Publications by authors named "Serge Batalov"

34 Publications

Concomitant diagnosis of immune deficiency and sepsis in a 19 month old with ecthyma gangrenosum by host whole-genome sequencing.

Cold Spring Harb Mol Case Stud 2018 12 17;4(6). Epub 2018 Dec 17.

Division of Infectious Disease, Department of Pediatrics, UCSD, San Diego, California 92093, USA.

X-linked agammaglobulinemia (XLA, OMIM#300300) is a rare monogenic primary immunodeficiency caused by mutations in the Bruton tyrosine kinase () gene. XLA is characterized by insufficient immunoglobulin levels and susceptibility to life-threatening bacterial infections. We report on a patient that presented with ecthyma gangrenosum and septicemia. Rapid trio whole-genome sequencing (rWGS) revealed an apparently de novo hemizygous pathogenic variant (c.726dupT; p.Ile243TyrfsTer15) in the gene. Metagenomic analysis of rWGS sequences that did not align to the human genome revealed 770 aligned to the PAO1 genome. The patient was diagnosed with XLA and pseudomonal sepsis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1101/mcs.a003244DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6318772PMC
December 2018

The NSIGHT1-randomized controlled trial: rapid whole-genome sequencing for accelerated etiologic diagnosis in critically ill infants.

NPJ Genom Med 2018 9;3. Epub 2018 Feb 9.

Rady Children's Institute for Genomic Medicine, San Diego, CA 92123 USA.

Genetic disorders are a leading cause of morbidity and mortality in infants in neonatal and pediatric intensive care units (NICU/PICU). While genomic sequencing is useful for genetic disease diagnosis, results are usually reported too late to guide inpatient management. We performed an investigator-initiated, partially blinded, pragmatic, randomized, controlled trial to test the hypothesis that rapid whole-genome sequencing (rWGS) increased the proportion of NICU/PICU infants receiving a genetic diagnosis within 28 days. The participants were families with infants aged <4 months in a regional NICU and PICU, with illnesses of unknown etiology. The intervention was trio rWGS. Enrollment from October 2014 to June 2016, and follow-up until November 2016. Of all, 26 female infants, 37 male infants, and 2 infants of undetermined sex were randomized to receive rWGS plus standard genetic tests ( = 32, cases) or standard genetic tests alone ( = 33, controls). The study was terminated early due to loss of equipoise: 73% (24) controls received genomic sequencing as standard tests, and 15% (five) controls underwent compassionate cross-over to receive rWGS. Nevertheless, intention to treat analysis showed the rate of genetic diagnosis within 28 days of enrollment (the primary end-point) to be higher in cases (31%, 10 of 32) than controls (3%, 1 of 33; difference, 28% [95% CI, 10-46%];  = 0.003). Among infants enrolled in the first 25 days of life, the rate of neonatal diagnosis was higher in cases (32%, 7 of 22) than controls (0%, 0 of 23; difference, 32% [95% CI, 11-53%]; = 0.004). Median age at diagnosis (25 days [range 14-90] in cases vs. 130 days [range 37-451] in controls) and median time to diagnosis (13 days [range 1-84] in cases, vs. 107 days [range 21-429] in controls) were significantly less in cases than controls ( = 0.04). In conclusion, rWGS increased the proportion of NICU/PICU infants who received timely diagnoses of genetic diseases.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41525-018-0045-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5807510PMC
February 2018

Rapid whole-genome sequencing identifies a novel variant associated with West syndrome.

Cold Spring Harb Mol Case Stud 2017 Sep 1;3(5). Epub 2017 Sep 1.

Rady Children's Institute of Genomic Medicine (RCIGM), San Diego, California 92123, USA.

A 9-mo-old infant was admitted with infantile spasms that improved on administration of topiramate and steroids. He also had developmental delay, esotropia, and hypsarrhythmia on interictal electroencephalogram (EEG), and normal brain magnetic resonance imaging (MRI). West syndrome is the triad of infantile spasms, interictal hypsarrhythmia, and mental retardation. Rapid trio whole-genome sequencing (WGS) revealed a novel, likely pathogenic, de novo variant in the gene encoding γ-aminobutyric acid (GABA) type A receptor, α1 polypeptide ( c.789G>A, p.Met263Ile) in the proband. mutations have been associated with early infantile epileptic encephalopathy type 19 (EIEE19). We suggest that p.Met263Ile is associated with a distinct West syndrome phenotype.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1101/mcs.a001776DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5593154PMC
September 2017

Identification of synthetic lethality of PRKDC in MYC-dependent human cancers by pooled shRNA screening.

BMC Cancer 2014 Dec 13;14:944. Epub 2014 Dec 13.

Genomics Institute of the Novartis Research Foundation, 10675 John Jay Hopkins Drive, San Diego, CA 92121, USA.

Background: MYC family members are among the most frequently deregulated oncogenes in human cancers, yet direct therapeutic targeting of MYC in cancer has been challenging thus far. Synthetic lethality provides an opportunity for therapeutic intervention of MYC-driven cancers.

Methods: A pooled kinase shRNA library screen was performed and next-generation deep sequencing efforts identified that PRKDC was synthetically lethal in cells overexpressing MYC. Genes and proteins of interest were knocked down or inhibited using RNAi technology and small molecule inhibitors, respectively. Quantitative RT-PCR using TaqMan probes examined mRNA expression levels and cell viability was assessed using CellTiter-Glo (Promega). Western blotting was performed to monitor different protein levels in the presence or absence of RNAi or compound treatment. Statistical significance of differences among data sets were determined using unpaired t test (Mann-Whitney test) or ANOVA.

Results: Inhibition of PRKDC using RNAi (RNA interference) or small molecular inhibitors preferentially killed MYC-overexpressing human lung fibroblasts. Moreover, inducible PRKDC knockdown decreased cell viability selectively in high MYC-expressing human small cell lung cancer cell lines. At the molecular level, we found that inhibition of PRKDC downregulated MYC mRNA and protein expression in multiple cancer cell lines. In addition, we confirmed that overexpression of MYC family proteins induced DNA double-strand breaks; our results also revealed that PRKDC inhibition in these cells led to an increase in DNA damage levels.

Conclusions: Our data suggest that the synthetic lethality between PRKDC and MYC may in part be due to PRKDC dependent modulation of MYC expression, as well as MYC-induced DNA damage where PRKDC plays a key role in DNA damage repair.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/1471-2407-14-944DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4320452PMC
December 2014

Mst1 directs Myosin IIa partitioning of low and higher affinity integrins during T cell migration.

PLoS One 2014 18;9(8):e105561. Epub 2014 Aug 18.

Department of Pathology and Immunology, Washington University, St. Louis, Missouri, United States of America; Departments of Pathology and Microbiology & Immunology, The Geisel School of Medicine at Dartmouth, Lebanon, New Hampshire, United States of America.

Chemokines promote T cell migration by transmitting signals that induce T cell polarization and integrin activation and adhesion. Mst1 kinase is a key signal mediator required for both of these processes; however, its molecular mechanism remains unclear. Here, we present a mouse model in which Mst1 function is disrupted by a hypomorphic mutation. Microscopic analysis of Mst1-deficient CD4 T cells revealed a necessary role for Mst1 in controlling the localization and activity of Myosin IIa, a molecular motor that moves along actin filaments. Using affinity specific LFA-1 antibodies, we identified a requirement for Myosin IIa-dependent contraction in the precise spatial distribution of low and higher affinity LFA-1 on the membrane of migrating T cells. Mst1 deficiency or Myosin inhibition resulted in multipolar cells, difficulties in uropod detachment and mis-localization of low affinity LFA-1. Thus, Mst1 regulates Myosin IIa dynamics to organize high and low affinity LFA-1 to the anterior and posterior membrane during T cell migration.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0105561PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4136924PMC
December 2015

Fitness costs of rifampicin resistance in Mycobacterium tuberculosis are amplified under conditions of nutrient starvation and compensated by mutation in the β' subunit of RNA polymerase.

Mol Microbiol 2014 Mar 26;91(6):1106-19. Epub 2014 Feb 26.

International Tuberculosis Research Center, Changwon, South Korea.

Rifampicin resistance, a defining attribute of multidrug-resistant tuberculosis, is conferred by mutations in the β subunit of RNA polymerase. Sequencing of rifampicin-resistant (RIF-R) clinical isolates of Mycobacterium tuberculosis revealed, in addition to RIF-R mutations, enrichment of potential compensatory mutations around the double-psi β-barrel domain of the β' subunit comprising the catalytic site and the exit tunnel for newly synthesized RNA. Sequential introduction of the resistance allele followed by the compensatory allele in isogenic Mycobacterium smegmatis showed that these mutations respectively caused and compensated a starvation enhanced growth defect by altering RNA polymerase activity. While specific combinations of resistance and compensatory alleles converged in divergent lineages, other combinations recurred among related isolates suggesting transmission of compensated RIF-R strains. These findings suggest nutrient poor growth conditions impose larger selective pressure on RIF-R organisms that results in the selection of compensatory mutations in a domain involved in catalysis and starvation control of RNA polymerase transcription.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/mmi.12520DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3951610PMC
March 2014

Whole-genome sequencing and microarray analysis of ex vivo Plasmodium vivax reveal selective pressure on putative drug resistance genes.

Proc Natl Acad Sci U S A 2010 Nov 29;107(46):20045-50. Epub 2010 Oct 29.

Department of Cell Biology, Institute for Childhood and Neglected Diseases 202, The Scripps Research Institute, La Jolla, CA 92037, USA.

Plasmodium vivax causes 25-40% of malaria cases worldwide, yet research on this human malaria parasite has been neglected. Nevertheless, the recent publication of the P. vivax reference genome now allows genomics and systems biology approaches to be applied to this pathogen. We show here that whole-genome analysis of the parasite can be achieved directly from ex vivo-isolated parasites, without the need for in vitro propagation. A single isolate of P. vivax obtained from a febrile patient with clinical malaria from Peru was subjected to whole-genome sequencing (30× coverage). This analysis revealed over 18,261 single-nucleotide polymorphisms (SNPs), 6,257 of which were further validated using a tiling microarray. Within core chromosomal genes we find that one SNP per every 985 bases of coding sequence distinguishes this recent Peruvian isolate, designated IQ07, from the reference Salvador I strain obtained in 1972. This full-genome sequence of an uncultured P. vivax isolate shows that the same regions with low numbers of aligned sequencing reads are also highly variable by genomic microarray analysis. Finally, we show that the genes containing the largest ratio of nonsynonymous-to-synonymous SNPs include two AP2 transcription factors and the P. vivax multidrug resistance-associated protein (PvMRP1), an ABC transporter shown to be associated with quinoline and antifolate tolerance in Plasmodium falciparum. This analysis provides a data set for comparative analysis with important potential for identifying markers for global parasite diversity and drug resistance mapping studies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.1003776107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2993397PMC
November 2010

Screening the mammalian extracellular proteome for regulators of embryonic human stem cell pluripotency.

Proc Natl Acad Sci U S A 2010 Feb 2;107(8):3552-7. Epub 2010 Feb 2.

Department of Chemistry, The Scripps Research Institute, La Jolla, CA 92037, USA.

Approximately 3,500 mammalian genes are predicted to be secreted or single-pass transmembrane proteins. The function of the majority of these genes is still unknown, and a number of the encoded proteins might find use as new therapeutic agents themselves or as targets for small molecule or antibody drug development. To analyze the physiological activities of the extracellular proteome, we developed a large-scale, high-throughput protein expression, purification, and screening platform. For this study, the complete human extracellular proteome was analyzed and prioritized based on genome-wide disease association studies to select 529 initial target genes. These genes were cloned into three expression vectors as native sequences and as N-terminal and C-terminal Fc fusions to create an initial collection of 806 purified secreted proteins. To determine its utility, this library was screened in an OCT4-based cellular assay to identify regulators of human embryonic stem-cell self-renewal. We found that the pigment epithelium-derived factor can promote long-term pluripotent growth of human embryonic stem cells without bFGF or TGFbeta/Activin/Nodal ligand supplementation. Our results further indicate that activation of the pigment epithelium-derived factor receptor-Erk1/2 signaling pathway by the pigment epithelium-derived factor is sufficient to maintain the self-renewal of pluripotent human embryonic stem cells. These experiments illustrate the potential for discovering novel biological functions by directly screening protein diversity in cell-based phenotypic or reporter assays.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.0914019107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2840467PMC
February 2010

BioGPS: an extensible and customizable portal for querying and organizing gene annotation resources.

Genome Biol 2009 17;10(11):R130. Epub 2009 Nov 17.

Genomics Institute of the Novartis Research Foundation, 10675 John Jay Hopkins Dr, San Diego, CA 92121, USA.

Online gene annotation resources are indispensable for analysis of genomics data. However, the landscape of these online resources is highly fragmented, and scientists often visit dozens of these sites for each gene in a candidate gene list. Here, we introduce BioGPS http://biogps.gnf.org, a centralized gene portal for aggregating distributed gene annotation resources. Moreover, BioGPS embraces the principle of community intelligence, enabling any user to easily and directly contribute to the BioGPS platform.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/gb-2009-10-11-r130DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3091323PMC
July 2010

Use of high-density tiling microarrays to identify mutations globally and elucidate mechanisms of drug resistance in Plasmodium falciparum.

Genome Biol 2009 Feb 13;10(2):R21. Epub 2009 Feb 13.

Department of Cell Biology, ICND 202, The Scripps Research Institute, North Torrey Pines Road, La Jolla, CA 92037, USA.

Background: The identification of genetic changes that confer drug resistance or other phenotypic changes in pathogens can help optimize treatment strategies, support the development of new therapeutic agents, and provide information about the likely function of genes. Elucidating mechanisms of phenotypic drug resistance can also assist in identifying the mode of action of uncharacterized but potent antimalarial compounds identified in high-throughput chemical screening campaigns against Plasmodium falciparum.

Results: Here we show that tiling microarrays can detect de novo a large proportion of the genetic changes that differentiate one genome from another. We show that we detect most single nucleotide polymorphisms or small insertion deletion events and all known copy number variations that distinguish three laboratory isolates using readily accessible methods. We used the approach to discover mutations that occur during the selection process after transfection. We also elucidated a mechanism by which parasites acquire resistance to the antimalarial fosmidomycin, which targets the parasite isoprenoid synthesis pathway. Our microarray-based approach allowed us to attribute in vitro derived fosmidomycin resistance to a copy number variation event in the pfdxr gene, which enables the parasite to overcome fosmidomycin-mediated inhibition of isoprenoid biosynthesis.

Conclusions: We show that newly emerged single nucleotide polymorphisms can readily be detected and that malaria parasites can rapidly acquire gene amplifications in response to in vitro drug pressure. The ability to define comprehensively genetic variability in P. falciparum with a single overnight hybridization creates new opportunities to study parasite evolution and improve the treatment and control of malaria.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/gb-2009-10-2-r21DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2688282PMC
February 2009

A conserved salt bridge in the G loop of multiple protein kinases is important for catalysis and for in vivo Lyn function.

Mol Cell 2009 Jan;33(1):43-52

Genomics Institute of the Novartis Research Foundation, 10675 John J. Hopkins Drive, San Diego, CA 92121, USA.

The glycine-rich G loop controls ATP binding and phosphate transfer in protein kinases. Here we show that the functions of Src family and Abl protein tyrosine kinases require an electrostatic interaction between oppositely charged amino acids within their G loops that is conserved in multiple other phylogenetically distinct protein kinases, from plants to humans. By limiting G loop flexibility, it controls ATP binding, catalysis, and inhibition by ATP-competitive compounds such as Imatinib. In WeeB mice, mutational disruption of the interaction results in expression of a Lyn protein with reduced catalytic activity, and in perturbed B cell receptor signaling. Like Lyn(-/-) mice, WeeB mice show profound defects in B cell development and function and succumb to autoimmune glomerulonephritis. This demonstrates the physiological importance of the conserved G loop salt bridge and at the same time distinguishes the in vivo requirement for the Lyn kinase activity from other potential functions of the protein.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.molcel.2008.12.024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2683036PMC
January 2009

Evolution of the human ion channel set.

Comb Chem High Throughput Screen 2009 Jan;12(1):2-23

The Scripps Research Institute, La Jolla, CA 92037, USA.

Ion channels are intimately involved in virtually every physiological process of consequence in humans. Their importance is underscored by the identification of numerous "channelopathies", human diseases caused by ion channel mutations. Ion Channels have consequently been viewed as fertile ground for drug discovery and, indeed, they represent one of the largest target classes for current medicines. The future prospects of ion channels as a target class are tied to the functional characterization of the human ion channel set on a genomic scale. The focus of this review is to describe the molecular diversity and conservation of human ion channels. The human genome contains at least 232 genes that encode the pore-forming subunits of plasma membrane ion channels. Comparative genome analysis shows that most human ion channel gene families have their origins in the earliest metazoans but the human genes are largely derived from duplications that took place in the vertebrate lineage. The mouse and human ion channel gene sets are virtually identical, but differ significantly from fish channel sets. Genome comparisons highlight a number of highly conserved channel families that do not yet have specifically defined functional roles in vivo. These channel families are likely to have non-redundant functions in metazoans and represent some of the best new opportunities for channel target prospecting. Furthermore, genome-wide patterns of sequence conservation can now be used to refine strategies for the identification of gene-specific channel probes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.2174/138620709787047957DOI Listing
January 2009

A gene wiki for community annotation of gene function.

PLoS Biol 2008 Jul;6(7):e175

San Diego State University, Bioinformatics and Medical Informatics Graduate Program, San Diego, California, USA.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.pbio.0060175DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2443188PMC
July 2008

Gene set enrichment in eQTL data identifies novel annotations and pathway regulators.

PLoS Genet 2008 May 9;4(5):e1000070. Epub 2008 May 9.

Genomics Institute of the Novartis Research Foundation, San Diego, California, United States of America.

Genome-wide gene expression profiling has been extensively used to generate biological hypotheses based on differential expression. Recently, many studies have used microarrays to measure gene expression levels across genetic mapping populations. These gene expression phenotypes have been used for genome-wide association analyses, an analysis referred to as expression QTL (eQTL) mapping. Here, eQTL analysis was performed in adipose tissue from 28 inbred strains of mice. We focused our analysis on "trans-eQTL bands", defined as instances in which the expression patterns of many genes were all associated to a common genetic locus. Genes comprising trans-eQTL bands were screened for enrichments in functional gene sets representing known biological pathways, and genes located at associated trans-eQTL band loci were considered candidate transcriptional modulators. We demonstrate that these patterns were enriched for previously characterized relationships between known upstream transcriptional regulators and their downstream target genes. Moreover, we used this strategy to identify both novel regulators and novel members of known pathways. Finally, based on a putative regulatory relationship identified in our analysis, we identified and validated a previously uncharacterized role for cyclin H in the regulation of oxidative phosphorylation. We believe that the specific molecular hypotheses generated in this study will reveal many additional pathway members and regulators, and that the analysis approaches described herein will be broadly applicable to other eQTL data sets.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.pgen.1000070DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2346558PMC
May 2008

Whole genome functional analysis identifies novel components required for mitotic spindle integrity in human cells.

Genome Biol 2008 26;9(2):R44. Epub 2008 Feb 26.

Genomics Institute of Novartis Research Foundation, John Jay Hopkins Drive, San Diego, California 92121, USA.

Background: The mitotic spindle is a complex mechanical apparatus required for accurate segregation of sister chromosomes during mitosis. We designed a genetic screen using automated microscopy to discover factors essential for mitotic progression. Using a RNA interference library of 49,164 double-stranded RNAs targeting 23,835 human genes, we performed a loss of function screen to look for small interfering RNAs that arrest cells in metaphase.

Results: Here we report the identification of genes that, when suppressed, result in structural defects in the mitotic spindle leading to bent, twisted, monopolar, or multipolar spindles, and cause cell cycle arrest. We further describe a novel analysis methodology for large-scale RNA interference datasets that relies on supervised clustering of these genes based on Gene Ontology, protein families, tissue expression, and protein-protein interactions.

Conclusion: This approach was utilized to classify functionally the identified genes in discrete mitotic processes. We confirmed the identity for a subset of these genes and examined more closely their mechanical role in spindle architecture.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/gb-2008-9-2-r44DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2374723PMC
May 2008

Genome-wide and functional annotation of human E3 ubiquitin ligases identifies MULAN, a mitochondrial E3 that regulates the organelle's dynamics and signaling.

PLoS One 2008 Jan 23;3(1):e1487. Epub 2008 Jan 23.

Department of Cell Biology, The Scripps Research Institute, La Jolla, California, USA.

Specificity of protein ubiquitylation is conferred by E3 ubiquitin (Ub) ligases. We have annotated approximately 617 putative E3s and substrate-recognition subunits of E3 complexes encoded in the human genome. The limited knowledge of the function of members of the large E3 superfamily prompted us to generate genome-wide E3 cDNA and RNAi expression libraries designed for functional screening. An imaging-based screen using these libraries to identify E3s that regulate mitochondrial dynamics uncovered MULAN/FLJ12875, a RING finger protein whose ectopic expression and knockdown both interfered with mitochondrial trafficking and morphology. We found that MULAN is a mitochondrial protein - two transmembrane domains mediate its localization to the organelle's outer membrane. MULAN is oriented such that its E3-active, C-terminal RING finger is exposed to the cytosol, where it has access to other components of the Ub system. Both an intact RING finger and the correct subcellular localization were required for regulation of mitochondrial dynamics, suggesting that MULAN's downstream effectors are proteins that are either integral to, or associated with, mitochondria and that become modified with Ub. Interestingly, MULAN had previously been identified as an activator of NF-kappaB, thus providing a link between mitochondrial dynamics and mitochondria-to-nucleus signaling. These findings suggest the existence of a new, Ub-mediated mechanism responsible for integration of mitochondria into the cellular environment.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0001487PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2198940PMC
January 2008

A coactivator trap identifies NONO (p54nrb) as a component of the cAMP-signaling pathway.

Proc Natl Acad Sci U S A 2007 Dec 11;104(51):20314-9. Epub 2007 Dec 11.

Departments of Cancer Biology and Molecular Therapeutics and Translational Research Institute, The Scripps Research Institute, 5353 Parkside Drive, Jupiter, FL 33458, USA.

Signal transduction pathways often use a transcriptional component to mediate adaptive cellular responses. Coactivator proteins function prominently in these pathways as the conduit to the basic transcriptional machinery. Here we present a high-throughput cell-based screening strategy, termed the "coactivator trap," to study the functional interactions of coactivators with transcription factors. We applied this strategy to the cAMP signaling pathway, which utilizes two families of coactivators, the cAMP response element binding protein (CREB) binding protein (CBP)/p300 family and the recently identified transducers of regulated CREB activity family (TORCs1-3). In addition to identifying numerous known interactions of these coactivators, this analysis identified NONO (p54(nrb)) as a TORC-interacting protein. RNA interference experiments demonstrate that NONO is necessary for cAMP-dependent activation of CREB target genes in vivo. Furthermore, TORC2 and NONO complex on cAMP-responsive promoters, and NONO acts as a bridge between the CREB/TORC complex and RNA polymerase II. These data demonstrate the utility of the coactivator trap by identification of a component of cAMP-mediated transcription.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.0707999105DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2154428PMC
December 2007

Genomewide association analysis in diverse inbred mice: power and population structure.

Genetics 2007 May 3;176(1):675-83. Epub 2007 Apr 3.

Genomics Institute of the Novartis Research Foundation, San Diego, California 92121, USA.

The discovery of quantitative trait loci (QTL) in model organisms has relied heavily on the ability to perform controlled breeding to generate genotypic and phenotypic diversity. Recently, we and others have demonstrated the use of an existing set of diverse inbred mice (referred to here as the mouse diversity panel, MDP) as a QTL mapping population. The use of the MDP population has many advantages relative to traditional F(2) mapping populations, including increased phenotypic diversity, a higher recombination frequency, and the ability to collect genotype and phenotype data in community databases. However, these methods are complicated by population structure inherent in the MDP and the lack of an analytical framework to assess statistical power. To address these issues, we measured gene expression levels in hypothalamus across the MDP. We then mapped these phenotypes as quantitative traits with our association algorithm, resulting in a large set of expression QTL (eQTL). We utilized these eQTL, and specifically cis-eQTL, to develop a novel nonparametric method for association analysis in structured populations like the MDP. These eQTL data confirmed that the MDP is a suitable mapping population for QTL discovery and that eQTL results can serve as a gold standard for relative measures of statistical power.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1534/genetics.106.066241DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1893038PMC
May 2007

Genome-wide functional analysis of human cell-cycle regulators.

Proc Natl Acad Sci U S A 2006 Oct 25;103(40):14819-24. Epub 2006 Sep 25.

The Skaggs Institute for Chemical Biology, and Department of Chemistry, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.

Human cells have evolved complex signaling networks to coordinate the cell cycle. A detailed understanding of the global regulation of this fundamental process requires comprehensive identification of the genes and pathways involved in the various stages of cell-cycle progression. To this end, we report a genome-wide analysis of the human cell cycle, cell size, and proliferation by targeting >95% of the protein-coding genes in the human genome using small interfering RNAs (siRNAs). Analysis of >2 million images, acquired by quantitative fluorescence microscopy, showed that depletion of 1,152 genes strongly affected cell-cycle progression. These genes clustered into eight distinct phenotypic categories based on phase of arrest, nuclear area, and nuclear morphology. Phase-specific networks were built by interrogating knowledge-based and physical interaction databases with identified genes. Genome-wide analysis of cell-cycle regulators revealed a number of kinase, phosphatase, and proteolytic proteins and also suggests that processes thought to regulate G(1)-S phase progression like receptor-mediated signaling, nutrient status, and translation also play important roles in the regulation of G(2)/M phase transition. Moreover, 15 genes that are integral to TNF/NF-kappaB signaling were found to regulate G(2)/M, a previously unanticipated role for this pathway. These analyses provide systems-level insight into both known and novel genes as well as pathways that regulate cell-cycle progression, a number of which may provide new therapeutic approaches for the treatment of cancer.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.0604320103DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1595435PMC
October 2006

A systematic map of genetic variation in Plasmodium falciparum.

PLoS Pathog 2006 Jun 23;2(6):e57. Epub 2006 Jun 23.

Department of Cell Biology, The Scripps Research Institute, La Jolla, California, USA.

Discovering novel genes involved in immune evasion and drug resistance in the human malaria parasite, Plasmodium falciparum, is of critical importance to global health. Such knowledge may assist in the development of new effective vaccines and in the appropriate use of antimalarial drugs. By performing a full-genome scan of allelic variability in 14 field and laboratory strains of P. falciparum, we comprehensively identified approximately 500 genes evolving at higher than neutral rates. The majority of the most variable genes have paralogs within the P. falciparum genome and may be subject to a different evolutionary clock than those without. The group of 211 variable genes without paralogs contains most known immunogens and a few drug targets, consistent with the idea that the human immune system and drug use is driving parasite evolution. We also reveal gene-amplification events including one surrounding pfmdr1, the P. falciparum multidrug-resistance gene, and a previously uncharacterized amplification centered around the P. falciparum GTP cyclohydrolase gene, the first enzyme in the folate biosynthesis pathway. Although GTP cyclohydrolase is not the known target of any current drugs, downstream members of the pathway are targeted by several widely used antimalarials. We speculate that an amplification of the GTP cyclohydrolase enzyme in the folate biosynthesis pathway may increase flux through this pathway and facilitate parasite resistance to antifolate drugs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.ppat.0020057DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1480597PMC
June 2006

Transcript annotation in FANTOM3: mouse gene catalog based on physical cDNAs.

PLoS Genet 2006 Apr;2(4):e62

Genome Science Laboratory, Discovery Research Institute, RIKEN Wako Institute, Wako, Japan.

The international FANTOM consortium aims to produce a comprehensive picture of the mammalian transcriptome, based upon an extensive cDNA collection and functional annotation of full-length enriched cDNAs. The previous dataset, FANTOM2, comprised 60,770 full-length enriched cDNAs. Functional annotation revealed that this cDNA dataset contained only about half of the estimated number of mouse protein-coding genes, indicating that a number of cDNAs still remained to be collected and identified. To pursue the complete gene catalog that covers all predicted mouse genes, cloning and sequencing of full-length enriched cDNAs has been continued since FANTOM2. In FANTOM3, 42,031 newly isolated cDNAs were subjected to functional annotation, and the annotation of 4,347 FANTOM2 cDNAs was updated. To accomplish accurate functional annotation, we improved our automated annotation pipeline by introducing new coding sequence prediction programs and developed a Web-based annotation interface for simplifying the annotation procedures to reduce manual annotation errors. Automated coding sequence and function prediction was followed with manual curation and review by expert curators. A total of 102,801 full-length enriched mouse cDNAs were annotated. Out of 102,801 transcripts, 56,722 were functionally annotated as protein coding (including partial or truncated transcripts), providing to our knowledge the greatest current coverage of the mouse proteome by full-length cDNAs. The total number of distinct non-protein-coding transcripts increased to 34,030. The FANTOM3 annotation system, consisting of automated computational prediction, manual curation, and final expert curation, facilitated the comprehensive characterization of the mouse transcriptome, and could be applied to the transcriptomes of other species.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.pgen.0020062DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1449903PMC
April 2006

Complex Loci in human and mouse genomes.

PLoS Genet 2006 Apr 28;2(4):e47. Epub 2006 Apr 28.

Computational Biology Unit, Bergen Center for Computational Science, University of Bergen, Bergen, Norway.

Mammalian genomes harbor a larger than expected number of complex loci, in which multiple genes are coupled by shared transcribed regions in antisense orientation and/or by bidirectional core promoters. To determine the incidence, functional significance, and evolutionary context of mammalian complex loci, we identified and characterized 5,248 cis-antisense pairs, 1,638 bidirectional promoters, and 1,153 chains of multiple cis-antisense and/or bidirectionally promoted pairs from 36,606 mouse transcriptional units (TUs), along with 6,141 cis-antisense pairs, 2,113 bidirectional promoters, and 1,480 chains from 42,887 human TUs. In both human and mouse, 25% of TUs resided in cis-antisense pairs, only 17% of which were conserved between the two organisms, indicating frequent species specificity of antisense gene arrangements. A sampling approach indicated that over 40% of all TUs might actually be in cis-antisense pairs, and that only a minority of these arrangements are likely to be conserved between human and mouse. Bidirectional promoters were characterized by variable transcriptional start sites and an identifiable midpoint at which overall sequence composition changed strand and the direction of transcriptional initiation switched. In microarray data covering a wide range of mouse tissues, genes in cis-antisense and bidirectionally promoted arrangement showed a higher probability of being coordinately expressed than random pairs of genes. In a case study on homeotic loci, we observed extensive transcription of nonconserved sequences on the noncoding strand, implying that the presence rather than the sequence of these transcripts is of functional importance. Complex loci are ubiquitous, host numerous nonconserved gene structures and lineage-specific exonification events, and may have a cis-regulatory impact on the member genes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.pgen.0020047DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1449890PMC
April 2006

Susceptibility and modifier genes in Portuguese transthyretin V30M amyloid polyneuropathy: complexity in a single-gene disease.

Hum Mol Genet 2005 Feb 13;14(4):543-53. Epub 2005 Jan 13.

Department of Molecular and Experimental Medicine, Division of Rheumatology Research and the WM Keck Autoimmune Disease Center, The Scripps Research Institute, La Jolla, CA 92037, USA.

Familial amyloid polyneuropathy type I is an autosomal dominant disorder caused by mutations in the transthyretin (TTR) gene; however, carriers of the same mutation exhibit variability in penetrance and clinical expression. We analyzed alleles of candidate genes encoding non-fibrillar components of TTR amyloid deposits and a molecule metabolically interacting with TTR [retinol-binding protein (RBP)], for possible associations with age of disease onset and/or susceptibility in a Portuguese population sample with the TTR V30M mutation and unrelated controls. We show that the V30M carriers represent a distinct subset of the Portuguese population. Estimates of genetic distance indicated that the controls and the classical-onset group were furthest apart, whereas the late-onset group appeared to differ from both. Importantly, the data also indicate that genetic interactions among the multiple loci evaluated, rather than single-locus effects, are more likely to determine differences in the age of disease onset. Multifactor dimensionality reduction indicated that the best genetic model for classical onset group versus controls involved the APCS gene, whereas for late-onset cases, one APCS variant (APCSv1) and two RBP variants (RBPv1 and RBPv2) are involved. Thus, although the TTR V30M mutation is required for the disease in Portuguese patients, different genetic factors may govern the age of onset, as well as the occurrence of anticipation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/hmg/ddi051DOI Listing
February 2005

The promise of genomics to identify novel therapeutic targets.

Expert Opin Ther Targets 2004 Dec;8(6):587-96

The Genomics Institute of the Novartis Research Foundation, 10675 John J. Hopkins Drive, San Diego, CA 92121, USA.

The cataloguing of the human genome has provided an unprecedented prospectus for target identification and drug discovery. A current analysis indicates that slightly more than 3000 unique protein encoding loci are potentially amenable to pharmacological intervention (the 'druggable genome', which can be queried at http://function.gnf.org/druggable). However, the assessment of genome sequence data has not resulted in the anticipated acceleration of novel therapeutic developments. The basis for this shortfall lies in the significant attrition rates endemic to preclinical/clinical development, as well as the often underestimated complexity of gene function in higher order biological systems. To address the latter issue, a number of strategies have emerged to facilitate genomics-driven target identification and validation, including cellular profiling of gene function, in silico modelling of gene networks, and systematic analyses of protein complexes. The expectation is that the integration of these and other systems-based technologies may enable the conversion of potential genomic targets into functionally validated molecules, and result in practicable gene-based drug discovery pipelines.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1517/14728222.8.6.587DOI Listing
December 2004

Use of a dense single nucleotide polymorphism map for in silico mapping in the mouse.

PLoS Biol 2004 Dec 9;2(12):e393. Epub 2004 Nov 9.

Genomics Institute of the Novartis Research Foundation, San Diego, California, USA.

Rapid expansion of available data, both phenotypic and genotypic, for multiple strains of mice has enabled the development of new methods to interrogate the mouse genome for functional genetic perturbations. In silico mapping provides an expedient way to associate the natural diversity of phenotypic traits with ancestrally inherited polymorphisms for the purpose of dissecting genetic traits. In mouse, the current single nucleotide polymorphism (SNP) data have lacked the density across the genome and coverage of enough strains to properly achieve this goal. To remedy this, 470,407 allele calls were produced for 10,990 evenly spaced SNP loci across 48 inbred mouse strains. Use of the SNP set with statistical models that considered unique patterns within blocks of three SNPs as an inferred haplotype could successfully map known single gene traits and a cloned quantitative trait gene. Application of this method to high-density lipoprotein and gallstone phenotypes reproduced previously characterized quantitative trait loci (QTL). The inferred haplotype data also facilitates the refinement of QTL regions such that candidate genes can be more easily identified and characterized as shown for adenylate cyclase 7.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.pbio.0020393DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC526179PMC
December 2004

A Plasmodium gene family encoding Maurer's cleft membrane proteins: structural properties and expression profiling.

Genome Res 2004 Jun 12;14(6):1052-9. Epub 2004 May 12.

Department of Biological, Geological and Environmental Sciences, Cleveland State University, Cleveland, Ohio 44115, USA.

Upon invasion of the erythrocyte cell, the malaria parasite remodels its environment; in particular, it establishes a complex membrane network, which connects the parasitophorous vacuole to the host plasma membrane and is involved in protein transport and trafficking. We have identified a novel subtelomeric gene family in Plasmodium falciparum that encodes 11 transmembrane proteins localized to the Maurer's clefts. Using coimmunoprecipitation and shotgun proteomics, we were able to enrich specifically for these proteins and detect distinct peptides, allowing us to conclude that four to 10 products were present at a given time. Nearly all of the Pfmc-2tm genes are transcribed during the trophozoite stage; this narrow time frame of transcription overlaps with the specific stevor and rif genes that are differentially expressed during the erythrocyte cycle. The description of the structural properties of the proteins led us to manually reannotate published sequences, and to detect potentially homologous gene families in both P. falciparum and Plasmodium yoelii yoelii, where no orthologs were predicted uniquely based on sequence similarity. These basic proteins with two transmembrane domains belong to a larger superfamily, which includes STEVORs and RIFINs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1101/gr.2126104DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC419783PMC
June 2004

A gene atlas of the mouse and human protein-encoding transcriptomes.

Proc Natl Acad Sci U S A 2004 Apr 9;101(16):6062-7. Epub 2004 Apr 9.

The Genomics Institute of the Novartis Research Foundation, 10675 John J. Hopkins Drive, San Diego, CA 92121, USA.

The tissue-specific pattern of mRNA expression can indicate important clues about gene function. High-density oligonucleotide arrays offer the opportunity to examine patterns of gene expression on a genome scale. Toward this end, we have designed custom arrays that interrogate the expression of the vast majority of protein-encoding human and mouse genes and have used them to profile a panel of 79 human and 61 mouse tissues. The resulting data set provides the expression patterns for thousands of predicted genes, as well as known and poorly characterized genes, from mice and humans. We have explored this data set for global trends in gene expression, evaluated commonly used lines of evidence in gene prediction methodologies, and investigated patterns indicative of chromosomal organization of transcription. We describe hundreds of regions of correlated transcription and show that some are subject to both tissue and parental allele-specific expression, suggesting a link between spatial expression and imprinting.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.0400782101DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC395923PMC
April 2004

Identification of modulators of TRAIL-induced apoptosis via RNAi-based phenotypic screening.

Mol Cell 2003 Sep;12(3):627-37

Genomics Institute of the Novartis Research Foundation, 10675 John J. Hopkins Drive, San Diego, CA 92121, USA.

New opportunities in mammalian functional genomics are emerging through the combination of high throughput technology and methods that allow manipulation of gene expression in living cells. Here we describe the application of an RNAi-based forward genomics approach toward understanding the biology and mechanism of TRAIL-induced apoptosis. TRAIL is a TNF superfamily member that induces selective cytotoxicity of tumor cells when bound to its cognate receptors. In addition to detecting well-characterized genes in the apoptosis pathway, we uncover several modulators including DOBI, a gene required for progression of the apoptotic signal through the intrinsic mitochondrial cell death pathway, and MIRSA, a gene that acts to limit TRAIL-induced apoptosis. Moreover, our data suggest a role for MYC and the WNT pathway in maintaining susceptibility to TRAIL. Collectively, these observations offer several insights on how TRAIL mediates the selective killing of tumor cells and demonstrate the utility of large-scale RNAi screens in mammalian cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/s1097-2765(03)00348-4DOI Listing
September 2003

Discovery of gene function by expression profiling of the malaria parasite life cycle.

Science 2003 Sep 31;301(5639):1503-8. Epub 2003 Jul 31.

Department of Cell Biology ICND202, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.

The completion of the genome sequence for Plasmodium falciparum, the species responsible for most malaria human deaths, has the potential to reveal hundreds of new drug targets and proteins involved in pathogenesis. However, only approximately 35% of the genes code for proteins with an identifiable function. The absence of routine genetic tools for studying Plasmodium parasites suggests that this number is unlikely to change quickly if conventional serial methods are used to characterize encoded proteins. Here, we use a high-density oligonucleotide array to generate expression profiles of human and mosquito stages of the malaria parasite's life cycle. Genes with highly correlated levels and temporal patterns of expression were often involved in similar functions or cellular processes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1126/science.1087025DOI Listing
September 2003

Ubiquitin-mediated sequestration of normal cellular proteins into polyglutamine aggregates.

Proc Natl Acad Sci U S A 2003 Jul 11;100(15):8892-7. Epub 2003 Jul 11.

Department of Cancer and Cell Biology, Genomics Institute of the Novartis Research Foundation (GNF), 10675 John J. Hopkins Drive, San Diego, CA 92121, USA.

A hallmark of most neurodegenerative diseases, including those caused by polyglutamine expansion, is the formation of ubiquitin (Ub)-positive protein aggregates in affected neurons. This finding suggests that the Ub system may be involved in common mechanisms underlying these otherwise unrelated diseases. Here we report the finding of ataxin-3 (Atx-3), whose mutation is implicated in the neurodegenerative disease spinocerebellar ataxia type 3, in a bioinformatics search of the human genome for components of the Ub system. We show that wild-type Atx-3 is a Ub-binding protein and that the interaction of Atx-3 with Ub is mediated by motifs homologous to those found in a proteasome subunit. Both wild-type Atx-3 and the otherwise unrelated Ub-binding protein p62/Sequestosome-1 have been shown to be sequestered into aggregates in affected neurons in several neurodegenerative diseases, but the mechanism for this recruitment has remained unclear. In this article, we show that functional Ub-binding motifs in Atx-3 and p62 proteins are required for the localization of both proteins into aggregates in a cell-based assay that recapitulates several features of polyglutamine disease. We propose that the Ub-mediated sequestration of essential Ub-binding protein(s) into aggregates may be a common mechanism contributing to the pathogenesis of neurodegenerative diseases.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.1530212100DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC166409PMC
July 2003
-->