37 results on '"Coulombe-Huntington J"'
Search Results
2. Comparison of Affymetrix Gene Array with the Exon Array shows potential application for detection of transcript isoform variation
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Coulombe-Huntington Jasmin, Ha Kevin CH, and Majewski Jacek
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Biotechnology ,TP248.13-248.65 ,Genetics ,QH426-470 - Abstract
Abstract Background The emergence of isoform-sensitive microarrays has helped fuel in-depth studies of the human transcriptome. The Affymetrix GeneChip Human Exon 1.0 ST Array (Exon Array) has been previously shown to be effective in profiling gene expression at the isoform level. More recently, the Affymetrix GeneChip Human Gene 1.0 ST Array (Gene Array) has been released for measuring gene expression and interestingly contains a large subset of probes from the Exon Array. Here, we explore the potential of using Gene Array probes to assess expression variation at the sub-transcript level. Utilizing datasets of the high quality Microarray Quality Control (MAQC) RNA samples previously assayed on the Exon Array and Gene Array, we compare the expression measurements of the two platforms to determine the performance of the Gene Array in detecting isoform variations. Results Overall, we show that the Gene Array is comparable to the Exon Array in making gene expression calls. Moreover, to examine expression of different isoforms, we modify the Gene Array probe set definition file to enable summarization of probe intensity values at the exon level and show that the expression profiles between the two platforms are also highly correlated. Next, expression calls of previously known differentially spliced genes were compared and also show concordant results. Splicing index analysis, representing estimates of exon inclusion levels, shows a lower but good correlation between platforms. As the Gene Array contains a significant subset of probes from the Exon Array, we note that, in comparison, the Gene Array overlaps with fewer but still a high proportion of splicing events annotated in the Known Alt Events UCSC track, with abundant coverage of cassette exons. We discuss the ability of the Gene Array to detect alternative splicing and isoform variation and address its limitations. Conclusion The Gene Array is an effective expression profiling tool at gene and exon expression level, the latter made possible by probe set annotation modifications. We demonstrate that the Gene Array is capable of detecting alternative splicing and isoform variation. As expected, in comparison to the Exon Array, it is limited by reduced gene content coverage and is not able to detect as wide a range of alternative splicing events. However, for the events that can be monitored by both platforms, we estimate that the selectivity and sensitivity levels are comparable. We hope our findings will shed light on the potential extension of the Gene Array to detect alternative splicing. It should be particularly suitable for researchers primarily interested in gene expression analysis, but who may be willing to look for splicing and isoform differences within their dataset. However, we do not suggest it to be an equivalent substitute to the more comprehensive Exon Array.
- Published
- 2009
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3. The anticancer potential of the CLK kinases inhibitors 1C8 and GPS167 revealed by their impact on the epithelial-mesenchymal transition and the antiviral immune response.
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Shkreta L, Toutant J, Delannoy A, Durantel D, Salvetti A, Ehresmann S, Sauvageau M, Delbrouck JA, Gravel-Trudeau A, Comeau C, Huard C, Coulombe-Huntington J, Tyers M, Grierson D, Boudreault PL, and Chabot B
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- Humans, Cell Line, Tumor, Antineoplastic Agents pharmacology, Protein Serine-Threonine Kinases metabolism, Protein Serine-Threonine Kinases genetics, Protein Serine-Threonine Kinases antagonists & inhibitors, Thiazoles pharmacology, Antiviral Agents pharmacology, HCT116 Cells, DEAD-box RNA Helicases metabolism, DEAD-box RNA Helicases genetics, Gene Expression Profiling, Epithelial-Mesenchymal Transition drug effects, Protein-Tyrosine Kinases metabolism, Protein-Tyrosine Kinases antagonists & inhibitors, Protein-Tyrosine Kinases genetics, Protein Kinase Inhibitors pharmacology, Cell Proliferation drug effects
- Abstract
The diheteroarylamide-based compound 1C8 and the aminothiazole carboxamide-related compound GPS167 inhibit the CLK kinases, and affect the proliferation of a broad range of cancer cell lines. A chemogenomic screen previously performed with GPS167 revealed that the depletion of components associated with mitotic spindle assembly altered sensitivity to GPS167. Here, a similar screen performed with 1C8 also established the impact of components involved in mitotic spindle assembly. Accordingly, transcriptome analyses of cells treated with 1C8 and GPS167 indicated that the expression and RNA splicing of transcripts encoding mitotic spindle assembly components were affected. The functional relevance of the microtubule connection was confirmed by showing that subtoxic concentrations of drugs affecting mitotic spindle assembly increased sensitivity to GPS167. 1C8 and GPS167 impacted the expression and splicing of transcripts in pathways relevant to tumor progression, including MYC targets and the epithelial mesenchymal transition (EMT). Finally, 1C8 and GPS167 altered the expression and alternative splicing of transcripts involved in the antiviral immune response. Consistent with this observation, depleting the double-stranded RNA sensor DHX33 suppressed GPS167-mediated cytotoxicity on HCT116 cells. Our study uncovered molecular mechanisms through which 1C8 and GPS167 affect cancer cell proliferation as well as processes critical for metastasis.
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- 2024
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4. Active growth signaling promotes senescence and cancer cell sensitivity to CDK7 inhibition.
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Wilson GA, Vuina K, Sava G, Huard C, Meneguello L, Coulombe-Huntington J, Bertomeu T, Maizels RJ, Lauring J, Kriston-Vizi J, Tyers M, Ali S, Bertoli C, and de Bruin RAM
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- Humans, Cyclin-Dependent Kinase-Activating Kinase, Signal Transduction, Cell Cycle, Enzyme Inhibitors, TOR Serine-Threonine Kinases genetics, TOR Serine-Threonine Kinases metabolism, Cell Line, Tumor, Cyclin-Dependent Kinases genetics, Cyclin-Dependent Kinases metabolism, Neoplasms drug therapy, Neoplasms genetics
- Abstract
Tumor growth is driven by continued cellular growth and proliferation. Cyclin-dependent kinase 7's (CDK7) role in activating mitotic CDKs and global gene expression makes it therefore an attractive target for cancer therapies. However, what makes cancer cells particularly sensitive to CDK7 inhibition (CDK7i) remains unclear. Here, we address this question. We show that CDK7i, by samuraciclib, induces a permanent cell-cycle exit, known as senescence, without promoting DNA damage signaling or cell death. A chemogenetic genome-wide CRISPR knockout screen identified that active mTOR (mammalian target of rapamycin) signaling promotes samuraciclib-induced senescence. mTOR inhibition decreases samuraciclib sensitivity, and increased mTOR-dependent growth signaling correlates with sensitivity in cancer cell lines. Reverting a growth-promoting mutation in PIK3CA to wild type decreases sensitivity to CDK7i. Our work establishes that enhanced growth alone promotes CDK7i sensitivity, providing an explanation for why some cancers are more sensitive to CDK inhibition than normally growing cells., Competing Interests: Declaration of interests S.A. is an inventor on a patent describing samuraciclib, which is licensed to Carrick Therapeutics Ltd. S.A. has received royalties from and has share ownership in Carrick Therapeutics. J.L. is currently a full-time employee of and owns stock in Janssen Research and Development and is entitled to receive royalty payments for commercial use of cell lines described in this work under a licensing agreement between Johns Hopkins University and Horizon Discovery, Ltd., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)
- Published
- 2023
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5. Genetic enhancers of partial PLK1 inhibition reveal hypersensitivity to kinetochore perturbations.
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Normandin K, Coulombe-Huntington J, St-Denis C, Bernard A, Bourouh M, Bertomeu T, Tyers M, and Archambault V
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- Humans, Polo-Like Kinase 1, Cell Cycle Proteins genetics, Kinetochores, Protein Serine-Threonine Kinases genetics, Enhancer Elements, Genetic
- Abstract
Polo-like kinase 1 (PLK1) is a serine/threonine kinase required for mitosis and cytokinesis. As cancer cells are often hypersensitive to partial PLK1 inactivation, chemical inhibitors of PLK1 have been developed and tested in clinical trials. However, these small molecule inhibitors alone are not completely effective. PLK1 promotes numerous molecular and cellular events in the cell division cycle and it is unclear which of these events most crucially depend on PLK1 activity. We used a CRISPR-based genome-wide screening strategy to identify genes whose inactivation enhances cell proliferation defects upon partial chemical inhibition of PLK1. Genes identified encode proteins that are functionally linked to PLK1 in multiple ways, most notably factors that promote centromere and kinetochore function. Loss of the kinesin KIF18A or the outer kinetochore protein SKA1 in PLK1-compromised cells resulted in mitotic defects, activation of the spindle assembly checkpoint and nuclear reassembly defects. We also show that PLK1-dependent CENP-A loading at centromeres is extremely sensitive to partial PLK1 inhibition. Our results suggest that partial inhibition of PLK1 compromises the integrity and function of the centromere/kinetochore complex, rendering cells hypersensitive to different kinetochore perturbations. We propose that KIF18A is a promising target for combinatorial therapies with PLK1 inhibitors., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Normandin et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)
- Published
- 2023
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6. Viral protein engagement of GBF1 induces host cell vulnerability through synthetic lethality.
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Navare AT, Mast FD, Olivier JP, Bertomeu T, Neal ML, Carpp LN, Kaushansky A, Coulombe-Huntington J, Tyers M, and Aitchison JD
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- Humans, Synthetic Lethal Mutations, Virus Replication, Gene Expression Regulation, Viral, Host-Pathogen Interactions, Guanine Nucleotide Exchange Factors metabolism, Poliovirus, Viral Core Proteins genetics, Viral Core Proteins metabolism
- Abstract
Viruses co-opt host proteins to carry out their lifecycle. Repurposed host proteins may thus become functionally compromised; a situation analogous to a loss-of-function mutation. We term such host proteins as viral-induced hypomorphs. Cells bearing cancer driver loss-of-function mutations have successfully been targeted with drugs perturbing proteins encoded by the synthetic lethal (SL) partners of cancer-specific mutations. Similarly, SL interactions of viral-induced hypomorphs can potentially be targeted as host-based antiviral therapeutics. Here, we use GBF1, which supports the infection of many RNA viruses, as a proof-of-concept. GBF1 becomes a hypomorph upon interaction with the poliovirus protein 3A. Screening for SL partners of GBF1 revealed ARF1 as the top hit, disruption of which selectively killed cells that synthesize 3A alone or in the context of a poliovirus replicon. Thus, viral protein interactions can induce hypomorphs that render host cells selectively vulnerable to perturbations that leave uninfected cells otherwise unscathed. Exploiting viral-induced vulnerabilities could lead to broad-spectrum antivirals for many viruses, including SARS-CoV-2., (© 2022 Navare et al.)
- Published
- 2022
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7. The microprotein Nrs1 rewires the G1/S transcriptional machinery during nitrogen limitation in budding yeast.
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Tollis S, Singh J, Palou R, Thattikota Y, Ghazal G, Coulombe-Huntington J, Tang X, Moore S, Blake D, Bonneil E, Royer CA, Thibault P, and Tyers M
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- Amino Acid Sequence, Cell Division genetics, DNA-Binding Proteins genetics, DNA-Binding Proteins metabolism, Immunoblotting, Protein Binding, RNA-Seq methods, Repressor Proteins genetics, Repressor Proteins metabolism, Saccharomyces cerevisiae metabolism, Saccharomyces cerevisiae Proteins metabolism, Sequence Homology, Amino Acid, Transcription Factors genetics, Transcription Factors metabolism, G1 Phase genetics, Gene Expression Regulation, Fungal, Nitrogen metabolism, S Phase genetics, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae Proteins genetics
- Abstract
Commitment to cell division at the end of G1 phase, termed Start in the budding yeast Saccharomyces cerevisiae, is strongly influenced by nutrient availability. To identify new dominant activators of Start that might operate under different nutrient conditions, we screened a genome-wide ORF overexpression library for genes that bypass a Start arrest caused by absence of the G1 cyclin Cln3 and the transcriptional activator Bck2. We recovered a hypothetical gene YLR053c, renamed NRS1 for Nitrogen-Responsive Start regulator 1, which encodes a poorly characterized 108 amino acid microprotein. Endogenous Nrs1 was nuclear-localized, restricted to poor nitrogen conditions, induced upon TORC1 inhibition, and cell cycle-regulated with a peak at Start. NRS1 interacted genetically with SWI4 and SWI6, which encode subunits of the main G1/S transcription factor complex SBF. Correspondingly, Nrs1 physically interacted with Swi4 and Swi6 and was localized to G1/S promoter DNA. Nrs1 exhibited inherent transactivation activity, and fusion of Nrs1 to the SBF inhibitor Whi5 was sufficient to suppress other Start defects. Nrs1 appears to be a recently evolved microprotein that rewires the G1/S transcriptional machinery under poor nitrogen conditions., Competing Interests: The authors have declared that no competing interests exist.
- Published
- 2022
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8. Vesicular trafficking is a key determinant of the statin response in acute myeloid leukemia.
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Krosl J, Bordeleau ME, Moison C, MacRae T, Boivin I, Mayotte N, Gracias D, Baccelli I, Lavallée VP, Bisaillon R, Lehnertz B, Mendoza-Sanchez R, Ruel R, Bertomeu T, Coulombe-Huntington J, Boucher G, Noronha N, Pabst C, Tyers M, Gendron P, Lemieux S, Barabé F, Marinier A, Hébert J, and Sauvageau G
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- Humans, Hydroxymethylglutaryl-CoA Reductase Inhibitors pharmacology, Hydroxymethylglutaryl-CoA Reductase Inhibitors therapeutic use, Leukemia, Myeloid, Acute drug therapy, Leukemia, Myeloid, Acute genetics
- Abstract
Cholesterol homeostasis has been proposed as one mechanism contributing to chemoresistance in AML and hence, inclusion of statins in therapeutic regimens as part of clinical trials in AML has shown encouraging results. Chemical screening of primary human AML specimens by our group led to the identification of lipophilic statins as potent inhibitors of AMLs from a wide range of cytogenetic groups. Genetic screening to identify modulators of the statin response uncovered the role of protein geranylgeranylation and of RAB proteins, coordinating various aspect of vesicular trafficking, in mediating the effects of statins on AML cell viability. We further show that statins can inhibit vesicle-mediated transport in primary human specimens, and that statins sensitive samples show expression signatures reminiscent of enhanced vesicular trafficking. Overall, this study sheds light into the mechanism of action of statins in AML and identifies a novel vulnerability for cytogenetically diverse AML., (© 2022 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)
- Published
- 2022
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9. Chemical Genetics Screen Identifies COPB2 Tool Compounds That Alters ER Stress Response and Induces RTK Dysregulation in Lung Cancer Cells.
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Saraon P, Snider J, Schormann W, Rai A, Radulovich N, Sánchez-Osuna M, Coulombe-Huntington J, Huard C, Mohammed M, Lima-Fernandes E, Thériault B, Halabelian L, Chan M, Joshi D, Drecun L, Yao Z, Pathmanathan S, Wong V, Lyakisheva A, Aboualizadeh F, Niu L, Li F, Kiyota T, Subramanian R, Joseph B, Aman A, Prakesch M, Isaac M, Mamai A, Poda G, Vedadi M, Marcellus R, Uehling D, Leighl N, Sacher A, Samaržija M, Jakopović M, Arrowsmith C, Tyers M, Tsao MS, Andrews D, Al-Awar R, and Stagljar I
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- Drug Screening Assays, Antitumor, ErbB Receptors antagonists & inhibitors, ErbB Receptors genetics, ErbB Receptors metabolism, Humans, Mutation, Protein Kinase Inhibitors pharmacology, Protein Processing, Post-Translational, Receptor Protein-Tyrosine Kinases metabolism, Signal Transduction drug effects, Coatomer Protein genetics, Coatomer Protein metabolism, Drug Discovery methods, Endoplasmic Reticulum Stress drug effects, Endoplasmic Reticulum Stress genetics, Gene Expression Regulation, Neoplastic drug effects, Receptor Protein-Tyrosine Kinases genetics
- Abstract
Activating mutations in the epidermal growth factor receptor (EGFR) are common driver mutations in non-small cell lung cancer (NSCLC). First, second and third generation EGFR tyrosine kinase inhibitors (TKIs) are effective at inhibiting mutant EGFR NSCLC, however, acquired resistance is a major issue, leading to disease relapse. Here, we characterize a small molecule, EMI66, an analog of a small molecule which we previously identified to inhibit mutant EGFR signalling via a novel mechanism of action. We show that EMI66 attenuates receptor tyrosine kinase (RTK) expression and signalling and alters the electrophoretic mobility of Coatomer Protein Complex Beta 2 (COPB2) protein in mutant EGFR NSCLC cells. Moreover, we demonstrate that EMI66 can alter the subcellular localization of EGFR and COPB2 within the early secretory pathway. Furthermore, we find that COPB2 knockdown reduces the growth of mutant EGFR lung cancer cells, alters the post-translational processing of RTKs, and alters the endoplasmic reticulum (ER) stress response pathway. Lastly, we show that EMI66 treatment also alters the ER stress response pathway and inhibits the growth of mutant EGFR lung cancer cells and organoids. Our results demonstrate that targeting of COPB2 with EMI66 presents a viable approach to attenuate mutant EGFR signalling and growth in NSCLC., Competing Interests: Declaration of interests I.S., P.S. and J.S. (in conjunction with the University of Toronto) are listed as inventors on a patent (publication number 20190091205) for the use of EMI1 (and structurally related analogues), midostaurin, gilteritinib and AZD7762 (and structurally related analogues) in the treatment of mutant EGFR-mediated non-small-cell lung cancer., (Copyright © 2021 Elsevier Ltd. All rights reserved.)
- Published
- 2021
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10. Identification and optimization of molecular glue compounds that inhibit a noncovalent E2 enzyme-ubiquitin complex.
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St-Cyr D, Ceccarelli DF, Orlicky S, van der Sloot AM, Tang X, Kelso S, Moore S, James C, Posternak G, Coulombe-Huntington J, Bertomeu T, Marinier A, Sicheri F, and Tyers M
- Abstract
Pharmacological control of the ubiquitin-proteasome system (UPS) is of intense interest in drug discovery. Here, we report the development of chemical inhibitors of the ubiquitin-conjugating (E2) enzyme CDC34A (also known as UBE2R1), which donates activated ubiquitin to the cullin-RING ligase (CRL) family of ubiquitin ligase (E3) enzymes. A FRET-based interaction assay was used to screen for novel compounds that stabilize the noncovalent complex between CDC34A and ubiquitin, and thereby inhibit the CDC34A catalytic cycle. An isonipecotamide hit compound was elaborated into analogs with ~1000-fold increased potency in stabilizing the CDC34A-ubiquitin complex. These analogs specifically inhibited CDC34A-dependent ubiquitination in vitro and stabilized an E2~ubiquitin thioester reaction intermediate in cells. The x-ray crystal structure of a CDC34A-ubiquitin-inhibitor complex uncovered the basis for analog structure-activity relationships. The development of chemical stabilizers of the CDC34A-ubiquitin complex illustrates a general strategy for de novo discovery of molecular glue compounds that stabilize weak protein interactions.
- Published
- 2021
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11. A novel class of inhibitors that target SRSF10 and promote p53-mediated cytotoxicity on human colorectal cancer cells.
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Sohail M, Shkreta L, Toutant J, Rabea S, Babeu JP, Huard C, Coulombe-Huntington J, Delannoy A, Placet M, Geha S, Gendron FP, Boudreau F, Tyers M, Grierson DS, and Chabot B
- Abstract
The elevated expression of the splicing regulator SRSF10 in metastatic colorectal cancer (CRC) stimulates the production of the pro-tumorigenic BCLAF1-L splice variant. We discovered a group of small molecules with an aminothiazole carboxamide core (GPS167, GPS192 and others) that decrease production of BCLAF1-L . While additional alternative splicing events regulated by SRSF10 are affected by GPS167/192 in HCT116 cells (e.g. in MDM4, WTAP, SLK1 and CLK1 ), other events are shifted in a SRSF10-independent manner (e.g. in MDM2, NAB2 and TRA2A ). GPS167/192 increased the interaction of SRSF10 with the CLK1 and CLK4 kinases, leading us to show that GPS167/192 can inhibit CLK kinases preferentially impacting the activity of SRSF10. Notably, GPS167 impairs the growth of CRC cell lines and organoids, inhibits anchorage-independent colony formation, cell migration, and promotes cytoxicity in a manner that requires SRSF10 and p53. In contrast, GPS167 only minimally affects normal colonocytes and normal colorectal organoids. Thus, GPS167 reprograms the tumorigenic activity of SRSF10 in CRC cells to elicit p53-dependent apoptosis., (© The Author(s) 2021. Published by Oxford University Press on behalf of NAR Cancer.)
- Published
- 2021
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12. A novel p53 regulator, C16ORF72/TAPR1, buffers against telomerase inhibition.
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Benslimane Y, Sánchez-Osuna M, Coulombe-Huntington J, Bertomeu T, Henry D, Huard C, Bonneil É, Thibault P, Tyers M, and Harrington L
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- Humans, Cell Line, Tumor, DNA Damage drug effects, Gene Knockout Techniques, Imidazoles pharmacology, Piperazines pharmacology, Proto-Oncogene Proteins c-mdm2 antagonists & inhibitors, Telomere metabolism, Transduction, Genetic, Up-Regulation genetics, Aminobenzoates pharmacology, Intercellular Signaling Peptides and Proteins genetics, Intercellular Signaling Peptides and Proteins metabolism, Naphthalenes pharmacology, Precursor Cell Lymphoblastic Leukemia-Lymphoma metabolism, Precursor Cell Lymphoblastic Leukemia-Lymphoma pathology, Signal Transduction drug effects, Signal Transduction genetics, Telomerase antagonists & inhibitors, Telomerase genetics, Telomerase metabolism, Tumor Suppressor Protein p53 metabolism
- Abstract
Telomere erosion in cells with insufficient levels of the telomerase reverse transcriptase (TERT), contributes to age-associated tissue dysfunction and senescence, and p53 plays a crucial role in this response. We undertook a genome-wide CRISPR screen to identify gene deletions that sensitized p53-positive human cells to telomerase inhibition. We uncovered a previously unannotated gene, C16ORF72, which we term Telomere Attrition and p53 Response 1 (TAPR1), that exhibited a synthetic-sick relationship with TERT loss. A subsequent genome-wide CRISPR screen in TAPR1-disrupted cells reciprocally identified TERT as a sensitizing gene deletion. Cells lacking TAPR1 or TERT possessed elevated p53 levels and transcriptional signatures consistent with p53 upregulation. The elevated p53 response in TERT- or TAPR1-deficient cells was exacerbated by treatment with the MDM2 inhibitor and p53 stabilizer nutlin-3a and coincided with a further reduction in cell fitness. Importantly, the sensitivity to treatment with nutlin-3a in TERT- or TAPR1-deficient cells was rescued by loss of p53. These data suggest that TAPR1 buffers against the deleterious consequences of telomere erosion or DNA damage by constraining p53. These findings identify C16ORF72/TAPR1 as new regulator at the nexus of telomere integrity and p53 regulation., (© 2021 The Authors. Aging Cell published by Anatomical Society and John Wiley & Sons Ltd.)
- Published
- 2021
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13. PRMT5 inhibition disrupts splicing and stemness in glioblastoma.
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Sachamitr P, Ho JC, Ciamponi FE, Ba-Alawi W, Coutinho FJ, Guilhamon P, Kushida MM, Cavalli FMG, Lee L, Rastegar N, Vu V, Sánchez-Osuna M, Coulombe-Huntington J, Kanshin E, Whetstone H, Durand M, Thibault P, Hart K, Mangos M, Veyhl J, Chen W, Tran N, Duong BC, Aman AM, Che X, Lan X, Whitley O, Zaslaver O, Barsyte-Lovejoy D, Richards LM, Restall I, Caudy A, Röst HL, Bonday ZQ, Bernstein M, Das S, Cusimano MD, Spears J, Bader GD, Pugh TJ, Tyers M, Lupien M, Haibe-Kains B, Artee Luchman H, Weiss S, Massirer KB, Prinos P, Arrowsmith CH, and Dirks PB
- Subjects
- Animals, Apoptosis, Brain Neoplasms drug therapy, Brain Neoplasms genetics, Brain Neoplasms pathology, Cell Cycle, Cell Line, Tumor, Cell Proliferation drug effects, Drug Discovery, Epigenomics, Female, Gene Expression Regulation, Neoplastic, Glioblastoma drug therapy, Glioblastoma genetics, Glioblastoma pathology, Humans, Mice, Neoplastic Stem Cells metabolism, Protein-Arginine N-Methyltransferases drug effects, Protein-Arginine N-Methyltransferases genetics, RNA Splicing, Xenograft Model Antitumor Assays, Antineoplastic Agents pharmacology, Brain Neoplasms metabolism, Glioblastoma metabolism, Protein-Arginine N-Methyltransferases metabolism
- Abstract
Glioblastoma (GBM) is a deadly cancer in which cancer stem cells (CSCs) sustain tumor growth and contribute to therapeutic resistance. Protein arginine methyltransferase 5 (PRMT5) has recently emerged as a promising target in GBM. Using two orthogonal-acting inhibitors of PRMT5 (GSK591 or LLY-283), we show that pharmacological inhibition of PRMT5 suppresses the growth of a cohort of 46 patient-derived GBM stem cell cultures, with the proneural subtype showing greater sensitivity. We show that PRMT5 inhibition causes widespread disruption of splicing across the transcriptome, particularly affecting cell cycle gene products. We identify a GBM splicing signature that correlates with the degree of response to PRMT5 inhibition. Importantly, we demonstrate that LLY-283 is brain-penetrant and significantly prolongs the survival of mice with orthotopic patient-derived xenografts. Collectively, our findings provide a rationale for the clinical development of brain penetrant PRMT5 inhibitors as treatment for GBM.
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- 2021
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14. UM171 Preserves Epigenetic Marks that Are Reduced in Ex Vivo Culture of Human HSCs via Potentiation of the CLR3-KBTBD4 Complex.
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Chagraoui J, Girard S, Spinella JF, Simon L, Bonneil E, Mayotte N, MacRae T, Coulombe-Huntington J, Bertomeu T, Moison C, Tomellini E, Thibault P, Tyers M, Marinier A, and Sauvageau G
- Subjects
- Histone Deacetylases metabolism, Humans, Co-Repressor Proteins genetics, Co-Repressor Proteins metabolism, Epigenesis, Genetic, Hematopoietic Stem Cells metabolism
- Abstract
Human hematopoietic stem cells (HSCs) exhibit attrition of their self-renewal capacity when cultured ex vivo, a process that is partially reversed upon treatment with epigenetic modifiers, most notably inhibitors of histone deacetylases (HDACs) or lysine-specific demethylase LSD1. A recent study showed that the human HSC self-renewal agonist UM171 modulates the CoREST complex, leading to LSD1 degradation, whose inhibition mimics the activity of UM171. The mechanism underlying the UM171-mediated loss of CoREST function remains undetermined. We now report that UM171 potentiates the activity of a CULLIN3-E3 ubiquitin ligase (CRL3) complex whose target specificity is dictated by the poorly characterized Kelch/BTB domain protein KBTBD4. CRL3
KBTBD4 targets components of the LSD1/RCOR1 corepressor complex for proteasomal degradation, hence re-establishing H3K4me2 and H3K27ac epigenetic marks, which are rapidly decreased upon ex vivo culture of human HSCs., Competing Interests: Declaration of interests Except for G.S., the authors declare no competing interests. G.S. is founder and CEO of ExCellThera, a small biotech that owns an exclusive license to UM171., (Copyright © 2020 Elsevier Inc. All rights reserved.)- Published
- 2021
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15. The BioGRID database: A comprehensive biomedical resource of curated protein, genetic, and chemical interactions.
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Oughtred R, Rust J, Chang C, Breitkreutz BJ, Stark C, Willems A, Boucher L, Leung G, Kolas N, Zhang F, Dolma S, Coulombe-Huntington J, Chatr-Aryamontri A, Dolinski K, and Tyers M
- Subjects
- Animals, COVID-19 virology, Humans, Mice, SARS-CoV-2 genetics, SARS-CoV-2 pathogenicity, User-Computer Interface, COVID-19 genetics, Databases, Factual, Protein Interaction Mapping, Proteins genetics
- Abstract
The BioGRID (Biological General Repository for Interaction Datasets, thebiogrid.org) is an open-access database resource that houses manually curated protein and genetic interactions from multiple species including yeast, worm, fly, mouse, and human. The ~1.93 million curated interactions in BioGRID can be used to build complex networks to facilitate biomedical discoveries, particularly as related to human health and disease. All BioGRID content is curated from primary experimental evidence in the biomedical literature, and includes both focused low-throughput studies and large high-throughput datasets. BioGRID also captures protein post-translational modifications and protein or gene interactions with bioactive small molecules including many known drugs. A built-in network visualization tool combines all annotations and allows users to generate network graphs of protein, genetic and chemical interactions. In addition to general curation across species, BioGRID undertakes themed curation projects in specific aspects of cellular regulation, for example the ubiquitin-proteasome system, as well as specific disease areas, such as for the SARS-CoV-2 virus that causes COVID-19 severe acute respiratory syndrome. A recent extension of BioGRID, named the Open Repository of CRISPR Screens (ORCS, orcs.thebiogrid.org), captures single mutant phenotypes and genetic interactions from published high throughput genome-wide CRISPR/Cas9-based genetic screens. BioGRID-ORCS contains datasets for over 1,042 CRISPR screens carried out to date in human, mouse and fly cell lines. The biomedical research community can freely access all BioGRID data through the web interface, standardized file downloads, or via model organism databases and partner meta-databases., (© 2020 The Authors. Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)
- Published
- 2021
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16. Viral protein engagement of GBF1 induces host cell vulnerability through synthetic lethality.
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Navare AT, Mast FD, Olivier JP, Bertomeu T, Neal M, Carpp LN, Kaushansky A, Coulombe-Huntington J, Tyers M, and Aitchison JD
- Abstract
Viruses co-opt host proteins to carry out their lifecycle. Repurposed host proteins may thus become functionally compromised; a situation analogous to a loss-of-function mutation. We term such host proteins viral-induced hypomorphs. Cells bearing cancer driver loss-of-function mutations have successfully been targeted with drugs perturbing proteins encoded by the synthetic lethal partners of cancer-specific mutations. Synthetic lethal interactions of viral-induced hypomorphs have the potential to be similarly targeted for the development of host-based antiviral therapeutics. Here, we use GBF1, which supports the infection of many RNA viruses, as a proof-of-concept. GBF1 becomes a hypomorph upon interaction with the poliovirus protein 3A. Screening for synthetic lethal partners of GBF1 revealed ARF1 as the top hit, disruption of which, selectively killed cells that synthesize poliovirus 3A. Thus, viral protein interactions can induce hypomorphs that render host cells vulnerable to perturbations that leave uninfected cells intact. Exploiting viral-induced vulnerabilities could lead to broad-spectrum antivirals for many viruses, including SARS-CoV-2., Summary: Using a viral-induced hypomorph of GBF1, Navare et al., demonstrate that the principle of synthetic lethality is a mechanism to selectively kill virus-infected cells.
- Published
- 2020
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17. Crippling life support for SARS-CoV-2 and other viruses through synthetic lethality.
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Mast FD, Navare AT, van der Sloot AM, Coulombe-Huntington J, Rout MP, Baliga NS, Kaushansky A, Chait BT, Aderem A, Rice CM, Sali A, Tyers M, and Aitchison JD
- Subjects
- Drug Discovery, Humans, Immunologic Factors pharmacology, Metabolic Networks and Pathways drug effects, Protein Interaction Maps, Proteolysis, RNA Viruses drug effects, RNA Viruses physiology, Virus Diseases genetics, Antiviral Agents pharmacology, Host Microbial Interactions drug effects, Virus Diseases drug therapy, Virus Replication drug effects
- Abstract
With the rapid global spread of SARS-CoV-2, we have become acutely aware of the inadequacies of our ability to respond to viral epidemics. Although disrupting the viral life cycle is critical for limiting viral spread and disease, it has proven challenging to develop targeted and selective therapeutics. Synthetic lethality offers a promising but largely unexploited strategy against infectious viral disease; as viruses infect cells, they abnormally alter the cell state, unwittingly exposing new vulnerabilities in the infected cell. Therefore, we propose that effective therapies can be developed to selectively target the virally reconfigured host cell networks that accompany altered cellular states to cripple the host cell that has been converted into a virus factory, thus disrupting the viral life cycle., (© 2020 Mast et al.)
- Published
- 2020
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18. Genome-Wide Screens Reveal that Resveratrol Induces Replicative Stress in Human Cells.
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Benslimane Y, Bertomeu T, Coulombe-Huntington J, McQuaid M, Sánchez-Osuna M, Papadopoli D, Avizonis D, Russo MST, Huard C, Topisirovic I, Wurtele H, Tyers M, and Harrington L
- Subjects
- CRISPR-Cas Systems, Cell Line, Drug Resistance genetics, Humans, Hydroxyurea pharmacology, Jurkat Cells, Nucleotides metabolism, S Phase Cell Cycle Checkpoints drug effects, Sirtuin 1 metabolism, Stilbenes pharmacology, Cell Proliferation drug effects, DNA Replication drug effects, Resveratrol pharmacology
- Abstract
Resveratrol is a natural product associated with wide-ranging effects in animal and cellular models, including lifespan extension. To identify the genetic target of resveratrol in human cells, we conducted genome-wide CRISPR-Cas9 screens to pinpoint genes that confer sensitivity or resistance to resveratrol. An extensive network of DNA damage response and replicative stress genes exhibited genetic interactions with resveratrol and its analog pterostilbene. These genetic profiles showed similarity to the response to hydroxyurea, an inhibitor of ribonucleotide reductase that causes replicative stress. Resveratrol, pterostilbene, and hydroxyurea caused similar depletion of nucleotide pools, inhibition of replication fork progression, and induction of replicative stress. The ability of resveratrol to inhibit cell proliferation and S phase transit was independent of the histone deacetylase sirtuin 1, which has been implicated in lifespan extension by resveratrol. These results establish that a primary impact of resveratrol on human cell proliferation is the induction of low-level replicative stress., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)
- Published
- 2020
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19. Imipridone Anticancer Compounds Ectopically Activate the ClpP Protease and Represent a New Scaffold for Antibiotic Development.
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Jacques S, van der Sloot AM, C Huard C, Coulombe-Huntington J, Tsao S, Tollis S, Bertomeu T, Culp EJ, Pallant D, Cook MA, Bonneil E, Thibault P, Wright GD, and Tyers M
- Subjects
- Bacillus subtilis drug effects, Binding Sites, Conserved Sequence, Depsipeptides metabolism, Endopeptidase Clp chemistry, Escherichia coli drug effects, Escherichia coli Proteins chemistry, Escherichia coli Proteins metabolism, HEK293 Cells, Humans, Metalloendopeptidases metabolism, Protein Binding, Rifampin pharmacology, Staphylococcus aureus drug effects, Anti-Bacterial Agents pharmacology, Antineoplastic Agents pharmacology, Endopeptidase Clp metabolism, Escherichia coli Proteins agonists, Imidazoles pharmacology, Proteolysis, Pyridines pharmacology, Pyrimidines pharmacology
- Abstract
Systematic genetic interaction profiles can reveal the mechanisms-of-action of bioactive compounds. The imipridone ONC201, which is currently in cancer clinical trials, has been ascribed a variety of different targets. To investigate the genetic dependencies of imipridone action, we screened a genome-wide clustered regularly interspaced short palindromic repeats (CRISPR) knockout library in the presence of either ONC201 or its more potent analog ONC212. Loss of the mitochondrial matrix protease CLPP or the mitochondrial intermediate peptidase MIPEP conferred strong resistance to both compounds. Biochemical and surrogate genetic assays showed that impridones directly activate CLPP and that MIPEP is necessary for proteolytic maturation of CLPP into a catalytically competent form. Quantitative proteomic analysis of cells treated with ONC212 revealed degradation of many mitochondrial as well as nonmitochondrial proteins. Prompted by the conservation of ClpP from bacteria to humans, we found that the imipridones also activate ClpP from Escherichia coli , Bacillus subtilis , and Staphylococcus aureus in biochemical and genetic assays. ONC212 and acyldepsipeptide-4 (ADEP4), a known activator of bacterial ClpP, caused similar proteome-wide degradation profiles in S. aureus ONC212 suppressed the proliferation of a number of Gram-positive ( S. aureus , B. subtilis , and Enterococcus faecium ) and Gram-negative species ( E. coli and Neisseria gonorrhoeae ). Moreover, ONC212 enhanced the ability of rifampin to eradicate antibiotic-tolerant S. aureus persister cells. These results reveal the genetic dependencies of imipridone action in human cells and identify the imipridone scaffold as a new entry point for antibiotic development., (Copyright © 2020 Jacques et al.)
- Published
- 2020
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20. Robust cullin-RING ligase function is established by a multiplicity of poly-ubiquitylation pathways.
- Author
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Hill S, Reichermeier K, Scott DC, Samentar L, Coulombe-Huntington J, Izzi L, Tang X, Ibarra R, Bertomeu T, Moradian A, Sweredoski MJ, Caberoy N, Schulman BA, Sicheri F, Tyers M, and Kleiger G
- Subjects
- Genome, Human genetics, Humans, Hypoxia-Inducible Factor 1, alpha Subunit genetics, Mass Spectrometry, Polyubiquitin genetics, Signal Transduction genetics, Ubiquitination genetics, Ubiquitin-Conjugating Enzymes genetics, Ubiquitin-Protein Ligases genetics
- Abstract
The cullin-RING ligases (CRLs) form the major family of E3 ubiquitin ligases. The prototypic CRLs in yeast, called SCF enzymes, employ a single E2 enzyme, Cdc34, to build poly-ubiquitin chains required for degradation. In contrast, six different human E2 and E3 enzyme activities, including Cdc34 orthologs UBE2R1 and UBE2R2, appear to mediate SCF-catalyzed substrate polyubiquitylation in vitro. The combinatorial interplay of these enzymes raises questions about genetic buffering of SCFs in human cells and challenges the dogma that E3s alone determine substrate specificity. To enable the quantitative comparisons of SCF-dependent ubiquitylation reactions with physiological enzyme concentrations, mass spectrometry was employed to estimate E2 and E3 levels in cells. In combination with UBE2R1/2, the E2 UBE2D3 and the E3 ARIH1 both promoted SCF-mediated polyubiquitylation in a substrate-specific fashion. Unexpectedly, UBE2R2 alone had negligible ubiquitylation activity at physiological concentrations and the ablation of UBE2R1/2 had no effect on the stability of SCF substrates in cells. A genome-wide CRISPR screen revealed that an additional E2 enzyme, UBE2G1, buffers against the loss of UBE2R1/2. UBE2G1 had robust in vitro chain extension activity with SCF, and UBE2G1 knockdown in cells lacking UBE2R1/2 resulted in stabilization of the SCF substrates p27 and CYCLIN E as well as the CUL2-RING ligase substrate HIF1α. The results demonstrate the human SCF enzyme system is diversified by association with multiple catalytic enzyme partners., Competing Interests: SH, DS, LS, JC, LI, XT, RI, TB, AM, MS, NC, BS, MT, GK No competing interests declared, KR is an employee of the Genetech Biotechnology Compnay, FS is a founder and consultant for Repare Therapeutics, (© 2019, Hill et al.)
- Published
- 2019
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21. Mubritinib Targets the Electron Transport Chain Complex I and Reveals the Landscape of OXPHOS Dependency in Acute Myeloid Leukemia.
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Baccelli I, Gareau Y, Lehnertz B, Gingras S, Spinella JF, Corneau S, Mayotte N, Girard S, Frechette M, Blouin-Chagnon V, Leveillé K, Boivin I, MacRae T, Krosl J, Thiollier C, Lavallée VP, Kanshin E, Bertomeu T, Coulombe-Huntington J, St-Denis C, Bordeleau ME, Boucher G, Roux PP, Lemieux S, Tyers M, Thibault P, Hébert J, Marinier A, and Sauvageau G
- Subjects
- Animals, Biomarkers, Cell Line, Tumor, Cell Survival drug effects, Disease Models, Animal, Dose-Response Relationship, Drug, Female, Hematopoiesis drug effects, Humans, Leukemia, Myeloid, Acute drug therapy, Leukemia, Myeloid, Acute genetics, Leukemia, Myeloid, Acute mortality, Mice, Models, Biological, Receptor, ErbB-2 antagonists & inhibitors, Antineoplastic Agents pharmacology, Electron Transport Complex I antagonists & inhibitors, Leukemia, Myeloid, Acute metabolism, Oxazoles pharmacology, Oxidative Phosphorylation drug effects, Protein Kinase Inhibitors pharmacology, Triazoles pharmacology
- Abstract
To identify therapeutic targets in acute myeloid leukemia (AML), we chemically interrogated 200 sequenced primary specimens. Mubritinib, a known ERBB2 inhibitor, elicited strong anti-leukemic effects in vitro and in vivo. In the context of AML, mubritinib functions through ubiquinone-dependent inhibition of electron transport chain (ETC) complex I activity. Resistance to mubritinib characterized normal CD34
+ hematopoietic cells and chemotherapy-sensitive AMLs, which displayed transcriptomic hallmarks of hypoxia. Conversely, sensitivity correlated with mitochondrial function-related gene expression levels and characterized a large subset of chemotherapy-resistant AMLs with oxidative phosphorylation (OXPHOS) hyperactivity. Altogether, our work thus identifies an ETC complex I inhibitor and reveals the genetic landscape of OXPHOS dependency in AML., (Copyright © 2019 Elsevier Inc. All rights reserved.)- Published
- 2019
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22. The BioGRID interaction database: 2019 update.
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Oughtred R, Stark C, Breitkreutz BJ, Rust J, Boucher L, Chang C, Kolas N, O'Donnell L, Leung G, McAdam R, Zhang F, Dolma S, Willems A, Coulombe-Huntington J, Chatr-Aryamontri A, Dolinski K, and Tyers M
- Subjects
- Animals, CRISPR-Cas Systems, Data Curation, Drug Discovery, Genes, Humans, Mice, Protein Interaction Mapping, Databases, Factual
- Abstract
The Biological General Repository for Interaction Datasets (BioGRID: https://thebiogrid.org) is an open access database dedicated to the curation and archival storage of protein, genetic and chemical interactions for all major model organism species and humans. As of September 2018 (build 3.4.164), BioGRID contains records for 1 598 688 biological interactions manually annotated from 55 809 publications for 71 species, as classified by an updated set of controlled vocabularies for experimental detection methods. BioGRID also houses records for >700 000 post-translational modification sites. BioGRID now captures chemical interaction data, including chemical-protein interactions for human drug targets drawn from the DrugBank database and manually curated bioactive compounds reported in the literature. A new dedicated aspect of BioGRID annotates genome-wide CRISPR/Cas9-based screens that report gene-phenotype and gene-gene relationships. An extension of the BioGRID resource called the Open Repository for CRISPR Screens (ORCS) database (https://orcs.thebiogrid.org) currently contains over 500 genome-wide screens carried out in human or mouse cell lines. All data in BioGRID is made freely available without restriction, is directly downloadable in standard formats and can be readily incorporated into existing applications via our web service platforms. BioGRID data are also freely distributed through partner model organism databases and meta-databases.
- Published
- 2019
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23. Interactome evolution: insights from genome-wide analyses of protein-protein interactions.
- Author
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Ghadie MA, Coulombe-Huntington J, and Xia Y
- Subjects
- Evolution, Molecular, Genetic Fitness, Genetic Variation, Humans, Species Specificity, Genome-Wide Association Study, Protein Interaction Mapping methods, Protein Interaction Maps, Proteins genetics, Proteins metabolism
- Abstract
We highlight new evolutionary insights enabled by recent genome-wide studies on protein-protein interaction (PPI) networks ('interactomes'). While most PPIs are mediated by a single sequence region promoting or inhibiting interactions, many PPIs are mediated by multiple sequence regions acting cooperatively. Most PPIs perform important functions maintained by negative selection: we estimate that less than ∼10% of the human interactome is effectively neutral upon perturbation (i.e. 'junk' PPIs), and the rest are deleterious upon perturbation; interfacial sites evolve more slowly than other sites; many conserved PPIs show signatures of co-evolution at the interface; PPIs evolve more slowly than protein sequence. At the same time, many PPIs undergo rewiring during evolution for lineage-specific adaptation. Finally, chaperone-protein and host-pathogen interactomes are governed by distinct evolutionary principles., (Copyright © 2017 Elsevier Ltd. All rights reserved.)
- Published
- 2018
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24. A High-Resolution Genome-Wide CRISPR/Cas9 Viability Screen Reveals Structural Features and Contextual Diversity of the Human Cell-Essential Proteome.
- Author
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Bertomeu T, Coulombe-Huntington J, Chatr-Aryamontri A, Bourdages KG, Coyaud E, Raught B, Xia Y, and Tyers M
- Subjects
- Cell Line, Tumor, Cell Survival genetics, Gene Library, Humans, Proteomics methods, CRISPR-Cas Systems, Genes, Essential genetics, Genome-Wide Association Study methods, Proteome genetics, Proteome metabolism
- Abstract
To interrogate genes essential for cell growth, proliferation and survival in human cells, we carried out a genome-wide clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 screen in a B-cell lymphoma line using a custom extended-knockout (EKO) library of 278,754 single-guide RNAs (sgRNAs) that targeted 19,084 RefSeq genes, 20,852 alternatively spliced exons, and 3,872 hypothetical genes. A new statistical analysis tool called robust analytics and normalization for knockout screens (RANKS) identified 2,280 essential genes, 234 of which were unique. Individual essential genes were validated experimentally and linked to ribosome biogenesis and stress responses. Essential genes exhibited a bimodal distribution across 10 different cell lines, consistent with a continuous variation in essentiality as a function of cell type. Genes essential in more lines had more severe fitness defects and encoded the evolutionarily conserved structural cores of protein complexes, whereas genes essential in fewer lines formed context-specific modules and encoded subunits at the periphery of essential complexes. The essentiality of individual protein residues across the proteome correlated with evolutionary conservation, structural burial, modular domains, and protein interaction interfaces. Many alternatively spliced exons in essential genes were dispensable and were enriched for disordered regions. Fitness defects were observed for 44 newly evolved hypothetical reading frames. These results illuminate the contextual nature and evolution of essential gene functions in human cells., (Copyright © 2017 American Society for Microbiology.)
- Published
- 2017
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25. Revealing the Determinants of Widespread Alternative Splicing Perturbation in Cancer.
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Li Y, Sahni N, Pancsa R, McGrail DJ, Xu J, Hua X, Coulombe-Huntington J, Ryan M, Tychhon B, Sudhakar D, Hu L, Tyers M, Jiang X, Lin SY, Babu MM, and Yi S
- Subjects
- Gene Regulatory Networks, Genetic Heterogeneity, Humans, Internet, Mutation genetics, Neoplasms immunology, Phenotype, Alternative Splicing genetics, Neoplasms genetics
- Abstract
It is increasingly appreciated that alternative splicing plays a key role in generating functional specificity and diversity in cancer. However, the mechanisms by which cancer mutations perturb splicing remain unknown. Here, we developed a network-based strategy, DrAS-Net, to investigate more than 2.5 million variants across cancer types and link somatic mutations with cancer-specific splicing events. We identified more than 40,000 driver variant candidates and their 80,000 putative splicing targets deregulated in 33 cancer types and inferred their functional impact. Strikingly, tumors with splicing perturbations show reduced expression of immune system-related genes and increased expression of cell proliferation markers. Tumors harboring different mutations in the same gene often exhibit distinct splicing perturbations. Further stratification of 10,000 patients based on their mutation-splicing relationships identifies subtypes with distinct clinical features, including survival rates. Our work reveals how single-nucleotide changes can alter the repertoires of splicing isoforms, providing insights into oncogenic mechanisms for precision medicine., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)
- Published
- 2017
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26. CLMSVault: A Software Suite for Protein Cross-Linking Mass-Spectrometry Data Analysis and Visualization.
- Author
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Courcelles M, Coulombe-Huntington J, Cossette É, Gingras AC, Thibault P, and Tyers M
- Subjects
- Algorithms, Amino Acid Sequence, Binding Sites, Humans, Mass Spectrometry, Models, Molecular, Protein Binding, Protein Interaction Domains and Motifs, Protein Interaction Mapping, Protein Structure, Secondary, Cross-Linking Reagents chemistry, Peptides chemistry, Software, Ubiquitin chemistry, Ubiquitin-Conjugating Enzymes chemistry
- Abstract
Protein cross-linking mass spectrometry (CL-MS) enables the sensitive detection of protein interactions and the inference of protein complex topology. The detection of chemical cross-links between protein residues can identify intra- and interprotein contact sites or provide physical constraints for molecular modeling of protein structure. Recent innovations in cross-linker design, sample preparation, mass spectrometry, and software tools have significantly improved CL-MS approaches. Although a number of algorithms now exist for the identification of cross-linked peptides from mass spectral data, a dearth of user-friendly analysis tools represent a practical bottleneck to the broad adoption of the approach. To facilitate the analysis of CL-MS data, we developed CLMSVault, a software suite designed to leverage existing CL-MS algorithms and provide intuitive and flexible tools for cross-platform data interpretation. CLMSVault stores and combines complementary information obtained from different cross-linkers and search algorithms. CLMSVault provides filtering, comparison, and visualization tools to support CL-MS analyses and includes a workflow for label-free quantification of cross-linked peptides. An embedded 3D viewer enables the visualization of quantitative data and the mapping of cross-linked sites onto PDB structural models. We demonstrate the application of CLMSVault for the analysis of a noncovalent Cdc34-ubiquitin protein complex cross-linked under different conditions. CLMSVault is open-source software (available at https://gitlab.com/courcelm/clmsvault.git ), and a live demo is available at http://democlmsvault.tyerslab.com/ .
- Published
- 2017
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27. Network Centrality Analysis in Fungi Reveals Complex Regulation of Lost and Gained Genes.
- Author
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Coulombe-Huntington J and Xia Y
- Subjects
- Evolution, Molecular, Gene Duplication, Gene Expression Regulation, Fungal, Genes, Fungal, Genome, Fungal, Genome-Wide Association Study, Phylogeny, Transcription Factors metabolism, Gene Regulatory Networks, Saccharomyces cerevisiae genetics, Schizosaccharomyces genetics
- Abstract
Gene gain and loss shape both proteomes and the networks they form. The increasing availability of closely related sequenced genomes and of genome-wide network data should enable a better understanding of the evolutionary forces driving gene gain, gene loss and evolutionary network rewiring. Using orthology mappings across 23 ascomycete fungi genomes, we identified proteins that were lost, gained or universally conserved across the tree, enabling us to compare genes across all stages of their life-cycle. Based on a collection of genome-wide network and gene expression datasets from baker's yeast, as well as a few from fission yeast, we found that gene loss is more strongly associated with network and expression features of closely related species than that of distant species, consistent with the evolutionary modulation of gene loss propensity through network rewiring. We also discovered that lost and gained genes, as compared to universally conserved "core" genes, have more regulators, more complex expression patterns and are much more likely to encode for transcription factors. Finally, we found that the relative rate of network integration of new genes into the different types of networks agrees with experimentally measured rates of network rewiring. This systems-level view of the life-cycle of eukaryotic genes suggests that the gain and loss of genes is tightly coupled to the gain and loss of network interactions, that lineage-specific adaptations drive regulatory complexity and that the relative rates of integration of new genes are consistent with network rewiring rates., Competing Interests: The authors have declared that no competing interests exist.
- Published
- 2017
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28. RNA-Seq identifies SPGs as a ventral skeletal patterning cue in sea urchins.
- Author
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Piacentino ML, Zuch DT, Fishman J, Rose S, Speranza EE, Li C, Yu J, Chung O, Ramachandran J, Ferrell P, Patel V, Reyna A, Hameeduddin H, Chaves J, Hewitt FB, Bardot E, Lee D, Core AB, Hogan JD, Keenan JL, Luo L, Coulombe-Huntington J, Blute TA, Oleinik E, Ibn-Salem J, Poustka AJ, and Bradham CA
- Subjects
- Animals, Body Patterning drug effects, Cation Transport Proteins metabolism, Cell Differentiation drug effects, Ectoderm drug effects, Ectoderm enzymology, Embryo, Nonmammalian drug effects, Embryo, Nonmammalian metabolism, Gene Expression Regulation, Developmental drug effects, Mesoderm cytology, Models, Biological, Nickel toxicity, Sea Urchins drug effects, Signal Transduction drug effects, Vascular Endothelial Growth Factor A metabolism, p38 Mitogen-Activated Protein Kinases antagonists & inhibitors, p38 Mitogen-Activated Protein Kinases metabolism, Body Patterning genetics, Proteoglycans metabolism, Sea Urchins embryology, Sea Urchins genetics, Sequence Analysis, RNA methods, Sulfates metabolism
- Abstract
The sea urchin larval skeleton offers a simple model for formation of developmental patterns. The calcium carbonate skeleton is secreted by primary mesenchyme cells (PMCs) in response to largely unknown patterning cues expressed by the ectoderm. To discover novel ectodermal cues, we performed an unbiased RNA-Seq-based screen and functionally tested candidates; we thereby identified several novel skeletal patterning cues. Among these, we show that SLC26a2/7 is a ventrally expressed sulfate transporter that promotes a ventral accumulation of sulfated proteoglycans, which is required for ventral PMC positioning and skeletal patterning. We show that the effects of SLC perturbation are mimicked by manipulation of either external sulfate levels or proteoglycan sulfation. These results identify novel skeletal patterning genes and demonstrate that ventral proteoglycan sulfation serves as a positional cue for sea urchin skeletal patterning., (© 2016. Published by The Company of Biologists Ltd.)
- Published
- 2016
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29. Widespread Expansion of Protein Interaction Capabilities by Alternative Splicing.
- Author
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Yang X, Coulombe-Huntington J, Kang S, Sheynkman GM, Hao T, Richardson A, Sun S, Yang F, Shen YA, Murray RR, Spirohn K, Begg BE, Duran-Frigola M, MacWilliams A, Pevzner SJ, Zhong Q, Wanamaker SA, Tam S, Ghamsari L, Sahni N, Yi S, Rodriguez MD, Balcha D, Tan G, Costanzo M, Andrews B, Boone C, Zhou XJ, Salehi-Ashtiani K, Charloteaux B, Chen AA, Calderwood MA, Aloy P, Roth FP, Hill DE, Iakoucheva LM, Xia Y, and Vidal M
- Subjects
- Animals, Cloning, Molecular, Evolution, Molecular, Humans, Models, Molecular, Open Reading Frames, Protein Interaction Domains and Motifs, Protein Interaction Maps, Proteome analysis, Alternative Splicing, Protein Isoforms metabolism, Proteome metabolism
- Abstract
While alternative splicing is known to diversify the functional characteristics of some genes, the extent to which protein isoforms globally contribute to functional complexity on a proteomic scale remains unknown. To address this systematically, we cloned full-length open reading frames of alternatively spliced transcripts for a large number of human genes and used protein-protein interaction profiling to functionally compare hundreds of protein isoform pairs. The majority of isoform pairs share less than 50% of their interactions. In the global context of interactome network maps, alternative isoforms tend to behave like distinct proteins rather than minor variants of each other. Interaction partners specific to alternative isoforms tend to be expressed in a highly tissue-specific manner and belong to distinct functional modules. Our strategy, applicable to other functional characteristics, reveals a widespread expansion of protein interaction capabilities through alternative splicing and suggests that many alternative "isoforms" are functionally divergent (i.e., "functional alloforms")., (Copyright © 2016 Elsevier Inc. All rights reserved.)
- Published
- 2016
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30. Widespread macromolecular interaction perturbations in human genetic disorders.
- Author
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Sahni N, Yi S, Taipale M, Fuxman Bass JI, Coulombe-Huntington J, Yang F, Peng J, Weile J, Karras GI, Wang Y, Kovács IA, Kamburov A, Krykbaeva I, Lam MH, Tucker G, Khurana V, Sharma A, Liu YY, Yachie N, Zhong Q, Shen Y, Palagi A, San-Miguel A, Fan C, Balcha D, Dricot A, Jordan DM, Walsh JM, Shah AA, Yang X, Stoyanova AK, Leighton A, Calderwood MA, Jacob Y, Cusick ME, Salehi-Ashtiani K, Whitesell LJ, Sunyaev S, Berger B, Barabási AL, Charloteaux B, Hill DE, Hao T, Roth FP, Xia Y, Walhout AJM, Lindquist S, and Vidal M
- Subjects
- DNA-Binding Proteins genetics, DNA-Binding Proteins metabolism, Genome-Wide Association Study, Humans, Open Reading Frames, Protein Folding, Protein Stability, Disease genetics, Mutation, Missense, Protein Interaction Maps, Proteins genetics, Proteins metabolism
- Abstract
How disease-associated mutations impair protein activities in the context of biological networks remains mostly undetermined. Although a few renowned alleles are well characterized, functional information is missing for over 100,000 disease-associated variants. Here we functionally profile several thousand missense mutations across a spectrum of Mendelian disorders using various interaction assays. The majority of disease-associated alleles exhibit wild-type chaperone binding profiles, suggesting they preserve protein folding or stability. While common variants from healthy individuals rarely affect interactions, two-thirds of disease-associated alleles perturb protein-protein interactions, with half corresponding to "edgetic" alleles affecting only a subset of interactions while leaving most other interactions unperturbed. With transcription factors, many alleles that leave protein-protein interactions intact affect DNA binding. Different mutations in the same gene leading to different interaction profiles often result in distinct disease phenotypes. Thus disease-associated alleles that perturb distinct protein activities rather than grossly affecting folding and stability are relatively widespread., (Copyright © 2015 Elsevier Inc. All rights reserved.)
- Published
- 2015
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31. A proteome-scale map of the human interactome network.
- Author
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Rolland T, Taşan M, Charloteaux B, Pevzner SJ, Zhong Q, Sahni N, Yi S, Lemmens I, Fontanillo C, Mosca R, Kamburov A, Ghiassian SD, Yang X, Ghamsari L, Balcha D, Begg BE, Braun P, Brehme M, Broly MP, Carvunis AR, Convery-Zupan D, Corominas R, Coulombe-Huntington J, Dann E, Dreze M, Dricot A, Fan C, Franzosa E, Gebreab F, Gutierrez BJ, Hardy MF, Jin M, Kang S, Kiros R, Lin GN, Luck K, MacWilliams A, Menche J, Murray RR, Palagi A, Poulin MM, Rambout X, Rasla J, Reichert P, Romero V, Ruyssinck E, Sahalie JM, Scholz A, Shah AA, Sharma A, Shen Y, Spirohn K, Tam S, Tejeda AO, Wanamaker SA, Twizere JC, Vega K, Walsh J, Cusick ME, Xia Y, Barabási AL, Iakoucheva LM, Aloy P, De Las Rivas J, Tavernier J, Calderwood MA, Hill DE, Hao T, Roth FP, and Vidal M
- Subjects
- Animals, Databases, Protein, Genome-Wide Association Study, Humans, Mice, Neoplasms metabolism, Protein Interaction Maps, Proteome metabolism
- Abstract
Just as reference genome sequences revolutionized human genetics, reference maps of interactome networks will be critical to fully understand genotype-phenotype relationships. Here, we describe a systematic map of ?14,000 high-quality human binary protein-protein interactions. At equal quality, this map is ?30% larger than what is available from small-scale studies published in the literature in the last few decades. While currently available information is highly biased and only covers a relatively small portion of the proteome, our systematic map appears strikingly more homogeneous, revealing a "broader" human interactome network than currently appreciated. The map also uncovers significant interconnectivity between known and candidate cancer gene products, providing unbiased evidence for an expanded functional cancer landscape, while demonstrating how high-quality interactome models will help "connect the dots" of the genomic revolution.
- Published
- 2014
- Full Text
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32. The Sam68 STAR RNA-binding protein regulates mTOR alternative splicing during adipogenesis.
- Author
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Huot MÉ, Vogel G, Zabarauskas A, Ngo CT, Coulombe-Huntington J, Majewski J, and Richard S
- Subjects
- Adaptor Proteins, Signal Transducing physiology, Animals, Body Composition genetics, Energy Metabolism genetics, Mechanistic Target of Rapamycin Complex 1, Mice, Multiprotein Complexes, NIH 3T3 Cells, Phenotype, Proteins metabolism, Proteins physiology, RNA-Binding Proteins physiology, Signal Transduction genetics, Adaptor Proteins, Signal Transducing genetics, Adipogenesis genetics, Alternative Splicing, RNA-Binding Proteins genetics, TOR Serine-Threonine Kinases physiology
- Abstract
We report that mice ablated for the Sam68 RNA-binding protein exhibit a lean phenotype as a result of increased energy expenditure, decreased commitment to early adipocyte progenitors, and defects in adipogenic differentiation. The Sam68(-/-) mice were protected from obesity, insulin resistance, and glucose intolerance induced with a high-fat diet. To identify the alternative splice events regulated by Sam68, genome-wide exon usage profiling in white adipose tissue was performed. Adipocytes from Sam68(-/-) mice retained intron 5 within the mTOR transcript introducing a premature termination codon, leading to an unstable mRNA. Consequently, Sam68-depleted cells had reduced mTOR levels resulting in lower levels of insulin-stimulated S6 and Akt phosphorylation leading to defects in adipogenesis, and this defect was rescued by the exogenous expression of full-length mTOR. Sam68 bound intronic splice elements within mTOR intron 5 required for the usage of the 5' splice site. We propose that Sam68 regulates alternative splicing during adipogenesis., (Copyright © 2012 Elsevier Inc. All rights reserved.)
- Published
- 2012
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33. Regulatory network structure as a dominant determinant of transcription factor evolutionary rate.
- Author
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Coulombe-Huntington J and Xia Y
- Subjects
- Codon, Fungal Proteins genetics, Fungal Proteins metabolism, Genomics, Mutation, Protein Interaction Maps genetics, Saccharomyces genetics, Saccharomyces metabolism, Transcription Factors metabolism, Evolution, Molecular, Gene Regulatory Networks, Transcription Factors genetics
- Abstract
The evolution of transcriptional regulatory networks has thus far mostly been studied at the level of cis-regulatory elements. To gain a complete understanding of regulatory network evolution we must also study the evolutionary role of trans-factors, such as transcription factors (TFs). Here, we systematically assess genomic and network-level determinants of TF evolutionary rate in yeast, and how they compare to those of generic proteins, while carefully controlling for differences of the TF protein set, such as expression level. We found significantly distinct trends relating TF evolutionary rate to mRNA expression level, codon adaptation index, the evolutionary rate of physical interaction partners, and, confirming previous reports, to protein-protein interaction degree and regulatory in-degree. We discovered that for TFs, the dominant determinants of evolutionary rate lie in the structure of the regulatory network, such as the median evolutionary rate of target genes and the fraction of species-specific target genes. Decomposing the regulatory network by edge sign, we found that this modular evolution of TFs and their targets is limited to activating regulatory relationships. We show that fast evolving TFs tend to regulate other TFs and niche-specific processes and that their targets show larger evolutionary expression changes than targets of other TFs. We also show that the positive trend relating TF regulatory in-degree and evolutionary rate is likely related to the species-specificity of the transcriptional regulation modules. Finally, we discuss likely causes for TFs' different evolutionary relationship to the physical interaction network, such as the prevalence of transient interactions in the TF subnetwork. This work suggests that positive and negative regulatory networks follow very different evolutionary rules, and that transcription factor evolution is best understood at a network- or systems-level.
- Published
- 2012
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34. Fine-scale variation and genetic determinants of alternative splicing across individuals.
- Author
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Coulombe-Huntington J, Lam KC, Dias C, and Majewski J
- Subjects
- Genetic Predisposition to Disease, Humans, Oligonucleotide Array Sequence Analysis, Polymerase Chain Reaction, Polymorphism, Single Nucleotide, RNA, Messenger genetics, Alternative Splicing
- Abstract
Recently, thanks to the increasing throughput of new technologies, we have begun to explore the full extent of alternative pre-mRNA splicing (AS) in the human transcriptome. This is unveiling a vast layer of complexity in isoform-level expression differences between individuals. We used previously published splicing sensitive microarray data from lymphoblastoid cell lines to conduct an in-depth analysis on splicing efficiency of known and predicted exons. By combining publicly available AS annotation with a novel algorithm designed to search for AS, we show that many real AS events can be detected within the usually unexploited, speculative majority of the array and at significance levels much below standard multiple-testing thresholds, demonstrating that the extent of cis-regulated differential splicing between individuals is potentially far greater than previously reported. Specifically, many genes show subtle but significant genetically controlled differences in splice-site usage. PCR validation shows that 42 out of 58 (72%) candidate gene regions undergo detectable AS, amounting to the largest scale validation of isoform eQTLs to date. Targeted sequencing revealed a likely causative SNP in most validated cases. In all 17 incidences where a SNP affected a splice-site region, in silico splice-site strength modeling correctly predicted the direction of the micro-array and PCR results. In 13 other cases, we identified likely causative SNPs disrupting predicted splicing enhancers. Using Fst and REHH analysis, we uncovered significant evidence that 2 putative causative SNPs have undergone recent positive selection. We verified the effect of five SNPs using in vivo minigene assays. This study shows that splicing differences between individuals, including quantitative differences in isoform ratios, are frequent in human populations and that causative SNPs can be identified using in silico predictions. Several cases affected disease-relevant genes and it is likely some of these differences are involved in phenotypic diversity and susceptibility to complex diseases., Competing Interests: The authors have declared that no competing interests exist.
- Published
- 2009
- Full Text
- View/download PDF
35. Comparison of Affymetrix Gene Array with the Exon Array shows potential application for detection of transcript isoform variation.
- Author
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Ha KCh, Coulombe-Huntington J, and Majewski J
- Subjects
- Gene Expression Profiling, Humans, Reproducibility of Results, Alternative Splicing genetics, Exons genetics, Oligonucleotide Array Sequence Analysis methods, RNA, Messenger analysis, RNA, Messenger genetics
- Abstract
Background: The emergence of isoform-sensitive microarrays has helped fuel in-depth studies of the human transcriptome. The Affymetrix GeneChip Human Exon 1.0 ST Array (Exon Array) has been previously shown to be effective in profiling gene expression at the isoform level. More recently, the Affymetrix GeneChip Human Gene 1.0 ST Array (Gene Array) has been released for measuring gene expression and interestingly contains a large subset of probes from the Exon Array. Here, we explore the potential of using Gene Array probes to assess expression variation at the sub-transcript level. Utilizing datasets of the high quality Microarray Quality Control (MAQC) RNA samples previously assayed on the Exon Array and Gene Array, we compare the expression measurements of the two platforms to determine the performance of the Gene Array in detecting isoform variations., Results: Overall, we show that the Gene Array is comparable to the Exon Array in making gene expression calls. Moreover, to examine expression of different isoforms, we modify the Gene Array probe set definition file to enable summarization of probe intensity values at the exon level and show that the expression profiles between the two platforms are also highly correlated. Next, expression calls of previously known differentially spliced genes were compared and also show concordant results. Splicing index analysis, representing estimates of exon inclusion levels, shows a lower but good correlation between platforms. As the Gene Array contains a significant subset of probes from the Exon Array, we note that, in comparison, the Gene Array overlaps with fewer but still a high proportion of splicing events annotated in the Known Alt Events UCSC track, with abundant coverage of cassette exons. We discuss the ability of the Gene Array to detect alternative splicing and isoform variation and address its limitations., Conclusion: The Gene Array is an effective expression profiling tool at gene and exon expression level, the latter made possible by probe set annotation modifications. We demonstrate that the Gene Array is capable of detecting alternative splicing and isoform variation. As expected, in comparison to the Exon Array, it is limited by reduced gene content coverage and is not able to detect as wide a range of alternative splicing events. However, for the events that can be monitored by both platforms, we estimate that the selectivity and sensitivity levels are comparable. We hope our findings will shed light on the potential extension of the Gene Array to detect alternative splicing. It should be particularly suitable for researchers primarily interested in gene expression analysis, but who may be willing to look for splicing and isoform differences within their dataset. However, we do not suggest it to be an equivalent substitute to the more comprehensive Exon Array.
- Published
- 2009
- Full Text
- View/download PDF
36. Intron loss and gain in Drosophila.
- Author
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Coulombe-Huntington J and Majewski J
- Subjects
- Alternative Splicing genetics, Animals, Conserved Sequence, Genes, Insect, Models, Genetic, Multivariate Analysis, RNA Splice Sites genetics, Selection, Genetic, Drosophila melanogaster genetics, Evolution, Molecular, Introns genetics
- Abstract
Although introns were first discovered almost 30 years ago, their evolutionary origin remains elusive. In this work, we used multispecies whole-genome alignments to map Drosophila melanogaster introns onto 10 other fully sequenced Drosophila genomes. We were able to find 1,944 sites where an intron was missing in one or more species. We show that for most (>80%) of these cases, there is no leftover intronic sequence or any missing exonic sequence, indicating exact intron loss or gain events. We used parsimony to classify these differences as 1,754 intron loss events and 213 gain events. We show that lost and gained introns are significantly shorter than average and flanked by longer than average exons. They also display quite distinct phase distributions and show greater than average similarity between the 5' splice site and its 3' partner splice site. Introns that have been lost in one or more species evolve faster than other introns, occur in slowly evolving genes, and are found adjacent to each other more often than would be expected for independent single losses. Our results support the cDNA recombination mechanism of intron loss, suggest that selective pressures affect site-specific loss rates, and show conclusively that intron gain has occurred within the Drosophila lineage, solidifying the "introns-middle" hypothesis and providing some hints about the gain mechanism.
- Published
- 2007
- Full Text
- View/download PDF
37. Characterization of intron loss events in mammals.
- Author
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Coulombe-Huntington J and Majewski J
- Subjects
- Animals, Base Sequence, DNA, Dogs, Genome, Human, Glyceraldehyde-3-Phosphate Dehydrogenases genetics, Humans, Mice, Molecular Sequence Data, Phylogeny, Rats, Sequence Alignment, Evolution, Molecular, Gene Deletion, Introns genetics, Mammals genetics
- Abstract
The exon/intron structure of eukaryotic genes differs extensively across species, but the mechanisms and relative rates of intron loss and gain are still poorly understood. Here, we used whole-genome sequence alignments of human, mouse, rat, and dog to perform a genome-wide analysis of intron loss and gain events in >17,000 mammalian genes. We found no evidence for intron gain and 122 cases of intron loss, most of which occurred within the rodent lineage. The majority (68%) of the deleted introns were extremely small (<150 bp), significantly smaller than average. The intron losses occurred almost exclusively within highly expressed, housekeeping genes, supporting the hypothesis that intron loss is mediated via germline recombination of genomic DNA with intronless cDNA. This study constitutes the largest scale analysis for intron dynamics in vertebrates to date and allows us to confirm and extend several hypotheses previously based on much smaller samples. Our results in mammals show that intron gain has not been a factor in the evolution of gene structure during the past 95 Myr and has likely been restricted to more ancient history.
- Published
- 2007
- Full Text
- View/download PDF
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