The following alphabetical list represents papers published in 2008 with at least one Whitehead author (in red). Not all of this work was done at the Whitehead Institute. Some of these papers are collaborations with scientists elsewhere. The papers are gathered from PubMed and from Science Citation Index (also known as the Web of Science) Preceding the bibliography is an alphabetical list of the titles of the papers followed by the first author.
P.S. The journal links only work if you have a license to those respective online journals.
AddoQuaye C., Eshoo, T.W., Bartel, D.P., and Axtell, M.J. (2008). Endogenous Sirna and Mirna Targets Identified by Sequencing of the Arabidopsis Degradome. Current Biology 18, 758-762. MicroRNAs (miRNAs) regulate the expression of target mRNAs in plants and animals [1]. Plant miRNA targets have been predicted on the basis of their extensive and often conserved complementarity to the miRNAs [2-4], as well as on miRNA overexpression experiments [5]; many of these target predictions have been confirmed by isolation of the products of miRNA-directed cleavage. Here, we present a transcriptome-wide experimental method, called "degradome sequencing," to directly detect cleaved miRNA targets without relying on predictions or overexpression. The 5' ends of polyadenylated, uncapped mRNAs from Arabidopsis were directly sampled, resulting in an empirical snapshot of the degradome. miRNA-mediated-cleavage products were easily discerned from an extensive background of degraded mRNAs, which collectively covered the majority of the annotated transcriptome. Many previously known Arabidopsis miRNA targets were confirmed, and several novel targets were also discovered. Quantification of cleavage fragments revealed that those derived from TAS transcripts, which are unusual in their production of abundant secondary small interfering RNAs (siRNAs), accumulated to very high levels. A subset of secondary siRNAs are also known to direct cleavage of targets in trans [6]; degradome sequencing revealed many cleaved targets of these trans-acting siRNAs (ta-siRNAs). This empirical method is broadly applicable to the discovery and quantification of cleaved targets of small RNAs without a priori predictions. Full Text.
Alper, H., and Stephanopoulos, G. (2008). Uncovering the Gene Knockout Landscape for Improved Lycopene Production in E. Coli. Applied Microbiology and Biotechnology 78, 801-810. Systematic and combinatorial genetic approaches for the identification of gene knockout and overexpression targets have been effectively employed in the improvement of cellular phenotypes. Previously, we demonstrated how two of these tools, metabolic modeling and transposon mutagenesis, can be combined to identify strains of interest spanning the metabolic landscape of recombinant lycopene production in Escherichia coli. However, it is unknown how to best select multiple-gene knockout targets. Hence, this study seeks to understand how the overall order of gene selection, or search trajectory, biases the exploration and topology of the metabolic landscape. In particular, transposon mutagenesis and selection were employed in the background of eight different knockout genotypes. Collectively, 800,000 mutants were analyzed in hopes of exhaustively identifying all advantageous gene knockout targets. Several interesting observations, including clusters of gene functions, recurrence, and divergent genotypes, demonstrate the complexity of mapping only one genotype to one phenotype. One particularly interesting mutant, the ?hnr?yliE genotype, exhibited a drastically improved lycopene production capacity in basic minimal medium in comparison to the best strains identified in previous studies. Full Text.
Baek, D., Villen, J., Shin, C., Camargo, F.D., Gygi, S.P., and Bartel, D.P. (2008). The Impact of Micrornas on Protein Output. Nature Jul 30. [Epub ahead of print]. MicroRNAs are endogenous approximately 23-nucleotide RNAs that can pair to sites in the messenger RNAs of protein-coding genes to downregulate the expression from these messages. MicroRNAs are known to influence the evolution and stability of many mRNAs, but their global impact on protein output had not been examined. Here we use quantitative mass spectrometry to measure the response of thousands of proteins after introducing microRNAs into cultured cells and after deleting mir-223 in mouse neutrophils. The identities of the responsive proteins indicate that targeting is primarily through seed-matched sites located within favourable predicted contexts in 3' untranslated regions. Hundreds of genes were directly repressed, albeit each to a modest degree, by individual microRNAs. Although some targets were repressed without detectable changes in mRNA levels, those translationally repressed by more than a third also displayed detectable mRNA destabilization, and, for the more highly repressed targets, mRNA destabilization usually comprised the major component of repression. The impact of microRNAs on the proteome indicated that for most interactions microRNAs act as rheostats to make fine-scale adjustments to protein output. Full Text.
BenPorath, I., Thomson, M.W., Carey, V.J., Ge, R., Bell, G.W., Regev, A., and Weinberg, R.A. (2008). An Embryonic Stem Cell-Like Gene Expression Signature in Poorly Differentiated Aggressive Human Tumors. Nat Genet 40, 499-507. Cancer cells possess traits reminiscent of those ascribed to normal stem cells. It is unclear, however, whether these phenotypic similarities reflect the activity of common molecular pathways. Here, we analyze the enrichment patterns of gene sets associated with embryonic stem (ES) cell identity in the expression profiles of various human tumor types. We find that histologically poorly differentiated tumors show preferential overexpression of genes normally enriched in ES cells, combined with preferential repression of Polycomb-regulated genes. Moreover, activation targets of Nanog, Oct4, Sox2 and c-Myc are more frequently overexpressed in poorly differentiated tumors than in well-differentiated tumors. In breast cancers, this ES-like signature is associated with high-grade estrogen receptor (ER)-negative tumors, often of the basal-like subtype, and with poor clinical outcome. The ES signature is also present in poorly differentiated glioblastomas and bladder carcinomas. We identify a subset of ES cell-associated transcription regulators that are highly expressed in poorly differentiated tumors. Our results reveal a previously unknown link between genes associated with ES cell identity and the histopathological traits of tumors and support the possibility that these genes contribute to stem cell-like phenotypes shown by many tumors. Full Text.
Brambrink, T., Foreman, R., Welstead, G.G., Lengner, C.J., Wernig, M., Suh, H., and Jaenisch, R. (2008). Sequential Expression of Pluripotency Markers During Direct Reprogramming of Mouse Somatic Cells. Cell Stem Cell 2, 151-159. Pluripotency can be induced in differentiated murine and human cells by retroviral transduction of Oct4, Sox2, Klf4, and c-Myc. We have devised a reprogramming strategy in which these four transcription factors are expressed from doxycycline (dox)-inducible lentiviral vectors. Using these inducible constructs, we derived induced pluripotent stem (PS) cells from mouse embryonic fibroblasts (MEFs) and found that transgene silencing is a prerequisite for normal cell differentiation. We have analyzed the timing of known pluripotency marker activation during mouse PS cell derivation and observed that alkaline phosphatase (AP) was activated first, followed by stage-specific embryonic antigen 1 (SSEA1). Expression of Nanog and the endogenous Oct4 gene, marking fully reprogrammed cells, was only observed late in the process. Importantly, the virally transduced cDNAs needed to be expressed for at least 12 days in order to generate PS cells. Our results are a step toward understanding some of the molecular events governing epigenetic reprogramming. Full Text .
Cheeseman, I.M., and Desai, A. (2008). Molecular architecture of the kinetochore-microtubule interface. Nat Rev Mol Cell Biol 9, 33-46. Segregation of the replicated genome during cell division in eukaryotes requires the kinetochore to link centromeric DNA to spindle microtubules. The kinetochore is composed of a number of conserved protein complexes that direct its specification and assembly, bind to spindle microtubules and regulate chromosome segregation. Recent studies have identified more than 80 kinetochore components, and are revealing how these proteins are organized into the higher order kinetochore structure, as well as how they function to achieve proper chromosome segregation. Full Text.
Cole, M.F., Johnstone, S.E., Newman, J.J., Kagey, M.H., and Young, R.A. (2008). Tcf3 Is an Integral Component of the Core Regulatory Circuitry of Embryonic Stem Cells. Genes Dev 22, 746-755. Embryonic stem (ES) cells have a unique regulatory circuitry, largely controlled by the transcription factors Oct4, Sox2, and Nanog, which generates a gene expression program necessary for pluripotency and self-renewal. How external signals connect to this regulatory circuitry to influence ES cell fate is not known. We report here that a terminal component of the canonical Wnt pathway in ES cells, the transcription factor T-cell factor-3 (Tcf3), co-occupies promoters throughout the genome in association with the pluripotency regulators Oct4 and Nanog. Thus, Tcf3 is an integral component of the core regulatory circuitry of ES cells, which includes an autoregulatory loop involving the pluripotency regulators. Both Tcf3 depletion and Wnt pathway activation cause increased expression of Oct4, Nanog, and other pluripotency factors and produce ES cells that are refractory to differentiation. Our results suggest that the Wnt pathway, through Tcf3, brings developmental signals directly to the core regulatory circuitry of ES cells to influence the balance between pluripotency and differentiation. Full Text.
Cong, Y., Topf, M., Sali, A., Matsudaira, P., Dougherty, M., Chiu, W., and Schmid, M.F. (2008). Crystallographic Conformers of Actin in a Biologically Active Bundle of Filaments. Journal of Molecular Biology 375, 331-336. Actin carries out many of its cellular functions through its filamentous form; thus, understanding the detailed structure of actin filaments is an essential step in achieving a mechanistic understanding of actin function. The acrosomal bundle in the Limulus sperm has been shown to be a quasi-crystalline array with an asymmetric unit composed of a filament with 14 actin-scruin pairs. The bundle in its true discharge state penetrates the jelly coat of the egg. Our previous electron crystallographic reconstruction demonstrated that the actin filament cross-linked by scruin in this acrosomal bundle state deviates significantly from a perfect F-actin helix. In that study, the tertiary structure of each of the 14 actin protomers in the asymmetric unit of the bundle filament was assumed to be constant. In the current study, an actin filament atomic model in the acrosomal bundle has been refined by combining rigid-body docking with multiple actin crystal structures from the Protein Data Bank and constrained energy minimization. Our observation demonstrates that actin protomers adopt different tertiary conformations when they form an actin filament in the bundle. The scruin and bundle packing forces appear to influence the tertiary and quaternary conformations of actin in the filament of this biologically active bundle. . Full Text.
Costantino, C.M., Hang, H.C., Kent, S.C., Hafler, D.A., and Ploegh, H.L. (2008). Lysosomal Cysteine and Aspartic Proteases Are Heterogeneously Expressed and Act Redundantly to Initiate Human Invariant Chain Degradation. Journal of Immunology 180, 2876-2885. Presentation of Ag by class II MHC is regulated by lysosomal proteases that not only destroy the class II invariant chain (Ii) chaperone but also generate the peptide Ag that is loaded onto the class II MHC dimer. We sought to determine the extent to which asparagine endopeptidase (AEP) influences human Ag and Ii processing. Our data confirm the constructive function of AEP in tetanus toxoid processing, but they are discordant with findings that suggest a destructive role for AEP in processing of the immunodominant myelin basic protein epitope. Furthermore, we observed no effect on invariant chain processing following AEP inhibition for several distinct allelic variants of human class II MHC products. We find that cysteine and aspartic proteases, as well as AEP, can act redundantly to initiate Ii processing. We detected considerable variation in lysosomal activity between different EBV-transformed B cell lines, but these differences do not result in altered regulation of invariant chain catabolism. We propose that, as for bound peptide Ag, the identity of the lysosomal enzyme that initiates invariant chain cleavage is dependent on the class II MHC allelic variants expressed.PDF
Demmel, L., Beck, M., Klose, C., Schlaitz, A.L., Gloor, Y., Hsu, P.P., Havlis, J., Shevchenko, A., Krause, E., Kalaidzidis, Y., et al. (2008). Nucleo-cytoplasmic Shuttling of the Golgi Phosphatidylinositol 4-Kinase Pik1 Is Regulated by 14-3-3 Proteins and Coordinates Golgi Function with Cell Growth. Mol Biol Cell. Jan 2 [Epub ahead of print] Monitoring Editor: Benjamin Glick The yeast phosphatidylinositol 4-kinase Pik1p is essential for proliferation and controls Golgi homeostasis and transport of newly synthesized proteins from this compartment. At the Golgi, phosphatidylinositol 4-phosphate recruits multiple cytosolic effectors involved in formation of post-Golgi transport vesicles. A second pool of catalytically active Pik1p localizes to the nucleus. The physiological significance and regulation of this dual localization of the lipid kinase remains unknown. Here we show that Pik1p binds to the redundant 14-3-3 proteins Bmh1p and Bmh2p. We provide evidence that nucleo-cytoplasmic shuttling of Pik1p involves phosphorylation and that 14-3-3 proteins bind Pik1p in the cytoplasm. Nutrient deprivation results in relocation of Pik1p from the Golgi to the nucleus and increases the amount of Pik1p - 14-3-3 complex, a process reversed upon restored nutrient supply. These data suggest a role of Pik1p nucleo-cytoplasmic shuttling in coordination of biosynthetic transport from the Golgi with nutrient signaling. PDF
Douglas, P.M., Treusch, S., Ren, H.Y., Halfmann, R., Duennwald, M.L., Lindquist, S., and Cyr, D.M. (2008). Chaperone-Dependent Amyloid Assembly Protects Cells from Prion Toxicity. Proceedings of the National Academy of Sciences of the United States of America 105, 7206-7211. Protein conformational diseases are associated with the aberrant accumulation of amyloid protein aggregates, but whether amyloid formation is cytotoxic or protective is unclear. To address this issue, we investigated a normally benign amyloid formed by the yeast prion [RNQ(+)]. Surprisingly, modest overexpression of Rnq1 protein was deadly, but only when preexisting Rnq1 was in the [RNQ(+)] prion conformation. Molecular chaperones protect against protein aggregation diseases and are generally believed to do so by solubilizing their substrates. The Hsp40 chaperone, Sis1, suppressed Rnq1 proteotoxicity, but instead of blocking Rnq1 protein aggregation, it stimulated conversion of soluble Rnq1 to [RNQ(+)] amyloid. Furthermore, interference with Sis1-mediated [RNQ(+)] amyloid formation exacerbated Rnq1 toxicity. These and other data establish that even subtle changes in the folding homeostasis of an amyloidogenic protein can create a severe proteotoxic gain-of-function phenotype and that chaperone-mediated amyloid assembly can be cytoprotective. The possible relevance of these findings to other phenomena, including prion-driven neurodegenerative diseases and heterokaryon incompatibility in fungi, is discussed. Full Text.
Fleming, J., Outeiro, T.F., Slack, M., Lindquist, S.L., and Bulawa, C.E. (2008). Detection of Compounds That Rescue Rab1-Synuclein Toxicity. In Small Gtpases in Disease, Pt B, pp. 339-351..
Fouse, S.D., Shen, Y., Pellegrini, M., Cole, S., Meissner, A., Van Neste, L., Jaenisch, R., and Fan, G.P. (2008). Promoter Cpg Methylation Contributes to Es Cell Gene Regulation in Parallel with Oct4/Nanog, Pcg Complex, and Histone H3k4/K27 Trimethylation. Cell Stem Cell 2, 160-169. We report here genome-wide mapping of DNA methylation patterns at proximal promoter regions in mouse embryonic stem (mES) cells. Most methylated genes are differentiation associated and repressed in mES cells. By contrast, the unmethylated gene set includes many housekeeping and pluripotency genes. By crossreferencing methylation patterns to genome-wide mapping of histone H3 lysine (K) 4/27 trimethylation and binding of Oct4, Nanog, and Polycomb proteins on gene promoters, we found that promoter DNA methylation is the only marker of this group present on similar to 30% of genes, many of which are silenced in mES cells. In demethylated mutant mES cells, we saw upregulation of a subset of X-linked genes and developmental genes that are methylated in wild-type mES cells, but lack either H3K4 and H3K27 trimethylation or association with Polycomb, Oct4, or Nanog. Our data suggest that in mES cells promoter methylation represents a unique epigenetic program that complements other regulatory mechanisms to ensure appropriate gene expression. Full Text.
Gitler, A.D., Bevis, B.J., Shorter, J., Strathearn, K.E., Hamamichi, S., Su, L.J., Caldwell, K.A., Caldwell, G.A., Rochet, J.C., McCaffery, J.M.,Charles Barlowe, and Susan Lindquist (2008). The Parkinson's disease protein alpha-synuclein disrupts cellular Rab homeostasis. Proc Natl Acad Sci U S A 105, 145-150. alpha-Synuclein (alpha-syn), a protein of unknown function, is the most abundant protein in Lewy bodies, the histological hallmark of Parkinson's disease (PD). In yeast alpha-syn inhibits endoplasmic reticulum (ER)-to-Golgi (ER-->Golgi) vesicle trafficking, which is rescued by overexpression of a Rab GTPase that regulates ER-->Golgi trafficking. The homologous Rab1 rescues alpha-syn toxicity in dopaminergic neuronal models of PD. Here we investigate this conserved feature of alpha-syn pathobiology. In a cell-free system with purified transport factors alpha-syn inhibited ER-->Golgi trafficking in an alpha-syn dose-dependent manner. Vesicles budded efficiently from the ER, but their docking or fusion to Golgi membranes was inhibited. Thus, the in vivo trafficking problem is due to a direct effect of alpha-syn on the transport machinery. By ultrastructural analysis the earliest in vivo defect was an accumulation of morphologically undocked vesicles, starting near the plasma membrane and growing into massive intracellular vesicular clusters in a dose-dependent manner. By immunofluorescence/immunoelectron microscopy, these clusters were associated both with alpha-syn and with diverse vesicle markers, suggesting that alpha-syn can impair multiple trafficking steps. Other Rabs did not ameliorate alpha-syn toxicity in yeast, but RAB3A, which is highly expressed in neurons and localized to presynaptic termini, and RAB8A, which is localized to post-Golgi vesicles, suppressed toxicity in neuronal models of PD. Thus, alpha-syn causes general defects in vesicle trafficking, to which dopaminergic neurons are especially sensitive. Full Text.
Godar, S., Ince, T.A., Bell, G.W., Feldser, D., Donaher, J.L., Bergh, J., Liu, A., Miu, K., Watnick, R.S., Reinhardt, F., McAllister SS, Jacks T, and Weinberg RA. (2008). Growth-Inhibitory and Tumor- Suppressive Functions of P53 Depend on Its Repression of Cd44 Expression. Cell 134, 62-73. The p53 tumor suppressor is a key mediator of cellular responses to various stresses. Here, we show that under conditions of basal physiologic and cell-culture stress, p53 inhibits expression of the CD44 cell-surface molecule via binding to a noncanonical p53-binding sequence in the CD44 promoter. This interaction enables an untransformed cell to respond to stress-induced, p53-dependent cytostatic and apoptotic signals that would otherwise be blocked by the actions of CD44. In the absence of p53 function, the resulting derepressed CD44 expression is essential for the growth and tumor-initiating ability of highly tumorigenic mammary epithelial cells. In both tumorigenic and nontumorigenic cells, CD44's expression is positively regulated by p63, a paralogue of p53. Our data indicate that CD44 is a key tumor-promoting agent in transformed tumor cells lacking p53 function. They also suggest that the derepression of CD44 resulting from inactivation of p53 can potentially aid the survival of immortalized, premalignant cells. Full Text.
Grotenbreg, G.M., Roan, N.R., Guillen, E., Meijers, R., Wang, J.H., Bell, G.W., Starnbach, M.N., and Ploegh, H.L. (2008). Discovery of Cd8+ T Cell Epitopes in Chlamydia Trachomatis Infection through Use of Caged Class I Mhc Tetramers. Proc Natl Acad Sci U S A. 2008 Feb 1 [Epub ahead of print]. Class I MHC tetramers allow direct phenotypic identification of CD8(+) T cell populations, but their production remains laborious. A peptide exchange strategy that employs class I MHC products loaded with conditional ligands (caged MHC molecules) provides a fast and straightforward method to obtain diverse arrays of class I MHC tetramers and facilitates CD8(+) T cell epitope discovery. Here, we describe the development of photocleavable analogs of the FAPGNYPAL (SV9) epitope that bind H-2K(b) and H-2D(b) with full retention of their structural and functional integrity. We ranked all possible H-2K(b) octameric and H-2D(b) nonameric epitopes that span the genome of Chlamydia trachomatis and prepared MHC tetramers from approximately 2,000 of the highest scoring peptides by replacement of the SV9 analog with the peptide of choice. The resulting 2,000-member class I MHC tetramer array allowed the discovery of two variants of an epitope derived from polymorphic membrane protein I (PmpI) and an assessment of the kinetics of emergence and the effector function of the corresponding CD8(+) T cells. PDF
Gutzman, J.H., Graeden, E.G., Lowery, L.A., Holley, H.S., and Sive, H. (2008). Formation of the Zebrafish Midbrain-Hindbrain Boundary Constriction Requires Laminin-Dependent Basal Constriction. Mechanisms of Development.Jul 18. [Epub ahead of print]The midbrain-hindbrain boundary (MHB) is a highly conserved fold in the vertebrate embryonic brain. We have termed the deepest point of this fold the MHB constriction (MHBC) and have begun to define the mechanisms by which it develops. In the zebrafish, the MHBC is formed soon after neural tube closure, concomitant with inflation of the brain ventricles. The MHBC is unusual, as it forms by bending the basal side of the neuroepithelium. At single cell resolution, we show that zebrafish MHBC formation involves two steps. The first is a shortening of MHB cells to approximately 75% of the length of surrounding cells. The second is basal constriction, and apical expansion, of a small group of cells that contribute to the MHBC. In the absence of inflated brain ventricles, basal constriction still occurs, indicating that the MHBC is not formed as a passive consequence of ventricle inflation. In laminin mutants, basal constriction does not occur, indicating an active role for the basement membrane in this process. Apical expansion also fails to occur in laminin mutants, suggesting that apical expansion may be dependent on basal constriction. This study demonstrates laminin-dependent basal constriction as a previously undescribed molecular mechanism for brain morphogenesis. PDF.
Hanahan, D., and Weinberg, R.A. (2008). Retrospective: Judah Folkman (1933-2008). Science 319, 1055. Full Text.
Hanna, J., Markoulaki, S., Schorderet, P., Carey, B.W., Beard, C., Wernig, M., Creyghton, M.P., Steine, E.J., Cassady, J.P., Foreman, R., Rudolph Jaenisch et al. (2008). Direct Reprogramming of Terminally Differentiated Mature B Lymphocytes to Pluripotency. Cell 133, 250-264. Pluripotent cells can be derived from fibroblasts by ectopic expression of defined transcription factors. A fundamental unresolved question is whether terminally differentiated cells can be reprogrammed to pluripotency. We utilized transgenic and inducible expression of four transcription factors (Oct4, Sox2, Klf4, and c-Myc) to reprogram mouse B lymphocytes. These factors were sufficient to convert nonterminally differentiated B cells to a pluripotent state. However, reprogramming of mature B cells required additional interruption with the transcriptional state maintaining B cell identity by either ectopic expression of the myeloid transcription factor CCAAT/enhancer-binding-protein-alpha (C/EBPalpha) or specific knockdown of the B cell transcription factor Pax5. Multiple iPS lines were clonally derived from both nonfully and fully differentiated B lymphocytes, which gave rise to adult chimeras with germline contribution, and to late-term embryos when injected into tetraploid blastocysts. Our study provides definite proof for the direct nuclear reprogramming of terminally differentiated adult cells to pluripotency. Full Text.
Hargreaves, R.H.J., David, C.L., Whitesell, L.J., LaBarbera, D.V., Jamil, A., Chapuis, J.C., and Skibo, E.B. (2008). Discovery of Quinolinediones Exhibiting a Heat Shock Response and Angiogenesis Inhibition. Journal of Medicinal Chemistry 51, 2492-2501. A series of substituted quinoline-5,8-diones were synthesized and evaluated as inhibitors of the chaperone protein Hsp90 using two assays: competition for binding to C-terminal ATP-binding site and competition for binding to N-terminal ATP-binding site. In addition, the ability of the compounds to induce the heat shock response was determined using a reporter fibroblast cell line. Of all the compounds assayed, only 6-aziridinyl-2-biphenylquinoline-5,8-dione induced a heat shock response and did so without interacting at the ATP binding sites of Hsp90. COMPARE analysis was carried out on quinoline-5,8-diones active in the National Cancer Institute's 60-cell line screen with the goal of discovering quinoline-5,8-dione structures that interact with other cellular targets (molecular targets) important for cancer chemotherapy. COMPARE analysis led to the discovery of a combretastatin- like quinoline-5,8-dione structure that, in fact, inhibited angiogenesis. Full Text.
Hochwagen, A. (2008). Meiosis. Current Biology 18, R641-645. Full Text.
Howard, G., Eiges, R., Gaudet, F., Jaenisch, R., and Eden, A. (2008). Activation and Transposition of Endogenous Retroviral Elements in Hypomethylation Induced Tumors in Mice. Oncogene 27, 404-408. Genomewide DNA hypomethylation is a consistent finding in human tumors, but the importance of this change for human tumorigenesis remains an open question. We have previously reported that mice carrying a hypomorphic allele for the maintenance DNA methyltransferase (Dnmt1(chip/-)) are hypomethylated and develop thymic lymphomas, demonstrating that genomewide DNA hypomethylation can induce tumors. Hypomethylated cells exhibit inherent chromosomal instability, which is revealed in the lymphomas as a consistent trisomy of chromosome 15. We now report another aspect of the molecular basis for tumor development upon DNA hypomethylation. Seven out of 16 hypomethylation-induced lymphomas were found to contain an intracisternal A particle (IAP) somatic insertion in the middle of the Notch1 genomic locus, leading to generation of an oncogenic form of Notch1 in the tumors. This finding suggests that the molecular basis for hypomethylation-induced tumors in this model involves chromosomal instability events accompanied by activation of endogenous retroviral elements. Our findings validate the proposed role of DNA methylation in suppression of transposable elements in mammalian cells and demonstrate the importance of DNA methylation for normal cell function as well as the potential consequences of spontaneously occurring or chemically induced DNA hypomethylation. Full Text.
Jaenisch R., and Young, R. (2008). Stem Cells, the Molecular Circuitry of Pluripotency and Nuclear Reprogramming. Cell 132, 567-582. Reprogramming of somatic cells to a pluripotent embryonic stem cell-like state has been achieved by nuclear transplantation of a somatic nucleus into an enucleated egg and most recently by introducing defined transcription factors into somatic cells. Nuclear reprogramming is of great medical interest, as it has the potential to generate a source of patient-specific cells. Here, we review strategies to reprogram somatic cells to a pluripotent embryonic state and discuss our understanding of the molecular mechanisms of reprogramming based on recent insights into the regulatory circuitry of the pluripotent state. Full Text.
Ji, P., Jayapal, S.R., and Lodish, H.F. (2008). Enucleation of Cultured Mouse Fetal Erythroblasts Requires Rac Gtpases and Mdia2. Nature Cell Biology. Published online: 10 February 2008 Mammalian erythroid cells undergo enucleation, an asymmetric cell division involving extrusion of a pycnotic nucleus enveloped by the plasma membrane. The mechanisms that power and regulate the enucleation process have remained obscure. Here, we show that deregulation of Rac GTPase during a late stage of erythropoiesis completely blocks enucleation of cultured mouse fetal erythroblasts without affecting their proliferation or differentiation. Formation of the contractile actin ring (CAR) on the plasma membrane of enucleating erythroblasts was disrupted by inhibition of Rac GTPases. Furthermore, we demonstrate that mDia2, a downstream effector of Rho GTPases and a formin protein required for nucleation of unbranched actin filaments, is also required for enucleation of mouse fetal erythroblasts. We show that Rac1 and Rac2 bind to mDia2 in a GTP-dependent manner and that downregulation of mDia2, but not mDia1, by small interfering RNA (siRNA) during the late stages of erythropoiesis blocked both CAR formation and erythroblast enucleation. Additionally, overexpression of a constitutively active mutant of mDia2 rescued the enucleation defects induced by the inhibition of Rac GTPases. These results reveal important roles for Rac GTPases and their effector mDia2 in enucleation of mammalian erythroblasts. Full Text.
Johnnidis, J.B., and Camargo, F.D. (2008). Isolation and Functional Characterization of Side Population Stem Cells. Methods Mol Biol 430, 183-193. The "side population" (SP) phenotype is a manifestation of primitive cells' ability to efficiently efflux the fluorescent DNA-staining dye Hoechst 33342 and can be used as the basis by which to isolate these cells using flow cytometry. In the bone marrow (BM), the SP defines a cell subset with a highly homogeneous content of hematopoietic stem cells (HSCs). In this chapter, we describe a protocol to reproducibly isolate murine BM SP cells, as well as analytic measures, such as single cell transplantation, that can be used to assess the functionality of SP-derived stem cells.
Johnnidis, J.B., Harris, M.H., Wheeler, R.T., Stehling-Sun, S., Lam, M.H., Kirak, O., Brummelkamp, T.R., Fleming, M.D., and Camargo, F.D. (2008). Regulation of Progenitor Cell Proliferation and Granulocyte Function by Microrna-223. Nature Feb 17 [Epub ahead of print]. MicroRNAs are abundant in animal genomes and have been predicted to have important roles in a broad range of gene expression programmes. Despite this prominence, there is a dearth of functional knowledge regarding individual mammalian microRNAs. Using a loss-of-function allele in mice, we report here that the myeloid-specific microRNA-223 (miR-223) negatively regulates progenitor proliferation and granulocyte differentiation and activation. miR-223 (also called Mirn223) mutant mice have an expanded granulocytic compartment resulting from a cell-autonomous increase in the number of granulocyte progenitors. We show that Mef2c, a transcription factor that promotes myeloid progenitor proliferation, is a target of miR-223, and that genetic ablation of Mef2c suppresses progenitor expansion and corrects the neutrophilic phenotype in miR-223 null mice. In addition, granulocytes lacking miR-223 are hypermature, hypersensitive to activating stimuli and display increased fungicidal activity. As a consequence of this neutrophil hyperactivity, miR-223 mutant mice spontaneously develop inflammatory lung pathology and exhibit exaggerated tissue destruction after endotoxin challenge. Our data support a model in which miR-223 acts as a fine-tuner of granulocyte production and the inflammatory response. Full Text.
Johnson, B.S., McCaffery, J.M., Lindquist, S., and Gitler, A.D. (2008). A Yeast Tdp-43 Proteinopathy Model: Exploring the Molecular Determinants of Tdr-43 Aggregation and Cellular Toxicity. Proceedings of the National Academy of Sciences of the United States of America 105, 6439-6444. Protein misfolding is intimately associated with devastating human neurodegenerative diseases, including Alzheimer's, Huntington's, and Parkinson's. Although disparate in their pathophysiology, many of these disorders share a common theme, manifested in the accumulation of insoluble protein aggregates in the brain. Recently, the major disease protein found in the pathological inclusions of two of these diseases, amyotrophic lateral sclerosis (ALS) and frontal temporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U), was identified as the 43-kDa TAR-DNA-binding protein (TDP-43), providing a molecular link between them. TDP-43 is a ubiquitously expressed nuclear protein that undergoes a pathological conversion to an aggregated cytoplasmic localization in affected regions of the nervous system. Whether TDP-43 itself can convey toxicity and whether its abnormal aggregation is a cause or consequence of pathogenesis remain unknown. We report a yeast model to define mechanisms governing TDP-43 subcellular localization and aggregation. Remarkably, this simple model recapitulates several salient features of human TDP-43 proteinopathies, including conversion from nuclear localization to cytoplasmic aggregation. We establish a connection between this aggregation and toxicity. The pathological features of TDP-43 are distinct from those of yeast models of other protein-misfolding diseases, such as polyglutamine. This suggests that the yeast model reveals specific aspects of the underlying biology of the disease protein rather than general cellular stresses associated with accumulating misfolded proteins. This work provides a mechanistic framework for investigating the toxicity of TDP-43 aggregation relevant to human disease and establishes a manipulable, high-throughput model for discovering potential therapeutic strategies. Full Text
Johnson, D.S., Li, W., Gordon, D.B., Bhattacharjee, A., Curry, B., Ghosh, J., Brizuela, L., Carroll, J.S., Brown, M., Flicek, P., Scott A. McCuine, et al. (2008). Systematic Evaluation of Variability in Chip-Chip Experiments Using Predefined DNA Targets. Genome Research 18, 393-403. The most widely used method for detecting genome-wide protein-DNA interactions is chromatin immunoprecipitation on tiling microarrays, commonly known as ChIP-chip. Here, we conducted the first objective analysis of tiling array platforms, amplification procedures, and signal detection algorithms in a simulated ChIP-chip experiment. Mixtures of human genomic DNA and "spike-ins" comprised of nearly 100 human sequences at various concentrations were hybridized to four tiling array platforms by eight independent groups. Blind to the number of spike-ins, their locations, and the range of concentrations, each group made predictions of the spike-in locations. We found that microarray platform choice is not the primary determinant of overall performance. In fact, variation in performance between labs, protocols, and algorithms within the same array platform was greater than the variation in performance between array platforms. However, each array platform had unique performance characteristics that varied with tiling resolution and the number of replicates, which have implications for cost versus detection power. Long oligonucleotide arrays were slightly more sensitive at detecting very low enrichment. On all platforms, simple sequence repeats and genome redundancy tended to result in false positives. LM-PCR and WGA, the most popular sample amplification techniques, reproduced relative enrichment levels with high fidelity. Performance among signal detection algorithms was heavily dependent on array platform. The spike-in DNA samples and the data presented here provide a stable benchmark against which future ChIP platforms, protocol improvements, and analysis methods can be evaluated. Full Text.
Kario, E., Tirosh, B., Ploegh, H.L., and Navon, A. (2008). N-Linked Glycosylation Does Not Impair Proteasomal Degradation but Affects Class I Major Histocompatibility Complex Presentation. Journal of Biological Chemistry 283, 244-254. The addition of N-linked glycans to nascent polypeptides occurs cotranslationally in the endoplasmic reticulum (ER). For many proteins the state of the glycans serves as an indicator, which allows the ER quality control system to monitor the conformation of polypeptides upon folding. Proteins that fail to fold in the ER are often dislocated to the cytoplasm, where they are subjected to proteasomal degradation. Although the addition of N-linked glycans occurs within the ER, non-lysosomal removal of the glycans occurs in the cytosol by the action of peptide N-glycanase (PNGase). In this study, we investigated the interplay between PNGase action and proteasomal degradation of ER misfolded proteins (i.e. whether PNGase acts prior to or following proteasomal degradation). Interestingly, we found that glycan removal from N-terminally extended peptides modulates the presentation of class I major histocompatibility complex-restricted epitopes. Our findings provide direct evidence that the proteasome is capable of degrading glycoproteins without prior removal of their glycans. This degradation is independent of either the identity of the glycosylated protein or the type and number of N-linked glycans it harbors. We also captured and characterized glycopeptides generated following proteasomal degradation of RNaseB. Although the carbohydrate moiety reduced the variability of the degradation products that include the glycosylated residue ( local effect), the overall global digestion pattern of RNaseB was unaffected. Together with earlier findings by others, our data support a model in which PNGase may act both upstream and downstream to proteasomal degradation and demonstrates its important role in class I major histocompatibility complex antigen presentation. Full Text.
Karnoub, A.E., and Weinberg, R.A. (2008). Ras Oncogenes: Split Personalities. Nat Rev Mol Cell Biol 9, 517-531. Extensive research on the Ras proteins and their functions in cell physiology over the past 30 years has led to numerous insights that have revealed the involvement of Ras not only in tumorigenesis but also in many developmental disorders. Despite great strides in our understanding of the molecular and cellular mechanisms of action of the Ras proteins, the expanding roster of their downstream effectors and the complexity of the signalling cascades that they regulate indicate that much remains to be learnt. Full Text.
Kim, Y.M., Brinkmann, M.M., Paquet, M.E., and Ploegh, H.L. (2008). Unc93b1 Delivers Nucleotide-Sensing Toll-Like Receptors to Endolysosomes. Nature Published online 27 February 2008. Signalling by means of toll-like receptors (TLRs) is essential for the development of innate and adaptive immune responses. UNC93B1, essential for signalling of TLR3, TLR7 and TLR9 in both humans and mice, physically interacts with these TLRs in the endoplasmic reticulum (ER). Here we show that the function of the polytopic membrane protein UNC93B1 is to deliver the nucleotide-sensing receptors TLR7 and TLR9 from the ER to endolysosomes. In dendritic cells of 3d mice, which express an UNC93B1 missense mutant (H412R) incapable of TLR binding, neither TLR7 nor TLR9 exits the ER. Furthermore, the trafficking and signalling defects of the nucleotide-sensing TLRs in 3d dendritic cells are corrected by expression of wild-type UNC93B1. However, UNC93B1 is dispensable for ligand recognition and signal initiation by TLRs. To our knowledge, UNC93B1 is the first protein to be identified as a molecule specifically involved in trafficking of nucleotide-sensing TLRs. By inhibiting the interaction between UNC93B1 and TLRs it should be possible to achieve specific regulation of the nucleotide-sensing TLRs without compromising signalling via the cell-surface-disposed TLRs. Full Text.
Lange, J., Skaletsky, H., Bell, G.W., and Page, D.C. (2008). Msy Breakpoint Mapper, a Database of Sequence-Tagged Sites Useful in Defining Naturally Occurring Deletions in the Human Y Chromosome. Nucleic Acids Res 36, D809-814. Y chromosome deletions arise frequently in human populations, where they cause sex reversal and Turner syndrome and predispose individuals to infertility and germ cell cancer. Knowledge of the nucleotide sequence of the male-specific region of the Y chromosome (MSY) makes it possible to precisely demarcate such deletions and the repertoires of genes lost, offering insights into mechanisms of deletion and the molecular etiologies of associated phenotypes. Such deletion mapping is usually conducted using polymerase chain reaction (PCR) assays for the presence or absence of a series of Y-chromosomal DNA markers, or sequence-tagged sites (STSs). In the course of mapping intact and aberrant Y chromosomes during the past two decades, we and our colleagues have developed robust PCR assays for 1287 Y-specific STSs. These PCR assays amplify 1698 loci at an average spacing of <14 kb across the MSY euchromatin. To facilitate mapping of deletions, we have compiled a database of these STSs, MSY Breakpoint Mapper (http://breakpointmapper.wi.mit.edu/). When queried, this online database provides regionally targeted catalogs of STSs and nearby genes. MSY Breakpoint Mapper is useful for efficiently and systematically defining the breakpoint(s) of virtually any naturally occurring Y chromosome deletion. Full Text.
Lengner, C.J., Welstead, G.G., and Jaenisch, R. (2008). The Pluripotency Regulator Oct4: A Role in Somatic Stem Cells? Cell Cycle 7(6) 15 March 2008 . Since its discovery as a critical regulator of pluripotency in embryonic stem (ES) cells and the inner cells mass of the developing blastocyst, the Pou domaincontaining transcription factor Oct4 has become a proxy for "stemness" in numerous studies of somatic stem cells as it's presence is often taken as evidence of pluripotency in these cells. Recent studies, however, have demonstrated that not only is Oct4 dispensable for maintaining potency in somatic stem cell compartments, but also that the methods applied to detect Oct4 and the interpretation of the resulting data may be flawed. Here we contrast pathways known to govern pluripotency in embryonic stem cells with those in adult stem cells and critically discuss the concept of pluripotency in adult stem cells of the mammalian soma.
LoBianco, C., Shorter, J., Regulier, E., Lashuel, H., Iwatsubo, T., Lindquist, S., and Aebischer, P. (2008). Hsp104 Antagonizes Alpha-Synuclein Aggregation and Reduces Dopaminergic Degeneration in a Rat Model of Parkinson Disease. Journal of Clinical Investigation(New Online) Parkinson disease (PD) is characterized by dopaminergic neurodegeneration and intracellular inclusions of alpha-synuclein amyloid fibers, which are stable and difficult to dissolve. Whether inclusions are neuroprotective or pathological remains controversial, because prefibrillar oligomers may be more toxic than amyloid inclusions. Thus, whether therapies should target inclusions, preamyloid oligomers, or both is a critically important issue. In yeast, the protein-remodeling factor Hsp104 cooperates with Hsp70 and Hsp40 to dissolve and reactivate aggregated proteins. Metazoans, however, have no Hsp104 ortholog. Here we introduced Hsp104 into a rat PD model. Remarkably, Hsp104 reduced formation of phosphorylated alpha-synuclein inclusions and prevented nigrostriatal dopaminergic neurodegeneration induced by PD-linked alpha-synuclein (A30P). An in vitro assay employing pure proteins revealed that Hsp104 prevented fibrillization of alpha-synuclein and PD-linked variants (A30P, A53T, E46K). Hsp104 coupled ATP hydrolysis to the disassembly of preamyloid oligomers and amyloid fibers composed of alpha-synuclein. Furthermore, the mammalian Hsp70 and Hsp40 chaperones, Hsc70 and Hdj2, enhanced alpha-synuclein fiber disassembly by Hsp104. Hsp104 likely protects dopaminergic neurons by antagonizing toxic alpha-synuclein assemblies and might have therapeutic potential for PD and other neurodegenerative amyloidoses. Full Text.
Lodish, H.F., Zhou, B., Liu, G., and Chen, C.Z. (2008). Micromanagement of the Immune System by Micrornas. Nat Rev Immunol.advance online publication, 21 January 2008 MicroRNAs (miRNAs) are an abundant class of evolutionarily conserved small non-coding RNAs that are thought to control gene expression by targeting mRNAs for degradation or translational repression. Emerging evidence suggests that miRNA-mediated gene regulation represents a fundamental layer of genetic programmes at the post-transcriptional level and has diverse functional roles in animals. Here, we provide an overview of the mechanisms by which miRNAs regulate gene expression, with specific focus on the role of miRNAs in regulating the development of immune cells and in modulating innate and adaptive immune responses. Full Text.
Lu, X.H., Huang, L.J.S., and Lodish, H.F. (2008). Dimerization by a Cytokine Receptor Is Necessary for Constitutive Activation of Jak2v617f. Journal of Biological Chemistry 283, 5258-5266. The majority of the BCR-ABL-negative myeloproliferative disorders express the mutant JAK2, JAK2V617F. Previously we showed that constitutive activation of this oncogenic JAK2 mutant in Ba/F3 or 32D cells requires coexpression of a cognate homodimeric cytokine receptor, such as the EpoR. However, overexpression of JAK2V617F in Ba/F3 cells renders them cytokine-independent for growth in the absence of an exogenous cytokine receptor. Here, we demonstrated that JAK2V617F domains required for receptor association are essential for cytokine-independent growth by overexpressed JAK2V617F, suggesting JAK2V617F is binding to an unknown endogenous cytokine receptor(s) for its activation. We further showed that disruption of EpoR dimerization by coexpressing a truncated EpoR disrupted JAK2V617F-mediated transformation, indicating that EpoR dimerization plays an essential role in the activation of JAK2V617F. Interestingly, coexpression of JAK2V617F with EpoR mutants that retain JAK2 binding but are defective in mediating Epo-dependent JAK2 activation due to mutations in a conserved juxtamembrane motif does lead to cytokine-independent activation of JAK2V617F. Overall, these findings confirm that JAK2V617F requires binding to a dimerized cytokine receptor for its activation, and that the key EpoR juxtamembrane regulatory motif essential for Epo-dependent JAK2 activation is not essential for the activation of JAK2V617F. The structure of the activated JAK2V617F is thus likely to be different from that of the activated wild-type JAK2, raising the possibility of developing a specifically targeted therapy for myeloproliferative disorders. Full Text.
Ma, L., and Weinberg, R.A. (2008). Micromanagers of Malignancy: Role of Micrornas in Regulating Metastasis. Trends in Genetics Jul 30. [Epub ahead of print] It has become evident that cancer pathogenesis involves, among other macromolecules, a class of small regulatory RNAs named microRNAs, and that microRNA expression profiles are associated with prognosis and therapeutic outcome in several human cancers. Although the oncogenic or tumor-suppressing functions of several microRNAs have been characterized, the mechanistic roles played by microRNAs specifically in mediating metastasis have been addressed only recently. In this review, we focus on our emerging understanding of the contributions of microRNAs to malignant progression, specifically their functions in mediating tumor invasion and metastasis. These findings illuminate the molecular basis of metastasis and begin to connect small-RNA discoveries to the development of novel clinical biomarkers and therapeutic targets in neoplastic diseases. PDF
Mani S.A., Guo, W., Liao, M.J., Eaton, E.N., Ayyanan, A., Zhou, A.Y., Brooks, M., Reinhard, F., Zhang, C.C., Shipitsin, M., Robert A Weinberg et al(2008). The Epithelial-Mesenchymal Transition Generates Cells with Properties of Stem Cells. Cell 133, 704-715. The epithelial-mesenchymal transition (EMT) is a key developmental program that is often activated during cancer invasion and metastasis. We here report that the induction of an EMT in immortalized human mammary epithelial cells (HMLEs) results in the acquisition of mesenchymal traits and in the expression of stem-cell markers. Furthermore, we show that those cells have an increased ability to form mammospheres, a property associated with mammary epithelial stem cells. Independent of this, stem cell-like cells isolated from HMLE cultures form mammospheres and express markers similar to those of HMLEs that have undergone an EMT. Moreover, stem-like cells isolated either from mouse or human mammary glands or mammary carcinomas express EMT markers. Finally, transformed human mammary epithelial cells that have undergone an EMT form mammospheres, soft agar colonies, and tumors more efficiently. These findings illustrate a direct link between the EMT and the gain of epithelial stem cell properties. Full Text.
Markoulaki, S., Meissner, A., and Jaenisch, R. (2008). Somatic Cell Nuclear Transfer and Derivation of Embryonic Stem Cells in the Mouse. Methods 45, 101-114. Addressing the fundamental questions of nuclear equivalence in somatic cells has fascinated scientists for decades and has resulted in the development of somatic cell nuclear transfer (SCNT) or animal cloning. SCNT involves the transfer of the nucleus of a somatic cell into the cytoplasm of an egg whose own chromosomes have been removed. In the mouse, SCNT has not only been successfully used to address the issue of nuclear equivalence, but has been used as a model system to test the hypothesis that embryonic stem cells (ESCs) derived from NT blastocysts have the potential to correct-through genetic manipulations-degenerative diseases. This paper aims to provide a comprehensive description of SCNT in the mouse and the derivation of ESCs from blastocysts generated by this technique. SCNT is a very challenging and inefficient procedure because it is technically complex, it bypasses the normal events of gamete interactions and egg activation, and it depends on adequate reprogramming of the somatic cell nucleus in vivo. Improvements in any or all those aspects may enhance the efficiency and applicability of SCNT. ESC derivation from SCNT blastocysts, on the other hand, requires the survival of only a few successfully reprogrammed cells, which have the capacity to proliferate indefinitely in vitro, maintain correct genetic and epigenetic status, and differentiate into any cell type in the body-characteristics that are essential for transplantation therapy or any other in vivo application. PDF
Marson, A., Levine, S.S., Cole, M.F., Frampton, G.M., Brambrink, T., Johnstone, S., Guenther, M.G., Johnston, W.K., Wernig, M., Newman, J.,Young RA et al. (2008). Connecting Microrna Genes to the Core Transcriptional Regulatory Circuitry of Embryonic Stem Cells. Cell 134, 521-533. MicroRNAs (miRNAs) are crucial for normal embryonic stem (ES) cell self-renewal and cellular differentiation, but how miRNA gene expression is controlled by the key transcriptional regulators of ES cells has not been established. We describe here the transcriptional regulatory circuitry of ES cells that incorporates protein-coding and miRNA genes based on high-resolution ChIP-seq data, systematic identification of miRNA promoters, and quantitative sequencing of short transcripts in multiple cell types. We find that the key ES cell transcription factors are associated with promoters for miRNAs that are preferentially expressed in ES cells and with promoters for a set of silent miRNA genes. This silent set of miRNA genes is co-occupied by Polycomb group proteins in ES cells and shows tissue-specific expression in differentiated cells. These data reveal how key ES cell transcription factors promote the ES cell miRNA expression program and integrate miRNAs into the regulatory circuitry controlling ES cell identity. Full Text.
McAllister, S.S., Gifford, A.M., Greiner, A.L., Kelleher, S.P., Saelzler, M.P., Ince, T.A., Reinhardt, F., Harris, L.N., Hylander, B.L., Repasky, E.A.,and Robert A Weinberg (2008). Systemic Endocrine Instigation of Indolent Tumor Growth Requires Osteopontin. Cell 133, 994-1005. The effects of primary tumors on the host systemic environment and resulting contributions of the host to tumor growth are poorly understood. Here, we find that human breast carcinomas instigate the growth of otherwise-indolent tumor cells, micrometastases, and human tumor surgical specimens located at distant anatomical sites. This systemic instigation is accompanied by incorporation of bone-marrow cells (BMCs) into the stroma of the distant, once-indolent tumors. We find that BMCs of hosts bearing instigating tumors are functionally activated prior to their mobilization; hence, when coinjected with indolent cells, these activated BMCs mimic the systemic effects imparted by instigating tumors. Secretion of osteopontin by instigating tumors is necessary for BMC activation and the subsequent outgrowth of the distant otherwise-indolent tumors. These results reveal that outgrowth of indolent tumors can be governed on a systemic level by endocrine factors released by certain instigating tumors, and hold important experimental and therapeutic implications. Full Text.
Meissner, A., Mikkelsen, T.S., Gu, H., Wernig, M., Hanna, J., Sivachenko, A., Zhang, X., Bernstein, B.E., Nusbaum, C., Jaffe, D.B., Wernig M, Jaenisch R et al(2008). Genome-Scale DNA Methylation Maps of Pluripotent and Differentiated Cells. Nature. Jul 6. [Epub ahead of print] DNA methylation is essential for normal development and has been implicated in many pathologies including cancer. Our knowledge about the genome-wide distribution of DNA methylation, how it changes during cellular differentiation and how it relates to histone methylation and other chromatin modifications in mammals remains limited. Here we report the generation and analysis of genome-scale DNA methylation profiles at nucleotide resolution in mammalian cells. Using high-throughput reduced representation bisulphite sequencing and single-molecule-based sequencing, we generated DNA methylation maps covering most CpG islands, and a representative sampling of conserved non-coding elements, transposons and other genomic features, for mouse embryonic stem cells, embryonic-stem-cell-derived and primary neural cells, and eight other primary tissues. Several key findings emerge from the data. First, DNA methylation patterns are better correlated with histone methylation patterns than with the underlying genome sequence context. Second, methylation of CpGs are dynamic epigenetic marks that undergo extensive changes during cellular differentiation, particularly in regulatory regions outside of core promoters. Third, analysis of embryonic-stem-cell-derived and primary cells reveals that 'weak' CpG islands associated with a specific set of developmentally regulated genes undergo aberrant hypermethylation during extended proliferation in vitro, in a pattern reminiscent of that reported in some primary tumours. More generally, the results establish reduced representation bisulphite sequencing as a powerful technology for epigenetic profiling of cell populations relevant to developmental biology, cancer and regenerative medicine. Full Text.
Mueller, J.L., Mahadevaiah, S.K., Park, P.J., Warburton, P.E., Page, D.C., and Turner, J.M. (2008). The Mouse X Chromosome Is Enriched for Multicopy Testis Genes Showing Postmeiotic Expression. Nature Genetics May 4 [Epub ahead of print]. According to the prevailing view, mammalian X chromosomes are enriched in spermatogenesis genes expressed before meiosis and deficient in spermatogenesis genes expressed after meiosis. The paucity of postmeiotic genes on the X chromosome has been interpreted as a consequence of meiotic sex chromosome inactivation (MSCI)-the complete silencing of genes on the XY bivalent at meiotic prophase. Recent studies have concluded that MSCI-initiated silencing persists beyond meiosis and that most genes on the X chromosome remain repressed in round spermatids. Here, we report that 33 multicopy gene families, representing approximately 273 mouse X-linked genes, are expressed in the testis and that this expression is predominantly in postmeiotic cells. RNA FISH and microarray analysis show that the maintenance of X chromosome postmeiotic repression is incomplete. Furthermore, X-linked multicopy genes exhibit a similar degree of expression as autosomal genes. Thus, not only is the mouse X chromosome enriched for spermatogenesis genes functioning before meiosis, but in addition, approximately 18% of mouse X-linked genes are expressed in postmeiotic cells. Full Text.
Muller, U.F., and Bartel, D.P. (2008). Improved Polymerase Ribozyme Efficiency on Hydrophobic Assemblies. RNA Jan 29 [Epub ahead of print]. During an early step in the evolution of life, RNA served both as genome and as catalyst, according to the RNA world hypothesis. For self-replication, the RNA organisms must have contained an RNA that catalyzes RNA polymerization. As a first step toward recapitulating an RNA world in the laboratory, a polymerase ribozyme was generated previously by in vitro evolution and design. However, the efficiency of this ribozyme is about 100-fold too low for self-replication because of a low affinity of the ribozyme to its primer/template substrate. To improve the substrate interactions by colocalizing ribozyme and substrate on micelles, we attached hydrophobic anchors to both RNAs. We show here that the hydrophobic anchors led to aggregates with the expected size of the corresponding micelles. The micelle formation increased the polymerization yield of full-length products by 3- to 20-fold, depending on substrates and reaction conditions. With the best-characterized substrate, the improvement in polymerization efficiency was primarily due to reduced sequence-specific stalling on partially extended substrates. We discuss how, during the origin of life, micellar ribozyme aggregates could have acted as precursors to membrane-encapsulated life forms. PDF. Okamura, K., Chung, W.J., Ruby, J.G., Guo, H.L., Bartel, D.P., and Lai, E.C. (2008). The Drosophila Hairpin Rna Pathway Generates Endogenous Short Interfering Rnas. Nature 453, 803-U808. In contrast to microRNAs and Piwi- associated RNAs, short interfering RNAs ( siRNAs) are seemingly dispensable for host- directed gene regulation in Drosophila. This notion is based on the fact that mutants lacking the core siRNA- generating enzyme Dicer- 2 or the predominant siRNA effector Argonaute 2 are viable, fertile and of relatively normal morphology(1,2). Moreover, endogenous Drosophila siRNAs have not yet been identified. Here we report that siRNAs derived from long hairpin RNA genes ( hpRNAs) programme Slicer complexes that can repress endogenous target transcripts. The Drosophila hpRNA pathway is a hybrid mechanism that combines canonical RNA interference factors ( Dicer- 2, Hen1 ( known as CG12367) and Argonaute 2) with a canonical microRNA factor ( Loquacious) to generate similar to 21- nucleotide siRNAs. These novel regulatory RNAs reveal unexpected complexity in the sorting of small RNAs, and open a window onto the biological usage of endogenous RNA interference in Drosophila. Full Text.
Okamura, K., Chung, W.J., Ruby, J.G., Guo, H.L., Bartel, D.P., and Lai, E.C. (2008). The Drosophila Hairpin Rna Pathway Generates Endogenous Short Interfering Rnas. Nature 453, 803-806 In contrast to microRNAs and Piwi- associated RNAs, short interfering RNAs ( siRNAs) are seemingly dispensable for host- directed gene regulation in Drosophila. This notion is based on the fact that mutants lacking the core siRNA- generating enzyme Dicer- 2 or the predominant siRNA effector Argonaute 2 are viable, fertile and of relatively normal morphology(1,2). Moreover, endogenous Drosophila siRNAs have not yet been identified. Here we report that siRNAs derived from long hairpin RNA genes ( hpRNAs) programme Slicer complexes that can repress endogenous target transcripts. The Drosophila hpRNA pathway is a hybrid mechanism that combines canonical RNA interference factors ( Dicer- 2, Hen1 ( known as CG12367) and Argonaute 2) with a canonical microRNA factor ( Loquacious) to generate similar to 21- nucleotide siRNAs. These novel regulatory RNAs reveal unexpected complexity in the sorting of small RNAs, and open a window onto the biological usage of endogenous RNA interference in Drosophila. Full Text.
Onder, T.T., Gupta, P.B., Mani, S.A., Yang, J., Lander, E.S., and Weinberg, R.A. (2008). Loss of E-Cadherin Promotes Metastasis Via Multiple Downstream Transcriptional Pathways. Cancer Res 68, 3645-3654. Loss of the epithelial adhesion molecule E-cadherin is thought to enable metastasis by disrupting intercellular contacts-an early step in metastatic dissemination. To further investigate the molecular basis of this notion, we use two methods to inhibit E-cadherin function that distinguish between E-cadherin's cell-cell adhesion and intracellular signaling functions. Whereas the disruption of cell-cell contacts alone does not enable metastasis, the loss of E-cadherin protein does, through induction of an epithelial-to-mesenchymal transition, invasiveness, and anoikis resistance. We find the E-cadherin binding partner beta-catenin to be necessary, but not sufficient, for induction of these phenotypes. In addition, gene expression analysis shows that E-cadherin loss results in the induction of multiple transcription factors, at least one of which, Twist, is necessary for E-cadherin loss-induced metastasis. These findings indicate that E-cadherin loss in tumors contributes to metastatic dissemination by inducing wide-ranging transcriptional and functional changes.. Full Text.
Pesin, J.A., and Orr-Weaver, T.L. (2008). Regulation
of Apc/C Activators in Mitosis and Meiosis. Annual Review of Cell and
Developmental Biology Vol. 24. The
anaphase-promoting complex/cyclosome
(APC/C) is a multisubunit E3 ubiquitin ligase that triggers the degradation
of multiple substrates during mitosis. Cdc20/Fizzy and Cdh1/Fizzy-related
activate the APC/C and confer substrate specificity through complex interactions
with
both the core APC/C and substrate proteins. The regulation of Cdc20 and Cdh1
is critical for proper APC/C activity and occurs in multiple ways: targeted
protein degradation, phosphorylation, and direct binding of inhibitory proteins.
During the specialized divisions of meiosis, the activity of the APC/C must
be modified to achieve proper chromosome segregation. Recent studies show
that one way in which APC/C activity is modified is through the use of
meiosis-specific
APC/C activators. Furthermore, regulation of the APC/C during meiosis is
carried out by both mitotic regulators of the APC/C as well as meiosisspecific
regulators.
Here, we review the regulation of APC/C activators during mitosis and the
role and regulation of the APC/C during female meiosis. Full
Text.
Petersen, C.P., and Reddien, P.W. (2008). Smed-Beta-Catenin-1 Is Required for Anteroposterior Blastema Polarity in Planarian Regeneration. Science 319, 327-330. Planarian flatworms can regenerate heads at anterior- facing wounds and tails at posterior- facing wounds throughout the body. How this regeneration polarity is specified has been a classic problem for more than a century. We identified a planarian gene, Smed-beta catenin-1, that controls regeneration polarity. Posterior- facing blastemas regenerate a head instead of a tail in Smed-beta catenin- 1( RNAi) animals. Smed-beta catenin- 1 is required after wounding and at any posterior- facing wound for polarity. Additionally, intact Smed-beta catenin- 1( RNAi) animals display anteriorization during tissue turnover. Five Wnt genes and a secreted Frizzled- related Wnt antagonist- like gene are expressed in domains along the anteroposterior axis that reset to new positions during regeneration, which suggests that Wnts control polarity through Smed-beta catenin-1. Our data suggest that beta-catenin specifies the posterior character of the anteroposterior axis throughout the Bilateria and specifies regeneration polarity in planarians. Full Text.
Reddien, P.W., Newmark, P.A., and Alvarado, A.S. (2008). Gene Nomenclature Guidelines for the Planarian Schmidtea Mediterranea. Developmental Dynamics Published Online: 15 Jul 2008. We describe a gene nomenclature system for the freshwater planarian Schmidtea mediterranea. Guidelines are specified for designating names for genes and proteins, as well as for describing RNA-mediated genetic interference (RNAi) experiments. The proposed conventions aim to avoid multiple names being ascribed to single genes and to establish a uniform, simple method for naming genes in S. mediterranea that is readily understood by researchers working on planarians and other organisms . Full Text.
Reiling, J.H., and Sabatini, D.M. (2008). Increased Mtorc1 Signaling Upregulates Stress. Mol Cell 29, 533-535. In this issue of Molecular Cell, Ozcan et al. (2008) show that the loss of the tuberous sclerosis tumor suppressor complex induces endoplasmic reticulum stress, leading to attenuation of insulin receptor signaling activity via the unfolded protein response.Full Text.
Sancak, Y., Peterson, T.R., Shaul, Y.D., Lindquist, R.A., Thoreen, C.C., Bar-Peled, L., and Sabatini, D.M. (2008). The Rag Gtpases Bind Raptor and Mediate Amino Acid Signaling to Mtorc1. Science.Published Online May 22, 2008 The multiprotein mTORC1 protein kinase complex is the central component of a pathway that promotes growth in response to insulin, energy levels, and amino acids, and is deregulated in common cancers. We find that the Rag proteins-a family of four related small guanosine triphosphatases (GTPases)-interact with mTORC1 in an amino acid-sensitive manner and are necessary for the activation of the mTORC1 pathway by amino acids. A Rag mutant that is constitutively bound to GTP interacted strongly with mTORC1, and its expression within cells made the mTORC1 pathway resistant to amino acid deprivation. Conversely, expression of a GDP-bound Rag mutant prevented stimulation of mTORC1 by amino acids. The Rag proteins do not directly stimulate the kinase activity of mTORC1, but, like amino acids, promote the intracellular localization of mTOR to a compartment that also contains its activator Rheb. Full Text.
Sangster, T.A., Salathia, N., Undurraga, S., Milo, R., Schellenberg, K., Lindquist, S., and Queitsch, C. (2008). Hsp90 Affects the Expression of Genetic Variation and Developmental Stability in Quantitative Traits. Proc Natl Acad Sci U S A 105, 2963-2968. Modulation of the activity of the molecular chaperone HSP90 has been extensively discussed as a means to alter phenotype in many traits and organisms. Such changes can be due to the exposure of cryptic genetic variation, which in some instances may also be accomplished by mild environmental alteration. Should such polymorphisms be widespread, natural selection may be more effective at producing phenotypic change in suboptimal environments. However, the frequency and identity of buffered polymorphisms in natural populations are unknown. Here, we employ quantitative genetic dissection of an Arabidopsis thaliana developmental response, hypocotyl elongation in the dark, to detail the underpinnings of genetic variation responsive to HSP90 modulation. We demonstrate that HSP90-dependent alleles occur in continuously distributed, environmentally responsive traits and are amenable to quantitative genetic mapping techniques. Furthermore, such alleles are frequent in natural populations and can have significant effects on natural phenotypic variation. We also find that HSP90 modulation has both general and allele-specific effects on developmental stability; that is, developmental stability is a phenotypic trait that can be affected by natural variation. However, effects of revealed variation on trait means outweigh effects of decreased developmental stability, and the HSP90-dependent trait alterations could be acted on by natural selection. Thus, HSP90 may centrally influence canalization, assimilation, and the rapid evolutionary alteration of phenotype through the concealment and exposure of cryptic genetic variation. Full Text.
Sangster, T.A., Salathia, N., Lee, H.N., Watanabe, E., Schellenberg, K., Morneau, K., Wang, H., Undurraga, S., Queitsch, C., and Lindquist, S. (2008). Hsp90-Buffered Genetic Variation Is Common in Arabidopsis Thaliana. Proc Natl Acad Sci U S A 105, 2969-2974. HSP90 is a protein chaperone particularly important in the maturation of a diverse set of proteins that regulate key steps in a multitude of biological processes. Alterations in HSP90 function produce altered phenotypes at low penetrance in natural populations. Previous work has shown that at least some of these phenotypes are due to genetic variation that remains phenotypically cryptic until it is revealed by the impairment of HSP90 function. Exposure of such "buffered" genetic polymorphisms can also be accomplished by environmental stress, linking the appearance of new phenotypes to defects in protein homeostasis. Should such polymorphisms be widespread, natural selection may be more effective at producing phenotypic change in suboptimal environments. In evaluating this hypothesis, a key unknown factor is the frequency with which HSP90-buffered polymorphisms occur in natural populations. Here, we present Arabidopsis thaliana populations suitable for genetic mapping that have constitutively reduced HSP90 levels. We employ quantitative genetic techniques to examine the HSP90-dependent polymorphisms affecting a host of plastic plant life-history traits. Our results demonstrate that HSP90-dependent natural variation is present at high frequencies in A. thaliana, with an expectation that at least one HSP90-dependent polymorphism will affect nearly every quantitative trait in progeny of two different wild lines. Hence, HSP90 is likely to occupy a central position in the translation of genotypic variation into phenotypic differences. Full Text.
Stark, A., Bushati, N., Jan, C.H., Kheradpour, P., Hodges, E., Brennecke, J., Bartel, D.P., Cohen, S.M., and Kellis, M. (2008). A Single Hox Locus in Drosophila Produces Functional Micrornas from Opposite DNA Strands. Genes & Development 22, 8-13. MicroRNAs (miRNAs) are similar to 22-nucleotide RNAs that are processed from characteristic precursor hairpins and pair to sites in messages of protein-coding genes to direct post-transcriptional repression. Here, we report that the miRNA iab-4 locus in the Drosophila Hox cluster is transcribed convergently from both DNA strands, giving rise to two distinct functional miRNAs. Both sense and antisense miRNA products target neighboring Hox genes via highly conserved sites, leading to homeotic transformations when ectopically expressed. We also report sense/antisense miRNAs in mouse and find antisense transcripts close to many miRNAs in both flies and mammals, suggesting that additional sense/antisense pairs exist. Full Text.
Upadhyaya, A., Baraban, M., Wong, J., Matsudaira, P., van Oudenaarden, A., and Mahadevan, L. (2008). Power-Limited Contraction Dynamics of Vorticella Convallaria: An Ultrafast Biological Spring. Biophysical Journal 94, 265-272. Vorticella convallaria is one of the fastest and most powerful cellular machines. The cell body is attached to a substrate by a slender stalk containing a polymeric structure - the spasmoneme. Helical coiling of the stalk results from rapid contraction of the spasmoneme, an event mediated by calcium binding to a negatively charged polymeric backbone. We use high speed imaging to measure the contraction velocity as a function of the viscosity of the external environment and find that the maximum velocity scales inversely with the square root of the viscosity. This can be explained if the rate of contraction is ultimately limited by the power delivered by the actively contracting spasmoneme. Microscopically, this scenario would arise if the mechanochemical wave that propagates along the spasmoneme is faster than the rate at which the cell body can respond due to its large hydrodynamic resistance. We corroborate this by using beads as markers on the stalk and find that the contraction starts at the cell body and proceeds down the stalk at a speed that exceeds the velocity of the cell body. Full Text.
Vyas, J.M., Van der Veen, A.G., and Ploegh, H.L. (2008). The Known Unknowns of Antigen Processing and Presentation. Nature Review Immunology. Jul 18. [Epub ahead of print] The principal components of both MHC class I and class II antigen processing and presentation pathways are well known. In dendritic cells, these pathways are tightly regulated by Toll-like-receptor signalling and include features, such as cross-presentation, that are not seen in other cell types. However, the exact mechanisms involved in the subcellular trafficking of antigens remain poorly understood and in some cases are controversial. Recent data suggest that diverse cellular machineries, including autophagy, participate in antigen processing and presentation, although their relative contributions remain to be fully elucidated. Here, we highlight some emerging themes of antigen processing and presentation that we think merit further attention. Full Text.
Wang, H., Duennwald, M.L., Roberts, B.E., Rozeboom, L.M., Zhang, Y.X.L., Steele, A.D., Krishnan, R., Su, L.J., Griffin, D., Mukhopadhyay, S., Lindquist S, and Shorter J.(2008). Direct and Selective Elimination of Specific Prions and Amyloids by 4,5-Dianilinophthalimide and Analogs. Proceedings of the National Academy of Sciences of the United States of America 105, 7159-7164. Mechanisms to safely eliminate amyloids and preamyloid oligomers associated with many devastating diseases are urgently needed. Biophysical principles dictate that small molecules are unlikely to perturb large intermolecular protein-protein interfaces, let alone extraordinarily stable amyloid interfaces. Yet 4,5-dianilinophthalimide (DAPH-1) reverses A beta 42 amyloidogenesis and neurotoxicity, which is associated with Alzheimer's disease. Here, we show that DAPH-1 and select derivatives are ineffective against several amyloidogenic proteins, including tau, a-synuclein, Ure2, and PrP, but antagonize the yeast prion protein, Sup35, in vitro and in vivo. This allowed us to exploit several powerful new tools created for studying the conformational transitions of Sup35 and decipher the mechanisms by which DAPH-1 and related compounds antagonize the prion state. During fibrillization, inhibitory DAPHs alter the folding of Sup35's amyloidogenic core, preventing amyloidogenic oligomerization and specific recognition events that nucleate prion assembly. Select DAPHs also are capable of attacking preformed amyloids. They remodel Sup35 prion-specific intermolecular interfaces to create morphologically altered aggregates with diminished infectivity and self-templating activity. Our studies provide mechanistic insights and reinvigorate hopes for small-molecule therapies that specifically disrupt intermolecular amyloid contacts. Full Text.
Weinberg, R.A. (2008). In Retrospect: The Chromosome Trail. Nature 453, 725. A new translation of Theodor Boveri's 1914 monograph brings the early origins of contemporary cancer research to a wider readership Full Text.
Weinberg, R.A. (2008). Coevolution in the Tumor Microenvironment. Nature Genetics 40, 494-495. The progression of carcinomas to high- grade malignancies is accompanied by profound histological changes in the tumor-associated stroma. Although previous studies have suggested that mesenchymal cells of the stroma undergo genetic alterations during this progression, a new study now provides evidence that strongly contradicts this theory of stromal cell coevolution. Full Text.
Weinberg, R.A. (2008). Mechanisms of Malignant Progression. Carcinogenesis.May 2 [Epub ahead of print] PDF
Welstead, G.G., Schorderet, P., and Boyer, L.A. (2008). The Reprogramming Language of Pluripotency. Current Opinion in Genetics and Development Mar 19; [Epub ahead of print]. In metazoans, lineage-specific transcription factors and epigenetic modifiers function to establish and maintain proper gene expression programs during development. Recent landmark studies in both mouse and human have defined a set of transcription factors whose ectopic expression by retroviral transduction is capable of reprogramming a somatic nucleus to the pluripotent state. The identification of factors that are sufficient for the induction of pluripotency suggests that rewiring transcriptional regulatory networks at the molecular level can be used to manipulate cell fate in vitro. These findings have broad implications for understanding development and disease and for the potential use of stem cells in therapeutic applications. Full Text.
Wernig, M., Lengner, C.J., Hanna, J., Lodato, M.A., Steine, E., Foreman, R., Staerk, J., Markoulaki, S., and Jaenisch, R. (2008). A Drug-Inducible Transgenic System for Direct Reprogramming of Multiple Somatic Cell Types. Nature Biotechnology.Jul 1. [Epub ahead of print] The study of induced pluripotency is complicated by the need for infection with high-titer retroviral vectors, which results in genetically heterogeneous cell populations. We generated genetically homogeneous 'secondary' somatic cells that carry the reprogramming factors as defined doxycycline (dox)-inducible transgenes. These cells were produced by infecting fibroblasts with dox-inducible lentiviruses, reprogramming by dox addition, selecting induced pluripotent stem cells and producing chimeric mice. Cells derived from these chimeras reprogram upon dox exposure without the need for viral infection with efficiencies 25- to 50-fold greater than those observed using direct infection and drug selection for pluripotency marker reactivation. We demonstrate that (i) various induction levels of the reprogramming factors can induce pluripotency, (ii) the duration of transgene activity directly correlates with reprogramming efficiency, (iii) cells from many somatic tissues can be reprogrammed and (iv) different cell types require different induction levels. This system facilitates the characterization of reprogramming and provides a tool for genetic or chemical screens to enhance reprogramming. Full Text.
Wernig, M., Zhao, J.P., Pruszak, J., Hedlund, E., Fu, D., Soldner, F., Broccoli, V., Constantine-Paton, M., Isacson, O., and Jaenisch, R. (2008). Neurons Derived from Reprogrammed Fibroblasts Functionally Integrate into the Fetal Brain and Improve Symptoms of Rats with Parkinson's Disease. Proc Natl Acad Sci U S A.Apr 7 [Epub ahead of print] The long-term goal of nuclear transfer or alternative reprogramming approaches is to create patient-specific donor cells for transplantation therapy, avoiding immunorejection, a major complication in current transplantation medicine. It was recently shown that the four transcription factors Oct4, Sox2, Klf4, and c-Myc induce pluripotency in mouse fibroblasts. However, the therapeutic potential of induced pluripotent stem (iPS) cells for neural cell replacement strategies remained unexplored. Here, we show that iPS cells can be efficiently differentiated into neural precursor cells, giving rise to neuronal and glial cell types in culture. Upon transplantation into the fetal mouse brain, the cells migrate into various brain regions and differentiate into glia and neurons, including glutamatergic, GABAergic, and catecholaminergic subtypes. Electrophysiological recordings and morphological analysis demonstrated that the grafted neurons had mature neuronal activity and were functionally integrated in the host brain. Furthermore, iPS cells were induced to differentiate into dopamine neurons of midbrain character and were able to improve behavior in a rat model of Parkinson's disease upon transplantation into the adult brain. We minimized the risk of tumor formation from the grafted cells by separating contaminating pluripotent cells and committed neural cells using fluorescence-activated cell sorting. Our results demonstrate the therapeutic potential of directly reprogrammed fibroblasts for neuronal cell replacement in the animal model. PDF
Wernig, M., Meissner, A., Cassady, J.P., and Jaenisch, R. (2008). C-Myc Is Dispensable for Direct Reprogramming of Mouse Fibroblasts. Cell Stem Cell 2, 10-12. Retroviral transduction of the four transcription factors Oct4, Sox2, Klf4, and c-Myc has been shown to initiate a reprogramming process that results in the transformation of mouse fibroblasts into embryonic stem (ES)-like cells designated as induced pluripotent stem (iPS) cells ([Maherali et al., 2007], [Meissner et al., 2007], [Okita et al., 2007], [Takahashi and Yamanaka, 2006] and [Wernig et al., 2007]). The promise of somatic reprogramming is the possibility to generate pluripotent stem cells that are patient specific and can be used as a unique source for autologous cell types for transplantation therapy ([Jaenisch, 2004] and [Yamanaka, 2007]). Many iPS cell-derived animals develop tumors due to the reactivation of the c-Myc virus (Okita et al., 2007), and this represents a major safety concern if we want to translate this approach to humans. It is thus of great importance to achieve reprogramming without this particular oncogene in the future. Here we show that fibroblasts can be reprogrammed to a pluripotent state by Oct4, Sox2, and Klf4 in the absence of c-Myc. Full Text.
Xie, F., and Orr-Weaver, T.L. (2008). Isolation of a Drosophila Amplification Origin Developmentally Activated by Transcription. Proc Natl Acad Sci U S A. We exploited the Drosophila Amplicon in Follicle Cells, DAFC-62D, to identify a new metazoan amplification origin, ori62. In addition to the origin, DAFC-62D contains two other developmental stage-specific binding regions for the Origin Recognition Complex (ORC) and the replicative helicase MCM2-7. All three of these regions are required for proper amplification. There are two rounds of amplification initiation at ori62, and the second round is preceded by transcription across ori62. We show by alpha-amanitin inhibition that RNA polymerase II (RNAPII) transcription is required to localize MCM2-7 (but not ORC) to permit the second round of origin firing. This role for transcription appears unique to DAFC-62D, because neither other DAFCs nor ectopic transposons with the DAFC-62D replication elements bounded by functional chromatin insulators are affected by alpha-amanitin. By sequential chromatin immunoprecipitation, we show that the MCM complex and RNAPII are bound to the same 100-500 bp pieces of chromatin during late origin firing. These results raise the possibility that RNAPII may recruit MCM2-7 at some metazoan replication origins. Full Text.
Yang, J., and Weinberg, R.A. (2008). Epithelial-Mesenchymal Transition: At the Crossroads of Development and Tumor Metastasis. Developmental Cell 14, 818-829. The epithelial-mesenchymal transition is a highly conserved cellular program that allows polarized, immotile epithelial cells to convert to motile mesenchymal cells. This important process was initially recognized during several critical stages of embryonic development and has more recently been implicated in promoting carcinoma invasion and metastasis. In this review, we summarize and compare major signaling pathways that regulate the epithelial-mesenchymal transitions during both development and tumor metastasis. Studies in both fields are critical for our molecular understanding of cell migration and morphogenesis.Full Text.
Yang, F., Gell, K., van der Heijden, G.W., Eckardt, S., Leu, N.A., Page, D.C., Benavente, R., Her, C., Hoog, C., McLaughlin, K.J., et al. (2008). Meiotic Failure in Male Mice Lacking an X-Linked Factor. Genes & Development 22, 682-691. Meiotic silencing of sex chromosomes may cause their depletion of meiosis-specific genes during evolution. Here, we challenge this hypothesis by reporting the identification of TEX11 as the first X-encoded meiosis-specific factor in mice. TEX11 forms discrete foci on synapsed regions of meiotic chromosomes and appears to be a novel constituent of meiotic nodules involved in recombination. Loss of TEX11 function causes chromosomal asynapsis and reduced crossover formation, leading to elimination of spermatocytes, respectively, at the pachytene and anaphase I stages. Specifically, TEX11-deficient spermatocytes with asynapsed autosomes undergo apoptosis at the pachytene stage, while those with only asynapsed sex chromosomes progress. However, cells that survive the pachytene stage display chromosome nondisjunction at the first meiotic division, resulting in cell death and male infertility. TEX11 interacts with SYCP2, which is an integral component of the synaptonemal complex lateral elements. Thus, TEX11 promotes initiation and/or maintenance of synapsis and formation of crossovers, and may provide a physical link between these two meiotic processes. Full Text.
Zhang, C.C., Kaba, M., Iizuka, S., Huynh, H., and Lodish, H.F. (2008). Angiopoietin-Like 5 and Igfbp2 Stimulate Ex Vivo Expansion of Human Cord Blood Hematopoietic Stem Cells as Assayed by Nod/Scid Transplantation. Blood.Jan 17;. [Epub ahead of print] Pluripotent hematopoietic stem cells (HSCs) are the basis of bone marrow transplantation and are attractive target cells for hematopoietic gene therapy, but these important clinical applications have been severely hampered by difficulties in ex vivo expansion of HSCs. In particular, the use of cord blood for adult transplantation is greatly limited by the number of HSCs. Previously we identified Angiopoietin-like proteins and IGF binding protein 2 (IGFBP2) as new hormones that, together with other factors, can expand mouse bone marrow HSCs in culture. Here we measure the activity of multipotent human SCID-repopulating cells (SRCs) by transplantation into the nonobese diabetic severe combined immunodeficiency (NOD/SCID) mice; secondary transplantation was performed to evaluate the self-renewal potential of SRCs. A serum- free medium containing SCF, TPO, and FGF-1 or Flt3-L cannot significantly support expansion of the SRCs present in human cord blood CD133(+) cells. Addition of either Angiopoietin-like 5, or IGF binding protein 2 to the cultures led to a sizable expansion of HSC numbers, as assayed by NOD/SCID transplantation. A serum-free culture containing SCF, TPO, FGF-1, Angiopoietin-like 5, and IGFBP2 supports a ~20 fold net expansion of repopulating human cord blood HSCs, a number potentially applicable to several clinical processes including HSC transplantation. PDF
8/18/08