboratory (RML) prions HSF1 KO mice had a dramatically shortened lifespan, succumbing to disease approximately 20% faster than controls. Surprisingly, both the onset of home-cage behavioral symptoms and pathological alterations occurred at a similar time in HSF1 KO and control mice. The accumulation of proteinase K (PK)-resistant PrP also occurred with similar kinetics and prion infectivity accrued at an equal or slower rate. Thus, HSF1 provides an important protective function that is specifically manifest after the onset of behavioral symptoms of prion disease. Full Text.

Story, C.M., Papa, E., Hu, C.C., Ronan, J.L., Herlihy, K., Ploegh, H.L., and Love, J.C. (2008). Profiling Antibody Responses by Multiparametric Analysis of Primary B Cells.PNAS 105(46) : 17902-17907. Determining the efficacy of a vaccine generally relies on measuring neutralizing antibodies in sera. This measure cannot elucidate the mechanisms responsible for the development of immunological memory at the cellular level, however. Quantitative profiles that detail the cellular origin, extent, and diversity of the humoral (antibody-based) immune response would improve both the assessment and development of vaccines. Here, we describe a novel approach to collect multiparametric datasets that describe the specificity, isotype, and apparent affinity of the antibodies secreted from large numbers of individual primary B cells ( approximately 10(3)-10(4)). The antibody/antigen binding curves obtained by this approach can be used to classify closely related populations of cells using algorithms for data clustering, and the relationships among populations can be visualized graphically using affinity heatmaps. The technique described was used to evaluate the diversity of antigen-specific antibody-secreting cells generated during an in vivo humoral response to a series of immunizations designed to mimic a multipart vaccination. Profiles correlating primary antibody-producing cells with the molecular characteristics of their secreted antibodies should facilitate both the evaluation of candidate vaccines and, broadly, studies on the repertoires of antibodies generated in response to infectious or autoimmune diseases.PDF

Timp, W., and Matsudaira, P. (2008). Chapter 14: Electron Microscopy of Hydrated Samples. Methods Cell Biol 89, 391-407. Conventional electron microscopy offers a substantial resolution advantage over light microscopy, but requires difficult and often destructive preparation techniques. Recent advances in electron microscopy allow for imaging of hydrated samples, retaining the resolution advantage while removing the difficulty in preparation. Two new techniques, environmental scanning electron microscopy and wet electron microscopy offer this advantage, allowing for new possibilities in biological imaging.

Tyedmers, J., Madariaga, M.L., and Lindquist, S. (2008). Prion Switching in Response to Environmental Stress. PLoS Biol 6, e294. Evolution depends on the manner in which genetic variation is translated into new phenotypes. There has been much debate about whether organisms might have specific mechanisms for "evolvability," which would generate heritable phenotypic variation with adaptive value and could act to enhance the rate of evolution. Capacitor systems, which allow the accumulation of cryptic genetic variation and release it under stressful conditions, might provide such a mechanism. In yeast, the prion [PSI(+)] exposes a large array of previously hidden genetic variation, and the phenotypes it thereby produces are advantageous roughly 25% of the time. The notion that [PSI(+)] is a mechanism for evolvability would be strengthened if the frequency of its appearance increased with stress. That is, a system that mediates even the haphazard appearance of new phenotypes, which have a reasonable chance of adaptive value would be beneficial if it were deployed at times when the organism is not well adapted to its environment. In an unbiased, high-throughput, genome-wide screen for factors that modify the frequency of [PSI(+)] induction, signal transducers and stress response genes were particularly prominent. Furthermore, prion induction increased by as much as 60-fold when cells were exposed to various stressful conditions, such as oxidative stress (H2O2) or high salt concentrations. The severity of stress and the frequency of [PSI(+)] induction were highly correlated. These findings support the hypothesis that [PSI(+)] is a mechanism to increase survival in fluctuating environments and might function as a capacitor to promote evolvability. Full Text.

Ueberfeld, J., McKenna, B., Rubin-Bejerano, I., Verstrepen, K., and Ehrlich, D.J. (2008). Reaction-Mapped Quantitative Multiplexed Polymerase Chain Reaction on a Microfluidic Device. Anal Chem 80, 7430-7436. We have applied multiple-time-point reaction mapping to generate high-dynamic-range quantitative data from PCR multiplexes. The approach measures, then compensates, numerous PCR slope nonidealities across the multiplex without prejudice. A multilane microelectophoresis device with a novel scanning detector that reports redundantly over more than six decades in signal strength was used to collect data with multiple readings for each amplification point and with double internal calibration (lane standards and gene standards). We investigated scaling properties and sensitivity for readout of 12plex PCR reactions. The sensitive detection, stemming from confocal optics, allowed reduction of the PCR cycle number by approximately five cycles compared to commercial fluorometric readout. This increased sensitivity appears to allow quantitative PCR over a dynamic range of >9 log2 abundance ratio in multiplex reactions exceeding 20plexes. We argue that the combination of mapping, multiplexing, and an internal standard, improves the per-well efficiency of quantitative expression analysis by a factor of 50-100 relative to fluorometric qPCR readout. Therefore, the approach is attractive for analysis of large gene networks at reduced cost. Full Text.

Upadhyaya, A., Baraban, M., Wong, J., Matsudaira, P., van Oudenaarden, A., and Mahadevan, L. (2008). Power-Limited Contraction Dynamics of Vorticella Convallaria: An Ultrafast Biological Spring. Biophysical Journal 94, 265-272. Vorticella convallaria is one of the fastest and most powerful cellular machines. The cell body is attached to a substrate by a slender stalk containing a polymeric structure - the spasmoneme. Helical coiling of the stalk results from rapid contraction of the spasmoneme, an event mediated by calcium binding to a negatively charged polymeric backbone. We use high speed imaging to measure the contraction velocity as a function of the viscosity of the external environment and find that the maximum velocity scales inversely with the square root of the viscosity. This can be explained if the rate of contraction is ultimately limited by the power delivered by the actively contracting spasmoneme. Microscopically, this scenario would arise if the mechanochemical wave that propagates along the spasmoneme is faster than the rate at which the cell body can respond due to its large hydrodynamic resistance. We corroborate this by using beads as markers on the stalk and find that the contraction starts at the cell body and proceeds down the stalk at a speed that exceeds the velocity of the cell body. Full Text.

Vyas, J.M., Van der Veen, A.G., and Ploegh, H.L. (2008). The Known Unknowns of Antigen Processing and Presentation. Nature Review Immunology. Jul 18. [Epub ahead of print] The principal components of both MHC class I and class II antigen processing and presentation pathways are well known. In dendritic cells, these pathways are tightly regulated by Toll-like-receptor signalling and include features, such as cross-presentation, that are not seen in other cell types. However, the exact mechanisms involved in the subcellular trafficking of antigens remain poorly understood and in some cases are controversial. Recent data suggest that diverse cellular machineries, including autophagy, participate in antigen processing and presentation, although their relative contributions remain to be fully elucidated. Here, we highlight some emerging themes of antigen processing and presentation that we think merit further attention. Full Text.

Walters, B.J., Campbell, S.L., Chen, P.C., Taylor, A.P., Schroeder, D.G., Dobrunz, L.E., Artavanis-Tsakonas, K., Ploegh, H.L., Wilson, J.A., Cox, G.A., et al. (2008). Differential Effects of Usp14 and Uch-L1 on the Ubiquitin Proteasome System and Synaptic Activity. Molecular and Cellular Neuroscience 39, 539-548. The ubiquitin proteasome pathway has been implicated in the pathogenesis of many neurodegenerative diseases, and alterations in two different deubiquitinating enzymes, Uch-L1 and Usp14, result in neurological phenotypes in mice. We identified a new Mutation in Uch-L1 and compared the roles of Uch-L1 and Usp14 in the ubiquitin proteasome system. Deficiencies in either Uch-L1 or Usp14 result in decreased levels of ubiquitin, Suggesting that they both regulate ubiquitin stability in the nervous system, However, the effect of ubiquitin depletion on viability and onset of symptoms is more severe in the Usp14-deficient mice, and changes in hippocampal synaptic transmission were only observed in Usp14-deficient mice. In addition, while Usp14 appears to function at the proteasome, Uch-L1 deficiency resulted in up-regulation of lysosomal components, indicating that Uch-L1 and Usp14 may differentially affect the ubiquitin proteasome system and synaptic activity by regulating different pools of ubiquitin in the cell.Full Text.

Wang, E.T., Sandberg, R., Luo, S.J., Khrebtukova, I., Zhang, L., Mayr, C., Kingsmore, S.F., Schroth, G.P., and Burge, C.B. (2008). Alternative Isoform Regulation in Human Tissue Transcriptomes. Nature 456, 470-476. Through alternative processing of pre- messenger RNAs, individual mammalian genes often produce multiple mRNA and protein isoforms that may have related, distinct or even opposing functions. Here we report an in- depth analysis of 15 diverse human tissue and cell line transcriptomes on the basis of deep sequencing of complementary DNA fragments, yielding a digital inventory of gene and mRNA isoform expression. Analyses in which sequence reads are mapped to exon - exon junctions indicated that 92 - 94% of human genes undergo alternative splicing,, 86% with a minor isoform frequency of 15% or more. Differences in isoform- specific read densities indicated that most alternative splicing and alternative cleavage and polyadenylation events vary between tissues, whereas variation between individuals was approximately twofold to threefold less common. Extreme or 'switch-like' regulation of splicing between tissues was associated with increased sequence conservation in regulatory regions and with generation of full- length open reading frames. Patterns of alternative splicing and alternative cleavage and polyadenylation were strongly correlated across tissues, suggesting coordinated regulation of these processes, and sequence conservation of a subset of known regulatory motifs in both alternative introns and 39 untranslated regions suggested common involvement of specific factors in tissue- level regulation of both splicing and polyadenylation. Full Text.

Wang, H., Duennwald, M.L., Roberts, B.E., Rozeboom, L.M., Zhang, Y.X.L., Steele, A.D., Krishnan, R., Su, L.J., Griffin, D., Mukhopadhyay, S., Lindquist S, and Shorter J.(2008). Direct and Selective Elimination of Specific Prions and Amyloids by 4,5-Dianilinophthalimide and Analogs. Proceedings of the National Academy of Sciences of the United States of America 105, 7159-7164. Mechanisms to safely eliminate amyloids and preamyloid oligomers associated with many devastating diseases are urgently needed. Biophysical principles dictate that small molecules are unlikely to perturb large intermolecular protein-protein interfaces, let alone extraordinarily stable amyloid interfaces. Yet 4,5-dianilinophthalimide (DAPH-1) reverses A beta 42 amyloidogenesis and neurotoxicity, which is associated with Alzheimer's disease. Here, we show that DAPH-1 and select derivatives are ineffective against several amyloidogenic proteins, including tau, a-synuclein, Ure2, and PrP, but antagonize the yeast prion protein, Sup35, in vitro and in vivo. This allowed us to exploit several powerful new tools created for studying the conformational transitions of Sup35 and decipher the mechanisms by which DAPH-1 and related compounds antagonize the prion state. During fibrillization, inhibitory DAPHs alter the folding of Sup35's amyloidogenic core, preventing amyloidogenic oligomerization and specific recognition events that nucleate prion assembly. Select DAPHs also are capable of attacking preformed amyloids. They remodel Sup35 prion-specific intermolecular interfaces to create morphologically altered aggregates with diminished infectivity and self-templating activity. Our studies provide mechanistic insights and reinvigorate hopes for small-molecule therapies that specifically disrupt intermolecular amyloid contacts. Full Text.

Weinberg, R.A. (2008). Leaving Home Early: Reexamination of the Canonical Models of Tumor Progression. Cancer Cell 14, 283-284. A recent report in Science from the Varmus laboratory (Podsypanina et al., 2008) puts an interesting twist on the origins of metastatic cells, suggesting that metastases can arise in ways that are very different from those widely believed. PDF.

Weinberg, R.A. (2008). The Many Faces of Tumor Dormancy. Apmis 116, 548-551. Full Text.

Weinberg, R.A. (2008). Twisted Epithelial-Mesenchymal Transition Blocks Senescence. Nature Cell Biology 10, 1021-1023. The epithelial-mesenchymal transition (EMT) is a cellular transdifferentiation program that enables epithelial cancer cells to acquire traits of high-grade malignancy, notably invasive and metastatic powers. A new study indicates that it may also function early in tumour progression by preventing oncogene-induced senescence. Full Text.

Weinberg, R.A. (2008). In Retrospect: The Chromosome Trail. Nature 453, 725. A new translation of Theodor Boveri's 1914 monograph brings the early origins of contemporary cancer research to a wider readership Full Text.

Weinberg, R.A. (2008). Coevolution in the Tumor Microenvironment. Nature Genetics 40, 494-495. The progression of carcinomas to high- grade malignancies is accompanied by profound histological changes in the tumor-associated stroma. Although previous studies have suggested that mesenchymal cells of the stroma undergo genetic alterations during this progression, a new study now provides evidence that strongly contradicts this theory of stromal cell coevolution. Full Text.

Weinberg, R.A. (2008). Mechanisms of Malignant Progression. Carcinogenesis.May 2 [Epub ahead of print] PDF

Welburn, J.P., and Cheeseman, I.M. (2008). Toward a Molecular Structure of the Eukaryotic Kinetochore. Dev Cell 15, 645-655. Chromosome segregation in eukaryotes requires a large molecular assembly termed the kinetochore to attach chromosomes to spindle microtubules. Recent work has made substantial progress in defining the composition and activities of the kinetochore, but much remains to be learned about its macromolecular structure. This commentary discusses recent insights into structural features of the kinetochore, how these inform our understanding of its biological function, and the key challenges for the future. Full Text.

Welstead, G.G., Schorderet, P., and Boyer, L.A. (2008). The Reprogramming Language of Pluripotency. Current Opinion in Genetics and Development Mar 19; [Epub ahead of print]. In metazoans, lineage-specific transcription factors and epigenetic modifiers function to establish and maintain proper gene expression programs during development. Recent landmark studies in both mouse and human have defined a set of transcription factors whose ectopic expression by retroviral transduction is capable of reprogramming a somatic nucleus to the pluripotent state. The identification of factors that are sufficient for the induction of pluripotency suggests that rewiring transcriptional regulatory networks at the molecular level can be used to manipulate cell fate in vitro. These findings have broad implications for understanding development and disease and for the potential use of stem cells in therapeutic applications. Full Text.

Wernig, M., Lengner, C.J., Hanna, J., Lodato, M.A., Steine, E., Foreman, R., Staerk, J., Markoulaki, S., and Jaenisch, R. (2008). A Drug-Inducible Transgenic System for Direct Reprogramming of Multiple Somatic Cell Types. Nature Biotechnology.Jul 1. [Epub ahead of print] The study of induced pluripotency is complicated by the need for infection with high-titer retroviral vectors, which results in genetically heterogeneous cell populations. We generated genetically homogeneous 'secondary' somatic cells that carry the reprogramming factors as defined doxycycline (dox)-inducible transgenes. These cells were produced by infecting fibroblasts with dox-inducible lentiviruses, reprogramming by dox addition, selecting induced pluripotent stem cells and producing chimeric mice. Cells derived from these chimeras reprogram upon dox exposure without the need for viral infection with efficiencies 25- to 50-fold greater than those observed using direct infection and drug selection for pluripotency marker reactivation. We demonstrate that (i) various induction levels of the reprogramming factors can induce pluripotency, (ii) the duration of transgene activity directly correlates with reprogramming efficiency, (iii) cells from many somatic tissues can be reprogrammed and (iv) different cell types require different induction levels. This system facilitates the characterization of reprogramming and provides a tool for genetic or chemical screens to enhance reprogramming. Full Text.

Wernig, M., Zhao, J.P., Pruszak, J., Hedlund, E., Fu, D., Soldner, F., Broccoli, V., Constantine-Paton, M., Isacson, O., and Jaenisch, R. (2008). Neurons Derived from Reprogrammed Fibroblasts Functionally Integrate into the Fetal Brain and Improve Symptoms of Rats with Parkinson's Disease. Proc Natl Acad Sci U S A.Apr 7 [Epub ahead of print] The long-term goal of nuclear transfer or alternative reprogramming approaches is to create patient-specific donor cells for transplantation therapy, avoiding immunorejection, a major complication in current transplantation medicine. It was recently shown that the four transcription factors Oct4, Sox2, Klf4, and c-Myc induce pluripotency in mouse fibroblasts. However, the therapeutic potential of induced pluripotent stem (iPS) cells for neural cell replacement strategies remained unexplored. Here, we show that iPS cells can be efficiently differentiated into neural precursor cells, giving rise to neuronal and glial cell types in culture. Upon transplantation into the fetal mouse brain, the cells migrate into various brain regions and differentiate into glia and neurons, including glutamatergic, GABAergic, and catecholaminergic subtypes. Electrophysiological recordings and morphological analysis demonstrated that the grafted neurons had mature neuronal activity and were functionally integrated in the host brain. Furthermore, iPS cells were induced to differentiate into dopamine neurons of midbrain character and were able to improve behavior in a rat model of Parkinson's disease upon transplantation into the adult brain. We minimized the risk of tumor formation from the grafted cells by separating contaminating pluripotent cells and committed neural cells using fluorescence-activated cell sorting. Our results demonstrate the therapeutic potential of directly reprogrammed fibroblasts for neuronal cell replacement in the animal model. PDF

Wernig, M., Meissner, A., Cassady, J.P., and Jaenisch, R. (2008). C-Myc Is Dispensable for Direct Reprogramming of Mouse Fibroblasts. Cell Stem Cell 2, 10-12. Retroviral transduction of the four transcription factors Oct4, Sox2, Klf4, and c-Myc has been shown to initiate a reprogramming process that results in the transformation of mouse fibroblasts into embryonic stem (ES)-like cells designated as induced pluripotent stem (iPS) cells ([Maherali et al., 2007], [Meissner et al., 2007], [Okita et al., 2007], [Takahashi and Yamanaka, 2006] and [Wernig et al., 2007]). The promise of somatic reprogramming is the possibility to generate pluripotent stem cells that are patient specific and can be used as a unique source for autologous cell types for transplantation therapy ([Jaenisch, 2004] and [Yamanaka, 2007]). Many iPS cell-derived animals develop tumors due to the reactivation of the c-Myc virus (Okita et al., 2007), and this represents a major safety concern if we want to translate this approach to humans. It is thus of great importance to achieve reprogramming without this particular oncogene in the future. Here we show that fibroblasts can be reprogrammed to a pluripotent state by Oct4, Sox2, and Klf4 in the absence of c-Myc. Full Text.

Wheeler, R.T., Kombe, D., Agarwala, S.D., and Fink, G.R. (2008). Dynamic, Morphotype-Specific Candida Albicans Beta-Glucan Exposure During Infection and Drug Treatment. PLoS Pathogens 4, e1000227. Candida albicans, a clinically important dimorphic fungal pathogen that can evade immune attack by masking its cell wall beta-glucan from immune recognition, mutes protective host responses mediated by the Dectin-1 beta-glucan receptor on innate immune cells. Although the ability of C. albicans to switch between a yeast- or hyphal-form is a key virulence determinant, the role of each morphotype in beta-glucan masking during infection and treatment has not been addressed. Here, we show that during infection of mice, the C. albicans beta-glucan is masked initially but becomes exposed later in several organs. At all measured stages of infection, there is no difference in beta-glucan exposure between yeast-form and hyphal cells. We have previously shown that sub-inhibitory doses of the anti-fungal drug caspofungin can expose beta-glucan in vitro, suggesting that the drug may enhance immune activity during therapy. This report shows that caspofungin also mediates beta-glucan unmasking in vivo. Surprisingly, caspofungin preferentially unmasks filamentous cells, as opposed to yeast form cells, both in vivo and in vitro. The fungicidal activity of caspofungin in vitro is also filament-biased, as corroborated using yeast-locked and hyphal-locked mutants. The uncloaking of filaments is not a general effect of anti-fungal drugs, as another anti-fungal agent does not have this effect. These results highlight the advantage of studying host-pathogen interaction in vivo and suggest new avenues for drug development. Full Text.

Wong, G.W., Krawczyk, S.A., Kitidis-Mitrokostas, C., Revett, T., Gimeno, R., and Lodish, H.F. (2008). Molecular, Biochemical and Functional Characterizations of C1q/Tnf Family Members: Adipose-Tissue-Selective Expression Patterns, Regulation by Ppar-Gamma Agonist, Cysteine-Mediated Oligomerizations, Combinatorial Associations and Metabolic Functions. Biochemical Journal 416, 161-177. The insulin-sensitizing hormone, adiponectin, belongs to the expanding Clq/TNF (tumour necrosis factor) family of proteins. We recently identified a family of adiponectin paralogues designated as CTRP ((C) under bar lq/(T) under bar NF-(r) under bar elated (p) under bar rotein) 1-7, and in the present study describe CTRP10. In the present Study, we show that CTRP1, CTRP2, CTRP3, CTRP5 and CTRP7 transcripts are expressed predominantly by adipose tissue. In contrast, placenta and eye expressed the highest levels of CTRP6 and CTRP10 transcripts respectively. Expression levels of CTRP1, CTRP2, CTRP3, CTRP6 and CTRP7 transcripts are up-regulated in 8-week-old obese (ob/ob) in ice relative to lean controls. Treatment of mice with a PPAR-gamma (peroxisome-proliferator-activated receptor-gamma) agonist, rosiglitazone, increased the expression of CTRP1 and decreased CTRP6 transcript levels. All CTRPs are secreted glycoproteins when expressed in mammalian cells. CTRP1, CTRP2, CTRP3, CTRP5 and CTRP6 circulate in the blood and are potential endocrine hormones; their serum levels vary according to the sex and genetic background of mice. Importantly, serum levels of CTRP1 and CTRP6 are increased in adiponectin-null mice. Like adiponectin, all secreted CTRP proteins form trimers its their basic structural units. CTRP3, CTRP5, CTRP6 and CTRP10 trimers are further assembled into higher-order oligorneric complexes via disulfide bonding mediated by their N-terminal cysteine residues. Besides forming homo-oligomners, CTRP1/CTRP6, CTRP2/CTRP7 and adiponectin/CTRP2 are secreted its heterotrimers, thus providing a mechanism to potentially generate functionally distinct ligands. Functional characterization of one such family member, CTRP1, showed that it specifically activates Akt and p44/42-MAPK (mitogen-activated protein kinase) signalling pathways in differentiated mouse myotubes. Moreover, injection of recombinant CTRP I into mice significantly reduced their serum glucose levels. Thus at least CTRP1 may be considered a novel adipokine. In summary, these molecular, biochemical and functional data provide an important framework to further address the physiological functions and mechanisms of the action of this family of secreted glycoproteins in normal and disease states.Full Text.

Wong, G.W., Krawczyk, S.A., Kitidis-Mitrokostas, C., Ge, G., Spooner, E., Hug, C., Gimeno, R., and Lodish, H.F. (2008). Identification and Characterization of Ctrp9, a Novel Secreted Glycoprotein, from Adipose Tissue That Reduces Serum Glucose in Mice and Forms Heterotrimers with Adiponectin. FASEB Journal Published Online 11 September. Adiponectin is a major insulin-sensitizing, multimeric hormone derived from adipose tissue that acts on muscle and liver to regulate whole-body glucose and lipid metabolism. Here, we describe a novel and highly conserved paralog of adiponectin designated as C1q/TNF-related protein (CTRP) 9. Of all the CTRP paralogs, CTRP9 shows the highest degree of amino acid identity to adiponectin in its globular C1q domain. CTRP9 is expressed predominantly in adipose tissue and females expresses higher levels of the transcript than males. Moreover, its expression levels in ob/ob mice changed in an age-dependent manner, with significant up-regulation in younger mice. CTRP9 is a secreted glycoprotein with multiple post-translational modifications in its collagen domain that include hydroxylated prolines and hydroxylated and glycosylated lysines. It is secreted as multimers (predominantly trimers) from transfected cells and circulates in the mouse serum with levels varying according to sex and metabolic state of mice. Furthermore, CTRP9 and adiponectin can be secreted as heterooligomers when cotransfected into mammalian cells, and in vivo, adiponectin/CTRP9 complexes can be reciprocally coimmunoprecipitated from the serum of adiponectin and CTRP9 transgenic mice. Biochemical analysis demonstrates that adiponectin and CTRP9 associate via their globular C1q domain, and this interaction does not require their conserved N-terminal cysteines or their collagen domains. Furthermore, we show that adiponectin and CTRP9 form heterotrimers. In cultured myotubes, CTRP9 specifically activates AMPK, Akt, and p44/42 MAPK signaling pathways. Adenovirus-mediated overexpression of CTRP9 in obese (ob/ob) mice significantly lowered serum glucose levels. Collectively, these results suggest that CTRP9 is a novel adipokine, and further study of CTRP9 will yield novel mechanistic insights into its physiological and metabolic function.. PDF

Xie, F., and Orr-Weaver, T.L. (2008). Isolation of a Drosophila Amplification Origin Developmentally Activated by Transcription. Proc Natl Acad Sci U S A. We exploited the Drosophila Amplicon in Follicle Cells, DAFC-62D, to identify a new metazoan amplification origin, ori62. In addition to the origin, DAFC-62D contains two other developmental stage-specific binding regions for the Origin Recognition Complex (ORC) and the replicative helicase MCM2-7. All three of these regions are required for proper amplification. There are two rounds of amplification initiation at ori62, and the second round is preceded by transcription across ori62. We show by alpha-amanitin inhibition that RNA polymerase II (RNAPII) transcription is required to localize MCM2-7 (but not ORC) to permit the second round of origin firing. This role for transcription appears unique to DAFC-62D, because neither other DAFCs nor ectopic transposons with the DAFC-62D replication elements bounded by functional chromatin insulators are affected by alpha-amanitin. By sequential chromatin immunoprecipitation, we show that the MCM complex and RNAPII are bound to the same 100-500 bp pieces of chromatin during late origin firing. These results raise the possibility that RNAPII may recruit MCM2-7 at some metazoan replication origins. Full Text.

Yang, J., and Weinberg, R.A. (2008). Epithelial-Mesenchymal Transition: At the Crossroads of Development and Tumor Metastasis. Developmental Cell 14, 818-829. The epithelial-mesenchymal transition is a highly conserved cellular program that allows polarized, immotile epithelial cells to convert to motile mesenchymal cells. This important process was initially recognized during several critical stages of embryonic development and has more recently been implicated in promoting carcinoma invasion and metastasis. In this review, we summarize and compare major signaling pathways that regulate the epithelial-mesenchymal transitions during both development and tumor metastasis. Studies in both fields are critical for our molecular understanding of cell migration and morphogenesis.Full Text.

Yang, F., Gell, K., van der Heijden, G.W., Eckardt, S., Leu, N.A., Page, D.C., Benavente, R., Her, C., Hoog, C., McLaughlin, K.J., et al. (2008). Meiotic Failure in Male Mice Lacking an X-Linked Factor. Genes & Development 22, 682-691. Meiotic silencing of sex chromosomes may cause their depletion of meiosis-specific genes during evolution. Here, we challenge this hypothesis by reporting the identification of TEX11 as the first X-encoded meiosis-specific factor in mice. TEX11 forms discrete foci on synapsed regions of meiotic chromosomes and appears to be a novel constituent of meiotic nodules involved in recombination. Loss of TEX11 function causes chromosomal asynapsis and reduced crossover formation, leading to elimination of spermatocytes, respectively, at the pachytene and anaphase I stages. Specifically, TEX11-deficient spermatocytes with asynapsed autosomes undergo apoptosis at the pachytene stage, while those with only asynapsed sex chromosomes progress. However, cells that survive the pachytene stage display chromosome nondisjunction at the first meiotic division, resulting in cell death and male infertility. TEX11 interacts with SYCP2, which is an integral component of the synaptonemal complex lateral elements. Thus, TEX11 promotes initiation and/or maintenance of synapsis and formation of crossovers, and may provide a physical link between these two meiotic processes. Full Text.

Yekta, S., Tabin, C.J., and Bartel, D.P. (2008). Micrornas in the Hox Network: An Apparent Link to Posterior Prevalence. Nature Reviews Genetics Sep 10. [Epub ahead of print] Homeobox (Hox) transcription factors confer anterior-posterior (AP) axial coordinates to vertebrate embryos. Hox genes are found in clusters that also contain genes for microRNAs (miRNAs). Our analysis of predicted miRNA targets indicates that Hox cluster-embedded miRNAs preferentially target Hox mRNAs. Moreover, the presumed Hox target genes are predominantly situated on the 3' side of each Hox miRNA locus. These results suggest that Hox miRNAs help repress more anterior programmes, thereby reinforcing posterior prevalence, which is the hierarchical dominance of posterior over anterior Hox gene function that is observed in bilaterians. In this way, miRNA-mediated regulation seems to recapitulate interactions at other levels of gene expression, some more ancestral, within a network under stabilizing selection. Full Text.

Zhang, C.C., Kaba, M., Iizuka, S., Huynh, H., and Lodish, H.F. (2008). Angiopoietin-Like 5 and Igfbp2 Stimulate Ex Vivo Expansion of Human Cord Blood Hematopoietic Stem Cells as Assayed by Nod/Scid Transplantation. Blood.Jan 17;. [Epub ahead of print] Pluripotent hematopoietic stem cells (HSCs) are the basis of bone marrow transplantation and are attractive target cells for hematopoietic gene therapy, but these important clinical applications have been severely hampered by difficulties in ex vivo expansion of HSCs. In particular, the use of cord blood for adult transplantation is greatly limited by the number of HSCs. Previously we identified Angiopoietin-like proteins and IGF binding protein 2 (IGFBP2) as new hormones that, together with other factors, can expand mouse bone marrow HSCs in culture. Here we measure the activity of multipotent human SCID-repopulating cells (SRCs) by transplantation into the nonobese diabetic severe combined immunodeficiency (NOD/SCID) mice; secondary transplantation was performed to evaluate the self-renewal potential of SRCs. A serum- free medium containing SCF, TPO, and FGF-1 or Flt3-L cannot significantly support expansion of the SRCs present in human cord blood CD133(+) cells. Addition of either Angiopoietin-like 5, or IGF binding protein 2 to the cultures led to a sizable expansion of HSC numbers, as assayed by NOD/SCID transplantation. A serum-free culture containing SCF, TPO, FGF-1, Angiopoietin-like 5, and IGFBP2 supports a ~20 fold net expansion of repopulating human cord blood HSCs, a number potentially applicable to several clinical processes including HSC transplantation. PDF

Zhang, J., Wang, J., Liu, Y., Sidik, H., Young, K.H., Lodish, H.F., and Fleming, M.D. (2008). Oncogenic Kras-Induced Leukemogeneis: Hematopoietic Stem Cells as the Initial Target and Lineage-Specific Progenitors as the Potential Targets for Final Leukemic Transformation. Blood (prepublished online December 9, 2008) K-ras is often mutated in human hematopoietic malignancies, including juvenile myelomonocytic leukemia (JMML) and T-cell lymphoblastic leukemia/lymphoma (TLL/L). However, the exact role and function of oncogenic K-ras mutations in the initiation and progression of JMML and TLL/L remain elusive. Here, we report the use of a mouse bone marrow transplantation model to study oncogenic Kras-induced leukemogenesis. We show that as the first genetic hit, oncogenic K-ras mutations initiate both JMML and TLL/L, but with different efficiencies. Limiting dilution analyses indicated that an oncogenic K-ras mutation alone is insufficient to produce frank malignancy. Rather, it co-operates with additional subsequent genetic event(s). Moreover, transplantation of highly purified hematopoietic stem cells (HSCs) and myeloid progenitors identified HSCs as the primary target for the oncogenic K-ras mutation. Karyotypic analysis further indicated that secondary genetic hit(s) target lineage-specific progenitors rather than HSCs for terminal tumor transformation into leukemic stem cells. Thus, we propose the cellular mechanism underlying oncogenic Kras-induced leukemogenesis, with HSCs being the primary target by the oncogenic K-ras mutations and lineage-committed progenitors being the final target for cancer stem cell transformation. Our model might be also applicable to other solid tumors harboring oncogenic K-ras mutations.PDF

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