The following alphabetical list represents papers published in 2007 with at least one Whitehead author (in red). Not all of this work was done at the Whitehead Institute. Some of these papers are collaborations with scientists elsewhere. The papers are gathered from PubMed and from Science Citation Index (also known as the Web of Science) Preceding the bibliography is an alphabetical list of the titles of the papers followed by the first author.

P.S. The journal links only work if you have a license to those respective online journals.

-Ablation of de novo DNA methyltransferase Dnmt3a in the nervous system leads to neuromuscular defects and shortened lifespan. Nguyen
-Abnormal sperm in mice lacking the Taf7l (TBP-associated factor 7 like) gene. Cheng
-Activation and transposition of endogenous retroviral elements in hypomethylation induced tumors in mice. Howard
-Adaptation versus selection: the origins of metastatic behavior.Scheel
-AID-/-{micro}s-/- Mice Are Agammaglobulinemic and Fail to Maintain B220-CD138+ Plasma Cells.Kumazak
-Alternative Assembly Pathways of the Amyloidogenic Yeast Prion Determinant Sup35-Nm. Hess
-Analysis of integrin signaling in genetically engineered mouse models of mammary tumor progression Pylayeva
-Antifungal hydrogels. Zumbuehl
-Apicomplexan UCHL3 retains dual specificity for ubiquitin and Nedd8 throughout evolution. Frickel
-Asymmetric deceleration of ClpB or Hsp104 ATPase activity unleashes protein-remodeling activity Doyle
-Atypical Aaa+ Subunit Packing Creates an Expanded Cavity for Disaggregation by the Protein-Remodeling Factor Hsp104 Wendler
-Automated discovery of functional generality of human gene expression programs. Gerber
-Biological consequences of tightly bent DNA: The other life of a macromolecular celebrity.Garcia
-BMP signaling regulates the dorsal planarian midline and is needed for asymmetric regeneration.Reddien
-Bridging B cell and T cell recognition of antigen. Ploegh
-CellProfiler: free, versatile software for automated biological image analysis. Lamprecht
-Chemical Probes for the Rapid Detection of Fatty-Acylated Proteins in Mammalian Cells. Hang
-Chemical biology - Dressed-up proteins Grotenbreg
-Chemokine networks and breast cancer metastasis.Karnoub
-A chromatin landmark and transcription initiation at most promoters in human cells Guenther
-A "classical" homodimeric erythropoietin receptor is essential for the antiapoptotic effects of erythropoietin on differentiated neuroblastoma SH-SY5Y and pheochromocytoma PC-12 cells. Um
-Coding tandem repeats generate genetic diversity in Aspergillus fumigatus genes Levdansky
-Commentary: isolated stem cells--patentable as cultural artifacts? Hansson
-Common functions for diverse small RNAs of land plants.Axtell
-Cytosolic Delivery of Membrane-Impermeable Molecules in Dendritic Cells Using pH-Responsive Core-Shell Nanoparticles Hu
-Developmental role and regulation of cortex, a meiosis-specific anaphase-promoting complex/cyclosome activator.Pesin
-Defining the Role of mTOR in Cancer.Guertin
-Development of Gastric Tumors in ApcMin/+ Mice by the Activation of the {beta}-Catenin/Tcf Signaling Pathway.Tomita
-Developmentally regulated histone modifications in Drosophila follicle cells: initiation of gene amplification is associated with histone H3 and H4 hyperacetylation and H1 phosphorylation.Hartl
-DGCR8 is essential for microRNA biogenesis and silencing of embryonic stem cell self-renewal. Wang
-Differential priming of CD8 and CD4 T-Cells in animal models of autoimmune hepatitis and cholangitis.Derkow
-Dimerization by a cytokine receptor is necessary for constitutive activation of JAK2V617F.Lu
-Diminishing apoptosis by deletion of Bax or overexpression of Bcl-2 does not protect against infectious prion toxicity in vivo.Steele
-Direct reprogramming of genetically unmodified fibroblasts into pluripotent stem cells. Meissner
-Disrupting the Pairing Between let-7 and Hmga2 Enhances Oncogenic Transformation Mayr
-Dnmt3b promotes tumorigenesis in vivo by gene-specific de novo methylation and transcriptional silencing. Linhart
-DPL-1 DP, LIN-35 Rb, and EFL-1 E2F act with the MCD-1 Zinc-finger protein to promote programmed cell death in C. elegans. Reddien
-Drosophila follicle cell amplicons as models for metazoan DNA replication: A cyclinE mutant exhibits increased replication fork elongation. Park
-The Drosophila PNG Kinase Complex Regulates the Translation of Cyclin B. Vardy
-Early TCR expression and aberrant T cell development in mice with endogenous prerearranged T cell receptor genes Serwold
-Empty class II major histocompatibility complex created by peptide photolysis establishes the role of DM in peptide association. Grotenbreg
-Endogenous K-ras signaling in erythroid differentiation. Zhang
-Enrichment of a Population of Mammary Gland Cells that Form Mammospheres and Have In vivo Repopulating Activity. Liao
-Evolution, biogenesis, expression, and target predictions of a substantially expanded set of Drosophila microRNAs. Ruby
-The evolution of bet-hedging adaptations to rare scenarios.King
-Expression of oncogenic K-ras from its endogenous promoter leads to a partial block of erythroid differentiation and hyperactivation of cytokine-dependent signaling pathways Zhang
-Force of an actin spring. Shin
-Foxp3 occupancy and regulation of key target genes during T-cell stimulation. Marson
-A functional ubiquitin-specific protease embedded in the large tegument protein (ORF64) of murine gammaherpesvirus 68 is active during the course of infection. Gredmark
-Genome-wide maps of chromatin state in pluripotent and lineage-committed cells. Mikkelsen
-Heat shock factor 1 is a powerful multifaceted modifier of carcinogenesis .Dai
-A Herpesvirus Ubiquitin-Specific Protease Is Critical for Efficient T Cell Lymphoma Formation. Jarosinski
-Heterogeneity of stromal fibroblasts in tumors. Orimo
-Histone deacetylase inhibitors affect dendritic cell differentiation and immunogenicity.Nencione
-HOXA10 is a critical regulator for hematopoietic stem cells and erythroid/megakaryocyte development.Magnusson
-Human and Simian immunodeficiency virus-mediated upregulation of the apoptotic factor TRAIL occurs in antigen presenting cells from AIDS-susceptible but not from AIDS-resistant species. Kim
-Identification of novel superior polycationic vectors for gene delivery by high-throughput synthesis and screening of a combinatorial library.Thomas
-In vitro erythropoiesis from bone marrow-derived progenitors provides a physiological assay for toxic and mutagenic compounds.Shuga
-In vitro reprogramming of fibroblasts into a pluripotent ES-cell-like state. Wernig
-Identification and analysis of functional elements in 1% of the human genome by the ENCODE pilot project. Birney
-Illuminating aggregate heterogeneity in neurodegenerative disease. Jackson
-Intronic microRNA precursors that bypass Drosha processing Ruby
-The interaction between the ER membrane protein UNC93B and TLR3, 7, and 9 is crucial for TLR signaling. Brinkmann
-JAK2 exon 12 mutations in polycythemia vera and idiopathic erythrocytosis.Scott
-The large tegument protein (ORF64) of the mouse {gamma} herpesvirus MHV-68 encodes a functional ubiquitin-specific protease, expressed and active in the course of infection Gredmark
-A lipid-based model for the creation of an escape hatch from the endoplasmic reticulum.Ploegh
-The loss of adaptive plasticity during long periods of environmental stasis. Masel
-Mapping of meiotic single-stranded DNA reveals double-strand-break hotspots near centromeres and telomeres. Blitzblau
-Mat formation in Saccharomyces cerevisiae requires nutrient and pH gradients. Reynolds
-Mechanisms, biology and inhibitors of deubiquitinating enzymes. Love
-Mesenchymal stem cells within tumour stroma promote breast cancer metastasis. Karnoub
-Mesenchyme Forkhead 1 (FOXC2) plays a key role in metastasis and is associated with aggressive basal-like breast cancers. Mani
-MicroRNA Targeting Specificity in Mammals: Determinants beyond Seed Pairing Grimson
-Micrornas in Malignant Progression. Ma
-miR-150, a microRNA expressed in mature B and T cells, blocks early B cell development when expressed prematurely. Zhou
-Moving education forward.Lewitter
-MSY Breakpoint Mapper, a database of sequence-tagged sites useful in defining naturally occurring deletions in the human Y chromosome.Lange
-A natively unfolded yeast prion monomer adopts an ensemble of collapsed and rapidly fluctuating structures. Mukhopadhyay
-Nucleic acid and protein mass mapping by live-cell deep-ultraviolet microscopy. Zeskind
-Oct4 expression is not required for mouse somatic stem cell self-renewal. Lengner
-Off-target effects associated with long dsRNAs in Drosophila RNAi screens. Moffat
-On the cloning of animals from terminally differentiated cells. Hochedlinger
-The Parkinson's disease protein {alpha}-synuclein disrupts cellular Rab homeostasis. Gitler
-Partial rescue of MeCP2 deficiency by postnatal activation of MeCP2 Giacometti
-The peptide cargo of class I molecules: not just passive passengers Ploegh
-Phagocytosis by Human Neutrophils Is Stimulated by a Unique Fungal Cell Wall Component RubinBejerano
-Positioning the extreme anterior in Xenopus: Cement gland, primary mouth and anterior pituitary.Dickinson
-The power of automated high-resolution behavior analysis revealed by its application to mouse models of Huntington's and prion diseases.Steele
-PRAS40 Is an Insulin-Regulated Inhibitor of the mTORC1 Protein Kinase. Sancak
-Prime time for alpha-synuclein Gitler
-Prion recognition elements govern nucleation, strain specificity and species barriers.Tessier
-Probing the role of PrP repeats in conformational conversion and amyloid assembly of chimeric yeast prions.Dong
-Protein kinase C delta stimulates antigen presentation by Class II MHC in murine dendritic cells. Majewski
-Rapamycin derivatives reduce mTORC2 signaling and inhibit AKT activation in AML Zeng
-Reconstructing dynamic regulatory maps Ernst
-Regulating translation of maternal messages: multiple repression mechanisms.Vardy
-Regulation of erythrocyte lifespan: do reactive oxygen species set the clock? Hattangadi-A Rhizosphere Fungus Enhances Arabidopsis Thermotolerance Through Production of an HSP90 Inhibitor.McLellan
-RNA polymerase is poised for activation across the genome. Muse
-RNA polymerase stalling at developmental control genes in the Drosophila melanogaster embryo Zeitlinger
-The role of CaMKII as an F-actin-bundling protein crucial for maintenance of dendritic spine structure.Okamoto
-Sequence and structure evolved separately in a ribosomal ubiquitin variant. Catic
-Signals emanating from the membrane proximal region of the thrombopoietin receptor (mpl) support hematopoietic stem cell self-renewal. Tong
-Smed-{beta}catenin-1 Is Required for Anteroposterior Blastema Polarity in Planarian Regeneration. Petersen
-Software opens the door to quantitative imaging. Carpenter
-Sortagging: a versatile method for protein labeling .Popp
-Specific and covalent labeling of a membrane protein with organic fluorochromes and quantum dots. Bonasio
-Split ends antagonizes the Notch and potentiates the EGFR signaling pathways during Drosophila eye development..Doroquez
-Sprouty-2 regulates oncogenic K-ras in lung development and tumorigenesis Shaw
-Study of Cell-Cell Signaling in 3d Bacterial Arrays Assembled Using Optical Tweezers. Mirsaidov
-Structure of a herpesvirus-encoded cysteine protease reveals a unique class of deubiquitinating enzymes.Schlieker
-Substituted organosiloxanes as potential therapeutics for treatment and prevention of neurodegenerative diseases.Friedman
-A suite of Gateway((R)) cloning vectors for high-throughput genetic analysis in Saccharomyces cerevisiae.Alberti
-Tissue-specific transcriptional regulation has diverged significantly between human and mouse.Odom
-TLRs bent into shape. Kim
-Treatment of Sickle Cell Anemia Mouse Model with iPS. Hanna
-Tumour invasion and metastasis initiated by microRNA-10b in breast cancer. Ma
-Transformation of Different Human Breast Epithelial Cell Types Leads to Distinct Tumor Phenotypes .Ince
-Tubulation of Class II MHC Compartments Is Microtubule Dependent and Involves Multiple Endolysosomal Membrane Proteins in Primary Dendritic Cells Vyas
-Urmylation controls Nil1p and Gln3p-dependent expression of nitrogen-catabolite repressed genes in Saccharomyces cerevisiae RubioTexeira
-Using maths to tackle cancer.Weinberg
-Vascular Progenitor Cells Isolated From Human Embryonic Stem Cells Give Rise to Endothelial and Smooth Muscle-Like Cells and Form Vascular Networks In Vivo.Ferreira
-When killers become helpers. Hanna
-Whitesnake/sfpq is required for cell survival and neuronal development in the zebrafish .Lowery
-Whole-genome ChIP-chip analysis of Dorsal, Twist, and Snail suggests integration of diverse patterning processes in the Drosophila embryo. Zeitlinger
-YAP1 Increases Organ Size and Expands Undifferentiated Progenitor Cells. Camargo

Alberti, S., Gitler, A.D., and Lindquist, S. (2007). A suite of Gateway((R)) cloning vectors for high-throughput genetic analysis in Saccharomyces cerevisiae. Yeast.Jun 21; [Epub ahead of print] In the post-genomic era, academic and biotechnological research is increasingly shifting its attention from single proteins to the analysis of complex protein networks. This change in experimental design requires the use of simple and experimentally tractable organisms, such as the unicellular eukaryote Saccharomyces cerevisiae, and a range of new high-throughput techniques. The Gateway((R)) system has emerged as a powerful high-throughput cloning method that allows for the in vitro recombination of DNA with high speed, accuracy and reliability. Two Gateway-based libraries of overexpression plasmids containing the entire complement of yeast open reading frames (ORFs) have recently been completed. In order to make use of these powerful resources, we adapted the widely used pRS series of yeast shuttle vectors for use in Gateway-based cloning. The resulting suite of 288 yeast Gateway vectors is based upon the two commonly used GPD and GAL1 promoter expression systems that enable expression of ORFs, either constitutively or under galactose-inducible conditions. In addition, proteins of interest can be fused to a choice of frequently used N- or C-terminal tags, such as EGFP, ECFP, EYFP, Cerulean, monomeric DsRed, HA or TAP. We have made this yeast Gateway((R)) vector kit available to the research community via the non-profit Addgene Plasmid Repository (http://www.addgene.org/yeast_gateway).PDF

Axtell, M.J., Snyder, J.A., and Bartell, D.P. (2007). Common functions for diverse small RNAs of land plants. Plant Cell 19, 1750-1769. Endogenous small RNAs, including microRNAs ( miRNAs) and short interfering RNAs (siRNAs), are critical components of plant gene regulation. Some abundant miRNAs involved in developmental control are conserved between anciently diverged plants, while many other less-abundant miRNAs appear to have recently emerged in the Arabidopsis thaliana lineage. Using large-scale sequencing of small RNAs, we extended the known diversity of miRNAs in basal plants to include 88 confidently annotated miRNA families in the moss Physcomitrella patens and 44 in the lycopod Selaginella moellendorffii. Cleavage of 29 targets directed by 14 distinct P. patens miRNA families and a trans-acting siRNA (ta-siRNA) was experimentally confirmed. Despite a core set of 12 miRNA families also expressed in angiosperms, weakly expressed and apparently lineage-specific miRNAs accounted for the majority of miRNA diversity in both species. Nevertheless, the molecular functions of several of these lineage-specific small RNAs matched those of angiosperms, despite dissimilarities in the small RNA sequences themselves, including small RNAs that mediated negative feedback regulation of the miRNA pathway and miR390-dependent ta-siRNAs that guided the cleavage of AUXIN RESPONSE FACTOR mRNAs. Diverse, lineage-specific, small RNAs can therefore perform common biological functions in plants. Full Text.

Birney, E., Stamatoyannopoulos, J.A., Dutta, A., Guigo, R., Gingeras, T.R., Margulies, E.H., Weng, Z.P., Snyder, M., Dermitzakis, E.T., Stamatoyannopoulos, J.A., et al.(The ENCODE Project Consortium) (2007). Identification and analysis of functional elements in 1% of the human genome by the ENCODE pilot project. Nature 447, 799-816.We report the generation and analysis of functional data from multiple, diverse experiments performed on a targeted 1% of the human genome as part of the pilot phase of the ENCODE Project. These data have been further integrated and augmented by a number of evolutionary and computational analyses. Together, our results advance the collective knowledge about human genome function in several major areas. First, our studies provide convincing evidence that the genome is pervasively transcribed, such that the majority of its bases can be found in primary transcripts, including non-protein-coding transcripts, and those that extensively overlap one another. Second, systematic examination of transcriptional regulation has yielded new understanding about transcription start sites, including their relationship to specific regulatory sequences and features of chromatin accessibility and histone modification. Third, a more sophisticated view of chromatin structure has emerged, including its inter-relationship with DNA replication and transcriptional regulation. Finally, integration of these new sources of information, in particular with respect to mammalian evolution based on inter- and intra-species sequence comparisons, has yielded new mechanistic and evolutionary insights concerning the functional landscape of the human genome. Together, these studies are defining a path for pursuit of a more comprehensive characterization of human genome function.Full Text

Blitzblau, H.G., Bell, G.W., Rodriguez, J., Bell, S.P., and Hochwagen, A. (2007). Mapping of meiotic single-stranded DNA reveals double-strand-break hotspots near centromeres and telomeres. Current Biology 17, 2003-2012. Background: Every chromosome requires at least one crossover to be faithfully segregated during meiosis. At least two levels of regulation govern crossover distribution: where the initiating DNA double-strand breaks (DSBs) occur and whether those DSBs are repaired as crossovers. Results: We mapped meiotic DSBs in budding yeast by identifying sites of DSB-associated single-stranded DNA (ssDNA) accumulation. These analyses revealed substantial DSB activity in pericentrometric regions, in which crossover formation is largely absent. Our data suggest that centromeric suppression of recombination occurs at the level of break repair rather than DSB formation. Additionally, we found an enrichment of DSBs within a similar to 100 kb region near the ends of all chromosomes. Introduction of new telomeres was sufficient for inducing large ectopic regions of increased DSB formation, thereby revealing a remarkable long-range effect of telomeres on DSB formation. The concentration of DSBs close to chromosome ends increases the relative DSB density on small chromosomes, providing an interference-independent mechanism that ensures that all chromosomes receive at least one crossover per homolog pair. Conclusions: Together, our results indicate that selective DSB repair accounts for crossover suppression near centromeres and suggest a simple telomere-guided mechanism that ensures sufficient DSB activity on all chromosomes. Full Text.

Bonasio, R., Carman, C.V., Kim, E., Sage, P.T., Love, K.R., Mempel, T.R., Springer, T.A., and von Andrian, U.H. (2007). Specific and covalent labeling of a membrane protein with organic fluorochromes and quantum dots. Proceedings of the National Academy of Sciences of the United States of America 104, 14753-14758. The real-time observation of protein dynamics in living cells and organisms is of fundamental importance for understanding biological processes. Most approaches to labeling proteins exploit noncovalent interactions, unsuitable to long-term studies, or genetic fusion to naturally occurring fluorescent proteins that often have unsatisfactory optical properties. Here we used the fungal enzyme cutinase and its suicide substrate p-nitrophenyl phosphonate to covalently attach a variety of labels to the integrin lymphocyte function-associated antigen-1 (LFA-1) on the surface of living cells. Cutinase was embedded in the extracellular domain of LFA-1 with no appreciable influence on integrin function and conformational regulation. p-nitrophenyl phosphonate-conjugated fluorochromes, including the very bright and stable quantum dots, bound efficiently and specifically to LFA-1/cutinase. The availability of a genetically encoded tag that binds covalently to quantum dots could foster the development of new experimental strategies for the study of protein dynamics in Vivo. Full Text.

Brinkmann, M.M., Spooner, E., Hoebe, K., Beutler, B., Ploegh, H.L., and Kim, Y.M. (2007). The interaction between the ER membrane protein UNC93B and TLR3, 7, and 9 is crucial for TLR signaling. J Cell Biol 177, 265-275. Toll-like receptors (TLRs) sense the presence of microbial and viral pathogens by signal transduction mechanisms that remain to be fully elucidated. A single point mutation (H412R) in the polytopic endoplasmic reticulum (ER)-resident membrane protein UNC93B abolishes signaling via TLR3, 7, and 9. We show that UNC93B specifically interacts with TLR3, 7, 9, and 13, whereas introduction of the point mutation H412R in UNC93B abolishes their interactions. We establish the physical interaction of the intracellular TLRs with UNC93B in splenocytes and bone marrow-derived dendritic cells. Further, by expressing chimeric TLRs, we show that TLR3 and 9 bind to UNC93B via their transmembrane domains. We propose that a physical association between UNC93B and TLRs in the ER is essential for proper TLR signaling. Full Text

Camargo, F.D., Gokhale, S., Johnnidis, J.B., Fu, D., Bell, G.W., Jaenisch, R., and Brummelkamp, T.R. (2007). YAP1 Increases Organ Size and Expands Undifferentiated Progenitor Cells. Current Biology Articles in Press 31 Oct.. The mechanisms that regulate mammalian organ size are poorly understood. It is unclear whether the pathways that control organ size also impinge on stem/progenitor cells. A highly expressed gene in stem cells is YAP1[1], the ortholog of Drosophila Yorkie, a downstream component of the Hippo pathway [2]. Mutations in components of this pathway produce tissue overgrowth phenotypes in the fly whereas mammalian orthologs, like salvador [3], merlin [4], LATS[5], and YAP1[6, 7], have been implicated in tumorigenesis. We report here that YAP1 increases organ size and causes aberrant tissue expansion in mice. YAP1 activation reversibly increases liver size more than 4-fold. In the intestine, expression of endogenous YAP1 is restricted to the progenitor/stem cell compartment, and activation of YAP1 expands multipotent undifferentiated progenitor cells, which differentiate upon cessation of YAP1 expression. YAP1 stimulates Notch signaling, and administration of gamma-secretase inhibitors suppressed the intestinal dysplasia caused by YAP1. Human colorectal cancers expressing higher levels of YAP1 share molecular aspects with YAP1-induced dysplastic growth in the mouse. Our data show that the Hippo signaling pathway regulates organ size in mammals and can act on stem cell compartments, indicating a potential link between stem/progenitor cells, organ size, and cancer. Full Text.

Carpenter, A. E. (2007) Software opens the door to quantitative imaging. Nat Methods. 4 120-1. Full Text

Catic, A., Sun, Z.Y., Ratner, D.M., Misaghi, S., Spooner, E., Samuelson, J., Wagner, G., and Ploegh, H.L. (2007). Sequence and structure evolved separately in a ribosomal ubiquitin variant. Embo J.advance online publication 28 June 2007 Encoded by a multigene family, ubiquitin is expressed in the form of three precursor proteins, two of which are fusions to the ribosomal subunits S27a and L40. Ubiquitin assists in ribosome biogenesis and also functions as a post-translational modifier after its release from S27a or L40. However, several species do not conserve the ribosomal ubiquitin domains. We report here the solution structure of a distant variant of ubiquitin, found at the N-terminus of S27a in Giardia lamblia, referred to as GlUbS27a. Despite the considerable evolutionary distance that separates ubiquitin from GlUbS27a, the structure of GlUbS27a is largely identical to that of ubiquitin. The variant domain remains attached to S27a and is part of the assembled holoribosome. Thus, conservation of tertiary structure suggests a role of this variant as a chaperone, while conservation of the primary structure—necessary for ubiquitin's function as a post-translational modifier—is no longer required. Based on these observations, we propose a model to explain the origin of the widespread ubiquitin superfold in eukaryotes. PDF

Cheng, Y., Buffone, M. G., Kouadio, M., Goodheart, M., Page, D. C., Gerton, G. L., Davidson, I. and Wang, P. J. (2007) Abnormal sperm in mice lacking the Taf7l (TBP-associated factor 7 like) gene. Mol Cell Biol.Jan 22; [Epub ahead of print] TFIID is a general transcription factor required for transcription of most protein-coding genes by RNA polymerase II. TAF7L is an X-linked germ cell-specific paralogue of TAF7, which is a generally expressed component of TFIID. Here we report the generation of Taf7l mutant mice by homologous recombination in embryonic stem cells using the Cre-loxP strategy. While spermatogenesis was completed in Taf7l(-/Y) mice, the weight of Taf7l(-/Y) testis decreased and the amount of sperm in the epididymides was sharply reduced. Mutant epididymal sperm exhibited abnormal morphology including folded tails. Sperm motility was significantly reduced, and Taf7l(-/Y) males were fertile with reduced litter size. Microarray profiling revealed that the abundance of six gene transcripts (including Fscn1) in Taf7l(-/Y) testes decreased by > 2-fold. In particular, FSCN1 is an F-action-bundling protein and thus may be critical for normal sperm morphology and sperm motility. Although deficiency of Taf7l may be compensated in part by Taf7, Taf7l has apparently evolved new specialized functions in the gene-selective transcription in male germ-cell differentiation. Our mouse studies suggest that mutations in human TAF7L gene might be implicated in X-linked oligozoospermia in men .PDF

Dai, C., Whitesell, L., Rogers, A.B., and Lindquist, S. (2007). Heat shock factor 1 is a powerful multifaceted modifier of carcinogenesis. Cell 130, 1005-1018. Heat shock factor 1 (HSF1) is the master regulator of the heat shock response in eukaryotes, a very highly conserved protective mechanism. HSF1 function increases survival under a great many pathophysiological conditions. How it might be involved in malignancy remains largely unexplored. We report that eliminating HSF1 protects mice from tumors induced by mutations of the RAS oncogene or a hot spot mutation in the tumor suppressor p53. In cell culture, HSF1 supports malignant transformation by orchestrating a network of core cellular functions including proliferation, survival, protein synthesis, and glucose metabolism. The striking effects of HSF1 on oncogenic transformation are not limited to mouse systems or tumor initiation; human cancer lines of diverse origins show much greater dependence on HSF1 function to maintain proliferation and survival than their nontransformed counterparts. While it enhances organismal survival and longevity under most circumstances, HSF1 has the opposite effect in supporting the lethal phenomenon of cancer. Full Text.

Derkow, K., Loddenkemper, C., Mintern, J., Kruse, N., Klugewitz, K., Berg, T., Wiedenmann, B., Ploegh, H.L., and Schott, E. (2007). Differential priming of CD8 and CD4 T-Cells in animal models of autoimmune hepatitis and cholangitis. Hepatology 46, 1155-1165. The pathogenesis of autoimmune liver diseases is poorly understood. Animal models are necessary to investigate antigen presentation and priming of T-cells in the context of autoimmunity in the liver. Transgenic mouse models were generated in which the model antigen ovalbumin is expressed in hepatocytes (TF-OVA) or cholangiocytes (ASBT-OVA). Transgenic OT-I (CD8) or OT-II (CD4) T-cells specific for ovalbumin were adoptively transferred into TF-OVA and ASBTOVA mice to induce in vivo priming of antigen-specific T-cells. T-cell migration and activation, as well as induction of liver inflammation, were studied. OT-I T-cells preferentially located to the liver of both mouse strains whereas no migration of OT-II T-cells to the liver was observed. OT-I T-cells proliferated in the liver of TF-OVA mice and the liver and liver draining lymph nodes of ASBT-OVA mice. OT-II CD4 T-cells were activated in spleen and liver draining lymph node of TF-OVA mice but not in ASBT-OVA mice. Transfer of OT-I T-cells led to histologically distinct inflammatory conditions in the liver of ASBT-OVA and TF-OVA mice and caused liver injury as determined by the elevation of serum alanine aminotransferase. Conclusion: An antigen expressed in hepatocytes is presented to CD8 and CD4 T-cells, whereas the same antigen expressed in cholangiocytes is presented to CD8 but not CD4 T-cells. In both models, activation of CD8 T-cells occurs within the liver and causes liver inflammation. The models presented here are valuable to investigate the priming of T-cells in the liver and their role in the development of autoimmune disease of the liver. Full Text.

Dickinson, A., and Sive, H. (2007). Positioning the extreme anterior in Xenopus: Cement gland, primary mouth and anterior pituitary. Semin Cell Dev Biol.Apr 19; [Epub ahead of print] The extreme anterior of the deuterostome embryo is unusual in that ectoderm and endoderm are directly juxtaposed, without intervening mesoderm. In all vertebrates, this region gives rise to the anterior pituitary, the primary mouth and, in most frogs, to the mucus-secreting cement gland. Using the frog Xenopus laevis as a paradigm, we suggest that, initially, the extreme anterior forms a homogenous domain characterized by expression of pitx genes. Subsequently, this domain becomes subdivided to form these three different structures under the influence of different inductive signals from surrounding tissues.Full Text

Dong, J., Bloom, J.D., Goncharov, V., Chattopadhyay, M., Millhauser, G.L., Lynn, D.G., Scheibel, T., and Lindquist, S. (2007). Probing the role of PrP repeats in conformational conversion and amyloid assembly of chimeric yeast prions. J Biol Chem.Sep 24; [Epub ahead of print] Oligopeptide repeats appear in many proteins that undergo conformational conversions to form amyloid, including the mammalian prion protein PrP and the yeast prion protein Sup35. While the repeats in PrP have been studied more exhaustively, interpretation of these studies is confounded by the fact that many details of the PrP prion conformational conversion are not well understood. On the other hand, there is now a relatively good understanding of the factors that guide the conformational conversion of the Sup35 prion protein. In order to provide a general model for studying the role of oligopeptide repeats in prion conformational conversion and amyloid formation, we have substituted various numbers of the PrP octarepeats for the endogenous Sup35 repeats. The resulting chimeric proteins can adopt the [PSI+] prion state in yeast, and the stability of the prion state depends on the number of repeats. In vitro, these chimeric proteins form amyloid fibers, with more repeats leading to shorter lag phases and faster assembly rates. Both pH and the presence of metal ions modulate assembly kinetics of the chimeric proteins, and the extent of modulation is highly sensitive to the number of PrP repeats. This work offers new insight into the properties of the PrP octarepeats in amyloid assembly and prion formation. It also reveals new features of the yeast prion protein, and provides a level of control over yeast prion assembly that will be useful for future structural studies and for creating amyloid-based biomaterials. PDF

Doroquez, D.B., Orr-Weaver, T.L., and Rebay, I. (2007). Split ends antagonizes the Notch and potentiates the EGFR signaling pathways during Drosophila eye development. Mechanisms of Development 124 (9-10) : 792-806. The Notch and Epidermal Growth Factor Receptor (EGFR) signaling pathways interact cooperatively and antagonistically to regulate many aspects of Drosophila development, including the eye. How output from these two signaling networks is fine-tuned to achieve the precise balance needed for specific inductive interactions and patterning events remains an open and important question. Previously, we reported that the gene split ends (spen) functions within or parallel to the EGFR pathway during midline glial cell development in the embryonic central nervous system. Here, we report that the cellular defects caused by loss of spen function in the developing eye imaginal disc place spen as both an antagonist of the Notch pathway and a positive contributor to EGFR signaling during retinal cell differentiation. Specifically, loss of spen results in broadened expression of Scabrous, ectopic activation of Notch signaling, and a corresponding reduction in Atonal expression at the morphogenetic furrow. Consistent with Spen's role in antagonizing Notch signaling, reduction of spen levels is sufficient to suppress Notch-dependent phenotypes. At least in part due to loss of Spen-dependent down-regulation of Notch signaling, loss of spen also dampens EGFR signaling as evidenced by reduced activity of MAP kinase (MAPK). This reduced MAPK activity in turn leads to a failure to limit expression of the EGFR pathway antagonist and the ETS-domain transcriptional repressor Yan and to a corresponding loss of cell fate specification in spen mutant ommatidia. We propose that Spen plays a role in modulating output from the Notch and EGFR pathways to ensure appropriate patterning during eye development. Full Text.

Doyle, S. M., Shorter, J., Zolkiewski, M., Hoskins, J. R., Lindquist, S. and Wickner, S. (2007) Asymmetric deceleration of ClpB or Hsp104 ATPase activity unleashes protein-remodeling activity. Nature Structural & Molecular Biology. 14 114-122. Two members of the AAA+ superfamily, ClpB and Hsp104, collaborate with Hsp70 and Hsp40 to rescue aggregated proteins. However, the mechanisms that elicit and underlie their protein-remodeling activities remain unclear. We report that for both Hsp104 and ClpB, mixtures of ATP and ATP-gamma S unexpectedly unleash activation, disaggregation and unfolding activities independent of cochaperones. Mutations reveal how remodeling activities are elicited by impaired hydrolysis at individual nucleotide-binding domains. However, for some substrates, mixtures of ATP and ATP-gamma S abolish remodeling, whereas for others, ATP binding without hydrolysis is sufficient. Remodeling of different substrates necessitates a diverse balance of polypeptide 'holding' (which requires ATP binding but not hydrolysis) and unfolding (which requires ATP hydrolysis). We suggest that this versatility in reaction mechanism enables ClpB and Hsp104 to reactivate the entire aggregated proteome after stress and enables Hsp104 to control prion inheritance.Full Text

Ernst, J., Vainas, O., Harbison, C. T., Simon, I. and Bar-Joseph, Z. (2007) Reconstructing dynamic regulatory maps. Molecular Systems Biology 3 : 74 Even simple organisms have the ability to respond to internal and external stimuli. This response is carried out by a dynamic network of protein-DNA interactions that allows the specific regulation of genes needed for the response. We have developed a novel computational method that uses an input-output hidden Markov model to model these regulatory networks while taking into account their dynamic nature. Our method works by identifying bifurcation points, places in the time series where the expression of a subset of genes diverges from the rest of the genes. These points are annotated with the transcription factors regulating these transitions resulting in a unified temporal map. Applying our method to study yeast response to stress, we derive dynamic models that are able to recover many of the known aspects of these responses. Predictions made by our method have been experimentally validated leading to new roles for Ino4 and Gcn4 in controlling yeast response to stress. The temporal cascade of factors reveals common pathways and highlights differences between master and secondary factors in the utilization of network motifs and in condition-specific regulation.Full Text

Ferreira, L.S., Gerecht, S., Shieh, H.F., Watson, N., Rupnick, M.A., Dallabrida, S.M., Vunjak-Novakovic, G., and Langer, R. (2007). Vascular Progenitor Cells Isolated From Human Embryonic Stem Cells Give Rise to Endothelial and Smooth Muscle-Like Cells and Form Vascular Networks In Vivo. Circ Res. Jun 14; [Epub ahead of print] PDF

Frickel, E. M., Quesada, V., Muething, L., Gubbels, M. J., Spooner, E., Ploegh, H. and Artavanis-Tsakonas, K. (2007) Apicomplexan UCHL3 retains dual specificity for ubiquitin and Nedd8 throughout evolution. Cell Microbiol.Feb 15; [Epub ahead of print] Post-translational modification of proteins by ubiquitin or ubiquitin-like polypeptides such as Nedd8 controls cellular functions including protein degradation, the cell cycle and transcription. Here we have used an activity-based chemical probe that covalently labels ubiquitin hydrolases. We identify four such enzymes from Toxoplasma gondii by mass spectrometry. The homologue of mammalian UCHL3 was cloned from both T. gondii and Plasmodium falciparum and we show that both enzymes possess deubiquitinating as well as deNeddylating activity. A phylogenetic analysis of the UCHL3 amino acid sequences from several eukaryotes suggests that dual specificity for ubiquitin and Nedd8 was present in the ancestral eukaryotic UCHL3 and has been conserved throughout evolution. Finally, the structural characterization of UCHL3 from T. gondii shows a unique insertion at the surface of this enzyme, which may be involved in novel interactions with other proteins. The characterization of these apicomplexan UCHL3s adds to our understanding of the ubiquitin and Nedd8 pathways in these parasites.Full Text

Friedman, O.M., Matsudaira, P., Reis, A.H., Simister, N., Correia, I., Kew, D., Wei, J.Y., and Pochapsky, T. (2007). Substituted organosiloxanes as potential therapeutics for treatment and prevention of neurodegenerative diseases. Journal of Alzheimers Disease 11, 291-300. Extensive testing of hydrolysates of commercially available organosilanes has identified a number of bifunctional organosiloxane compounds that show potential as therapeutics for treatment of diseases characterized by amyloid deposition such as Alzheimer's disease (AD). All of these compounds protect from and/or reverse the metal-induced aggregation of amyloid A beta(1-42) peptide in dynamic light scattering (DLS) assays in trifluoroethanol (TFE) solutions, protect from and/or reverse the metal-induced loss of a-helical structure in TFE solutions of amyloid A beta (1-42) as measured by circular dichroism (CD), and are able to cross blood-brain barrier models at rates above background using Caco-2 and MDCK cell permeation assays. Based on these studies, we conclude that members of this class of bifunctional organosiloxanes are promising candidates for testing in treatment and/or prevention of AD and other diseases characterized by amyloid deposition.

Garcia, H. G., Grayson, P., Han, L., Inamdar, M., Kondev, J., Nelson, P. C., Phillips, R., Widom, J. and Wiggins, P. A. (2007) Biological consequences of tightly bent DNA: The other life of a macromolecular celebrity. Biopolymers. 85 115-130. The mechanical properties of DNA play a critical role in many biological functions. For example, DNA packing in viruses involves confining the viral genome in a volume (the viral capsid) with dimensions that are comparable to the DNA persistence length. Similarly, eukaryotic DNA is packed in DNA-protein complexes (nucleosomes), in which DNA is tightly bent around protein spools. DNA is also tightly bent by many proteins that regulate transcription, resulting in a variation in gene expression that is amenable to quantitative analysis. In these cases, DNA loops are formed with lengths that are comparable to or smaller than the DNA persistence length. The aim of this review is to describe the physical forces associatede with tightly bent DNA in all of these settings and to explore the biological consequences of such bending, as increasingly accessible by single-molecule techniques.Full Text

Gerber, G.K., Dowell, R.D., Jaakkola, T.S., and Gifford, D.K. (2007). Automated discovery of functional generality of human gene expression programs. Plos Computational Biology 3, 1426-1440. An important research problem in computational biology is the identification of expression programs, sets of coexpressed genes orchestrating normal or pathological processes, and the characterization of the functional breadth of these programs. The use of human expression data compendia for discovery of such programs presents several challenges including cellular inhomogeneity within samples, genetic and environmental variation across samples, uncertainty in the numbers of programs and sample populations, and temporal behavior. We developed GeneProgram, a new unsupervised computational framework based on Hierarchical Dirichlet Processes that addresses each of the above challenges. GeneProgram uses expression data to simultaneously organize tissues into groups and genes into overlapping programs with consistent temporal behavior, to produce maps of expression programs, which are sorted by generality scores that exploit the automatically learned groupings. Using synthetic and real gene expression data, we showed that GeneProgram outperformed several popular expression analysis methods. We applied GeneProgram to a compendium of 62 short time- series gene expression datasets exploring the responses of human cells to infectious agents and immune- modulating molecules. GeneProgram produced a map of 104 expression programs, a substantial number of which were significantly enriched for genes involved in key signaling pathways and/ or bound by NF- kappa B transcription factors in genome- wide experiments. Further, GeneProgram discovered expression programs that appear to implicate surprising signaling pathways or receptor types in the response to infection, including Wnt signaling and neurotransmitter receptors. We believe the discovered map of expression programs involved in the response to infection will be useful for guiding future biological experiments; genes from programs with low generality scores might serve as new drug targets that exhibit minimal " cross- talk,'' and genes from high generality programs may maintain common physiological responses that go awry in disease states. Further, our method is multipurpose, and can be applied readily to novel compendia of biological data. Full Text

Giacometti, E., Luikenhuis, S., Beard, C. and Jaenisch, R. (2007) Partial rescue of MeCP2 deficiency by postnatal activation of MeCP2. Proc Natl Acad Sci U S A. Feb 6;104(6):1931-6 In humans, mutations in the X-linked MECP2 gene, are the cause of Rett syndrome (RTT), a neurodevelopmental disorder that affects mainly girls. MeCP2 binds to methylated CpGs and is thought to act as a transcriptional repressor. In male mice, deletion or targeted mutation of Mecp2 leads to lethality and causes a neuronal phenotype. Selective mutation of Mecp2 in postnatal neurons results in a similar, although delayed, phenotype, suggesting that the symptoms are caused by MeCP2 deficiency in postmitotic neurons. In agreement with this idea, expression of a Mecp2 transgene in postmitotic neurons of Mecp2-null mutant mice resulted in the phenotypical rescue of the symptoms. To assess whether postnatal activation of MeCP2 in mutant animals could also affect the progression of the disorder, we constructed a conditionally active Mecp2 "rescue transgene" that was activated between P0 and P30. The Mecp2 transgene was under the control of the CAGGS promoter and was activated by using brain specific Cre-mediated recombination. Our results indicate that postnatal, neuron-specific activation of MeCP2 as late as 2-4 weeks of age significantly prolonged the lifespan of mutant animals and delayed the onset of neurologic symptoms.PDF

Gitler, A. D. and Shorter, J. (2007) Prime time for alpha-synuclein. J Neurosci. 27 2433-4. Full Text

Gitler, A.D., Bevis, B.J., Shorter, J., Strathearn, K.E., Hamamichi, S., Su, L.J., Caldwell, K.A., Caldwell, G.A., Rochet, J.C., McCaffery, J.M., Barlowe C, and Lindquisst S. The Parkinson's disease protein {alpha}-synuclein disrupts cellular Rab homeostasis. Proc Natl Acad Sci U S A.Dec 27; [Epub ahead of print] alpha-Synuclein (alpha-syn), a protein of unknown function, is the most abundant protein in Lewy bodies, the histological hallmark of Parkinson's disease (PD). In yeast alpha-syn inhibits endoplasmic reticulum (ER)-to-Golgi (ER-->Golgi) vesicle trafficking, which is rescued by overexpression of a Rab GTPase that regulates ER-->Golgi trafficking. The homologous Rab1 rescues alpha-syn toxicity in dopaminergic neuronal models of PD. Here we investigate this conserved feature of alpha-syn pathobiology. In a cell-free system with purified transport factors alpha-syn inhibited ER-->Golgi trafficking in an alpha-syn dose-dependent manner. Vesicles budded efficiently from the ER, but their docking or fusion to Golgi membranes was inhibited. Thus, the in vivo trafficking problem is due to a direct effect of alpha-syn on the transport machinery. By ultrastructural analysis the earliest in vivo defect was an accumulation of morphologically undocked vesicles, starting near the plasma membrane and growing into massive intracellular vesicular clusters in a dose-dependent manner. By immunofluorescence/immunoelectron microscopy, these clusters were associated both with alpha-syn and with diverse vesicle markers, suggesting that alpha-syn can impair multiple trafficking steps. Other Rabs did not ameliorate alpha-syn toxicity in yeast, but RAB3A, which is highly expressed in neurons and localized to presynaptic termini, and RAB8A, which is localized to post-Golgi vesicles, suppressed toxicity in neuronal models of PD. Thus, alpha-syn causes general defects in vesicle trafficking, to which dopaminergic neurons are especially sensitive.PDF.

Gredmark, S., Schlieker, C., Quesada, V., Spooner, E., and Ploegh, H.L. (2007). A functional ubiquitin-specific protease embedded in the large tegument protein (ORF64) of murine gammaherpesvirus 68 is active during the course of infection. Journal of Virology 81, 10300-10309. All herpesviruses contain a ubiquitin (Ub)-specific cysteine protease domain embedded within their large tegument protein, based on homology with the corresponding sequences of UL36 from herpes simplex virus type 1 and M48 from murine cytomegalovirus. This type of activity has yet to be demonstrated for cells infected with a gammaherpesvirus. By activity-based profiling, we show that the large tegument protein of murine gammaherpesvirus (MHV-68) ORF64 (273 kDa) is a functional deubiquitinating protease, as assessed by tandem mass spectrometry of adducts in extracts from MHV-68-infected cells that had been labeled with ubiquitin vinylmethylester, a ubiquitin-based active site-directed probe. The recombinantly expressed aminoterminal segment of ORF64 displays deubiquitinating activity toward Ub C-terminal 7-amido-4-methylcoumarin in vitro. The findings reported here for MHV-68 ORF64 extend those made for the alpha- and betaherpesvirus families and are consistent with an important, conserved enzymatic function of the tegument protein. Full Text.

Gredmark, S., Schlieker, C., Quesada, V., Spooner, E., and Ploegh, H.L. (2007). The large tegument protein (ORF64) of the mouse {gamma} herpesvirus MHV-68 encodes a functional ubiquitin-specific protease, expressed and active in the course of infection. J Virology Jul 18; [Epub ahead of print] All herpes viruses contain a ubiquitin (Ub)-specific cysteine protease domain embedded within their large tegument protein, based on homology with the corresponding sequences of UL36 from HSV-1 and M48 from MCMV. This type of activity has yet to be demonstrated for cells infected with a gamma herpes virus. By activity-based profiling we show that the large tegument protein encoded by the murine gamma herpes virus (MHV-68) ORF64 (273 kDa) is a functional deubiquitinating protease, as assessed by tandem mass spectrometry of adducts in extracts from MHV-68 infected cells that had been labeled with ubiquitin vinylmethylester, a ubiquitin-based active site-directed probe. The recombinantly expressed amino terminal segment of ORF64 displays deubiquitinating activity towards Ub-AMC in vitro. The findings reported here for MHV-68 ORF64 extend those made for the alpha and the beta herpesvirus families, and are consistent with an important, conserved enzymatic function of the tegument protein PDF

Grimson, A., Farh, K.K., Johnston, W.K., Garrett-Engele, P., Lim, L.P., and Bartel, D.P. (2007). MicroRNA Targeting Specificity in Mammals: Determinants beyond Seed Pairing. Mol Cell 27, 91-105. Mammalian microRNAs (miRNAs) pair to 3?UTRs of mRNAs to direct their posttranscriptional repression. Important for target recognition are not, vert, similar7 nt sites that match the seed region of the miRNA. However, these seed matches are not always sufficient for repression, indicating that other characteristics help specify targeting. By combining computational and experimental approaches, we uncovered five general features of site context that boost site efficacy: AU-rich nucleotide composition near the site, proximity to sites for coexpressed miRNAs (which leads to cooperative action), proximity to residues pairing to miRNA nucleotides 13–16, positioning within the 3?UTR at least 15 nt from the stop codon, and positioning away from the center of long UTRs. A model combining these context determinants quantitatively predicts site performance both for exogenously added miRNAs and for endogenous miRNA-message interactions. Because it predicts site efficacy without recourse to evolutionary conservation, the model also identifies effective nonconserved sites and siRNA off-targets. Full Text

Grotenbreg, G.M., Nicholson, M.J., Fowler, K.D., Wilbuer, K., Octavio, L., Yang, M.X., Chakraborty, A.K., Ploegh, H.L., and Wucherpfennig, K.W. (2007). Empty class II major histocompatibility complex created by peptide photolysis establishes the role of DM in peptide association. Journal of Biological Chemistry 282, 21425-21436. DM catalyzes the exchange of peptides bound to Class II major histocompatibility complex (MHC) molecules. Because the dissociation and association components of the overall reaction are difficult to separate, a detailed mechanism of DM catalysis has long resisted elucidation. UV irradiation of DR molecules loaded with a photocleavable peptide (caged Class II MHC molecules) enabled synchronous and verifiable evacuation of the peptide-binding groove and tracking of early binding events in real time by fluorescence polarization. Empty DR molecules generated by photocleavage rapidly bound peptide but quickly resolved into species with substantially slower binding kinetics. DM formed a complex with empty DR molecules that bound peptide with even faster kinetics than empty DR molecules just having lost their peptide cargo. Mathematical models demonstrate that the peptide association rate of DR molecules is substantially higher in the presence of DM. We therefore unequivocally establish that DM contributes directly to peptide association through formation of a peptide-loading complex between DM and empty Class II MHC. This complex rapidly acquires a peptide analogous to the MHC class I peptide-loading complex. Full Text.

Grotenbreg, G., and Ploegh, H. (2007). Chemical biology - Dressed-up proteins. Nature 446, 993-995. Proteins aren't just defined by their constituent amino acids — structural modifications can yield complex mixtures of protein forms. An approach that controls the addition of such modifications may help to define their role.Full Text

Guenther, M.G., Levine, S.S., Boyer, L.A., Jaenisch, R., and Young, R.A. (2007). A chromatin landmark and transcription initiation at most promoters in human cells. Cell 130, 77-88 We describe the results of a genome-wide analysis of human cells that suggests that most protein-coding genes, including most genes thought to be transcriptionally inactive, experience transcription initiation. We found that nucleosomes with H3K4me3 and H3K9,14Ac modifications, together with RNA polymerase II, occupy the promoters of most protein-coding genes in human embryonic stem cells. Only a subset of these genes produce detectable full-length transcripts and are occupied by nucleosomes with H3K36me3 modifications, a hallmark of elongation. The other genes experience transcription initiation but show no evidence of elongation, suggesting that they are predominantly regulated at postinitiation steps. Genes encoding most developmental regulators fall into this group. Our results also identify a class of genes that are excluded from experiencing transcription initiation, at which mechanisms that prevent initiation must predominate. These observations extend to differentiated cells, suggesting that transcription initiation at most genes is a general phenomenon in human cells.. Full Text

Guertin, D.A., and Sabatini, D.M. (2007). Defining the Role of mTOR in Cancer. Cancer Cell 12, 9-22. The mammalian target of rapamycin (mTOR) has emerged as a critical effector in cell-signaling pathways commonly deregulated in human cancers. This has led to the prediction that mTOR inhibitors may be useful in oncology, and derivatives of one such molecule, rapamycin (from which mTOR derives its name), are currently in clinical development. In this review, we discuss recent progress in understanding mTOR signaling, paying particular attention to its relevance in cancer. We further discuss the use of rapamycin in oncology and conclude with a discussion on the future of mTOR-targeted therapy.Full Text

Hang, H. C., Geutjes, E. J., Grotenbreg, G., Pollington, A. M., Bijlmakers, M. J. and Ploegh, H. L. (2007) Chemical Probes for the Rapid Detection of Fatty-Acylated Proteins in Mammalian Cells. J Am Chem Soc. 2007 Feb 17; [Epub ahead of print] The attachment of lipids onto proteins modulates the activity of proteins in many biological settings. The analysis of protein lipidation, however, is challenging due to the relatively few methods for the detection of lipid-modified proteins. Here we describe the synthesis of omega-azido-fatty acids as non-radioactive chemical probes for the rapid visualization of fatty-acylated proteins in mammalian cells. Following metabolic installation of the omega-azido-fatty acids onto target proteins by cellular enzymes, fatty-acylated proteins are selectively biotinylated with a phosphine-biotin reagent via the Staudinger ligation and visualized by streptavidin blotting. Depending on the chain length of the omega-azido-fatty acids, N-myristoylated and S-palmitoylated proteins can be visualized selectively in cell lysates and on specific proteins. These chemical probes provide new tools to analyze fatty acylation of proteins in living cells.Full Text

Hanna, J., Wernig, M., Markoulaki, S., Sun, C.W., Meissner, A., Cassady, J.P., Beard, C., Brambrink, T., Wu, L.C., Townes, T.M., and Jaenisch R. (2007). Treatment of Sickle Cell Anemia Mouse Model with iPS Cells Generated from Autologous Skin. Science Express Advance of Print 12.6.07. It has recently been demonstrated that mouse and human fibroblasts can be reprogrammed into an embryonic stem cell-like state by introducing combinations of four transcription factors. However, the therapeutic potential of such induced pluripotent stem (iPS) cells remained undefined. By using a humanized sickle cell anemia mouse model, we show that mice can be rescued after transplantation with hematopoietic progenitors obtained in vitro from autologous iPS cells. This was achieved after correction of the human sickle hemoglobin allele by gene-specific targeting. Our results provide proof of principle for using transcription factor-induced reprogramming combined with gene and cell therapy for disease treatment in mice. The problems associated with using retroviruses and oncogenes for reprogramming need to be resolved before iPS cells can be considered for human therapy.PDF.

Hanna, J., and Mandelboim, O. (2007). When killers become helpers. Trends in Immunology.Mar 31; [Epub ahead of print] Since their initial characterization by Kiessling over 35 years ago, natural killer (NK) cells continue to constitute an area of intensive discovery in the immunology field. Although most of the research efforts concentrated on characterizing the role of NK cells in tumor prevention and fighting infection through the killing of dangerous cells, several recent findings highlight unexpected non-cytolytic functions of human and mouse NK cells. Such functions include promoting placental tissue development, antigen presentation and stimulation of T cells, priming of macrophages and dendritic cells, reducing transplant tissue rejection and several others.

Hansson, M.G., Helgesson, G., Wessman, R., and Jaenisch, R. (2007). Commentary: isolated stem cells--patentable as cultural artifacts? Stem Cells 25, 1507-1510. This article argues that an isolated embryonic stem cell basically represents a cultural artifact that has no equivalent to cells of the embryo, and that it is likely that the isolation of adult stem cells has a similar consequence. An isolated stem cell could thus be distinguished as something other than the stem cell existing as part of a human body. Since isolation of stem cells implies modification, product patents should, where the results carry enough novelty, inventive step, and potential for industrial application, as a matter of principle be a viable option for patent authorities. Questions of morality, which may affect the patentability, should also be viewed in light of the distinction between isolated result and body part. At the same time, it is essential that patent authorities do not accept broad patent claims that will be detrimental to research. Disclosure of potential conflicts of interest is found at the end of this article. Full Text.

Hartl, T., Boswell, C., Orr-Weaver, T.L., and Bosco, G. (2007). Developmentally regulated histone modifications in Drosophila follicle cells: initiation of gene amplification is associated with histone H3 and H4 hyperacetylation and H1 phosphorylation. Chromosoma 116, 197-214. We have used gene amplification in Drosophila follicle cells as a model of metazoan DNA replication to address whether changes in histone modifications are associated with replication origin activation. We observe that replication initiation is associated with distinct histone modifications. Acetylated lysines K5, K8, and K12 on histone H4 and K14 on histone H3 are specifically enriched during replication initiation at the amplification origins. Strikingly, H4 acetylation persists at an amplification origin well after replication forks have progressed significantly outward from the origin, indicating that H4 acetylation is associated with origin regulation and not histone deposition at the replication forks. Origin recognition complex subunit 2 (orc2) mutants with severe amplification defects do not abolish H4 acetylation, whereas the dup/cdt1 mutant delays the appearance of acetylation foci, and mutants in rbf result in temporal persistence. These data indicate that core histone acetylation is associated with origin activity. Furthermore, follicle cells undergoing gene amplification exhibit high levels of histone H1 phosphorylation. The patterns of H1 phosphorylation provide insights into cell cycle states during amplification, as H1 kinase activity in follicle cells is responsive to high Cyclin E activity, and it can be abolished by overexpressing the retinoblastoma homolog, Rbf, that represses Cyclin E. These data suggest that amplification origins are able to initiate when the cells are in a late S-phase, when the genome is normally not licensed for replication Full Text

Hattangadi, S.M., and Lodish, H.F. (2007). Regulation of erythrocyte lifespan: do reactive oxygen species set the clock? J Clin Invest 117, 2075-2077.The forkhead box O (Foxo) subfamily of transcription factors regulates expression of genes important for many cellular processes, ranging from initiation of cell cycle arrest and apoptosis to induction of DNA damage repair. Invertebrate Foxo orthologs such as DAF-16 also regulate longevity. Cellular responses inducing resistance to ROS are important for cellular survival and organism lifespan, but until recently, mammalian factors regulating resistance to oxidative stress have not been well characterized. Marinkovic and colleagues demonstrate in this issue of the JCI that Foxo3 is specifically required for induction of proteins that regulate the in vivo oxidative stress response in murine erythrocytes (see the related article beginning on page 2133). Their work offers the interesting hypothesis that in so doing, Foxo3 may regulate the lifespan of red blood cells, and underlies the importance of understanding the direct targets of this transcription factor and its regulation.Full Text

Hess, S., Lindquist, S.L., and Scheibel, T. (2007). Alternative Assembly Pathways of the Amyloidogenic Yeast Prion Determinant Sup35-Nm. Embo Reports 8, 1196-1201. The self-perpetuating conformational change of the translation termination factor Sup35 is associated with a prion phenomenon of Saccharomyces cerevisiae. In vitro, the prion-determining region (NM) of Sup35 assembles into amyloid-like fibres through a mechanism of nucleated conformational conversion. Here, we describe an alternative assembly pathway of NM that produces filaments that are composed of b-strands and random coiled regions with several-fold smaller diameters than the amyloid fibres. NM filaments are not detectable with either thioflavin T or Congo Red and do not show SDS or protease resistance. As filaments do not self-convert into fibres and do not act as seed, they are not intermediates of amyloid fibre formation. Instead, they represent a stable off-pathway form. Similar to mammalian prion proteins, Sup35 contains oligopeptide repeats located in the NM region. We found that the number of repeats determines the partitioning of the protein between filaments and amyloid-like fibres. Low numbers of repeats favour the formation of the filamentous structure, whereas high numbers of repeats favour the formation of amyloid-like fibres. Full Text.

Hochedlinger, K. and Jaenisch, R. (2007) On the cloning of animals from terminally differentiated cells. Nature Genetics. 39 136-137. Full Text

Howard, G., Eiges, R., Gaudet, F., Jaenisch, R., and Eden, A. (2007). Activation and transposition of endogenous retroviral elements in hypomethylation induced tumors in mice. Oncogene. Advance online publication, 9 July 2007 Genomewide DNA hypomethylation is a consistent finding in human tumors, but the importance of this change for human tumorigenesis remains an open question. We have previously reported that mice carrying a hypomorphic allele for the maintenance DNA methyltransferase (Dnmt1(chip/-)) are hypomethylated and develop thymic lymphomas, demonstrating that genomewide DNA hypomethylation can induce tumors. Hypomethylated cells exhibit inherent chromosomal instability, which is revealed in the lymphomas as a consistent trisomy of chromosome 15. We now report another aspect of the molecular basis for tumor development upon DNA hypomethylation. Seven out of 16 hypomethylation-induced lymphomas were found to contain an intracisternal A particle (IAP) somatic insertion in the middle of the Notch1 genomic locus, leading to generation of an oncogenic form of Notch1 in the tumors. This finding suggests that the molecular basis for hypomethylation-induced tumors in this model involves chromosomal instability events accompanied by activation of endogenous retroviral elements. Our findings validate the proposed role of DNA methylation in suppression of transposable elements in mammalian cells and demonstrate the importance of DNA methylation for normal cell function as well as the potential consequences of spontaneously occurring or chemically induced DNA hypomethylation.. Full Text.

Hu, Y., Litwin, T., Nagaraja, A.R., Kwong, B., Katz, J., Watson, N., and Irvine, D.J. (2007). Cytosolic Delivery of Membrane-Impermeable Molecules in Dendritic Cells Using pH-Responsive Core-Shell Nanoparticles. Nano Letters (Articles ASAP). Polycations that absorb protons in response to the acidification of endosomes can theoretically disrupt these vesicles via the "proton sponge" effect. To exploit this mechanism, we created nanoparticles with a segregated core-shell structure for efficient, noncytotoxic intracellular drug delivery. Cross-linked polymer nanoparticles were synthesized with a pH-responsive core and hydrophilic charged shell designed to disrupt endosomes and mediate drug/cell binding, respectively. By sequestering the relatively hydrophobic pH-responsive core component within a more hydrophilic pH-insensitive shell, nontoxic delivery of small molecules and proteins to the cytosol was achieved in dendritic cells, a key cell type of interest in the context of vaccines and immunotherapy. Full Text.

Ince, T.A., Richardson, A.L., Bell, G.W., Saitoh, M., Godar, S., Karnoub, A.E., Iglehart, J.D., and Weinberg, R.A. (2007). Transformation of Different Human Breast Epithelial Cell Types Leads to Distinct Tumor Phenotypes. Cancer Cell 12, 160-170. We investigated the influence of normal cell phenotype on the neoplastic phenotype by comparing tumors derived from two different normal human mammary epithelial cell populations, one of which was isolated using a new culture medium. Transformation of these two cell populations with the same set of genetic elements yielded cells that formed tumor xenografts exhibiting major differences in histopathology, tumorigenicity, and metastatic behavior. While one cell type (HMECs) yielded squamous cell carcinomas, the other cell type (BPECs) yielded tumors closely resembling human breast adenocarcinomas. Transformed BPECs gave rise to lung metastases and were up to 10(4)-fold more tumorigenic than transformed HMECs, which are nonmetastatic. Hence, the pre-existing differences between BPECs and HMECs strongly influence the phenotypes of their transformed derivatives. Full Text

Jackson, W.S., and Lindquist, S. (2007). Illuminating aggregate heterogeneity in neurodegenerative disease. Nat Methods 4(12) : 1000-1001 .Luminescent conjugated polymers (LCPs) bind to prion aggregates and emit different fluorescent spectra depending on their binding conformation. As such, they are promising tools for investigating the biophysical basis of prion strains. Full Text.

Jarosinski, K., Kattenhorn, L., Kaufer, B., Ploegh, H., and Osterrieder, N. (2007). A Herpesvirus Ubiquitin-Specific Protease Is Critical for Efficient T Cell Lymphoma Formation. Proceedings of the National Academy of Sciences of the United States of America 104, 20025-20030. The herpesvirus ubiquitin-specific protease (USP) family, whose founding member was discovered as a protease domain embedded in the large tegument protein of herpes simplex virus 1 (HSV-1), is conserved across all members of the Herpesviridae. Whether this conservation is indicative of an essential function of the enzyme in vivo has not yet been established. As reported here, USP activity is conserved in Marek's disease virus (MDV), a tumorigenic alpha-herpesvirus. A single amino acid substitution that abolishes the USP activity of the MDV large tegument protein diminishes MDV replication in vivo, and severely limits the oncogenic potential of the virus. Expression of the USP transcripts in MDV-transformed cell lines further substantiates this hypothesis. The herpesvirus USP thus appears to be required not only to maintain a foothold in the immunocompetent host, but also to contribute to malignant out-growths. Full Text.

Karnoub, A.E., Dash, A.B., Vo, A.P., Sullivan, A., Brooks, M.W., Bell, G.W., Richardson, A.L., Polyak, K., Tubo, R., and Weinberg, R.A. (2007). Mesenchymal stem cells within tumour stroma promote breast cancer metastasis. Nature 449, 557-563. Mesenchymal stem cells have been recently described to localize to breast carcinomas, where they integrate into the tumour-associated stroma. However, the involvement of mesenchymal stem cells (or their derivatives) in tumour pathophysiology has not been addressed. Here, we demonstrate that bone-marrow-derived human mesenchymal stem cells, when mixed with otherwise weakly metastatic human breast carcinoma cells, cause the cancer cells to increase their metastatic potency greatly when this cell mixture is introduced into a subcutaneous site and allowed to form a tumour xenograft. The breast cancer cells stimulate de novo secretion of the chemokine CCL5 (also called RANTES) from mesenchymal stem cells, which then acts in a paracrine fashion on the cancer cells to enhance their motility, invasion and metastasis. This enhanced metastatic ability is reversible and is dependent on CCL5 signalling through the chemokine receptor CCR5. Collectively, these data demonstrate that the tumour microenvironment facilitates metastatic spread by eliciting reversible changes in the phenotype of cancer cells. Full Text.

Kim, N., Dabrowska, A., Jenner, R.G., and Aldovini, A. (2007). Human and Simian immunodeficiency virus-mediated upregulation of the apoptotic factor TRAIL occurs in antigen presenting cells from AIDS-susceptible but not from AIDS-resistant species. J Virol. May 9; [Epub ahead of print] PDF

Kim, Y.M., Brinkmann, M.M., and Ploegh, H.L. (2007). TLRs bent into shape. Nat Immunol 8, 675-677. Toll-like receptors respond to ligands embedded in pathogen-derived macromolecules to induce immune responses. Binding of a stimulatory ligand to preexisting dimers of Toll-like receptor 9 induces conformational changes that lead to their full activation.Full Text

King, O.D., and Masel, J. (2007). The evolution of bet-hedging adaptations to rare scenarios. Theoretical Population Biology (Articles in Press 31 August) When faced with a variable environment, organisms may switch between different strategies according to some probabilistic rule. In an infinite population, evolution is expected to favor the rule that maximizes geometric mean fitness. If some environments are encountered only rarely, selection may not be strong enough for optimal switching probabilities to evolve. Here we calculate the evolution of switching probabilities in a finite population by analyzing fixation probabilities of alleles specifying switching rules. We calculate the conditions required for the evolution of phenotypic switching as a form of bet-hedging as a function of the population size N, the rate theta at which a rare environment is encountered, and the selective advantage s associated with switching in the rare environment. We consider a simplified model in which environmental switching and phenotypic switching are one-way processes, and mutation is symmetric and rare with respect to the timescale of fixation events. In this case, the approximate requirements for bet-hedging to be favored by a ratio of at least R are that sN>log(R) and thetaN>R .

Kumazaki, K., Tirosh, B., Maehr, R., Boes, M., Honjo, T. and Ploegh, H. L. (2007) AID-/-{micro}s-/- Mice Are Agammaglobulinemic and Fail to Maintain B220-CD138+ Plasma Cells. J Immunol. 178 2192-203. The terminal stage of B cell differentiation culminates in the formation of plasma cells (PC), which secrete large quantities of Igs. Despite recent progress in understanding the molecular aspect of PC differentiation and maintenance, the requirement for the synthesis of secretory Igs as a contributing factor has not been explored. To address this issue, we generated activation-induced cytidine deaminase (AID)/secretory mu-chain (mus) double-knockout mice, in which a normally diverse repertoire of B cell receptors is retained, yet B cells are unable to synthesize secretory Igs. These mice possess polyclonal B cells but have no serum Igs. Following immunization in vivo, PCs, identified by CD138 expression and loss of the B220 marker, were starkly reduced in number in spleen and bone marrow of AID(-/-)mus(-/-) agammaglobulinemic mice compared with wild-type mice. Upon mitogenic stimulation in vitro, AID(-/-)mus(-/-) B cells differentiated into plasmablasts to some extent, but showed reduced survival compared with wild-type B cells. We found no evidence that this reduced survival was attributable to accumulation of membrane IgM. Our results indicate that the synthesis of secretory Igs is a requirement for maintenance of B220(-)CD138(+) PCs. Full Text

Lamprecht, M. R., Sabatini, D. M. and Carpenter, A. E. (2007) CellProfiler: free, versatile software for automated biological image analysis. Biotechniques. 42 71-5. Careful visual examination of biological samples is quite powerful, but many visual analysis tasks done in the laboratory are repetitive, tedious, and subjective. Here we describe the use of the open-source software, CellProfiler, to automatically identify and measure a variety of biological objects in images. The applications demonstrated here include yeast colony counting and classifying, cell microarray annotation, yeast patch assays, mouse tumor quantification, wound healing assays, and tissue topology measurement. The software automatically identifies objects in digital images, counts them, and records a full spectrum of measurements for each object, including location within the image, size, shape, color intensity, degree of correlation between colors, texture (smoothness), and number of neighbors. Small numbers of images can be processed automatically on a personal computer and hundreds of thousands can be analyzed using a computing cluster. This free, easy-to-use software enables biologists to comprehensively and quantitatively address many questions that previously would have required custom programming, thereby facilitating discovery in a variety of biological fields of study. Full Text

Lange, J., Skaletsky, H., Bell, G.W., and Page, D.C. (2007). MSY Breakpoint Mapper, a database of sequence-tagged sites useful in defining naturally occurring deletions in the human Y chromosome. Nucleic Acids Research Advance Access published online on October 26, 2007. Y chromosome deletions arise frequently in human populations, where they cause sex reversal and Turner syndrome and predispose individuals to infertility and germ cell cancer. Knowledge of the nucleotide sequence of the male-specific region of the Y chromosome (MSY) makes it possible to precisely demarcate such deletions and the repertoires of genes lost, offering insights into mechanisms of deletion and the molecular etiologies of associated phenotypes. Such deletion mapping is usually conducted using polymerase chain reaction (PCR) assays for the presence or absence of a series of Y-chromosomal DNA markers, or sequence-tagged sites (STSs). In the course of mapping intact and aberrant Y chromosomes during the past two decades, we and our colleagues have developed robust PCR assays for 1287 Y-specific STSs. These PCR assays amplify 1698 loci at an average spacing of <14 kb across the MSY euchromatin. To facilitate mapping of deletions, we have compiled a database of these STSs, MSY Breakpoint Mapper (http://breakpointmapper.wi.mit.edu/). When queried, this online database provides regionally targeted catalogs of STSs and nearby genes. MSY Breakpoint Mapper is useful for efficiently and systematically defining the breakpoint(s) of virtually any naturally occurring Y chromosome deletion. Full Text.

Lengner, C.J., Camargo, F.D., Hochedlinger, K., Welstead, G.G., Zaidi, S., Gokhale, S., Scholer, H.R., Tomilin, A., and Jaenisch, R. (2007). Oct4 expression is not required for mouse somatic stem cell self-renewal. Cell Stem Cell 1, 403-415. The Pou domain containing transcription factor Oct4 is a well-established regulator of pluripotency in the inner cell mass of the mammalian blastocyst as well as in embryonic stem cells. While it has been shown that the Oct4 gene is inactivated through a series of epigenetic modifications following implantation, recent studies have detected Oct4 activity in a variety of somatic stem cells and tumor cells. Based on these observations it has been suggested that Oct4 may also function in maintaining self-renewal of somatic stem cells and, in addition, may promote tumor formation. We employed a genetic approach to determine whether Oct4 is important for maintaining pluripotency in the stem cell compartments of several somatic tissues including the intestinal epithelium, bone marrow (hematopoietic and mesenchymal lineages), hair follicle, brain, and liver. Oct4 gene ablation in these tissues revealed no abnormalities in homeostasis or regenerative capacity. We conclude that Oct4 is dispensable for both self-renewal and maintenance of somatic stem cells in the adult mammal. Full Text.

Levdansky, E., Romano, J., Shadkchan, Y., Sharon, H., Verstrepen, K.J., Fink, G.R., and Osherov, N. (2007). Coding tandem repeats generate genetic diversity in Aspergillus fumigatus genes. Eukaryot Cell Jun 8; [Epub ahead of print]. PDF

Lewitter, F. (2007) Moving education forward. PLoS Comput Biol. 3 e19. Full Text

Liao, M.J., Zhang, C.C., Zhou, B., Zimonjic, D.B., Mani, S.A., Kaba, M., Gifford, A., Reinhardt, F., Popescu, N.C., Guo, W., et al. (2007). Enrichment of a Population of Mammary Gland Cells that Form Mammospheres and Have In vivo Repopulating Activity. Cancer Res 67, 8131-8138. The identification of mammary gland stem cells (MGSC) or progenitors is important for the study of normal breast development and tumorigenesis. Based on their immunophenotype, we have isolated a population of mouse mammary gland cells that are capable of forming "mammospheres" in vitro. Importantly, mammospheres are enriched for cells that regenerate an entire mammary gland on implantation into a mammary fat pad. We also undertook cytogenetic analyses of mammosphere-forming cells after prolonged culture, which provided preliminary insight into the genomic stability of these cells. Our identification of new cell surface markers for enriching mammosphere-initiating cells, including endoglin and prion protein, will facilitate the elucidation of the cell biology of MGSC. Full Text.

Linhart, H.G., Lin, H., Yamada, Y., Moran, E., Steine, E.J., Gokhale, S., Lo, G., Cantu, E., Ehrich, M., He, T., Meissner A, and Jaenisch R. (2007). Dnmt3b promotes tumorigenesis in vivo by gene-specific de novo methylation and transcriptional silencing. Genes Dev 21, 3110-3122. Increased methylation of CpG islands and silencing of affected target genes is frequently found in human cancer; however, in vivo the question of causality has only been addressed by loss-of-function studies. To directly evaluate the role and mechanism of de novo methylation in tumor development, we overexpressed the de novo DNA methyltransferases Dnmt3a1 and Dnmt3b1 in Apc(Min/+) mice. We found that Dnmt3b1 enhanced the number of colon tumors in Apc(Min/+) mice approximately twofold and increased the average size of colonic microadenomas, whereas Dnmt3a1 had no effect. The overexpression of Dnmt3b1 caused loss of imprinting and increased expression of Igf2 as well as methylation and transcriptional silencing of the tumor suppressor genes Sfrp2, Sfrp4, and Sfrp5. Importantly, we found that Dnmt3b1 but not Dnmt3a1 efficiently methylates the same set of genes in tumors and in nontumor tissues, demonstrating that de novo methyltransferases can initiate methylation and silencing of specific genes in phenotypically normal cells. This suggests that DNA methylation patterns in cancer are the result of specific targeting of at least some tumor suppressor genes rather than of random, stochastic methylation followed by clonal selection due to a proliferative advantage caused by tumor suppressor gene silencing. Full Text.

Love, K.R., Catic, A., Schlieker, C., and Ploegh, H.L. (2007). Mechanisms, biology and inhibitors of deubiquitinating enzymes. Nat Chem Biol 3, 697-705. The addition of ubiquitin (Ub) and ubiquitin-like (Ubl) modifiers to proteins serves to modulate function and is a key step in protein degradation, epigenetic modification and intracellular localization. Deubiquitinating enzymes and Ubl-specific proteases, the proteins responsible for the removal of Ub and Ubls, act as an additional level of control over the ubiquitin-proteasome system. Their conservation and widespread occurrence in eukaryotes, prokaryotes and viruses shows that these proteases constitute an essential class of enzymes. Here, we discuss how chemical tools, including activity-based probes and suicide inhibitors, have enabled (i) discovery of deubiquitinating enzymes, (ii) their functional profiling, crystallographic characterization and mechanistic classification and (iii) development of molecules for therapeutic purposes. Full Text.

Lowery, L. A., Rubin, J. and Sive, H. (2007) Whitesnake/sfpq is required for cell survival and neuronal development in the zebrafish. Developmental Dynamics Published Online: 28 Mar 2007 Organogenesis involves both the development of specific cell types and their organization into a functional three-dimensional structure. We are using the zebrafish to assess the genetic basis for brain organogenesis. We show that the whitesnake mutant corresponds to the sfpq (splicing factor, proline/glutamine rich) gene, encoding the PSF protein (polypyrimidine tract-binding protein-associated splicing factor). In vitro studies have shown that PSF is important for RNA splicing and transcription and is a candidate brain-specific splicing factor, however, the in vivo function of this gene is unclear. sfpq is expressed throughout development and in the adult zebrafish, with strong expression in the developing brain, particularly in regions enriched for neuronal progenitors. In the whitesnake mutant, a brain phenotype is visible by 28 hr after fertilization, when it becomes apparent that the midbrain and hindbrain are abnormally shaped. Neural crest, heart, and muscle development or function is also abnormal. sfpq function appears to be required in two distinct phases during development. First, loss of sfpq gene function leads to increased cell death throughout the early embryo, suggesting that cell survival requires functional PSF protein. Second, sfpq function is required for differentiation, but not for determination, of specific classes of brain neurons. These data indicate that, in vertebrates, sfpq plays a key role in neuronal development and is essential for normal brain development.Full Text

Lu, X., Huang, L.J., and Lodish, H.F. (2007). Dimerization by a cytokine receptor is necessary for constitutive activation of JAK2V617F. J Biol Chem.Dec 23 [Epub ahead of print] The majority of the BCR-ABL negative myeloproliferative disorders express the mutant JAK2, JAK2V617F. Previously we showed that constitutive activation of this oncogenic JAK2 mutant in Ba/F3 or 32D cells requires coexpression of a cognate homodimeric cytokine receptor, such as the EpoR. However, overexpression of JAK2V617F in Ba/F3 cells renders them cytokine-independent for growth in the absence of an exogenous cytokine receptor. Here, we demonstrated that JAK2V617F domains required for receptor association are essential for cytokine-independent growth by overexpressed JAK2V617F, suggesting JAK2V617F is binding to an unknown endogenous cytokine receptor(s) for its activation. We further showed that disruption of EpoR dimerization by coexpressing a truncated EpoR disrupted JAK2V617F-mediated transformation, indicating that EpoR dimerization plays an essential role in the activation of JAK2V617F. Interestingly, coexpression of JAK2V617F with EpoR mutants that retain JAK2 binding but are defective in mediating Epo-dependent JAK2 activation due to mutations in a conserved juxtamembrane motif, does lead to cytokine-independent activation of JAK2V617F. Overall, these findings confirm that JAK2V617F requires binding to a dimerized cytokine receptor for its activation, and that the key EpoR juxtamembrane regulatory motif essential for Epo-dependent JAK2 activation is not essential for the activation of JAK2V617F. The structure of the activated JAK2V617F is thus likely to be different from that of the activated wild-type JAK2, raising the possibility of developing a specifically targeted therapy for myeloproliferative disorders. PDF

Ma, L., and Weinberg, R.A. (2007). Micrornas in Malignant Progression. Cell Cycle 31 Dec. Epub ahead of publication 7(5) : A growing body of evidence suggests that certain microRNAs (miRNAs) play causal roles in tumorigenesis by regulating genes that control cellular processes such as cell cycle, apoptosis, and differentiation. Lately, work in our laboratory has revealed that a specific miRNA can cause cancer cells to invade and metastasize. Here we review recent progress made in several laboratories concerning the roles played by miRNAs in cancer pathogenesis and metastatic spread, with the focus on the pro-metastatic microRNA, miR-10b, and discuss the future directions and possibilities for clinical applications. PDF not available yet.

Ma, L., Teruya-Feldstein, J., and Weinberg, R.A. (2007). Tumour invasion and metastasis initiated by microRNA-10b in breast cancer. Nature.Sep 26; [Epub ahead of print] MicroRNAs have been implicated in regulating diverse cellular pathways. Although there is emerging evidence that some microRNAs can function as oncogenes or tumour suppressors, the role of microRNAs in mediating cancer metastasis remains unexplored. Here we show, using a combination of mouse and human cells, that microRNA-10b (miR-10b) is highly expressed in metastatic breast cancer cells and positively regulates cell migration and invasion. Overexpression of miR-10b in otherwise non-metastatic breast tumours initiates robust invasion and metastasis. Expression of miR-10b is induced by the transcription factor Twist, which binds directly to the putative promoter of mir-10b (MIRN10B). The miR-10b induced by Twist proceeds to inhibit translation of the messenger RNA encoding homeobox D10, resulting in increased expression of a well-characterized pro-metastatic gene, RHOC. Significantly, the level of miR-10b expression in primary breast carcinomas correlates with clinical progression. These findings suggest the workings of an undescribed regulatory pathway, in which a pleiotropic transcription factor induces expression of a specific microRNA, which suppresses its direct target and in turn activates another pro-metastatic gene, leading to tumour cell invasion and metastasis. Full Text.

Magnusson, M., Brun, A.C.M., Miyake, N., Larsson, J., Ehinger, M., Bjornsson, J.M., Wutz, A., Sigvardsson, M., and Karlsson, S. (2007). HOXA10 is a critical regulator for hematopoietic stem cells and erythroid/megakaryocyte development. Blood 109, 3687-3696. The Homeobox (Hox) transcription factors are important regulators of normal and malignant hematopoiesis because they control proliferation, differentiation, and self-renewal of hematopoietic cells at different levels of the hematopoietic hierarchy. In transgenic mice we show that the expression of HOXA10 is tightly regulated by doxycycline. Intermediate concentrations of HOXA10 induced a 15-fold increase in the repopulating capacity of hematopoietic stem cells (HSCs) after 13 days of in vitro culture. Notably, the proliferation induction of HSC by HOXA10 was dependent on the HOXA10 concentration, because high levels of HOXA10 had no effect on HSC proliferation. Furthermore, high levels of HOXA10 blocked erythroid and megakaryocyte development, demonstrating that tight regulation of HOXA10 is critical for normal development of the erythroid and megakaryocytic lineages. The HOXA10-mediated effects on hematopoietic cells were associated with altered expression of genes that govern stem-cell self-renewal and lineage commitment (eg, hepatic leukemia factor [HlF], Dickkopf-1 [Dkk-1], growth factor independent-1 [Gfi-1], and Gata-1). Interestingly, binding sites for HOXA10 were found in HLF, Dkk-1, and Gata-1, and Dkk-1 and Gfi-1 were transcriptionally activated by HOXA10. These findings reveal novel molecular pathways that act downstream of HOXA10 and identify HOXA10 as a master regulator of postnatal hematopoietic development. Full Text

Majewski, M., Bose, T.O., Sille, F.C.M., Pollington, A.M., Fiebiger, E., and Boes, M. (2007). Protein kinase C delta stimulates antigen presentation by Class II MHC in murine dendritic cells. International Immunology 19, 719-732. Maturation of dendritic cells (DCs) regulates protein sorting in endosomal compartments to promote the surface expression of molecules involved in T cell activation. MHC Class II complexes are mobilized to the surface via intracellular effector molecules that remain largely unknown. We here show that protein kinase C (PKC) stimulates Class II antigen surface expression, using knock-in mice that express a Class II-green fluorescent protein fusion protein as a read out. Selective inhibition of PKC delta counteracts the ability of DCs to stimulate Class II MHC-restricted antigen-specific T cells. Activation of PKC does not affect antigen uptake, peptide loading and surface display of Class 1 MHC and transferrin receptor in DCs. We show that activation-induced Class 11 MHC surface expression is dependent on activation of PKC delta and conclude that this event is pivotal for optimal CD4 T cell activation. Full Text.

Mani, S.A., Yang, J., Brooks, M., Schwaninger, G., Zhou, A.,Miura, N., Kutok, J.L., Hartwell, K., Richardson, A.L., and Weinberg, R.A. (2007). Mesenchyme Forkhead 1 (FOXC2) plays a key role in metastasis and is associated with aggressive basal-like breast cancers. Proc Natl Acad Sci U S A.May 30; [Epub ahead of print] The metastatic spread of epithelial cancer cells from the primary tumor to distant organs mimics the cell migrations that occur during embryogenesis. Using gene expression profiling, we have found that the FOXC2 transcription factor, which is involved in specifying mesenchymal cell fate during embryogenesis, is associated with the metastatic capabilities of cancer cells. FOXC2 expression is required for the ability of murine mammary carcinoma cells to metastasize to the lung, and overexpression of FOXC2 enhances the metastatic ability of mouse mammary carcinoma cells. We show that FOXC2 expression is induced in cells undergoing epithelial-mesenchymal transitions (EMTs) triggered by a number of signals, including TGF-beta1 and several EMT-inducing transcription factors, such as Snail, Twist, and Goosecoid. FOXC2 specifically promotes mesenchymal differentiation during an EMT and may serve as a key mediator to orchestrate the mesenchymal component of the EMT program. Expression of FOXC2 is significantly correlated with the highly aggressive basal-like subtype of human breast cancers. These observations indicate that FOXC2 plays a central role in promoting invasion and metastasis and that it may prove to be a highly specific molecular marker for human basal-like breast cancers.PDF

Marson, A., Kretschmer, K., Frampton, G. M., Jacobsen, E. S., Polansky, J. K., Macisaac, K. D., Levine, S. S., Fraenkel, E., von Boehmer, H. and Young, R. A. (2007) Foxp3 occupancy and regulation of key target genes during T-cell stimulation. Nature.Jan 21; [Epub ahead of print] Foxp3(+)CD4(+)CD25(+) regulatory T (T(reg)) cells are essential for the prevention of autoimmunity. T(reg) cells have an attenuated cytokine response to T-cell receptor stimulation, and can suppress the proliferation and effector function of neighbouring T cells. The forkhead transcription factor Foxp3 (forkhead box P3) is selectively expressed in T(reg) cells, is required for T(reg) development and function, and is sufficient to induce a T(reg) phenotype in conventional CD4(+)CD25(-) T cells. Mutations in Foxp3 cause severe, multi-organ autoimmunity in both human and mouse. FOXP3 can cooperate in a DNA-binding complex with NFAT (nuclear factor of activated T cells) to regulate the transcription of several known target genes. However, the global set of genes regulated directly by Foxp3 is not known and consequently, how this transcription factor controls the gene expression programme for T(reg) function is not understood. Here we identify Foxp3 target genes and report that many of these are key modulators of T-cell activation and function. Remarkably, the predominant, although not exclusive, effect of Foxp3 occupancy is to suppress the activation of target genes on T-cell stimulation. Foxp3 suppression of its targets appears to be crucial for the normal function of T(reg) cells, because overactive variants of some target genes are known to be associated with autoimmune disease. Full Text.

Masel, J., King, O. D. and Maughan, H. (2007) The loss of adaptive plasticity during long periods of environmental stasis. American Naturalist. 169 38-46. Adaptive plasticity allows populations to adjust rapidly to environmental change. If this is useful only rarely, plasticity may undergo mutational degradation and be lost from a population. We consider a population of constant size N undergoing loss of plasticity at functional mutation rate m and with selective advantage s associated with loss. Environmental change events occur at rate theta per generation, killing all individuals that lack plasticity. The expected time until loss of plasticity in a fluctuating environment is always at least (tau) over bar, the expected time until loss of plasticity in a static environment . When and, we find that plasticity will be mN > 1 and N theta >> 1, we find that plasticity will be maintained for an average of at least 10(8) generations in a single population, provided (tau) over bar > 18/theta. In a metapopulation, plasticity is retained under the more lenient condition (tau) over bar > 1.3/theta, irrespective of mN, for a modest number of demes. We calculate both exact and approximate solutions for (tau) over bar and find that it is linearly dependent only on the logarithm of N, and so, surprisingly, both the population size and the number of demes in the metapopulation make little difference to the retention of plasticity. Instead, (tau) over bar is dominated by the term 1/(m + s/2) .Full Text

Mayr, C., Hemann, M. T. and Bartel, D. P. (2007) Disrupting the Pairing Between let-7 and Hmga2 Enhances Oncogenic Transformation. Science. Feb 22; [Epub ahead of print] MicroRNAs (miRNAs) are ~22-nt RNAs that can pair to sites within mRNAs to specify posttranscriptional repression of these messages. Aberrant miRNA expression can contribute to tumorigenesis, but which of the many miRNA-target relationships are relevant to this process has been unclear. Here we report that chromosomal translocations previously associated with human tumors disrupt repression of High Mobility Group A2 (Hmga2) by let-7 miRNA. This disrupted repression promotes anchorage-independent growth, a characteristic of oncogenic transformation. Thus, losing miRNA-directed repression of an oncogene provides a mechanism for tumorigenesis, and disrupting a single miRNA-target interaction can produce an observable phenotype in mammalian cells. Full Text

McLellan, C.A., Turbyville, T.J., Wijeratne, E.M., Kerschen, A., Vierling, E., Queitsch, C., Whitesell, L., and Gunatilaka, A.A. (2007). A Rhizosphere Fungus Enhances Arabidopsis Thermotolerance Through Production of an HSP90 Inhibitor. Plant Physiology First published on July 13, 2007 The molecular chaperone Heat Shock Protein 90 (HSP90) is essential for the maturation of key regulatory proteins in eukaryotes and for the response to temperature stress. Earlier we have reported that fungi living in association with plants of the Sonoran desert produce small molecule inhibitors of mammalian HSP90. Here, we address whether elaboration of the HSP90 inhibitor monocillin I [MON] by the rhizosphere fungus Paraphaeosphaeria quadriseptata affects plant HSP90 and plant environmental responsiveness. We demonstrate that MON binds Arabidopsis HSP90 and can inhibit the function of HSP90 in lysates of wheat (Triticum aestivum) germ. MON treatment of Arabidopsis seedlings induced HSP101 and HSP70, conserved components of the stress response. Application of MON, or growth in the presence of MON, allowed Arabidopsis wild type, but not AtHSP101 knock-out mutant seedlings to survive otherwise lethal temperature stress. Finally, co-cultivation of P. quadriseptata with Arabidopsis enhanced plant heat stress tolerance. These data demonstrate that HSP90-inhibitory compounds produced by fungi can influence plant growth and responses to the environment.. PDF

Meissner, A., Wernig, M., and Jaenisch, R. (2007). Direct reprogramming of genetically unmodified fibroblasts into pluripotent stem cells. Nat Biotechnology Aug 27; [Epub ahead of print] In vitro reprogramming of somatic cells into a pluripotent embryonic stem cell-like state has been achieved through retroviral transduction of murine fibroblasts with Oct4, Sox2, c-myc and Klf4 (refs. ). In these experiments, the rare 'induced pluripotent stem' (iPS) cells were isolated by stringent selection for activation of a neomycin-resistance gene inserted into the endogenous Oct4 (also known as Pou5f1) or Nanog loci. Direct isolation of pluripotent cells from cultured somatic cells is of potential therapeutic interest, but translation to human systems would be hindered by the requirement for transgenic donors in the present iPS isolation protocol. Here we demonstrate that reprogrammed pluripotent cells can be isolated from genetically unmodified somatic donor cells solely based upon morphological criteria. Full Text.

Mikkelsen, T.S., Ku, M., Jaffe, D.B., Issac, B., Lieberman, E., Giannoukos, G., Alvarez, P., Brockman, W., Kim, T.K., Koche, R.P., Meissner A, Wernig M, Jaenisch R et al. (2007). Genome-wide maps of chromatin state in pluripotent and lineage-committed cells. Nature 448, 553-560. We report the application of single-molecule-based sequencing technology for high-throughput profiling of histone modifications in mammalian cells. By obtaining over four billion bases of sequence from chromatin immunoprecipitated DNA, we generated genome-wide chromatin-state maps of mouse embryonic stem cells, neural progenitor cells and embryonic fibroblasts. We find that lysine 4 and lysine 27 trimethylation effectively discriminates genes that are expressed, poised for expression, or stably repressed, and therefore reflect cell state and lineage potential. Lysine 36 trimethylation marks primary coding and non-coding transcripts, facilitating gene annotation. Trimethylation of lysine 9 and lysine 20 is detected at satellite, telomeric and active long-terminal repeats, and can spread into proximal unique sequences. Lysine 4 and lysine 9 trimethylation marks imprinting control regions. Finally, we show that chromatin state can be read in an allele-specific manner by using single nucleotide polymorphisms. This study provides a framework for the application of comprehensive chromatin profiling towards characterization of diverse mammalian cell populations. Full Text.

Mirsaidov, U., Timp, W., Mir, M., Matsudaira, P., and Timp, G. (2007). Study of Cell-Cell Signaling in 3d Bacterial Arrays Assembled Using Optical Tweezers. Nanomedicine-Nanotechnology Biology and Medicine 3, 347-347..

Moffat, J., Reiling, J. H. and Sabatini, D. M. (2007) Off-target effects associated with long dsRNAs in Drosophila RNAi screens. Trends Pharmacol Sci. Mar 8; [Epub ahead of print] Evidence of off-target effects (OTEs) associated with small interfering (si)RNAs (19-29bp) in mammalian cells has existed for several years. Two recent articles demonstrate that short sequences within long double-stranded (ds)RNAs frequently cause undesirable OTEs in cultured Drosophila cells. These results reveal the potential for high false-positive rates in RNA interference (RNAi) screens using long dsRNAs and highlight the need for screening with multiple, non-overlapping long dsRNAs or siRNAs. Discovering multiple potent siRNAs with minimal off-target profiles for each target transcript will be invaluable for genome-based studies of gene function and for personalized RNAi therapeutics.Full Text

Mukhopadhyay, S., Krishnan, R., Lemke, E. A., Lindquist, S. and Deniz, A. A. (2007) A natively unfolded yeast prion monomer adopts an ensemble of collapsed and rapidly fluctuating structures. Proceedings of the National Academy of Sciences of the United States of America. 104 2649-2654. The yeast prion protein Sup35 is a translation termination factor, whose activity is modulated by sequestration into a self-perpetuating amyloid. The prion-determining domain, NM, consists of two distinct regions: an amyloidogenic N terminus domain (N) and a charged solubilizing middle region (M). To gain insight into prion conversion, we used single-molecule fluorescence resonance energy transfer (SM-FRET) and fluorescence correlation spectroscopy to investigate the structure and dynamics of monomeric NM. Low protein concentrations in these experiments prevented the formation of obligate on-pathway oligomers, allowing us to study early folding intermediates in isolation from higher-order species. SM-FRET experiments on a dual-labeled amyloid core variant (N21C/S121C, retaining wild-type prion behavior) indicated that the N region of NM adopts a collapsed form similar to "burst-phase" intermediates formed during the folding of many globular proteins, even though it lacks a typical hydrophobic core. The mean distance between residues 21 and 121 was approximate to 43 A. This increased with denaturant in a noncooperative fashion to approximate to 63 A, suggesting a multitude of interconverting species rather than a small number of discrete monomeric conformers. Fluorescence correlation spectroscopy analysis of singly labeled NM revealed fast conformational fluctuations on the 20- to 300-ns time scale. Quenching from proximal and distal tyrosines resulted in distinct fast and slower fluctuations. Our results indicate that native monomeric NM is composed of an ensemble of structures, having a collapsed and rapidly fluctuating N region juxtaposed with a more extended M region. The stability of such ensembles is likely to play a key role in prion conversion. Full Text

Muse, G.W., Gilchrist, D.A., Nechaev, S., Shah, R., Parker, J.S., Grissom, S.F., Zeitlinger, J., and Adelman, K. (2007). RNA polymerase is poised for activation across the genome. Nature Genetics 39, 1507-1511. Regulation of gene expression is integral to the development and survival of all organisms. Transcription begins with the assembly of a pre-initiation complex at the gene promoter(1), followed by initiation of RNA synthesis and the transition to productive elongation(2-4). In many cases, recruitment of RNA polymerase II ( Pol II) to a promoter is necessary and sufficient for activation of genes. However, there are a few notable exceptions to this paradigm, including heat shock genes and several proto-oncogenes, whose expression is attenuated by regulated stalling of polymerase elongation within the promoter-proximal region(5-13). To determine the importance of polymerase stalling for transcription regulation, we carried out a genome-wide search for Drosophila melanogaster genes with Pol II stalled within the promoter-proximal region. Our data show that stalling is widespread, occurring at hundreds of genes that respond to stimuli and developmental signals. This finding indicates a role for regulation of polymerase elongation in the transcriptional responses to dynamic environmental and developmental cues. Full Text.

Nencioni, A., Beck, J., Werth, D., Grunebach, F., Patrone, F., Ballestrero, A., and Brossart, P. (2007). Histone deacetylase inhibitors affect dendritic cell differentiation and immunogenicity. Clinical Cancer Research 13, 3933-3941. Purpose: Histone deacetylases (HDAC) modulate gene transcription and chromatin assembly by modifying histones at the posttranscriptional level. HDAC inhibitors have promising antitumor activity and are presently explored in clinical studies. Cumulating evidence in animal models of immune disorders also suggests immunosuppressive properties for these small molecules, although the underlying mechanisms remain at present poorly understood. Here, we have evaluated the effects of two HDAC inhibitors currently in clinical use, sodium valproate and MS-275, on human monocyte-derived DCs. Experimental Design: DCs were generated from monocytes through incubation with granulocyte macrophage colony-stimulating factor and interleukin-4. DC maturation was induced by addition of polyinosinic-polycytidylic acid. DC phenotype, immunostimulatory capacity, cytokine secretion, and migratory capacity were determined by flow cytometry, mixed leukocyte reaction, ELISA, and Transwell migration assay, respectively. Nuclear translocation of RelB, IFN regulatory factor (IRF)-3, and IRF-8 were determined by immunoblotting. Results: HDAC inhibition skews DC differentiation by preventing the acquisition of the DC hallmark CD1a and by affecting the expression of costimulation and adhesion molecules. In addition, macrophage inflammatory protein-3 beta/chemokine, motif CC, ligand 19-induced migration, immunostimulatory capacity, and cytokine secretion by DCs are also profoundly impaired. The observed defects in DC function on exposure to HDAC inhibitors seem to reflect the obstruction of signaling through nuclear factor-kappa B, IRF-3, and IRF-8. Conclusions: HDAC inhibitors exhibit strong immunomodulatory properties in human DCs. Our results support the evaluation of HDAC inhibitors in inflammatory and autoimmune disorders. Full Text.

Nguyen, S., Meletis, K., Fu, D., Jhaveri, S., and Jaenisch, R. (2007). Ablation of de novo DNA methyltransferase Dnmt3a in the nervous system leads to neuromuscular defects and shortened lifespan. Developmental Dynamics.May 3; [Epub ahead of print] DNA methylation is an epigenetic mechanism involved in gene regulation and implicated in the functioning of the nervous system. The de novo DNA methyltransferase Dnmt3a is expressed in neurons, but its specific role has not been clarified. Dnmt3a is activated around embryonic day 10.5 in mouse neuronal precursor cells and remains active in postmitotic neurons in the adult. We assessed the role of neuronal Dnmt3a by conditional gene targeting. Mice lacking functional Dnmt3a in the nervous system were born healthy, but degenerated in adulthood and died prematurely. Mutant mice were hypoactive, walked abnormally, and underperformed on tests of neuromuscular function and motor coordination. Loss of Dnmt3a also led to fewer motor neurons in the hypoglossal nucleus and more fragmented endplates in neuromuscular junctions of the diaphragm muscle. Our results implicate a role for Dnmt3a in the neuromuscular control of motor movement. Full Text

Odom, D.T., Dowell, R.D., Jacobsen, E.S., Gordon, W., Danford, T.W., Macisaac, K.D., Rolfe, P.A., Conboy, C.M., Gifford, D.K., and Fraenkel, E. (2007). Tissue-specific transcriptional regulation has diverged significantly between human and mouse. Nat Genet.39(6):730-732. We demonstrate that the binding sites for highly conserved transcription factors vary extensively between human and mouse. We mapped the binding of four tissue-specific transcription factors (FOXA2, HNF1A, HNF4A and HNF6) to 4,000 orthologous gene pairs in hepatocytes purified from human and mouse livers. Despite the conserved function of these factors, from 41% to 89% of their binding events seem to be species specific. When the same protein binds the promoters of orthologous genes, approximately two-thirds of the binding sites do not align.Full Text

Okamoto, K.I., Narayanan, R., Lee, S.H., Murata, K., and Hayashi, Y. (2007). The role of CaMKII as an F-actin-bundling protein crucial for maintenance of dendritic spine structure. Proceedings of the National Academy of Sciences of the United States of America 104, 6418-6423 Ca2+-calmodulin-dependent protein kinase II (CaMKII) is a serine/threonine protein kinase critically involved in synaptic plasticity in the brain. It is highly concentrated in the postsynaptic density fraction, exceeding the amount of any other signal transduction molecules. Because kinase signaling can be amplified by catalytic reaction, why CaMKII exists in such a large quantity has been a mystery. Here, we provide biochemical evidence that CaMKII is capable of bundling F-actin through a stoichiometric interaction. Consistent with this evidence, in hippocampal neurons, RNAi-mediated down-regulation of CaMKII leads to a reduction in the volume of dendritic spine head that is mediated by F-actin dynamics. An overexpression of CaMKII slowed down the actin turnover in the spine head. This activity was associated with beta subunit of CaMKII in a manner requiring its actin-binding and association domains but not the kinase domain. This finding indicates that CaMKII serves as a central signaling molecule in both functional and structural changes during synaptic plasticity. Full Text

Orimo, A., and Weinberg, R.A. (2007). Heterogeneity of stromal fibroblasts in tumors. Cancer Biology & Therapy 6, 618-619

Park, E.A., Macalpine, D.M., and Orr-Weaver, T.L. (2007). Drosophila follicle cell amplicons as models for metazoan DNA replication: A cyclinE mutant exhibits increased replication fork elongation. Proc Natl Acad Sci U S A. Oct 11; [Epub ahead of print] Gene clusters amplified in the ovarian follicle cells of Drosophila serve as powerful models for metazoan DNA replication. In response to developmental signals, specific genomic regions undergo amplification by repeated firing of replication origins and bidirectional movement of replication forks for approximately 50 kb in each direction. Previous work focused on initiation of amplification, defining replication origins, establishing the role of the prereplication complex and origin recognition complex (ORC), and uncovering regulatory functions for the Myb, E2F1, and Rb transcription factors. Here, we exploit follicle cell amplification to investigate the control of DNA replication fork progression and termination, poorly understood processes in metazoans. We identified a mutant in which, during gene amplification, the replication forks move twice as far from the origin compared with wild type. This phenotype is the result of an amino acid substitution mutation in the cyclinE gene, cyclinE(1f36). The rate of oogenesis is normal in cyclinE(1f36)/cyclinE(Pz8) mutant ovaries, indicating that increased replication fork progression is due to increased replication fork speed, possibly from increased processivity. The increased amplification domains observed in the mutant imply that there are not replication fork barriers preventing replication forks from progressing beyond the normal 100-kb amplified region. These results reveal a previously unrecognized role for CyclinE in controlling replication fork movement. PDF.

Pesin, J.A., and Orr-Weaver, T.L. (2007). Developmental role and regulation of cortex, a meiosis-specific anaphase-promoting complex/cyclosome activator. Plos Genetics 3, 2208-2220. During oogenesis in metazoans, the meiotic divisions must be coordinated with development of the oocyte to ensure successful fertilization and subsequent embryogenesis. The ways in which the mitotic machinery is specialized for meiosis are not fully understood. cortex, which encodes a putative female meiosis-specific anaphase-promoting complex/cyclosome (APC/C) activator, is required for proper meiosis in Drosophila. We demonstrate that CORT physically associates with core subunits of the APC/C in ovaries. APC/C CORT targets Cyclin A for degradation prior to the metaphase I arrest, while Cyclins B and B3 are not targeted until after egg activation. We investigate the regulation of CORT and find that CORT protein is specifically expressed during the meiotic divisions in the oocyte. Polyadenylation of cort mRNA is correlated with appearance of CORT protein at oocyte maturation, while deadenylation of cort mRNA occurs in the early embryo. CORT protein is targeted for degradation by the APC/C following egg activation, and this degradation is dependent on an intact D- box in the C terminus of CORT. Our studies reveal the mechanism for developmental regulation of an APC/C activator and suggest it is one strategy for control of the female meiotic cell cycle in a multicellular organism. Full Text.

Petersen, C.P., and Reddien, P.W. (2007). Smed-{beta}catenin-1 Is Required for Anteroposterior Blastema Polarity in Planarian Regeneration. Science Express Advance of Print.12.6.07. Planarian flatworms can regenerate heads at anterior-facing wounds and tails at posterior-facing wounds throughout the body. How this regeneration polarity is specified has been a classic problem for over a century. We identified a planarian gene, Smed-betacatenin-1, that controls regeneration polarity. Posterior-facing blastemas regenerate a head instead of a tail in Smed-betacatenin-1(RNAi) animals. Smed-betacatenin-1 is required after wounding and at any posterior-facing wound for polarity. Additionally, intact Smed-betacatenin-1(RNAi) animals display anteriorization during tissue turnover. Five Wnt genes and a secreted Frizzled-related Wnt antagonist-like gene are expressed in domains along the anteroposterior axis that reset to new positions during regeneration, suggesting Wnts control polarity through Smed-betacatenin-1. Our data suggest beta-catenin specifies the posterior character of the anteroposterior axis throughout the Bilateria and specifies regeneration polarity in planarians. PDF.

Ploegh, H.L. (2007). Bridging B cell and T cell recognition of antigen. Journal of Immunology 179, 7193-7193. Full Text.

Ploegh, H.L. (2007). The peptide cargo of class I molecules: not just passive passengers. J Immunol 179, 4299-4300. Full Text..

Ploegh, H.L. (2007). A lipid-based model for the creation of an escape hatch from the endoplasmic reticulum. Nature 448, 435-438. Lipids are not encoded by a DNA template and therefore cannot be mutated, knocked out or knocked down. This by no means renders them impotent from a cell biological perspective. Here I propose a model for the involvement of lipid rearrangements in the execution of crucial steps in (glyco)protein quality control. Full Text

Popp, M.W., Antos, J.M., Grotenbreg, G.M., Spooner, E., and Ploegh, H.L. (2007). Sortagging: a versatile method for protein labeling. Nature Chemical Biology.Advance online publication 23 September. Genetically encoded reporter constructs that yield fluorescently labeled fusion proteins are a powerful tool for observing cell biological phenomena, but they have limitations. Sortagging (sortase-mediated transpeptidation) is a versatile chemoenzymatic system for site-specific labeling of proteins with small (<2 kDa) probes. Sortagging combines the precision of a genetically encoded tag with the specificity of an enzymatic reaction and the ease and chemical versatility of peptide synthesis. Here we apply this technique to proteins in vitro and on the surface of living cells. Full Text.

Pylayeva, Y., Guo, W., and Giancotti, F.G. (2007). Analysis of integrin signaling in genetically engineered mouse models of mammary tumor progression. Methods Enzymol 426, 439-461. Cancer progression-the evolution of malignant tumors towards metastatic dissemination-is a complex, multistep process orchestrated by neoplastic cells but aided by elements of the tumor microenvironment such as macrophages, activated fibroblasts, and endothelial cells. During tumor progression, cancer cells acquire a number of traits, such as the ability to undergo unrestrained proliferation, to resist pro-apoptotic insults, and to invade through tissue boundaries. Genetic and epigenetic changes conspire to drive the emergence of these traits against the backdrop of host selection. It is becoming increasingly clear that certain integrins and integrin-signaling components amplify oncogenic signaling to promote tumor progression. Mouse models of cancer provide useful, if not necessary, experimental systems to study tumor initiation and progression in vivo and to test novel therapeutic approaches. We have utilized mouse models of mammary tumorigenesis to examine the role of integrin alpha6beta4 signaling in tumor progression in vivo. In this chapter, we describe a collection of cell biological and genetic methods that may aid in characterizing the roles of integrin signals in mammary tumorigenesis .Full Text.

Reddien, P.W., Bermange, A.L., Kicza, A.M., and Sanchez Alvarado, A. (2007). BMP signaling regulates the dorsal planarian midline and is needed for asymmetric regeneration. Development 134, 4043-4051. Planarians can be cut into irregularly shaped fragments capable of regenerating new and complete organisms. Such regenerative capacities involve a robust ability to restore bilateral symmetry. We have identified three genes needed for bilaterally asymmetric fragments to regenerate missing body parts. These genes are candidate components of a signaling pathway that controls the dorsal-ventral patterning of many animal embryos: a BMP1/Tolloid-like gene (smedolloid-1), a SMAD4-like gene (smedsmad4-1), and a BMP2/4/DPP-like gene (smedbmp4-1). BMP signaling was involved in the formation of new tissues at the midline of regeneration, the dorsal-ventral patterning of new tissues, and the maintenance of the dorsal-ventral pattern of existing adult tissue in homeostasis. smedbmp4-1 was normally expressed at the dorsal midline. Asymmetric fragments lacking a midline displayed new smedbmp4-1 expression prior to formation of a regenerative outgrowth (blastema). Asymmetric fragments containing the midline displayed expanded smedbmp4-1 expression towards the wound. We suggest injured animals that lack left-right symmetry reset their midline through modulation of BMP activity as an early and necessary event in regeneration. Full Text.

Reddien, P. W., Andersen, E. C., Huang, M. and Horvitz, R. (2007) DPL-1 DP, LIN-35 Rb, and EFL-1 E2F act with the MCD-1 Zinc-finger protein to promote programmed cell death in C. elegans. Genetics.Jan 21; [Epub ahead of print] The genes egl-1, ced-9, ced-4, and ced-3 play major roles in programmed cell death in C. elegans. To identify genes that have more subtle activities, we sought mutations that confer strong cell-death defects in a genetically sensitized mutant background. Specifically, we screened for mutations that enhance the cell-death defects caused by a partial loss-of-function allele of the ced-3 caspase gene. We identified mutations in two genes not previously known to affect cell death, dpl-1 and mcd-1 (modifier of cell death). dpl-1 encodes the C. elegans homolog of DP, the human E2F-heterodimerization partner. By testing genes known to interact with dpl-1, we identified roles in cell death for four additional genes: efl-1 E2F, lin-35 Rb, lin-37 Mip40, and lin-52 dLin52. mcd-1 encodes a novel protein that contains one zinc finger and that is synthetically required with lin-35 Rb for animal viability. dpl-1 and mcd-1 act with efl-1 E2F and lin-35 Rb to promote programmed cell death and do so by regulating the killing process rather than by affecting the decision between survival and death. We propose that the DPL-1 DP, MCD-1 Zinc finger, EFL-1 E2F, LIN-35 Rb, LIN-37 Mip40, and LIN-52 dLin52 proteins act together in transcriptional regulation to promote programmed cell death .PDF

Reynolds, T.B., Jansen, A., Peng, X., and Fink, G.R. (2007). Mat formation in Saccharomyces cerevisiae requires nutrient and pH gradients. Eukaryot Cell. 19 October New Accepted Manuscripts The ability of Saccharomyces cerevisiae to form morphologically complex colony-like structures called mats requires expression of the cell surface glycoprotein Flo11p and growth on a semi-solid surface. As it grows, the mat forms two visually distinct populations called the rim (edge of the mat) and the hub (interior of the mat), which can be physically separated from one another based on their agar-adherence properties. Here we show that growth of the mat on a semi-solid agar surface creates concentric glucose and pH gradients in the medium that are required for the differentiation of the hub and rim. Disruption of pathways that respond to changing levels of glucose block mat formation by decreasing FLO11 expression. However, in wild-type cells Flo11p is expressed in both portions of the structure. The difference in adherence between the rim and hub appears to be a consequence of the reduced adherence of Flo11p at the elevated pH of the rim. PDF

RubinBejerano, I., Abeijon, C., Magnelli, P., Grisafi, P., and Fink, G.R. (2007). Phagocytosis by Human Neutrophils Is Stimulated by a Unique Fungal Cell Wall Component. Cell Host Microbe 2, 55-67. Innate immunity depends upon recognition of surface features common to broad groups of pathogens. The glucose polymer beta-glucan has been implicated in fungal immune recognition. Fungal walls have two kinds of beta-glucan: beta-1,3-glucan and beta-1,6-glucan. Predominance of beta-1,3-glucan has led to the presumption that it is the key immunological determinant for neutrophils. Examining various beta-glucans for their ability to stimulate human neutrophils, we find that the minor cell wall component beta-1,6-glucan mediates neutrophil activity more efficiently than beta-1,3-glucan, as measured by engulfment, production of reactive oxygen species, and expression of heat shock proteins. Neutrophils rapidly ingest beads coated with beta-1,6-glucan while ignoring those coated with beta-1,3-glucan. Complement factors C3b/C3d are deposited on beta-1,6-glucan more readily than on beta-1,3-glucan. beta-1,6-glucan is also important for efficient engulfment of the human pathogen Candida albicans. These unique stimulatory effects offer potential for directed stimulation of neutrophils in a therapeutic context. Full Text.

RubioTexeira, M. (2007) Urmylation controls Nil1p and Gln3p-dependent expression of nitrogen-catabolite repressed genes in Saccharomyces cerevisiae. FEBS Lett Volume 581, Issue 3, 6 February 2007, Pages 541-550. Urm1 is a modifier protein that is conjugated to substrate proteins through thioester formation with the E1-like enzyme, Uba4. Here is shown that the lack of urmylation causes derepression of the GAP1 gene (encoding a nitrogen-regulated broad-spectrum amino acid-scavenging permease) in the presence of rich nitrogen sources, and simultaneous inhibition of the expression of CIT2, a TCA-cycle gene involved in the production of glutamate and glutamine. This effect is dependent on the TORC1- and nutrient-regulated transcriptional factors, Nil1p and Gln3p. Evidence is provided that, in the absence of urmylation, nuclear/cytosolic shuffling of both transcriptional factors is altered, ultimately leading to inability to repress GAP1 gene in the presence of a rich nitrogen source. Altogether, the data presented here indicate an important role of the urmylation pathway in regulating the expression of genes involved in sensing and controlling amino acids levels. Full Text

Ruby, J.G., Stark, A., Johnston, W.K., Kellis, M., Bartel, D.P., and Lai, E.C. (2007). Evolution, biogenesis, expression, and target predictions of a substantially expanded set of Drosophila microRNAs. Genome Res. 2007 Nov 7; [Epub ahead of print] MicroRNA (miRNA) genes give rise to small regulatory RNAs in a wide variety of organisms. We used computational methods to predict miRNAs conserved among Drosophila species and large-scale sequencing of small RNAs from Drosophila melanogaster to experimentally confirm and complement these predictions. In addition to validating 20 of our top 45 predictions for novel miRNA loci, the large-scale sequencing identified many miRNAs that had not been predicted. In total, 59 novel genes were identified, increasing our tally of confirmed fly miRNAs to 148. The large-scale sequencing also refined the identities of previously known miRNAs and provided insights into their biogenesis and expression. Many miRNAs were expressed in particular developmental contexts, with a large cohort of miRNAs expressed primarily in imaginal discs. Conserved miRNAs typically were expressed more broadly and robustly than were nonconserved miRNAs, and those conserved miRNAs with more restricted expression tended to have fewer predicted targets than those expressed more broadly. Predicted targets for the expanded set of microRNAs substantially increased and revised the miRNA-target relationships that appear conserved among the fly species. Insights were also provided into miRNA gene evolution, including evidence for emergent regulatory function deriving from the opposite arm of the miRNA hairpin, exemplified by mir-10, and even the opposite strand of the DNA, exemplified by mir-iab-4. PDF

Ruby, J.G., Jan, C.H., and Bartel, D.P. (2007). Intronic microRNA precursors that bypass Drosha processing. Nature 448, 83-U87. MicroRNAs (miRNAs) are approximately 22-nucleotide endogenous RNAs that often repress the expression of complementary messenger RNAs. In animals, miRNAs derive from characteristic hairpins in primary transcripts through two sequential RNase III-mediated cleavages; Drosha cleaves near the base of the stem to liberate a approximately 60-nucleotide pre-miRNA hairpin, then Dicer cleaves near the loop to generate a miRNA:miRNA* duplex. From that duplex, the mature miRNA is incorporated into the silencing complex. Here we identify an alternative pathway for miRNA biogenesis, in which certain debranched introns mimic the structural features of pre-miRNAs to enter the miRNA-processing pathway without Drosha-mediated cleavage. We call these pre-miRNAs/introns 'mirtrons', and have identified 14 mirtrons in Drosophila melanogaster and another four in Caenorhabditis elegans (including the reclassification of mir-62). Some of these have been selectively maintained during evolution with patterns of sequence conservation suggesting important regulatory functions in the animal. The abundance of introns comparable in size to pre-miRNAs appears to have created a context favourable for the emergence of mirtrons in flies and nematodes. This suggests that other lineages with many similarly sized introns probably also have mirtrons, and that the mirtron pathway could have provided an early avenue for the emergence of miRNAs before the advent of Drosha Full Text

Sancak, Y., Thoreen, C. C., Peterson, T. R., Lindquist, R. A., Kang, S. A., Spooner, E., Carr, S. A. and Sabatini, D. M. (2007) PRAS40 Is an Insulin-Regulated Inhibitor of the mTORC1 Protein Kinase. Mol Cell. 25 903-15. The heterotrimeric mTORC1 protein kinase nucleates a signaling network that promotes cell growth in response to insulin and becomes constitutively active in cells missing the TSC1 or TSC2 tumor suppressors. Insulin stimulates the phosphorylation of S6K1, an mTORC1 substrate, but it is not known how mTORC1 kinase activity is regulated. We identify PRAS40 as a raptor-interacting protein that binds to mTORC1 in insulin-deprived cells and whose in vitro interaction with mTORC1 is disrupted by high salt concentrations. PRAS40 inhibits cell growth, S6K1 phosphorylation, and rheb-induced activation of the mTORC1 pathway, and in vitro it prevents the great increase in mTORC1 kinase activity induced by rheb1-GTP. Insulin stimulates Akt/PKB-mediated phosphorylation of PRAS40, which prevents its inhibition of mTORC1 in cells and in vitro. We propose that the relative strengths of the rheb- and PRAS40-mediated inputs to mTORC1 set overall pathway activity and that insulin activates mTORC1 through the coordinated regulation of both. Full Text

Scheel, C., Onder, T., Karnoub, A., and Weinberg, R.A. (2007). Adaptation versus selection: the origins of metastatic behavior. Cancer Res 67, 11476-11479; discussion 11479-11480. Full Text.

Schlieker, C., Weihofen, W. A., Frijns, E., Kattenhorn, L. M. Gaudet, R. and Ploegh, H. L. (2007) Structure of a herpesvirus-encoded cysteine protease reveals a unique class of deubiquitinating enzymes. Mol Cell. 25 677-87. All members of the herpesviridae contain within their large tegument protein a cysteine protease module that displays deubiquitinating activity. We report the crystal structure of the cysteine protease domain of murine cytomegalovirus M48 (M48(USP)) in a complex with a ubiquitin (Ub)-based suicide substrate. M48(USP) adopts a papain-like fold, with the active-site cysteine forming a thioether linkage to the suicide substrate. The Ub core participates in an extensive hydrophobic interaction with an exposed beta hairpin loop of M48(USP). This Ub binding mode contributes to Ub specificity and is distinct from that observed in other deubiquitinating enzymes. Both the arrangement of active-site residues and the architecture of the interface with Ub lead us to classify this domain as the founding member of a previously unknown class of deubiquitinating enzymes.Full Text

Scott, L. M., Tong, W., Levine, R. L., Scott, M. A., Beer, P. A., Stratton, M. R., Futreal, P. A., Erber, W. N., McMullin, M. F., Harrison, C. N., Warren, A. J., Gilliland, D. G., Lodish, H. F. and Green, A. R. (2007) JAK2 exon 12 mutations in polycythemia vera and idiopathic erythrocytosis. New England Journal of Medicine. 356 459-468. BACKGROUND: The V617F mutation, which causes the substitution of phenylalanine for valine at position 617 of the Janus kinase (JAK) 2 gene (JAK2), is often present in patients with polycythemia vera, essential thrombocythemia, and idiopathic myelofibrosis. However, the molecular basis of these myeloproliferative disorders in patients without the V617F mutation is unclear. METHODS: We searched for new mutations in members of the JAK and signal transducer and activator of transcription (STAT) gene families in patients with V617F-negative polycythemia vera or idiopathic erythrocytosis. The mutations were characterized biochemically and in a murine model of bone marrow transplantation. RESULTS: We identified four somatic gain-of-function mutations affecting JAK2 exon 12 in 10 V617F-negative patients. Those with a JAK2 exon 12 mutation presented with an isolated erythrocytosis and distinctive bone marrow morphology, and several also had reduced serum erythropoietin levels. Erythroid colonies could be grown from their blood samples in the absence of exogenous erythropoietin. All such erythroid colonies were heterozygous for the mutation, whereas colonies homozygous for the mutation occur in most patients with V617F-positive polycythemia vera. BaF3 cells expressing the murine erythropoietin receptor and also carrying exon 12 mutations could proliferate without added interleukin-3. They also exhibited increased phosphorylation of JAK2 and extracellular regulated kinase 1 and 2, as compared with cells transduced by wild-type JAK2 or V617F JAK2. Three of the exon 12 mutations included a substitution of leucine for lysine at position 539 of JAK2. This mutation resulted in a myeloproliferative phenotype, including erythrocytosis, in a murine model of retroviral bone marrow transplantation. CONCLUSIONS: JAK2 exon 12 mutations define a distinctive myeloproliferative syndrome that affects patients who currently receive a diagnosis of polycythemia vera or idiopathic erythrocytosis. Full Text

Serwold, T., Hochedlinger, K., Inlay, M.A., Jaenisch, R., and Weissman, I.L. (2007). Early TCR expression and aberrant T cell development in mice with endogenous prerearranged T cell receptor genes. J Immunol 179, 928-938. The factors that regulate the rate of production of T cells by the thymus remain incompletely defined. To test whether generation of functional T cell receptors limits the rate of thymic T cell export, we made use of a line of mice, LN3alphabeta, that have endogenously prerearranged TCR genes. The prerearranged TCR genes were expressed abnormally early in hemopoietic development, indicating that RAG-mediated recombination, rather than transcription factor expression, is the key determinant of the initiation of robust TCR transcription. Thymic T cell export rates were similar between wild-type (wt) and LN3alphabeta mice, indicating that T cell maturation rates in these mice are determined by factors other than TCR gene rearrangement. In competitive bone marrow chimeras, however, LN3alphabeta thymocytes were out-competed by wt cells and failed to develop beyond the double-negative 4 stage. Furthermore, wt progenitors transplanted intrathymically into LN3alphabeta mice proliferated excessively, suggesting that increased proliferative signals in the LN3alphabeta thymus compensate for faulty T cell development driven by early TCR expression. Full Text.

Shaw, A.T., Meissner, A., Dowdle, J.A., Crowley, D., Magendantz, M., Ouyang, C., Parisi, T., Rajagopal, J., Blank, L.J., Bronson, R.T., Jaenisch, R et al. (2007). Sprouty-2 regulates oncogenic K-ras in lung development and tumorigenesis. Genes Dev 21, 694-707. Somatic activation of Ras occurs frequently in human cancers, including one-third of lung cancers. Activating Ras mutations also occur in the germline, leading to complex developmental syndromes. The precise mechanism by which Ras activation results in human disease is uncertain. Here we describe the phenotype of a mouse engineered to harbor a germline oncogenic K-rasG12D mutation. This mouse exhibits early embryonic lethality due to a placental trophoblast defect. Reconstitution with a wild-type placenta rescues the early lethality, but mutant embryos still succumb to cardiovascular and hematopoietic defects. In addition, mutant embryos demonstrate a profound defect in lung branching morphogenesis associated with striking up-regulation of the Ras/mitogen-activated protein kinase (MAPK) antagonist Sprouty-2 and abnormal localization of MAPK activity within the lung epithelium. This defect can be significantly suppressed by lentiviral short hairpin RNA (shRNA)-mediated knockdown of Sprouty-2 in vivo. Furthermore, in the context of K-rasG12D-mediated lung tumorigenesis, Sprouty-2 is also up-regulated and functions as a tumor suppressor to limit tumor number and overall tumor burden. These findings indicate that in the lung, Sprouty-2 plays a critical role in the regulation of oncogenic K-ras, and implicate counter-regulatory mechanisms in the pathogenesis of Ras-based disease. Full Text.

Shin, J.H., Tam, B.K., Brau, R.R., Lang, M.J., Mahadevan, L., and Matsudaira, P. (2007). Force of an actin spring. Biophys Journal 92, 3729-3733.Cellular movements are produced by forces. Typically, cytoskeletal proteins such as microtubules and actin filaments generate forces via polymerization or in conjunction with molecular motors. However, the fertilization of a Limulus polyphemus egg involves a third type of actin-based cellular engine—a biological spring. During the acrosome reaction, a 60-µm long coiled and twisted bundle of actin filaments straightens and extends from a sperm cell, penetrating the vitelline layer surrounding the egg. A subtle overtwist of 0.2°/subunit underlies the mechanochemical basis for the extension of this actin spring. Upon calcium activation, this conformational strain energy is converted to mechanical work, generating the force required to extend the bundle through the vitelline layer. In this article, we stall the extension of the acrosome bundle in agarose gels of different concentrations. From the stall forces, we estimate a maximum force of 2 nN and a puncturing pressure of 1.6 MPa. We show the maximum force of extension is three times larger than the force required to puncture the vitelline layer. Thus, the elastic strain energy stored in the acrosome bundle is more than sufficient to power the acrosome reaction through the egg envelope.Full Text

Shuga, J., Zhang, J., Samson, L.D., Lodish, H.F., and Griffith, L.G. (2007). In vitro erythropoiesis from bone marrow-derived progenitors provides a physiological assay for toxic and mutagenic compounds. Proc Natl Acad Sci U S A.May 14; [Epub ahead of print] The goal of this study was to create an in vitro cell culture system that captures essential features of the in vivo erythroid micronucleus (MN) genotoxicity assay, thus enabling increased throughput and controlled studies of the hematopoietic DNA damage response. We show that adult bone marrow (BM) cultures respond to erythropoietin, the principal hormone that stimulates erythropoiesis, with physiological erythropoietic proliferation, differentiation, and enucleation. We then show that this in vitro erythropoietic system clearly signals exposure to genotoxicants through erythroid MN formation. Furthermore, we determined that DNA repair-deficient (MGMT(-/-)) BM displayed sensitivity to genotoxic exposure in vivo compared with WT BM and that this phenotypic response was reflected in erythropoietic cultures. These findings suggest that this in vitro erythroid MN assay is capable of screening for genotoxicity on BM in a physiologically reflective manner. Finally, responses to genotoxicants during erythroid differentiation varied with exposure time, demonstrating that this system can be used to study the effect of DNA damage at specific developmental stages. PDF

Steele, A.D., King, O.D., Jackson, W.S., Hetz, C.A., Borkowski, A.W., Thielen, P., Wollmann, R., and Lindquist, S. (2007). Diminishing apoptosis by deletion of Bax or overexpression of Bcl-2 does not protect against infectious prion toxicity in vivo. J Neurosci 27, 13022-13027. B-cell lymphoma protein 2 (Bcl-2) and Bcl-2-associated X protein (Bax), key antiapoptotic and proapoptotic proteins, respectively, have important roles in acute and chronic models of neurologic disease. Several studies have implicated Bax and Bcl-2 in mediating neurotoxicity in prion diseases. To determine whether diminishing apoptotic cell death is protective in an infectious prion disease model we inoculated mice that either were null for proapoptotic Bax or overexpressed antiapoptotic Bcl-2. Interestingly, genetic manipulation of apoptosis did not lessen the clinical severity of disease. Moreover, some disease parameters, such as behavioral alterations and death, occurred slightly earlier in mice that are null for Bax or overexpress Bcl-2. These results suggest that Bax and Bcl-2 mediated apoptotic pathways are not the major contributing factor to the clinical or pathological features of infectious prion disease. Full Text.

Steele, A. D., Jackson, W. S., King, O. D. and Lindquist, S. (2007) The power of automated high-resolution behavior analysis revealed by its application to mouse models of Huntington's and prion diseases. Proc Natl Acad Sci U S A.Jan 29; [Epub ahead of print] Automated analysis of mouse behavior will be vital for elucidating the genetic determinants of behavior, for comprehensive analysis of human disease models, and for assessing the efficacy of various therapeutic strategies and their unexpected side effects. We describe a video-based behavior-recognition technology to analyze home-cage behaviors and demonstrate its power by discovering previously unrecognized features of two already extensively characterized mouse models of neurodegenerative disease. The severe motor abnormalities in Huntington's disease mice manifested in our analysis by decreased hanging, jumping, stretching, and rearing. Surprisingly, behaviors such as resting and grooming were also affected. Unexpectedly, mice with infectious prion disease showed profound increases in activity at disease onset: rearing increased 2.5-fold, walking 10-fold and jumping 30-fold. Strikingly, distinct behaviors were altered specifically during day or night hours. We devised a systems approach for multiple-parameter phenotypic characterization and applied it to defining disease onset robustly and at early time points. PDF

Tessier, P.M., and Lindquist, S. (2007). Prion recognition elements govern nucleation, strain specificity and species barriers. Nature.May 9; [Epub ahead of print] Prions are proteins that can switch to self-perpetuating, infectious conformations. The abilities of prions to replicate, form structurally distinct strains, and establish and overcome transmission barriers between species are poorly understood. We exploit surface-bound peptides to overcome complexities of investigating such problems in solution. For the yeast prion Sup35, we find that the switch to the prion state is controlled with exquisite specificity by small elements of primary sequence. Strikingly, these same sequence elements govern the formation of distinct self-perpetuating conformations (prion strains) and determine species-specific seeding activities. A Sup35 chimaera that traverses the transmission barrier between two yeast species possesses the critical sequence elements from both. Using this chimaera, we show that the influence of environment and mutations on the formation of species-specific strains is driven by selective recognition of either sequence element. Thus, critical aspects of prion conversion are enciphered by subtle differences between small, highly specific recognition elements.Full Text

Thomas, M., Lu, J.J., Zhang, C.C., Chen, J.Z., and Klibanov, A.M. (2007). Identification of novel superior polycationic vectors for gene delivery by high-throughput synthesis and screening of a combinatorial library. Pharmaceutical Research 24, 1564-1571. Purpose. Low efficiency and toxicity are two major drawbacks of current non-viral gene delivery vectors. Since DNA delivery to mammalian cells is a multi-step process, generating and searching combinatorial libraries of vectors employing high-throughput synthesis and screening methods is an attractive strategy for the development of new improved vectors because it increases the chance of identifying the most overall optimized vectors. Materials and Methods. Based on the rationale that increasing the effective molecular weight of small PEIs, which are poor vectors compared to the higher molecular weight homologues but less toxic, raises their transfection efficiency due to better DNA binding, we synthesized a library of 144 biodegradable derivatives from two small PEIs and 24 bi- and oligo-acrylate esters. A 423-Da linear PEI and its 1: 1 (w/w) mixture with a 1.8-kDa branched PEI were cross-linked with the acrylates at three molar ratios in DMSO. The resulting polymers were screened for their efficiency in delivering a beta-galactosidase expressing plasmid to COS-7 monkey kidney cells. Selected most potent polymers from the initial screen were tested for toxicity in A549 human lung cancer cells, and in vivo in a systemic gene delivery model in mice employing a firefly luciferase expressing plasmid. Results. Several polycations that exhibited high potency and low toxicity in vitro were identified from the library. The most potent derivative of the linear 423-Da PEI was that cross-linked with tricycle[5.2.1.0]-decane- dimethanol diacrylate (diacrylate 14), which exhibited an over 3,600-fold enhancement in efficiency over the parent. The most potent mixed PEI was that cross-linked with ethylene glycol diacrylate (diacrylate 4) which was over 850-fold more efficient than the physically mixed parent PEIs. The relative efficiencies of these polymers were even up to over twice as high as that of the linear 22-kDa PEI, considered the "gold standard" for in vitro and systemic gene delivery. The potent cross-linked polycations identified were also less toxic than the 22-kDa PEI. The optimal vector in vivo was the mixed PEI cross-linked with propylene glycol glycerolate diacrylate ( diacrylate 7); it mediated the highest gene expression in the lungs, followed by the spleen, with the expression in the former being 53-fold higher compared to the latter. In contrast, the parent PEIs mediated no gene expression at all under similar conditions, and injection of the polyplexes of the 22-kDa PEI at its optimal N/P of 10 prepared under identical conditions killed half of the mice injected. Conclusions. High-throughput synthesis and transfection assay of a cross-linked library of biodegradable PEIs was proven effective in identifying highly transfecting vectors. The identified vectors exhibited dramatically superior efficiency compared to their parents both in vitro and in an in vivo systemic gene delivery model. The majority of these vectors mediated preferential gene delivery to the lung, and their in vivo toxicity paralleled that in vitro. Full Text.

Tomita, H., Yamada, Y., Oyama, T., Hata, K., Hirose, Y., Hara, A., Kunisada, T., Sugiyama, Y., Adachi, Y., Linhart, H., et al. (2007). Development of Gastric Tumors in ApcMin/+ Mice by the Activation of the {beta}-Catenin/Tcf Signaling Pathway. Cancer Res 67, 4079-4087. Although several lines of evidence suggest the involvement of the Wnt pathway in the development of gastric cancers, the functional significance of the pathway in gastric carcinogenesis is still poorly defined. To examine the role of the Apc/beta-catenin signaling pathway in the development of gastric cancers, we investigated the gastric mucosa of the Apc(Min/+) mouse, which is a murine model for familial adenomatous polyposis, carrying a germ-line mutation at codon 850 of Apc. We found that aged Apc(Min/+) mice spontaneously develop multiple tumors in the stomach, which are accompanied by loss of heterozygosity of Apc. Such tumors consisted of adenomatous glands with strong nuclear accumulation of beta-catenin. Even a single adenomatous gland already showed nuclear accumulation of beta-catenin, suggesting that Apc/beta-catenin pathway is an initiating event in gastric tumorigenesis in Apc(Min/+) mice. Myc and cyclin D1 expressions, which are transcriptional targets of beta-catenin/Tcf, increased in the adenomatous lesions. Furthermore, beta-catenin/Tcf reporter transgenic mice with Apc(Min) allele showed higher levels of the transcriptional activity of beta-catenin/Tcf in the gastric tumors. We also treated Apc(Min/+) and wild-type mice with N-methyl-N-nitrosourea (MNU), an alkylating agent that induces adenomas and adenocarcinomas in the stomach. Consequently, MNU-treated Apc(Min/+) mice significantly enhanced the tumor development in comparison with Apc(Min/+) mice or MNU-treated wild-type mice. Several gastric tumors in MNU-treated Apc(Min/+) mice showed invasion into the submucosal layer. These results indicate that the Apc/beta-catenin pathway may play an important role in at least subset of gastric carcinomas. In addition, Apc(Min/+) mice combined with MNU could be a useful short-term model to investigate multistage carcinogenesis in the stomach.Full Text

Tong, W., Ibarra, Y.M., and Lodish, H.F. (2007). Signals emanating from the membrane proximal region of the thrombopoietin receptor (mpl) support hematopoietic stem cell self-renewal. Experimental Hematology Jul 14; [Epub ahead of print]. Studies using thrombopoietin -/- (TPO-/-) or TPO receptor, mpl-/- mice have established a critical role for TPO/mpl signaling in hematopoietic stem cell (HSC) development. In this study, we further dissected mpl signaling in both megakaryopoiesis and HSC function, using mice bearing a truncated mpl receptor lacking the distal 60 amino acids (?60). This deletion removes three major signaling tyrosines on the mpl cytoplasmic domain, but retains the membrane proximal Box1 and Box2 domains required for JAK2 activation. Full Text

Um, M., Gross, A. W. and Lodish, H. F. (2007) A "classical" homodimeric erythropoietin receptor is essential for the antiapoptotic effects of erythropoietin on differentiated neuroblastoma SH-SY5Y and pheochromocytoma PC-12 cells. Cellular Signalling. 19 634-645. 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 I-125-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 I 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 P 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

Vardy, L., and Orr-Weaver, T.L. (2007). Regulating translation of maternal messages: multiple repression mechanisms. Trends Cell Biol. Oct 26; [Epub ahead of print] The dowry of mRNAs and proteins that mothers provide their progeny as part of a common developmental strategy to permit rapid embryogenesis necessitates precise translational regulation of the deposited mRNAs. Recent studies with Drosophila uncovered diverse mechanisms to control translation of the transcripts for genes that control the cell cycle and embryonic patterning. The newly delineated mechanisms include: alternative ways to disrupt eIF4E action and the formation of the preinitiation complex by the eIF4E homologous protein, d4EHP; recruitment of the deadenylase complex by the SMAUG and PUMILIO proteins; both poly(A)-dependent and -independent promotion of translation by the PNG kinase complex; and 5' cap-independent translational regulation by BRUNO. Full Text.

Vardy, L. and Orr-Weaver, T. L. (2007) The Drosophila PNG Kinase Complex Regulates the Translation of Cyclin B. Dev Cell. 12 157-66. The Drosophila PAN GU (PNG) kinase complex regulates the developmental translation of cyclin B. cyclin B mRNA becomes unmasked during oogenesis independent of PNG activity, but PNG is required for translation from egg activation. We find that although polyadenylation of cyclin B augments translation, it is not essential, and a fully elongated poly(A) is not required for translation to proceed. In fact, changes in poly(A) tail length are not sufficient to account for PNG-mediated control of cyclin B translation and of the early embryonic cell cycles. We present evidence that PNG functions instead as an antagonist of PUMILIO-dependent translational repression. Our data argue that changes in poly(A) tail length are not a universal mechanism governing embryonic cell cycles, and that PNG-mediated derepression of translation is an important alternative mechanism in Drosophila .Full Text

Vyas, J.M., Kim, Y.M., Artavanis-Tsakonas, K., Love, J.C., Van der Veen, A.G., and Ploegh, H.L. (2007). Tubulation of Class II MHC Compartments Is Microtubule Dependent and Involves Multiple Endolysosomal Membrane Proteins in Primary Dendritic Cells. J Immunol 178, 7199-7210. Immature dendritic cells (DCs) capture exogenous Ags in the periphery for eventual processing in endolysosomes. Upon maturation by TLR agonists, DCs deliver peptide-loaded class II MHC molecules from these compartments to the cell surface via long tubular structures (endolysosomal tubules). The nature and rules that govern the movement of these DC compartments are unknown. In this study, we demonstrate that the tubules contain multiple proteins including the class II MHC molecules and LAMP1, a lysosomal resident protein, as well as CD63 and CD82, members of the tetraspanin family. Endolysosomal tubules can be stained with acidotropic dyes, indicating that they are extensions of lysosomes. However, the proper trafficking of class II MHC molecules themselves is not necessary for endolysosomal tubule formation. DCs lacking MyD88 can also form endolysosomal tubules, demonstrating that MyD88-dependent TLR activation is not necessary for the formation of this compartment. Endolysosomal tubules in DCs exhibit dynamic and saltatory movement, including bidirectional travel. Measured velocities are consistent with motor-based movement along microtubules. Indeed, nocodazole causes the collapse of endolysosomal tubules. In addition to its association with microtubules, endolysosomal tubules follow the plus ends of microtubules as visualized in primary DCs expressing end binding protein 1 (EB1)-enhanced GFP. Full Text

Wang, Y. M., Medvid, R., Melton, C., Jaenisch, R. and Blelloch, R. (2007) DGCR8 is essential for microRNA biogenesis and silencing of embryonic stem cell self-renewal. Nature Genetics. 39 380-385. The molecular controls that govern the differentiation of embryonic stem (ES) cells remain poorly understood. DGCR8 is an RNA-binding protein that assists the RNase III enzyme Drosha in the processing of microRNAs (miRNAs), a subclass of small RNAs. Here we study the role of miRNAs in ES cell differentiation by generating a Dgcr8 knockout model. Analysis of mouse knockout ES cells shows that DGCR8 is essential for biogenesis of miRNAs. On the induction of differentiation, DGCR8-deficient ES cells do not fully downregulate pluripotency markers and retain the ability to produce ES cell colonies; however, they do express some markers of differentiation. This phenotype differs from that reported for Dicer1 knockout cells, suggesting that Dicer has miRNA-independent roles in ES cell function. Our findings indicate that miRNAs function in the silencing of ES cell self-renewal that normally occurs with the induction of differentiation. Full Text

Weinberg, R.A. (2007). Using maths to tackle cancer. Nature 449, 978-981. Full Text.

Wendler, P., Shorter, J., Plisson, C., Cashikar, A.G., Lindquist, S., and Saibil, H.R. (2007). Atypical Aaa+ Subunit Packing Creates an Expanded Cavity for Disaggregation by the Protein-Remodeling Factor Hsp104. Cell 131, 1366-1377. Hsp104, a yeast protein-remodeling factor of the AAA+ (ATPases associated with various cellular activities) superfamily, and its homologs in bacteria and plants mediate cell recovery after severe stress by disaggregating denatured proteins through a poorly understood mechanism. Here, we present cryo-electron microscopy maps and domain fitting of Hsp104 hexamers, revealing an unusual arrangement of AAA+ modules with the prominent coiled-coil domain intercalated between the AAA+ domains. This packing results in a greatly expanded cavity, which is capped at either end by N- and C-terminal domains. The fitted structures as well as mutation of conserved coiled-coil arginines suggest that the coiled-coil domain plays a major role in the extraction of proteins from aggregates, providing conserved residues for key functions in ATP hydrolysis and potentially for substrate interaction. The large cavity could enable the uptake of polypeptide loops without a requirement for exposed N or C termini. Full Text.

Wernig, M., Meissner, A., Foreman, R., Brambrink, T., Ku, M., Hochedlinger, K., Bernstein, B.E., and Jaenisch, R. (2007). In vitro reprogramming of fibroblasts into a pluripotent ES-cell-like state. Nature Advance online publication Published online 6 June. Nuclear transplantation can reprogramme a somatic genome back into an embryonic epigenetic state, and the reprogrammed nucleus can create a cloned animal or produce pluripotent embryonic stem cells. One potential use of the nuclear cloning approach is the derivation of 'customized' embryonic stem (ES) cells for patient-specific cell treatment, but technical and ethical considerations impede the therapeutic application of this technology. Reprogramming of fibroblasts to a pluripotent state can be induced in vitro through ectopic expression of the four transcription factors Oct4 (also called Oct3/4 or Pou5f1), Sox2, c-Myc and Klf4. Here we show that DNA methylation, gene expression and chromatin state of such induced reprogrammed stem cells are similar to those of ES cells. Notably, the cells—derived from mouse fibroblasts—can form viable chimaeras, can contribute to the germ line and can generate live late-term embryos when injected into tetraploid blastocysts. Our results show that the biological potency and epigenetic state of in-vitro-reprogrammed induced pluripotent stem cells are indistinguishable from those of ES cells.Full Text

Zeitlinger, J., Stark, A., Kellis, M., Hong, J.W., Nechaev, S., Adelman, K., Levine, M., and Young, R.A. (2007). RNA polymerase stalling at developmental control genes in the Drosophila melanogaster embryo. Nature Genetics Published online: 11 November 2007 It is widely assumed that the key rate-limiting step in gene activation is the recruitment of RNA polymerase II (Pol II) to the core promoter. Although there are well-documented examples in which Pol II is recruited to a gene but stalls, a general role for Pol II stalling in development has not been established. We have carried out comprehensive Pol II chromatin immunoprecipitation microarray (ChIP-chip) assays in Drosophila embryos and identified three distinct Pol II binding behaviors: active (uniform binding across the entire transcription unit), no binding, and stalled (binding at the transcription start site). The notable feature of the approximately 10% genes that are stalled is that they are highly enriched for developmental control genes, which are either repressed or poised for activation during later stages of embryogenesis. We propose that Pol II stalling facilitates rapid temporal and spatial changes in gene activity during development. Full Text.

Zeitlinger, J., Zinzen, R. P., Stark, A., Kellis, M., Zhang, H., Young, R. A. and Levine, M. (2007) Whole-genome ChIP-chip analysis of Dorsal, Twist, and Snail suggests integration of diverse patterning processes in the Drosophila embryo. Genes Dev. 21 385-90. Genetic studies have identified numerous sequence-specific transcription factors that control development, yet little is known about their in vivo distribution across animal genomes. We determined the genome-wide occupancy of the dorsoventral (DV) determinants Dorsal, Twist, and Snail in the Drosophila embryo using chromatin immunoprecipitation coupled with microarray analysis (ChIP-chip). The in vivo binding of these proteins correlate tightly with the limits of known enhancers. Our analysis predicts substantially more target genes than previous estimates, and includes Dpp signaling components and anteroposterior (AP) segmentation determinants. Thus, the ChIP-chip data uncover a much larger than expected regulatory network, which integrates diverse patterning processes during development.Full Text

Zeng, Z.H., Sarbassov, D.D., Samudio, I.J., Yee, K.W.L., Munsell, M.F., Jackson, C.E., Giles, F.J., Sabatini, D.M., Andreeff, M., and Konopleva, M. (2007). Rapamycin derivatives reduce mTORC2 signaling and inhibit AKT activation in AML. Blood 109, 3509-3512.The mTOR complex 2 (mTORC2) containing mTOR and rictor is thought to be rapamycin insensitive and was recently shown to regulate the prosurvival kinase AKT by phosphorylation on Ser473. We investigated the molecular effects of mTOR inhibition by the rapamycin derivatives (RDs) temsirolimus (CCI-779) and everolimus (RAD001) in acute myeloid leukemia (AML) cells. Unexpectedly, RDs not only inhibited the mTOR complex 1 (mTORC1) containing mTOR and raptor with decreased p70S6K, 4EPB1 phosphorylation, and GLUT1 mRNA, but also blocked AKT activation via inhibition of mTORC2 formation. This resulted in suppression of phosphorylation of the direct AKT substrate FKHR and decreased transcription of D-cyclins in AML cells. Similar observations were made in samples from patients with hematologic malignancies who received RDs in clinical studies. Our study provides the first evidence that rapamycin derivatives inhibit AKT signaling in primary AML cells both in vitro and in vivo, and supports the therapeutic potential of mTOR inhibition strategies in leukemias. Full Text

Zeskind, B.J., Jordan, C.D., Timp, W., Trapani, L., Waller, G., Horodincu, V., Ehrlich, D.J., and Matsudaira, P. (2007). Nucleic acid and protein mass mapping by live-cell deep-ultraviolet microscopy. Nat Methods. June 3; [Epub ahead of print] We developed a deep-ultraviolet (UV) microscope capable of imaging cell mitosis and motility at 280 nm for 45 min with minimal UV-induced toxicity, and for 6 h before the onset of visible cell death in cultured human and mouse cells. Combined with computational methods that convert the intensity of each pixel into an estimate of mass, deep-UV microscopy images generate maps of nucleic acid mass, protein mass and fluorescence yield in unlabeled cells.Full Text

Zhang, J., and Lodish, H.F. (2007). Endogenous K-ras signaling in erythroid differentiation. Cell Cycle 6, 1970-1973. K-ras is one of the most frequently mutated genes in virtually all types of human cancers. Using mouse fetal liver erythroid progenitors as a model system, we studied the role of endogenous K-ras signaling in erythroid differentiation. When oncogenic K-ras is expressed from its endogenous promoter, it hyperactivates cytokine-dependent signaling pathways and results in a partial block in erythroid differentiation. In erythroid progenitors deficient in K-ras, cytokine-dependent Akt activation is greatly reduced, leading to delays in erythroid differentiation. Thus, both loss- and gain-of-Kras functions affect erythroid differentiation through modulation of cytokine signaling. These results support the notion that in human cancer patients oncogenic Ras signaling might be controlled by antagonizing essential cytokines. PDF

Zhang, J., Liu, Y., Beard, C., Tuveson, D. A., Jaenisch, R., Jacks, T. E. and Lodish, H. F. (2007) Expression of oncogenic K-ras from its endogenous promoter leads to a partial block of erythroid differentiation and hyperactivation of cytokine-dependent signaling pathways. Blood.Feb 22; [Epub ahead of print] When overexpressed in primary erythroid progenitors, oncogenic Ras leads to the constitutive activation of its downstream signaling pathways, severe block of terminal erythroid differentiation, and cytokine-independent growth of primary erythroid progenitors. However, whether high-level expression of oncogenic Ras is required for these phenotypes is unknown. To address this issue, we expressed oncogenic K-ras (K-ras(G12D) ) from its endogenous promoter using a tetracycline-inducible system. We show that endogenous K-ras(G12D) leads to a partial block of terminal erythroid differentiation in vivo. In contrast to results obtained when oncogenic Ras was overexpressed from retroviral vectors, endogenous levels of K-ras(G12D) fail to constitutively activate but rather hyperactivates cytokine-dependent signaling pathways, including Stat5, Akt, and p44/42 MAPK, in primary erythroid progenitors. This explains previous observations that hematopoietic progenitors expressing endogenous K-ras(G12D) display hypersensitivity to cytokine stimulation in various colony assays. Our results support efforts to modulate Ras signaling for treating hematopoietic malignancies. PDF

Zhou, B., Wang, S., Mayr, C., Bartel, D.P., and Lodish, H.F. (2007). miR-150, a microRNA expressed in mature B and T cells, blocks early B cell development when expressed prematurely. Proc Natl Acad Sci U S A.Apr 16; [Epub ahead of print] MicroRNAs (miRNAs) are a family of approximately 22-nt noncoding RNAs that can posttranscriptionally regulate gene expression. Several miRNAs are specifically expressed in hematopoietic cells. Here we show that one such miRNA, miR-150, is mainly expressed in the lymph nodes and spleen and is highly up-regulated during the development of mature T and B cells; expression of miR-150 is sharply up-regulated at the immature B cell stage. Overexpression of miR-150 in hematopoietic stem cells, followed by bone marrow transplantation, had little effect on the formation of either mature CD8- and CD4-positive T cells or granulocytes or macrophages, but the formation of mature B cells was greatly impaired. Furthermore, premature expression of miR-150 blocked the transition from the pro-B to the pre-B stage. Our results indicate that miR-150 most likely down-regulates mRNAs that are important for pre- and pro-B cell formation or function, and its ectopic expression in these cells blocks further development of B cells. PDF

Zumbuehl, A., Ferreira, L., Kuhn, D., Astashkina, A., Long, L., Yeo, Y., Iaconis, T., Ghannoum, M., Fink, G.R., Langer, R., et al. (2007). Antifungal hydrogels. Proc Natl Acad Sci U S A. 2007 Jul 30; [Epub ahead of print] Fungi are increasingly identified as major pathogens in bloodstream infections, often involving indwelling devices. Materials with antifungal properties may provide an important deterrent to these infections. Here we describe amphogel, a dextran-based hydrogel into which amphotericin B is adsorbed. Amphogel kills fungi within 2 h of contact and can be reused for at least 53 days without losing its effectiveness against Candida albicans. The antifungal material is biocompatible in vivo and does not cause hemolysis in human blood. Amphogel inoculated with C. albicans and implanted in mice prevents fungal infection. Amphogel also mitigates fungal biofilm formation. An antifungal matrix with these properties could be used to coat a variety of medical devices such as catheters as well as industrial surfaces. PDF

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