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1.  ISCB Ebola Award for Important Future Research on the Computational Biology of Ebola Virus 
PLoS Computational Biology  2015;11(1):e1004087.
Speed is of the essence in combating Ebola; thus, computational approaches should form a significant component of Ebola research. As for the development of any modern drug, computational biology is uniquely positioned to contribute through comparative analysis of the genome sequences of Ebola strains as well as 3-D protein modeling. Other computational approaches to Ebola may include large-scale docking studies of Ebola proteins with human proteins and with small-molecule libraries, computational modeling of the spread of the virus, computational mining of the Ebola literature, and creation of a curated Ebola database. Taken together, such computational efforts could significantly accelerate traditional scientific approaches. In recognition of the need for important and immediate solutions from the field of computational biology against Ebola, the International Society for Computational Biology (ISCB) announces a prize for an important computational advance in fighting the Ebola virus. ISCB will confer the ISCB Fight against Ebola Award, along with a prize of US$2,000, at its July 2016 annual meeting (ISCB Intelligent Systems for Molecular Biology [ISMB] 2016, Orlando, Florida).
doi:10.1371/journal.pcbi.1004087
PMCID: PMC4310586  PMID: 25633975
2.  Gateways to the FANTOM5 promoter level mammalian expression atlas 
Genome Biology  2015;16(1):22.
The FANTOM5 project investigates transcription initiation activities in more than 1,000 human and mouse primary cells, cell lines and tissues using CAGE. Based on manual curation of sample information and development of an ontology for sample classification, we assemble the resulting data into a centralized data resource (http://fantom.gsc.riken.jp/5/). This resource contains web-based tools and data-access points for the research community to search and extract data related to samples, genes, promoter activities, transcription factors and enhancers across the FANTOM5 atlas.
Electronic supplementary material
The online version of this article (doi:10.1186/s13059-014-0560-6) contains supplementary material, which is available to authorized users.
doi:10.1186/s13059-014-0560-6
PMCID: PMC4310165
3.  A genome-wide siRNA screen identifies proteasome addiction as a vulnerability of basal-like triple-negative breast cancer cells 
Cancer cell  2013;24(2):182-196.
Summary
Basal-like triple negative breast cancers (TNBC) have poor prognosis. To identify basal-like TNBC dependencies, a genome-wide siRNA lethality screen compared two human breast epithelial cell lines transformed with the same genes - basal-like BPLER and myoepithelial HMLER. Expression of the screen’s 154 BPLER dependency genes correlated with poor prognosis in breast, but not lung or colon, cancer. Proteasome genes were overrepresented hits. Basal-like TNBC lines were selectively sensitive to proteasome inhibitor drugs relative to normal epithelial, luminal and mesenchymal TNBC lines. Proteasome inhibition reduced growth of established basal-like TNBC tumors in mice and blocked tumor-initiating cell function and macrometastasis. Proteasome addiction in basal-like TNBCs was mediated by NOXA and linked to MCL-1 dependence.
doi:10.1016/j.ccr.2013.07.008
PMCID: PMC3773329  PMID: 23948298
4.  A Network of Epigenetic Regulators Guide Developmental Hematopoiesis In Vivo 
Nature cell biology  2013;15(12):1516-1525.
The initiation of cellular programs is orchestrated by key transcription factors and chromatin regulators that activate or inhibit target gene expression. To generate a compendium of chromatin factors that establish the epigenetic code during developmental hematopoiesis, a large-scale reverse genetic screen was conducted targeting orthologs of 425 human chromatin factors in zebrafish. A set of chromatin regulators was identified that target different stages of primitive and definitive blood formation, including factors not previously implicated in hematopoiesis. We identified 15 factors that regulate development of primitive erythroid progenitors and 29 factors that regulate development of definitive stem and progenitor cells. These chromatin factors are associated with SWI/SNF and ISWI chromatin remodeling, SET1 methyltransferase, CBP/P300/HBO1/NuA4 acetyltransferase, HDAC/NuRD deacetylase, and Polycomb repressive complexes. Our work provides a comprehensive view of how specific chromatin factors and their associated complexes play a major role in the establishment of hematopoietic cells in vivo.
doi:10.1038/ncb2870
PMCID: PMC3959952  PMID: 24240475
5.  Divergent LIN28-mRNA associations result in translational suppression upon the initiation of differentiation 
Nucleic Acids Research  2014;42(12):7997-8007.
LIN28 function is fundamental to the activity and behavior of human embryonic stem cells (hESCs) and induced pluripotent stem cells. Its main roles in these cell types are the regulation of translational efficiency and let-7 miRNA maturation. However, LIN28-associated mRNA cargo shifting and resultant regulation of translational efficiency upon the initiation of differentiation remain unknown. An RNA-immunoprecipitation and microarray analysis protocol, eRIP, that has high specificity and sensitivity was developed to test endogenous LIN28-associated mRNA cargo shifting. A combined eRIP and polysome analysis of early stage differentiation of hESCs with two distinct differentiation cues revealed close similarities between the dynamics of LIN28 association and translational modulation of genes involved in the Wnt signaling, cell cycle, RNA metabolism and proteasomal pathways. Our data demonstrate that change in translational efficiency is a major contributor to early stages of differentiation of hESCs, in which LIN28 plays a central role. This implies that eRIP analysis of LIN28-associated RNA cargoes may be used for rapid functional quality control of pluripotent stem cells under manufacture for therapeutic applications.
doi:10.1093/nar/gku430
PMCID: PMC4081066  PMID: 24860167
7.  Comparison of Illumina and 454 Deep Sequencing in Participants Failing Raltegravir-Based Antiretroviral Therapy 
PLoS ONE  2014;9(3):e90485.
Background
The impact of raltegravir-resistant HIV-1 minority variants (MVs) on raltegravir treatment failure is unknown. Illumina sequencing offers greater throughput than 454, but sequence analysis tools for viral sequencing are needed. We evaluated Illumina and 454 for the detection of HIV-1 raltegravir-resistant MVs.
Methods
A5262 was a single-arm study of raltegravir and darunavir/ritonavir in treatment-naïve patients. Pre-treatment plasma was obtained from 5 participants with raltegravir resistance at the time of virologic failure. A control library was created by pooling integrase clones at predefined proportions. Multiplexed sequencing was performed with Illumina and 454 platforms at comparable costs. Illumina sequence analysis was performed with the novel snp-assess tool and 454 sequencing was analyzed with V-Phaser.
Results
Illumina sequencing resulted in significantly higher sequence coverage and a 0.095% limit of detection. Illumina accurately detected all MVs in the control library at ≥0.5% and 7/10 MVs expected at 0.1%. 454 sequencing failed to detect any MVs at 0.1% with 5 false positive calls. For MVs detected in the patient samples by both 454 and Illumina, the correlation in the detected variant frequencies was high (R2 = 0.92, P<0.001). Illumina sequencing detected 2.4-fold greater nucleotide MVs and 2.9-fold greater amino acid MVs compared to 454. The only raltegravir-resistant MV detected was an E138K mutation in one participant by Illumina sequencing, but not by 454.
Conclusions
In participants of A5262 with raltegravir resistance at virologic failure, baseline raltegravir-resistant MVs were rarely detected. At comparable costs to 454 sequencing, Illumina demonstrated greater depth of coverage, increased sensitivity for detecting HIV MVs, and fewer false positive variant calls.
doi:10.1371/journal.pone.0090485
PMCID: PMC3946168  PMID: 24603872
8.  Cell-specific translational profiling in acute kidney injury 
The Journal of Clinical Investigation  2014;124(3):1242-1254.
Acute kidney injury (AKI) promotes an abrupt loss of kidney function that results in substantial morbidity and mortality. Considerable effort has gone toward identification of diagnostic biomarkers and analysis of AKI-associated molecular events; however, most studies have adopted organ-wide approaches and have not elucidated the interplay among different cell types involved in AKI pathophysiology. To better characterize AKI-associated molecular and cellular events, we developed a mouse line that enables the identification of translational profiles in specific cell types. This strategy relies on CRE recombinase–dependent activation of an EGFP-tagged L10a ribosomal protein subunit, which allows translating ribosome affinity purification (TRAP) of mRNA populations in CRE-expressing cells. Combining this mouse line with cell type–specific CRE-driver lines, we identified distinct cellular responses in an ischemia reperfusion injury (IRI) model of AKI. Twenty-four hours following IRI, distinct translational signatures were identified in the nephron, kidney interstitial cell populations, vascular endothelium, and macrophages/monocytes. Furthermore, TRAP captured known IRI-associated markers, validating this approach. Biological function annotation, canonical pathway analysis, and in situ analysis of identified response genes provided insight into cell-specific injury signatures. Our study provides a deep, cell-based view of early injury-associated molecular events in AKI and documents a versatile, genetic tool to monitor cell-specific and temporal-specific biological processes in disease modeling.
doi:10.1172/JCI72126
PMCID: PMC3938273  PMID: 24569379
9.  A comprehensive promoter landscape identifies a novel promoter for CD133 in restricted tissues, cancers, and stem cells 
Frontiers in Genetics  2013;4:209.
PROM1 is the gene encoding prominin-1 or CD133, an important cell surface marker for the isolation of both normal and cancer stem cells. PROM1 transcripts initiate at a range of transcription start sites (TSS) associated with distinct tissue and cancer expression profiles. Using high resolution Cap Analysis of Gene Expression (CAGE) sequencing we characterize TSS utilization across a broad range of normal and developmental tissues. We identify a novel proximal promoter (P6) within CD133+ melanoma cell lines and stem cells. Additional exon array sampling finds P6 to be active in populations enriched for mesenchyme, neural stem cells and within CD133+ enriched Ewing sarcomas. The P6 promoter is enriched with respect to previously characterized PROM1 promoters for a HMGI/Y (HMGA1) family transcription factor binding site motif and exhibits different epigenetic modifications relative to the canonical promoter region of PROM1.
doi:10.3389/fgene.2013.00209
PMCID: PMC3810939  PMID: 24194746
PROM1 protein; human; AC133 antigen; transcription start site; promoter regions; genetic; melanoma; cancer stem cells
10.  Chronic Endotoxin Exposure Produces Airflow Obstruction and Lung Dendritic Cell Expansion 
Little is known about the mechanisms of persistent airflow obstruction that result from chronic occupational endotoxin exposure. We sought to analyze the inflammatory response underlying persistent airflow obstruction as a result of chronic occupational endotoxin exposure. We developed a murine model of daily inhaled endotoxin for periods of 5 days to 8 weeks. We analyzed physiologic lung dysfunction, lung histology, bronchoalveolar lavage fluid and total lung homogenate inflammatory cell and cytokine profiles, and pulmonary gene expression profiles. We observed an increase in airway hyperresponsiveness as a result of chronic endotoxin exposure. After 8 weeks, the mice exhibited an increase in bronchoalveolar lavage and lung neutrophils that correlated with an increase in proinflammatory cytokines. Detailed analyses of inflammatory cell subsets revealed an expansion of dendritic cells (DCs), and in particular, proinflammatory DCs, with a reduced percentage of macrophages. Gene expression profiling revealed the up-regulation of a panel of genes that was consistent with DC recruitment, and lung histology revealed an accumulation of DCs in inflammatory aggregates around the airways in 8-week–exposed animals. Repeated, low-dose LPS inhalation, which mirrors occupational exposure, resulted in airway hyperresponsiveness, associated with a failure to resolve the proinflammatory response, an inverted macrophage to DC ratio, and a significant rise in the inflammatory DC population. These findings point to a novel underlying mechanism of airflow obstruction as a result of occupational LPS exposure, and suggest molecular and cellular targets for therapeutic development.
doi:10.1165/rcmb.2011-0447OC
PMCID: PMC3423463  PMID: 22517795
airway resistance; inhalation; neutrophils; macrophages; dendritic cells; endotoxin
11.  Pathprinting: An integrative approach to understand the functional basis of disease 
Genome Medicine  2013;5(7):68.
New strategies to combat complex human disease require systems approaches to biology that integrate experiments from cell lines, primary tissues and model organisms. We have developed Pathprint, a functional approach that compares gene expression profiles in a set of pathways, networks and transcriptionally regulated targets. It can be applied universally to gene expression profiles across species. Integration of large-scale profiling methods and curation of the public repository overcomes platform, species and batch effects to yield a standard measure of functional distance between experiments. We show that pathprints combine mouse and human blood developmental lineage, and can be used to identify new prognostic indicators in acute myeloid leukemia. The code and resources are available at http://compbio.sph.harvard.edu/hidelab/pathprint
doi:10.1186/gm472
PMCID: PMC3971351  PMID: 23890051
12.  Integrating Murine Gene Expression Studies to Understand Obstructive Lung Disease Due to Chronic Inhaled Endotoxin 
PLoS ONE  2013;8(5):e62910.
Rationale
Endotoxin is a near ubiquitous environmental exposure that that has been associated with both asthma and chronic obstructive pulmonary disease (COPD). These obstructive lung diseases have a complex pathophysiology, making them difficult to study comprehensively in the context of endotoxin. Genome-wide gene expression studies have been used to identify a molecular snapshot of the response to environmental exposures. Identification of differentially expressed genes shared across all published murine models of chronic inhaled endotoxin will provide insight into the biology underlying endotoxin-associated lung disease.
Methods
We identified three published murine models with gene expression profiling after repeated low-dose inhaled endotoxin. All array data from these experiments were re-analyzed, annotated consistently, and tested for shared genes found to be differentially expressed. Additional functional comparison was conducted by testing for significant enrichment of differentially expressed genes in known pathways. The importance of this gene signature in smoking-related lung disease was assessed using hierarchical clustering in an independent experiment where mice were exposed to endotoxin, smoke, and endotoxin plus smoke.
Results
A 101-gene signature was detected in three murine models, more than expected by chance. The three model systems exhibit additional similarity beyond shared genes when compared at the pathway level, with increasing enrichment of inflammatory pathways associated with longer duration of endotoxin exposure. Genes and pathways important in both asthma and COPD were shared across all endotoxin models. Mice exposed to endotoxin, smoke, and smoke plus endotoxin were accurately classified with the endotoxin gene signature.
Conclusions
Despite the differences in laboratory, duration of exposure, and strain of mouse used in three experimental models of chronic inhaled endotoxin, surprising similarities in gene expression were observed. The endotoxin component of tobacco smoke may play an important role in disease development.
doi:10.1371/journal.pone.0062910
PMCID: PMC3652821  PMID: 23675439
13.  Novel Developmental Analyses Identify Longitudinal Patterns of Early Gut Microbiota that Affect Infant Growth 
PLoS Computational Biology  2013;9(5):e1003042.
It is acknowledged that some obesity trajectories are set early in life, and that rapid weight gain in infancy is a risk factor for later development of obesity. Identifying modifiable factors associated with early rapid weight gain is a prerequisite for curtailing the growing worldwide obesity epidemic. Recently, much attention has been given to findings indicating that gut microbiota may play a role in obesity development. We aim at identifying how the development of early gut microbiota is associated with expected infant growth. We developed a novel procedure that allows for the identification of longitudinal gut microbiota patterns (corresponding to the gut ecosystem developing), which are associated with an outcome of interest, while appropriately controlling for the false discovery rate. Our method identified developmental pathways of Staphylococcus species and Escherichia coli that were associated with expected growth, and traditional methods indicated that the detection of Bacteroides species at day 30 was associated with growth. Our method should have wide future applicability for studying gut microbiota, and is particularly important for translational considerations, as it is critical to understand the timing of microbiome transitions prior to attempting to manipulate gut microbiota in early life.
Author Summary
Some obesity trajectories are set early in life, with rapid weight gain being a risk factor for later development of obesity. Recently, much attention has been given to findings indicating that gut microbiota may play a role in obesity development. The existence of time-dependent exposure windows, which rely on stimuli from the gut to initiate healthy development, gives the evolution of early life gut microbiota a critical role in human health. We identified children that followed their expected growth trajectories at six months of life, and those that had deviated. We then developed a novel statistical approach that allowed the identification of longitudinal gut microbiota patterns (e.g. a particular species was detected at days 4, 10, and 30 and not detected at day 120) that were associated with expected growth, while appropriately restricting the false discovery rate. We further identified when a deviation from the proposed longitudinal gut microbiota patterns would result in an abnormal growth outcome (either rapid or decreased growth at six months of life). We found developmental pathways of Staphylococcus species and Escherichia coli that were associated with expected growth, as well as indications that Bacteroides species at day 30 was associated with growth.
doi:10.1371/journal.pcbi.1003042
PMCID: PMC3650000  PMID: 23671411
14.  The Stem Cell Commons: an exemplar for data integration in the biomedical domain driven by the ISA framework 
Comparisons of stem cell experiments at both molecular and semantic levels remain challenging due to inconsistencies in results, data formats, and descriptions among biomedical research discoveries. The Harvard Stem Cell Institute (HSCI) has created the Stem Cell Commons (stemcellcommons.org), an open, community-based approach to data sharing. Experimental information is integrated using the Investigation-Study-Assay tabular format (ISA-Tab) used by over 30 organizations (ISA Commons, isacommons.org). The early adoption of this format permitted the novel integration of three independent systems to facilitate stem cell data storage, exchange and analysis: the Blood Genomics Repository, the Stem Cell Discovery Engine, and the new Refinery platform that links the Galaxy analytical engine to data repositories.
PMCID: PMC3814497  PMID: 24303302
15.  A functional genomic screen reveals novel host genes that mediate interferon-alpha's effects against hepatitis C virus 
Journal of Hepatology  2011;56(2):326-333.
Background & Aims
The precise mechanisms by which IFN exerts its antiviral effect against HCV have not yet been elucidated. We sought to identify host genes that mediate the antiviral effect of IFN-α by conducting a whole-genome siRNA library screen.
Methods
High throughput screening was performed using an HCV genotype 1b replicon, pRep-Feo. Those pools with replicate robust Z scores ≥ 2.0 entered secondary validation in full-length OR6 replicon cells. Huh7.5.1 cells infected with JFH1 were then used to validate the rescue efficacy of selected genes for HCV replication under IFN-α treatment.
Results
We identified and confirmed 93 human genes involved in the IFN-α anti-HCV effect using a whole-genome siRNA library. Gene ontology analysis revealed that mRNA processing (23 genes, P=2.756e-22), translation initiation (9 genes, P=2.42e-6), and IFN signaling (5 genes, P=1.00e-3) were the most enriched functional groups. Nine genes were components of U4/U6.U5 tri-snRNP. We confirmed that silencing squamous cell carcinoma antigen recognized by T cells (SART1), a specific factor of tri-snRNP, abrogates IFN-α's suppressive effects against HCV in both replicon cells and JFH1 infectious cells. We further found that SART1 was not an IFN-α inducible, and its anti-HCV effector in the JFH1 infectious model was through regulation of interferon stimulated genes (ISGs) with or without IFN-α.
Conclusions
We identified 93 genes that mediate the anti-HCV effect of IFN-α through genome-wide siRNA screening; 23 and 9 genes were involved in mRNA processing and translation initiation, respectively. These findings reveal an unexpected role for mRNA processing in generation of the antiviral state, and suggest a new avenue for therapeutic development in HCV.
doi:10.1016/j.jhep.2011.07.026
PMCID: PMC3261326  PMID: 21888876
Hepatitis C Virus, HCV; Interferon-α, IFN-α; Small interfering RNA, siRNA; Squamous cell carcinoma Antigen Recognized by T cells, SART1; U4/U6.U5 tri-small nuclear ribonucleoproteins, U4/U6.U5 tri-snRNP
16.  Toward interoperable bioscience data 
Nature genetics  2012;44(2):121-126.
To make full use of research data, the bioscience community needs to adopt technologies and reward mechanisms that support interoperability and promote the growth of an open ‘data commoning’ culture. Here we describe the prerequisites for data commoning and present an established and growing ecosystem of solutions using the shared ‘Investigation-Study-Assay’ framework to support that vision.
doi:10.1038/ng.1054
PMCID: PMC3428019  PMID: 22281772
17.  Prenatal Lead Levels, Plasma Amyloid β Levels, and Gene Expression in Young Adulthood 
Environmental Health Perspectives  2012;120(5):702-707.
Background: Animal studies suggest that early-life lead exposure influences gene expression and production of proteins associated with Alzheimer’s disease (AD).
Objectives: We attempted to assess the relationship between early-life lead exposure and potential biomarkers for AD among young men and women. We also attempted to assess whether early-life lead exposure was associated with changes in expression of AD-related genes.
Methods: We used sandwich enzyme-linked immunosorbent assays (ELISA) to measure plasma concentrations of amyloid β proteins Aβ40 and Aβ42 among 55 adults who had participated as newborns and young children in a prospective cohort study of the effects of lead exposure on development. We used RNA microarray techniques to analyze gene expression.
Results: Mean plasma Aβ42 concentrations were lower among 13 participants with high umbilical cord blood lead concentrations (≥ 10 μg/dL) than in 42 participants with lower cord blood lead concentrations (p = 0.08). Among 10 participants with high prenatal lead exposure, we found evidence of an inverse relationship between umbilical cord lead concentration and expression of ADAM metallopeptidase domain 9 (ADAM9), reticulon 4 (RTN4), and low-density lipoprotein receptor-related protein associated protein 1 (LRPAP1) genes, whose products are believed to affect Aβ production and deposition. Gene network analysis suggested enrichment in gene sets involved in nerve growth and general cell development.
Conclusions: Data from our exploratory study suggest that prenatal lead exposure may influence Aβ-related biological pathways that have been implicated in AD onset. Gene network analysis identified further candidates to study the mechanisms of developmental lead neurotoxicity.
doi:10.1289/ehp.1104474
PMCID: PMC3346789  PMID: 22313790
Alzheimer’s disease; children; fetal basis of adult disease; human; lead
18.  Population Differences in Transcript-Regulator Expression Quantitative Trait Loci 
PLoS ONE  2012;7(3):e34286.
Gene expression quantitative trait loci (eQTL) are useful for identifying single nucleotide polymorphisms (SNPs) associated with diseases. At times, a genetic variant may be associated with a master regulator involved in the manifestation of a disease. The downstream target genes of the master regulator are typically co-expressed and share biological function. Therefore, it is practical to screen for eQTLs by identifying SNPs associated with the targets of a transcript-regulator (TR). We used a multivariate regression with the gene expression of known targets of TRs and SNPs to identify TReQTLs in European (CEU) and African (YRI) HapMap populations. A nominal p-value of <1×10−6 revealed 234 SNPs in CEU and 154 in YRI as TReQTLs. These represent 36 independent (tag) SNPs in CEU and 39 in YRI affecting the downstream targets of 25 and 36 TRs respectively. At a false discovery rate (FDR) = 45%, one cis-acting tag SNP (within 1 kb of a gene) in each population was identified as a TReQTL. In CEU, the SNP (rs16858621) in Pcnxl2 was found to be associated with the genes regulated by CREM whereas in YRI, the SNP (rs16909324) was linked to the targets of miRNA hsa-miR-125a. To infer the pathways that regulate expression, we ranked TReQTLs by connectivity within the structure of biological process subtrees. One TReQTL SNP (rs3790904) in CEU maps to Lphn2 and is associated (nominal p-value = 8.1×10−7) with the targets of the X-linked breast cancer suppressor Foxp3. The structure of the biological process subtree and a gene interaction network of the TReQTL revealed that tumor necrosis factor, NF-kappaB and variants in G-protein coupled receptors signaling may play a central role as communicators in Foxp3 functional regulation. The potential pleiotropic effect of the Foxp3 TReQTLs was gleaned from integrating mRNA-Seq data and SNP-set enrichment into the analysis.
doi:10.1371/journal.pone.0034286
PMCID: PMC3313997  PMID: 22479588
20.  The Stem Cell Discovery Engine: an integrated repository and analysis system for cancer stem cell comparisons 
Nucleic Acids Research  2011;40(Database issue):D984-D991.
Mounting evidence suggests that malignant tumors are initiated and maintained by a subpopulation of cancerous cells with biological properties similar to those of normal stem cells. However, descriptions of stem-like gene and pathway signatures in cancers are inconsistent across experimental systems. Driven by a need to improve our understanding of molecular processes that are common and unique across cancer stem cells (CSCs), we have developed the Stem Cell Discovery Engine (SCDE)—an online database of curated CSC experiments coupled to the Galaxy analytical framework. The SCDE allows users to consistently describe, share and compare CSC data at the gene and pathway level. Our initial focus has been on carefully curating tissue and cancer stem cell-related experiments from blood, intestine and brain to create a high quality resource containing 53 public studies and 1098 assays. The experimental information is captured and stored in the multi-omics Investigation/Study/Assay (ISA-Tab) format and can be queried in the data repository. A linked Galaxy framework provides a comprehensive, flexible environment populated with novel tools for gene list comparisons against molecular signatures in GeneSigDB and MSigDB, curated experiments in the SCDE and pathways in WikiPathways. The SCDE is available at http://discovery.hsci.harvard.edu.
doi:10.1093/nar/gkr1051
PMCID: PMC3245064  PMID: 22121217
21.  Capture of MicroRNA–Bound mRNAs Identifies the Tumor Suppressor miR-34a as a Regulator of Growth Factor Signaling 
PLoS Genetics  2011;7(11):e1002363.
A simple biochemical method to isolate mRNAs pulled down with a transfected, biotinylated microRNA was used to identify direct target genes of miR-34a, a tumor suppressor gene. The method reidentified most of the known miR-34a regulated genes expressed in K562 and HCT116 cancer cell lines. Transcripts for 982 genes were enriched in the pull-down with miR-34a in both cell lines. Despite this large number, validation experiments suggested that ∼90% of the genes identified in both cell lines can be directly regulated by miR-34a. Thus miR-34a is capable of regulating hundreds of genes. The transcripts pulled down with miR-34a were highly enriched for their roles in growth factor signaling and cell cycle progression. These genes form a dense network of interacting gene products that regulate multiple signal transduction pathways that orchestrate the proliferative response to external growth stimuli. Multiple candidate miR-34a–regulated genes participate in RAS-RAF-MAPK signaling. Ectopic miR-34a expression reduced basal ERK and AKT phosphorylation and enhanced sensitivity to serum growth factor withdrawal, while cells genetically deficient in miR-34a were less sensitive. Fourteen new direct targets of miR-34a were experimentally validated, including genes that participate in growth factor signaling (ARAF and PIK3R2) as well as genes that regulate cell cycle progression at various phases of the cell cycle (cyclins D3 and G2, MCM2 and MCM5, PLK1 and SMAD4). Thus miR-34a tempers the proliferative and pro-survival effect of growth factor stimulation by interfering with growth factor signal transduction and downstream pathways required for cell division.
Author Summary
microRNAs (miRNAs) are small RNAs that regulate gene expression by binding to mRNAs bearing a partially complementary sequence. miRNAs decrease the stability or translation of mRNA targets, leading to reduced protein expression. Understanding the biological function of a miRNA requires identifying its targets. Here we developed a sensitive and specific biochemical method to identify candidate microRNA targets that are enriched by pull-down with a tagged, transfected microRNA mimic. The method was applied to miR-34a, a miRNA that inhibits cell proliferation. We found that miR-34a can potentially regulate hundreds of genes. Computational analysis of these genes suggested a novel function for miR-34a—suppression of the pro-proliferative response to diverse growth factors. This function complements the previously known role of miR-34a in blocking cell cycle progression. Thus, by reducing the expression of an extensive network of genes, miR-34a dampens growth factor signaling as well as its downstream consequences, promotion of cell survival and proliferation.
doi:10.1371/journal.pgen.1002363
PMCID: PMC3213160  PMID: 22102825
22.  Twelve type 2 diabetes susceptibility loci identified through large-scale association analysis 
Voight, Benjamin F | Scott, Laura J | Steinthorsdottir, Valgerdur | Morris, Andrew P | Dina, Christian | Welch, Ryan P | Zeggini, Eleftheria | Huth, Cornelia | Aulchenko, Yurii S | Thorleifsson, Gudmar | McCulloch, Laura J | Ferreira, Teresa | Grallert, Harald | Amin, Najaf | Wu, Guanming | Willer, Cristen J | Raychaudhuri, Soumya | McCarroll, Steve A | Langenberg, Claudia | Hofmann, Oliver M | Dupuis, Josée | Qi, Lu | Segrè, Ayellet V | van Hoek, Mandy | Navarro, Pau | Ardlie, Kristin | Balkau, Beverley | Benediktsson, Rafn | Bennett, Amanda J | Blagieva, Roza | Boerwinkle, Eric | Bonnycastle, Lori L | Boström, Kristina Bengtsson | Bravenboer, Bert | Bumpstead, Suzannah | Burtt, Noisël P | Charpentier, Guillaume | Chines, Peter S | Cornelis, Marilyn | Couper, David J | Crawford, Gabe | Doney, Alex S F | Elliott, Katherine S | Elliott, Amanda L | Erdos, Michael R | Fox, Caroline S | Franklin, Christopher S | Ganser, Martha | Gieger, Christian | Grarup, Niels | Green, Todd | Griffin, Simon | Groves, Christopher J | Guiducci, Candace | Hadjadj, Samy | Hassanali, Neelam | Herder, Christian | Isomaa, Bo | Jackson, Anne U | Johnson, Paul R V | Jørgensen, Torben | Kao, Wen H L | Klopp, Norman | Kong, Augustine | Kraft, Peter | Kuusisto, Johanna | Lauritzen, Torsten | Li, Man | Lieverse, Aloysius | Lindgren, Cecilia M | Lyssenko, Valeriya | Marre, Michel | Meitinger, Thomas | Midthjell, Kristian | Morken, Mario A | Narisu, Narisu | Nilsson, Peter | Owen, Katharine R | Payne, Felicity | Perry, John R B | Petersen, Ann-Kristin | Platou, Carl | Proença, Christine | Prokopenko, Inga | Rathmann, Wolfgang | Rayner, N William | Robertson, Neil R | Rocheleau, Ghislain | Roden, Michael | Sampson, Michael J | Saxena, Richa | Shields, Beverley M | Shrader, Peter | Sigurdsson, Gunnar | Sparsø, Thomas | Strassburger, Klaus | Stringham, Heather M | Sun, Qi | Swift, Amy J | Thorand, Barbara | Tichet, Jean | Tuomi, Tiinamaija | van Dam, Rob M | van Haeften, Timon W | van Herpt, Thijs | van Vliet-Ostaptchouk, Jana V | Walters, G Bragi | Weedon, Michael N | Wijmenga, Cisca | Witteman, Jacqueline | Bergman, Richard N | Cauchi, Stephane | Collins, Francis S | Gloyn, Anna L | Gyllensten, Ulf | Hansen, Torben | Hide, Winston A | Hitman, Graham A | Hofman, Albert | Hunter, David J | Hveem, Kristian | Laakso, Markku | Mohlke, Karen L | Morris, Andrew D | Palmer, Colin N A | Pramstaller, Peter P | Rudan, Igor | Sijbrands, Eric | Stein, Lincoln D | Tuomilehto, Jaakko | Uitterlinden, Andre | Walker, Mark | Wareham, Nicholas J | Watanabe, Richard M | Abecasis, Gonçalo R | Boehm, Bernhard O | Campbell, Harry | Daly, Mark J | Hattersley, Andrew T | Hu, Frank B | Meigs, James B | Pankow, James S | Pedersen, Oluf | Wichmann, H-Erich | Barroso, Inês | Florez, Jose C | Frayling, Timothy M | Groop, Leif | Sladek, Rob | Thorsteinsdottir, Unnur | Wilson, James F | Illig, Thomas | Froguel, Philippe | van Duijn, Cornelia M | Stefansson, Kari | Altshuler, David | Boehnke, Michael | McCarthy, Mark I
Nature genetics  2010;42(7):579-589.
By combining genome-wide association data from 8,130 individuals with type 2 diabetes (T2D) and 38,987 controls of European descent and following up previously unidentified meta-analysis signals in a further 34,412 cases and 59,925 controls, we identified 12 new T2D association signals with combinedP < 5 × 10−8. These include a second independent signal at the KCNQ1 locus; the first report, to our knowledge, of an X-chromosomal association (near DUSP9); and a further instance of overlap between loci implicated in monogenic and multifactorial forms of diabetes (at HNF1A). The identified loci affect both beta-cell function and insulin action, and, overall, T2D association signals show evidence of enrichment for genes involved in cell cycle regulation. We also show that a high proportion of T2D susceptibility loci harbor independent association signals influencing apparently unrelated complex traits.
doi:10.1038/ng.609
PMCID: PMC3080658  PMID: 20581827
23.  miR-24 inhibits cell proliferation by suppressing expression of E2F2, MYC and other cell cycle regulatory genes by binding to “seedless” 3′UTR microRNA recognition elements 
Molecular cell  2009;35(5):610-625.
Summary
miR-24, up-regulated during terminal differentiation of multiple lineages, inhibits cell cycle progression. Antagonizing miR-24 restores post-mitotic cell proliferation and enhances fibroblast proliferation, while over-expressing miR-24 increases the G1 compartment. The 248 mRNAs down-regulated upon miR-24 over-expression are highly enriched for DNA repair and cell cycle regulatory genes that form a direct interaction network with prominent nodes at genes that enhance (MYC, E2F2, CCNB1, CDC2) or inhibit (p27Kip1, VHL) cell cycle progression. miR-24 directly regulates MYC and E2F2 and some genes they transactivate. Enhanced proliferation from antagonizing miR-24 is abrogated by knocking down E2F2, but not MYC, and cell proliferation, inhibited by miR-24 over-expression, is rescued by miR-24-insensitive E2F2. Therefore, E2F2 is a critical miR-24 target. The E2F2 3′UTR lacks a predicted miR-24 recognition element. In fact, miR-24 regulates expression of E2F2, MYC, AURKB, CCNA2, CDC2, CDK4 and FEN1 by recognizing seedless, but highly complementary, sequences.
doi:10.1016/j.molcel.2009.08.020
PMCID: PMC2757794  PMID: 19748357
24.  ISA software suite: supporting standards-compliant experimental annotation and enabling curation at the community level 
Bioinformatics  2010;26(18):2354-2356.
Summary: The first open source software suite for experimentalists and curators that (i) assists in the annotation and local management of experimental metadata from high-throughput studies employing one or a combination of omics and other technologies; (ii) empowers users to uptake community-defined checklists and ontologies; and (iii) facilitates submission to international public repositories.
Availability and Implementation: Software, documentation, case studies and implementations at http://www.isa-tools.org
Contact: isatools@googlegroups.com
doi:10.1093/bioinformatics/btq415
PMCID: PMC2935443  PMID: 20679334

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