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Showing papers by "Cold Spring Harbor Laboratory published in 2010"


Journal ArticleDOI
12 Aug 2010-Nature
TL;DR: It is demonstrated that mesenchymal stem cells (MSCs), identified using nestin expression, constitute an essential HSC niche component and are indicative of a unique niche in the bone marrow made of heterotypic stem-cell pairs.
Abstract: The cellular constituents forming the haematopoietic stem cell (HSC) niche in the bone marrow are unclear, with studies implicating osteoblasts, endothelial and perivascular cells. Here we demonstrate that mesenchymal stem cells (MSCs), identified using nestin expression, constitute an essential HSC niche component. Nestin(+) MSCs contain all the bone-marrow colony-forming-unit fibroblastic activity and can be propagated as non-adherent 'mesenspheres' that can self-renew and expand in serial transplantations. Nestin(+) MSCs are spatially associated with HSCs and adrenergic nerve fibres, and highly express HSC maintenance genes. These genes, and others triggering osteoblastic differentiation, are selectively downregulated during enforced HSC mobilization or beta3 adrenoreceptor activation. Whereas parathormone administration doubles the number of bone marrow nestin(+) cells and favours their osteoblastic differentiation, in vivo nestin(+) cell depletion rapidly reduces HSC content in the bone marrow. Purified HSCs home near nestin(+) MSCs in the bone marrow of lethally irradiated mice, whereas in vivo nestin(+) cell depletion significantly reduces bone marrow homing of haematopoietic progenitors. These results uncover an unprecedented partnership between two distinct somatic stem-cell types and are indicative of a unique niche in the bone marrow made of heterotypic stem-cell pairs.

3,012 citations


Journal ArticleDOI
TL;DR: It is shown that the majority of the newborn cells undergo death by apoptosis in the first 1 to 4 days of their life, during the transition from amplifying neuroprogenitors to neuroblasts, which suggests a new role for microglia in maintaining the homeostasis of the baseline neurogenic cascade.

1,322 citations


Journal ArticleDOI
Sushmita Roy1, Jason Ernst1, Peter V. Kharchenko2, Pouya Kheradpour1, Nicolas Nègre3, Matthew L. Eaton4, Jane M. Landolin5, Christopher A. Bristow1, Lijia Ma3, Michael F. Lin1, Stefan Washietl6, Bradley I. Arshinoff7, Ferhat Ay8, Patrick E. Meyer9, Nicolas Robine10, Nicole L. Washington5, Luisa Di Stefano2, Eugene Berezikov11, Christopher D. Brown3, Rogerio Candeias6, Joseph W. Carlson5, Adrian Carr12, Irwin Jungreis1, Daniel Marbach1, Rachel Sealfon1, Michael Y. Tolstorukov2, Sebastian Will6, Artyom A. Alekseyenko2, Carlo G. Artieri13, Benjamin W. Booth5, Angela N. Brooks14, Qi Dai10, Carrie A. Davis15, Michael O. Duff16, X. Feng, Andrey A. Gorchakov2, Tingting Gu17, Jorja G. Henikoff10, Philipp Kapranov18, Renhua Li13, Heather K. MacAlpine4, John H. Malone13, Aki Minoda5, Jared T. Nordman6, Katsutomo Okamura10, Marc D. Perry7, Sara K. Powell4, Nicole C. Riddle17, Akiko Sakai2, Anastasia Samsonova2, Jeremy E. Sandler5, Yuri B. Schwartz2, Noa Sher6, Rebecca Spokony3, David Sturgill13, Marijke J. van Baren17, Kenneth H. Wan5, Li Yang16, Charles Yu5, Elise A. Feingold13, Peter J. Good13, Mark S. Guyer13, Rebecca F. Lowdon13, Kami Ahmad2, Justen Andrews19, Bonnie Berger1, Steven E. Brenner14, Michael R. Brent17, Lucy Cherbas19, Sarah C. R. Elgin17, Thomas R. Gingeras18, Robert L. Grossman3, Roger A. Hoskins5, Thomas C. Kaufman19, W. J. Kent20, Mitzi I. Kuroda2, Terry L. Orr-Weaver6, Norbert Perrimon2, Vincenzo Pirrotta21, James W. Posakony22, Bing Ren22, Steven Russell12, Peter Cherbas19, Brenton R. Graveley16, Suzanna E. Lewis5, Gos Micklem12, Brian Oliver13, Peter J. Park2, Susan E. Celniker5, Steven Henikoff23, Gary H. Karpen14, Eric C. Lai10, David M. MacAlpine4, Lincoln Stein7, Kevin P. White3, Manolis Kellis1 
24 Dec 2010-Science
TL;DR: The Drosophila Encyclopedia of DNA Elements (modENCODE) project as mentioned in this paper has been used to map transcripts, histone modifications, chromosomal proteins, transcription factors, replication proteins and intermediates, and nucleosome properties across a developmental time course and in multiple cell lines.
Abstract: To gain insight into how genomic information is translated into cellular and developmental programs, the Drosophila model organism Encyclopedia of DNA Elements (modENCODE) project is comprehensively mapping transcripts, histone modifications, chromosomal proteins, transcription factors, replication proteins and intermediates, and nucleosome properties across a developmental time course and in multiple cell lines. We have generated more than 700 data sets and discovered protein-coding, noncoding, RNA regulatory, replication, and chromatin elements, more than tripling the annotated portion of the Drosophila genome. Correlated activity patterns of these elements reveal a functional regulatory network, which predicts putative new functions for genes, reveals stage- and tissue-specific regulators, and enables gene-expression prediction. Our results provide a foundation for directed experimental and computational studies in Drosophila and related species and also a model for systematic data integration toward comprehensive genomic and functional annotation.

1,102 citations


Journal ArticleDOI
TL;DR: Understanding the complex ways in which cancer cells interact with their surroundings, both locally in the tumor organ and systemically in the body as a whole, has implications for effective cancer prevention and therapy.

1,069 citations


Journal ArticleDOI
Mark Gerstein1, Zhi John Lu1, Eric L. Van Nostrand2, Chao Cheng1, Bradley I. Arshinoff3, Tao Liu4, Kevin Y. Yip1, R. Robilotto1, Andreas Rechtsteiner5, Kohta Ikegami6, P. Alves1, A. Chateigner, Marc D. Perry7, Mitzi Morris8, Raymond K. Auerbach1, X. Feng9, Jing Leng1, A. Vielle10, Wei Niu1, Kahn Rhrissorrakrai8, Ashish Agarwal1, Roger P. Alexander1, Galt P. Barber5, Cathleen M. Brdlik2, J. Brennan6, Jeremy Brouillet2, Adrian Carr, Ming Sin Cheung10, Hiram Clawson5, Sergio Contrino, Luke Dannenberg11, Abby F. Dernburg12, Arshad Desai13, L. Dick14, Andréa C. Dosé12, Jiang Du1, Thea A. Egelhofer5, Sevinc Ercan6, Ghia Euskirchen1, Brent Ewing15, Elise A. Feingold16, Reto Gassmann13, Peter J. Good16, Philip Green15, Francois Gullier, M. Gutwein8, Mark S. Guyer16, Lukas Habegger1, Ting Han17, Jorja G. Henikoff18, Stefan R. Henz19, Angie S. Hinrichs5, H. Holster11, Tony Hyman19, A. Leo Iniguez11, J. Janette1, M. Jensen6, Masaomi Kato1, W. James Kent5, E. Kephart7, Vishal Khivansara17, Ekta Khurana1, John Kim17, P. Kolasinska-Zwierz10, Eric C. Lai20, Isabel J. Latorre10, Amber Leahey15, Suzanna E. Lewis12, Paul Lloyd7, Lucas Lochovsky1, Rebecca F. Lowdon16, Yaniv Lubling21, Rachel Lyne, Michael J. MacCoss15, Sebastian D. Mackowiak22, Marco Mangone8, Sheldon J. McKay23, D. Mecenas8, Gennifer E. Merrihew15, David M. Miller24, A. Muroyama13, John I. Murray15, Siew Loon Ooi18, Hoang Pham12, T. Phippen5, Elicia Preston15, Nikolaus Rajewsky22, Gunnar Rätsch19, Heidi Rosenbaum11, Joel Rozowsky1, Kim Rutherford, P. Ruzanov7, Mihail Sarov19, Rajkumar Sasidharan1, Andrea Sboner1, P. Scheid8, Eran Segal21, Hyunjin Shin4, C. Shou1, Frank J. Slack1, C. Slightam2, Richard J.H. Smith, William C. Spencer24, Eo Stinson12, S. Taing4, Teruaki Takasaki5, D. Vafeados15, Ksenia Voronina13, Guilin Wang1, Nicole L. Washington12, Christina M. Whittle6, Beijing Wu2, Koon-Kiu Yan1, Georg Zeller, Z. Zha7, Mei Zhong1, Xingliang Zhou6, Julie Ahringer10, Susan Strome5, Kristin C. Gunsalus25, Gos Micklem, X. Shirley Liu4, Valerie Reinke1, Stuart K. Kim2, LaDeana W. Hillier15, Steven Henikoff18, Fabio Piano25, Michael Snyder1, Lincoln Stein23, Jason D. Lieb6, Robert H. Waterston15 
24 Dec 2010-Science
TL;DR: These studies identified regions of the nematode and fly genomes that show highly occupied targets (or HOT) regions where DNA was bound by more than 15 of the transcription factors analyzed and the expression of related genes were characterized, providing insights into the organization, structure, and function of the two genomes.
Abstract: We systematically generated large-scale data sets to improve genome annotation for the nematode Caenorhabditis elegans, a key model organism. These data sets include transcriptome profiling across a developmental time course, genome-wide identification of transcription factor-binding sites, and maps of chromatin organization. From this, we created more complete and accurate gene models, including alternative splice forms and candidate noncoding RNAs. We constructed hierarchical networks of transcription factor-binding and microRNA interactions and discovered chromosomal locations bound by an unusually large number of transcription factors. Different patterns of chromatin composition and histone modification were revealed between chromosome arms and centers, with similarly prominent differences between autosomes and the X chromosome. Integrating data types, we built statistical models relating chromatin, transcription factor binding, and gene expression. Overall, our analyses ascribed putative functions to most of the conserved genome.

978 citations


Journal ArticleDOI
TL;DR: How abnormal expression, proteolysis and structure of these collagens influence cellular functions to elicit multiple effects on tumors, including proliferation, initiation, invasion, metastasis, and therapy response is discussed.

744 citations


Journal ArticleDOI
TL;DR: The current version of Chronux includes software for signal processing of neural time-series data including several specialized mini-packages for spike-sorting, local regression, audio segmentation, and other data-analysis tasks typically encountered by a neuroscientist.

734 citations


Journal ArticleDOI
TL;DR: It is shown, using a mouse model of liver cancer, that miR-221 overexpression stimulates growth of tumorigenic murine hepatic progenitor cells, and DNA damage-inducible transcript 4 (DDIT4), a modulator of mTOR pathway, is identified as a bona fide target of miR -221.
Abstract: MicroRNA (miRNAs) are negative regulators of gene expression and can function as tumor suppressors or oncogenes. Expression patterns of miRNAs and their role in the pathogenesis of hepatocellular carcinoma (HCC) are still poorly understood. We profiled miRNA expression in tissue samples (104 HCC, 90 adjacent cirrhotic livers, 21 normal livers) as well as in 35 HCC cell lines. A set of 12 miRNAs (including miR-21, miR-221/222, miR-34a, miR-519a, miR-93, miR-96, and let-7c) was linked to disease progression from normal liver through cirrhosis to full-blown HCC. miR-221/222, the most up-regulated miRNAs in tumor samples, are shown to target the CDK inhibitor p27 and to enhance cell growth in vitro. Conversely, these activities can be efficiently inhibited by an antagomiR specific for miR-221. In addition, we show, using a mouse model of liver cancer, that miR-221 overexpression stimulates growth of tumorigenic murine hepatic progenitor cells. Finally, we identified DNA damage-inducible transcript 4 (DDIT4), a modulator of mTOR pathway, as a bona fide target of miR-221. Taken together, these data reveal an important contribution for miR-221 in hepatocarcinogenesis and suggest a role for DDIT4 dysregulation in this process. Thus, the use of synthetic inhibitors of miR-221 may prove to be a promising approach to liver cancer treatment.

717 citations


Journal ArticleDOI
Emek Demir1, Emek Demir2, Michael P. Cary1, Suzanne M. Paley3, Ken Fukuda, Christian Lemer4, Imre Vastrik, Guanming Wu5, Peter D'Eustachio6, Carl F. Schaefer7, Joanne S. Luciano, Frank Schacherer, Irma Martínez-Flores8, Zhenjun Hu9, Verónica Jiménez-Jacinto8, Geeta Joshi-Tope10, Kumaran Kandasamy11, Alejandra López-Fuentes8, Huaiyu Mi3, Elgar Pichler, Igor Rodchenkov12, Andrea Splendiani13, Andrea Splendiani14, Sasha Tkachev15, Jeremy Zucker16, Gopal R. Gopinath17, Harsha Rajasimha18, Harsha Rajasimha7, Ranjani Ramakrishnan19, Imran Shah20, Mustafa H Syed21, Nadia Anwar1, Özgün Babur2, Özgün Babur1, Michael L. Blinov22, Erik Brauner23, Dan Corwin, Sylva L. Donaldson12, Frank Gibbons23, Robert N. Goldberg24, Peter Hornbeck15, Augustin Luna7, Peter Murray-Rust25, Eric K. Neumann, Oliver Reubenacker22, Matthias Samwald26, Matthias Samwald27, Martijn P. van Iersel28, Sarala M. Wimalaratne29, Keith Allen30, Burk Braun, Michelle Whirl-Carrillo31, Kei-Hoi Cheung32, Kam D. Dahlquist33, Andrew Finney, Marc Gillespie34, Elizabeth M. Glass21, Li Gong31, Robin Haw5, Michael Honig35, Olivier Hubaut4, David W. Kane36, Shiva Krupa37, Martina Kutmon38, Julie Leonard30, Debbie Marks23, David Merberg39, Victoria Petri40, Alexander R. Pico41, Dean Ravenscroft42, Liya Ren10, Nigam H. Shah31, Margot Sunshine7, Rebecca Tang30, Ryan Whaley30, Stan Letovksy43, Kenneth H. Buetow7, Andrey Rzhetsky44, Vincent Schächter45, Bruno S. Sobral18, Ugur Dogrusoz2, Shannon K. McWeeney19, Mirit I. Aladjem7, Ewan Birney, Julio Collado-Vides8, Susumu Goto46, Michael Hucka47, Nicolas Le Novère, Natalia Maltsev21, Akhilesh Pandey11, Paul Thomas3, Edgar Wingender, Peter D. Karp3, Chris Sander1, Gary D. Bader12 
TL;DR: Thousands of interactions, organized into thousands of pathways, from many organisms are available from a growing number of databases, and this large amount of pathway data in a computable form will support visualization, analysis and biological discovery.
Abstract: Biological Pathway Exchange (BioPAX) is a standard language to represent biological pathways at the molecular and cellular level and to facilitate the exchange of pathway data. The rapid growth of the volume of pathway data has spurred the development of databases and computational tools to aid interpretation; however, use of these data is hampered by the current fragmentation of pathway information across many databases with incompatible formats. BioPAX, which was created through a community process, solves this problem by making pathway data substantially easier to collect, index, interpret and share. BioPAX can represent metabolic and signaling pathways, molecular and genetic interactions and gene regulation networks. Using BioPAX, millions of interactions, organized into thousands of pathways, from many organisms are available from a growing number of databases. This large amount of pathway data in a computable form will support visualization, analysis and biological discovery.

673 citations


Journal ArticleDOI
TL;DR: DNA microarray analysis in Malat1‐depleted neuroblastoma cells indicates that Malat 1 controls the expression of genes involved not only in nuclear processes, but also in synapse function, suggesting that Mal at1 regulates synapse formation by modulating the expressionof genes involved in synapses formation and/or maintenance.
Abstract: A growing number of long nuclear-retained non-coding RNAs (ncRNAs) have recently been described. However, few functions have been elucidated for these ncRNAs. Here, we have characterized the function of one such ncRNA, identified as metastasis-associated lung adenocarcinoma transcript 1 (Malat1). Malat1 RNA is expressed in numerous tissues and is highly abundant in neurons. It is enriched in nuclear speckles only when RNA polymerase II-dependent transcription is active. Knock-down studies revealed that Malat1 modulates the recruitment of SR family pre-mRNA-splicing factors to the transcription site of a transgene array. DNA microarray analysis in Malat1-depleted neuroblastoma cells indicates that Malat1 controls the expression of genes involved not only in nuclear processes, but also in synapse function. In cultured hippocampal neurons, knock-down of Malat1 decreases synaptic density, whereas its over-expression results in a cell-autonomous increase in synaptic density. Our results suggest that Malat1 regulates synapse formation by modulating the expression of genes involved in synapse formation and/or maintenance.

641 citations


Journal ArticleDOI
TL;DR: A highly reliable functional interaction network upon expert-curated pathways is built and applied to the analysis of two genome-wide GBM and several other cancer data sets, suggesting common mechanisms in the cancer biology.
Abstract: One challenge facing biologists is to tease out useful information from massive data sets for further analysis. A pathway-based analysis may shed light by projecting candidate genes onto protein functional relationship networks. We are building such a pathway-based analysis system. We have constructed a protein functional interaction network by extending curated pathways with non-curated sources of information, including protein-protein interactions, gene coexpression, protein domain interaction, Gene Ontology (GO) annotations and text-mined protein interactions, which cover close to 50% of the human proteome. By applying this network to two glioblastoma multiforme (GBM) data sets and projecting cancer candidate genes onto the network, we found that the majority of GBM candidate genes form a cluster and are closer than expected by chance, and the majority of GBM samples have sequence-altered genes in two network modules, one mainly comprising genes whose products are localized in the cytoplasm and plasma membrane, and another comprising gene products in the nucleus. Both modules are highly enriched in known oncogenes, tumor suppressors and genes involved in signal transduction. Similar network patterns were also found in breast, colorectal and pancreatic cancers. We have built a highly reliable functional interaction network upon expert-curated pathways and applied this network to the analysis of two genome-wide GBM and several other cancer data sets. The network patterns revealed from our results suggest common mechanisms in the cancer biology. Our system should provide a foundation for a network or pathway-based analysis platform for cancer and other diseases.

Journal ArticleDOI
TL;DR: This work introduces Quake, a program to detect and correct errors in DNA sequencing reads using a maximum likelihood approach incorporating quality values and nucleotide specific miscall rates, which achieves the highest accuracy on realistically simulated reads.
Abstract: We introduce Quake, a program to detect and correct errors in DNA sequencing reads. Using a maximum likelihood approach incorporating quality values and nucleotide specific miscall rates, Quake achieves the highest accuracy on realistically simulated reads. We further demonstrate substantial improvements in de novo assembly and SNP detection after using Quake. Quake can be used for any size project, including more than one billion human reads, and is freely available as open source software from http://www.cbcb.umd.edu/software/quake.

Journal ArticleDOI
TL;DR: It is concluded that this MOE ASO is a promising drug candidate for SMA therapy, and, more generally, that ASOs can be used to efficiently redirect alternative splicing of target genes in the CNS.
Abstract: Increasing survival of motor neuron 2, centromeric (SMN2) exon 7 inclusion to express more full-length SMN protein in motor neurons is a promising approach to treat spinal muscular atrophy (SMA), a genetic neurodegenerative disease. Previously, we identified a potent 29-O-(2-methoxyethyl) (MOE) phosphorothioate-modified antisense oligonucleotide (ASO) that blocks an SMN2 intronic splicing silencer element and efficiently promotes exon 7 inclusion in transgenic mouse peripheral tissues after systemic administration. Here we address its efficacy in the spinal cord—a prerequisite for disease treatment—and its ability to rescue a mild SMA mouse model that develops tail and ear necrosis, resembling the distal tissue necrosis reported in some SMA infants. Using a microosmotic pump, we directly infused the ASO into a lateral cerebral ventricle in adult mice expressing a human SMN2 transgene; the ASO gave a robust and long-lasting increase in SMN2 exon 7 inclusion measured at both the mRNA and protein levels in spinal cord motor neurons. A single embryonic or neonatal intracerebroventricular ASO injection strikingly rescued the tail and ear necrosis in SMA mice. We conclude that this MOE ASO is a promising drug candidate for SMA therapy, and, more generally, that ASOs can be used to efficiently redirect alternative splicing of target genes in the CNS.

Journal ArticleDOI
TL;DR: The data support a two-hit model in which the 16p12.1 microdeletion both predisposes to neuropsychiatric phenotypes as a single event and exacerbates neurodevelopmental phenotypes in association with other large deletions or duplications.
Abstract: We report the identification of a recurrent, 520-kb 16p12.1 microdeletion associated with childhood developmental delay. The microdeletion was detected in 20 of 11,873 cases compared with 2 of 8,540 controls (P = 0.0009, OR = 7.2) and replicated in a second series of 22 of 9,254 cases compared with 6 of 6,299 controls (P = 0.028, OR = 2.5). Most deletions were inherited, with carrier parents likely to manifest neuropsychiatric phenotypes compared to non-carrier parents (P = 0.037, OR = 6). Probands were more likely to carry an additional large copy-number variant when compared to matched controls (10 of 42 cases, P = 5.7 x 10(-5), OR = 6.6). The clinical features of individuals with two mutations were distinct from and/or more severe than those of individuals carrying only the co-occurring mutation. Our data support a two-hit model in which the 16p12.1 microdeletion both predisposes to neuropsychiatric phenotypes as a single event and exacerbates neurodevelopmental phenotypes in association with other large deletions or duplications. Analysis of other microdeletions with variable expressivity indicates that this two-hit model might be more generally applicable to neuropsychiatric disease.

Journal ArticleDOI
20 Oct 2010-PLOS ONE
TL;DR: It is reported that released miRNAs do not necessarily reflect the abundance of miRNA in the cell of origin, and the existence of a cellular selection mechanism for miRNA release is suggested and it is indicated that the extracellular and cellular miRNA profiles differ.
Abstract: MicroRNAs (miRNAs) in body fluids are candidate diagnostics for a variety of conditions and diseases, including breast cancer. One premise for using extracellular miRNAs to diagnose disease is the notion that the abundance of the miRNAs in body fluids reflects their abundance in the abnormal cells causing the disease. As a result, the search for such diagnostics in body fluids has focused on miRNAs that are abundant in the cells of origin. Here we report that released miRNAs do not necessarily reflect the abundance of miRNA in the cell of origin. We find that release of miRNAs from cells into blood, milk and ductal fluids is selective and that the selection of released miRNAs may correlate with malignancy. In particular, the bulk of miR-451 and miR-1246 produced by malignant mammary epithelial cells was released, but the majority of these miRNAs produced by non-malignant mammary epithelial cells was retained. Our findings suggest the existence of a cellular selection mechanism for miRNA release and indicate that the extracellular and cellular miRNA profiles differ. This selective release of miRNAs is an important consideration for the identification of circulating miRNAs as biomarkers of disease.

Journal ArticleDOI
TL;DR: An alterative working model for cancer development is presented in which subtle reductions in the dose of TSGs predispose to tumorigenesis in a tissue-specific manner and subtle downregulation of Pten altered the steady-state biology of the mammary tissues and the expression profiles of genes involved in cancer cell proliferation.
Abstract: Cancer susceptibility has been attributed to at least one heterozygous genetic alteration in a tumor suppressor gene (TSG). It has been hypothesized that subtle variations in TSG expression can promote cancer development. However, this hypothesis has not yet been definitively supported in vivo. Pten is a TSG frequently lost in human cancer and mutated in inherited cancer-predisposition syndromes. Here we analyze Pten hypermorphic mice (Pten(hy/+)), expressing 80% normal levels of Pten. Pten(hy/+) mice develop a spectrum of tumors, with breast tumors occurring at the highest penetrance. All breast tumors analyzed here retained two intact copies of Pten and maintained Pten levels above heterozygosity. Notably, subtle downregulation of Pten altered the steady-state biology of the mammary tissues and the expression profiles of genes involved in cancer cell proliferation. We present an alterative working model for cancer development in which subtle reductions in the dose of TSGs predispose to tumorigenesis in a tissue-specific manner.

Journal ArticleDOI
01 Feb 2010-Proteins
TL;DR: It is shown that gain and loss of predicted ubiquitination sites may likely represent a molecular mechanism behind a number of disease‐associatedmutations.
Abstract: Ubiquitination plays an important role in many cellular processes and is implicated in many diseases. Experimental identification of ubiquitination sites is challenging due to rapid turnover of ubiquitinated proteins and the large size of the ubiquitin modifier. We identified 141 new ubiquitination sites using a combination of liquid chromatography, mass spectrometry, and mutant yeast strains. Investigation of the sequence biases and structural preferences around known ubiquitination sites indicated that their properties were similar to those of intrinsically disordered protein regions. Using a combined set of new and previously known ubiquitination sites, we developed a random forest predictor of ubiquitination sites, UbPred. The class-balanced accuracy of UbPred reached 72%, with the area under the ROC curve at 80%. The application of UbPred showed that high confidence Rsp5 ubiquitin ligase substrates and proteins with very short half-lives were significantly enriched in the number of predicted ubiquitination sites. Proteome-wide prediction of ubiquitination sites in Saccharomyces cerevisiae indicated that highly ubiquitinated substrates were prevalent among transcription/enzyme regulators and proteins involved in cell cycle control. In the human proteome, cytoskeletal, cell cycle, regulatory, and cancer-associated proteins display higher extent of ubiquitination than proteins from other functional categories. We show that gain and loss of predicted ubiquitination sites may likely represent a molecular mechanism behind a number of disease-associatedmutations. UbPred is available at http://www.ubpred.org.

Journal ArticleDOI
25 Mar 2010-Nature
TL;DR: The results show that AGO9-dependent sRNA silencing is crucial to specify cell fate in the Arabidopsis ovule, and that epigenetic reprogramming in companion cells is necessary for sRNA–dependent silencing in plant gametes.
Abstract: In the ovules of most sexual flowering plants female gametogenesis is initiated from a single surviving gametic cell, the functional megaspore, formed after meiosis of the somatically derived megaspore mother cell (MMC). Because some mutants and certain sexual species exhibit more than one MMC, and many others are able to form gametes without meiosis (by apomixis), it has been suggested that somatic cells in the ovule are competent to respond to a local signal likely to have an important function in determination. Here we show that the Arabidopsis protein ARGONAUTE 9 (AGO9) controls female gamete formation by restricting the specification of gametophyte precursors in a dosage-dependent, non-cell-autonomous manner. Mutations in AGO9 lead to the differentiation of multiple gametic cells that are able to initiate gametogenesis. The AGO9 protein is not expressed in the gamete lineage; instead, it is expressed in cytoplasmic foci of somatic companion cells. Mutations in SUPPRESSOR OF GENE SILENCING 3 and RNA-DEPENDENT RNA POLYMERASE 6 exhibit an identical defect to ago9 mutants, indicating that the movement of small RNA (sRNAs) silencing out of somatic companion cells is necessary for controlling the specification of gametic cells. AGO9 preferentially interacts with 24-nucleotide sRNAs derived from transposable elements (TEs), and its activity is necessary to silence TEs in female gametes and their accessory cells. Our results show that AGO9-dependent sRNA silencing is crucial to specify cell fate in the Arabidopsis ovule, and that epigenetic reprogramming in companion cells is necessary for sRNA-dependent silencing in plant gametes.

Journal ArticleDOI
TL;DR: In polygenomic tumors, it is shown that heterogeneity can be ascribed to a few clonal subpopulations, rather than a series of gradual intermediates, and inferred pathways of cancer progression and the organization of tumor growth are inferred.
Abstract: Cancer progression in humans is difficult to infer because we do not routinely sample patients at multiple stages of their disease. However, heterogeneous breast tumors provide a unique opportunity to study human tumor progression because they still contain evidence of early and intermediate subpopulations in the form of the phylogenetic relationships. We have developed a method we call Sector-Ploidy-Profiling (SPP) to study the clonal composition of breast tumors. SPP involves macro-dissecting tumors, flow-sorting genomic subpopulations by DNA content, and profiling genomes using comparative genomic hybridization (CGH). Breast carcinomas display two classes of genomic structural variation: (1) monogenomic and (2) polygenomic. Monogenomic tumors appear to contain a single major clonal subpopulation with a highly stable chromosome structure. Polygenomic tumors contain multiple clonal tumor subpopulations, which may occupy the same sectors, or separate anatomic locations. In polygenomic tumors, we show that heterogeneity can be ascribed to a few clonal subpopulations, rather than a series of gradual intermediates. By comparing multiple subpopulations from different anatomic locations, we have inferred pathways of cancer progression and the organization of tumor growth.

Journal ArticleDOI
TL;DR: It is reported that heterozygosity for tomato loss-of-function alleles of SINGLE FLOWER TRUSS (SFT), which is the genetic originator of the flowering hormone florigen, increases yield by up to 60%.
Abstract: Intercrossing different varieties of plants frequently produces hybrid offspring with superior vigor and increased yields, in a poorly understood phenomenon known as heterosis. One classical unproven model for heterosis is overdominance, which posits in its simplest form that improved vigor can result from a single heterozygous gene. Here we report that heterozygosity for tomato loss-of-function alleles of SINGLE FLOWER TRUSS (SFT), which is the genetic originator of the flowering hormone florigen, increases yield by up to 60%. Yield overdominance from SFT heterozygosity is robust, occurring in distinct genetic backgrounds and environments. We show that several traits integrate pleiotropically to drive heterosis in a multiplicative manner, and these effects derive from a suppression of growth termination mediated by SELF PRUNING (SP), an antagonist of SFT. Our findings provide the first example of a single overdominant gene for yield and suggest that single heterozygous mutations may improve productivity in other agricultural organisms.

Journal ArticleDOI
TL;DR: A rapid peptide screening approach was used to determine consensus phosphorylation site motifs targeted by 61 of the 122 kinases in Saccharomyces cerevisiae, and previously unappreciated rules for determining specificity within the kinase family were uncovered.
Abstract: Phosphorylation is a universal mechanism for regulating cell behavior in eukaryotes. Although protein kinases target short linear sequence motifs on their substrates, the rules for kinase substrate recognition are not completely understood. We used a rapid peptide screening approach to determine consensus phosphorylation site motifs targeted by 61 of the 122 kinases in Saccharomyces cerevisiae. By correlating these motifs with kinase primary sequence, we uncovered previously unappreciated rules for determining specificity within the kinase family, including a residue determining P-3 arginine specificity among members of the CMGC [CDK (cyclin-dependent kinase), MAPK (mitogen-activated protein kinase), GSK (glycogen synthase kinase), and CDK-like] group of kinases. Furthermore, computational scanning of the yeast proteome enabled the prediction of thousands of new kinase-substrate relationships. We experimentally verified several candidate substrates of the Prk1 family of kinases in vitro and in vivo and identified a protein substrate of the kinase Vhs1. Together, these results elucidate how kinase catalytic domains recognize their phosphorylation targets and suggest general avenues for the identification of previously unknown kinase substrates across eukaryotes.

Journal ArticleDOI
TL;DR: It is argued that both tumor cell-autonomous mechanisms and/or activation of the tumor microenvironment could contribute to primary and acquired erlotinib resistance, and as such, treatments based on EGFR inhibition may not be sufficient for the effective treatment of lung-cancer patients harboring mutant EGFR.
Abstract: The epidermal growth-factor receptor (EGFR) tyrosine kinase inhibitor erlotinib has been proven to be highly effective in the treatment of nonsmall cell lung cancer (NSCLC) harboring oncogenic EGFR mutations. The majority of patients, however, will eventually develop resistance and succumb to the disease. Recent studies have identified secondary mutations in the EGFR (EGFR T790M) and amplification of the N-Methyl-N′-nitro-N-nitroso-guanidine (MNNG) HOS transforming gene (MET) oncogene as two principal mechanisms of acquired resistance. Although they can account for approximately 50% of acquired resistance cases together, in the remaining 50%, the mechanism remains unknown. In NSCLC-derived cell lines and early-stage tumors before erlotinib treatment, we have uncovered the existence of a subpopulation of cells that are intrinsically resistant to erlotinib and display features suggestive of epithelial-to-mesenchymal transition (EMT). We showed that activation of TGF-β–mediated signaling was sufficient to induce these phenotypes. In particular, we determined that an increased TGF-β–dependent IL-6 secretion unleashed previously addicted lung tumor cells from their EGFR dependency. Because IL-6 and TGF-β are prominently produced during inflammatory response, we used a mouse model system to determine whether inflammation might impair erlotinib sensitivity. Indeed, induction of inflammation not only stimulated IL-6 secretion but was sufficient to decrease the tumor response to erlotinib. Our data, thus, argue that both tumor cell-autonomous mechanisms and/or activation of the tumor microenvironment could contribute to primary and acquired erlotinib resistance, and as such, treatments based on EGFR inhibition may not be sufficient for the effective treatment of lung-cancer patients harboring mutant EGFR.

Journal ArticleDOI
TL;DR: It is demonstrated that RB preferentially associates with E2F target genes involved in DNA replication and is uniquely required to repress these genes during senescence but not other growth states, which leads to RB loss leading to inappropriate DNA synthesis following a senescences trigger and enables extensive proliferation and rampant genomic instability.

Journal ArticleDOI
TL;DR: It is shown that miR-19 is sufficient to promote leukaemogenesis in Notch1-induced T-cell acute lymphoblastic leukaemia (T-ALL) in vivo and an unbiased, functional genomics approach reveals a coordinate clampdown on several regulators of phosphatidylinositol-3-OH kinase-related survival signals by theLeukaemogenic miR -19.
Abstract: MicroRNAs (miRNAs) have emerged as novel cancer genes. In particular, the miR-17-92 cluster, containing six individual miRNAs, is highly expressed in haematopoietic cancers and promotes lymphomagenesis in vivo. Clinical use of these findings hinges on isolating the oncogenic activity within the 17-92 cluster and defining its relevant target genes. Here we show that miR-19 is sufficient to promote leukaemogenesis in Notch1-induced T-cell acute lymphoblastic leukaemia (T-ALL) in vivo. In concord with the pathogenic importance of this interaction in T-ALL, we report a novel translocation that targets the 17-92 cluster and coincides with a second rearrangement that activates Notch1. To identify the miR-19 targets responsible for its oncogenic action, we conducted a large-scale short hairpin RNA screen for genes whose knockdown can phenocopy miR-19. Strikingly, the results of this screen were enriched for miR-19 target genes, and include Bim (Bcl2L11), AMP-activated kinase (Prkaa1) and the phosphatases Pten and PP2A (Ppp2r5e). Hence, an unbiased, functional genomics approach reveals a coordinate clampdown on several regulators of phosphatidylinositol-3-OH kinase-related survival signals by the leukaemogenic miR-19.

Journal ArticleDOI
07 Jan 2010-Nature
TL;DR: It is shown that the amino terminal serine/threonine-rich domain of Mcm4 has both inhibitory and facilitating roles in DNA replication control and that the sole essential function of DDK is to relieve an inhibitory activity residing within the NSD.
Abstract: Eukaryotic DNA replication uses kinase regulatory pathways to facilitate coordination with other processes during cell division cycles and response to environmental cues. At least two cell cycle-regulated protein kinase systems, the S-phase-specific cyclin-dependent protein kinases (S-CDKs) and the Dbf4-Cdc7 kinase (DDK, Dbf4-dependent protein kinase) are essential activators for initiation of DNA replication. Although the essential mechanism of CDK activation of DNA replication in Saccharomyces cerevisiae has been established, exactly how DDK acts has been unclear. Here we show that the amino terminal serine/threonine-rich domain (NSD) of Mcm4 has both inhibitory and facilitating roles in DNA replication control and that the sole essential function of DDK is to relieve an inhibitory activity residing within the NSD. By combining an mcm4 mutant lacking the inhibitory activity with mutations that bypass the requirement for CDKs for initiation of DNA replication, we show that DNA synthesis can occur in G1 phase when CDKs and DDK are limited. However, DDK is still required for efficient S phase progression. In the absence of DDK, CDK phosphorylation at the distal part of the Mcm4 NSD becomes crucial. Moreover, DDK-null cells fail to activate the intra-S-phase checkpoint in the presence of hydroxyurea-induced DNA damage and are unable to survive this challenge. Our studies establish that the eukaryote-specific NSD of Mcm4 has evolved to integrate several protein kinase regulatory signals for progression through S phase.

Journal ArticleDOI
TL;DR: In this paper, the genome-wide binding sites of 6 insulator-associated proteins (dCTCF, CP190, BEAF-32, Su(Hw), Mod(mdg4), and GAF) were studied to obtain the first comprehensive map of insulator elements in Drosophila embryos.
Abstract: Insulators are DNA sequences that control the interactions among genomic regulatory elements and act as chromatin boundaries. A thorough understanding of their location and function is necessary to address the complexities of metazoan gene regulation. We studied by ChIP–chip the genome-wide binding sites of 6 insulator-associated proteins—dCTCF, CP190, BEAF-32, Su(Hw), Mod(mdg4), and GAF—to obtain the first comprehensive map of insulator elements in Drosophila embryos. We identify over 14,000 putative insulators, including all classically defined insulators. We find two major classes of insulators defined by dCTCF/CP190/BEAF-32 and Su(Hw), respectively. Distributional analyses of insulators revealed that particular sub-classes of insulator elements are excluded between cis-regulatory elements and their target promoters; divide differentially expressed, alternative, and divergent promoters; act as chromatin boundaries; are associated with chromosomal breakpoints among species; and are embedded within active chromatin domains. Together, these results provide a map demarcating the boundaries of gene regulatory units and a framework for understanding insulator function during the development and evolution of Drosophila.

Journal ArticleDOI
TL;DR: Recent advances that promise greatly to accelerate the use of S. viridis as a genetic system are summarized, including recent successful efforts at regenerating plants from seed callus, establishing a transient transformation system, and developing stable transformation.
Abstract: C(4) photosynthesis drives productivity in several major food crops and bioenergy grasses, including maize (Zea mays), sugarcane (Saccharum officinarum), sorghum (Sorghum bicolor), Miscanthus x giganteus, and switchgrass (Panicum virgatum). Gains in productivity associated with C(4) photosynthesis include improved water and nitrogen use efficiencies. Thus, engineering C(4) traits into C(3) crops is an attractive target for crop improvement. However, the lack of a small, rapid cycling genetic model system to study C(4) photosynthesis has limited progress in dissecting the regulatory networks underlying the C(4) syndrome. Setaria viridis is a member of the Panicoideae clade and is a close relative of several major feed, fuel, and bioenergy grasses. It is a true diploid with a relatively small genome of ~510 Mb. Its short stature, simple growth requirements, and rapid life cycle will greatly facilitate genetic studies of the C(4) grasses. Importantly, S. viridis uses an NADP-malic enzyme subtype C(4) photosynthetic system to fix carbon and therefore is a potentially powerful model system for dissecting C(4) photosynthesis. Here, we summarize some of the recent advances that promise greatly to accelerate the use of S. viridis as a genetic system. These include our recent successful efforts at regenerating plants from seed callus, establishing a transient transformation system, and developing stable transformation.

Journal ArticleDOI
TL;DR: Over 70% of the CNV/PAV examples are identified in multiple genotypes, and the majority of events are observed in both maize and teosinte, suggesting that these variants predate domestication and that there is not strong selection acting against them.
Abstract: Individuals of the same species are generally thought to have very similar genomes. However, there is growing evidence that structural variation in the form of copy number variation (CNV) and presence-absence variation (PAV) can lead to variation in the genome content of individuals within a species. Array comparative genomic hybridization (CGH) was used to compare gene content and copy number variation among 19 diverse maize inbreds and 14 genotypes of the wild ancestor of maize, teosinte. We identified 479 genes exhibiting higher copy number in some genotypes (UpCNV) and 3410 genes that have either fewer copies or are missing in the genome of at least one genotype relative to B73 (DownCNV/PAV). Many of these DownCNV/PAV are examples of genes present in B73, but missing from other genotypes. Over 70% of the CNV/PAV examples are identified in multiple genotypes, and the majority of events are observed in both maize and teosinte, suggesting that these variants predate domestication and that there is not strong selection acting against them. Many of the genes affected by CNV/PAV are either maize specific (thus possible annotation artifacts) or members of large gene families, suggesting that the gene loss can be tolerated through buffering by redundant functions encoded elsewhere in the genome. While this structural variation may not result in major qualitative variation due to genetic buffering, it may significantly contribute to quantitative variation.

Journal ArticleDOI
25 Feb 2010-Nature
TL;DR: Findings reveal that microRNA evolution and the establishment of tissue identities were closely coupled in bilaterian evolution, and outline a minimum set of cell types and tissues that existed in the protostome–deuterostome ancestor.
Abstract: The spectacular escalation in complexity in early bilaterian evolution correlates with a strong increase in the number of microRNAs. To explore the link between the birth of ancient microRNAs and body plan evolution, we set out to determine the ancient sites of activity of conserved bilaterian microRNA families in a comparative approach. We reason that any specific localization shared between protostomes and deuterostomes (the two major superphyla of bilaterian animals) should probably reflect an ancient specificity of that microRNA in their last common ancestor. Here, we investigate the expression of conserved bilaterian microRNAs in Platynereis dumerilii, a protostome retaining ancestral bilaterian features, in Capitella, another marine annelid, in the sea urchin Strongylocentrotus, a deuterostome, and in sea anemone Nematostella, representing an outgroup to the bilaterians. Our comparative data indicate that the oldest known animal microRNA, miR-100, and the related miR-125 and let-7 were initially active in neurosecretory cells located around the mouth. Other sets of ancient microRNAs were first present in locomotor ciliated cells, specific brain centres, or, more broadly, one of four major organ systems: central nervous system, sensory tissue, musculature and gut. These findings reveal that microRNA evolution and the establishment of tissue identities were closely coupled in bilaterian evolution. Also, they outline a minimum set of cell types and tissues that existed in the protostome-deuterostome ancestor.

Journal ArticleDOI
07 May 2010-Science
TL;DR: It is shown that hybridization capture on microarrays can successfully recover more than a megabase of target regions from Neandertal DNA even in the presence of ~99.8% microbial DNA.
Abstract: Neandertals, our closest relatives, ranged across Europe and Southwest Asia before their extinction approximately 30,000 years ago. Green et al. (p. [710][1]) report a draft sequence of the Neandertal genome, created from three individuals, and compare it with genomes of five modern humans. The results suggest that ancient genomes of human relatives can be recovered with acceptably low contamination from modern human DNA. Because ancient DNA can be contaminated with microbial DNA, Burbano et al. (p. [723][2]) developed a target sequence capture approach to obtain 14 kilobases of Neandertal DNA from a fairly poorly preserved sample with a high microbial load. A number of genomic regions and genes were revealed as candidates for positive selection early in modern human history. The genomic data suggest that Neandertals mixed with modern human ancestors some 120,000 years ago, leaving traces of Neandertal DNA in contemporary humans. [1]: /lookup/doi/10.1126/science.1188021 [2]: /lookup/doi/10.1126/science.1188046