The following alphabetical list represents papers published in 2009 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.
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Alberti, S., Halfmann, R., King, O., Kapila, A., and Lindquist, S. (2009). A Systematic Survey Identifies Prions and Illuminates Sequence Features of Prionogenic Proteins. Cell 137, 146-158. Prions are proteins that convert between structurally and functionally distinct states, one or more of which is transmissible. In yeast, this ability allows them to act as non-Mendelian elements of phenotypic inheritance. To further our understanding of prion biology, we conducted a bioinformatic proteome-wide survey for prionogenic proteins in S. cerevisiae, followed by experimental investigations of 100 prion candidates. We found an unexpected amino acid bias in aggregation-prone candidates and discovered that 19 of these could also form prions. At least one of these prion proteins, Mot3, produces a bona fide prion in its natural context that increases population-level phenotypic heterogeneity. The self-perpetuating states of these proteins present a vast source of heritable phenotypic variation that increases the adaptability of yeast populations to diverse environments. Full Text.
Antos, J. M., G. L. Chew, Carla P. Guimaraes, Nicholas C. Yoder, Gijsbert M. Grotenbreg, Maximilian Wei-Lin Popp and Hidde L. Ploegh (2009). Site-Specific N- and C-Terminal Labeling of a Single Polypeptide Using Sortases of Different Specificity. Journal of the American Chemical Society Jul 17. [Epub ahead of print].The unique reactivity of two sortase enzymes, SrtA(staph) from Staphylococcus aureus and SrtA(strep) from Streptococcus pyogenes, is exploited for site-specific labeling of a single polypeptide with different labels at its N and C termini. SrtA(strep) is used to label the protein's C terminus at an LPXTG site with a fluorescently labeled dialanine nucleophile. Selective N-terminal labeling of proteins containing N-terminal glycine residues is achieved using SrtA(staph) and LPXT derivatives. The generality of N-terminal labeling with SrtA(staph) is demonstrated by near-quantitative labeling of multiple protein substrates with excellent site specificity.Full Text
Antos, J.M., Popp, M.W., Ernst, R., Chew, G.L., Spooner, E., and Ploegh, H.L. (2009). A Straight Path to Circular Proteins. Journal of Biological Chemistry.Apr 9. [Epub ahead of print] Folding and stability are parameters that control protein behavior. The possibility of conferring additional stability on proteins has implications for their use in vivo, and for their structural analysis in the laboratory. Cyclic polypeptides ranging in size from 14 to 78 amino acids occur naturally and often show enhanced resistance toward denaturation and proteolysis when compared to their linear counterparts. Native chemical ligation and intein-based methods allow production of circular derivatives of larger proteins, resulting in improved stability and refolding properties. Here we show that circular proteins can be made reversibly with excellent efficiency by means of a sortase-catalyzed cyclization reaction, requiring only minimal modification of the protein to be circularized PDF.
Anwar, M., Aytur, T., and Matsudaira, P. (2009). A Wireless-Compatible Optics-Free Fluorescent Array Reader. Applied Physics Letters 94 111102 (2009). Optical-based bioassays, particularly fluorescent-based arrays, form the basis for biological detection in both biological and clinical assays, yet their use is restricted by the need for sensitive optical detectors and optics. To address this, we developed an integrated circuit-based fluorescent imaging platform that functions without the use of optics while directly integrating electronics, enabling a low-cost integrated solution. Additionally, we incorporate a wireless interface, enabling optical detection within any biological environment. As a proof of concept, we demonstrate the detection of 20 nM of Streptavidin on an array without the use of optical filters and lenses. Full Text.
Bagby, S.C., Bergman, N.H., Shechner, D.M., Yen, C., and Bartel, D.P. (2009). A Class I Ligase Ribozyme with Reduced Mg2+ Dependence: Selection, Sequence Analysis, and Identification of Functional Tertiary Interactions. RNA 15, 2129-2146. The class I ligase was among the first ribozymes to have been isolated from random sequences and represents the catalytic core of several RNA-directed RNA polymerase ribozymes. The ligase is also notable for its catalytic efficiency and structural complexity. Here, we report an improved version of this ribozyme, arising from selection that targeted the kinetics of the chemical step. Compared with the parent ribozyme, the improved ligase achieves a modest increase in rate enhancement under the selective conditions and shows a sharp reduction in [Mg(2+)] dependence. Analysis of the sequences and kinetics of successful clones suggests which mutations play the greatest part in these improvements. Moreover, backbone and nucleobase interference maps of the parent and improved ligase ribozymes complement the newly solved crystal structure of the improved ligase to identify the functionally significant interactions underlying the catalytic ability and structural complexity of the ligase ribozyme.Full Text.
Barbie, D.A., Tamayo, P., Boehm, J.S., Kim, S.Y., Moody, S.E., Dunn, I.F., Schinzel, A.C., Sandy, P., Meylan, E., Scholl, C., Sabatini DM, et al. (2009). Systematic Rna Interference Reveals That Oncogenic Kras-Driven Cancers Require Tbk1. Nature 462, 108-U122. The proto-oncogene KRAS is mutated in a wide array of human cancers, most of which are aggressive and respond poorly to standard therapies. Although the identification of specific oncogenes has led to the development of clinically effective, molecularly targeted therapies in some cases, KRAS has remained refractory to this approach. A complementary strategy for targeting KRAS is to identify gene products that, when inhibited, result in cell death only in the presence of an oncogenic allele(1,2). Here we have used systematic RNA interference to detect synthetic lethal partners of oncogenic KRAS and found that the non-canonical I kappa B kinase TBK1 was selectively essential in cells that contain mutant KRAS. Suppression of TBK1 induced apoptosis specifically in human cancer cell lines that depend on oncogenic KRAS expression. In these cells, TBK1 activated NF-kappa B anti-apoptotic signals involving c-Rel and BCL-XL (also known as BCL2L1) that were essential for survival, providing mechanistic insights into this synthetic lethal interaction. These observations indicate that TBK1 and NF-kappa B signalling are essential in KRAS mutant tumours, and establish a general approach for the rational identification of co-dependent pathways in cancer. Full Text.
Bartel, D.P. (2009). Micrornas: Target Recognition and Regulatory Functions. Cell 136, 215-233. MicroRNAs (miRNAs) are endogenous approximately 23 nt RNAs that play important gene-regulatory roles in animals and plants by pairing to the mRNAs of protein-coding genes to direct their posttranscriptional repression. This review outlines the current understanding of miRNA target recognition in animals and discusses the widespread impact of miRNAs on both the expression and evolution of protein-coding genes. Full Text.
Beauregard, P.B., Guerin, R., Turcotte, C., Lindquist, S., and Rokeach, L.A. (2009). A Nucleolar Protein Allows Viability in the Absence of the Essential Er-Residing Molecular Chaperone Calnexin. Journal of Cell Science 122, 1342-1351. In fission yeast, the ER-residing molecular chaperone calnexin is normally essential for viability. However, a specific mutant of calnexin that is devoid of chaperone function(Delta hcd_Cnx1p) induces an epigenetic state that allows growth of Schizosaccharomyces pombe without calnexin. This calnexin-independent (Cin) state was previously shown to be mediated via a non-chromosomal element exhibiting some prion-like features. Here, we report the identification of a gene whose overexpression induces the appearance of stable Cin cells. This gene, here named cif1(+) for calnexin-independence factor 1, encodes an uncharacterized nucleolar protein. The Cin cells arising from cif1(+) overexpression (Cin(cif1) cells) are genetically and phenotypically distinct from the previously characterized Cin(Delta hcd_cnx1) cells, which spontaneously appear in the presence of the Delta hcd_Cnx1p mutant. Moreover, cif1(+) is not required for the induction or maintenance of the Cin(Delta hcd_cnx1) state. These observations argue for different pathways of induction and/or maintenance of the state of calnexin independence. Nucleolar localization of Cif1p is required to induce the Cin(cif1) state, thus suggesting an unexpected interaction between the vital cellular role of calnexin and a function of the nucleolus. Full Text.
Bell, G.W., and Lewitter, F. (2009). Resources for Small Regulatory Rnas. Curr Protoc Mol Biol Chapter 19, Unit 19.8. In the past fifteen years, new classes of regulatory RNAs have been discovered, previously hidden in the transcriptome mostly due to their small size. These small regulatory RNAs include small interfering RNAs (siRNAs), microRNAs (miRNAs), and Piwi-interacting RNAs (piRNAs). Numerous databases have been developed to store information about these small regulatory RNAs, and many tools have been developed to work with the data. This overview introduces the reader to the many resources available for working with small regulatory RNAs. Fulltext
Bilodeau, S., Kagey, M.H., Frampton, G.M., Rahl, P.B., and Young, R.A. (2009). Setdb1 Contributes to Repression of Genes Encoding Developmental Regulators and Maintenance of Es Cell State. Genes Dev 23, 2484-2489. Transcription factors that play key roles in regulating embryonic stem (ES) cell state have been identified, but the chromatin regulators that help maintain ES cells are less well understood. A high-throughput shRNA screen was used to identify novel chromatin regulators that influence ES cell state. Loss of histone H3 Lys 9 (H3K9) methyltransferases, particularly SetDB1, had the most profound effects on ES cells. Chromatin immunoprecipitation (ChIP) coupled with massively parallel DNA sequencing (ChIP-Seq) and functional analysis revealed that SetDB1 and histone H3K9-methylated nucleosomes occupy and repress genes encoding developmental regulators. These SetDB1-occupied genes are a subset of the "bivalent" genes, which contain nucleosomes with H3K4me3 (H3K4 trimethylation) and H3K27me3 modifications catalyzed by Trithorax and Polycomb group proteins, respectively. These genes are subjected to repression by both Polycomb group proteins and SetDB1, and loss of either regulator can destabilize ES cell state. Full Text.
Brandon, N.J., Millar, J.K., Korth, C., Sive, H., Singh, K.K., and Sawa, A. (2009). Understanding the Role of Disc1 in Psychiatric Disease and During Normal Development. J Neurosci 29, 12768-12775. The biology of schizophrenia is complex with multiple hypotheses (dopamine, glutamate, neurodevelopmental) well supported to underlie the disease. Pathways centered on the risk factor "disrupted in schizophrenia 1" (DISC1) may be able to explain and unite these disparate hypotheses and will be the topic of this mini-symposium preview. Nearly a decade after its original identification at the center of a translocation breakpoint in a large Scottish family that was associated with major psychiatric disease, we are starting to obtain credible insights into its function and role in disease etiology. This preview will highlight a number of exciting areas of current DISC1 research that are revealing roles for DISC1 during normal brain development and also in the disease state. Together these different threads will provide a timely and exciting overview of the DISC1 field and its potential in furthering our understanding of psychiatric diseases and in developing new therapies. Full Text.
Brar, G.A., Hochwagen, A., Ee, L.S., and Amon, A. (2009). The Multiple Roles of Cohesin in Meiotic Chromosome Morphogenesis and Pairing. Molecular Biology of the Cell 20, 1030-1047. 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 their 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 mutations 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. Full Text
Broske, A.M., Vockentanz, L., Kharazi, S., Huska, M.R., Mancini, E., Scheller, M., Kuhl, C., Enns, A., Prinz, M., Jaenisch, R., et al. (2009). DNA Methylation Protects Hematopoietic Stem Cell Multipotency from Myeloerythroid Restriction. Nature Genetics 41, 1207-U1269. DNA methylation is a dynamic epigenetic mark that undergoes extensive changes during differentiation of self-renewing stem cells. However, whether these changes are the cause or consequence of stem cell fate remains unknown. Here, we show that alternative functional programs of hematopoietic stem cells (HSCs) are governed by gradual differences in methylation levels. Constitutive methylation is essential for HSC self-renewal but dispensable for homing, cell cycle control and suppression of apoptosis. Notably, HSCs from mice with reduced DNA methyltransferase 1 activity cannot suppress key myeloerythroid regulators and thus can differentiate into myeloerythroid, but not lymphoid, progeny. A similar methylation dosage effect controls stem cell function in leukemia. These data identify DNA methylation as an essential epigenetic mechanism to protect stem cells from premature activation of predominant differentiation programs and suggest that methylation dynamics determine stem cell functions in tissue homeostasis and cancer. Full Text.
Brown, J.C., and Lindquist, S. (2009). A Heritable Switch in Carbon Source Utilization Driven by an Unusual Yeast Prion. Genes Dev 23, 2320-2332. Several well-characterized fungal proteins act as prions, proteins capable of multiple conformations, each with different activities, at least one of which is self-propagating. Through such self-propagating changes in function, yeast prions act as protein-based elements of phenotypic inheritance. We report a prion that makes cells resistant to the glucose-associated repression of alternative carbon sources, [GAR(+)] (for "resistant to glucose-associated repression," with capital letters indicating dominance and brackets indicating its non-Mendelian character). [GAR(+)] appears spontaneously at a high rate and is transmissible by non-Mendelian, cytoplasmic inheritance. Several lines of evidence suggest that the prion state involves a complex between a small fraction of the cellular complement of Pma1, the major plasma membrane proton pump, and Std1, a much lower-abundance protein that participates in glucose signaling. The Pma1 proteins from closely related Saccharomyces species are also associated with the appearance of [GAR(+)]. This allowed us to confirm the relationship between Pma1, Std1, and [GAR(+)] by establishing that these proteins can create a transmission barrier for prion propagation and induction in Saccharomyces cerevisiae. The fact that yeast cells employ a prion-based mechanism for heritably switching between distinct carbon source utilization strategies, and employ the plasma membrane proton pump to do so, expands the biological framework in which self-propagating protein-based elements of inheritance operate. Full Text.
Bryan, A.W., Menke, M., Cowen, L.J., Lindquist, S.L., and Berger, B. (2009). Betascan: Probable Beta-Amyloids Identified by Pairwise Probabilistic Analysis. Plos Computational Biology 5(3): e1000333.. Amyloids and prion proteins are clinically and biologically important beta-structures, whose supersecondary structures are difficult to determine by standard experimental or computational means. In addition, significant conformational heterogeneity is known or suspected to exist in many amyloid fibrils. Recent work has indicated the utility of pairwise probabilistic statistics in beta-structure prediction. We develop here a new strategy for beta-structure prediction, emphasizing the determination of beta-strands and pairs of beta-strands as fundamental units of beta-structure. Our program, BETASCAN, calculates likelihood scores for potential beta-strands and strand-pairs based on correlations observed in parallel beta-sheets. The program then determines the strands and pairs with the greatest local likelihood for all of the sequence's potential beta-structures. BETASCAN suggests multiple alternate folding patterns and assigns relative a priori probabilities based solely on amino acid sequence, probability tables, and pre-chosen parameters. The algorithm compares favorably with the results of previous algorithms (BETAPRO, PASTA, SALSA, TANGO, and Zyggregator) in beta-structure prediction and amyloid propensity prediction. Accurate prediction is demonstrated for experimentally determined amyloid beta-structures, for a set of known beta-aggregates, and for the parallel beta-strands of beta-helices, amyloid-like globular proteins. BETASCAN is able both to detect beta-strands with higher sensitivity and to detect the edges of beta-strands in a richly beta-like sequence. For two proteins ( Ab and Het-s), there exist multiple sets of experimental data implying contradictory structures; BETASCAN is able to detect each competing structure as a potential structure variant. The ability to correlate multiple alternate beta-structures to experiment opens the possibility of computational investigation of prion strains and structural heterogeneity of amyloid. BETASCAN is publicly accessible on the Web at http://betascan.csail.mit.edu. Full Text
Bumgarner, S.L., Dowell, R.D., Grisafi, P., Gifford, D.K., and Fink, G.R. (2009). Toggle Involving Cis-Interfering Noncoding Rnas Controls Variegated Gene Expression in Yeast. Proc Natl Acad Sci U S A.Sep 30. [Epub ahead of print] The identification of specific functional roles for the numerous long noncoding (nc)RNAs found in eukaryotic transcriptomes is currently a matter of intense study amid speculation that these ncRNAs have key regulatory roles. We have identified a pair of cis-interfering ncRNAs in yeast that contribute to the control of variegated gene expression at the FLO11 locus by implementing a regulatory circuit that toggles between two stable states. These capped, polyadenylated ncRNAs are transcribed across the large intergenic region upstream of the FLO11 ORF. As with mammalian long intervening (li)ncRNAs, these yeast ncRNAs (ICR1 and PWR1) are themselves regulated by transcription factors (Sfl1 and Flo8) and chromatin remodelers (Rpd3L) that are key elements in phenotypic transitions in yeast. The mechanism that we describe explains the unanticipated role of a histone deacetylase complex in activating gene expression, because Rpd3L mutants force the ncRNA circuit into a state that silences the expression of the adjacent variegating gene. Full Text.
Carette, J.E., Guimaraes, C.P., Varadarajan, M., Park, A.S., Wuethrich, I., Godarova, A., Kotecki, M., Cochran, B.H., Spooner, E., Ploegh, H.L., and Brummelkamp TR (2009). Haploid Genetic Screens in Human Cells Identify Host Factors Used by Pathogens. Science 326, 1231-1235. Loss-of-function genetic screens in model organisms have elucidated numerous biological processes, but the diploid genome of mammalian cells has precluded large-scale gene disruption. We used insertional mutagenesis to develop a screening method to generate null alleles in a human cell line haploid for all chromosomes except chromosome 8. Using this approach, we identified host factors essential for infection with influenza and genes encoding important elements of the biosynthetic pathway of diphthamide, which are required for the cytotoxic effects of diphtheria toxin and exotoxin A. We also identified genes needed for the action of cytolethal distending toxin, including a cell-surface protein that interacts with the toxin. This approach has both conceptual and practical parallels with genetic approaches in haploid yeast. Full Text.
Carey, B.W., Markoulaki, S., Beard, C., Hanna, J., and Jaenisch, R. (2009). Single-Gene Transgenic Mouse Strains for Reprogramming Adult Somatic Cells. Nature Methods [Epub ahead of print]. We report transgenic mouse models in which three or four reprogramming factors are expressed from a single genomic locus using a drug-inducible transgene. Multiple somatic cell types can be directly reprogrammed to generate induced pluripotent stem cells (iPSCs) by culture in doxycycline. Because reprogramming factors are carried on a single polycistronic construct, the mice can be easily maintained, and the transgene can be easily transferred into other genetic backgrounds. Full Text.
Carey, B.W., Markoulaki, S., Hanna, J., Saha, K., Gao, Q., Mitalipova, M., and Jaenisch, R. (2009). Reprogramming of Murine and Human Somatic Cells Using a Single Polycistronic Vector. Proceedings of the National Academy of Sciences of the United States of America 106, 157-162. 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.
Costantino, C.M., Ploegh, H.L., and Hafler, D.A. (2009). Cathepsin S Regulates Class Ii Mhc Processing in Human Cd4(+) Hla-Dr+ T Cells. Journal of Immunology 183, 945-952. Although it has long been known that human CD4(+) T cells can express functional class II MHC molecules, the role of lysosomal proteases in the T cell class II MHC processing and presentation pathway is unknown. Using CD4(+) T cell clones that constitutively express class II MHC, we determined that cathepsin S is necessary for invariant chain proteolysis in T cells. CD4(+)HLA-DR+ T cells down-regulated cathepsin S expression and activity 18 h after activation, thereby ceasing nascent class II MHC product formation. This blockade resulted in the loss of the invariant chain fragment CLIP from the cell surface, suggesting that-like professional APC-CD4(+) HLA-DR+ cells modulate self-Ag presentation as a consequence of activation. Furthermore, cathepsin S expression and activity, and concordantly cell surface CLIP expression, was reduced in HLA-DR+ CD4(+) T cells as compared with B cells both in vitro and ex vivo. Full Text.
Cowen, L.E., Singh, S.D., Kohler, J.R., Collins, C., Zaas, A.K., Schell, W.A., Aziz, H., Mylonakis, E., Perfect, J.R., Whitesell, L., and Susan Lindquist (2009). Harnessing Hsp90 Function as a Powerful, Broadly Effective Therapeutic Strategy for Fungal Infectious Disease. Proc Natl Acad Sci U S A.Online Pub 5 Feb. Invasive fungal infections are a leading cause of mortality among immunocompromised individuals. Treatment is notoriously difficult with the limited armamentarium of antifungal drugs, whose efficacy is compromised by host toxicity, a limited activity spectrum, or the emergence of drug resistance. We previously established that the molecular chaperone Hsp90 enables the emergence and maintenance of fungal drug resistance. For the most prevalent fungal pathogen of humans, Candida albicans, Hsp90 mediates resistance to azoles, which inhibit ergosterol biosynthesis and are the most widely deployed antifungals in the clinic. For the emerging opportunistic pathogen Aspergillus terreus, Hsp90 is required for basal resistance to echinocandins, which inhibit beta(1, 3)-glucan synthesis and are the only new class of antifungals to reach the clinic in decades. Here, we explore the therapeutic potential of Hsp90 inhibitors in fungal disease using a tractable host-model system, larvae of the greater wax moth Galleria mellonella, and a murine model of disseminated disease. Combination therapy with Hsp90 inhibitors that are well tolerated in humans and an azole rescued larvae from lethal C. albicans infections. Combination therapy with an Hsp90 inhibitor and an echinocandin rescued larvae from infections with the most lethal mold, Aspergillus fumigatus. In a murine model of disseminated candidiasis, genetic compromise of C. albicans HSP90 expression enhanced the therapeutic efficacy of an azole. Thus, harnessing Hsp90 provides a much-needed strategy for improving the treatment of fungal disease because it enhances the efficacy of existing antifungals, blocks the emergence of drug resistance, and exerts broad- spectrum activity against diverse fungal pathogens.PDF
Daley, G.Q., Lensch, M.W., Jaenisch, R., Meissner, A., Plath, K., and Yamanaka, S. (2009). Broader Implications of Defining Standards for the Pluripotency of Ipscs. Cell Stem Cell 4, 200-201. The field of factor-based reprogramming is in its infancy. For the foreseeable future, as we are learning more about what signaling pathways and epigenetic modifications distinguish one particular pluripotent stem cell state from another, and until such time as reliable molecular surrogates of the reprogrammed state can be validated, we believe it is best to encourage everyone to characterize their cells according to the most stringent test of pluripotency available. We need to understand the behavior of iPSCs in a standard set of assays to enable cross-lab comparisons and to move toward a deeper understanding of the molecular basis of pluripotency and lineage restriction. Full Text.
Dickinson, A.J., and Sive, H.L. (2009). The Wnt Antagonists Frzb-1 and Crescent Locally Regulate Basement Membrane Dissolution in the Developing Primary Mouth. Development 136, 1071-1081. The primary mouth forms from ectoderm and endoderm at the extreme anterior of the embryo, a conserved mesoderm-free region. In Xenopus, a very early step in primary mouth formation is loss of the basement membrane between the ectoderm and endoderm. In an unbiased microarray screen, we defined genes encoding the sFRPs Frzb-1 and Crescent as transiently and locally expressed in the primary mouth anlage. Using antisense oligonucleotides and ;face transplants', we show that frzb-1 and crescent expression is specifically required in the primary mouth region at the time this organ begins to form. Several assays indicate that Frzb-1 and Crescent modulate primary mouth formation by suppressing Wnt signaling, which is likely to be mediated by beta-catenin. First, a similar phenotype (no primary mouth) is seen after loss of Frzb-1/Crescent function to that seen after temporally and spatially restricted overexpression of Wnt-8. Second, overexpression of either Frzb-1 or Dkk-1 results in an enlarged primary mouth anlage. Third, overexpression of Dkk-1 can restore a primary mouth to embryos in which Frzb-1/Crescent expression has been inhibited. We show that Frzb-1/Crescent function locally promotes basement membrane dissolution in the primary mouth primordium. Consistently, Frzb-1 overexpression decreases RNA levels of the essential basement membrane genes fibronectin and laminin, whereas Wnt-8 overexpression increases the levels of these RNAs. These data are the first to connect Wnt signaling and basement membrane integrity during primary mouth development, and suggest a general paradigm for the regulation of basement membrane remodeling. Full Text.
Distefano, G., Boca, M., Rowe, I., Claas, W., Ma, L., Piontek, K.B., Germino, G.G., Pandolfi, P.P., and Boletta, A. (2009). Polycystin-1 Regulates Erks-Dependent Phosphorylation of Tuberin to Control Cell Size through Mtor and Its Downstream Effectors S6k and 4ebp1. Molecular Cell BiologyMar 2. [Epub ahead of print] Autosomal Dominant Polycystic Kidney Disease (ADPKD) is a common genetic disease characterized by bilateral renal cyst formation. Both hyperproliferation and hypertrophy have been previously observed in ADPKD kidneys. Polycystin-1 (PC-1), a large orphan receptor encoded by the PKD1 gene and mutated in 85% of all cases, is able to inhibit proliferation and apoptosis. Here we show that overexpression of PC-1 in renal epithelial cells inhibits cell growth (size) in a cell-cycle independent manner due to the downregulation of mTOR, S6K1 and 4EBP1. Upregulation of the same pathway leads to increased cell size, as found in MEFs derived from Pkd1-/- mice. We show that PC-1 controls the mTOR pathway in a Tsc2-dependent manner, by inhibiting the ERKs-mediated phosphorylation of Tuberin in Ser664. We provide a detailed molecular mechanism by which PC-1 can inhibit the mTOR pathway and regulate cell size.PDF
Drinnenberg, I.A., Weinberg, D.E., Xie, K.T., Mower, J.P., Wolfe, K.H., Fink, G.R., and Bartel, D.P. (2009). Rnai in Budding Yeast. Science Sep 10. [Epub ahead of print] RNA interference (RNAi), a gene-silencing pathway triggered by double-stranded RNA, is conserved in diverse eukaryotic species but has been lost in the model budding yeast, Saccharomyces cerevisiae. Here, we show that RNAi is present in other budding-yeast species, including Saccharomyces castellii and Candida albicans. These species use noncanonical Dicer proteins to generate small interfering RNAs, which mostly correspond to transposable elements and Y' subtelomeric repeats. In S. castellii, RNAi mutants are viable but have excess Y' mRNA levels. In S. cerevisiae, introducing Dicer and Argonaute of S. castellii restores RNAi, and the reconstituted pathway silences endogenous retrotransposons. These results identify a novel class of Dicer proteins, bring the tool of RNAi to the study of budding yeasts, and bring the tools of budding yeast to the study of RNAi. Full Text.
Efeyan, A., and Sabatini, D.M. (2009). Mtor and Cancer: Many Loops in One Pathway. Current Opinion in Cell Biology Nov 27. [Epub ahead of print]. The mammalian target of rapamycin (mTOR) is a master regulator of cell growth and division that responds to a variety of stimuli, including nutrient, energy, and growth factors. In the last years, a significant number of pieces have been added to the puzzle of how mTOR coordinates and executes its functions. Extensive research on mTOR has also uncovered a complex network of regulatory loops that impact the therapeutic approaches aimed at targeting mTOR. PDF.
Ernst, R., Mueller, B., Ploegh, H.L., and Schlieker, C. (2009). The Otubain Yod1 Is a Deubiquitinating Enzyme That Associates with P97 to Facilitate Protein Dislocation from the Er. Molecular Cell 36, 28-38. YOD1 is a highly conserved deubiquitinating enzyme of the ovarian tumor (otubain) family, whose function has yet to be assigned in mammalian cells. YOD1 is a constituent of a multiprotein complex with p97 as its nucleus, suggesting a functional link to a pathway responsible for the dislocation of misfolded proteins from the endoplasmic reticulum. Expression of a YOD1 variant deprived of its deubiquitinating activity imposes a halt on the dislocation reaction, as judged by the stabilization of various dislocation substrates. Accordingly, we observe an increase in polyubiquitinated dislocation intermediates in association with p97 in the cytosol. This dominant-negative effect is dependent on the UBX and Zinc finger domains, appended to the N and C terminus of the catalytic otubain core domain, respectively. The assignment of a p97-associated ubiquitin processing function to YOD1 adds to our understanding of p97's role in the dislocation process. PDF
Faas, H., Jackson, W.S., Borkowski, A.W., Wang, X.H., Ma, J.Y., Lindquist, S., and Jasanoff, A. Context-Dependent Perturbation of Neural Systems in Transgenic Mice Expressing a Cytosolic Prion Protein. Neuroimage 49, 2607-2617. We analyzed the relationship between pathogenic protein expression and perturbations to brain anatomy and physiology in a genetic model of prion disease In this model, the mouse line 1D4, neuropathology is promoted by accumulation of a cytosolic form of the prion protein (cyPrP) CyPrP distribution was determined and compared with anatomical magnetic resonance imaging (MRI) data, a form of functional MRI based on manganese labeling, and immediate early gene mapping with an antibody to c-Fos. Significant discrepancies between 1D4 and control mice became apparent well in advance of overt behavioral pathology in the mutant mice Alterations to brain structure and function in the mutants varied among brain regions, however, and differed strikingly even among regions with the highest levels of cyPrP expression In the cerebellum, gross neurodegeneration was accompanied by increased Mn2+-enhanced MRI signal, raising the possibility that compensatory mechanisms act to preserve cerebellar function in the face of massive atrophy In the hippocampus of IN mice, no significant structural alterations were observed, but both Mn2+-enhanced MRI and c-Fos data indicated perturbations to neurophysiology. In the neocortex, there were no clear neural activity differences between 1D4 and control animals, but mutant mice showed significant reduction in cortical thickness Our finding that distinct combinations of anatomical and functional abnormalities accompanied cyPrP overexpression in different parts of the brain indicates the importance of context in conditioning effects of protein pathogens. and exemplifies the notion that neurodegenerative phenotypes extend beyond cell death and the immediate consequences of atrophy for particular neural system .Full Text.
Festuccia, W.T., Blanchard, P.G., Turcotte, V., Laplante, M., Sariahmetoglu, M., Brindley, D.N., and Deshaies, Y. (2009). Depot-Specific Effects of the Ppar Gamma Agonist Rosiglitazone on Adipose Tissue Glucose Uptake and Metabolism. Journal of Lipid Research 50, 1185-1194. We investigated mechanisms whereby peroxisome proliferator-activated receptor gamma (PPAR gamma) agonism redistributes lipid from visceral (VF) toward subcutaneous fat (SF) by studying the impact of PPARg activation on VF and SF glucose uptake and metabolism, lipogenesis, and enzymes involved in triacylglycerol (TAG) synthesis. VF (retroperitoneal) and SF (inguinal) of rats treated or not for 7 days with rosiglitazone (15 mg/kg/day) were evaluated in vivo for glucose uptake and lipogenesis and in vitro for glucose metabolism, gene expression, and activities of glycerolphosphate acyltransferase (GPAT), phosphatidate phosphatase-1 (or lipin-1), and diacylglycerol acyltransferase. Rosiglitazone increased SF glucose uptake, GLUT4 mRNA, and insulin-stimulated glucose oxidation, conversion to lactate, glycogen, and the glycerol and fatty acid components of TAG. In VF, only glucose incorporation into TAG-glycerol was stimulated by rosiglitazone and less so than in SF (1.5- vs. 3-fold). mRNA levels of proteins involved in glycolysis, Krebs cycle, glycogen synthesis, and lipogenesis were markedly upregulated by rosiglitazone in SF and again less so in VF. Rosiglitazone activated TAG-glycerol synthesis in vivo (2.8- vs. 1.9-fold) and lipin activity (4.6- vs. 1.5-fold) more strongly in SF than VF, whereas GPAT activity was increased similarly in both depots. The preferential increase in glucose uptake and intracellular metabolism in SF contributes to the PPAR gamma-mediated redistribution of TAG from VF to SF, which in turn favors global insulin sensitization.-Festuccia, W. T., P-G. Blanchard, V. Turcotte, M. Laplante, M. Sariahmetoglu, D. N. Brindley, and Y. Deshaies. Depot-specific effects of the PPARg agonist rosiglitazone on adipose tissue glucose uptake and metabolism.. Full Text.
Fink, G.R. (2009). Getting Along with a Little Help from My Friends. Journal of Biological Chemistry.Jun 10. [Epub ahead of print] PDF
Foudi, A., Hochedlinger, K., Van Buren, D., Schindler, J.W., Jaenisch, R., Carey, V., and Hock, H. (2009). Analysis of Histone 2b-Gfp Retention Reveals Slowly Cycling Hematopoietic Stem Cells. Nature Biotechnology 27, 84-90. Hematopoietic stem cells (HSCs) are thought to divide infrequently based on their resistance to cytotoxic injury targeted at rapidly cycling cells(1,2) and have been presumed to retain labels such as the thymidine analog 5-bromodeoxyuridine (BrdU). However, BrdU retention is neither a sensitive nor specific marker for HSCs3. Here we show that transient, transgenic expression of a histone 2B (H2B)-green fluorescent protein (GFP) fusion protein in mice has several advantages for label-retention studies over BrdU, including rapid induction of H2B-GFP in virtually all HSCs, higher labeling intensity and the ability to prospectively study label-retaining cells, which together permit a more precise analysis of division history. Mathematical modeling of H2B-GFP dilution in HSCs, identified with a stringent marker combination (L-K(+)S(+)CD48(-)CD150(+))(4), revealed unexpected heterogeneity in their proliferation rates and showed that similar to 20% of HSCs divide at an extremely low rate (<= 0.8-1.8% per day). Full Text.
Friedman, R.C., Farh, K.K.H., Burge, C.B., and Bartel, D.P. (2009). Most Mammalian Mrnas Are Conserved Targets of Micrornas. Genome Research 19, 92-105. MicroRNAs (miRNAs) are small endogenous RNAs that pair to sites in mRNAs to direct post-transcriptional repression. Many sites that match the miRNA seed (nucleotides 2-7), particularly those in 3' untranslated regions (3'UTRs), are preferentially conserved. Here, we overhauled our tool for finding preferential conservation of sequence motifs and applied it to the analysis of human 3'UTRs, increasing by nearly threefold the detected number of preferentially conserved miRNA target sites. The new tool more efficiently incorporates new genomes and more completely controls for background conservation by accounting for mutational biases, dinucleotide conservation rates, and the conservation rates of individual UTRs. The improved background model enabled preferential conservation of a new site type, the "offset 6mer," to be detected. In total, >45,000 miRNA target sites within human 3'UTRs are conserved above background levels, and >60% of human protein-coding genes have been under selective pressure to maintain pairing to miRNAs. Mammalian-specific miRNAs have far fewer conserved targets than do the more broadly conserved miRNAs, even when considering only more recently emerged targets. Although pairing to the 3' end of miRNAs can compensate for seed mismatches, this class of sites constitutes less than 2% of all preferentially conserved sites detected. The new tool enables statistically powerful analysis of individual miRNA target sites, with the probability of preferentially conserved targeting (P-CT) correlating with experimental measurements of repression. Our expanded set of target predictions (including conserved 3'-compensatory sites), are available at the TargetScan website, which displays the P-CT for each site and each predicted target. Full Text.
GabeauLacet, D., Engler, D., Gupta, S., Scangas, G.A., Betensky, R.A., Barker, F.G., Loeffler, J.S., Louis, D.N., and Mohapatra, G. (2009). Genomic Profiling of Atypical Meningiomas Associates Gain of 1q with Poor Clinical Outcome. Journal of Neuropathology and Experimental Neurology 68, 1155-1165. Atypical meningiomas exhibit heterogeneous clinical outcomes. It is unclear which atypical meningiomas require aggressive multimodality treatment with surgery and radiation therapy versus surgery alone to prevent recurrence. Detailed molecular-genetic characterization of these neoplasms is necessary to understand their pathogenesis and identify clinically relevant genetic markers. Oligonucleotide array comparative genomic hybridization was used to identify frequent genetic alterations in 47 primary atypical meningiomas resected at Massachusetts General Hospital between August 1987 and September 2006. Eighty-five percent of samples exhibited loss of 22q, including the neurofibromatosis type 2 gene. The second most frequent regions of loss were confined to the short arm of chromosome 1, particularly 1p33-p36.2 (70%) and 1p13.2 (64%). Other frequent regions of loss, detected in more than 50% of samples, included 14q, 10q, 8q, 7p, 21q, 19, 9q34, and 4p16. Frequent regions of gain were detected along 1q (59%), 17q (44%), 9q34 (30%), and 7q36 (26%). Univariate marker-by-marker analysis of all frequently identified copy number alterations showed potential correlation between gain of 1q and shorter progression-free survival. Given the heterogeneous treatment outcomes of atypical meningioma, investigation of large-scale and focal genomic alterations in multi-institutional efforts may help clarify molecular-genetic signatures of clinical use..
Gitler, A.D., Chesi, A., Geddie, M.L., Strathearn, K.E., Hamamichi, S., Hill, K.J., Caldwell, K.A., Caldwell, G.A., Cooper, A.A., Rochet, J.C., and Susan Lindquist (2009). Alpha-Synuclein Is Part of a Diverse and Highly Conserved Interaction Network That Includes Park9 and Manganese Toxicity. Nature Genetics Feb 1. [Epub ahead of print]. Parkinson's disease (PD), dementia with Lewy bodies and multiple system atrophy, collectively referred to as synucleinopathies, are associated with a diverse group of genetic and environmental susceptibilities. The best studied of these is PD. alpha-Synuclein (alpha-syn) has a key role in the pathogenesis of both familial and sporadic PD, but evidence linking it to other predisposition factors is limited. Here we report a strong genetic interaction between alpha-syn and the yeast ortholog of the PD-linked gene ATP13A2 (also known as PARK9). Dopaminergic neuron loss caused by alpha-syn overexpression in animal and neuronal PD models is rescued by coexpression of PARK9. Further, knockdown of the ATP13A2 ortholog in Caenorhabditis elegans enhances alpha-syn misfolding. These data provide a direct functional connection between alpha-syn and another PD susceptibility locus. Manganese exposure is an environmental risk factor linked to PD and PD-like syndromes. We discovered that yeast PARK9 helps to protect cells from manganese toxicity, revealing a connection between PD genetics (alpha-syn and PARK9) and an environmental risk factor (PARK9 and manganese). Finally, we show that additional genes from our yeast screen, with diverse functions, are potent modifiers of alpha-syn-induced neuron loss in animals, establishing a diverse, highly conserved interaction network for alpha-syn. Full Text.
Glazer, A., Wilkinson, A., Backer, C.B., Lapan, S., Gutzman, J.H., Cheeseman, I.M., and Reddien, P.W. (2009). The Zn Finger Protein Iguana Impacts Hedgehog Signaling by Promoting Ciliogenesis. Developmental Biology.Oct 20. [Epub ahead of print] Hedgehog signaling is critical for metazoan development and requires cilia for pathway activity. The gene iguana was discovered in zebrafish as required for Hedgehog signaling, and encodes a novel Zn finger protein. Planarians are flatworms with robust regenerative capacities and that utilize epidermal cilia for locomotion. RNA interference of Smed-iguana in the planarian S. mediterranea caused cilia loss and failure to regenerate new cilia, but did not cause defects similar to those observed in hedgehog(RNAi) animals. Smed-iguana gene expression was also similar in pattern to the expression of multiple other ciliogenesis genes, but was not required for expression of these ciliogenesis genes. iguana-defective zebrafish had too few motile cilia in pronephric ducts and in Kupffer's vesicle. Kupffer's vesicle promotes left-right asymmetry and iguana mutant embryos had left-right asymmetry defects. Finally, human Iguana proteins (dZIP1 and dZIP1L) localize to the basal bodies of primary cilia and, together, are required for primary cilia formation. Our results indicate that a critical and broadly conserved function for Iguana is in ciliogenesis and that this function has come to be required for Hedgehog signaling in vertebrates. Full Text.
Graeden, E., and Sive, H. (2009). Live Imaging of the Zebrafish Embryonic Brain by Confocal Microscopy. Journal of Visualized Experiments 2009 26. In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the developing zebrafish brain (Gutzman et al, 2008). Such analysis affords a new understanding of the underlying cell biology required for development of the 3D structure of the vertebrate brain, and significantly increases our ability to study neural tube morphogenesis. The embryonic zebrafish brain is shaped beginning at 18 hours post fertilization (hpf) as the ventricles within the neuroepithelium inflate. By 24 hpf, the initial steps of neural tube morphogenesis are complete. Using the method described here, embryos at the one cell stage are injected with mRNA encoding membrane-targeted green fluorescent protein (memGFP). After injection and incubation, the embryo, now between 18 and 24 hpf, is mounted, inverted, in agarose and imaged by confocal microscopy. Notably, the zebrafish embryo is transparent making it an ideal system for fluorescent imaging. While our analyses have focused on the midbrain-hindbrain boundary and the hindbrain, this method could be extended for analysis of any region in the zebrafish to a depth of 80-100 microm. Full Text.
GredmarkRuss, S., Isaacson, M.K., Kattenhorn, L., Cheung, E.J., Watson, N., and Ploegh, H.L. (2009). A Gammaherpesvirus Ubiquitin-Specific Protease Is Involved in the Establishment of Mhv-68 Infection. Journal of Virology (published online ahead of print on 12 August 2009) Murine gammaherpesvirus 68 (MHV-68) contains a ubiquitin (Ub)-specific cysteine protease (USP) domain embedded within the large tegument protein, ORF64, as do all other herpes viruses. The biological role of this protease is still unclear, but for the alpha herpes virus MDV its USP is involved in T cell lymphoma formation. We here study the role of the MHV-68 USP, encoded by ORF64. By constructing a mutant virus with a single cysteine to alanine replacement in the active site of ORF64, we demonstrate that the USP activity of ORF64 is abolished. The mutant virus replicates less efficiently in vitro, and plaque size is reduced compared to a revertant virus. Electron microscopy of infected cells did not reveal any obvious differences in virion morphogenesis or differences in egress for the mutant and revertant viruses. Intra-peritoneal infection of C57/BL6 mice demonstrates that the mutant virus is generally cleared by day 7, indicating a role for the USP in the persistence of MHV-68 infection or efficient replication. However, the USP activity in MHV-68 is unlikely to be involved in the establishment of latency or reactivation, since we observed no significant difference in viral DNA genome copy number in the spleen or in the number of cells that reactivate MHV-68 from latency. Our results for MHV-68 ORF64 are consistent with an enzymatic function of the tegument protein that is beneficial to the virus during acute infection, particularly in vivo. PDF
Green, N.M., Laws, A., Kiefer, K., Busconi, L., Kim, Y.M., Brinkmann, M.M., Trail, E.H., Yasuda, K., Christensen, S.R., Shlomchik, M.J.,Stefanie Vogel||, John H. Connor, Hidde Ploegh, Dan Eilat, Ian R. Rifkin, Jean Maguire van Seventer and Ann Marshak-Rothstein (2009). Murine B Cell Response to Tlr7 Ligands Depends on an Ifn-Beta Feedback Loop. Journal of Immunology 183, 1569-1576. Type I IFNs play an important, yet poorly characterized, role in systemic lupus erythematosus. To better understand the interplay between type I IFNs and the activation of autoreactive B cells, we evaluated the effect of type I IFN receptor (IFNAR) deficiency in murine B cell responses to common TLR ligands. In comparison to wild-type B cells, TLR7-stimulated IFNAR(-/-) B cells proliferated significantly less well and did not up-regulate costimulatory molecules. By contrast, IFNAR1(-/-) B cells did not produce cytokines, but did proliferate and up-regulate activation markers in response to other TLR ligands. These defects were not due to a difference in the distribution of B cell populations or a failure to produce a soluble factor other than a type I IFN. Instead, the compromised response pattern reflected the disruption of an IFN-beta feedback loop and constitutively low expression of TLR7 in the IFNAR1(-/-) B cells. These results highlight subtle differences in the IFN dependence of TLR7 responses compared with other TLR-mediated B cell responses. Full Text.
Guan, J.S., Haggarty, S.J., Giacometti, E., Dannenberg, J.H., Joseph, N., Gao, J., Nieland, T.J.F., Zhou, Y., Wang, X.Y., Mazitschek, R., James E. Bradner, Ronald A. DePinho, Rudolf Jaenisch & Li-Huei Tsai1 (2009). Hdac2 Negatively Regulates Memory Formation and Synaptic Plasticity. Nature 459, 55-60. Chromatin modifications, especially histone-tail acetylation, have been implicated in memory formation. Increased histone-tail acetylation induced by inhibitors of histone deacetylases (HDACis) facilitates learning and memory in wild-type mice as well as in mouse models of neurodegeneration. Harnessing the therapeutic potential of HDACis requires knowledge of the specific HDAC family member(s) linked to cognitive enhancement. Here we show that neuron-specific overexpression of HDAC2, but not that of HDAC1, decreased dendritic spine density, synapse number, synaptic plasticity and memory formation. Conversely, Hdac2 deficiency resulted in increased synapse number and memory facilitation, similar to chronic treatment with HDACis in mice. Notably, reduced synapse number and learning impairment of HDAC2-overexpressing mice were ameliorated by chronic treatment with HDACis. Correspondingly, treatment with HDACis failed to further facilitate memory formation in Hdac2-deficient mice. Furthermore, analysis of promoter occupancy revealed an association of HDAC2 with the promoters of genes implicated in synaptic plasticity and memory formation. Taken together, our results suggest that HDAC2 functions in modulating synaptic plasticity and long-lasting changes of neural circuits, which in turn negatively regulates learning and memory. These observations encourage the development and testing of HDAC2-selective inhibitors for human diseases associated with memory impairment. Full Text.
Guertin, D.A., and Sabatini, D.M. (2009). The Pharmacology of Mtor Inhibition. Sci Signal 2, pe24. A flurry of reports indicates that we are entering a new phase in the development of mammalian target of rapamycin (mTOR)-based therapies for oncology. Here, we summarize exciting findings regarding mTOR signaling and the outlook for mTOR inhibitors as tools to study the mTOR pathway and as drugs in the clinic. Full Text.
Guertin, D.A., Stevens, D.M., Saitoh, M., Kinkel, S., Crosby, K., Sheen, J.H., Mullholland, D.J., Magnuson, M.A., Wu, H., and Sabatini, D.M. (2009). Mtor Complex 2 Is Required for the Development of Prostate Cancer Induced by Pten Loss in Mice. Cancer Cell 15, 148-159. mTOR complex 2 (mTORC2) contains the mammalian target of rapamycin (mTOR) kinase and the Rictor regulatory protein and phosphorylates Akt. Whether this function of mTORC2 is critical for cancer progression is unknown. Here, we show that transformed human prostate epithelial cells lacking PTEN require mTORC2 to form tumors when injected into nude mice. Furthermore, we find that Rictor is a haploinsufficient gene and that deleting one copy protects Pten heterozygous mice from prostate cancer. Finally, we show that the development of prostate cancer caused by Pten deletion specifically in prostate epithelium requires mTORC2, but that for normal prostate epithelial cells, mTORC2 activity is nonessential. The selective requirement for mTORC2 in tumor development suggests that mTORC2 inhibitors may be of substantial clinical utility. Full Text.
Gupta, P.B., Chaffer, C.L., and Weinberg, R.A. (2009). Cancer Stem Cells: Mirage or Reality? Nature Medicine 15, 1010-1012. The similarities and differences between normal tissue stem cells and cancer stem cells (CSCs) have been the source of much contention, with some recent studies calling into question the very existence of CSCs. An examination of the literature indicates, however, that the CSC model rests on firm experimental foundations and that differences in the observed frequencies of CSCs within tumors reflect the various cancer types and hosts used to assay these cells. Studies of stem cells and the differentiation program termed the epithelial-mesenchymal transition (EMT) point to the possible existence of plasticity between stem cells and their more differentiated derivatives. If present, such plasticity would have major implications for the CSC model and for future therapeutic approaches. Full Text.
Gupta, P.B., Onder, T.T., Jiang, G., Tao, K., Kuperwasser, C., Weinberg, R.A., and Lander, E.S. (2009). Identification of Selective Inhibitors of Cancer Stem Cells by High-Throughput Screening. Cell. Aug 12. [Epub ahead of print]. Screens for agents that specifically kill epithelial cancer stem cells (CSCs) have not been possible due to the rarity of these cells within tumor cell populations and their relative instability in culture. We describe here an approach to screening for agents with epithelial CSC-specific toxicity. We implemented this method in a chemical screen and discovered compounds showing selective toxicity for breast CSCs. One compound, salinomycin, reduces the proportion of CSCs by >100-fold relative to paclitaxel, a commonly used breast cancer chemotherapeutic drug. Treatment of mice with salinomycin inhibits mammary tumor growth in vivo and induces increased epithelial differentiation of tumor cells. In addition, global gene expression analyses show that salinomycin treatment results in the loss of expression of breast CSC genes previously identified by analyses of breast tissues isolated directly from patients. This study demonstrates the ability to identify agents with specific toxicity for epithelial CSCs. Full Text.
Guttman, M., Amit, I., Garber, M., French, C., Lin, M.F., Feldser, D., Huarte, M., Zuk, O., Carey, B.W., Cassady, J.P.,Cabili MN, Jaenisch R, et al. (2009). Chromatin Signature Reveals over a Thousand Highly Conserved Large Non-Coding Rnas in Mammals. Nature.Feb 1. [Epub ahead of print]. There is growing recognition that mammalian cells produce many thousands of large intergenic transcripts. However, the functional significance of these transcripts has been particularly controversial. Although there are some well-characterized examples, most (>95%) show little evidence of evolutionary conservation and have been suggested to represent transcriptional noise. Here we report a new approach to identifying large non-coding RNAs using chromatin-state maps to discover discrete transcriptional units intervening known protein-coding loci. Our approach identified approximately 1,600 large multi-exonic RNAs across four mouse cell types. In sharp contrast to previous collections, these large intervening non-coding RNAs (lincRNAs) show strong purifying selection in their genomic loci, exonic sequences and promoter regions, with greater than 95% showing clear evolutionary conservation. We also developed a functional genomics approach that assigns putative functions to each lincRNA, demonstrating a diverse range of roles for lincRNAs in processes from embryonic stem cell pluripotency to cell proliferation. We obtained independent functional validation for the predictions for over 100 lincRNAs, using cell-based assays. In particular, we demonstrate that specific lincRNAs are transcriptionally regulated by key transcription factors in these processes such as p53, NFkappaB, Sox2, Oct4 (also known as Pou5f1) and Nanog. Together, these results define a unique collection of functional lincRNAs that are highly conserved and implicated in diverse biological processes. Full Text.
Gutzman, J.H., and Sive, H. (2009). Zebrafish Brain Ventricle Injection. Journal of Visualized Experiments : JoVE.Apr 6;(26). Proper brain ventricle formation during embryonic brain development is required for normal brain function. Brain ventricles are the highly conserved cavities within the brain that are filled with cerebrospinal fluid. In zebrafish, after neural tube formation, the neuroepithelium undergoes a series of constrictions and folds while it fills with fluid resulting in brain ventricle formation. In order to understand the process of ventricle formation, and the neuroepithelial shape changes that occur at the same time, we needed a way to visualize the ventricle space in comparison to the brain tissue. However, the nature of transparent zebrafish embryos makes it difficult to differentiate the tissue from the ventricle space. Therefore, we developed a brain ventricle injection technique where the ventricle space is filled with a fluorescent dye and imaged by brightfield and fluorescent microscopy. The brightfield and the fluorescent images are then processed and superimposed in Photoshop. This technique allows for visualization of the ventricle space with the fluorescent dye, in comparison to the shape of the neuroepithelium in the brightfield image. Brain ventricle injection in zebrafish can be employed from 18 hours post fertilization through early larval stages. We have used this technique extensively in our studies of brain ventricle formation and morphogenesis as well as in characterizing brain morphogenesis mutants (1-3). Full Text.
Han, D.W., Do, J.T., Arauzo-Bravo, M.J., Lee, S.H., Meissner, A., Lee, H.T., Jaenisch, R., and Scholer, H.R. (2009). Epigenetic Hierarchy Governing Nestin Expression. Stem Cells 27, 1088-1097. Nestin is an intermediate. lament protein expressed specifically in neural stem cells and progenitor cells of the central nervous system. DNA demethylation and histone modi. cations are two types of epigenetic modifications working in a coordinate or synergistic manner to regulate the expression of various genes. This study investigated and elucidated the epigenetic regulation of Nestin gene expression during embryonic differentiation along the neural cell lineage. Nestin exhibits differential DNA methylation and histone acetylation patterns in Nestin-expressing and nonexpressing cells. In P19 embryonic carcinoma cells, activation of Nestin expression is mediated by both trichostatin A and 5-aza-2'-deoxycytidine treatment, concomitant with histone acetylation, but not with DNA demethylation. Nestin transcription is also mediated by treatment with retinoic acid, again in the absence of DNA demethylation. Thus, histone acetylation is sufficient to mediate the activation of Nestin transcription. This study proposed that the regulation of Nestin gene expression can be used as a model to study the epigenetic regulation of gene expression mediated by histone acetylation, but not by DNA demethylation.. Full Text.
Hanna, J., Saha, K., Pando, B., van Zon, J., Lengner, C.J., Creyghton, M.P., van Oudenaarden, A., and Jaenisch, R. (2009). Direct Cell Reprogramming Is a Stochastic Process Amenable to Acceleration. Nature.Nov 8. [Epub ahead of print] Direct reprogramming of somatic cells into induced pluripotent stem (iPS) cells can be achieved by overexpression of Oct4, Sox2, Klf4 and c-Myc transcription factors, but only a minority of donor somatic cells can be reprogrammed to pluripotency. Here we demonstrate that reprogramming by these transcription factors is a continuous stochastic process where almost all mouse donor cells eventually give rise to iPS cells on continued growth and transcription factor expression. Additional inhibition of the p53/p21 pathway or overexpression of Lin28 increased the cell division rate and resulted in an accelerated kinetics of iPS cell formation that was directly proportional to the increase in cell proliferation. In contrast, Nanog overexpression accelerated reprogramming in a predominantly cell-division-rate-independent manner. Quantitative analyses define distinct cell-division-rate-dependent and -independent modes for accelerating the stochastic course of reprogramming, and suggest that the number of cell divisions is a key parameter driving epigenetic reprogramming to pluripotency. Full Text.
Hanna, J., Markoulaki, S., Mitalipova, M., Cheng, A.W., Cassady, J.P., Staerk, J., Carey, B.W., Lengner, C.J., Foreman, R., Love, J., Qing Gao, Jongpil Kim and Rudolf Jaenisch. (2009). Metastable Pluripotent States in Nod-Mouse-Derived Escs. Cell Stem Cell. May 6. [Epub ahead of print] Embryonic stem cells (ESCs) are isolated from the inner cell mass (ICM) of blastocysts, whereas epiblast stem cells (EpiSCs) are derived from the postimplantation epiblast and display a restricted developmental potential. Here we characterize pluripotent states in the nonobese diabetic (NOD) mouse strain, which prior to this study was considered "nonpermissive" for ESC derivation. We find that NOD stem cells can be stabilized by providing constitutive expression of Klf4 or c-Myc or small molecules that can replace these factors during in vitro reprogramming. The NOD ESCs and iPSCs appear to be "metastable," as they acquire an alternative EpiSC-like identity after removal of the exogenous factors, while their reintroduction converts the cells back to ICM-like pluripotency. Our findings suggest that stem cells from different genetic backgrounds can assume distinct states of pluripotency in vitro, the stability of which is regulated by endogenous genetic determinants and can be modified by exogenous factors. Full Text.
Hockemeyer, D., Soldner, F., Beard, C., Gao, Q., Mitalipova, M., Dekelver, R.C., Katibah, G.E., Amora, R., Boydston, E.A., Zeitler, B., Meng X, Miller JC, Zhang L, Rebar EJ, Gregory PD, Urnov FD, Jaenisch R (2009). Efficient Targeting of Expressed and Silent Genes in Human Escs and Ipscs Using Zinc-Finger Nucleases. Nature Biotechnoly.Aug 13. [Epub ahead of print] Realizing the full potential of human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) requires efficient methods for genetic modification. However, techniques to generate cell type-specific lineage reporters, as well as reliable tools to disrupt, repair or overexpress genes by gene targeting, are inefficient at best and thus are not routinely used. Here we report the highly efficient targeting of three genes in human pluripotent cells using zinc-finger nuclease (ZFN)-mediated genome editing. First, using ZFNs specific for the OCT4 (POU5F1) locus, we generated OCT4-eGFP reporter cells to monitor the pluripotent state of hESCs. Second, we inserted a transgene into the AAVS1 locus to generate a robust drug-inducible overexpression system in hESCs. Finally, we targeted the PITX3 gene, demonstrating that ZFNs can be used to generate reporter cells by targeting non-expressed genes in hESCs and hiPSCs. Full Text.
Hochwagen, A. (2009).
Meiosis: Making a Synaptonemal Complex Just Got Easier. Curr
Biol 19, R849-851. In preparation for meiosis, chromosomes go
through several massive structural transitions, including chromosome
fragmentation, pairing and
synapsis. A checkpoint factor and a SUMO ligase collaborate to keep things
in order. Full Text.
Hu, C.C., Dougan, S.K., Winter, S.V., Paton, A.W., Paton, J.C., and Ploegh, H.L. (2009). Subtilase Cytotoxin Cleaves Newly Synthesized Bip and Blocks Antibody Secretion in B Lymphocytes. J Exp Med.Oct 7. [Epub ahead of print] Shiga-toxigenic Escherichia coli (STEC) use subtilase cytotoxin (SubAB) to interfere with adaptive immunity. Its inhibition of immunoglobulin secretion is both rapid and profound. SubAB favors cleavage of the newly synthesized immunoglobulin heavy chain-binding protein (BiP) to yield a C-terminal fragment that contains BiP's substrate-binding domain. In the absence of its regulatory nucleotide-binding domain, the SubAB-cleaved C-terminal BiP fragment remains tightly bound to newly synthesized immunoglobulin light chains, resulting in retention of light chains in the endoplasmic reticulum (ER). Immunoglobulins are thus detained in the ER, making impossible the secretion of antibodies by SubAB-treated B cells. The inhibitory effect of SubAB is highly specific for antibody secretion, because other secretory proteins such as IL-6 are released normally from SubAB-treated B cells. Although SubAB also causes BiP cleavage in HepG2 hepatoma cells, (glyco)protein secretion continues unabated in SubAB-exposed HepG2 cells. This specific block in antibody secretion is a novel means of immune evasion for STEC. The differential cleavage of newly synthesized versus "aged" BiP by SubAB in the ER provides insight into the architecture of the ER compartments involved. Full Text.
Hu, C.C., Dougan, S.K., McGehee, A.M., Love, J.C., and Ploegh, H.L. (2009). Xbp-1 Regulates Signal Transduction, Transcription Factors and Bone Marrow Colonization in B Cells. EMBO J Apr 30. [Epub ahead of print]. XBP-1, a transcription factor that drives the unfolded protein response (UPR), is activated in B cells when they differentiate to plasma cells. Here, we show that in the B cells, whose capacity to secrete IgM has been eliminated, XBP-1 is induced normally on induction of differentiation, suggesting that activation of XBP-1 in B cells is a differentiation-dependent event, but not the result of a UPR caused by the abundant synthesis of secreted IgM. Without XBP-1, B cells fail to signal effectively through the B-cell receptor. The signalling defects lead to aberrant expression of the plasma cell transcription factors IRF4 and Blimp-1, and altered levels of activation-induced cytidine deaminase and sphingosine-1-phosphate receptor. Using XBP-1-deficient/Blimp-1-GFP transgenic mice, we find that XBP-1-deficient B cells form antibody-secreting plasmablasts in response to initial immunization; however, these plasmablasts respond ineffectively to CXCL12. They fail to colonize the bone marrow and do not sustain antibody production. These findings define the role of XBP-1 in normal plasma cell development and have implications for management of B-cell malignancies. Full Text.
Isaacson, M.K., and Ploegh, H.L. (2009). Ubiquitination, Ubiquitin-Like Modifiers, and Deubiquitination in Viral Infection. Cell Host Microbe 5, 559-570. Ubiquitin is important for nearly every aspect of cellular physiology. All viruses rely extensively on host machinery for replication; therefore, it is not surprising that viruses connect to the ubiquitin pathway at many levels. Viral involvement with ubiquitin occurs either adventitiously because of the unavoidable usurpation of cellular processes, or for some specific purpose selected for by the virus to enhance viral replication. Here, we review current knowledge of how the ubiquitin pathway alters viral replication and how viruses influence the ubiquitin pathway to enhance their own replication. Full Text.
Jackson, W.S., Borkowski, A.W., Faas, H., Steele, A.D., King, O.D., Watson, N., Jasanoff, A., and Lindquist, S. (2009). Spontaneous Generation of Prion Infectivity in Fatal Familial Insomnia Knockin Mice. Neuron 63, 438-450. A crucial tenet of the prion hypothesis is that misfolding of the prion protein (PrP) induced by mutations associated with familial prion disease is, in an otherwise normal mammalian brain, sufficient to generate the infectious agent. Yet this has never been demonstrated. We engineered knockin mice to express a PrP mutation associated with a distinct human prion disease, fatal familial insomnia (FFI). An additional substitution created a strong transmission barrier against pre-existing prions. The mice spontaneously developed a disease distinct from that of other mouse prion models and highly reminiscent of FFI. Unique pathology was transmitted from FFI mice to mice expressing wild-type PrP sharing the same transmission barrier. FFI mice were highly resistant to infection by pre-existing prions, confirming infectivity did not arise from contaminating agents. Thus, a single amino acid change in PrP is sufficient to induce a distinct neurodegenerative disease and the spontaneous generation of prion infectivity.Full Text.
Jaenisch, R. (2009). Stem Cells, Pluripotency and Nuclear Reprogramming. Journal of Thrombosis and Haemostasis 7, S1 :21-23. 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. This short review summarizes strategies to reprogram somatic cells to a pluripotent embryonic state and discuss the implications of this technology for transplantation medicine. Full Text.
Jenner, R.G., Townsend, M.J., Jackson, I., Sun, K.M., Bouwman, R.D., Young, R.A., Glimcher, L.H., and Lord, G.M. (2009). The Transcription Factors T-Bet and Gata-3 Control Alternative Pathways of T-Cell Differentiation through a Shared Set of Target Genes. Proceedings of the National Academy of Sciences of the United States of America 106, 17876-17881. Upon detection of antigen, CD4(+) T helper (Th) cells can differentiate into a number of effector types that tailor the immune response to different pathogens. Alternative Th1 and Th2 cell fates are specified by the transcription factors T-bet and GATA-3, respectively. Only a handful of target genes are known for these two factors and because of this, the mechanism through which T-bet and GATA-3 induce differentiation toward alternative cell fates is not fully understood. Here, we provide a genomic map of T-bet and GATA-3 binding in primary human T cells and identify their target genes, most of which are previously unknown. In Th1 cells, T-bet associates with genes of diverse function, including those with roles in transcriptional regulation, chemotaxis and adhesion. GATA-3 occupies genes in both Th1 and Th2 cells and, unexpectedly, shares a large proportion of targets with T-bet. Re-complementation of T-bet alters the expression of these genes in a manner that mirrors their differential expression between Th1 and Th2 lineages. These data show that the choice between Th1 and Th2 lineage commitment is the result of the opposing action of T-bet and GATA-3 at a shared set of target genes and may provide a general paradigm for the interaction of lineage-specifying transcription factors. Full Text.
Jones, T.R., Carpenter, A.E., Lamprecht, M.R., Moffat, J., Silver, S.J., Grenier, J.K., Castoreno, A.B., Eggert, U.S., Root, D.E., Golland, P.and David M. Sabatini. (2009). Scoring Diverse Cellular Morphologies in Image-Based Screens with Iterative Feedback and Machine Learning. Proceedings of the National Academy of Sciences of the United States of America 106, 1826-1831. Many biological pathways were first uncovered by identifying mutants with visible phenotypes and by scoring every sample in a screen via tedious and subjective visual inspection. Now, automated image analysis can effectively score many phenotypes. In practical application, customizing an image-analysis algorithm or finding a sufficient number of example cells to train a machine learning algorithm can be infeasible, particularly when positive control samples are not available and the phenotype of interest is rare. Here we present a supervised machine learning approach that uses iterative feedback to readily score multiple subtle and complex morphological phenotypes in high-throughput, image-based screens. First, automated cytological profiling extracts hundreds of numerical descriptors for every cell in every image. Next, the researcher generates a rule (i.e., classifier) to recognize cells with a phenotype of interest during a short, interactive training session using iterative feedback. Finally, all of the cells in the experiment are automatically classified and each sample is scored based on the presence of cells displaying the phenotype. By using this approach, we successfully scored images in RNA interference screens in 2 organisms for the prevalence of 15 diverse cellular morphologies, some of which were previously intractable. Full Text.
Juan, A.H., Kumar, R.M., Marx, J.G., Young, R.A., and Sartorelli, V. (2009). Mir-214-Dependent Regulation of the Polycomb Protein Ezh2 in Skeletal Muscle and Embryonic Stem Cells. Molecular Cell 36, 61-74. Polycomb group (PcG) proteins exert essential functions in the most disparate biological processes. The contribution of PcG proteins to cell commitment and differentiation relates to their ability to repress transcription of developmental regulators in embryonic stem (ES) cells and in committed cell lineages, including skeletal muscle cells (SMC). PcG proteins are preferentially removed from transcribed regions, but the underlying mechanisms remain unclear. Here, PcG proteins are found to occupy and repress transcription from an intronic region containing the microRNA miR-214 in undifferentiated SMC. Differentiation coincides with PcG disengagement, recruitment of the developmental regulators MyoD and myogenin, and activation of miR-214 transcription. Once transcribed, miR-214 negatively feeds back on PcG by targeting the Ezh2 3'UTR the catalytic subunit of the PRC2 complex. miR-214-mediated Ezh2 protein reduction accelerates SMC differentiation and promotes unscheduled transcription of developmental regulators in ES cells. Thus, miR-214 and Ezh2 establish a regulatory loop controlling PcG-dependent gene expression during differentiation. Full Text.
Kalaany, N.Y., and Sabatini, D.M. (2009). Tumours with Pi3k Activation Are Resistant to Dietary Restriction. Nature. Mar 11. [Epub ahead of print] Dietary restriction delays the incidence and decreases the growth of various types of tumours, but the mechanisms underlying the sensitivity of tumours to food restriction remain unknown. Here we show that certain human cancer cell lines, when grown as tumour xenografts in mice, are highly sensitive to the anti-growth effects of dietary restriction, whereas others are resistant. Cancer cells that form dietary-restriction-resistant tumours carry mutations that cause constitutive activation of the phosphatidylinositol-3-kinase (PI3K) pathway and in culture proliferate in the absence of insulin or insulin-like growth factor 1. Substitution of an activated mutant allele of PI3K with wild-type PI3K in otherwise isogenic cancer cells, or the restoration of PTEN expression in a PTEN-null cancer cell line, is sufficient to convert a dietary-restriction-resistant tumour into one that is dietary-restriction-sensitive. Dietary restriction does not affect a PTEN-null mouse model of prostate cancer, but it significantly decreases tumour burden in a mouse model of lung cancer lacking constitutive PI3K signalling. Thus, the PI3K pathway is an important determinant of the sensitivity of tumours to dietary restriction, and activating mutations in the pathway may influence the response of cancers to dietary restriction-mimetic therapies. Full Text.
Kalinkovich, A., Spiegel, A., Shivtiel, S., Kollet, O., Jordaney, N., Piacibello, W., and Lapidot, T. (2009). Blood-Forming Stem Cells Are Nervous: Direct and Indirect Regulation of Immature Human Cd34(+) Cells by the Nervous System. Brain Behavior and Immunity 23, 1059-1065. The nervous system regulates immunity through hormonal and neuronal routes as part of host defense and repair mechanism. Here, we review the emerging evidence for regulation of human hematopoietic stem and progenitor cells (HSPC) by the nervous system both directly and indirectly via their bone marrow (BM) niche-supporting stromal cells. Functional expression of several neurotransmitter receptors was demonstrated on HSPC, mainly on the more primitive CD34(+)/CD38(-/low) fraction. The myeloid cytokines, G-CSF and GM-CSF, dynamically upregulate neuronal receptor expression on human HSPC. This is followed by an increased response to neurotransmitters, leading to enhanced proliferation and motility of human CD34(+) progenitors, repopulation of the murine BM and their egress to the circulation. Importantly, recent observations showed rapid mobilization of human HSPC to high SDF-1 expressing ischemic tissues of stroke individuals followed by neoangiogenesis. neurological and functional recovery. Along with decreased levels of circulating immature CD34(+) cells and SDF-1 blood levels found in patients with early-stage Alzheimer's disease, these findings suggest a possible involvement of human HSPC in brain homeostasis and thus their potential clinical applications in neuropathology. Full Text.
Kim, J., and Bartel, D.P. (2009). Allelic Imbalance Sequencing Reveals That Single-Nucleotide Polymorphisms Frequently Alter Microrna-Directed Repression. Nature Biotechnology Published online: 26 April 2009. Genetic changes that help explain the differences between two individuals might create or disrupt sites complementary to microRNAs (miRNAs), but the extent to which such polymorphic sites influence miRNA-mediated repression is unknown. Here, we describe a method to measure mRNA allelic imbalances associated with a regulatory site found in mRNA transcribed from one allele but not found in that transcribed from the other. Applying this method, called allelic imbalance sequencing, to sites for three miRNAs (miR-1, miR-133 and miR-122) provided quantitative measurements of repression in vivo without altering either the miRNAs or their targets. A substantial fraction of polymorphic sites mediated repression in tissues that expressed the cognate miRNA, and downregulation was correlated with site type and site context. Extrapolating these results to the other broadly conserved miRNAs suggests that when comparing two mouse strains (or two human individuals), polymorphic miRNA sites cause expression of many genes (often hundreds) to differ. Full Text.
Kotani, A., Ha, D., Hsieh, J., Rao, P.K., Schotte, D., den Boer, M.L., Armstrong, S.A., and Lodish, H.F. (2009). Mir-128b Is a Potent Glucocorticoid Sensitizer in Mll-Af4 Acute Lymphocytic Leukemia Cells and Exerts Cooperative Effects with Mir-221. Blood Sep 11. [Epub ahead of print] MLL-AF4 Acute Lymphocytic Leukemia (ALL) has a poor prognosis. MicroRNAs (miRNA) are small noncoding RNAs that post-transcriptionally regulate expression of target mRNAs. Our analysis of data published by Lu etc. (1), showed that expression of miR-128b and miR-221 is downregulated in MLL rearranged ALL relative to other types of ALL. Re-expression of these miRNAs cooperatively sensitizes two cultured lines of MLL-AF4 ALL cells to glucocorticoids. Target genes downregulated by miR-128b include MLL, AF4, and both MLL-AF4 and AF4-MLL fusion genes; miR-221 downregulates CDKN1B. These results demonstrate that downregulation of miR-128b and miR-221 is implicated in glucocorticoid resistance, that restoration of their levels is a potentially promising therapeutic in MLL-AF4 ALL. PDF
Kritzer, J.A., Hamamichi, S., McCaffery, J.M., Santagata, S., Naumann, T.A., Caldwell, K.A., Caldwell, G.A., and Lindquist, S. (2009). Rapid Selection of Cyclic Peptides That Reduce Alpha-Synuclein Toxicity in Yeast and Animal Models. Nature Chemical Biology.Published online: 13 July 2009. Phage display has demonstrated the utility of cyclic peptides as general protein ligands but cannot access proteins inside eukaryotic cells. Expanding a new chemical genetics tool, we describe the first expressed library of head-to-tail cyclic peptides in yeast (Saccharomyces cerevisiae). We applied the library to selections in a yeast model of alpha-synuclein toxicity that recapitulates much of the cellular pathology of Parkinson's disease. From a pool of 5 million transformants, we isolated two related cyclic peptide constructs that specifically reduced the toxicity of human alpha-synuclein. These expressed cyclic peptide constructs also prevented dopaminergic neuron loss in an established Caenorhabditis elegans Parkinson's model. This work highlights the speed and efficiency of using libraries of expressed cyclic peptides for forward chemical genetics in cellular models of human disease. Full Text.
Kuzmenkin, A., Liang, H.M., Xu, G.X., Pfannkuche, K., Eichhorn, H., Fatima, A., Luo, H.Y., Saric, T., Wernig, M., Jaenisch, R. and Hescheler J. (2009). Functional Characterization of Cardiomyocytes Derived from Murine Induced Pluripotent Stem Cells in Vitro. Faseb Journal 23, 4168-4180. Several types of terminally differentiated somatic cells can be reprogrammed into a pluripotent state by ectopic expression of Klf4, Oct3/4, Sox2, and c-Myc. Such induced pluripotent stem (iPS) cells have great potential to serve as an autologous source of cells for tissue repair. In the process of developing iPS-cell-based therapies, the major goal is to determine whether differentiated cells derived from iPS cells, such as cardiomyocytes (CMs), have the same functional properties as their physiological in vivo counterparts. Therefore, we differentiated murine iPS cells to CMs in vitro and characterized them by RTPCR, immunocytochemistry, and electrophysiology. As key markers of cardiac lineages, transcripts for Nkx2.5, alpha MHC, Mlc2v, and cTnT could be identified. Immunocytochemical stainings revealed the presence of organized sarcomeric actinin but the absence of mature atrial natriuretic factor. We examined characteristics and developmental changes of action potentials, as well as functional hormonal regulation and sensitivity to channel blockers. In addition, we determined expression patterns and functionality of cardiac-specific voltage-gated Na+, Ca2+, and K+ channels at early and late differentiation stages and compared them with CMs derived from murine embryonic stem cells (ESCs) as well as with fetal CMs. We conclude that iPS cells give rise to functional CMs in vitro, with established hormonal regulation pathways and functionally expressed cardiac ion channels; CMs generated from iPS cells have a ventricular phenotype; and cardiac development of iPS cells is delayed compared with maturation of native fetal CMs and of ESC-derived CMs. This difference may reflect the incomplete reprogramming of iPS cells and should be critically considered in further studies to clarify the suitability of the iPS model for regenerative medicine of heart disorders.-Kuzmenkin, A., Liang, H., Xu, G., Pfannkuche, K., Eichhorn, H., Fatima, A., Luo, H., S. aric, T., Wernig, M., Jaenisch, R., Hescheler, J. Functional characterization of cardiomyocytes derived from murine induced pluripotent stem cells in vitro. FASEB J. 23, 4168-4180 (2009). www.fasebj.org. Full Text.
Lange, J., Skaletsky, H., van Daalen, S.K., Embry, S.L., Korver, C.M., Brown, L.G., Oates, R.D., Silber, S., Repping, S., and Page, D.C. (2009). Isodicentric Y Chromosomes and Sex Disorders as Byproducts of Homologous Recombination That Maintains Palindromes. Cell 138, 855-869. Massive palindromes in the human Y chromosome harbor mirror-image gene pairs essential for spermatogenesis. During evolution, these gene pairs have been maintained by intrapalindrome, arm-to-arm recombination. The mechanism of intrapalindrome recombination and risk of harmful effects are unknown. We report 51 patients with isodicentric Y (idicY) chromosomes formed by homologous crossing over between opposing arms of palindromes on sister chromatids. These ectopic recombination events occur at nearly all Y-linked palindromes. Based on our findings, we propose that intrapalindrome sequence identity is maintained via noncrossover pathways of homologous recombination. DNA double-strand breaks that initiate these pathways can be alternatively resolved by crossing over between sister chromatids to form idicY chromosomes, with clinical consequences ranging from spermatogenic failure to sex reversal and Turner syndrome. Our observations imply that crossover and noncrossover pathways are active in nearly all Y-linked palindromes, exposing an Achilles' heel in the mechanism that preserves palindrome-borne genes. Full Text.
Laplante, M., and Sabatini, D.M. (2009). An Emerging Role of Mtor in Lipid Biosynthesis. Curr Biol 19, R1046-1052. Lipid biosynthesis is essential for the maintenance of cellular homeostasis. The lipids produced by cells (glycerolipids, fatty acids, phospholipids, cholesterol, and sphingolipids) are used as an energy source/reserve, as building blocks for membrane biosynthesis, as precursor molecules for the synthesis of various cellular products, and as signaling molecules. Defects in lipid synthesis or processing contribute to the development of many diseases, including obesity, insulin resistance, type 2 diabetes, non-alcoholic fatty liver disease, and cancer. Studies published over the last few years have shown that the target of rapamycin (TOR), a conserved serine/threonine kinase with an important role in regulating cell growth, controls lipid biosynthesis through various mechanisms. Here, we review these findings and briefly discuss their potential relevance for human health and disease. PDF
Laplante, M., and Sabatini, D.M. (2009). Mtor Signaling at a Glance. Journal of Cell Science 122, 3589-3594. The mammalian target of rapamycin (mTOR) signaling pathway integrates both intracellular and extracellular signals and serves as a central regulator of cell metabolism, growth, proliferation and survival. Discoveries that have been made over the last decade show that the mTOR pathway is activated during various cellular processes (e.g. tumor formation and angiogenesis, insulin resistance, adipogenesis and T-lymphocyte activation) and is deregulated in human diseases such as cancer and type 2 diabetes. These observations have attracted broad scientific and clinical interest in mTOR. This is highlighted by the growing use of mTOR inhibitors [rapamycin and its analogues (rapalogues)] in pathological settings, including the treatment of solid tumors, organ transplantation, coronary restenosis and rheumatoid arthritis. Here, we highlight and summarize the current understanding of how mTOR nucleates distinct multi-protein complexes, how intra- and extracellular signals are processed by the mTOR complexes, and how such signals affect cell metabolism, growth, proliferation and survival. Full Text.
Le, M.T., Xie, H., Zhou, B., Chia, P.H., Rizk, P., Um, M., Udolph, G., Yang, H., Lim, B., and Lodish, H.F. (2009). Microrna-125b Promotes Neuronal Differentiation in Human Cells by Repressing Multiple Targets. Molecular Cell Biology lJul 27. [Epub ahead of print]. microRNAs are a class of small non-coding RNAs that regulate gene expression at the post-transcriptional level. Research on microRNAs has highlighted their importance in neural development but the specific functions of neural-enriched microRNAs remain poorly understood. We report here the expression profile of microRNAs during neuronal differentiation in the human neuroblastoma cell line SH-SY5Y. Six microRNAs were significantly upregulated during differentiation induced by all-trans-retinoic acid and brain-derived neurotrophic factor. We demonstrated that ectopic expression of either miR-124a or miR-125b increases the percentage of differentiated SH-SY5Y cells with neurite outgrowth. Subsequently, we focused our functional analysis on miR-125b and demonstrated the important role of this miRNA in both spontaneous and induced differentiation of SH-SH5Y cells. miR-125b is also upregulated during differentiation of human neural progenitor ReNcell VM cells, and miR-125b ectopic expression significantly promotes neurite outgrowth of these cells. To identify the targets of miR-125b regulation, we profiled the global changes in gene expression following miR-125b ectopic expression in SH-SY5Y cells. miR-125b represses 164 genes that contain the seed match sequence of the microRNA and/or predicted to be direct targets of miR-125b by conventional methods. Pathway analysis suggests that a subset of miR-125b-repressed targets antagonize neuronal genes in several neurogenic pathways, thereby mediating the positive effect of miR-125b on neuronal differentiation. We have further validated the binding of miR-125b to the microRNA response elements of ten selected mRNA targets. Together, we report here for the first time the important role of miR-125b in human neuronal differentiation. Full Text.
Le, M.T., Teh, C., Shyh-Chang, N., Xie, H., Zhou, B., Korzh, V., Lodish, H.F., and Lim, B. (2009). Microrna-125b Is a Novel Negative Regulator of P53. Genes & Development ( published online March 17) The p53 transcription factor is a key tumor suppressor and a central regulator of the stress response. To ensure a robust and precise response to cellular signals, p53 gene expression must be tightly regulated from the transcriptional to the post-translational levels. Computational predictions suggest that several microRNAs are involved in the post-transcriptional regulation of p53. Here we demonstrate that miR-125b, a brain-enriched microRNA, is a bona fide negative regulator of p53 in both zebrafish and humans. miR-125b-mediated down-regulation of p53 is strictly dependent on the binding of miR-125b to a microRNA response element in the 3' untranslated region of p53 mRNA. Overexpression of miR-125b represses the endogenous level of p53 protein and suppresses apoptosis in human neuroblastoma cells and human lung fibroblast cells. In contrast, knockdown of miR-125b elevates the level of p53 protein and induces apoptosis in human lung fibroblasts and in the zebrafish brain. This phenotype can be rescued significantly by either an ablation of endogenous p53 function or ectopic expression of miR-125b in zebrafish. Interestingly, miR-125b is down-regulated when zebrafish embryos are treated with gamma-irradiation or camptothecin, corresponding to the rapid increase in p53 protein in response to DNA damage. Ectopic expression of miR-125b suppresses the increase of p53 and stress-induced apoptosis. Together, our study demonstrates that miR-125b is an important negative regulator of p53 and p53-induced apoptosis during development and during the stress response. Full Text.
Lewitter, F., Rebhan, M., Richter, B., and Sexton, D. (2009). The Need for Centralization of Computational Biology Resources. PLoS Comput Biol 5, e1000372. Full Text.
Lewitter, F., and Rebhan, M. (2009). Establishing a Successful Bioinformatics Core Facility Team. PLoS Comput Biol 5, e1000368. Full Text.
Lin, Y.L., Sengupta, S., Gurdziel, K., Bell, G.W., Jacks, T., and Flores, E.R. (2009). P63 and P73 Transcriptionally Regulate Genes Involved in DNA Repair. Plos Genetics 5(10): e1000680. The p53 family activates many of the same genes in response to DNA damage. Because p63 and p73 have structural differences from p53 and play distinct biological functions in development and metastasis, it is likely that they activate a unique transcriptional network. Therefore, we performed a genome-wide analysis using cells lacking the p53 family members after treatment with DNA damage. We identified over 100 genes involved in multiple pathways that were uniquely regulated by p63 or p73, and not p53. Further validation indicated that BRCA2, Rad51, and mre11 are direct transcriptional targets of p63 and p73. Additionally, cells deficient for p63 and p73 are impaired in DNA repair and p63+/-; p73+/- mice develop mammary tumors suggesting a novel mechanism whereby p63 and p73 suppress tumorigenesis. Full Text.
Love, K. R., R. K. Pandya, Spooner E, Ploegh HL.(2009). Ubiquitin C-Terminal Electrophiles Are Activity-Based Probes for Identification and Mechanistic Study of Ubiquitin Conjugating Machinery.ACS Chemical Biology Mar 26. [Epub ahead of print] Protein modification by ubiquitin (Ub) and ubiquitin-like modifiers (Ubl) requires the action of activating (E1), conjugating (E2), and ligating (E3) enzymes and is a key step in the specific destruction of proteins. Deubiquitinating enzymes (DUBs) deconjugate substrates modified with Ub/Ubl's and recycle Ub inside the cell. Genome mining based on sequence homology to proteins with known function has assigned many enzymes to this pathway without confirmation of either conjugating or DUB activity. Function-dependent methodologies are still the most useful for rapid identification or assessment of biological activity of expressed proteins from cells. Activity-based protein profiling uses chemical probes that are active-site-directed for the classification of protein activities in complex mixtures. Here we show that the design and use of an expanded set of Ub-based electrophilic probes allowed us to recover and identify members of each enzyme class in the ubiquitin-proteasome system, including E3 ligases and DUBs with previously unverified activity. We show that epitope-tagged Ub-electrophilic probes can be used as activity-based probes for E3 ligase identification by in vitro labeling and activity studies of purified enzymes identified from complex mixtures in cell lysate. Furthermore, the reactivity of our probe with the HECT domain of the E3 Ub ligase ARF-BP1 suggests that multiple cysteines may be in the vicinity of the E2-binding site and are capable of the transfer of Ub to self or to a substrate protein.
Lowery, L.A., and Sive, H. (2009). Totally Tubular: The Mystery Behind Function and Origin of the Brain Ventricular System. Bioessays Mar 9. [Epub ahead of print]. A unique feature of the vertebrate brain is the ventricular system, a series of connected cavities which are filled with cerebrospinal fluid (CSF) and surrounded by neuroepithelium. While CSF is critical for both adult brain function and embryonic brain development, neither development nor function of the brain ventricular system is fully understood. In this review, we discuss the mystery of why vertebrate brains have ventricles, and whence they originate. The brain ventricular system develops from the lumen of the neural tube, as the neuroepithelium undergoes morphogenesis. The molecular mechanisms underlying this ontogeny are described. We discuss possible functions of both adult and embryonic brain ventricles, as well as major brain defects that are associated with CSF and brain ventricular abnormalities. We conclude that vertebrates have taken advantage of their neural tube to form the essential brain ventricular system. Full Text.
Markoulaki, S., Hanna, J., Beard, C., Carey, B.W., Cheng, A.W., Lengner, C.J., Dausman, J.A., Fu, D., Gao, Q., Wu, S., Cassady, JP, & Rudolf Jaenisch (2009). Transgenic Mice with Defined Combinations of Drug-Inducible Reprogramming Factors. Nature Biotechnology Published online: 18 January 2009 Proviruses carrying drug-inducible Oct4, Sox2, Klf4 and c-Myc used to derive 'primary' induced pluripotent stem (iPS) cells were segregated through germline transmission, generating mice and cells carrying subsets of the reprogramming factors. Drug treatment produced 'secondary' iPS cells only when the missing factor was introduced. This approach creates a defined system for studying reprogramming mechanisms and allows screening of genetically homogeneous cells for compounds that can replace any transcription factor required for iPS cell derivation.Full Text.
Mayr, C., and Bartel, D.P. (2009). Widespread Shortening of 3 ' Utrs by Alternative Cleavage and Polyadenylation Activates Oncogenes in Cancer Cells. Cell 138, 673-684. In cancer cells, genetic alterations can activate proto-oncogenes, thereby contributing to tumorigenesis. However, the protein products of oncogenes are sometimes overexpressed without alteration of the proto-oncogene. Helping to explain this phenomenon, we found that when compared to similarly proliferating nontransformed cell lines, cancer cell lines often expressed substantial amounts of mRNA isoforms with shorter 30 untranslated regions (UTRs). These shorter isoforms usually resulted from alternative cleavage and polyadenylation (APA). The APA had functional consequences, with the shorter mRNA isoforms exhibiting increased stability and typically producing ten-fold more protein, in part through the loss of microRNA-mediated repression. Moreover, expression of the shorter mRNA isoform of the proto-oncogene IGF2BP1/IMP-1 led to far more oncogenic transformation than did expression of the full-length, annotated mRNA. The high incidence of APA in cancer cells, with consequent loss of 3'UTR repressive elements, suggests a pervasive role for APA in oncogene activation without genetic alteration. Full Text.
McGehee, A.M., Dougan, S.K., Klemm, E.J., Shui, G., Park, B., Kim, Y.M., Watson, N., Wenk, M.R., Ploegh, H.L., and Hu, C.C. (2009). Xbp-1-Deficient Plasmablasts Show Normal Protein Folding but Altered Glycosylation and Lipid Synthesis. Journal of Immunology.Aug 26. [Epub ahead of print] The accumulation of misfolded secreted IgM in the endoplasmic reticulum (ER) of X-box binding protein 1 (XBP-1)-deficient B cells has been held responsible for the inability of such cells to yield plasma cells, through the failure to mount a proper unfolded protein response. LPS-stimulated B cells incapable of secreting IgM still activate the XBP-1 axis normally, as follows: XBP-1 is turned on by cues that trigger differentiation and not in response to accumulation of unfolded IgM, but the impact of XBP-1 deficiency on glycoprotein folding and assembly has not been explored. The lack of XBP-1 compromised neither the formation of functional hen egg lysozyme-specific IgM nor the secretion of free kappa-chains. Although XBP-1 deficiency affects the synthesis of some ER chaperones, including protein disulfide isomerase, their steady state levels do not drop below the threshold required for proper assembly and maturation of the Igalpha/Igbeta heterodimer and MHC molecules. Intracellular transport and surface display of integral membrane proteins are unaffected by XBP-1 deficiency. Given the fact that we failed to observe any defects in folding of a variety of glycoproteins, we looked for other means to explain the requirement for XBP-1 in plasma cell development. We observed significantly reduced levels of phosphatidylcholine, sphingomyelin, and phosphatidylinositol in total membranes of XBP-1-deficient B cells, and reduced ER content. Terminal N-linked glycosylation of IgM and class I MHC was altered in these cells. XBP-1 hence has important roles beyond folding proteins in the ER. PDF
McGlinn, E., Yekta, S., Mansfield, J.H., Soutschek, J., Bartel, D.P., and Tabin, C.J. (2009). In Ovo Application of Antagomirs Indicates a Role for Mir-196 in Patterning the Chick Axial Skeleton through Hox Gene Regulation. Proceedings of the National Academy of Sciences of the United States of America 106, 18616-18621. Patterning of the vertebrate axial skeleton requires precise spatial and temporal control of Hox gene expression during embryonic development. MicroRNAs (miRNAs) are recently described modulators of gene activity, and members of the miR-196 and miR-10 families have been shown to target several Hox genes in vivo. Testing miRNA function in mice is complicated by potential redundancy between family members. To circumvent this, we have developed protocols for introducing modified antisense oligonucleotides (antagomiRs) in ovo during chick development. Using this approach, we identify a layer of regulatory control provided by the miR-196 family in defining the boundary of Hox gene expression along the anterior-posterior (A-P) embryonic axis. Following knockdown of miR-196, we observe a homeotic transformation of the last cervical vertebrae toward a thoracic identity. This phenotypic alteration is, in part, due to an anterior expansion of Hoxb8 gene expression and consolidates the in vivo relevance of post-transcriptional Hox gene regulation provided by miRNAs in the complex hierarchies governing axial pattering. Full Text.
McKenna, B.K., Selim, A.A., Bringhurst, F.R., and Ehrlich, D.J. (2009). 384-Channel Parallel Microfluidic Cytometer for Rare-Cell Screening. Lab on a 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.
Missiuro, P.V., Liu, K.S., Zou, L.H., Ross, B.C., Zhao, G.Y., Liu, J.S., and Ge, H. (2009). Information Flow Analysis of Interactome Networks. Plos Computational Biology 5(4):e1000350. Recent studies of cellular networks have revealed modular organizations of genes and proteins. For example, in interactome networks, a module refers to a group of interacting proteins that form molecular complexes and/or biochemical pathways and together mediate a biological process. However, it is still poorly understood how biological information is transmitted between different modules. We have developed information flow analysis, a new computational approach that identifies proteins central to the transmission of biological information throughout the network. In the information flow analysis, we represent an interactome network as an electrical circuit, where interactions are modeled as resistors and proteins as interconnecting junctions. Construing the propagation of biological signals as flow of electrical current, our method calculates an information flow score for every protein. Unlike previous metrics of network centrality such as degree or betweenness that only consider topological features, our approach incorporates confidence scores of protein-protein interactions and automatically considers all possible paths in a network when evaluating the importance of each protein. We apply our method to the interactome networks of Saccharomyces cerevisiae and Caenorhabditis elegans. We find that the likelihood of observing lethality and pleiotropy when a protein is eliminated is positively correlated with the protein's information flow score. Even among proteins of low degree or low betweenness, high information scores serve as a strong predictor of loss-of-function lethality or pleiotropy. The correlation between information flow scores and phenotypes supports our hypothesis that the proteins of high information flow reside in central positions in interactome networks. We also show that the ranks of information flow scores are more consistent than that of betweenness when a large amount of noisy data is added to an interactome. Finally, we combine gene expression data with interaction data in C. elegans and construct an interactome network for muscle-specific genes. We find that genes that rank high in terms of information flow in the muscle interactome network but not in the entire network tend to play important roles in muscle function. This framework for studying tissue-specific networks by the information flow model can be applied to other tissues and other organisms as well. Full Text.
Nosho, K., Shima, K., Irahara, N., Kure, S., Baba, Y., Kirkner, G.J., Chen, L., Gokhale, S., Hazra, A., Spiegelman, D.,Giovannucci EL, Jaenisch R, Fuchs CS, and Ogino S. (2009). Dnmt3b Expression Might Contribute to Cpg Island Methylator Phenotype in Colorectal Cancer. Clin Cancer Research Published Online First May 26, 2009. PURPOSE: DNA methyltransferase-3B (DNMT3B) plays an important role in de novo CpG island methylation. Dnmt3b can induce colon tumor in mice with methylation in specific CpG islands. We hypothesized that cellular DNMT3B level might influence the occurrence of widespread CpG island methylation (i.e., the CpG island methylator phenotype, CIMP) in colon cancer.EXPERIMENTAL DESIGN: Utilizing 765 colorectal cancers in two cohort studies, we detected DNMT3B expression in 116 (15%) tumors by immunohistochemistry. We assessed microsatellite instability, quantified DNA methylation in repetitive long interspersed nucleotide element-1 (LINE-1) by Pyrosequencing, eight CIMP-specific promoters [CACNA1G, CDKN2A (p16), CRABP1, IGF2, MLH1, NEUROG1, RUNX3, and SOCS1], and eight other CpG islands (CHFR, HIC1, IGFBP3, MGMT, MINT1, MINT31, p14, and WRN) by real-time PCR (MethyLight).RESULTS: Tumoral DNMT3B overexpression was significantly associated with CIMP-high [>/=6/8 methylated CIMP-specific promoters; odds ratio (OR), 3.34; 95% confidence interval, 2.11-5.29; P < 0.0001]. The relations between DNMT3B and methylation in 16 individual CpG islands varied substantially (OR, 0.80-2.96), suggesting variable locus-to-locus specificities of DNMT3B activity. DNMT3B expression was not significantly related with LINE-1 hypomethylation. In multivariate logistic regression, the significant relation between DNMT3B and CIMP-high persisted (OR, 2.39; 95% confidence interval, 1.11-5.14; P = 0.026) after adjusting for clinical and other molecular features, including p53, beta-catenin, LINE-1, microsatellite instability, KRAS, PIK3CA, and BRAF. DNMT3B expression was unrelated with patient outcome, survival, or prognosis.CONCLUSIONS: Tumoral DNMT3B overexpression is associated with CIMP-high in colorectal cancer. Our data support a possible role of DNMT3B in nonrandom de novo CpG island methylation leading to colorectal cancer. Full Text.
Oresic, K., Mueller, B., and Tortorella, D. (2009). Cln6 Mutants Associated with Neuronal Ceroid Lipofuscinosis Are Degraded in a Proteasome-Dependent Manner. Bioscience Reports 29, 173-181. NCLs (neuronal ceroid lipofuscinoses), a group of inherited neurodegenerative lysosomal storage diseases that predominantly affect children, are the result of autosomal recessive mutations within one of the nine cln genes. The wild-type cln gene products are composed of membrane and soluble proteins that localize to the lysosome or the ER (endoplasmic reticulum). However, the destiny of the Cln variants has not been fully characterized. To explore a possible link between ER quality control and processing of Cln mutants, we investigated the fate of two NCL-related Cln6 mutants found in patient samples (Cln6(G123D) and Cln(M241T)) in neuronal-derived human cells. The point mutations are predicted to be in the putative transmembrane domains and most probably generate misfolded membrane proteins that are subjected to ER quality control. Consistent with this paradigm, both mutants underwent rapid proteasome-mediated degradation and complexed with components of the ER extraction apparatus, Derlin-1 and p97. In addition, knockdown of SEL1L [sel-1 suppressor of lin-12-like (Caenorhabditis elegans)], a member of an E3 ubiquitin ligase complex involved in ER protein extraction, rescued significant amounts of Cln6(G123D) and Cln6(M241T) polypeptides. The results implicate ER quality control in the instability of the Cln variants that probably contributes to the development of NCL. Full Text.
Pallasch, C.P., Patz, M., Park, Y.J., Hagist, S., Eggle, D., Claus, R., Debey-Pascher, S., Schulz, A., Frenzel, L.P., Claasen, J, Nadine Kutsch, Günter Krause, Christine Mayr, Andreas Rosenwald, Christoph Plass, Joachim L. Schultze, Michael Hallek, and Clemens-Martin Wendtner (2009). Mirna Deregulation by Epigenetic Silencing Disrupts Suppression of the Oncogene Plag1 in Chronic Lymphocytic Leukemia. Blood 114, 3255-3264. MicroRNAs (miRNA) play a key role in cellular regulation and, if deregulated, in the development of neoplastic disorders including chronic lymphocytic leukemia (CLL). RNAs from primary cells of 50 treatment-naive CLL patients and peripheral B cells of 14 healthy donors were applied to miRNA expression profiling using bead chip technology. In CLL cells, a set of 7 up-and 19 down-regulated miRNAs was identified. Among the miRNAs down-regulated in CLL cells, 6 of 10 miRNA promoters examined showed gain of methylation compared with normal B-cell controls. Subsequent target prediction of deregulated miRNAs revealed a highly significant binding prediction at the 3' untranslated region of the pleomorphic adenoma gene 1 (PLAG1) oncogene. Luciferase reporter assays including site-directed mutagenesis of binding sites revealed a significant regulation of PLAG1 by miR-181a, miR-181b, miR-107, and miR-424. Although expression of PLAG1 mRNA was not affected, PLAG1 protein expression was shown to be significantly elevated in CLL cells compared with the levels in healthy donor B cells. In summary, we could demonstrate disruption of miRNA-mediated translational control, partly due to epigenetic transcriptional silencing of miRNAs, with subsequent overexpression of the oncogenic transcription factor PLAG1 as a putative novel mechanism of CLL pathogenesis.. Full Text.
Pandya, R.K., Partridge, J.R., Love, K.R., Schwartz, T.U., and Ploegh, H.L. (2009). A Structural Element within the Huwe1 Hect Domain Modulates Self- and Substrate Ubiquitination Activities. Journal of Biological Chemistry [Epub ahead of print] E3 ubiquitin ligases catalyze the final step of ubiquitin conjugation and regulate numerous cellular processes. The HECT class of E3 Ub ligases directly transfers Ub from bound E2 enzyme to a myriad of substrates. The catalytic domain of HECT Ub ligases has a bilobal architecture that separates the E2-binding region and catalytic site. An important question regarding HECT domain function is the control of ligase activity and specificity. Here we present a functional analysis of the HECT domain of the E3 ligase HUWE1, based on crystal structures, and show that a single, N-terminal helix significantly stabilizes the HECT domain. We observe that this element modulates HECT domain activity, as measured by self-ubiquitination induced in the absence of this helix, as distinct from its effects on Ub conjugation of substrate Mcl-1. Such subtle changes to the protein may be at the heart of the vast spectrum of substrate specificities displayed by HECT domain E3 ligases. PDF.
Petersen, C.P., and Reddien, P.W. (2009). Wnt Signaling and the Polarity of the Primary Body Axis. Cell 139, 1056-1068. How animals establish and pattern the primary body axis is one of the most fundamental problems in biology. Data from diverse deuterostomes (frog, fish, mouse, and amphioxus) and from planarians (protostomes) suggest that Wnt signaling through beta-catenin controls posterior identity during body plan formation in most bilaterally symmetric animals. Wnt signaling also influences primary axis polarity of pre-bilaterian animals, indicating that an axial patterning role for Wnt signaling predates the evolution of bilaterally symmetric animals. The use of posterior Wnt signaling and anterior Wnt inhibition might be a unifying principle of body plan development in most animals. Full Text.
Petersen, C.P., and Reddien, P.W. (2009). A Wound-Induced Wnt Expression Program Controls Planarian Regeneration Polarity. Proc Natl Acad Sci U S A.Sep 14. [Epub ahead of print]Regeneration requires specification of the identity of new tissues to be made. Whether this process relies only on intrinsic regulative properties of regenerating tissues or whether wound signaling provides input into tissue repatterning is not known. The head-versus-tail regeneration polarity decision in planarians, which requires Wnt signaling, provides a paradigm to study the process of tissue identity specification during regeneration. The Smed-wntP-1 gene is required for regeneration polarity and is expressed at the posterior pole of intact animals. Surprisingly, wntP-1 was expressed at both anterior- and posterior-facing wounds rapidly after wounding. wntP-1 expression was induced by all types of wounds examined, regardless of whether wounding prompted tail regeneration. Regeneration polarity was found to require new expression of wntP-1. Inhibition of the wntP-2 gene enhanced the polarity phenotype due to wntP-1 inhibition, with new expression of wntP-2 in regeneration occurring subsequent to expression of wntP-1 and localized only to posterior-facing wounds. New expression of wntP-2 required wound-induced wntP-1. Finally, wntP-1 and wntP-2 expression changes occurred even in the absence of neoblast stem cells, which are required for regeneration, suggesting that the role of these genes in polarity is independent of and instructive for tail formation. These data indicate that wound-induced input is involved in resetting the normal polarized features of the body axis during regeneration. Full Text.
Peterson, T.R., Laplante, M., Thoreen, C.C., Sancak, Y., Kang, S.A., Kuehl, W.M., Gray, N.S., and Sabatini, D.M. (2009). Deptor Is an Mtor Inhibitor Frequently Overexpressed in Multiple Myeloma Cells and Required for Their Survival.Cell. 2009 May 13. [Epub ahead of print] The mTORC1 and mTORC2 pathways regulate cell growth, proliferation, and survival. We identify DEPTOR as an mTOR-interacting protein whose expression is negatively regulated by mTORC1 and mTORC2. Loss of DEPTOR activates S6K1, Akt, and SGK1, promotes cell growth and survival, and activates mTORC1 and mTORC2 kinase activities. DEPTOR overexpression suppresses S6K1 but, by relieving feedback inhibition from mTORC1 to PI3K signaling, activates Akt. Consistent with many human cancers having activated mTORC1 and mTORC2 pathways, DEPTOR expression is low in most cancers. Surprisingly, DEPTOR is highly overexpressed in a subset of multiple myelomas harboring cyclin D1/D3 or c-MAF/MAFB translocations. In these cells, high DEPTOR expression is necessary to maintain PI3K and Akt activation and a reduction in DEPTOR levels leads to apoptosis. Thus, we identify a novel mTOR-interacting protein whose deregulated overexpression in multiple myeloma cells represents a mechanism for activating PI3K/Akt signaling and promoting cell survival. Full Text.
Pfannkuche, K., Liang, H., Hannes, T., Xi, J., Fatima, A., Nguemo, F., Matzkies, M., Wernig, M., Jaenisch, R., Pillekamp, F., et al. (2009). Cardiac Myocytes Derived from Murine Reprogrammed Fibroblasts: Intact Hormonal Regulation, Cardiac Ion Channel Expression and Development of Contractility. Cell Physiol Biochem 24, 73-86. AIMS: Induced pluripotent stem (iPS) cells have a developmental potential similar to that of blastocyst-derived embryonic stem (ES) cells and may serve as an autologous source of cells for tissue repair, in vitro disease modelling and toxicity assays. Here we aimed at generating iPS cell-derived cardiomyocytes (CMs) and comparing their molecular and functional characteristics with CMs derived from native murine ES cells. METHODS AND RESULTS: Beating cardiomyocytes were generated using a mass culture system from murine N10 and O9 iPS cells as well as R1 and D3 ES cells. Transcripts of the mesoderm specification factor T-brachyury and non-atrial cardiac specific genes were expressed in differentiating iPS EBs. Using immunocytochemistry to determine the expression and intracellular organisation of cardiac specific structural proteins we demonstrate strong similarity between iPS-CMs and ES-CMs. In line with a previous study electrophysiological analyses showed that hormonal response to beta-adrenergic and muscarinic receptor stimulation was intact. Action potential (AP) recordings suggested that most iPS-CMs measured up to day 23 of differentiation are of ventricular-like type. Application of lidocaine, Cs+, SEA0400 and verapamil+ nifedipine to plated iPS-EBs during multi-electrode array (MEA) measurements of extracellular field potentials and intracellular sharp electrode recordings of APs revealed the presence of I(Na), I(f), I(NCX), and I(CaL), respectively, and suggested their involvement in cardiac pacemaking, with I(CaL) being of major importance. Furthermore, iPS-CMs developed and conferred force to avitalized ventricular tissue that was responsive to beta-adrenergic stimulation. CONCLUSIONS: Our data demonstrate that the cardiogenic potential of iPS cells is comparable to that of ES cells and that iPS-CMs possess all fundamental functional elements of a typical cardiac cell, including spontaneous beating, hormonal regulation, cardiac ion channel expression and contractility. Therefore, iPS-CMs can be regarded as a potentially valuable source of cells for in vitro studies and cellular cardiomyoplasty. Full Text.
Phillips, R., Ursell, T., Wiggins, P., and Sens, P. (2009). Emerging Roles
for Lipids in Shaping Membrane-Protein Function. Nature 459, 379-385. Studies
of membrane proteins have revealed a direct link between the lipid environment
and the structure and function of some of these proteins. Although some of
these effects involve specific chemical interactions between lipids and protein
residues, many can be understood in terms of protein-induced perturbations
to the membrane shape. The free-energy cost of such perturbations can be
estimated quantitatively, and measurements of channel gating in model systems
of membrane
proteins with their lipid partners are now confirming predictions of simple
models. Full
Text.
Ploegh, H., and Amigorena, S. (2009). Editorial Overview. Current Opinion in Immunology (Article in Press) For those of us who entered the field relatively early (some 30 years ago), it is nothing short of amazing that one may now start to approach the subject of antigen presentation from a molecular perspective: beginning with phenomena as basic as the control of protein synthesis and ubiquitination, then continue through a truly mechanistic explanation of how antigenic peptides traverse the membrane of the endoplasmic reticulum, and how the functional organization of the phagocytic pathway determines peptide loading on class II MHC molecules, to finish with the most recent insights in how particular antigens contribute to pathology and the migratory properties of dendritic cells. The speed with which some of the major questions in the field have been resolved is nothing short of remarkable, and the reviews presented in this volume provide a glimpse of where new discoveries are likely to have their impact. Full Text.
Polyak, K., and Weinberg, R.A. (2009). Transitions between Epithelial and Mesenchymal States: Acquisition of Malignant and Stem Cell Traits. Nature Reviews Cancer 9, 265-273. Transitions between epithelial and mesenchymal states have crucial roles in embryonic development. Emerging data suggest a role for these processes in regulating cellular plasticity in normal adult tissues and in tumours, where they can generate multiple, distinct cellular subpopulations contributing to intratumoural heterogeneity. Some of these subpopulations may exhibit more differentiated features, whereas others have characteristics of stem cells. Owing to the importance of these tumour-associated phenotypes in metastasis and cancer-related mortality, targeting the products of such cellular plasticity is an attractive but challenging approach that is likely to lead to improved clinical management of cancer patients. Full Text.
Popp, M.W., Antos, J.M., and Ploegh, H.L. (2009). Site-Specific Protein Labeling Via Sortase-Mediated Transpeptidation. Curr Protoc Protein Sci Chapter 15, Unit 15 13. Creation of functional protein bioconjugates demands methods for attaching a diverse array of probes to target proteins with high specificity, under mild conditions. The sortase A transpeptidase enzyme from Staphylococcus aureus catalyzes the cleavage of a short 5-aa recognition sequence (LPXTG) with the concomitant formation of an amide linkage between an oligoglycine peptide and the target protein. By functionalizing the oligoglycine peptide, it is possible to incorporate reporters into target proteins in a site-specific fashion. This reaction is applicable to proteins in solution and on the living cell surface. The method described in this unit only requires incubation of the target protein, which has been engineered to contain a sortase recognition site either at the C terminus or within solvent-accessible loops, with a purified sortase enzyme and a suitably functionalized oligoglycine peptide. Full Text.
Popp, M.W., Artavanis-Tsakonas, K., and Ploegh, H.L. (2009). Substrate Filtering by the Active Site Crossover Loop in Uchl3 Revealed by Sortagging and Gain-of-Function Mutations. Journal of Biological Chemistry 284, 3593-3602. 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. Full Text.
Rai, P., Onder, T.T., Young, J.J., McFaline, J.L., Pang, B., Dedon, P.C., and Weinberg, R.A. (2009). Continuous Elimination of Oxidized Nucleotides Is Necessary to Prevent Rapid Onset of Cellular Senescence. Proceedings of the National Academy of Sciences of the United States of America 106, 169-174. 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.Full Text.
Rao, P.K., Toyama, Y., Chiang, H.R., Gupta, S., Bauer, M., Medvid, R., Reinhardt, F., Liao, R., Krieger, M., Jaenisch, R., Lodish, Harvey F.and Blelloch, Robert (2009). Loss of Cardiac Microrna-Mediated Regulation Leads to Dilated Cardiomyopathy and Heart Failure. Circulation Research.Aug 13. [Epub ahead of print] Rationale: Heart failure is a deadly and devastating disease that places immense costs on an aging society. To develop therapies aimed at rescuing the failing heart, it is important to understand the molecular mechanisms underlying cardiomyocyte structure and function. Objective: microRNAs are important regulators of gene expression, and we sought to define the global contributions made by microRNAs toward maintaining cardiomyocyte integrity. Methods and Results: First, we performed deep sequencing analysis to catalog the miRNA population in the adult heart. Second, we genetically deleted, in cardiac myocytes, an essential component of the machinery that is required to generate miRNAs. Deep sequencing of miRNAs from the heart revealed the enrichment of a small number of microRNAs with one, miR-1, accounting for 40% of all microRNAs. Cardiomyocyte-specific deletion of dgcr8, a gene required for microRNA biogenesis, revealed a fully penetrant phenotype that begins with left ventricular malfunction progressing to a dilated cardiomyopathy and premature lethality. Conclusions: These observations reveal a critical role for microRNAs in maintaining cardiac function in mature cardiomyocytes and raise the possibility that only a handful of microRNAs may ultimately be responsible for the dramatic cardiac phenotype seen in the absence of dgcr8. Full Text.
Rappley, I., Gitler, A.D., Selvy, P.E., Lavoie, M.J., Levy, B.D., Brown, H.A., Lindquist, S., and Selkoe, D.J. (2009). Evidence That Alpha-Synuclein Does Not Inhibit Phospholipase D (Dagger). Biochemistry.Articles ASAP 15 January alpha-Synuclein (alphaSyn) is a small cytosolic protein of unknown function, which is highly enriched in the brain. It is genetically linked to Parkinson's disease (PD) in that missense mutations or multiplication of the gene encoding alphaSyn causes early onset familial PD. Furthermore, the neuropathological hallmarks of both sporadic and familial PD, Lewy bodies and Lewy neurites, contain insoluble aggregates of alphaSyn. Several studies have reported evidence that alphaSyn can inhibit phospholipase D (PLD), which hydrolyzes phosphatidylcholine to form phosphatidic acid and choline. Although various hypotheses exist regarding the roles of alphaSyn in health and disease, no other specific biochemical function for this protein has been reported to date. Because PLD inhibition could represent an important function of alphaSyn, we sought to extend existing reports on this interaction. Using purified proteins, we tested the ability of alphaSyn to inhibit PLD activity in cell-free assays. We also examined several cell lines and transfection conditions to assess whether alphaSyn inhibits endogenous or overexpressed PLD in cultured mammalian cells. In yeast, we extended our previous report of an interaction between alphaSyn and PLD-dependent phenotypes, for which PLD activity is absolutely necessary. Despite testing a range of experimental conditions, including those previously published, we observed no significant inhibition of PLD by alphaSyn in any of these systems. We propose that the previously reported effects of alphaSyn on PLD activity could be due to increased endoplasmic reticulum-related stress associated with alphaSyn overexpression in cells, but are not likely due to a specific and direct interaction between alphaSyn and PLD. Full Text.
Ronan, J.L., Story, C.M., Papa, E., and Love, J.C. (2009). Optimization of the Surfaces Used to Capture Antibodies from Single Hybridomas Reduces the Time Required for Microengraving. Journal of Immunological Methods 340, 164-169. 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
Rozen, S., Marszalek, J.D., Alagappan, R.K., Skaletsky, H., and Page, D.C. (2009). Remarkably Little Variation in Proteins Encoded by the Y Chromosome's Single-Copy Genes, Implying Effective Purifying Selection. Am J Hum Genet 85, 923-928. Y-linked single-nucleotide polymorphisms (SNPs) have served as powerful tools for reconstructing the worldwide genealogy of human Y chromosomes and for illuminating patrilineal relationships among modern human populations. However, there has been no systematic, worldwide survey of sequence variation within the protein-coding genes of the Y chromosome. Here we report and analyze coding sequence variation among the 16 single-copy "X-degenerate" genes of the Y chromosome. We examined variation in these genes in 105 men representing worldwide diversity, resequencing in each man an average of 27 kb of coding DNA, 40 kb of intronic DNA, and, for comparison, 15 kb of DNA in single-copy Y-chromosomal pseudogenes. There is remarkably little variation in X-degenerate protein sequences: two chromosomes drawn at random differ on average by a single amino acid, with half of these differences arising from a single, conservative Asp-->Glu mutation that occurred approximately 50,000 years ago. Further analysis showed that nucleotide diversity and the proportion of variant sites are significantly lower for nonsynonymous sites than for synonymous sites, introns, or pseudogenes. These differences imply that natural selection has operated effectively in preserving the amino acid sequences of the Y chromosome's X-degenerate proteins during the last approximately 100,000 years of human history. Thus our findings are at odds with prominent accounts of the human Y chromosome's imminent demise. Full Text.
Saha, K., and Jaenisch, R. (2009). Technical Challenges in Using Human Induced Pluripotent Stem Cells to Model Disease. Cell Stem Cell 5, 584-595. Reprogramming of human somatic cells uses readily accessible tissue, such as skin or blood, to generate embryonic-like induced pluripotent stem cells (iPSCs). This procedure has been applied to somatic cells from patients who are classified into a disease group, thus creating "disease-specific" iPSCs. Here, we examine the challenges and assumptions in creating a disease model from a single cell of the patient. Both the kinetics of disease onset and progression as well as the spatial localization of disease in the patient's body are challenges to disease modeling. New tools in genetic modification, reprogramming, biomaterials, and animal models can be used for addressing these challenges. PDF
Sancak, Y., and Sabatini, D.M. (2009). Rag Proteins Regulate Amino-Acid-Induced Mtorc1 Signalling. Biochem Soc Trans 37, 289-290. The serum- and nutrient-sensitive protein kinase mTOR (mammalian target of rapamycin) is a master regulator of cell growth and survival. The mechanisms through which nutrients regulate mTOR have been one of the major unanswered questions in the mTOR field. Identification of the Rag (Ras-related GTPase) family of GTPases as mediators of amino acid signalling to mTOR is an important step towards our understanding of this mechanism. Full Text.
Shechner, D.M., Grant, R.A., Bagby, S.C., Koldobskaya, Y., Piccirilli, J.A., and Bartel, D.P. (2009). Crystal Structure of the Catalytic Core of an Rna-Polymerase Ribozyme. Science 326, 1271-1275. Primordial organisms of the putative RNA world would have required polymerase ribozymes able to replicate RNA. Known ribozymes with polymerase activity best approximating that needed for RNA replication contain at their catalytic core the class I RNA ligase, an artificial ribozyme with a catalytic rate among the fastest of known ribozymes. Here we present the 3.0 angstrom crystal structure of this ligase. The architecture resembles a tripod, its three legs converging near the ligation junction. Interacting with this tripod scaffold through a series of 10 minor-groove interactions (including two A-minor triads) is the unpaired segment that contributes to and organizes the active site. A cytosine nucleobase and two backbone phosphates abut the ligation junction; their location suggests a model for catalysis resembling that of proteinaceous polymerases. Full Text.
Sieff, C.A., Yang, J., Merida-Long, L.B., and Lodish, H.F. (2009). Pathogenesis of the Erythroid Failure in Diamond Blackfan Anaemia. British Journal of Haematology .Dec 1. [Epub ahead of print] Diamond Blackfan anaemia (DBA) is a severe congenital failure of erythropoiesis. Despite mutations in one of several ribosome protein genes, including RPS19, the cause of the erythroid specificity is still a mystery. We hypothesized that, because the chromatin of late erythroid cells becomes condensed and transcriptionally inactive prior to enucleation, the rapidly proliferating immature cells require very high ribosome synthetic rates. RNA biogenesis was measured in primary mouse fetal liver erythroid progenitor cells; during the first 24 h, cell number increased three to fourfold while, remarkably, RNA content increased sixfold, suggesting an accumulation of an excess of ribosomes during early erythropoiesis. Retrovirus infected siRNA RPS19 knockdown cells showed reduced proliferation but normal differentiation, and cell cycle analysis showed a G1/S phase delay. p53 protein was increased in the knockdown cells, and the mRNA level for p21, a transcriptional target of p53, was increased. Furthermore, we show that RPS19 knockdown decreased MYB protein, and Kit mRNA was reduced, as was the amount of cell surface KIT protein. Thus, in this small hairpin RNA murine model of DBA, RPS19 insufficient erythroid cells may proliferate poorly because of p53-mediated cell cycle arrest, and also because of decreased expression of the key erythroid signalling protein KIT. Full Text.
Shibue, T., and Weinberg, R.A. (2009). Integrin {Beta}1-Focal Adhesion Kinase Signaling Directs the Proliferation of Metastatic Cancer Cells Disseminated in the Lungs. Proc Natl Acad Sci U S A. Jun 5. [Epub ahead of print]The development of metastases is an extended and inefficient process involving multiple steps. The last of these involves the growth of micrometastases into macroscopic tumors. We show here that intravenously injected, nonmetastatic cancer cells cease proliferating after extravasating into the parenchyma of the lungs; this response is attributable to the cells inability to trigger adhesion-related signaling events when they are scattered sparsely within the extracellular matrix (ECM) of the parenchyma. We recapitulate this situation by culturing these nonmetastatic cells at low seeding density in ECM-derived gels in vitro, in which they undergo cell-cycle arrest resulting, in part, from insufficient activation of focal adhesion kinase (FAK). Metastatic cancer cells, in contrast, show sufficient FAK activation to enable their proliferation within ECM gels in vitro and continue cell-cycle progression within the lung parenchyma in vivo. Activation of FAK in these metastatic cells depends on expression of the beta(1) subunit of integrins, and proliferation of these cells after extravasation in the lungs is diminished by knocking down the expression of either FAK or integrin beta(1). These results demonstrate the critical role of integrin beta(1)-FAK signaling axis in controlling the initial proliferation of micrometastatic cancer cells disseminated in the lungs.PDF
Shkumatava, A., Stark, A., Sive, H., and Bartel, D.P. (2009). Coherent but Overlapping Expression of Micrornas and Their Targets During Vertebrate Development. Genes Dev 23, 466-481. MicroRNAs (miRNAs) are small noncoding RNAs that direct post-transcriptional repression of protein-coding genes. In vertebrates, each highly conserved miRNA typically regulates hundreds of target mRNAs. However, the precise relationship between expression of the miRNAs and that of their targets has remained unclear, in part because of the scarcity of quantitative expression data at cellular resolution. Here we report quantitative analyses of mRNA levels in miRNA-expressing cells of the zebrafish embryo, capturing entire miRNA expression domains, purified to cellular resolution using fluorescent-activated cell sorting (FACS). Focus was on regulation by miR-206 and miR-133 in the developing somites and miR-124 in the developing central nervous system. Comparison of wild-type embryos and those lacking miRNAs revealed predicted targets that responded to the miRNAs and distinguished miRNA-mediated mRNA destabilization from other regulatory effects. For all three miRNAs examined, expression of the miRNAs and that of their predicted targets usually overlapped. A few targets were expressed at higher levels in miRNA-expressing cells than in the rest of the embryo, demonstrating that miRNA-mediated repression can act in opposition to other regulatory processes. However, for most targets expression was lower in miRNA-expressing cells than in the rest of the embryo, indicating that miRNAs usually operate in concert with the other regulatory machinery of the cell. Full Text.
Skelley, A.M., Kirak, O., Suh, H., Jaenisch, R., and Voldman, J. (2009). Microfluidic Control of Cell Pairing and Fusion. Nat Methods 6, 147-152. Cell fusion has been used for many different purposes, including generation of hybridomas and reprogramming of somatic cells. The fusion step is the key event in initiation of these procedures. Standard fusion techniques, however, provide poor and random cell contact, leading to low yields. We present here a microfluidic device to trap and properly pair thousands of cells. Using this device, we paired different cell types, including fibroblasts, mouse embryonic stem cells and myeloma cells, achieving pairing efficiencies up to 70%. The device is compatible with both chemical and electrical fusion protocols. We observed that electrical fusion was more efficient than chemical fusion, with membrane reorganization efficiencies of up to 89%. We achieved greater than 50% properly paired and fused cells over the entire device, fivefold greater than with a commercial electrofusion chamber and observed reprogramming in hybrids between mouse embryonic stem cells and mouse embryonic fibroblasts. Full Text.
Soldner, F., Hockemeyer, D., Beard, C., Gao, Q., Bell, G.W., Cook, E.G., Hargus, G., Blak, A., Cooper, O., Mitalipova, M., Isacson O, and Jaenisch R.. (2009). Parkinson's Disease Patient-Derived Induced Pluripotent Stem Cells Free of Viral Reprogramming Factors. Cell Mar 6 136, 964-977. Induced pluripotent stem cells (iPSCs) derived from somatic cells of patients represent a powerful tool for biomedical research and may provide a source for replacement therapies. However, the use of viruses encoding the reprogramming factors represents a major limitation of the current technology since even low vector expression may alter the differentiation potential of the iPSCs or induce malignant transformation. Here, we show that fibroblasts from five patients with idiopathic Parkinson's disease can be efficiently reprogrammed and subsequently differentiated into dopaminergic neurons. Moreover, we derived hiPSCs free of reprogramming factors using Cre-recombinase excisable viruses. Factor-free hiPSCs maintain a pluripotent state and show a global gene expression profile, more closely related to hESCs than to hiPSCs carrying the transgenes. Our results indicate that residual transgene expression in virus-carrying hiPSCs can affect their molecular characteristics and that factor-free hiPSCs therefore represent a more suitable source of cells for modeling of human disease. Full Text.
Spies, N., Nielsen, C.B., Padgett, R.A., and Burge, C.B. (2009). Biased Chromatin Signatures around Polyadenylation Sites and Exons. Mol Cell 36, 245-254. Core RNA-processing reactions in eukaryotic cells occur cotranscriptionally in a chromatin context, but the relationship between chromatin structure and pre-mRNA processing is poorly understood. We observed strong nucleosome depletion around human polyadenylation sites (PAS) and nucleosome enrichment just downstream of PAS. In genes with multiple alternative PAS, higher downstream nucleosome affinity was associated with higher PAS usage, independently of known PAS motifs that function at the RNA level. Conversely, exons were associated with distinct peaks in nucleosome density. Exons flanked by long introns or weak splice sites exhibited stronger nucleosome enrichment, and incorporation of nucleosome density data improved splicing simulation accuracy. Certain histone modifications, including H3K36me3 and H3K27me2, were specifically enriched on exons, suggesting active marking of exon locations at the chromatin level. Together, these findings provide evidence for extensive functional connections between chromatin structure and RNA processing. Full Text.
Steele, A.D., Zhou, Z., Jackson,
W.S., Zhu, C., Auluck,
P., Moskowitz, M.A.,
Chesselet, M.F., and Lindquist, S. (2009). Context Dependent Neuroprotective
Properties of Prion Protein (Prp). Prion Oct 16;3(4). [Epub ahead of
print]. Although it has been known for more
than twenty years that an aberrant conformation of the prion protein (PrP)
is the causative agent in prion diseases, the role of PrP in normal biology
is undetermined.
Numerous studies have suggested a protective function for PrP, including protection
from ischemic and excitotoxic lesions and several apoptotic insults. On the
other hand, many observations have suggested the contrary, linking changes
in PrP localization
or domain structure-independent of infectious prion conformation-to severe
neuronal damage. Surprisingly, a recent report suggests that PrP is a receptor
for toxic
oligomeric species of a-beta, a pathogenic fragment of the amyloid precursor
protein, and likely contributes to disease pathogenesis of Alzheimer's disease.
We sought to access the role of PrP in diverse neurological disorders. First,
we confirmed that PrP confers protection against ischemic damage using an acute
stroke model, a well characterized association. After ischemic insult, PrP
knockouts had dramatically increased infarct volumes and decreased behavioral
performance
compared to controls. To examine the potential of PrP's neuroprotective or
neurotoxic properties in the context of other pathologies, we deleted PrP from
several transgenic
models of neurodegenerative disease. Deletion of PrP did not substantially
alter the disease phenotypes of mouse models of Parkinson's disease or tauopathy.
Deletion
of PrP in one of two Huntington's disease models tested, R6/2, modestly slowed
motor deterioration as measured on an accelerating rotarod but otherwise did
not alter other major features of the disease. Finally, transgenic overexpression
of PrP did not exacerbate the Huntington's motor phenotype. These results suggest
that PrP has a context-dependent neuroprotective function and does not broadly
contribute to the disease models tested herein. Full Text.
StehlingSun, S., Dade, J., Nutt, S.L., Dekoter, R.P., and Camargo, F.D. (2009). Regulation of Lymphoid Versus Myeloid Fate 'Choice' by the Transcription Factor Mef2c. Nature Immunology Jan 25. [Epub ahead of print]. Despite advances in the identification of lymphoid-restricted progenitor cells, the transcription factors essential for their generation remain to be identified. Here we describe an unexpected function for the myeloid oncogene product Mef2c in lymphoid development. Mef2c deficiency was associated with profound defects in the production of B cells, T cells, natural killer cells and common lymphoid progenitor cells and an enhanced myeloid output. In multipotent progenitors, Mef2c was required for the proper expression of several key lymphoid regulators and restriction of the myeloid fate. Our studies also show that Mef2c was a critical transcriptional target of the transcription factor PU.1 during lymphopoiesis. Thus, Mef2c is a crucial component of the transcriptional network that regulates cell fate 'choice' in multipotent progenitors. Full Text.
Su, L.J., Auluck, P.K., Outeiro, T.F., Yeger-Lotem, E., Kritzer, J.A., Tardiff, D.F., Strathearn, K.E., Liu, F., Cao, S., Hamamichi, S., et al and Susan Lindquist. (2009). Compounds from an Unbiased Chemical Screen Reverse Both Er-to-Golgi Trafficking Defects and Mitochondrial Dysfunction in Parkinson's Disease Models. Disease Models and Mechanisms ePress 28 Dec. 2009 alpha-Synuclein (alpha-syn) is a small lipid-binding protein involved in vesicle trafficking whose function is poorly characterized. It is of great interest to human biology and medicine because alpha-syn dysfunction is associated with several neurodegenerative disorders, including Parkinson's disease (PD). We previously created a yeast model of alpha-syn pathobiology, which established vesicle trafficking as a process that is particularly sensitive to alpha-syn expression. We also uncovered a core group of proteins with diverse activities related to alpha-syn toxicity that is conserved from yeast to mammalian neurons. Here, we report that a yeast strain expressing a somewhat higher level of alpha-syn also exhibits strong defects in mitochondrial function. Unlike our previous strain, genetic suppression of endoplasmic reticulum (ER)-to-Golgi trafficking alone does not suppress alpha-syn toxicity in this strain. In an effort to identify individual compounds that could simultaneously rescue these apparently disparate pathological effects of alpha-syn, we screened a library of 115,000 compounds. We identified a class of small molecules that reduced alpha-syn toxicity at micromolar concentrations in this higher toxicity strain. These compounds reduced the formation of alpha-syn foci, re-established ER-to-Golgi trafficking and ameliorated alpha-syn-mediated damage to mitochondria. They also corrected the toxicity of alpha-syn in nematode neurons and in primary rat neuronal midbrain cultures. Remarkably, the compounds also protected neurons against rotenone-induced toxicity, which has been used to model the mitochondrial defects associated with PD in humans. That single compounds are capable of rescuing the diverse toxicities of alpha-syn in yeast and neurons suggests that they are acting on deeply rooted biological processes that connect these toxicities and have been conserved for a billion years of eukaryotic evolution. Thus, it seems possible to develop novel therapeutic strategies to simultaneously target the multiple pathological features of PD. Full Text.
Tam, B.K., Shin, J.H., Pfeiffer, E., Matsudaira, P., and Mahadevan, L. (2009). Calcium Regulation of an Actin Spring. Biophysical Journal 97, 1125-1129. Calcium is essential for many biological processes involved in cellular motility. However, the pathway by which calcium influences motility, in processes such as muscle contraction and neuronal growth, is often indirect and complex. We establish a simple and direct mechanochemical link that shows how calcium quantitatively regulates the dynamics of a primitive motile system, the actin-based acrosomal bundle of horseshoe crab sperm. The extension of this bundle requires the continuous presence of external calcium. Furthermore, the extension rate increases with calcium concentration, but at a given concentration, we find that the volumetric rate of extension is constant. Our experiments and theory suggest that calcium sequentially binds to calmodulin molecules decorating the actin filaments. This binding leads to a collective wave of untwisting of the actin filaments that drives bundle extension. Full Text.
Tao, J., Hu, K., Chang, Q., Wu, H., Sherman, N.E., Martinowich, K., Klose, R.J., Schanen, C., Jaenisch, R., Wang, W., et al. (2009). Phosphorylation of Mecp2 at Serine 80 Regulates Its Chromatin Association and Neurological Function. Proc Natl Acad Sci U S A Feb 18. [Epub ahead of print]. Mutations of MECP2 (Methyl-CpG Binding Protein 2) cause Rett syndrome. As a chromatin-associated multifunctional protein, how MeCP2 integrates external signals and regulates neuronal function remain unclear. Although neuronal activity-induced phosphorylation of MeCP2 at serine 421 (S421) has been reported, the full spectrum of MeCP2 phosphorylation together with the in vivo function of such modifications are yet to be revealed. Here, we report the identification of several MeCP2 phosphorylation sites in normal and epileptic brains from multiple species. We demonstrate that serine 80 (S80) phosphorylation of MeCP2 is critical as its mutation into alanine (S80A) in transgenic knock-in mice leads to locomotor deficits. S80A mutation attenuates MeCP2 chromatin association at several gene promoters in resting neurons and leads to transcription changes of a small number of genes. Calcium influx in neurons causes dephosphorylation at S80, potentially contributing to its dissociation from the chromatin. We postulate that phosphorylation of MeCP2 modulates its dynamic function in neurons transiting between resting and active states within neural circuits that underlie behaviors.PDF.
Thoreen, C.C., and Sabatini, D.M. (2009). Rapamycin Inhibits Mtorc1, but Not Completely. Autophagy Jul 22;5(5). [Epub ahead of print]. Rapamycin is widely used as a complete inhibitor of the mTORC1 nutrient-sensitive signaling complex. Using a novel ATP-competitive inhibitor named Torin1, we have found that many mTORC1 functions that regulate cap-dependent translation and autophagy are resistant to inhibition by rapamycin.
Thoreen, C.C., Kang, S.A., Chang, J.W., Liu, Q., Zhang, J., Gao, Y., Reichling, L.J., Sim, T., Sabatini, D.M., and Gray, N.S. (2009). An Atp-Competitive Mtor Inhibitor Reveals Rapamycin-Insensitive Functions of Mtorc1. J Biol Chem. Jan 15. [Epub ahead of print]The mTOR kinase is the catalytic subunit of two functionally distinct complexes, mTORC1 and mTORC2, that coordinately promote cell growth, proliferation, and survival. Rapamycin is a potent allosteric mTORC1 inhibitor with clinical applications as an immunosuppressant and anti-cancer agent. Here, we find that Torin1, a highly potent and selective ATP-competitive mTOR inhibitor that directly inhibits both complexes, impairs cell growth and proliferation to a far greater degree than rapamycin. Surprisingly, these effects are independent of mTORC2 inhibition and are instead due to suppression of rapamycin-resistant functions of mTORC1 that are necessary for cap-dependent translation and suppression of autophagy. These effects are at least partly mediated by mTORC1-dependent and rapamycin-resistant phosphorylation of 4E-BP1. Our findings challenge the assumption that rapamycin completely inhibits mTORC1 and indicate that direct inhibitors of mTORC1 kinase activity may be more successful than rapamycin at inhibiting tumors that depend on mTORC1.PDF
Timp, W., Mirsaidov, U., Matsudaira, P., and Timp, G. (2009). Jamming Prokaryotic Cell-to-Cell Communications in a Model Biofilm. Lab Chip 9, 925-934. We report on the physical parameters governing prokaryotic cell-to-cell signaling in a model biofilm. The model biofilm is comprised of bacteria that are genetically engineered to transmit and receive quorum-sensing (QS) signals. The model is formed using arrays of time-shared, holographic optical traps in conjunction with microfluidics to precisely position bacteria, and then encapsulated within a hydrogel that mimics the extracellular matrix. Using fluorescent protein reporters functionally linked to QS genes, we assay the intercellular signaling. We find that there isn't a single cell density for which QS-regulated genes are induced or repressed. On the contrary, cell-to-cell signaling is largely governed by diffusion, and is acutely sensitive to mass-transfer to the surroundings and the cell location. These observations are consistent with the view that QS-signals act simply as a probe measuring mixing, flow, or diffusion in the microenvironment of the cell. Full Text.
Treusch, S., Cyr, D.M., and Lindquist, S. (2009). Amyloid Deposits: Protection against Toxic Protein Species? Cell Cycle Jun 20;8(11). [Epub ahead of print]. Neurodegenerative diseases ranging from Alzheimer disease and polyglutamine diseases to transmissible spongiform encephalopathies are associated with the aggregation and accumulation of misfolded proteins. In several cases the intracellular and extracellular protein deposits contain a fibrillar protein species called amyloid. However while amyloid deposits are hallmarks of numerous neurodegenerative diseases, their actual role in disease progression remains unclear. Especially perplexing is the often poor correlation between these deposits and other markers of neurodegeneration. As a result the question remains whether amyloid deposits are the disease-causing species, the consequence of cellular disease pathology or even the result of a protective cellular response to misfolded protein species. Here we highlight studies that suggest that accumulation and sequestration of misfolded protein in amyloid inclusion bodies and plaques can serve a protective function. Furthermore, we discuss how exceeding the cellular capacity for protective deposition of misfolded proteins may contribute to the formation of toxic protein species. Full Text
Tropea, D., Giacometti, E., Wilson, N.R., Beard, C., McCurry, C., Fu, D.D., Flannery, R., Jaenisch, R., and Sur, M. (2009). Partial Reversal of Rett Syndrome-Like Symptoms in Mecp2 Mutant Mice. Proc Natl Acad Sci U S A 106, 2029-2034. Rett Syndrome (RTT) is a severe form of X-linked mental retardation caused by mutations in the gene coding for methyl CpG-binding protein 2 (MECP2). Mice deficient in MeCP2 have a range of physiological and neurological abnormalities that mimic the human syndrome. Here we show that systemic treatment of MeCP2 mutant mice with an active peptide fragment of Insulin-like Growth Factor 1 (IGF-1) extends the life span of the mice, improves locomotor function, ameliorates breathing patterns, and reduces irregularity in heart rate. In addition, treatment with IGF-1 peptide increases brain weight of the mutant mice. Multiple measurements support the hypothesis that RTT results from a deficit in synaptic maturation in the brain: MeCP2 mutant mice have sparse dendritic spines and reduced PSD-95 in motor cortex pyramidal neurons, reduced synaptic amplitude in the same neurons, and protracted cortical plasticity in vivo. Treatment with IGF-1 peptide partially restores spine density and synaptic amplitude, increases PSD-95, and stabilizes cortical plasticity to wild-type levels. Our results thus strongly suggest IGF-1 as a candidate for pharmacological treatment of RTT and potentially of other CNS disorders caused by delayed synapse maturation. Full Text.
Valastyan, S., Benaich, N., Chang, A., Reinhardt, F., and Weinberg, R.A. (2009). Concomitant Suppression of Three Target Genes Can Explain the Impact of a Microrna on Metastasis. Genes & Development Oct 29. [Epub ahead of print]. It remains unclear whether a microRNA (miRNA) affects a given phenotype via concomitant down-regulation of its entire repertoire of targets or instead by suppression of only a modest subset of effectors. We demonstrate that inhibition of breast cancer metastasis by miR-31-a miRNA predicted to modulate >200 mRNAs-can be entirely explained by miR-31's pleiotropic regulation of three targets. Thus, concurrent re-expression of integrin-alpha5, radixin, and RhoA abrogates miR-31-imposed metastasis suppression. These effectors influence distinct steps of the metastatic process. Our findings have implications concerning the importance of pleiotropy for the biological actions of miRNAs and provide mechanistic insights into metastasis. PDF
Ueberfeld, J., and Ehrlich, D.J. (2009). Scaling of Nucleic Acid Assays on
Microelectrophoresis Array Devices: High-Dynamic Range Multi-Gene Readout
from Less Than Ten Transcripts.
Electrophoresis. [Epub ahead of print] In this paper we
describe progress in using the prodigious data-collecting ability of multilane
microelectrophoresis instruments to
bear on problems in
scaled nucleic acid assays. We emphasize compound stacking and solid-support
loading as means to concentrate <100 pg samples for direct injection.
Reaction Mapping is applied to readout quantitative polymerase chain reaction
gene-expression
and as a way to practically overcome difficulty in interpreting amplification
curves of multiplexed quantitative polymerase chain reaction at 20-50 gene/well
complexity. We demonstrate multiplexed readout of gene expression over an
abundancy range of 9 Log 2 units starting with reverse-transcribed samples
as small as
five molecules in each sample. Full
Text.
Valastyan, S., and Weinberg, R.A. (2009). Assaying Microrna Loss-of-Function Phenotypes in Mammalian Cells: Emerging Tools and Their Potential Therapeutic Utility. RNA Biology Nov 14;6(5). [Epub ahead of print]. MicroRNAs are small, non-coding RNAs that are increasingly appreciated to play critical roles in the modulation of gene expression. In mammalian cells, our knowledge regarding the full impact of microRNAs on cellular behavior remains fragmentary. This has been due, in significant part, to the limited availability of experimental tools for studying microRNA loss-of-function phenotypes. Recently, several strategies for achieving this goal have been developed. Here, we discuss these methodologies for inhibiting specific microRNAs in mammalian cells both in vitro and in vivo, compare and contrast the strengths and weaknesses of these approaches, and speculate regarding the future impact of these antagonists on the treatment of human diseases such as cancer. These emerging techniques enable the attenuation of microRNA function in a manner that is quite sequence-specific, relatively long-lasting and increasingly cost-effective. As such, some of these advances hold great promise in terms of their eventual utility as therapeutic agents.
Valastyan, S., and Weinberg, R.A. (2009). Micrornas: Crucial Multi-Tasking Components in the Complex Circuitry of Tumor Metastasis. Cell Cycle Nov 13;8(21). [Epub ahead of print]. Distant metastases are the underlying cause of patient mortality in an overwhelming majority of human carcinomas. Certain microRNAs have recently been found capable of regulating the process of tumor metastasis. In this review, we highlight advances within this rapidly emerging field, endeavor to connect known microRNA pathways with recent conceptual advances in the larger field of metastasis research, and speculate regarding the future utility of microRNAs in the diagnosis and treatment of human cancers. Assessed collectively, current evidence suggests that the pleiotropic activities of microRNAs endow them with the capacity to function as crucial, yet previously unappreciated, nodes within already-identified metastasis regulatory circuitry. This has important implications for our understanding of the pathogenesis of high-grade malignancies. PDF
Valastyan, Scott , Ferenc Reinhardt, Nathan Benaich,
Diana Calogrias, Attila M. Szász, Zhigang C. Wang, Jane E. Brock, Andrea
L. Richardson and Robert
A. Weinberg. (2009) A Pleiotropically Acting MicroRNA, miR-31, Inhibits
Breast Cancer Metastasis.Cell Vol. 137, Issue 6 Pages 1032-1046. MicroRNAs
are well suited to regulate tumor metastasis because of their capacity to
coordinately
repress numerous
target genes, thereby potentially enabling their intervention at multiple steps
of the invasion-metastasis cascade. We identify a microRNA exemplifying these
attributes, miR-31, whose expression correlates inversely with metastasis in
human breast cancer patients. Overexpression of miR-31 in otherwise-aggressive
breast tumor cells suppresses metastasis. We deploy a stable microRNA sponge
strategy to inhibit miR-31 in vivo; this allows otherwise-nonaggressive breast
cancer cells to metastasize. These phenotypes do not involve confounding influences
on primary tumor development and are specifically attributable to miR-31-mediated
inhibition of several steps of metastasis, including local invasion, extravasation
or initial survival at a distant site, and metastatic colonization. Such pleiotropy
is achieved via coordinate repression of a cohort of metastasis-promoting genes,
including RhoA. Indeed, RhoA re-expression partially reverses miR-31-imposed
metastasis suppression. These findings indicate that miR-31 uses multiple mechanisms
to oppose metastasis. Fulltext
Vardy, L., Pesin, J.A., and Orr-Weaver, T.L. (2009). Regulation of Cyclin a Protein in Meiosis and Early Embryogenesis. Proc Natl Acad Sci U S A.Published online 30 January. In contrast to the extensive analysis of the regulation of Cyclin B protein levels during developmental progression through meiosis in oogenesis, little is known about Cyclin A. Repression of cyclin A translation early in prophase I in Drosophila is important to maintain the oocyte in meiosis, and this has been shown to be mediated by deadenylation of the mRNA and inhibition by the Bruno repressor. We find that at oocyte maturation as meiosis resumes, Cyclin A protein reappears, coincident with polyadenylation of the mRNA and loss of Bruno repressor. Cyclin A is multiphosphorylated in a pattern consistent with autophosphorylation, and this form accumulates aberrantly in metaphase I if the Cortex form of the Anaphase Promoting Complex/Cyclosome is inactive. The PAN GU (PNG) kinase positively promotes translation of Cyclin A, beginning in oogenesis, an earlier onset than previously recognized. After egg activation and the completion of meiosis, PNG promotes further polyadenylation of cyclin A mRNA and appears to antagonize repression of translation by the PUMILIO inhibitor. Epistasis studies with png; apc mutants indicate that PNG acts solely to promote translation, rather than having a parallel function to inhibit degradation. These studies reveal multiple levels of posttranscriptional regulation of Cyclin A protein by translational and proteolytic control during oocyte maturation and the onset of embryogenesis.PDF
Vasudevan, K.M., Barbie, D.A., Davies, M.A., Rabinovsky, R., McNear, C.J., Kim, J.J., Hennessy, B.T., Tseng, H., Pochanard, P., Kim, S.Y., Reiling JH et al. (2009). Akt-Independent Signaling Downstream of Oncogenic Pik3ca Mutations in Human Cancer. Cancer Cell 16, 21-32. Dysregulation of the phosphatidylinositol 3-kinase (PI3K) signaling pathway occurs frequently in human cancer. PTEN tumor suppressor or PIK3CA oncogene mutations both direct PI3K-dependent tumorigenesis largely through activation of the AKT/PKB kinase. However, here we show through phosphoprotein profiling and functional genomic studies that many PIK3CA mutant cancer cell lines and human breast tumors exhibit only minimal AKT activation and a diminished reliance on AKT for anchorage-independent growth. Instead, these cells retain robust PDK1 activation and membrane localization and exhibit dependency on the PDK1 substrate SGK3. SGK3 undergoes PI3K- and PDK1-dependent activation in PIK3CA mutant cancer cells. Thus, PI3K may promote cancer through both AKT-dependent and AKT-independent mechanisms. Knowledge of differential PI3K/PDK1 signaling could inform rational therapeutics in cancers harboring PIK3CA mutations. Full Text.
Verstrepen, K.J., and Fink, G.R. (2009). Genetic and Epigenetic Mechanisms Underlying Cell-Surface Variability in Protozoa and Fungi. Annual Review of Genetics 43, 1-24. Eukaryotic microorganisms have evolved ingenious mechanisms to generate variability at their cell surface, permitting differential adherence, rapid adaptation to changing environments, and evasion of immune surveillance. Fungi such as Saccharomyces cerevisiae and the pathogen Candida albicans carry a family Of mucin and adhesin genes that allow adhesion to various Surfaces and tissues. Trypanosoma cruzi, T. brucei, and Plasmodium falciparum likewise contain large arsenals of different cell surface adhesion genes. In both yeasts and protozoa, silencing and differential expression of the gene family results in surface variability. Here, we discuss unexpected similarities in the structure and genomic location of the cell surface genes, the role of repeated DNA sequences, and the genetic and epigenetic mechanisms-all of which contribute to the remarkable cell surface variability in these highly divergent microbes. Full Text.
Wacker, J.L., Huang, S.Y., Steele, A.D., Aron, R., Lotz, G.P., Nguyen, Q., Giorgini, F., Roberson, E.D., Lindquist, S., Masliah, E., et al. (2009). Loss of Hsp70 Exacerbates Pathogenesis but Not Levels of Fibrillar Aggregates in a Mouse Model of Huntington's Disease. Journal of Neuroscience 29, 9104-9114. Endogenous protein quality control machinery has long been suspected of influencing the onset and progression of neurodegenerative diseases characterized by accumulation of misfolded proteins. Huntington's disease (HD) is a fatal neurodegenerative disorder caused by an expansion of a polyglutamine (polyQ) tract in the protein huntingtin (htt), which leads to its aggregation and accumulation in inclusion bodies. Here, we demonstrate in a mouse model of HD that deletion of the molecular chaperones Hsp70.1 and Hsp70.3 significantly exacerbated numerous physical, behavioral and neuropathological outcome measures, including survival, body weight, tremor, limb clasping and open field activities. Deletion of Hsp70.1 and Hsp70.3 significantly increased the size of inclusion bodies formed by mutant htt exon 1, but surprisingly did not affect the levels of fibrillar aggregates. Moreover, the lack of Hsp70s significantly decreased levels of the calcium regulated protein c-Fos, a marker for neuronal activity. In contrast, deletion of Hsp70s did not accelerate disease in a mouse model of infectious prion-mediated neurodegeneration, ruling out the possibility that the Hsp70.1/70.3 mice are nonspecifically sensitized to all protein misfolding disorders. Thus, endogenous Hsp70s are a critical component of the cellular defense against the toxic effects of misfolded htt protein in neurons, but buffer toxicity by mechanisms independent of the deposition of fibrillar aggregates. Full Text.
Welburn, J.P., Grishchuk, E.L., Backer, C.B., Wilson-Kubalek, E.M., Yates, J.R., 3rd, and Cheeseman, I.M. (2009). The Human Kinetochore Ska1 Complex Facilitates Microtubule Depolymerization-Coupled Motility. Dev Cell 16, 374-385. Mitotic chromosome segregation requires that kinetochores attach to microtubule polymers and harness microtubule dynamics to drive chromosome movement. In budding yeast, the Dam1 complex couples kinetochores with microtubule depolymerization. However, a metazoan homolog of the Dam1 complex has not been identified. To identify proteins that play a corresponding role at the vertebrate kinetochore-microtubule interface, we isolated a three subunit human Ska1 complex, including the previously uncharacterized protein Rama1 that localizes to the outer kinetochore and spindle microtubules. Depletion of Ska1 complex subunits severely compromises proper chromosome segregation. Reconstituted Ska1 complex possesses two separable biochemical activities: direct microtubule binding through the Ska1 subunit, and microtubule-stimulated oligomerization imparted by the Rama1 subunit. The full Ska1 complex forms assemblies on microtubules that can facilitate the processive movement of microspheres along depolymerizing microtubules. In total, these results demonstrate a critical role for the Ska1 complex in interacting with dynamic microtubules at the outer kinetochore.Full Text.
Wendler, P., Shorter, J., Snead, D., Plisson, C., Clare, D.K., Lindquist, S., and Saibil, H.R. (2009). Motor Mechanism for Protein Threading through Hsp104. Molecular Cell 34, 81-92. The protein-remodeling machine Hsp104 dissolves amorphous aggregates as well as ordered amyloid assemblies such as yeast prions. Force generation originates from a tandem AAA+ (ATPases associated with various cellular activities) cassette, but the mechanism and allostery of this action remain to be established. Our cryoelectron microscopy maps of Hsp104 hexamers reveal substantial domain movements upon ATP binding and hydrolysis in the first nucleotide-binding domain (NBD1). Fitting atomic models of Hsp104 domains to the EM density maps plus supporting biochemical measurements show how the domain movements displace sites bearing the substrate-binding tyrosine loops. This provides the structural basis for N- to C-terminal substrate threading through the central cavity, enabling a clockwise handover of substrate in the NBD1 ring and coordinated substrate binding between NBD1 an NBD2. Asymmetric reconstructions of Hsp104 in the presence of ATP gamma S or ATP support sequential rather than concerted ATP hydrolysis in the NBD1 ring. Full Text.
Whitesell, L., and Lindquist, S. (2009). Inhibiting the Transcription Factor Hsf1 as an Anticancer Strategy. Expert Opinion on Therapeutic Targets 13, 469-478. BACKGROUND: In mammals, the cytoprotective heat-shock response is regulated primarily by heat shock factor 1 (HSF1). Unfortunately, the effects of HSF1 also support the ability of cancer cells to accommodate imbalances in signaling and alterations in DNA, protein and energy metabolism associated with oncogenesis. The malignant lifestyle confers dependence on this 'non-oncogene', suggesting a therapeutic role for HSF1 inhibitors. OBJECTIVE/METHODS: We begin with an overview of how HSF1 affects cancer biology and how its activity is regulated. We then summarize progress in discovery and development of HSF1 inhibitors, their current limitations and potential as anticancer agents with a fundamentally different scope of action from other clinically validated modulators of protein homeostasis. RESULTS/CONCLUSIONS: It is likely that within the next 5 years usable inhibitors of HSF1 will be identified and in early pre-clinical evaluation.
Wiggins, P.A., Dame, R.T., Noom, M.C., and Wuite, G.J. (2009). Protein-Mediated Molecular Bridging: A Key Mechanism in Biopolymer Organization. Biophys J 97, 1997-2003. Protein-mediated bridging is ubiquitous and essential for shaping cellular structures in all organisms. Here we dissect this mechanism for a model system: the Histone-like Nucleoid-Structuring protein (H-NS). We present data from two complementary single-molecule assays that probe the H-NS-DNA interaction: a dynamic optical-trap-driven unzipping assay and an equilibrium H-NS-mediated DNA looping scanning force microscopy imaging assay. To quantitatively analyze and compare these assays, we employ what we consider a novel theoretical framework that describes the bridging motif. The interplay between the experiments and our theoretical model not only infers the effective interaction free energy, the bridging conformation and the duplex-duplex spacing, but also reveals a second, unresolved, cis-binding mode that challenges our current understanding of the role of bridging proteins in chromatin structure. We expect that this theoretical framework for describing protein-mediated bridging will be applicable to proteins acting in chromatin and cytoskeletal organization. Full Text.
Wood, A.D., Chen, E., Donaldson, I.J., Hattangadi, S., Burke, K.A., Dawson, M.A., Miranda-Saavedra, D., Lodish, H.F., Green, A.R., and Gottgens, B. (2009). Id1 Promotes Expansion and Survival of Primary Erythroid Cells and Is a Target of Jak2v617f-Stat5 Signaling. Blood 114, 1820-1830. The discovery of JAK2V617F as an acquired mutation in the majority of patients with myeloproliferative disorders (MPDs) and the key role of the JAK2-STAT5 signaling cascade in normal hematopoiesis has focused attention on the downstream transcriptional targets of STAT5. Despite evidence of its vital role in normal erythropoiesis and its ability to recapitulate many of the features of myeloid malignancies, including the MPDs, few functionally validated targets of STAT5 have been described. Here we used a combination of comparative genomics and chromatin immunoprecipitation assays to identify ID1 as a novel target of the JAK2-STAT5 signaling axis in erythroid cells. STAT5 binds and transactivates a downstream enhancer of ID1, and ID1 expression levels correlate with the JAK2V617F mutation in both retrovirally transfected fetal liver cells and polycythemia vera patients. Knockdown and overexpression studies in a well-characterized erythroid differentiation assay from primary murine fetal liver cells demonstrated a survival-promoting action of ID1. This hitherto unrecognized function implicates ID1 in the expansion of erythroblasts during terminal differentiation and suggests that ID1 plays an important role in the pathogenesis of polycythemia vera. Furthermore, our findings contribute to an increasing body of evidence implicating ID proteins in a wider range of cellular functions than initially appreciated. (Blood. 2009;114:1820-1830). Full Text.
Wood, K.C., and Sabatini, D.M. (2009). Growth Signaling at the Nexus of Stem Cell Life and Death. Cell Stem Cell 5, 232-234. Stress can activate tumor-suppressive mechanisms, causing the loss of adult stem cell function with age. In this issue of Cell Stem Cell and in Nature, Castilho et al. (2009) and Harrison et al. (2009) highlight the importance of mTOR signaling in stem cell exhaustion and mammalian aging, respectively.Full Text
Wu, S., Hu, Y.C., Liu, H., and Shi, Y. (2009). Loss of Yy1 Impacts the Heterochromatic State and Meiotic Double Strand Breaks During Mouse Spermatogenesis. Molecular and Cellular Biology (28 September Manuscripts published ahead of print) The progression of spermatogenesis involves global changes in chromatin structure and conformation. However, our understanding of the regulation of chromatin changes in germ cells remains limited. Here we describe both in vivo RNAi and genetic mouse knockout studies that identify a critical role for Yin Yang 1 (YY1) in mammalian spermatogenesis. In the YY1-deficient spermatocytes, we find a significant decrease in the global level of the heterochromatin markers (H3K9me3 and HP1-gamma) and a concomitant increase in the double-strand break signals on chromosomes (gamma-H2AX, TUNEL and Rad51) at the lepotene/zygotene stages of spermatocytes. These findings support a link between chromatin modifications and meiotic DSB formation, as has been seen in other model organisms. We propose that a depletion of YY1 may alter the structural integrity of heterochromatin rendering it more accessible to the DSB machinery. In addition, YY1-deficient spermatocytes show univalent formation, increased aneuploidy and pachytene cell death, which are likely due to defects in the DNA repair. Taken together, this study identifies an important role for YY1 in mouse meiosis and provides new insight into mechanisms that regulate mammalian spermatogenesis.PDF
Xie, H., Sun, L., and Lodish, H.F. (2009). Targeting Micrornas in Obesity. Expert Opinion on Therapeutic Targets. 2009 Aug 4. [Epub ahead of print] Obesity is a serious health problem worldwide associated with an increased risk of life-threatening diseases such as type 2 diabetes, atherosclerosis, and certain types of cancer. Fundamental for the development of novel therapeutics for obesity and its associated metabolic syndromes is an understanding of the regulation of fat cell development. Recent computational and experimental studies have shown that microRNAs (miRNAs) play a role in metabolic tissue development, lipid metabolism and glucose homeostasis. In addition, many miRNAs are dysregulated in metabolic tissues from obese animals and humans, which potentially contributes to the pathogenesis of obesity-associated complications. In this review we summarize the current state of understanding of the roles of miRNAs in metabolic tissues under normal development and obese conditions, and discuss the potential use of miRNAs as therapeutic targets.
Xie, H., Lim, B., and Lodish, H.F. (2009). Micrornas Induced During Adipogenesis That Accelerate Fat Cell Development Are Downregulated in Obesity. Diabetes.Feb 2. [Epub ahead of print] Objective: We investigated the regulation and involvement of microRNAs in fat cell development and obesity. Research Design and Methods: Using miRNA microarrays, we profiled the expression of more than 370 miRNAs during adipogenesis of preadipocyte 3T3-L1 cells and adipocytes from leptin deficient ob/ob and diet-induced obese mice. Changes in key miRNAs were validated by RT-PCR. We further assessed the contribution of the chronic inflammatory environment in obese adipose tissue to the dysregulated miRNA expression by TNFalpha treatment of adipocytes. We functionally characterized two adipocyte-enriched miRNAs, miR-103 and miR-143 by a gain-of-function approach. Results: Similar miRNAs were differentially regulated during in vitro and in vivo adipogenesis. Importantly, miRNAs that were induced during adipogenesis were downregulated in adipocytes from both types of obese mice, and vice versa. These changes are likely associated with the chronic inflammatory environment as they were mimicked by TNFalpha treatment of differentiated adipocytes. Ectopic expression of miR-103 or miR-143 in preadipocytes accelerated adipogenesis, as measured both by the upregulation of many adipogenesis markers and by an increase in triglyceride accumulation at an early stage of adipogenesis. Conclusions: Our results provide the first experimental evidence for miR-103 function in adipose biology. The remarkable inverse regulatory pattern for many miRNAs during adipogenesis and obesity has important implications for the understanding adipose tissue dysfunction in obese mice and humans and the link between chronic inflammation and obesity with insulin resistance.
YegerLotem, E., Riva, L., Su, L.J., Gitler, A.D., Cashikar, A.G., King, O.D., Auluck, P.K., Geddie, M.L., Valastyan, J.S., Karger, D.R., Susan Lindquist & Ernest Fraenkel. (2009). Bridging High-Throughput Genetic and Transcriptional Data Reveals Cellular Responses to Alpha-Synuclein Toxicity. Nature Genetics. Feb 22. [Epub ahead of print] Cells respond to stimuli by changes in various processes, including signaling pathways and gene expression. Efforts to identify components of these responses increasingly depend on mRNA profiling and genetic library screens. By comparing the results of these two assays across various stimuli, we found that genetic screens tend to identify response regulators, whereas mRNA profiling frequently detects metabolic responses. We developed an integrative approach that bridges the gap between these data using known molecular interactions, thus highlighting major response pathways. We used this approach to reveal cellular pathways responding to the toxicity of alpha-synuclein, a protein implicated in several neurodegenerative disorders including Parkinson's disease. For this we screened an established yeast model to identify genes that when overexpressed alter alpha-synuclein toxicity. Bridging these data and data from mRNA profiling provided functional explanations for many of these genes and identified previously unknown relations between alpha-synuclein toxicity and basic cellular pathways. Full Text.
Yen, J., Golan, R., and Rubins, K. (2009). Vaccinia Virus Infection & Temporal Analysis of Virus Gene Expression:. Journal of Visualized Experiments 2009 26 , 1-4. The family Poxviridae consists of large double-stranded DNA containing viruses that replicate exclusively in the cytoplasm of infected cells. Members of the orthopox genus include variola, the causative agent of human small pox, monkeypox, and vaccinia (VAC), the prototypic member of the virus family. Within the relatively large (approximately 200 kb) vaccinia genome, three classes of genes are encoded: early, intermediate, and late. While all three classes are transcribed by virally-encoded RNA polymerases, each class serves a different function in the life cycle of the virus. Poxviruses utilize multiple strategies for modulation of the host cellular environment during infection. In order to understand regulation of both host and virus gene expression, we have utilized genome-wide approaches to analyze transcript abundance from both virus and host cells. Here, we demonstrate time course infections of HeLa cells with Vaccinia virus and sampling RNA at several time points post-infection. Both host and viral total RNA is isolated and amplified for hybridization to microarrays for analysis of gene expression. Part 1 Part 2.Part 3
Zhang, J., Wang, J., Liu, Y.G., Sidik, H., Young, K.H., Lodish, H.F., and Fleming, M.D. (2009). Oncogenic Kras-Induced Leukemogeneis: Hematopoietic Stem Cells as the Initial Target and Lineage-Specific Progenitors as the Potential Targets for Final Leukemic Transformation. Blood 113, 1304-1314. KRAS 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 KRAS 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 Kras mutations initiate both JMML and TLL/L, but with different efficiencies. Limiting dilution analyses indicated that an oncogenic Kras mutation alone is insufficient to produce frank malignancy. Instead, it cooperates 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 Kras 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 as the primary target by the oncogenic Kras mutations and lineage-committed progenitors as the final target for cancer stem cell transformation. Our model might be also applicable to other solid tumors harboring oncogenic Kras mutations.Full Text
Zhang, Y.L., Radhakrishnan, M.L., Lu, X., Gross, A.W., Tidor, B., and Lodish, H.F. (2009). Symmetric Signaling by an Asymmetric 1 Erythropoietin: 2 Erythropoietin Receptor Complex. Mol Cell 33, 266-274. Via sites 1 and 2, erythropoietin binds asymmetrically to two identical receptor monomers, although it is unclear how asymmetry affects receptor activation and signaling. Here we report the design and validation of two mutant erythropoietin receptors that probe the role of individual members of the receptor dimer by selectively binding either site 1 or site 2 on erythropoietin. Ba/F3 cells expressing either mutant receptor do not respond to erythropoietin, but cells co-expressing both receptors respond to erythropoietin by proliferation and activation of the JAK2-Stat5 pathway. A truncated receptor with only one cytosolic tyrosine (Y343) is sufficient for signaling in response to erythropoietin, regardless of the monomer on which it is located. Similarly, only one receptor in the dimer needs a juxtamembrane hydrophobic L253 or W258 residue, essential for JAK2 activation. We conclude that despite asymmetry in the ligand-receptor interaction, both sides are competent for signaling, and appear to signal equally. Full Text.
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