Akselrod, G. M., Timp, W., Mirsaidov, U., Zhao, Q., Li, C., Timp, R., Timp, K., Matsudaira, P. and Timp, G. (2006) Laser-guided assembly of heterotypic three-dimensional living cell microarrays. Biophysical Journal. 91 3465-3473. We have assembled three-dimensional heterotypic networks of living cells in hydrogel without loss of viability using arrays of time-multiplexed, holographic optical traps. The hierarchical control of the cell positions is achieved with, to our knowledge, unprecedented submicron precision, resulting in arrays with an intercell separation < 400 nm. In particular, we have assembled networks of Swiss 3T3. broblasts surrounded by a ring of bacteria. We have also demonstrated the ability to manipulate hundreds of Pseudomonas aeruginosa simultaneously into two- and three-dimensional arrays with a time-averaged power < 2 mW per trap. This is the first time to our knowledge that living cell arrays of such complexity have been synthesized, and it represents a milestone in synthetic biology and tissue engineering.Full Text

ArtavanisTsakonas, K., Love, J. C., Ploegh, H. L. and Vyas, J. M. (2006) Recruitment of CD63 to Cryptococcus neoformans phagosomes requires acidification. Proc Natl Acad Sci U S A.Oct 24;103(43):15945-50 The subcellular localization of the cluster of differentiation 63 (CD63) tetraspanin and its interaction with the class II MHC antigen presentation pathway were examined in the context of phagocytosis by live cell imaging, by using monomeric red fluorescent protein-tagged mouse CD63 expressed in primary bone marrow-derived cell cultures. Upon phagocytosis of Cryptococcus neoformans and polystyrene beads, CD63 was recruited selectively to C. neoformans-containing phagosomes in a MyD88-independent acidification-dependent manner. Bead-containing phagosomes, within a C. neoformans-containing cell, acidified to a lesser extent and failed to recruit CD63 to a level detectable by microscopy. CD63 recruitment to yeast phagosomes occurred independently of class II MHC and LAMP-1. These observations indicate that the composition of distinct phagosomal compartments within the same cell is determined by phagosomal cargo and may affect the outcome of antigen processing and presentation.Full Text

ArtavanisTsakonas, K., Misaghi, S., Comeaux, C. A., Catic, A., Spooner, E., Duraisingh, M. T. and Ploegh, H. L. (2006) Identification by functional proteomics of a deubiquitinating/deNeddylating enzyme in Plasmodium falciparum. Mol Microbiol. 61 1187-95. Ubiquitination is a post-translational modification implicated in a variety of cellular functions, including transcriptional regulation, protein degradation and membrane protein trafficking. Ubiquitin and the enzymes that act on it, although conserved and essential in eukaryotes, have not been well studied in parasites, despite sequencing of several parasite genomes. Several putative ubiquitin hydrolases have been identified in Plasmodium falciparum based on sequence homology alone, with no evidence of expression or function. Here we identify the first deubiquitinating enzyme in P. falciparum, PfUCH54, by its activity. We show that PfUCH54 also has deNeddylating activity, as assayed by a mammalian Nedd8-based probe. This activity is absent from mammalian homologues of PfUCH54. Given the importance of parasitic membrane protein trafficking as well as protein degradation in the virulence of this parasite, this family of enzymes may represent a target for pharmacological intervention with this disease Full Text

Axtell, M. J., Jan, C., Rajagopalan, R. and Bartel, D. P. (2006) A Two-Hit Trigger for siRNA Biogenesis in Plants. Cell. 127 565-77. In Arabidopsis, microRNA-directed cleavage can define one end of RNAs that then generate phased siRNAs. However, most miRNA-targeted RNAs do not spawn siRNAs, suggesting the existence of additional determinants within those that do. We find that in moss, phased siRNAs arise from regions flanked by dual miR390 cleavage sites. AtTAS3, an siRNA locus important for development and conserved among higher plants, also has dual miR390 complementary sites. Both sites bind miR390 in vitro and are functionally required in Arabidopsis, but cleavage is undetectable at the 5' site-demonstrating that noncleavable sites can be functional in plants. Phased siRNAs also emanate from the bounded regions of every Arabidopsis gene with two known microRNA/siRNA complementary sites, but only rarely from genes with single sites. Therefore, two "hits,"-often, but not always, two cleavage events-constitute a conserved trigger for siRNA biogenesis, a finding with implications for recognition and silencing of aberrant RNA.Full Text

Bailey, S. N., Ali, S. M., Carpenter, A. E., Higgins, C. O. and Sabatini, D. M. (2006) Microarrays of lentiviruses for gene function screens in immortalized and primary cells. Nat Methods. 3 117-22. Here we describe lentivirus-infected cell microarrays for the high-throughput screening of gene function in mammalian cells. To create these arrays, we cultured mammalian cells on glass slides 'printed' with lentiviruses pseudotyped as vesicular stomatitis virus glycoprotein, which encode short hairpin RNA or cDNA. Cells that land on the printed 'features' become infected with lentivirus, creating a living array of stably transduced cell clusters within a monolayer of uninfected cells. The small size of the features of the microarrays (300 mum in diameter) allows high-density spotting of lentivirus, permitting thousands of distinct parallel infections on a single glass slide. Because lentiviruses have a wide cellular tropism, including primary cells, lentivirus-infected cell microarrays can be used as a platform for high-throughput screening in a variety of cell types. Full Text

Ballabio, A., Nelson, D. and Rozen, S. (2006) The sex chromosomes and human disease. Current Opinion in Genetics & Development. 16 209-212. Full Text

Bard, K. A., Todd, B. K., Bernier, C., Love, J. and Leavens, D. A. (2006) Self-awareness in human and chimpanzee infants: What is measured and what is meant by the mark and mirror test? Infancy. 9 191-219. The objective study of self-recognition, with a mirror and a mark applied to the face, was conducted independently by Gallup (1970) for use with chimpanzees and monkeys, and by Amsterdam (1972) for use with infant humans. Comparative psychologists have followed the model (and assumptions) set by Gallup, whereas developmental psychologists have followed a different model (e.g., Lewis & Brooks-Gunn, 1979). This article explores the assumptions in the definitions and methods of self-recognition assessments in the 30 years since these initial studies, and charts the divergence between the developmental mark test and the comparative mark test. Two new studies, 1 with infant chimpanzees and I with infant humans, illustrate a reconciliation of the 2 approaches. Overt application of the mark, or other procedures related to how the mark is discovered, did not enhance mirror self-recognition. In contrast, maternal scaffolding appears to enhance performance, perhaps by eliciting well-rehearsed verbal responses (i.e., naming self). When comparable testing procedures and assessment criteria are used, chimpanzee and human infants perform comparably. A combined developmental comparative approach allows us to suggest that mirror self-recognition may be based on a specific aspect of mental representation, the cognitive ability to symbolize.

Baltus, A. E., Menke, D. B., Hu, Y. C., Goodheart, M. L., Carpenter, A. E., de Rooij, D. G. and Page, D. C. (2006) In germ cells of mouse embryonic ovaries, the decision to enter meiosis precedes premeiotic DNA replication. Nature Genetics 38 (12) The transition from mitosis to meiosis is a defining juncture in the life cycle of sexually reproducing organisms. In yeast, the decision to enter meiosis is made before the single round of DNA replication that precedes the two meiotic divisions. We present genetic evidence of an analogous decision point in the germ line of a multicellular organism. The mouse Stra8 gene is expressed in germ cells of embryonic ovaries, where meiosis is initiated, but not in those of embryonic testes, where meiosis does not begin until after birth. Here we report that in female embryos lacking Stra8 gene function, the early, mitotic development of germ cells is normal, but these cells then fail to undergo premeiotic DNA replication, meiotic chromosome condensation, cohesion, synapsis and recombination. Combined with previous findings, these genetic data suggest that active differentiation of ovarian germ cells commences at a regulatory point upstream of premeiotic DNA replication.Full Text

Beard, C., Hochedlinger, K., Plath, K., Wutz, A. and Jaenisch, R. (2006) Efficient method to generate single-copy transgenic mice by site-specific integration in embryonic stem cells. Genesis. 44 23-28. Transgenic and gene-targeted mutant mice provide powerful tools for analysis of the cellular processes involved in early development and in the pathogenesis of many diseases. Here we describe a transgene integration strategy mediated by site-specific recombination that allows establishment of multiple embryonic stem (ES) cell lines carrying tetracycline-inducible genes targeted to a specific locus to assure predictable temporal and spatial expression in ES cells and mice. Using homologous recombination we inserted an frt homing site into which tetracycline-inducible transgenes can be integrated efficiently in the presence of FLPe recombinase. This strategy and the vectors described here are generally applicable to any locus in ES cells and should allow for the rapid production of mice with transgenes efficiently targeted to a defined site. Full Text.

Bell, G. W. and Lewitter, F. (2006) Visualizing networks. Methods Enzymol. 411 408-21. An interrelated set of genes or proteins can be represented effectively as a network that describes physical interactions, regulatory relationships, or metabolic pathways. Visualizing a network can be a helpful method to extract biological meaning and to generate testable hypotheses about large-scale biological data. This chapter describes some potential rationales for visualizing networks of microarray and other data types, which can be integrated and filtered to show potentially significant relationships. It also presents a practical introduction to Osprey and Cytoscape, two software platforms that are powerful tools for visualizing, integrating, and manipulating networks.Full Text

Bernstein, B. E., Mikkelsen, T. S., Xie, X., Kamal, M., Huebert, D. J., Cuff, J., Fry, B., Meissner, A., Wernig, M., Plath, K., Jaenisch, R., Wagschal, A., Feil, R., Schreiber, S. L. and Lander, E. S. (2006) A bivalent chromatin structure marks key developmental genes in embryonic stem cells. Cell. 125 315-26. The most highly conserved noncoding elements (HCNEs) in mammalian genomes cluster within regions enriched for genes encoding developmentally important transcription factors (TFs). This suggests that HCNE-rich regions may contain key regulatory controls involved in development. We explored this by examining histone methylation in mouse embryonic stem (ES) cells across 56 large HCNE-rich loci. We identified a specific modification pattern, termed "bivalent domains," consisting of large regions of H3 lysine 27 methylation harboring smaller regions of H3 lysine 4 methylation. Bivalent domains tend to coincide with TF genes expressed at low levels. We propose that bivalent domains silence developmental genes in ES cells while keeping them poised for activation. We also found striking correspondences between genome sequence and histone methylation in ES cells, which become notably weaker in differentiated cells. These results highlight the importance of DNA sequence in defining the initial epigenetic landscape and suggest a novel chromatin-based mechanism for maintaining pluripotency.Full Text

Blelloch, R., Wang, Z., Meissner, A., Pollard, S., Smith, A. and Jaenisch, R. (2006) Reprogramming Efficiency following Somatic Cell Nuclear Transfer is Influenced by the Differentiation and Methylation State of the Donor Nucleus. Stem Cells.May 18;. [Epub ahead of print] Reprogramming of a differentiated cell nucleus by somatic cell nuclear transplantation is an inefficient process. Following nuclear transfer, the donor nucleus often fails to express early embryonic genes and establish a normal embryonic pattern of chromatin modifications. These defects correlate with the low number of cloned embryos able to produce embryonic stem cells or develop into adult animals. Here, we show that the differentiation and methylation state of the donor cell influence the efficiency of genomic reprogramming. First, neural stem cells, when used as donors for nuclear transplantation, produce embryonic stem cells at a higher efficiency than blastocysts derived from terminally differentiated neuronal donor cells demonstrating a correlation between the state of differentiation and cloning efficiency. Second, using a hypomorphic allele of DNA methlytransferase-I, we found that global hypomethylation of a differentiated cell genome improved cloning efficiency. Our results provide functional evidence that the differentiation and epigenetic state of the donor nucleus influences reprogramming efficiency.PDF

Boyer, L. A., Mathur, D. and Jaenisch, R. (2006) Molecular control of pluripotency. Curr Opin Genet Dev Aug 18; [Epub ahead of print] . Transcriptional regulators and epigenetic modifiers play crucial roles throughout development to ensure that proper gene expression patterns are established and maintained in any given cell type. Recent genome-wide studies have begun to unravel how genetic and epigenetic factors maintain the undifferentiated state of embryonic stem cells while allowing these cells to remain poised to differentiate into somatic cells in response to developmental cues. These studies provide a conceptual framework for understanding pluripotency and lineage-specification at the molecular level. Full Text

Boyer, L. A., Plath, K., Zeitlinger, J., Brambrink, T., Medeiros, L. A., Lee, T. I., Levine, S. S., Wernig, M., Tajonar, A., Ray, M. K., Bell, G. W., Otte, A. P., Vidal, M., Gifford, D. K., Young, R. A. and Jaenisch, R. (2006) Polycomb complexes repress developmental regulators in murine embryonic stem cells. Nature Apr 19; [Epub ahead of print] . The mechanisms by which embryonic stem (ES) cells self-renew while maintaining the ability to differentiate into virtually all adult cell types are not well understood. Polycomb group (PcG) proteins are transcriptional repressors that help to maintain cellular identity during metazoan development by epigenetic modification of chromatin structure. PcG proteins have essential roles in early embryonic development and have been implicated in ES cell pluripotency, but few of their target genes are known in mammals. Here we show that PcG proteins directly repress a large cohort of developmental regulators in murine ES cells, the expression of which would otherwise promote differentiation. Using genome-wide location analysis in murine ES cells, we found that the Polycomb repressive complexes PRC1 and PRC2 co-occupied 512 genes, many of which encode transcription factors with important roles in development. All of the co-occupied genes contained modified nucleosomes (trimethylated Lys 27 on histone H3). Consistent with a causal role in gene silencing in ES cells, PcG target genes were de-repressed in cells deficient for the PRC2 component Eed, and were preferentially activated on induction of differentiation. Our results indicate that dynamic repression of developmental pathways by Polycomb complexes may be required for maintaining ES cell pluripotency and plasticity during embryonic development.Full Text

Brambrink, T., Hochedlinger, K., Bell, G. and Jaenisch, R. (2006) ES cells derived from cloned and fertilized blastocysts are transcriptionally and functionally indistinguishable. PNAS Jan 24;103(4):933-8.Reproductive cloning is uniformly rejected as a valid technology in humans because of the severely abnormal phenotypes seen in cloned animals. Gene expression aberrations observed in tissues of cloned animals have also raised concerns regarding the therapeutic application of "customized" embryonic stem (ES) cells derived by nuclear transplantation (NT) from a patient's somatic cells. Although previous experiments in mice have demonstrated that the developmental potential of ES cells derived from cloned blastocysts (NT-ES cells) is identical to that of ES cells derived from fertilized blastocysts, a systematic molecular characterization of NT-ES cell lines is lacking. To investigate whether transcriptional aberrations, similar to those observed in tissues of cloned mice, also occur in NT-ES cells, we have compared transcriptional profiles of 10 mouse NT- and fertilization-derived-ES cell lines. We report here that the ES cell lines derived from cloned and fertilized mouse blastocysts are indistinguishable based on their transcriptional profiles, consistent with their normal developmental potential. Our results indicate that, in contrast to embryonic and fetal development of clones, the process of NT-ES cell derivation rigorously selects for those immortal cells that have erased the "epigenetic memory" of the donor nucleus and, thus, become functionally equivalent. Our findings support the notion that ES cell lines derived from cloned or fertilized blastocysts have an identical therapeutic potential PDF

Brinkmann, M. M. and Schulz, T. F. (2006) Regulation of intracellular signalling by the terminal membrane proteins of members of the Gammaherpesvirinae. Journal of General Virology. 87 1047-1074. The human gamma(1)-herpesvirus Epstein-Barr virus (EBV) and the gamma(2)-herpesviruses Kaposi's sarcoma-associated herpesvirus (KSHV), rhesus rhadinovirus (RRV), herpesvirus saimiri (HVS) and herpesvirus ateles (HVA) all contain genes located adjacent to the terminal-repeat region of their genomes, encoding membrane proteins involved in signal transduction. Designated 'terminal membrane proteins' (TMPs) because of their localization in the viral genome, they interact with a variety of cellular signalling molecules, such as non-receptor protein tyrosine kinases, tumour-necrosis factor receptor-associated factors, Ras and Janus kinase (JAK), thereby initiating further downstream signalling cascades, such as the MAPK, PI3K/Akt, NF-kappa B and JAK/STAT pathways. In the case of TMPs expressed during latent persistence of EBV and HVS (LMP1, LMP2A, Stp and Tip), their modulation of intracellular signalling pathways has been linked to the provision of survival signals to latently infected cells and, hence, a contribution to occasional cellular transformation. In contrast, activation of similar pathways by TMPs of KSHV (K1 and K15) and RRV (R1), expressed during lytic replication, may extend the lifespan of virus-producing cells, alter their migration and/or modulate antiviral immune responses. Whether R1 and K1 contribute to the oncogenic properties of KSHV and RRV has not been established satisfactorily, despite their transforming qualities in experimental settings.Full Text

Brummelkamp, T., Fabius, A., Mullenders, J., Madiredjo, M., Velds, A., Kerkhoven, R., Bernards, R. and Beijersbergen, R. (2006) An shRNA barcode screen provides insight into cancer cell vulnerability to MDM2 inhibitors. Nat Chem Biol. 2006 Feb 13; [Epub ahead of print] . The identification of the cellular targets of small molecules with anticancer activity is crucial to their further development as drug candidates. Here, we present the application of a large-scale RNA interference-based short hairpin RNA (shRNA) barcode screen to gain insight in the mechanism of action of nutlin-3 (1). Nutlin-3 is a small-molecule inhibitor of MDM2, which can activate the p53 pathway. Nutlin-3 shows strong antitumor effects in mice, with surprisingly few side effects on normal tissues 1 . Aside from p53, we here identify 53BP1 as a critical mediator of nutlin-3-induced cytotoxicity. 53BP1 is part of a signaling network induced by DNA damage that is frequently activated in cancer but not in healthy tissues 2. Our results suggest that nutlin-3's tumor specificity may result from its ability to turn a cancer cell-specific property (activated DNA damage signaling 3) into a weakness that can be exploited therapeutically.Full Text

Burdick, M. M., Chu, J. T., Godar, S. and Sackstein, R. (2006) HCELL is the major E- and L-selectin ligand expressed on LS174T colon carcinoma cells. Journal of Biological Chemistry. 281 13899-13905. Engagement of vascular E-selectin and leukocyte L-selectin with relevant counter-receptors expressed on tumor cells contributes to the hematogenous spread of colon carcinoma. We recently demonstrated that the LS174T colon carcinoma cell line expresses the CD44 glycoform known as hematopoietic cell E-/L-selectin ligand (HCELL), which functions as a high affinity E- and L-selectin ligand on these cells. To define the contribution of HCELL to selectin-mediated adhesion on intact tumor cells, we measured the binding of LS174T cells transduced with CD44 short interfering RNA (siRNA) or with vector alone to 6-h interleukin-1 beta-stimulated human umbilical vein endothelial cells (HUVEC) and to human peripheral blood mononuclear cells (PBMC) under physiological flow conditions. LS174T cell attachment to HUVEC was entirely E-selectin-dependent, and PBMC tethering to tumor cell monolayers was completely L-selectin-dependent. At physiological shear stress, CD44 siRNA transduction led to an similar to 50% decrease in the number of LS174T cells binding to stimulated HUVEC relative to vector alone-transduced cells. CD44 siRNA-transduced cells also rolled significantly faster than vector-transduced cells on HUVEC, indicating prominent HCELL participation in stabilizing tumor cell-endothelial adhesive interactions against fluid shear. Furthermore, HCELL was identified as the principal L-selectin ligand on LS174T cells, as PBMC binding to CD44 siRNA-transduced tumor cells was reduced similar to 80% relative to vector-transduced cells. These data indicate that expression of HCELL confers robust and predominant tumor cell binding to E- and L-selectin, highlighting a central role for HCELL in promoting shear-resistant adhesive interactions essential for hematogenous cancer dissemination. Full Text

Camargo, F. D. and Goodell, M. A. (2006) The Hoechst low-fluorescent profile of the side population: clonogenicity versus dye retention - Response. Blood. 108 1774-1775. Full Text

Camargo, F. D., Chambers, S. M., Drew, E., McNagny, K. M. and Goodell, M. A. (2006) Hematopoietic stem cells do not engraft with absolute efficiencies. Blood. 107 501-7. Hematopoietic stem cells (HSCs) can be isolated from murine bone marrow by their ability to efflux the Hoechst 33342 dye. This method defines an extremely small and hematopoietically potent subset of cells known as the side population (SP). Recent studies suggest that transplanted single SP cells are capable of lymphohematopoietic repopulation at near absolute efficiencies. Here, we carefully reevaluate the hematopoietic potential of individual SP cells and find substantially lower rates of reconstitution. Our strategy involved the cotransplantation of single SP cells along with different populations of competitor cells that varied in their self-renewal capacity. Even with minimized HSC competition, SP cells were only able to reconstitute up to 35% of recipient mice. Furthermore, through immunophenotyping and clonal in vitro assays we find that SP cells are virtually homogeneous. Isolation of HSCs on the basis of Hoechst exclusion and a single cell-surface marker allows enrichment levels similar to that obtained with complex multicolor strategies. Altogether, our results indicate that even an extremely homogeneous HSC population, based on phenotype and dye efflux, cannot reconstitute mice at absolute efficiencies.Full Text

Cao, Y., Kumar, R. M., Penn, B. H., Berkes, C. A., Kooperberg, C., Boyer, L. A., Young, R. A. and Tapscott, S. J. (2006) Global and gene-specific analyses show distinct roles for Myod and Myog at a common set of promoters. Embo Journal. 25 502-511. We used a combination of genome-wide and promoter-specific DNA binding and expression analyses to assess the functional roles of Myod and Myog in regulating the program of skeletal muscle gene expression. Our findings indicate that Myod and Myog have distinct regulatory roles at a similar set of target genes. At genes expressed throughout the program of myogenic differentiation, Myod can bind and recruit histone acetyltransferases. At early targets, Myod is sufficient for near full expression, whereas, at late expressed genes, Myod initiates regional histone modification but is not sufficient for gene expression. At these late genes, Myog does not bind efficiently without Myod; however, transcriptional activation requires the combined activity of Myod and Myog. Therefore, the role of Myog in mediating terminal differentiation is, in part, to enhance expression of a subset of genes previously initiated by Myod. Full Text

Carpenter, A. E., Jones, T. R., Lamprecht, M. R., Clarke, C., Kang, I. H., Friman, O., Guertin, D. A., Chang, J. H., Lindquist, R. A., Moffat, J., Golland, P. and Sabatini, D. M. (2006) CellProfiler: image analysis software for identifying and quantifying cell phenotypes. Genome Biol. 7 R100. Biologists can now prepare and image thousands of samples per day using automation, enabling chemical screens and functional genomics (for example, using RNA interference). Here we describe the first free, open-source system designed for flexible, high-throughput cell image analysis, CellProfiler. CellProfiler can address a variety of biological questions quantitatively, including standard assays (for example, cell count, size, per-cell protein levels) and complex morphological assays (for example, cell/organelle shape or subcellular patterns of DNA or protein staining) Full Text

Chang, Q., Khare, G., Dani, V., Nelson, S. and Jaenisch, R. (2006) The disease progression of mecp2 mutant mice is affected by the level of BDNF expression. Neuron. 49 341-8. Mutations in the MECP2 gene cause Rett syndrome (RTT). Bdnf is a MeCP2 target gene; however, its role in RTT pathogenesis is unknown. We examined Bdnf conditional mutant mice for RTT-relevant pathologies and observed that loss of BDNF caused smaller brain size, smaller CA2 neurons, smaller glomerulus size, and a characteristic hindlimb-clasping phenotype. BDNF protein level was reduced in Mecp2 mutant mice, and deletion of Bdnf in Mecp2 mutants caused an earlier onset of RTT-like symptoms. To assess whether this interaction was functional and potentially therapeutically relevant, we increased BDNF expression in the Mecp2 mutant brain with a conditional Bdnf transgene. BDNF overexpression extended the lifespan, rescued a locomotor defect, and reversed an electrophysiological deficit observed in Mecp2 mutants. Our results provide in vivo evidence for a functional interaction between Mecp2 and Bdnf and demonstrate the physiological significance of altered BDNF expression/signaling in RTT disease progression.Full Text

Chappell, C., Beard, C., Altman, J., Jaenisch, R. and Jacob, J. (2006) DNA methylation by DNA methyltransferase 1 is critical for effector CD8 T cell expansion. Journal of Immunology. 176 4562-4572. Transcriptional silencing mediated by DNA methylation is a critical component of epigenetic regulation during early embryonic development in animals. However, the requirement for DNA methylation during activation and differentiation of mature CD8(+) T cells into effector and memory cells is not clear. Using cre-mediated deletion of DNA methyltransferase 1 (Dnmt1)at the time of CD8(+) T cell activation, we investigated the obligation for maintaining patterns of DNA methylation during the generation of Ag-specific effector and memory CD8(+) T cells in response to acute viral infection of mice with lymphocytic choriomeningitis virus. Dnmt1(-/-) CD8(+) T cells failed to undergo the massive CD8(+) T cell expansion characteristic of lymphocytic choriomeningitis virus infection, leading to > 80% reductions in Ag-specific effector CD8(+) T cells at the height of the response. Despite this, Dnmt1(-/-)CD8(+) T cells efficiently controlled the viral infection. Interestingly, the number of Ag-specific Dnmt1(-/-) memory CD8(+) T cells was moderately reduced compared with the reductions seen at day 8 postinfection. Our data suggest that ablation of Dnmt1 and subsequent DNA methylation affect the finite proliferative potential of Ag-specific CD8(+) T cells with moderate effects on their differentiation to effector and memory CD8(+) T cells. Full Text

Chen, D., Steele, A., Lindquist, S. and Guarente, L. (2006) Smaller, hungrier mice - Response. Science. 311 1553-1554. Full Text

Chen, H. and Fink, G. R. (2006) Feedback control of morphogenesis in fungi by aromatic alcohols. Genes Dev Apr 17; [Epub ahead of print] Many fungi undergo a developmental transition from a unicellular yeast form to an invasive filamentous form in response to environmental cues. Here we describe a quorum signaling pathway that links environmental sensing to morphogenesis in Saccharomyces cerevisiae. Saccharomyces cells secrete aromatic alcohols that stimulate morphogenesis by inducing the expression of FLO11 through a Tpk2p-dependent mechanism. Mutants defective in synthesis of these alcohols show reduced filamentous growth, which is partially suppressed by the addition of these aromatic alcohols. The production of these autosignaling alcohols is regulated by nitrogen: High ammonia restricts it by repressing the expression of their biosynthetic pathway, whereas nitrogen-poor conditions activate it. Moreover, the production of these aromatic alcohols is controlled by cell density and subjected to positive feedback regulation, which requires the transcription factor Aro80p. These interactions define a quorum-sensing circuit that allows Saccharomyces to respond to both cell density and the nutritional state of the environment. These same autoregulatory molecules do not evoke the morphological switch in Candida albicans, suggesting that these molecular signals are species-specific.PDF

Chu, F, Nusinow, D. A., Chalkely, R. J., Plath, K., Panning, B. and Burlingame, A. L. (2006) Mapping post-translational modifications of the histone variant macroH2A1 using tandem mass spectrometry. Molecular & Cellular Proteomics. 5 194-203. Post-translational histone modifications modulate chromatin-templated processes and therefore affect cellular proliferation, growth, and development. Although posttranslational modifications on the core histones have been under intense investigation for several years, the modifications on variant histones are poorly understood. We used tandem mass spectrometry to identify covalent modifications on a histone H2A variant, macroH2A1.2. MacroH2A1.2 can be monoubiquitinated; however, the site of monoubiquitination has not been documented. In this study we used green fluorescent protein-tagged macroH2A1.2 to determine that Lys(115) is a site of ubiquitination. In addition, we found that this variant H2A is methylated on the epsilon amino group of lysine residues Lys(17), Lys(122), and Lys(238) and phosphorylated on Thr(128). Three of these modifications were also found to be present in the endogenous protein by mass spectrometric analysis. These results provide the first direct evidence that multiple post-translational modifications are imposed on macroH2A1.2, suggesting that, like canonical H2A, this variant H2A is subject to regulation by combinatorial use of covalent modifications.Full Text

Clarke, A. and Orr-Weaver, T. L. (2006) Sister Chromatid Cohesion at the Centromere: Confrontation between Kinases and Phosphatases? Dev Cell. 10 544-7. Accurate chromosome segregation in mitosis and meiosis requires that the cohesin complex be protected at the centromere by the Shugoshin/MEI-S332 protein family. Recent studies show that Sgo directly binds the phosphatase PP2A, tethering it to the centromere where it can protect cohesin subunits from phosphorylation, and that localization of Sgo/MEI-S332 itself is regulated by phosphorylation. Full Text

Cooper, A. A., Gitler, A. D., Cashikar, A., Haynes, C. M., Hill, K. J., Bhullar, B., Liu, K., Xu, K., Strathearn, K. E., Liu, F., Cao, S., Caldwell, K. A., Caldwell, G. A., Marsischky, G., Kolodner, R. D., Labaer, J., Rochet, J. C., Bonini, N. M. and Lindquist, S. (2006) {alpha}-Synuclein Blocks ER-Golgi Traffic and Rab1 Rescues Neuron Loss in Parkinson's Models. Science.Published online 22 June.Alpha-synuclein (alphaSyn) misfolding is associated with several devastating neurodegenerative disorders including Parkinson's Disease (PD). In yeast cells and in neurons alphaSyn accumulation is cytotoxic, but little is known about its normal function or pathobiology. The earliest defect following alphaSyn expression in yeast was a block in endoplasmic reticulum (ER) to Golgi vesicular trafficking. In a genome-wide screen, the largest class of toxicity modifiers were proteins functioning at this same step, including the Rab GTPase Ypt1p, which associated with cytoplasmic alphaSyn inclusions. Elevated expression of Rab1, the mammalian YPT1 homolog, protected against alphaSyn-induced dopaminergic neuron loss in animal models of PD. Thus synucleinopathies may result from disruptions in basic cellular functions that interface with the unique biology of particular neurons to make them especially vulnerable.PDF

Cowen, L. E., Carpenter, A. E., Matangkasombut, O., Fink, G. R. and Lindquist, S. (2006) Genetic Architecture of Hsp90-Dependent Drug Resistance. Eukaryot Cell. Oct 20; [Epub ahead of print] Hsp90 potentiates the evolution of azole resistance in the model yeast Saccharomyces cerevisiae and the opportunistic pathogen Candida albicans via calcineurin. Here, we explored effectors downstream of calcineurin regulating this Hsp90-dependent trait. Using S. cerevisiae erg3 mutants as a model, we determined that both Crz1 and Hph1 modulate azole resistance.PDF

Cullinan, S. B. and Whitesell, L. (2006) Heat shock protein 90: a unique chemotherapeutic target. Semin Oncol. 33 457-65. A large body of work spanning the past decade has identified the molecular chaperone heat shock protein 90 (Hsp90) as a critical modulator of an extensive network of cellular signaling pathways. Many of the processes overseen by Hsp90 are deregulated in tumor cells, including cell cycle control, gene transcription, and apoptotic signaling. Hsp90 inhibition offers the potential of accomplishing what most molecularly targeted anticancer therapies do not-the simultaneous disruption of multiple signaling events critical to tumor cell growth and survival. Indeed, small molecule inhibitors of Hsp90 function are actively being evaluated in the clinic as anticancer agents. In this review, we highlight the current understanding of Hsp90 biology as it relates to cancer and discuss the discovery, development, and clinical status of Hsp90 inhibitors as anticancer drugs.

DiBacco, A., Ouyang, J., Lee, H. Y., Catic, A., Ploegh, H. and Gill, G. (2006) The SUMO-specific protease SENP5 is required for cell division. Molecular and Cellular Biology. 26 4489-4498. Posttranslational modification of substrates by the small ubiquitin-like modifier, SUMO, regulates diverse biological processes, including transcription, DNA repair, nucleocytoplasmic trafficking, and chromosome segregation. SUMOylation is reversible, and several mammalian homollogs of the yeast SUMO-specific protease Ulp1, termed SENPs, have been identified. We demonstrate here that SENP5, a previously uncharacterized Ulp1 homolog, has SUMO C-terminal hydrollase and SUMO isopeptidase activities. In contrast to other SENPs, the C-terminall catalytic domain of SENP5 preferentially processed SUMO-3 compared to SUMO-1 precursors and preferentially removed SUMO-2 and SUMO-3 from SUNIO-modified RanGAP1 in vitro. In cotransfection assays, SENP5 preferentially reduced high-molecular-weight conjugates of SUNIO-2 compared to SUMO-1 in vivo. Full-length SENP5 localized to the nucleolus. Deletion of the noncatalytic N-terminal domain led to loss of nucleolar localization and increased de-SUMOylation activity in vivo. Knockdown of SENP5 by RNA interference resulted in increased levels of SUMO-1 and SUMO-2/3 conjugates, inhibition of cell proliferation, defects in nuclear morphology, and appearance of binucleate cells, revealing an essential role for SENP5 in mitosis and/or cytokinesis. These findings establish SENP5 as a SUMO-specific protease required for cell division and suggest that mechanisms involving both the catalytic and noncatalytic domains determine the distinct substrate specificities of the mammalian SUMO-specific proteases. Full Text

Dickinson, A. J. and Sive, H. (2006) Development of the primary mouth in Xenopus laevis. Dev Biol.Apr 6; [Epub ahead of print] The initial opening between the gut and the outside of the deuterostome embryo breaks through at the extreme anterior. This region is unique in that ectoderm and endoderm are directly juxtaposed, without intervening mesoderm. This opening has been called the stomodeum, buccopharyngeal membrane or oral cavity at various stages of its formation, however, in order to clarify its function, we have termed this the "primary mouth". In vertebrates, the neural crest grows around the primary mouth to form the face and a "secondary mouth" forms. The primary mouth then becomes the pharyngeal opening. In order to establish a molecular understanding of primary mouth formation, we have begun to examine this process during Xenopus laevis development. An early step during this process occurs at tailbud and involves dissolution of the basement membrane between the ectoderm and endoderm. This is followed by ectodermal invagination to create the stomodeum. A subsequent step involves localized cell death in the ectoderm, which may lead to ectodermal thinning. Subsequently, ectoderm and endoderm apparently intercalate to generate one to two cell layers. The final step is perforation, where (after hatching) the primary mouth opens. Fate mapping has defined the ectodermal and endodermal regions that will form the primary mouth. Extirpations and transplants of these and adjacent regions indicate that, at tailbud, the oral ectoderm is not specifically required for primary mouth formation. In contrast, underlying endoderm and surrounding regions are crucial, presumably sources of necessary signals. This study indicates the complexity of primary mouth formation, and lays the groundwork for future molecular analyses of this important structure. Full Text

Doroquez, D. B. and Rebay, I. (2006) Signal Integration During Development: Mechanisms of EGFR and Notch Pathway Function and Cross-Talk. Crit Rev Biochem Mol Biol. 41 339-385. Metazoan development relies on a highly regulated network of interactions between conserved signal transduction pathways to coordinate all aspects of cell fate specification, differentiation, and growth. In this review, we discuss the intricate interplay between the epidermal growth factor receptor (EGFR; Drosophila EGFR/DER) and the Notch signaling pathways as a paradigm for signal integration during development. First, we describe the current state of understanding of the molecular architecture of the EGFR and Notch signaling pathways that has resulted from synergistic studies in vertebrate, invertebrate, and cultured cell model systems. Then, focusing specifically on the Drosophila eye, we discuss how cooperative, sequential, and antagonistic relationships between these pathways mediate the spatially and temporally regulated processes that generate this sensory organ. The common themes underlying the coordination of the EGFR and Notch pathways appear to be broadly conserved and should, therefore, be directly applicable to elucidating mechanisms of information integration and signaling specificity in vertebrate systems.Full Text

Duennwald, M. L., Jagadish, S., Muchowski, P. J. and Lindquist, S. (2006) Flanking sequences profoundly alter polyglutamine toxicity in yeast. Proc Natl Acad Sci U S A. 103 11045-50. Protein misfolding is the molecular basis for several human diseases. How the primary amino acid sequence triggers misfolding and determines the benign or toxic character of the misfolded protein remains largely obscure. Among proteins that misfold, polyglutamine (polyQ) expansion proteins provide an interesting case: Each causes a distinct neurodegenerative disease that selectively affects different neurons. However, all are broadly expressed and most become toxic when the glutamine expansion exceeds approximately 39 glutamine residues. The disease-causing polyQ expansion proteins differ profoundly in the amino acids flanking the polyQ region. We therefore hypothesized that these flanking sequences influence the specific toxic character of each polyQ expansion protein. Using a yeast model, we find that sequences flanking the polyQ region of human huntingtin exon I can convert a benign protein to a toxic species and vice versa. Further, we observe that flanking sequences can direct polyQ misfolding to at least two morphologically distinct types of polyQ aggregates. Very tight aggregates always are benign, whereas amorphous aggregates can be toxic. We thereby establish a previously undescribed systematic characterization of the influence of flanking amino acid sequences on polyQ toxicity.Full Text

Duennwald, M. L., Jagadish, S., Giorgini, F., Muchowski, P. J. and Lindquist, S. (2006) A network of protein interactions determines polyglutamine toxicity. Proc Natl Acad Sci Jul 18;103(29):. Several neurodegenerative diseases are associated with the toxicity of misfolded proteins. This toxicity must arise from a combination of the amino acid sequences of the misfolded proteins and their interactions with other factors in their environment. A particularly compelling example of how profoundly these intramolecular and intermolecular factors can modulate the toxicity of a misfolded protein is provided by the polyglutamine (polyQ) diseases. All of these disorders are caused by glutamine expansions in proteins that are broadly expressed, yet the nature of the proteins that harbor the glutamine expansions and the particular pathologies they produce are very different. We find, using a yeast model, that amino acid sequences that modulate polyQ toxicity in cis can also do so in trans. Furthermore, the prion conformation of the yeast protein Rnq1 and the level of expression of a suite of other glutamine-rich proteins profoundly affect polyQ toxicity. They can convert polyQ expansion proteins from toxic to benign and vice versa. Our work presents a paradigm for how a complex, dynamic interplay between intramolecular features of polyQ proteins and intermolecular factors in the cellular environment might determine the unique pathobiologies of polyQ expansion proteins.Full Text

Ehrnhoefer, D. E., Duennwald, M., Markovic, P., Wacker, J. L., Engemann, S., Roark, M., Legleiter, J., Marsh, J. L., Thompson, L. M., Lindquist, S., Muchowski, P. J. and Wanker, E. E. (2006) Green tea (-)-epigallocatechin-gallate modulates early events in huntingtin misfolding and reduces toxicity in Huntington's disease models. Human Molecular Genetics. 15 2743-2751. Huntington's disease (HD) is a progressive neurodegenerative disorder for which only symptomatic treatments of limited effectiveness are available. Preventing early misfolding steps and thereby aggregation of the polyglutamine (polyQ)-containing protein huntingtin (htt) in neurons of patients may represent an attractive therapeutic strategy to postpone the onset and progression of HD. Here, we demonstrate that the green tea polyphenol (-)-epigallocatechin-3-gallate (EGCG) potently inhibits the aggregation of mutant htt exon 1 protein in a dose-dependent manner. Dot-blot assays and atomic force microscopy studies revealed that EGCG modulates misfolding and oligomerization of mutant htt exon 1 protein in vitro, indicating that it interferes with very early events in the aggregation process. Also, EGCG significantly reduced polyQ-mediated htt protein aggregation and cytotoxicity in an yeast model of HD. When EGCG was fed to transgenic HD flies overexpressing a pathogenic htt exon 1 protein, photoreceptor degeneration and motor function improved. These results indicate that modulators of htt exon 1 misfolding and oligomerization like EGCG are likely to reduce polyQ-mediated toxicity in vivo. Our studies may provide the basis for the development of a novel pharmacotherapy for HD and related polyQ disorders.Full Text

ElDifrawy, S. A., Srivastava, A., Gismondi, E. A., McKenna, B. K. and Ehrlich, D. J. (2006) Numerical model for DNA loading in microdevices: Stacking and autogating effects. Electrophoresis.Sep 7; [Epub ahead of print] Many electrophoresis-based DNA sequencing and genotyping microdevices rely on field-driven effects to load and preconcentrate the sample. A quantitative model is developed for a broad class of electrophoresis-based microfabricated sample injectors. Quantitative predictions of DNA preconcentration are compared with experimental data and are shown to qualitatively reproduce the detailed time-evolving sample distribution in the injector. The model provides practical guidance on device and protocol design, in order to optimize this critical aspect of microfluidic devices. PDF

Elliott, S., Busse, L., Bass, M. B., Lu, H., Sarosi, I., Sinclair, A. M., Spahr, C., Um, M., Van, G. and Begley, C. G. (2006) Anti-Epo receptor antibodies do not predict Epo receptor expression. Blood. 107 1892-1895. Investigators using anti-EpoR antibodies for immunoblotting and immunostaining have reported erythropoietin receptor (EpoR) expression in nonhematopoietic tissues including human tumors. However, these antibodies detected proteins of 66 to 78 kDa, significantly larger than the predicted molecular weight of EpoR (56-57 kDa). We investigated the specificity of these antibodies and showed that they all detected non-EpoR proteins. C-20 detected 3 proteins in tumor cell lines (35, 66, and 100 kDa). Sequences obtained from preparative gels had similarity to the C-20-immunizing peptide. The 66-kDa protein was a heat shock protein (HSP70) to which antibody binding was abrogated in peptide competition experiments. Antibody M-20 readily identified a 59-kDa EpoR protein. However, neither M-20 nor C-20 was suitable for detection of EpoR using immunohistochemical methods. We concluded that these antibodies have limited utility for detecting EpoR. Thus, reports of EpoR expression in tumor cells using these antibodies should be viewed with caution. Full Text

Evans, J. G. and Matsudaira, P. (2006) Structure and dynamics of macrophage podosomes. Eur J Cell Biol. 85 145-9. Podosomes are short-lived, dynamic actin-rich adhesions classically formed in macrophages, osteoclasts and Src-transformed fibroblasts. Though related to the more commonly studied focal adhesions, sharing several structural and regulatory proteins, podosomes have distinct functional, structural, and dynamic characteristics. Here, we discuss current understanding of the function of podosomes in disparate cell types and how this relates to their structure and dynamics.Full Text

Frias, M. A., Thoreen, C. C., Jaffe, J. D., Schroder, W., Sculley, T., Carr, S. A. and Sabatini, D. M. (2006) mSin1 Is Necessary for Akt/PKB Phosphorylation, and Its Isoforms Define Three Distinct mTORC2s. Curr Biol. Aug 15; [Epub ahead of print] The mammalian target of rapamycin (mTOR) is a serine/threonine kinase that participates in at least two distinct multiprotein complexes, mTORC1 and mTORC2 . These complexes play important roles in the regulation of cell growth, proliferation, survival, and metabolism. mTORC2 is a hydrophobic motif kinase for the cell-survival protein Akt/PKB and, here, we identify mSin1 as a component of mTORC2 but not mTORC1. mSin1 is necessary for the assembly of mTORC2 and for its capacity to phosphorylate Akt/PKB. Alternative splicing generates at least five isoforms of the mSin1 protein , three of which assemble into mTORC2 to generate three distinct mTORC2s. Even though all mTORC2s can phosphorylate Akt/PKB in vitro, insulin regulates the activity of only two of them. Thus, we propose that cells contain several mTORC2 flavors that may phosphorylate Akt/PKB in response to different signals.Full Text

Ge, H., Player, C. M. and Zou, L. (2006) Toward a global picture of development: Lessons from genome-scale analysis in Caenorhabditis elegans embryonic development. Developmental Dynamics Early View June 15. Development is the result of complex events, including cascades of transcriptional programs and numerous molecular interactions. Traditionally, research focus has been given to the characterization of individual mutants, regulators, or interactions. With the availability of complete genome sequences and high-throughput (HT) experimental techniques, probing development on a system level has become feasible. Pioneering work initiated in invertebrate model systems such as Caenorhabditis elegans has provided first drafts of catalogs of essential components, transcriptional regulatory diagrams and molecular interaction networks underlying developmental processes. Integrating these drafts approximates a system-level picture of development and provides local models for protein/gene functions. Here we summarize the progress toward elucidating developmental processes on a system level, including the applications of genomic technologies and computational analyses. We discuss C. elegans embryonic development in case studies to illustrate how various HT approaches can be integrated and how biological insights can be gained from these approaches.Full Text

Gemelli, T., Berton, O., Nelson, E. D., Perrotti, L. I., Jaenisch, R. and Monteggia, L. M. (2006) Postnatal loss of methyl-CpG binding protein 2 in the Forebrain is sufficient to mediate behavioral aspects of Rett syndrome in mice. Biological Psychiatry. 59 468-476. Mutations in the methyl-CpG binding protein 2 (MeCP2) gene cause Rett syndrome (RTT), a neurodevelopmental disorder that is accompanied by a broad array of behavioral phenotypes, mainly affecting females. Methyl-CpG binding protein 2 is a transcriptional repressor that is widely expressed in all tissues. To investigate whether the postnatal loss of MeCP2 in the forebrain is sufficient to produce the behavioral phenotypes observed in RTT, we have generated conditional MeCP2 knockout mice. These mice display behavioral abnormailities similar to RTT phenotypes, including hindlimb clasping, impaired motor coordination, increased anxiety, and abnormal social behavior with other mice. These mice, however, have normal locomotor activity and unimpaired context-dependent fear conditioning, suggesting that the behavioral deficits observed are the result of loss of MeCP2 function in postnatal forebrain and not the result of generalized global deficits. These data highlight the important role of MeCP2 in the forebrain and suggest that even partial loss of MeCP2 expression in these brain regions is sufficient to recapitulate features of RTT. Full Text

Ghaffari, S., Kitidis, C., Zhao, W., Marinkovic, D., Fleming, M. D., Luo, B., Marszalek, J. and Lodish, H. F. (2006) AKT induces erythroid-cell maturation of JAK2-deficient fetal liver progenitor cells and is required for Epo regulation of erythroid-cell differentiation. Blood. 107 1888-1891. AKT serine threonine kinase of the protein kinase B (PKB) family plays essential roles in cell survival, growth, metabolism, and differentiation. In the erythroid system, AKT is known to be rapidly phosphorylated and activated in response to erythropoletin (Epo) engagement of Epo receptor (EpoR) and to sustain survival signals in cultured erythroid cells. Here we demonstrate that activated AKT complements EpoR signaling and supports erythroid-cell differentiation in wild-type and JAK2-deficient fetal liver cells. We show that erythroid maturation of AKT-transduced cells is not solely dependent on AKT-induced cell survival or proliferation signals, suggesting that AKT transduces also a differentiation-specific signal downstream of EpoR in erythroid cells. Downregulation of expression of AKT kinase by RNA interference, or AKT activity by expression of dominant negative forms, inhibits significantly fetal liver-derived erythroid-cell colony formation and gene expression, demonstrating that AKT is required for Epo regulation of erythroid-cell maturation. Full Text

Goedecke, N., McKenna, B., El-Difrawy, S., Gismondi, E., Swenson, A., Carey, L., Matsudaira, P. and Ehrlich, D. J. (2006) Microdevice DNA forensics by the simple tandem repeat method. J Chromatogr A. 1111 206-13. We review recent experiments on DNA forensics by the simple tandem repeat (STR) method using a 16-lane micromachined device as the active separation element. Separations by linear polyacrylamide matrices show very high data quality metrics when evaluated with statistically significant data sets. Full 16-locus multiplexes are verified on the multilane system. Multi-donor mixed samples are studied in the context of the limits of the laser-induced fluorescence detector and data-reduction software. The microdevice appears to be posed to outperform current capillary arrays in terms of stability and, through specialized sample loading, in the interpretation of complex mixtures.Full Text

Gross, A. W. and Lodish, H. F. (2006) Cellular trafficking and degradation of erythropoietin and novel erythropoiesis stimulating protein (NESP). Journal of Biological Chemistry. 281 2024-2032. Erythropoietin (Epo) is essential for the production of mature red blood cells, and recombinant Epo is commonly used to treat anemia, but how Epo is degraded and cleared from the body is not understood. Glycosylation of Epo is required for its in vivo bioactivity, although not for invitro receptor binding or stimulation of Epo-dependent celllines; Epoglycosylation actually reduces the affinity of Epo for the Epo receptor (EpoR). Interestingly, a hyperglycosylated analog of Epo, called novel erythropoiesis-stimulating protein (NESP), has a lower affinity than Epo for the EpoR but has greater in vivo activity and a longer serum half-life than Epo. We hypothesize that a major mechanism for degradation of Epo in the body occurs in cells expressing the Eporeceptor, through receptor-mediated endocytosis of Epo followed by degradation in lysosomes, and therefore investigated the trafficking and degradation of Epo and NESP by EpoR-expressing cells. We show that Epo and NESP are degraded only by cultured cells that express the EpoR, and their receptor binding, dissociation, and trafficking properties determine their rates of intracellular degradation. Epo binds surface EpoR faster than NESP (k(on) approximate to 5.0 x 10(8) M-1 min(-1) versus 1.1 x 10(8) M-1 min(-1)) but dissociates slower (koff = 0.029 min(-1) versus 0.042 min(-1)). Surface-bound Epo and NESP are internalized at the same rate (k(in) = 0.06 min(-1)), and after internalization 60% of each ligand is resecreted intact and 40% degraded. Our kinetic model of Epo and NESP receptor binding, intracellular trafficking, and degradation explains why Epo is degraded faster than NESP at the cellular level. Full Text.

Guertin, D. A., Stevens, D. M., Thoreen, C. C., Burds, A. A., Kalaany, N. Y., Moffat, J., Brown, M., Fitzgerald, K. J. and Sabatini, D. M. (2006) Ablation in Mice of the mTORC Components raptor, rictor, or mLST8 Reveals that mTORC2 Is Required for Signaling to Akt-FOXO and PKCalpha, but Not S6K1. Dev Cell. 11 859-71. The mTOR kinase controls cell growth, proliferation, and survival through two distinct multiprotein complexes, mTORC1 and mTORC2. mTOR and mLST8 are in both complexes, while raptor and rictor are part of only mTORC1 and mTORC2, respectively. To investigate mTORC1 and mTORC2 function in vivo, we generated mice deficient for raptor, rictor, or mLST8. Like mice null for mTOR, those lacking raptor die early in development. However, mLST8 null embryos survive until e10.5 and resemble embryos missing rictor. mLST8 is necessary to maintain the rictor-mTOR, but not the raptor-mTOR, interaction, and both mLST8 and rictor are required for the hydrophobic motif phosphorylation of Akt/PKB and PKCalpha, but not S6K1. Furthermore, insulin signaling to FOXO3, but not to TSC2 or GSK3beta, requires mLST8 and rictor. Thus, mTORC1 function is essential in early development, mLST8 is required only for mTORC2 signaling, and mTORC2 is a necessary component of the Akt-FOXO and PKCalpha pathways.Full Text

Guertin, D. A., Guntur, K. V., Bell, G. W., Thoreen, C. C. and Sabatini, D. M. (2006) Functional Genomics Identifies TOR-Regulated Genes that Control Growth and Division. Curr Biol. 16 958-70. BACKGROUND: The TOR (target of rapamycin) ser/thr protein kinase is the central component of a eukaryotic signaling pathway that regulates growth and is the direct target of the clinically useful drug rapamycin. Recent efforts have identified at least two multiprotein complexes that contain TOR, but little is known in higher eukaryotes about the genes downstream of TOR that control growth. RESULTS: By combining the use of a small molecule inhibitor (rapamycin), transcriptional profiling, and RNA interference in Drosophila tissue culture cells, we identified genes whose expression responds to Drosophila TOR (dTOR) inhibition and that regulate cell size. Several of the dTOR-regulated genes that function in cell size control have additional roles in cell division. Most of these genes are conserved in mammals and several are linked to human disease. This set of genes is highly enriched for regulators of ribosome biogenesis, which emphasizes the importance of TOR-dependent transcription in building the protein synthesis machinery in higher eukaryotes. In addition, we identify two dTOR-regulated genes, CG3071 and CG6677, whose human orthologs, SAW and ASH2L, are also under TOR-dependent transcriptional control and encode proteins with conserved functional roles in growth. CONCLUSIONS: We conclude that combining RNA interference with genomic analysis approaches, such as transcriptional profiling, is an effective way to identify genes functioning in a particular biological process. Moreover, this strategy, if applied in model systems with simpler genomes, can identify genes with conserved functions in mammals. Full Text

Handwerger, K. E. and Gall, J. G. (2006) Subnuclear organelles: new insights into form and function. Trends in Cell Biology. 16 19-26. The cell nucleus is a complex and highly dynamic environment with many functionally specialized regions of substructure that form and maintain themselves in the absence of membranes. Relatively little is known about the basic physical properties of the nuclear interior or how domains within the nucleus are structurally and functionally organized and interrelated. Here, we summarize recent data that shed light on the structural and functional properties of three prominent subnuclear organelles - nucleoli, Cajal bodies (CBs) and speckles. We discuss how these findings impact our understanding of the guiding principles of nuclear organization and various types of human disease. Full Text

Hang, H. C., Loureiro, J., Spooner, E., van der Velden, A. W., Kim, Y. M., Pollington, A. M., Maehr, R., Starnbach, M. N. and Ploegh, H. L. (2006) Mechanism-based probe for the analysis of cathepsin cysteine proteases in living cells. ACS Chem Biol. 1 713-23. Mechanism-based probes are providing new tools to evaluate the enzymatic activities of protein families in complex mixtures and to assign protein function. The application of these chemical probes for the visualization of protein labeling in cells and proteomic analysis is still challenging. As a consequence, imaging and proteomic analysis often require different sets of chemical probes. Here we describe a mechanism-based probe, azido-E-64, that can be used for both imaging and proteomics. Azido-E-64 covalently modifies active Cathepsin (Cat) B in living cells, an abundant cysteine protease involved in microbial infections, apoptosis, and cancer. Furthermore, azido-E-64 contains an azide chemical handle that can be selectively derivatized with phosphine reagents via the Staudinger ligation, which enables the imaging and proteomic analysis of Cat B. We have utilized azido-E-64 to visualize active Cat B during infection of primary macrophages with Salmonella typhimurium , an facultative intracellular bacterial pathogen. These studies demonstrated that active Cat B is specifically excluded from Salmonella -containing vacuoles, which suggests that inhibition of protease activity within bacteria-containing vacuoles may contribute to bacterial virulence.Full Text

Hartwell, K. A., Muir, B., Reinhardt, F., Carpenter, A. E., Sgroi, D. C. and Weinberg, R. A. (2006) The Spemann organizer gene, Goosecoid, promotes tumor metastasis. Proc Natl Acad Sci U S A. Dec 1; [Epub ahead of print] The process of invasion and metastasis during tumor progression is often reminiscent of cell migration events occurring during embryonic development. We hypothesized that genes controlling cellular changes in the Spemann organizer at gastrulation might be reactivated in tumors. The Goosecoid homeobox transcription factor is a known executer of cell migration from the Spemann organizer. We found that indeed Goosecoid is overexpressed in a majority of human breast tumors. Ectopic expression of Goosecoid in human breast cells generated invasion-associated cellular changes, including an epithelial-mesenchymal transition. TGF-beta signaling, known to promote metastasis, induced Goosecoid expression in human breast cells. Moreover, Goosecoid significantly enhanced the ability of breast cancer cells to form pulmonary metastases in mice. These results demonstrate that Goosecoid promotes tumor cell malignancy and suggest that other conserved organizer genes may function similarly in human cancer.PDF

Hill, A. E., Lander, E. S. and Nadeau, J. H. (2006) Chromosome substitution strains: a new way to study genetically complex traits. Methods Mol Med. 128 153-72. Many biological traits and heritable diseases are multifactorial, involving combinations of genetic variants and environmental factors. To dissect the genetic basis for these traits and to characterize their functional consequences, mouse models are widely used, not only because of their genetic and physiological similarity to humans, but also because an extraordinary variety of genetic resources enable rigorous functional studies. Chromosome substitution strains (CSSs) are a powerful complement to existing resources for studying multigenic traits. By partitioning the genome into a panel of new inbred strains with single chromosome substitutions, one strain for each of the autosomes, the X and Y chromosome, and the mitochondria, unique experimental designs and considerable statistical power are possible. Multigenic trait genes (or quantitative trait loci [QTLs]) with weak effects are easily detected, linkage and congenic crosses can be quickly made, gene interactions are readily characterized, and discovery of QTLs is greatly accelerated. Several published studies demonstrate the considerable utility of these strains and new applications for CSSs continue to be discovered.

Hochedlinger, K. and Jaenisch, R. (2006) Nuclear reprogramming and pluripotency. Nature. 441 1061-1067. The cloning of mammals from differentiated donor cells has refuted the old dogma that development is an irreversible process. It has demonstrated that the oocyte can reprogramme an adult nucleus into an embryonic state that can direct development of a new organism. The prospect of deriving patient-specific embryonic stem cells by nuclear transfer underscores the potential use of this technology in regenerative medicine. The future challenge will be to study alternatives to nuclear transfer in order to recapitulate reprogramming in a Petri dish without the use of oocytes.Full Text

Hongay, C. F., Grisafi, P. L., Galitski, T. and Fink, G. R. (2006) Antisense Transcription Controls Cell Fate in Saccharomyces cerevisiae. Cell. 127 735-745. Entry into meiosis is a key developmental decision. We show here that meiotic entry in Saccharomyces cerevisiae is controlled by antisense-mediated regulation of IME4, a gene required for initiating meiosis. In MAT a/alpha diploids the antisense IME4 transcript is repressed by binding of the a1/alpha2 heterodimer at a conserved site located downstream of the IME4 coding sequence. MAT a/alpha diploids that produce IME4 antisense transcript have diminished sense transcription and fail to initiate meiosis. Haploids that produce the sense transcript have diminished antisense transcription and manifest several diploid phenotypes. Our data are consistent with transcription interference as a regulatory mechanism at the IME4 locus that determines cell fate Full Text

Houstis, N., Rosen, E. D. and Lander, E. S. (2006) Reactive oxygen species have a causal role in multiple forms of insulin resistance. Nature. 440 944-8. Insulin resistance is a cardinal feature of type 2 diabetes and is characteristic of a wide range of other clinical and experimental settings. Little is known about why insulin resistance occurs in so many contexts. Do the various insults that trigger insulin resistance act through a common mechanism? Or, as has been suggested, do they use distinct cellular pathways? Here we report a genomic analysis of two cellular models of insulin resistance, one induced by treatment with the cytokine tumour-necrosis factor-alpha and the other with the glucocorticoid dexamethasone. Gene expression analysis suggests that reactive oxygen species (ROS) levels are increased in both models, and we confirmed this through measures of cellular redox state. ROS have previously been proposed to be involved in insulin resistance, although evidence for a causal role has been scant. We tested this hypothesis in cell culture using six treatments designed to alter ROS levels, including two small molecules and four transgenes; all ameliorated insulin resistance to varying degrees. One of these treatments was tested in obese, insulin-resistant mice and was shown to improve insulin sensitivity and glucose homeostasis. Together, our findings suggest that increased ROS levels are an important trigger for insulin resistance in numerous settings.Full Text

Hughes, J. F., Skaletsky, H., Rozen, S., Wilson, R. K. and Page, D. C. (2006) Has the chimpanzee Y chromosome been sequenced? Nat Genet. 38 853-4.Full Text

Ivanovska, I. and Orr-Weaver, T. L. (2006) Histone Modifications and the Chromatin Scaffold for Meiotic Chromosome Architecture. Cell Cycle.Sep 15;5(18) [Epub ahead of print] 5 Chromosomes are capable of remarkable structural adaptability that enables their diverse functions. Histone modifications play pivotal roles in conferring structural diversity to chromosomes by influencing the compactness of chromatin. Several multi-protein complexes bind to chromatin and affect chromosome dynamics, including cohesin, condensin, the chromosome passenger complex, and the synaptonemal complex. The roles of these complexes in promoting chromosome functions include cohesion, condensation and synapsis. It is now crucial to define the relationship between the protein complexes that affect chromosome architecture and the underlying state of the chromatin. During meiosis chromosomes undergo striking morphological changes, including alignment of homologous chromosomes, double-strand break formation and repair, and establishment of meiosis-specific chromosome structures. These dynamic chromosome arrangements are accompanied by the recruitment and expulsion of multi-protein complexes from chromatin. Meiotic chromosome dynamics ensure proper chromosome segregation and production of healthy gametes. Meiosis thus affords an excellent opportunity to determine how histone modifications impact higher order chromosome dynamics by affecting localization and function of chromosome protein complexes. A meiotic mutation in the Drosophila histone kinase, NHK-1, uncovered a critical requirement for histone modifications in chromosome architecture, underscoring the power of this approach.

Jaenisch, R., Meissner, A. and Solter, D. (2006) Politically correct human embryonic stem cells? N Engl J Med. 354 1208-9. Full Text

JonesRhoades, M. W., Bartel, D. P. and Bartel, B. (2006) MicroRNAs and Their Regulatory Roles in Plants. Annu Rev Plant Biol. 57: 19-53 MicroRNAs (miRNAs) are small, endogenous RNAs that regulate gene expression in plants and animals. In plants, these approximately 21-nucleotide RNAs are processed from stem-loop regions of long primary transcripts by a Dicer-like enzyme and are loaded into silencing complexes, where they generally direct cleavage of complementary mRNAs. Although plant miRNAs have some conserved functions extending beyond development, the importance of miRNA-directed gene regulation during plant development is now particularly clear. Identified in plants less than four years ago, miRNAs are already known to play numerous crucial roles at each major stage of development-typically at the cores of gene regulatory networks, targeting genes that that are themselves regulators, such as those encoding transcription factors and F-box proteins.Full Text

Kalaany, N. Y. and Mangelsdorf, D. J. (2006) LXRs AND FXR: The Yin and Yang of cholesterol and fat metabolism. Annual Review of Physiology. 68 159-191. Liver X receptors (LXRs) and farnesoid X receptor (FXR) are nuclear receptors that function as intracellular sensors for sterols and bile acids, respectively. In response to their ligands, these receptors induce transcriptional responses that maintain a balanced, finely tuned regulation of cholesterol and bile acid metabolism. LXRs also permit the efficient storage of carbohydrate- and fat-derived energy, whereas FXR activation results in an overall decrease in triglyceride levels and modulation of glucose metabolism. The elegant, dual interplay between these two receptor systems Suggests that they coevolved to Constitute a highly sensitive and efficient system for the maintenance of total body fat and cholesterol homeostasis. Emerging evidence suggests that the tissue-specific action of these receptors is also Crucial for the proper function of the cardiovascular, immune, reproductive, endocrine pancreas, renal, and central nervous systems. Together, LXRs and FXR represent potential therapeutic targets for the treatment and prevention of numerous metabolic and lipid-related diseases Full Text

Kassir, Y., Rubin-Bejerano, I. and Mandel-Gutfreund, Y. (2006) The Saccharomyces cerevisiae GSK-3 beta homologs. Current Drug Targets. 7 1455-1465. Yeast cells carry four homologs of GSK-3 beta, RIM11, MCK1, MRK1 and YGK3. The significant homologs are RIM11 and MCK1 that presumably arose from a recent genome duplication followed by a rapid divergence. Accordingly, these homologs phosphorylate specific substrates. Rim I I is essential for entry into meiosis, whereas Mck1 is essential for growth at elevated and low temperatures. Both kinases transmit nutrient signals, but Mck1 transmits additional signals including stress signals such as, temperature, osmotic shock and Ca2+. Consequently, Mck1 plays a role in multiple functions, including cell wall integrity, meiosis and centromere function. The other two homologs, MRK1 and YGK3 that belong to the RIM11 and MCK1 phylogenetic trees, respectively, show no distinct phenotype. These paralogs posses redundant roles, though less important, with Rim11 and Mck1 functions. This review summarizes the cellular roles of these kinases, their mode of regulation, and the signals that they transmit.

Kim, Y. M., Pan, J. Y., Korbel, G. A., Peperzak, V., Boes, M. and Ploegh, H. L. (2006) Monovalent ligation of the B cell receptor induces receptor activation but fails to promote antigen presentation. Proc Natl Acad Sci U S A. Published online before print February 21, 2006, We explored the role of antigen valency in B cell receptor (BCR) activation and rearrangement of intracellular MHC class II compartments as factors that contribute to the efficacy of antigen presentation. Using primary B cells that express a hen egg lysozyme (HEL)-specific BCR, we found that oligomeric HEL more efficiently promoted both BCR activation and internalization than did monovalent HEL, although monovalent HEL, unlike monovalent Fab fragments of anti-Ig, readily triggered the BCR. Nonetheless, oligovalent ligation positions the BCR in a membrane microdomain that is distinct from one engaged in the course of monovalent ligation, as judged by detergent extraction of the BCR. Furthermore, oligovalent HEL induced more pronounced rearrangement of MHC class II-containing antigen-processing compartments. Using videomicroscopy we observed in real time the rearrangement of MHC class II compartments as well as delivery of antigen in primary B cells. The observed increase in rearrangement of MHC class II-positive compartments and the disposition of antigen-bound BCRs therein correlates with improved presentation of a HEL-derived epitope. Although monomeric HEL efficiently engages the BCR, presentation of HEL-derived epitopes is impaired compared to oligovalent antigens. This trait may help explain the known ability of soluble, disaggregated antigen to induce a state of B cell tolerance.Full Text.

Koch, K. S., Son, K. H., Maehr, R., Pellicciotta, I., Ploegh, H. L., Zanetti, M., Sell, S. and Leffert, H. L. (2006) Immune-privileged embryonic Swiss mouse STO and STO cell-derived progenitor cells: major histocompatibility complex and cell differentiation antigen expression patterns resemble those of human embryonic stem cell lines. Immunology. 119 98-115. Embryonic mouse STO (S, SIM; T, 6-thioguanine resistant; O, ouabain resistant) and 3(8)21-enhanced green fluorescent protein (EGFP) cell lines exhibit long-term survival and hepatic progenitor cell behaviour after xenogeneic engraftment in non-immunosuppressed inbred rats, and were previously designated major histocompatibility complex (MHC) class I- and class II-negative lines. To determine the molecular basis for undetectable MHC determinants, the expression and haplotype of H-2K, H-2D, H-2L and I-A proteins were reassessed by reverse transcriptase-polymerase chain reaction (RT-PCR), cDNA sequencing, RNA hybridization, immunoblotting, quantitative RT-PCR (QPCR), immunocytochemistry and flow cytometry. To detect cell differentiation (CD) surface antigens characteristic of stem cells, apoptotic regulation or adaptive immunity that might facilitate progenitor cell status or immune privilege, flow cytometry was also used to screen untreated and cytokine [interferon (IFN)-gamma]-treated cultures. Despite prior PCR genotyping analyses suggestive of H-2(q) haplotypes in STO, 3(8)21-EGFP and parental 3(8)21 cells, all three lines expressed H-2K cDNA sequences identical to those of d-haplotype BALB/c mice, as well as constitutive and cytokine-inducible H-2K(d) determinants. In contrast, apart from H-2L(d[LOW]) display in 3(8)21 cells, H-2D(d), H-2L(d) and I-A(d) determinants were undetectable. All three lines expressed constitutive and cytokine-inducible CD34; however, except for inducible CD117([LOW]) expression in 3(8)21 cells, no expression of CD45, CD117, CD62L, CD80, CD86, CD90.1 or CD95L/CD178 was observed. Constitutive and cytokine-inducible CD95([LOW]) expression was detected in STO and 3(8)21 cells, but not in 3(8)21-EGFP cells. MHC (class I+[LOW]/class II-) and CD (CD34(+)/CD80(-)/CD86(-)/CD95L(-)) expression patterns in STO and STO cell-derived progenitor cells resemble patterns reported for human embryonic stem cell lines. Whether these patterns reflect associations with mechanisms that are regulatory of immune privilege or functional tissue-specific plasticity is unknown.Full Text

Koubova, J., Menke, D. B., Zhou, Q., Capel, B., Griswold, M. D. and Page, D. C. (2006) Retinoic acid regulates sex-specific timing of meiotic initiation in mice. Proc Natl Acad Sci U S A.Feb 6; [Epub ahead of print] This contribution is part of the special series of Inaugural Articles by members of the National Academy of Sciences elected on May 3, 2005. In mammals, meiosis is initiated at different time points in males and females, but the mechanism underlying this difference is unknown. Female germ cells begin meiosis during embryogenesis. In males, embryonic germ cells undergo G0/G1 mitotic cell cycle arrest, and meiosis begins after birth. In mice, the Stimulated by Retinoic Acid Gene 8 (Stra8) has been found to be required for the transition into meiosis in both female and male germ cells. Stra8 is expressed in embryonic ovaries just before meiotic initiation, whereas its expression in testes is first detected after birth. Here we examine the mechanism underlying the sex-specific timing of Stra8 expression and meiotic initiation in mice. Our work shows that signaling by retinoic acid (RA), an active derivative of vitamin A, is required for Stra8 expression and thereby meiotic initiation in embryonic ovaries. We also discovered that RA is sufficient to induce Stra8 expression in embryonic testes and in vitamin A-deficient adult testes in vivo. Finally, our results show that cytochrome p450 (CYP)-mediated RA metabolism prevents premature Stra8 expression in embryonic testes. Treatment with an inhibitor specific to RA-metabolizing enzymes indicates that a cytochrome p450 from the 26 family (CYP26) is responsible for delaying Stra8 expression in embryonic testes. Sex-specific regulation of RA signaling thus plays an essential role in meiotic initiation in embryonic ovaries and precludes its occurrence in embryonic testes. Because RA signaling regulates Stra8 expression in both embryonic ovaries and adult testes, this portion of the meiotic initiation pathway may be identical in both sexes.PDF

Kritzer, J. A. (2006) When undergraduates ask "why," chemical biology answers. ACS Chem Biol. 1 411-3.PDF

Lau, N. C., Seto, A. G., Kim, J., Kuramochi-Miyagawa, S., Nakano, T., Bartel, D. P. and Kingston, R. E. (2006) Characterization of the piRNA complex from rat testes. Science. 313 363-367. Small noncoding RNAs regulate processes essential for cell growth and development, including mRNA degradation, translational repression, and transcriptional gene silencing (TGS). During a search for candidate mammalian factors for TGS, we purified a complex that contains small RNAs and Riwi, the rat homolog to human Piwi. The RNAs, frequently 29 to 30 nucleotides in length, are called Piwi-interacting RNAs (piRNAs), 94% of which map to 100 defined (<= 101 kb) genomic regions. Within these regions, the piRNAs generally distribute across only one genomic strand or distribute on two strands but in a divergent, nonoverlapping manner. Preparations of piRNA complex ( piRC) contain rRecQ1, which is homologous to qde-3 from Neurospora, a gene implicated in silencing pathways. Piwi has been genetically linked to TGS in flies, and slicer activity cofractionates with the purified complex. These results are consistent with a gene-silencing role for piRC in mammals Full Text

Lee, T. I., Jenner, R. G., Boyer, L. A., Guenther, M. G., Levine, S. S., Kumar, R. M., Chevalier, B., Johnstone, S. E., Cole, M. F., Isono, K., Koseki, H., Fuchikami, T., Abe, K., Murray, H. L., Zucker, J. P., Yuan, B., Bell, G. W., Herbolsheimer, E., Hannett, N. M., Sun, K., Odom, D. T., Otte, A. P., Volkert, T. L., Bartel, D. P., Melton, D. A., Gifford, D. K., Jaenisch, R. and Young, R. A. (2006) Control of developmental regulators by polycomb in human embryonic stem cells. Cell. 125 301-13. Polycomb group proteins are essential for early development in metazoans, but their contributions to human development are not well understood. We have mapped the Polycomb Repressive Complex 2 (PRC2) subunit SUZ12 across the entire nonrepeat portion of the genome in human embryonic stem (ES) cells. We found that SUZ12 is distributed across large portions of over two hundred genes encoding key developmental regulators. These genes are occupied by nucleosomes trimethylated at histone H3K27, are transcriptionally repressed, and contain some of the most highly conserved noncoding elements in the genome. We found that PRC2 target genes are preferentially activated during ES cell differentiation and that the ES cell regulators OCT4, SOX2, and NANOG cooccupy a significant subset of these genes. These results indicate that PRC2 occupies a special set of developmental genes in ES cells that must be repressed to maintain pluripotency and that are poised for activation during ES cell differentiation. Full Text

Lin, H., Yamada, Y., Nguyen, S., Linhart, H., Jackson-Grusby, L., Meissner, A., Meletis, K., Lo, G. and Jaenisch, R. (2006) Suppression of intestinal neoplasia by deletion of dnmt3b. Mol Cell Biol. 26 2976-83. Aberrant gene silencing accompanied by DNA methylation is associated with neoplastic progression in many tumors that also show global loss of DNA methylation. Using conditional inactivation of de novo methyltransferase Dnmt3b in Apc(Min/+) mice, we demonstrate that the loss of Dnmt3b has no impact on microadenoma formation, which is considered the earliest stage of intestinal tumor formation. Nevertheless, we observed a significant decrease in the formation of macroscopic colonic adenomas. Interestingly, many large adenomas showed regions with Dnmt3b inactivation, indicating that Dnmt3b is required for initial outgrowth of macroscopic adenomas but is not required for their maintenance. These results support a role for Dnmt3b in the transition stage between microadenoma formation and macroscopic colonic tumor growth and further suggest that Dnmt3b, and by extension de novo methylation, is not required for maintaining tumor growth after this transition stage has occurred.Full Text

Loureiro, J. and Ploegh, H. L. (2006) Antigen presentation and the ubiquitin-proteasome system in host-pathogen interactions. Adv Immunol. 92 225-305. Relatively small genomes and high replication rates allow viruses and bacteria to accumulate mutations. This continuously presents the host immune system with new challenges. On the other side of the trenches, an increasingly well-adjusted host immune response, shaped by coevolutionary history, makes a pathogen's life a rather complicated endeavor. It is, therefore, no surprise that pathogens either escape detection or modulate the host immune response, often by redirecting normal cellular pathways to their advantage. For the purpose of this chapter, we focus mainly on the manipulation of the class I and class II major histocompatibility complex (MHC) antigen presentation pathways and the ubiquitin (Ub)-proteasome system by both viral and bacterial pathogens. First, we describe the general features of antigen presentation pathways and the Ub-proteasome system and then address how they are manipulated by pathogens. We discuss the many human cytomegalovirus (HCMV)-encoded immunomodulatory genes that interfere with antigen presentation (immunoevasins) and focus on the HCMV immunoevasins US2 and US11, which induce the degradation of class I MHC heavy chains by the proteasome by catalyzing their export from the endoplasmic reticulum (ER)-membrane into the cytosol, a process termed ER dislocation. US2- and US11-mediated subversion of ER dislocation ensures proteasomal degradation of class I MHC molecules and presumably allows HCMV to avoid recognition by cytotoxic T cells, whilst providing insight into general aspects of ER-associated degradation (ERAD) which is used by eukaryotic cells to purge their ER of defective proteins. We discuss the similarities and differences between the distinct pathways co-opted by US2 and US11 for dislocation and degradation of human class I MHC molecules and also a putatively distinct pathway utilized by the murine herpes virus (MHV)-68 mK3 immunoevasin for ER dislocation of murine class I MHC. We speculate on the implications of the three pathogen-exploited dislocation pathways to cellular ER quality control. Moreover, we discuss the ubiquitin (Ub)-proteasome system and its position at the core of antigen presentation as proteolysis and intracellular trafficking rely heavily on Ub-dependent processes. We add a few examples of manipulation of the Ub-proteasome system by pathogens in the context of the immune system and such diverse aspects of the host-pathogen relationship as virus budding, bacterial chromosome integration, and programmed cell death, to name a few. Finally, we speculate on newly found pathogen-encoded deubiquitinating enzymes (DUBs) and their putative roles in modulation of host-pathogen interactions.

Loureiro, J., Lilley, B. N., Spooner, E., Noriega, V., Tortorella, D. and Ploegh, H. L. (2006) Signal peptide peptidase is required for dislocation from the endoplasmic reticulum. Nature.(advance online publication 31 May 2006) Human cytomegalovirus (HCMV) prevents the display of class I major histocompatibility complex (MHC) peptide complexes at the surface of infected cells as a means of escaping immune detection. Two HCMV-encoded immunoevasins, US2 and US11, induce the dislocation of class I MHC heavy chains from the endoplasmic reticulum membrane and target them for proteasomal degradation in the cytosol. Although the outcome of the dislocation reactions catalysed is similar, US2 and US11 operate differently: Derlin-1 is a key component of the US11 but not the US2 pathway. So far, proteins essential for US2-dependent dislocation have not been identified. Here we compare interacting partners of wild-type US2 with those of a dislocation-incompetent US2 mutant, and identify signal peptide peptidase (SPP) as a partner for the active form of US2. We show that a decrease in SPP levels by RNA-mediated interference inhibits heavy-chain dislocation by US2 but not by US11. Our data implicate SPP in the US2 pathway and indicate the possibility of a previously unknown function for this intramembrane-cleaving aspartic protease in dislocation from the endoplasmic reticulum.Full Text

Love, J. C., Ronan, J. L., Grotenbreg, G. M., van der Veen, A. G. and Ploegh, H. L. (2006) A microengraving method for rapid selection of single cells producing antigen-specific antibodies. Nature Biotechnology .Published online: 14 May 2006 Monoclonal antibodies that recognize specific antigens of interest are used as therapeutic agents and as tools for biomedical research. Discovering a single monoclonal antibody requires retrieval of an individual hybridoma from polyclonal mixtures of cells producing antibodies with a variety of specificities. The time required to isolate hybridomas by a limiting serial-dilution, however, has restricted the diversity and breadth of available antibodies. Here we present a soft lithographic method based on intaglio printing to generate microarrays comprising the secreted products of single cells. These engraved arrays enable a rapid (<12 h) and high-throughput (>100,000 individual cells) system for identification, recovery and clonal expansion of cells producing antigen-specific antibodies. This method can be adapted, in principle, to detect any secreted product in a multiplexed manner.Full Text

Lu, X., Gross, A. W. and Lodish, H. F. (2006) Active conformation of the erythropoietin receptor: Random and cysteine-scanning mutagenesis of the extracellular juxtamembrane and transmembrane domains. Journal of Biological Chemistry 281 (11): 7002-7011 MAR 17 2006. In the absence of erythropoietin (Epo) cell surface Epo receptors (EpoR) are dimeric; dimerization is mediated mainly by the transmembrane domain. Binding of Epo changes the orientation of the two receptor subunits. This conformational change is transmitted through the juxtamembrane and transmembrane domains, leading to activation of JAK kinases and induction of proliferation and survival signals. To define the active EpoR conformation(s) we screened libraries of EpoRs with random mutations in the transmembrane domain and identified several point mutations that activate the EpoR in the absence of ligand, including changes of either of the first two transmembrane domain residues (L226 and I227) to cysteine. Following this discovery, we performed cysteine-scanning mutagenesis in the EpoR juxtamembrane and transmembrane domains. Many mutants formed disulfide-linked receptor dimers, but only EpoR dimers linked by cysteines at positions 223, 226 or 227 activated EpoR signal transduction pathways and supported proliferation of Ba/F3 cells in the absence of cytokines. These data suggest that activation of dimeric EpoR by Epo binding is achieved by reorienting the EpoR transmembrane and the connected cytosolic domains and that certain disulfide-bonded dimers represent the activated dimeric conformation of the EpoR, constitutively activating downstream signaling. Based on our data and the previously determined structure of Epo bound to a dimer of the EpoR extracellular domain, we present a model of the active and inactive conformations of the Epo receptor.PDF

MacIsaac, K. D., Gordon, D. B., Nekludova, L., Odom, D. T., Schreiber, J., Gifford, D. K., Young, R. A. and Fraenkel, E. (2006) A hypothesis-based approach for identifying the binding specificity of regulatory proteins from chromatin immunoprecipitation data. Bioinformatics. 22 423-429. Motivation: Genome-wide chromatin-immunoprecipitation (ChIP-chip) detects binding of transcriptional regulators to DNA in vivo at low resolution. Motif discovery algorithms can be used to discover sequence patterns in the bound regions that may be recognized by the immunoprecipitated protein. However, the discovered motifs often do not agree with the binding specificity of the protein, when it is known. Results: We present a powerful approach to analyzing ChIP- chip data, called THEME, that tests hypotheses concerning the sequence specificity of a protein. Hypotheses are refined using constrained local optimization. Cross-validation provides a principled standard for selecting the optimal weighting of the hypothesis and the ChIP-chip data and for choosing the best refined hypothesis. We demonstrate how to derive hypotheses for proteins from 36 domain families. Using THEME together with these hypotheses, we analyze ChIP-chip datasets for 14 human and mouse proteins. In all the cases the identified motifs are consistent with the published data with regard to the binding specificity of the proteins.Full Text.

Mallory, A. C. and Vaucheret, H. (2006) Functions of microRNAs and related small RNAs in plants. Nat Genet. 38 Suppl 1 S31-6. MicroRNAs (miRNAs) and short interfering RNAs (siRNAs), 20- to 27-nt in length, are essential regulatory molecules that act as sequence-specific guides in several processes in most eukaryotes (with the notable exception of the yeast Saccharomyces cerevisiae). These processes include DNA elimination, heterochromatin assembly, mRNA cleavage and translational repression. This review focuses on the regulatory roles of plant miRNAs during development, in the adaptive response to stresses and in the miRNA pathway itself. This review also covers the regulatory roles of two classes of endogenous plant siRNAs, ta-siRNAs and nat-siRNAs, which participate in post-transcriptional control of gene expression. Full Text

Meissner, A. and Jaenisch, R. (2006) Mammalian nuclear transfer. Dev Dyn. Early View 31 July During development, the genetic content of each cell remains, with a few exceptions, identical to that of the zygote. Differentiated cells, therefore, retain all the genetic information necessary to generate an entire organism (nuclear totipotency). Nuclear transfer (NT) was initially developed to test experimentally this concept by cloning animals from differentiated cells. It has, since then, been used to study the role of genetic and epigenetic alterations during development and disease. In this review, we highlight some of the milestones in mammalian NT reached in the 50 years after the first nuclear transplantations in frogs. We also address problems associated with mammalian nuclear transfer and provide a survey on current NT and stem cell technology. In the long term, nuclear transfer or alternative strategies aim to generate customized pluripotent cells, which would be invaluable to medical research and therapy.Full Text

Meissner, A. and Jaenisch, R. (2006) Generation of nuclear transfer-derived pluripotent ES cells from cloned Cdx2-deficient blastocysts. Nature. 439 212-215. The derivation of embryonic stem (ES) cells by nuclear transfer holds great promise for research and therapy but involves the destruction of cloned human blastocysts. Proof of principle experiments have shown that 'customized' ES cells derived by nuclear transfer (NT-ESCs) can be used to correct immunodeficiency in mice. Importantly, the feasibility of the approach has been demonstrated recently in humans, bringing the clinical application of NT- ESCs within reach. Altered nuclear transfer (ANT) has been proposed as a variation of nuclear transfer because it would create abnormal nuclear transfer blastocysts that are inherently unable to implant into the uterus but would be capable of generating customized ES cells. To assess the experimental validity of this concept we have used nuclear transfer to derive mouse blastocysts from donor fibroblasts that carried a short hairpin RNA construct targeting Cdx2. Cloned blastocysts were morphologically abnormal, lacked functional trophoblast and failed to implant into the uterus. However, they efficiently generated pluripotent embryonic stem cells when explanted into culture. Full Text

Merzdorf, C. S. and Sive, H. L. (2006) The zic1 gene is an activator of Wnt signaling. Int J Dev Biol. 50 611-7. The zic1 gene plays an important role in early patterning of the Xenopus neurectoderm. While Zic1 does not act as a neural inducer, it synergizes with the neural inducing factor Noggin to activate expression of posterior neural genes, including the midbrain/hindbrain boundary marker engrailed-2. Since the Drosophila homologue of zic1, odd-paired (opa), regulates expression of the wingless and engrailed genes and since Wnt proteins posteriorize neural tissue in Xenopus, we asked whether Xenopus Zic1 acted through the Wnt pathway. Using Wnt signaling inhibitors, we demonstrate that an active Wnt pathway is required for activation of en-2 expression by zic1. Consistent with this result, Zic1 induces expression of several wnt genes, including wnt1, wnt4 and wnt8b. wnt1 gene expression activates expression of engrailed in various organisms, including Xenopus, as demonstrated here. Together, our data suggest that zic1 is an upstream regulator of several wnt genes and that the regulatory relationships between opa, wingless and engrailed seen in Drosophila are also present in vertebrates.

Mintern, J. D., Klemm, E. J., Wagner, M., Paquet, M. E., Napier, M. D., Kim, Y. M., Koszinowski, U. H. and Ploegh, H. L. (2006) Viral interference with B7-1 costimulation: A new role for murine cytomegalovirus Fc receptor-1. Journal of Immunology. 177 8422-8431. Murine CMV (MCNIV), a beta-herpesvirus, infects dendritic cells (DC) and impairs their function. The underlying events are poorly described. In this study, we identify MCMV m138 as the viral gene responsible for promoting the rapid disappearance of the costimulatory molecule B7-1 (CD80) from the cell surface of DC. This was unexpected, as m138 was previously identified as fcr-1, a putative virus-encoded FcR. m138 impaired the ability of DC to activate CD8(+) T cells. Biochemical analysis and immunocytochemistry showed that m138 targets B7-1 in the secretory pathway and reroutes it to lysosomal associated membrane glyco-protein-1(+) compartments. These results show a novel function for m138 in MCNIV infection and identify the first viral protein to target B7-1 Full Text

Misaghi, S., Balsara, Z. R., Catic, A., Spooner, E., Ploegh, H. L. and Starnbach, M. N. (2006) Chlamydia trachomatis-derived deubiquitinating enzymes in mammalian cells during infection. Molecular Microbiology. 61 142-150. Chlamydia trachomatis is an obligate intracellular bacterium that causes a variety of diseases in humans. C. trachomatis has a complex developmental cycle that depends on host cells for replication, during which gene expression is tightly regulated. Here we identify two C. trachomatis proteases that possess deubiquitinating and deneddylating activities. We have designated these proteins ChlaDub1 and ChlaDub2. The genes encoding ChlaDub1 and ChlaDub2 are present in all Chlamydia species except for Chlamydia pneumoniae, and their catalytic domains bear similarity to the catalytic domains of other eukaryotic ubiquitin-like proteases (Ulp). The C. trachomatis DUBs react with activity-based probes and hydrolyse ubiquitinated and neddylated substrates. ChlaDub1 and ChlaDub2 represent the first known bacterial DUBs that possess both deubiquitinating and deneddylating activities.Full Text

Moffat, J., Grueneberg, D. A., Yang, X., Kim, S. Y., Kloepfer, A. M., Hinkle, G., Piqani, B., Eisenhaure, T. M., Luo, B., Grenier, J. K., Carpenter, A. E., Foo, S. Y., Stewart, S. A., Stockwell, B. R., Hacohen, N., Hahn, W. C., Lander, E. S., Sabatini, D. M. and Root, D. E. (2006) A Lentiviral RNAi Library for Human and Mouse Genes Applied to an Arrayed Viral High-Content Screen. Cell. 124 1283-98. To enable arrayed or pooled loss-of-function screens in a wide range of mammalian cell types, including primary and nondividing cells, we are developing lentiviral short hairpin RNA (shRNA) libraries targeting the human and murine genomes. The libraries currently contain 104,000 vectors, targeting each of 22,000 human and mouse genes with multiple sequence-verified constructs. To test the utility of the library for arrayed screens, we developed a screen based on high-content imaging to identify genes required for mitotic progression in human cancer cells and applied it to an arrayed set of 5,000 unique shRNA-expressing lentiviruses that target 1,028 human genes. The screen identified several known and approximately 100 candidate regulators of mitotic progression and proliferation; the availability of multiple shRNAs targeting the same gene facilitated functional validation of putative hits. This work provides a widely applicable resource for loss-of-function screens, as well as a roadmap for its application to biological discovery.Full Text

Moffat, J. and Sabatini, D. M. (2006) Building mammalian signalling pathways with RNAi screens. Nat Rev Mol Cell Biol. 7 177-187. Technological advances in mammalian systems are providing new tools to identify the molecular components of signalling pathways. Foremost among these tools is the ability to knock down gene function through the use of RNA interference (RNAi). The fact that RNAi can be scaled up for use in high-throughput techniques has motivated the creation of genome-wide RNAi reagents. We are now at the brink of being able to harness the power of RNAi for large-scale functional discovery in mammalian cells.Full Text

Mueller, B., Lilley, B. N. and Ploegh, H. L. (2006) SEL1L, the homologue of yeast Hrd3p, is involved in protein dislocation from the mammalian ER. J Cell Biol. Epub 2006 Oct 16 Protein quality control in the endoplasmic reticulum (ER) involves recognition of misfolded proteins and dislocation from the ER lumen into the cytosol, followed by proteasomal degradation. Viruses have co-opted this pathway to destroy proteins that are crucial for host defense. Examination of dislocation of class I major histocompatibility complex (MHC) heavy chains (HCs) catalyzed by the human cytomegalovirus (HCMV) immunoevasin US11 uncovered a conserved complex of the mammalian dislocation machinery. We analyze the contributions of a novel complex member, SEL1L, mammalian homologue of yHrd3p, to the dislocation process. Perturbation of SEL1L function discriminates between the dislocation pathways used by US11 and US2, which is a second HCMV protein that catalyzes dislocation of class I MHC HCs. Furthermore, reduction of the level of SEL1L by small hairpin RNA (shRNA) inhibits the degradation of a misfolded ribophorin fragment (RI(332)) independently of the presence of viral accessories. These results allow us to place SEL1L in the broader context of glycoprotein degradation, and imply the existence of multiple independent modes of extraction of misfolded substrates from the mammalian ER. Full Text

Muller, U. F. (2006) Re-creating an RNA world. Cell Mol Life Sci.May 2; [Epub ahead of print] The RNA world hypothesis states that life originated via a system based on RNA genomes and RNA catalysts. Researchers have been trying to develop such a system since catalytic RNAs (ribozymes) were discovered in 1982. This review summarizes the recent progress made in that endeavor and outlines the obstacles that remain to be overcome. After giving a short background on prebiotic chemistry and in vitro evolution, the discussion focuses on the generation of three important components of an RNA world: a sufficient polymerase ribozyme, self-replicating membrane compartments and ribozymes that are capable of performing basic metabolic processes. Full Text

Nencioni, A., Grunebach, F., Patrone, F., Ballestrero, A. and Brossart, P. (2006) The proteasome and its inhibitors in immune regulation and immune disorders. Crit Rev Immunol. 26 487-98. The ubiquitin-proteasome pathway is a well-characterized mechanism deputed to the degradation of intracellular proteins. Proteasomal degradation intervenes in the regulation of numerous cellular functions including signal transduction, apoptosis, cell cycle, and antigen presentation. In vitro and in vivo studies have shown that both normal and malignant cells of the immune system are exquisitely affected by inhibition of proteasome activity. This property is currently exploited in the treatment of multiple myeloma and mantle cell lymphoma, two B-cell malignancies that respond to treatment with the proteasome inhibitor bortezomib. Pharmacological inhibitors of the proteasome also affect function and survival of B and T lymphocytes and of dendritic cells and were shown to reduce autoimmune and inflammatory manifestations in several models of immune-mediated disorders. The present review offers an overview of the mechanisms implicated in the immunomodulatory effects of proteasome inhibitors and discusses prospective future applications for these small molecules in immune and inflammatory diseases.

Odom, D. T., Dowell, R. D., Jacobsen, E. S., Nekludova, L., Rolfe, P. A., Danford, T. W., Gifford, D. K., Fraenkel, E., Bell, G. I. and Young, R. A. (2006) Core transcriptional regulatory circuitry in human hepatocytes. Mol Syst Biol. 2 2006 0017. We mapped the transcriptional regulatory circuitry for six master regulators in human hepatocytes using chromatin immunoprecipitation and high-resolution promoter microarrays. The results show that these regulators form a highly interconnected core circuitry, and reveal the local regulatory network motifs created by regulator-gene interactions. Autoregulation was a prominent theme among these regulators. We found that hepatocyte master regulators tend to bind promoter regions combinatorially and that the number of transcription factors bound to a promoter corresponds with observed gene expression. Our studies reveal portions of the core circuitry of human hepatocytes.Full Text

Orimo, A. and Weinberg, R. A. (2006) Stromal Fibroblasts in Cancer: A Novel Tumor-Promoting Cell Type. Cell Cycle.Aug 1;5(15) [Epub ahead of print] Tumors are highly complex tissues composed of neoplastic cells and, in the case of carcinomas, stromal cell compartments containing a variety of mesenchymal cells, notably fibroblasts, myofibroblasts, endothelial cells, pericytes, and a variety of inflammatory cells associated with the immune system. Fibroblasts and myofibroblasts often represent the majority of the stromal cells within various types of human carcinomas, yet the specific contributions of these cells to tumor growth are poorly understood. Recent work has demonstrated that stromal fibroblast fractions, named carcinoma-associated fibroblasts (CAFs), that have been extracted from a number of invasive human breast carcinomas are more competent to promote the growth of mammary carcinoma cells and to enhance tumor angiogenesis than are comparable cells derived from outside of these tumor masses. CAFs include large populations of myofibroblasts that secrete elevated levels of stromal cell-derived factor 1 (SDF-1), also called CXCL12, which plays a central role in the promotion of tumor growth and angiogenesis; CAF-derived SDF-1 not only stimulates carcinoma cell growth directly through the CXCR4 receptor displayed on tumor cells but also serves to recruit endothelial progenitor cells (EPCs) into tumors, thereby furthering neoangiogenesis. In this review, we highlight the importance of this SDF-1-CXCR4 signaling pathway in the tumor microenvironment and discuss the mechanisms by which stromal fibroblasts within mammary carcinomas enhance tumor growth.

Outeiro, TF. and Giorgini, F. (2006) Yeast as a drug discovery platform in Huntington's and Parkinson's diseases. Biotechnol J. 1 258-69. The high degree of conservation of cellular and molecular processes between the budding yeast Saccharomyces cerevisiae and higher eukaryotes have made it a valuable system for numerous studies of the basic mechanisms behind devastating illnesses such as cancer, infectious disease, and neurodegenerative disorders. Several studies in yeast have already contributed to our basic understanding of cellular dysfunction in both Huntington's and Parkinson's disease. Functional genomics approaches currently being undertaken in yeast may lead to novel insights into the genes and pathways that modulate neuronal cell dysfunction and death in these diseases. In addition, the budding yeast constitutes a valuable system for identification of new drug targets, both via target-based and non-target-based drug screening. Importantly, yeast can be used as a cellular platform to analyze the cellular effects of candidate compounds, which is critical for the development of effective therapeutics. While the molecular mechanisms that underlie neurodegeneration will ultimately have to be tested in neuronal and animal models, there are several distinct advantages to using simple model organisms to elucidate fundamental aspects of protein aggregation, amyloid toxicity, and cellular dysfunction. Here, we review recent studies that have shown that amyloid formation by disease-causing proteins and many of the resulting cellular deficits can be faithfully recapitulated in yeast. In addition, we discuss new yeast-based techniques for screening candidate therapeutic compounds for Huntington's and Parkinson's diseases.

Palomero, T., Lim, W. K., Odom, D. T., Sulis, M. L., Real, P. J., Margolin, A., Barnes, K. C., O'Neil, J., Neuberg, D., Weng, A. P., Aster, J. C., Sigaux, F., Soulier, J., Look, A. T., Young, R. A., Califano, A. and Ferrando, A. A. (2006) NOTCH1 directly regulates c-MYC and activates a feed-forward-loop transcriptional network promoting leukemic cell growth. Proc Natl Acad Sci U S A. 103 (48) : 18261-18266 The NOTCH1 signaling pathway directly links extracellular signals with transcriptional responses in the cell nucleus and plays a critical role during T cell development and in the pathogenesis over 50% of human T cell lymphoblastic leukemia (T-ALL) cases. However, little is known about the transcriptional programs activated by NOTCH1. Using an integrative systems biology approach we show that NOTCH1 controls a feed-forward-loop transcriptional network that promotes cell growth. Inhibition of NOTCH1 signaling in T-ALL cells led to a reduction in cell size and elicited a gene expression signature dominated by down-regulated biosynthetic pathway genes. By integrating gene expression array and ChIP-on-chip data, we show that NOTCH1 directly activates multiple biosynthetic routes and induces c-MYC gene expression. Reverse engineering of regulatory networks from expression profiles showed that NOTCH1 and c-MYC govern two directly interconnected transcriptional programs containing common target genes that together regulate the growth of primary T-ALL cells. These results identify c-MYC as an essential mediator of NOTCH1 signaling and integrate NOTCH1 activation with oncogenic signaling pathways upstream of c-MYC. PDF

Palomero, T., Odom, D., O'Neil, J., Ferrando, A. A., Margolin, A., Neuberg, D. S., Winter, S. S., Larson, R. S., Li, W., Liu, X. S., Young, R. A. and Look, A. T. (2006) Transcriptional regulatory networks downstream of TAL1/SCL in T-cell acute lymphoblastic leukemia. Blood. 108 986-992. Aberrant expression of 1 or more transcription factor oncogenes is a critical component of the molecular pathogenesis of human T-cell acute lymphoblastic leukemia (T-ALL); however, oncogenic transcriptional programs downstream of T-ALL oncogenes are mostly unknown. TAL1/SCL is a basic helix-loop-helix (bHLH) transcription factor oncogene aberrantly expressed in 60% of human T-ALLs. We used chromatin immunoprecipitation (ChIP) on chip to identify 71 direct transcriptional targets of TAL1/SCL. Promoters occupied by TAL1 were also frequently bound by the class I bHLH proteins E2A and HEB, suggesting that TAL1/ E2A as well as TAL1/HEB heterodimers play a role in transformation of T-cell precursors. Using RNA interference, we demonstrated that TAL1 is required for the maintenance of the leukemic phenotype in Jurkat cells and showed that TAL1 binding can be associated with either repression or activation of genes whose promoters occupied by TAL1, E2A, and HEB. In addition, oligonucleotide microarray analysis of RNA from 47 primary T-ALL samples showed specific expression signatures involving TAL1 targets in TAL1-expressing compared with -nonexpressing human T-ALLs. Our results indicate that TAL1 may act as a bifunctional transcriptional regulator (activator and repressor) at the top of complex regulatory network that disrupts normal T-cell homeostasis and contributes to leukemogenesis. Full Text

Petrasek, J., Mravec, J., Bouchard, R., Blakeslee, J. J., Abas, M., Seifertova, D., Wisniewska, J., Tadele, Z., Kubes, M., Covanova, M., Dhonukshe, P., Skupa, P., Benkova, E., Perry, L., Krecek, P., Lee, O. R., Fink, G. R., Geisler, M., Murphy, A. S., Luschnig, C., Zazimalova, E. and Friml, J. (2006) PIN proteins perform a rate-limiting function in cellular auxin efflux. Science. 312 914-918. Intercellular flow of the phytohormone auxin underpins multiple developmental processes in plants. Plant-specific pin-formed ( PIN) proteins and several phosphoglycoprotein (PGP) transporters are crucial factors in auxin transport - related development, yet the molecular function of PINs remains unknown. Here, we show that PINs mediate auxin efflux from mammalian and yeast cells without needing additional plant-specific factors. Conditional gain-of-function alleles and quantitative measurements of auxin accumulation in Arabidopsis and tobacco cultured cells revealed that the action of PINs in auxin efflux is distinct from PGP, rate-limiting, specific to auxins, and sensitive to auxin transport inhibitors. This suggests a direct involvement of PINs in catalyzing cellular auxin efflux. Full Text

Pokholok, D. K., Zeitlinger, J., Hannett, N. M., Reynolds, D. B. and Young, R. A. (2006) Activated signal transduction kinases frequently occupy target genes. Science. 313 533-536. Cellular signal transduction pathways modify gene expression programs in response to changes in the environment, but the mechanisms by which these pathways regulate populations of genes under their control are not entirely understood. We present evidence that most mitogen-activated protein kinases and protein kinase A subunits become physically associated with the genes that they regulate in the yeast ( Saccharomyces cerevisiae) genome. The ability to detect this interaction of signaling kinases with target genes can be used to more precisely and comprehensively map the regulatory circuitry that eukaryotic cells use to respond to their environment.Full Text

Qi, Y. A. and Ge, H. (2006) Modularity and dynamics of cellular networks. Plos Computational Biology. 2 1502-1510.Full Text

Rajagopalan, R., Vaucheret, H., Trejo, J. and Bartel, D. P. (2006) A diverse and evolutionarily fluid set of microRNAs in Arabidopsis thaliana. Genes Dev. 20 3407-25. To better understand the diversity of small silencing RNAs expressed in plants, we employed high-throughput pyrosequencing to obtain 887,000 reads corresponding to Arabidopsis thaliana small RNAs. They represented 340,000 unique sequences, a substantially greater diversity than previously obtained in any species. Most of the small RNAs had the properties of heterochromatic small interfering RNAs (siRNAs) associated with DNA silencing in that they were preferentially 24 nucleotides long and mapped to intergenic regions. Their density was greatest in the proximal and distal pericentromeric regions, with only a slightly preferential propensity to match repetitive elements. Also present were 38 newly identified microRNAs (miRNAs) and dozens of other plausible candidates. One miRNA mapped within an intron of DICER-LIKE 1 (DCL1), suggesting a second homeostatic autoregulatory mechanism for DCL1 expression; another defined the phase for siRNAs deriving from a newly identified trans-acting siRNA gene (TAS4); and two depended on DCL4 rather than DCL1 for their accumulation, indicating a second pathway for miRNA biogenesis in plants. More generally, our results revealed the existence of a layer of miRNA-based control beyond that found previously that is evolutionarily much more fluid, employing many newly emergent and diverse miRNAs, each expressed in specialized tissues or at low levels under standard growth conditions.Full Text

Rao, P. K., Kumar, R. M., Farkhondeh, M., Baskerville, S. and Lodish, H. F. (2006) Myogenic factors that regulate expression of muscle-specific microRNAs. Proc Natl Acad Sci U S A. 103 8721-8726 Since their discovery as key regulators of early animal development, microRNAs now are recognized as widespread regulators of gene expression. Despite their abundance, little is known regarding the regulation of microRNA biogenesis. We show that three highly conserved muscle-specific microRNAs, miR-1, miR-133 and miR-206, are robustly induced during the myoblast-myotube transition, both in primary human myoblasts and in the mouse mesenchymal C2C12 stem cell line. These microRNAs were not induced during osteogenic conversion of C2C12 cells. Moreover, both loci encoding miR-1, miR-1-1, and miR-1-2, and two of the three encoding miR-133, miR-133a-1 and miR-133a-2, are strongly induced during myogenesis. Some of the induced microRNAs are in intergenic regions, whereas two are transcribed in the opposite direction to the nonmuscle-specific gene in which they are embedded. By using CHIP analysis, we demonstrate that the myogenic factors Myogenin and MyoD bind to regions upstream of these microRNAs and, therefore, are likely to regulate their expression. Because miR-1 and miR-206 are predicted to repress similar mRNA targets, our work suggests that induction of these microRNAs is important in regulating the expression of muscle-specific proteins.PDF

Reiling, J. H. and Sabatini, D. M. (2006) Stress and mTORture signaling. Oncogene. 25 6373-83. The TOR (target of rapamycin) pathway is an evolutionarily conserved signaling module regulating cell growth (accumulation of mass) in response to a variety of environmental cues such as nutrient availability, hypoxia, DNA damage and osmotic stress. Its pivotal role in cellular and organismal homeostasis is reflected in the fact that unrestrained signaling activity in mammals is associated with the occurrence of disease states including inflammation, cancer and diabetes. The existence of TOR homologs in unicellular organisms whose growth is affected by environmental factors, such as temperature, nutrients and osmolarity, suggests an ancient role for the TOR signaling network in the surveillance of stress conditions. Here, we will summarize recent advances in the TOR signaling field with special emphasis on how stress conditions impinge on insulin/insulin-like growth factor signaling/TOR signaling.Full Text

Repping, S., van Daalen, S. K., Brown, L. G., Korver, C. M., Lange, J., Marszalek, J. D., Pyntikova, T., van der Veen, F., Skaletsky, H., Page, D. C. and Rozen, S. (2006) High mutation rates have driven extensive structural polymorphism among human Y chromosomes. Nat Genet.Feb 26; [Epub ahead of print] Although much structural polymorphism in the human genome has been catalogued, the kinetics of underlying change remain largely unexplored. Because human Y chromosomes are clonally inherited, it has been possible to capture their detailed relationships in a robust, worldwide genealogical tree. Examination of structural variation across this tree opens avenues for investigating rates of underlying mutations. We selected one Y chromosome from each of 47 branches of this tree and searched for large-scale variation. Four chromosomal regions showed extensive variation resulting from numerous large-scale mutations. Within the tree encompassed by the studied chromosomes, the distal-Yq heterochromatin changed length >/=12 times, the TSPY gene array changed length >/=23 times, the 3.6-Mb IR3/IR3 region changed orientation >/=12 times and the AZFc region was rearranged >/=20 times. After determining the total time spanned by all branches of this tree ( approximately 1.3 million years or 52,000 generations), we converted these mutation counts to lower bounds on rates: >/=2.3 x 10(-4), >/=4.4 x 10(-4), >/=2.3 x 10(-4) and >/=3.8 x 10(-4) large-scale mutations per father-to-son Y transmission, respectively. Thus, high mutation rates have driven extensive structural polymorphism among human Y chromosomes. At the same time, we found limited variation in the copy number of Y-linked genes, which raises the possibility of selective constraints Full Text

Resnick, T. D., Satinover, D. L., Macisaac, F., Stukenberg, P. T., Earnshaw, W. C., Orr-Weaver, T. L. and Carmena, M. (2006) INCENP and Aurora B Promote Meiotic Sister Chromatid Cohesion through Localization of the Shugoshin MEI-S332 in Drosophila. Dev Cell. 11 57-68. The chromosomal passenger complex protein INCENP is required in mitosis for chromosome condensation, spindle attachment and function, and cytokinesis. Here, we show that INCENP has an essential function in the specialized behavior of centromeres in meiosis. Mutations affecting Drosophila incenp profoundly affect chromosome segregation in both meiosis I and II, due, at least in part, to premature sister chromatid separation in meiosis I. INCENP binds to the cohesion protector protein MEI-S332, which is also an excellent in vitro substrate for Aurora B kinase. A MEI-S332 mutant that is only poorly phosphorylated by Aurora B is defective in localization to centromeres. These results implicate the chromosomal passenger complex in directly regulating MEI-S332 localization and, therefore, the control of sister chromatid cohesion in meiosis.Full Text

Root, D. E., Hacohen, N., Hahn, W. C., Lander, E. S. and Sabatini, D. M. (2006) Genome-scale loss-of-function screening with a lentiviral RNAi library. Nature Methods. 3 715-719. The discovery that RNA interference (RNAi) is functional in mammalian cells led us to form The RNAi Consortium (TRC) with the goal of enabling large-scale loss-of-function screens through the development of genome-scale RNAi libraries and methodologies for their use. These resources form the basis of a loss-of-function screening platform created at the Broad Institute. Our human and mouse libraries currently contain > 135,000 lentiviral clones targeting 27,000 genes. Initial screening efforts have demonstrated that these libraries and methods are practical and powerful tools for high-throughput lentivirus RNAi screens.Full Text

Ruby, J. G., Jan, C., Player, C., Axtell, M. J., Lee, W., Nusbaum, C., Ge, H. and Bartel, D. P. (2006) Large-Scale Sequencing Reveals 21U-RNAs and Additional MicroRNAs and Endogenous siRNAs in C. elegans. Cell. 127 1193-207. We sequenced approximately 400,000 small RNAs from Caenorhabditis elegans. Another 18 microRNA (miRNA) genes were identified, thereby extending to 112 our tally of confidently identified miRNA genes in C. elegans. Also observed were thousands of endogenous siRNAs generated by RNA-directed RNA polymerases acting preferentially on transcripts associated with spermatogenesis and transposons. In addition, a third class of nematode small RNAs, called 21U-RNAs, was discovered. 21U-RNAs are precisely 21 nucleotides long, begin with a uridine 5'-monophosphate but are diverse in their remaining 20 nucleotides, and appear modified at their 3'-terminal ribose. 21U-RNAs originate from more than 5700 genomic loci dispersed in two broad regions of chromosome IV-primarily between protein-coding genes or within their introns. These loci share a large upstream motif that enables accurate prediction of additional 21U-RNAs. The motif is conserved in other nematodes, presumably because of its importance for producing these diverse, autonomously expressed, small RNAs (dasRNAs).Full Text

Sabatini, D. M. (2006) mTOR and cancer: insights into a complex relationship. Nature Reviews Cancer Advance online publication, 17 August 2006. mTOR (mammalian target of rapamycin) has come a long way since its humble beginnings as a kinase of unknown function. As part of the mTORC1 and mTORC2 complexes mTOR has key roles in several pathways that are involved in human cancer, stimulating interest in mTOR inhibitors and placing it on the radar of the pharmaceutical industry. Here, I discuss the rationale for the use of drugs that target mTOR, the unexpectedly complex mechanism of action of existing mTOR inhibitors and the potential benefits of developing drugs that function through different mechanisms. The purpose is not to cover all aspects of mTOR history and signalling, but rather to foster discussion by presenting some occasionally provocative ideas.Full Text

Sarbassov, D. D., Ali, S. M., Sengupta, S., Sheen, J. H., Hsu, P. P., Bagley, A. F., Markhard, A. L. and Sabatini, D. M. (2006) Prolonged Rapamycin Treatment Inhibits mTORC2 Assembly and Akt/PKB. Mol Cell. 22 159-68 The drug rapamycin has important uses in oncology, cardiology, and transplantation medicine, but its clinically relevant molecular effects are not understood. When bound to FKBP12, rapamycin interacts with and inhibits the kinase activity of a multiprotein complex composed of mTOR, mLST8, and raptor (mTORC1). The distinct complex of mTOR, mLST8, and rictor (mTORC2) does not interact with FKBP12-rapamycin and is not thought to be rapamycin sensitive. mTORC2 phosphorylates and activates Akt/PKB, a key regulator of cell survival. Here we show that rapamycin inhibits the assembly of mTORC2 and that, in many cell types, prolonged rapamycin treatment reduces the levels of mTORC2 below those needed to maintain Akt/PKB signaling. The proapoptotic and antitumor effects of rapamycin are suppressed in cells expressing an Akt/PKB mutant that is rapamycin resistant. Our work describes an unforeseen mechanism of action for rapamycin that suggests it can be used to inhibit Akt/PKB in certain cell types.Full Text

Savarese, F., Flahndorfer, K., Jaenisch, R., Busslinger, M. and Wutz, A. (2006) Hematopoietic precursor cells transiently reestablish permissiveness for X inactivation. Mol Cell Biol. 26 7167-77. Xist is the trigger for X inactivation in female mammals. The long noncoding Xist RNA localizes along one of the two female X chromosomes and initiates chromosome-wide silencing in the early embryo. In differentiated cells, Xist becomes dispensable for the maintenance of the inactive X, and its function for initiation of silencing is lost. How Xist mediates gene repression remains an open question. Here, we use an inducible Xist allele in adult mice to identify cells in which Xist can cause chromosome-wide silencing. We show that Xist has the ability to initiate silencing in immature hematopoietic precursor cells. In contrast, hematopoietic stem cells and mature blood cells are unable to initiate ectopic X inactivation. This indicates that pathways critical for silencing are transiently activated in hematopoietic differentiation. Xist-responsive cell types in normal female mice show a change of chromatin marks on the inactive X. However, dosage compensation is maintained throughout hematopoiesis. Therefore, Xist can initiate silencing in precursors with concomitant maintenance of dosage compensation. This suggests that Xist function is restricted in development by the limited activity of epigenetic pathways rather than by a change in the responsiveness of chromatin between embryonic and differentiated cell types.Full Text

Scacheri, P. C., Davis, S., Odom, D. T., Crawford, G. E., Perkins, S., Halawi, M. J., Agarwal, S. K., Marx, S. J., Spiegel, A. M., Meltzer, P. S. and Collins, F. S. (2006) Genome-wide analysis of menin binding provides insights into MEN1 tumorigenesis. Plos Genetics. 2 406-419. Multiple endocrine neoplasia type I ( MEN1) is a familial cancer syndrome characterized primarily by tumors of multiple endocrine glands. The gene for MEN1 encodes a ubiquitously expressed tumor suppressor protein called menin. Menin was recently shown to interact with several components of a trithorax family histone methyltransferase complex including ASH2, Rbbp5, WDR5, and the leukemia proto-oncoprotein MLL. To elucidate menin's role as a tumor suppressor and gain insights into the endocrine-specific tumor phenotype in MEN1, we mapped the genomic binding sites of menin, MLL1, and Rbbp5, to approximately 20,000 promoters in HeLa S3, HepG2, and pancreatic islet cells using the strategy of chromatin-immunoprecipitation coupled with microarray analysis. We found that menin, MLL1, and Rbbp5 localize to the promoters of thousands of human genes but do not always bind together. These data suggest that menin functions as a general regulator of transcription. We also found that factor occupancy generally correlates with high gene expression but that the loss of menin does not result in significant changes in most transcript levels. One exception is the developmentally programmed transcription factor, HLXB9, which is overexpressed in islets in the absence of menin. Our findings expand the realm of menin- targeted genes several hundred- fold beyond that previously described and provide potential insights to the endocrine tumor bias observed in MEN1 patients.Full Text

Schreiber, J., Jenner, R. G., Murray, H. L., Gerber, G. K., Gifford, D. K. and Young, R. A. (2006) Coordinated binding of NF-{kappa}B family members in the response of human cells to lipopolysaccharide. Proc Natl Acad Sci U S A.Apr 4; [Epub ahead of print] The NF-kappaB family of transcription factors plays a critical role in numerous cellular processes, particularly the immune response. Our understanding of how the different NF-kappaB subunits act coordinately to regulate gene expression is based on a limited set of genes. We used genome-scale location analysis to identify targets of all five NF-kappaB proteins before and after stimulation of monocytic cells with bacterial lipopolysaccharide (LPS). In unstimulated cells, p50 and p52 bound to a large number of gene promoters that were also occupied by RNA polymerase II. After LPS stimulation, additional NF-kappaB subunits bound to these genes and to other genes. Genes that became bound by multiple NF-kappaB subunits were the most likely to show increases in RNA polymerase II occupancy and gene expression. This study identifies NF-kappaB target genes, reveals how the different NF-kappaB proteins coordinate their activity, and provides an initial map of the transcriptional regulatory network that underlies the host response to infection.Full Text

Shibue, T. and Taniguchi, T. (2006) BH3-only proteins: Integrated control point of apoptosis. International Journal of Cancer. 119 2036-2043. Bcl-2 family proteins play central roles in the regulation of most, if not all, apoptotic pathways, and hence this family plays a critical role in oncogenesis. The Bcl-2 homology 3 (BH3)-only members of this family are "proapoptotic," promoting apoptosis by sensing cellular stresses: that is, they are activated or induced in response to stress stimuli. These BH3-only proteins then interfere with the function of "prosurvival" Bcl-2 family members, thereby promoting the progression of apoptosis. It has long been recognized that the induction and activation of each of the BH3-only proteins are subject to the type of stress stimulus. Although it was originally assumed that all the BH3-only proteins exert similar effects on the downstream apoptotic machinery, recent studies have uncovered their distinct functional properties, indicating the operation of more intricate, versatile control mechanisms of apoptosis. In this review, we will summarize recent findings on the diversity in the activation and function of BH3-only proteins. In particular, we focus on the overlapping and individual roles of 2 BH3-only proteins,. Puma and Noxa, in the context of the apoptotic response induced by the tumor suppressor p53. Full Text

Shorter, J. and Lindquist, S. (2006) Destruction or potentiation of different prions catalyzed by similar hsp104 remodeling activities. Mol Cell. 23 425-38. Yeast prions are protein-based genetic elements that self-perpetuate changes in protein conformation and function. A protein-remodeling factor, Hsp104, controls the inheritance of several yeast prions, including those formed by Sup35 and Ure2. Perplexingly, deletion of Hsp104 eliminates Sup35 and Ure2 prions, whereas overexpression of Hsp104 purges cells of Sup35 prions, but not Ure2 prions. Here, we used pure components to dissect how Hsp104 regulates prion formation, growth, and division. For both Sup35 and Ure2, Hsp104 catalyzes de novo prion nucleation from soluble, native protein. Using a distinct mechanism, Hsp104 fragments both prions to generate new prion assembly surfaces. For Sup35, the fragmentation endpoint is an ensemble of noninfectious, amyloid-like aggregates and soluble protein that cannot replicate conformation. In vivid distinction, the endpoint of Ure2 fragmentation is short prion fibers with enhanced infectivity and self-replicating ability. These advances explain the distinct effects of Hsp104 on the inheritance of the two prions. Full Text

Steele, A. D. and Yi, C. H. (2006) Neuromuscular denervation: Bax up against the wall in amyotrophic lateral sclerosis. Journal of Neuroscience. 26 12849-12851. Steele, A. D. and Yi, C. H. (2006) Neuromuscular denervation: Bax up against the wall in amyotrophic lateral sclerosis. Full Text.

Steele, A. D., Emsley, J. G., Ozdinler, P. H., Lindquist, S. and Macklis, J. D. (2006) Prion protein (PrPc) positively regulates neural precursor proliferation during developmental and adult mammalian neurogenesis. Proc Natl Acad Sci U S A.Feb 21; [Epub ahead of print] The misfolding of the prion protein (PrP(c)) is a central event in prion diseases, yet the normal function of PrP(c) remains unknown. PrP(c) has putative roles in many cellular processes including signaling, survival, adhesion, and differentiation. Given the abundance of PrP(c) in the developing and mature mammalian CNS, we investigated the role of PrP(c) in neural development and in adult neurogenesis, which occurs constitutively in the dentate gyrus (DG) of the hippocampus and in the olfactory bulb from precursors in the subventricular zone (SVZ)/rostral migratory stream. In vivo, we find that PrP(c) is expressed immediately adjacent to the proliferative region of the SVZ but not in mitotic cells. In vivo and in vitro studies further find that PrP(c) is expressed in multipotent neural precursors and mature neurons but is not detectable in glia. Loss- and gain-of-function experiments demonstrate that PrP(c) levels correlate with differentiation of multipotent neural precursors into mature neurons in vitro and that PrP(c) levels positively influence neuronal differentiation in a dose-dependent manner. PrP(c) also increases cellular proliferation in vivo; in the SVZ, PrP(c) overexpresser (OE) mice have more proliferating cells compared with wild-type (WT) or knockout (KO) mice; in the DG, PrP(c) OE and WT mice have more proliferating cells compared with KO mice. Our results demonstrate that PrP(c) plays an important role in neurogenesis and differentiation. Because the final number of neurons produced in the DG is unchanged by PrP(c) expression, other factors must control the ultimate fate of new neurons.PDF

Timp, W., Mirsaidov, U., Timp, K., Timp, G. and Matsudaira, P. (2006) Laser-guided assembly of 3D living cell microarrays. Nanomedicine. 2 316.

Timp, W., Watson, N., Sabban, A., Zik, O. and Matsudaira, P. (2006) Wet electron microscopy with quantum dots. Biotechniques. 41 295-8. Wet electron microscopy (EM) is a new imaging method with the potential to allow higher spatial resolution of samples. In contrast to most EM methods, it requires little time to perform and does not require complicated equipment or difficult steps. We used this method on a common murine macrophage cell line, IC-21, in combination with various stains and preparations, to collect high resolution images of the actin cytoskeleton. Most importantly, we demonstrated the use of quantum dots in conjunction with this technique to perform light/electron correlation microscopy. We found that wet EM is a useful tool that fits into a niche between the simplicity of light microscopy and the high spatial resolution of EM.Full Text

Tirosh, B., Iwakoshi, N. N., Glimcher, L. H. and Ploegh, H. L. (2006) Rapid turnover of unspliced Xbp-1 as a factor that modulates the unfolded protein response. Journal of Biological Chemistry. 281 5852-5860. The mammalian and yeast unfolded protein responses (UPR) share the characteristic of rapid elimination of unspliced Xbp-1 (Xbp-1u) and unspliced Hac1p, respectively. These polypeptides derive from mRNAs, whose splicing is induced upon onset of the UPR, so as to allow synthesis of transcription factors essential for execution of the UPR itself. Whereas in yeast translation of unspliced Hac1p is blocked, mammalian Xbp-1u is synthesized constitutively and eliminated by rapid proteasomal degradation. Here we show that the rate of Xbp-1u degradation approaches its rate of synthesis. The C terminus of XBP-1u ensures its trafficking to the cytoplasm, and is sufficient to impose rapid degradation. Degradation of XBP-1u involves both ubiquitin-dependent and ubiquitin-independent mechanisms, which might explain its unusually rapid turnover. Xbp-1(-/-) mouse embryonic fibroblasts reconstituted with mutants of XBP-1u that show improved stability differentially activate UPR target genes. Unexpectedly, we found that one of the mutants activates transcription of both Xbp-1-specific and non-Xbp-1-dependent UPR targets in response to tunicamycin treatment, even more potently than does wild type Xbp-1. We suggest that the degradation of Xbp-1u is required to prevent uncontrolled activation of the UPR while allowing short dwell times for initiation of this response. Full Text

Tong, W., Sulahian, R., Gross, A. W., Hendon, N., Lodish, H. F. and Huang, L. J. (2006) The membrane-proximal region of the thrombopoietin receptor confers its high surface expression by JAK2-dependent and -independent mechanisms. J Biol Chem. 281 38930-40. Janus tyrosine kinase 2 (JAK2) is essential for signaling by the thrombopoietin (TpoR) and erythropoietin (EpoR) receptors. In the absence of JAK2 most EpoR molecules are retained in the endoplasmic reticulum in an Endo H-sensitive form. In contrast, we show that in the absence of JAK2 a large fraction of the TpoR is processed to the mature Endo H-resistant form and reaches the cell surface. By studying chimeras of the TpoR and EpoR we show that high surface expression of the TpoR is entirely conferred by the membrane-proximal region of the intracellular domain that includes the juxtamembrane, Box 1, and Box 2 regions. The TpoR intracellular domain shows similar effects on receptor endocytosis rate as that of the EpoR, but does stabilize the mature receptor isoform from degradation. Co-expression of JAK2 further stabilizes mature TpoR and thus further increases its surface expression. This JAK2 effect depends on the Box 1 region, the only JAK2 interacting site in the TpoR. By contrast, EpoR requires Box 1 as well as the flanking 20 residues on the C-terminal side for JAK2 interaction and JAK2-dependent surface expression. Our study suggests that whereas cell surface expression of type I cytokine receptors requires their cognate JAKs, the mechanisms governing receptor-JAK interactions differ among receptors interacting with the same JAK protein.Full Text

Tropepe, V., Li, S. H., Dickinson, A., Gamse, J. T. and Sive, H. L. (2006) Identification of a BMP inhibitor-responsive promoter module required for expression of the early neural gene zic1. Developmental Biology. 289 517-529. Expression of the transcription factor zic1 at the onset of gastrulation is one of the earliest molecular indicators of neural fate determination in Xenopus. Inhibition of bone morphogenetic protein (BMP) signaling is critical for activation of zic1 expression and fundamental for establishing neural identity in both vertebrates and invertebrates. The mechanism by which interruption of BMP signaling activates neural-specific gene expression is not understood. Here, we report identification of a 215 bp genomic module that is both necessary and sufficient to activate Xenopus zic1 transcription upon interruption of BMP signaling. Transgenic analyses demonstrate that this BMP inhibitory response module (BIRM) is required for expression in the whole embryo. Multiple consensus binding sites for specific transcription factor families within the BIRM are required for its activity and some of these regions are phylogenetically conserved between orthologous vertebrate zic1 genes. These data suggest that interruption of BMP signaling facilitates neural determination via a complex mechanism, involving multiple regulatory factors that cooperate to control zic1 expression. Full Text

Ueberfeld, J., El-Difrawy, S. A., Ramdhanie, K. and Ehrlich, D. J. (2006) Solid-support sample loading for DNA sequencing. Anal Chem. 78 3632-7. We present a new method for simplified low-quantity DNA loading onto microelectrophoresis devices. The method is based on combined solid-phase extraction, purification, and transport of DNA reversibly bound on paramagnetic microspheres. DNA is adsorbed onto the microspheres, captured with a magnetized permalloy wire, and then directly injected as a highly focused sample plug into the separation channel. This method circumvents both the minimum volume requirement of pipettors (since only solid beads are transported) and the timing complications of double-T microfluidic injection. Injections from Sanger samples of <100 pg total suspended weight match the signal strength of our previous conventional injections at >10-times the starting DNA sample. Sequencing traces show a resolution that matches or exceeds double-T injections. A kinetic model reproduces the time-dependence of the injection signals and proves that total nonidealities in the method produce injection-broadened plugs of approximately 1-s duration. The method should be broadly extendable to DNA and protein separations in both microdevice and capillary electrophoresis.Full Text

Um, M., Gross, A. W. and Lodish, H. F. (2006) A "classical" homodimeric erythropoietin receptor is essential for the antiapoptotic effects of erythropoietin on differentiated neuroblastoma SH-SY5Y and pheochromocytoma PC-12 cells. Cell Signal. The hematopoietic cytokine erythropoietin (Epo) exerts cytoprotective effects on several types of neuronal cells both in vivo and in culture. Detailed molecular mechanisms underlying this phenomenon have not been elucidated and even the identity of the cytoprotective Epo receptors in neuronal cells is controversial. Here we show that Epo prevents staurosporine-induced apoptosis of differentiated human neuroblastoma SH-SY5Y cells, and activates the STAT5, AKT and MAPK signaling pathways. Differentiated SH-SY5Y cells have fewer than 50 high affinity Epo surface binding sites per cell, which could not be detected by standard assays measuring binding of (125)I-labeled Epo. However, by measuring endocytosis of (125)I-Epo, we could reliably quantify very small numbers of high-affinity Epo surface binding sites. Using SH-SY5Y cells stably expressing an Epo receptor (EpoR) shRNA and thus lacking detectable EpoR expression, we show that high affinity binding of Epo to these neuronal cells is mediated by the hematopoietic EpoR, and that this EpoR is also essential for the antiapoptotic activity of Epo. In contrast, a mutant Epo that has an intact binding site 1 but a non-functional binding site 2 and hence binds only to one cell surface EpoR molecule ("site 2" Epo mutant) displays significantly lower antiapoptotic activity than wild-type Epo. Furthermore, expression of the GM-CSF/IL-3/IL-5 receptor common beta chain, which was proposed to be responsible for the cytoprotective activity of Epo on certain types of neuronal cells, was undetectable in differentiated SH-SY5Y cells. Epo also alleviated staurosporine-induced apoptosis of rat PC-12 pheochromocytoma cells while the R103A "site 2" Epo mutant did not, and we could not detect expression of the common beta chain in PC-12 cells. Together our results indicate that Epo exerts its antiapoptotic effects on differentiated SH-SY5Y and PC-12 cells through the standard stoichiometry of one molecule of Epo binding to two EpoR subunits, comprising the "classical" Epo receptor signaling complex Full Text

Um, M. and Lodish, H. F. (2006) Antiapoptotic effects of erythropoietin in differentiated neuroblastoma SH-SY5Y cells require activation of both the STAT5 and AKT signaling pathways. Journal of Biological Chemistry. 281 5648-5656. The hematopoietic cytokine erythropoietin (Epo) prevents neuronal death during ischemic events in the brain and in neurodegenerative diseases, presumably through its antiapoptotic effects. To explore the role of different signaling pathways in Epo-mediated antiapoptotic effects in differentiated human neuroblastoma SH-SY5Y cells, we employed a prolactin receptor (PrlR)/ erythropoietin receptor (EpoR) chimera system, in which binding of prolactin (Prl) to the extracellular domain activates EpoR signaling in the cytosol. On induction of apoptosis by staurosporine, Prl supports survival of the SH-SY5Y cells expressing the wild-type PrlR/ EpoR chimera. In these cells Prl treatment strongly activates the STAT5, AKT, and MAPK signaling pathways and induces weak activation of the p65 NF-kappa B factor. Selective mutation of the eight tyrosine residues of the EpoR cytoplasmic domain results in impaired or absent activation of either STAT5 ( mutation of Tyr(343)) or AKT ( mutation of Tyr(479)) or both ( mutation of all eight tyrosine residues). Most interestingly, Prl treatment does not prevent apoptosis in cells expressing mutant PrlR/ EpoR chimeras in which either the STAT5 or the AKT signaling pathways are not activated. In contrast, ERK 1/2 is fully activated by all mutant PrlR/ EpoR chimeras, comparable with the level seen with the wild-type PrlR/ EpoR chimera, implying that activation of the MAPK signaling pathway per se is not sufficient for antiapoptotic activity. Therefore, the antiapoptotic effects of Epo in neuronal cells require the combinatorial activation of multiple signaling pathways, including STAT5, AKT, and potentially MAPK as well, in a manner similar to that observed in hematopoietic cells. Full Text

Vaucheret, H., Mallory, A. C. and Bartel, D. P. (2006) AGO1 Homeostasis Entails Coexpression of MIR168 and AGO1 and Preferential Stabilization of miR168 by AGO1. Mol Cell. 22 129-36. Arabidopsis ARGONAUTE1 (AGO1) encodes the RNA slicer enzyme of the microRNA (miRNA) pathway and is regulated by miR168-programmed, AGO1-catalyzed mRNA cleavage. Here, we describe two additional regulatory processes required for AGO1 homeostasis: transcriptional coregulation of MIR168 and AGO1 genes, and posttranscriptional stabilization of miR168 by AGO1. Disrupting any of these regulatory processes by using mutations or transgenes disturbs a proper functioning of the miRNA pathway. In contrast, minor perturbation leads to fine-tuned posttranscriptional adjustment of miR168 and AGO1 levels, thereby maintaining a proper balance of other miRNAs, which, together with AGO1, control the mRNA levels of miRNA targets. We suggest that miR168 stabilization occurs at the level of silencing-complex assembly and that modulating the efficiency of assembling miRNA-programmed silencing complexes will also be important in other contexts. Full Text

Vishwanath, M., Nishibu, A., Saeland, S., Ward, B. R., Mizumoto, N., Ploegh, H. L., Boes, M. and Takashima, A. (2006) Development of intravital intermittent confocal imaging system for studying Langerhans cell turnover. Journal of Investigative Dermatology. 126 2452-2457. Although several studies have suggested relatively slow turnover of Langerhans cells (LCs), their actual lifespan remains elusive. Here we report the development of a new intravital imaging system for studying LC efflux and influx. Epidermal LCs expressing enhanced green fluorescent protein (EGFP) were visualized in anesthetized I-A beta-EGFP knock-in mice by confocal microscopy. By overlaying two sets of EGFP(+) LC images recorded in the same microscopic fields at time 0 and 24 hours later, we identified LC subpopulations that had disappeared from or newly emerged in the epidermis during that period. Of > 10,000 LCs analyzed in this manner, an overwhelming majority (97.8 +/- 0.2%) of LCs showed no significant changes in the x-y locations, whereas 1.3 +/- 0.1% of the LCs that were found at time 0 became undetectable 24 hours later, representing LC efflux. Conversely, 0.9 +/- 0.1% of the LCs that were found at time 24 hours were not detectable at time 0, representing LC influx. From these frequencies, we estimated the half-life of epidermal LCs to range from 53 to 78 days, providing new insights into the immunobiology of LCs. Our intermittent imaging approach may be regarded as a technical breakthrough enabling direct visual assessment of LC turnover in living animals.

Voynov, V., Verstrepen, K. J., Jansen, A., Runner, V. M., Buratowski, S. and Fink, G. R. (2006) Genes with internal repeats require the THO complex for transcription. Proc Natl Acad Sci U S A. Sep 18; [Epub ahead of print] The evolutionarily conserved multisubunit THO complex, which is recruited to actively transcribed genes, is required for the efficient expression of FLO11 and other yeast genes that have long internal tandem repeats. FLO11 transcription elongation in Tho(-) mutants is hindered in the region of the tandem repeats, resulting in a loss of function. Moreover, the repeats become genetically unstable in Tho(-) mutants. A FLO11 gene without the tandem repeats is transcribed equally well in Tho(+) or Tho(-) strains. The Tho(-) defect in transcription is suppressed by overexpression of topoisomerase I, suggesting that the THO complex functions to rectify aberrant structures that arise during transcription .PDF

Wang, J. L., Loveland, A. N., Kattenhorn, L. M., Ploegh, H. L. and Gibson, W. (2006) High-molecular-weight protein (pUL48) of human cytomegalovirus is a competent deubiquitinating protease: Mutant viruses altered in its active-site cysteine or histidine are viable. Journal of Virology. 80 6003-6012. We show here that the high-molecular-weight protein (HMWP or pUL48; 253 kDa) of human cytomegalovirus (HCMV) is a functionally competent deubiquitinating protease (DUB). By using a suicide substrate probe specific for ubiquitin-binding cysteine proteases (DUB probe) to screen lysates of HCMV-infected cells, we found just one infected-cell- specific DUB. Characteristics of this protein, including its large size, expression at late times of infection, presence in extracellular virus particles, and reactivity with an antiserum to the HMWP, identified it as the HMWP. This was confirmed by constructing mutant viruses with substitutions in two of the putative active-site residues, Cys24IIe and His162Ala. HMWP with these mutations either failed to bind the DUB probe (C241) or had significantly reduced reactivity with it (H162A). More compellingly, the deubiquitinating activity detected in wild-type virus particles was completely abolished in both the C241 and H162A mutants, thereby directly linking HMWP with deubiquitinating enzyme activity. Mutations in these active-site residues were not lethal to virus replication but slowed production of infectious virus relative to wild type and mutations of other conserved residues. Initial studies, by electron microscopy, of cells infected with the mutants revealed no obvious differences at late times of replication in the general appearance of the cells or in the distribution, relative numbers, or appearance of virus particles in the cytoplasm or nucleus.Full Text

Wardle, F. C., Odom, D. T., Bell, G. W., Yuan, B. B., Danford, T. W., Wiellette, E. L., Herbolsheimer, E., Sive, H. L., Young, R. A. and Smith, J. C. (2006) Zebrafish promoter microarrays identify actively transcribed embryonic genes. Genome Biology.7(8):R71 We have designed a zebrafish genomic microarray to identify DNA-protein interactions in the proximal promoter regions of over 11,000 zebrafish genes. Using these microarrays, together with chromatin immunoprecipitation with an antibody directed against tri-methylated lysine 4 of Histone H3, we demonstrate the feasibility of this method in zebrafish. This approach will allow investigators to determine the genomic binding locations of DNA interacting proteins during development and expedite the assembly of the genetic networks that regulate embryogenesis.Full Text

Weinberg, R. A. (2006) A lost generation. Cell. 126 9-10. The funding policies of the NIH have made it increasingly difficult for young researchers to procure research funds. This threatens to drive a whole generation of young people away from careers in basic biomedical research.Full Text

Weinberg, R. and Mihich, E. (2006) Eighteenth Annual Pezcoller Symposium: Tumor Microenvironment and Heterotypic Interactions. Cancer Research 2006; 66(24): 11550-3 This symposium was held in Trento, Italy, from June 27 to 29, 2006, and was co-chaired by Robert Weinberg and Enrico Mihich. The interactions between tumor cells and their microenvironment were discussed with particular emphasis on their molecular mechanisms. The roles of transforming growth factor beta signaling, urokinase, and matrix metalloproteinases in matrix remodeling; the effects of matrix-tumor interactions on cell proliferation and migration; the tumor-promoting effects of inflammation and of related host cell and cytokine functions; the signaling mechanisms affecting the biology of the stroma; and the mechanisms governing angiogenesis were discussed. PDF

Wheeler, R. T. and Fink, G. R. (2006) A drug-sensitive genetic network masks fungi from the immune system. PLoS Pathog. 2(4):e35. Fungal pathogens can be recognized by the immune system via their beta-glucan, a potent proinflammatory molecule that is present at high levels but is predominantly buried beneath a mannoprotein coat and invisible to the host. To investigate the nature and significance of "masking" this molecule, we characterized the mechanism of masking and consequences of unmasking for immune recognition. We found that the underlying beta-glucan in the cell wall of Candida albicans is unmasked by subinhibitory doses of the antifungal drug caspofungin, causing the exposed fungi to elicit a stronger immune response. Using a library of bakers' yeast (Saccharomyces cerevisiae) mutants, we uncovered a conserved genetic network that is required for concealing beta-glucan from the immune system and limiting the host response. Perturbation of parts of this network in the pathogen C. albicans caused unmasking of its beta-glucan, leading to increased beta-glucan receptor-dependent elicitation of key proinflammatory cytokines from primary mouse macrophages. By creating an anti-inflammatory barrier to mask beta-glucan, opportunistic fungi may promote commensal colonization and have an increased propensity for causing disease. Targeting the widely conserved gene network required for creating and maintaining this barrier may lead to novel broad-spectrum antimycotics. Full Text

Whetstine, J. R., Nottke, A., Lan, F., Huarte, M., Smolikov, S., Chen, Z. Z., Spooner, E., Li, E., Zhang, G. Y., Colaiacovo, M. and Shi, Y. (2006) Reversal of histone lysine trimethylation by the JMJD2 family of histone demethylases. Cell. 125 467-481. Histone methylation regulates chromatin structure, transcription, and epigenetic state of the cell. Histone methylation is dynamically regulated by histone methylases and demethylases such as LSD1 and JHDM1, which mediate demethylation of di- and monomethylated histones. It has been unclear whether demethylases exist that reverse lysine trimethylation. We show the JmjC domain-containing protein JMJD2A reversed trimethylated H3-K9/K36 to di- but not mono- or unmethylated products. Overexpression of JMJD2A but not a catalytically inactive mutant reduced H3-K9/K36 trimethylation levels in cultured cells. In contrast, RNAi depletion of the C. elegans JMJD2A homolog resulted in an increase in general H3-K9Me3 and localized H3-K36Me3 levels on meiotic chromosomes and triggered p53-dependent germline apoptosis. Additionally, other human JMJD2 subfamily members also functioned as trimethylation-specific demethylases, converting H3-K9Me3 to H3-K9Me2 and H3-K9Me1, respectively. Our finding that this family of demethylases generates different methylated states at the same lysine residue provides a mechanism for fine-tuning histone methylation. Full Text

Wiggins, P. A., Van der Heijden, T., Moreno-Herrero, F., Spakowitz, A., Phillips, R., Widom, J., Dekker, C. and Nelson, P. C. (2006) High flexibility of DNA on short length scales probed by atomic force microscopy. Nature Nanotechnology. 1 137-141. The mechanics of DNA bending on intermediate length scales ( 5 - 100 nm) plays a key role in many cellular processes, and is also important in the fabrication of artificial DNA structures, but previous experimental studies of DNA mechanics have focused on longer length scales than these. We use high-resolution atomic force microscopy on individual DNA molecules to obtain a direct measurement of the bending energy function appropriate for scales down to 5 nm. Our measurements imply that the elastic energy of highly bent DNA conformations is lower than predicted by classical elasticity models such as the worm-like chain (WLC) model. For example, we found that on short length scales, spontaneous large-angle bends are many times more prevalent than predicted by the WLC model. We test our data and model with an interlocking set of consistency checks. Our analysis also shows how our model is compatible with previous experiments, which have sometimes been viewed as confirming the WLC. Full Text

Wong, K. A. and Lodish, H. F. (2006) A revised model for AMPK structure: The alpha -subunit binds to both the beta - and gamma -subunits but there is no direct binding between beta - and gamma -subunits. J Biol Chem. 2006 Sep 29; [Epub ahead of print] The 5'-AMP-activated protein kinase (AMPK) is a master sensor for cellular metabolic energy state. It is activated by a high AMP/ATP ratio and leads to metabolic changes that conserve energy and utilize alternative cellular fuel sources. The kinase is composed of a heterotrimeric protein complex containing a catalytic alpha-subunit, an AMP-binding gamma-subunit, and a scaffolding beta-subunit thought to bind directly both the alpha- and gamma-subunits. Here, we use coimmunoprecipitation of proteins in transiently transfected cells to show that the alpha2-subunit binds directly not only to the beta-subunit, confirming previous work, but also to the gamma1-subunit. Deletion analysis of the alpha2-subunit reveals that the C-terminal 386-552 residues are sufficient to bind to the beta-subunit. The gamma1-subunit binds directly to the alpha2-subunit at two interaction sites, one within the catalytic domain consisting of alpha2 amino acids 1-312 and a second within residues 386-552. Binding of the alpha2 and the gamma1-subunits was not affected by 400microM AMP or ATP. Furthermore, we show that the beta-subunit C-terminus is essential for binding to the alpha2-subunit but, in contrast to previous work, the beta-subunit does not bind directly to the gamma1-subunit. Taken together, this study presents a new model for AMPK heterotrimer structure where through its C-terminus the beta-subunit binds to the alpha-subunit that, in turn, binds to the gamma-subunit. There is no direct interaction between the beta- and gamma-subunits.PDF

Workman, C. T., Mak, H. C., McCuine, S., Tagne, J. B., Agarwal, M., Ozier, O., Begley, T. J., Samson, L. D. and Ideker, T. (2006) A systems approach to mapping DNA damage response pathways. Science. 312 1054-1059. Failure of cells to respond to DNA damage is a primary event associated with mutagenesis and environmental toxicity. To map the transcriptional network controlling the damage response, we measured genomewide binding locations for 30 damage-related transcription factors (TFs) after exposure of yeast to methyl-methanesulfonate (MMS). The resulting 5272 TF-target interactions revealed extensive changes in the pattern of promoter binding and identified damage-specific binding motifs. As systematic functional validation, we identified interactions for which the target changed expression in wild-type cells in response to MMS but was nonresponsive in cells lacking the TF. Validated interactions were assembled into causal pathway models that provide global hypotheses of how signaling, transcription, and phenotype are integrated after damage.Full Text

Yang, J., Mani, S. A. and Weinberg, R. A. (2006) Exploring a new twist on tumor metastasis. Cancer Res. 66 4549-52. Unraveling the genetic programs that drive -metastasis may offer insights into how to limit or prevent this deadly aspect of cancer progression. Our recent studies indicate that tumor cell metastasis involves the activity of the transcription factor, Twist, which regulates epithelial-mesenchymal transition and early embryonic morphogenesis. Here, we review the Twist signaling pathway during normal development and discuss how the transcription factor Twist and the epithelial-mesenchymal transition program impinge their biological functions during tumor metastasis.Full Text

Yang, S, Yang, H. J., Grisafi, P., Sanchatjate, S., Fink, G. R., Sun, Q. and Hua, J. (2006) The BON/CPN gene family represses cell death and promotes cell growth in Arabidopsis. Plant Journal. 45 166-179. Copines are calcium-dependent membrane-binding proteins that are highly conserved among protozoa, plants, nematodes and mammals. Although they are implicated in membrane trafficking and signal transduction, the functions of these proteins are not well understood. The Arabidopsis copine gene BON1/CPN1 was previously shown to negatively regulate a disease resistance ( R) gene SNC1. Here we report that in Arabidopsis, as in other organisms, there is a family of copine genes, BON1, 2 and 3. Using double and triple mutant combinations we show that these three copine genes have overlapping functions essential for the viability of plants. The loss of function of BON1 combined with that of BON2 or BON3 leads to extensive cell death phenotypes resembling the hypersensitive response ( HR) in defense responses. The resulting lethality can be suppressed by mutations in PAD4 or EDS1 which are required for R gene signaling and cell death control. Accession-dependent phenotypes of the mutant combinations suggest that the BON/CPN genes may together repress several R genes other than SNC1. Moreover, the mutant combinations exhibit developmental defects when R-gene-mediated defense responses are largely suppressed in pad4 and eds1 mutants. Thus, the copine family in Arabidopsis may have effects in promoting growth and development in addition to repressing cell death, and these two processes might be intricately intertwined. Full Text

Zaman, M. H., Trapani, L. M., Siemeski, A., Mackellar, D., Gong, H., Kamm, R. D., Wells, A., Lauffenburger, D. A. and Matsudaira, P. (2006) Migration of tumor cells in 3D matrices is governed by matrix stiffness along with cell-matrix adhesion and proteolysis. Proc Natl Acad Sci U S A. 103 10889-94. Cell migration on 2D surfaces is governed by a balance between counteracting tractile and adhesion forces. Although biochemical factors such as adhesion receptor and ligand concentration and binding, signaling through cell adhesion complexes, and cytoskeletal structure assembly/disassembly have been studied in detail in a 2D context, the critical biochemical and biophysical parameters that affect cell migration in 3D matrices have not been quantitatively investigated. We demonstrate that, in addition to adhesion and tractile forces, matrix stiffness is a key factor that influences cell movement in 3D. Cell migration assays in which Matrigel density, fibronectin concentration, and beta1 integrin binding are systematically varied show that at a specific Matrigel density the migration speed of DU-145 human prostate carcinoma cells is a balance between tractile and adhesion forces. However, when biochemical parameters such as matrix ligand and cell integrin receptor levels are held constant, maximal cell movement shifts to matrices exhibiting lesser stiffness. This behavior contradicts current 2D models but is predicted by a recent force-based computational model of cell movement in a 3D matrix. As expected, this 3D motility through an extracellular environment of pore size much smaller than cellular dimensions does depend on proteolytic activity as broad-spectrum matrix metalloproteinase (MMP) inhibitors limit the migration of DU-145 cells and also HT-1080 fibrosarcoma cells. Our experimental findings here represent, to our knowledge, discovery of a previously undescribed set of balances of cell and matrix properties that govern the ability of tumor cells to migration in 3D environments.PDF

Zhang, C. C., Steele, A. D., Lindquist, S. and Lodish, H. F. (2006) Prion protein is expressed on long-term repopulating hematopoietic stem cells and is important for their self-renewal. Proc Natl Acad Sci U S A. 2006 Feb 14;103(7):2184-9.. Although the wild-type prion protein (PrP) is abundant and widely expressed in various types of tissues and cells, its physiological function(s) remain unknown, and PrP knockout mice do not exhibit overt and undisputed phenotypes. Here we showed that PrP is expressed on the surface of several bone marrow cell populations successively enriched in long-term (LT) hematopoietic stem cells (HSCs) using flow cytometry analysis. Affinity purification of the PrP-positive and -negative fractions from these populations, followed by competitive bone marrow reconstitution assays, shows that all LT HSCs express PrP. HSCs from PrP-null bone marrow exhibited impaired self-renewal in serial transplantation of lethally irradiated mouse recipients both in the presence and absence of competitors. When treated with a cell cycle-specific myelotoxic agent, the animals reconstituted with PrP-null HSCs exhibit increased sensitivity to hematopoietic cell depletion. Ectopic expression of PrP in PrP-null bone marrow cells by retroviral infection rescued the defective hematopoietic engraftment during serial transplantation. Therefore, PrP is a marker for HSCs and supports their self-renewal. Full Text

Zhang, C. C., Kaba, M., Ge, G., Xie, K., Tong, W., Hug, C. and Lodish, H. F. (2006) Angiopoietin-like proteins stimulate ex vivo expansion of hematopoietic stem cells. Nature Medicine Jan 22; [Epub ahead of print]. Successful ex vivo expansion of hematopoietic stem cells (HSCs) would greatly benefit the treatment of disease and the understanding of crucial questions of stem cell biology. Here we show, using microarray studies, that the HSC-supportive mouse fetal liver CD3(+) cells specifically express the proteins angiopoietin-like 2 (Angptl2) and angiopoietin-like 3 (Angptl3). We observed a 24- or 30-fold net expansion of long-term HSCs by reconstitution analysis when we cultured highly enriched HSCs for 10 days in the presence of Angptl2 or Angptl3 together with saturating levels of other growth factors. The coiled-coil domain of Angptl2 was capable of stimulating expansion of HSCs. Furthermore, angiopoietin-like 5, angiopoietin-like 7 and microfibril-associated glycoprotein 4 also supported expansion of HSCs in culture. Full Text

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