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Recent Scientific Papers
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An Embryonic Stem Cell-Like Gene Expression Signature in Poorly Differentiated Aggressive Human Tumors.
Nat Genet. 2008. May;40(5):499-507.
Ben-Porath, I.*, Thomson, M.W., Carey, V.J., Ge, R*., Bell, G.W.*, Regev, A., and Weinberg, R.A.*
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.
Direct Reprogramming of Terminally Differentiated Mature B Lymphocytes to Pluripotency.
Cell. 2008. Apr 18;133(2):250-64.
Hanna, J.*, Markoulaki, S.*, Schorderet, P.*, Carey, B.W.*, Beard, C.*, Wernig, M.*, Creyghton, M.P.*, Steine, E.J.*, Cassady, J.P.*, Foreman, R.*, Lengner, C.J.*, Dausman, J.A.*, Jaenisch, R.*
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.
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. 2008. Apr 15;105(15):5856-61. Epub 2008 Apr 7.
Wernig, M*., Zhao, J.P., Pruszak, J., Hedlund, E., Fu, D., Soldner, F.*, Broccoli, V., Constantine-Paton, M., Isacson, O., and Jaenisch, R.*
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.
Uncovering the Gene Knockout Landscape for Improved Lycopene Production in E. Coli.
Applied Microbiology and Biotechnology. 2008. Apr;78(5):801-10. Epub 2008 Feb 1.
Alper, H.*, and Stephanopoulos, G.
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 exhibited a drastically improved lycopene production capacity in basic minimal medium in comparison to the best strains identified in previous studies.
Isolation and Functional Characterization of Side Population Stem Cells.
Methods Mol Biol. 2008. 430:183-193.
Johnnidis, J.B.*, and Camargo, F.D.*
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.
Meiotic Failure in Male Mice Lacking an X-Linked Factor.
Genes Dev. 2008. Mar 1;22(5):682-91.
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., and Wang PJ.
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.
Dimerization by a Cytokine Receptor Is Necessary for Constitutive Activation of Jak2v617f.
J Biol Chem. 2008. Feb 29;283(9):5258-66. Epub 2007 Dec 23.
Lu, X.H.*, Huang, L.J.S., and Lodish, H.F.*
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.
Systematic Evaluation of Variability in Chip-Chip Experiments Using Predefined DNA Targets.
Genome Res. 2008. Mar;18(3):393-403. Epub 2008 Feb 7.
Johnson, D.S., Li, W., Gordon, D.B., Bhattacharjee, A., Curry, B., Ghosh, J., Brizuela, L., Carroll, J.S., Brown, M., Flicek, P., Koch, C.M., Dunham, I., Bieda, M., Xu, X., Farnham, P.J., Kapranov, P., Nix, D.A., Gingeras, T.R., Zhang, X., Holster, H., Jiang, N., Green, R.D., Song, J.S., McCuine, S.A.*, Anton, E., Nguyen, L., Trinklein, N.D., Ye, Z., Ching, K., Hawkins, D., Ren, B., Scacheri, P.C., Rozowsky, J., Karpikov, A., Euskirchen, G., Weissman, S., Gerstein, M., Snyder, M., Yang, A., Moqtaderi, Z., Hirsch, H., Shulha, H.P., Fu, Y., Weng, Z., Struhl, K., Myers, R.M., Lieb, J.D., Liu, X.S.
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.
The Reprogramming Language of Pluripotency.
Curr Opin Genet Dev. 2008. Mar 19.
Welstead, G.G.*, Schorderet, P.*, and Boyer, L.A.*
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.
Tcf3 Is an Integral Component of the Core Regulatory Circuitry of Embryonic Stem Cells.
Genes Dev. 2008. Mar 15;22(6):746-55.
Cole, M.F.*, Johnstone, S.E.*, Newman, J.J.*, Kagey, M.H.*, and Young, R.A.*
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.
*Whitehead Institute for Biomedical Research
Last updated May 2, 2008. |
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