Publications by authors named "Lawrence E Heisler"

34 Publications

Prospective observational study and serosurvey of SARS-CoV-2 infection in asymptomatic healthcare workers at a Canadian tertiary care center.

PLoS One 2021 16;16(2):e0247258. Epub 2021 Feb 16.

University Health Network, Toronto, Ontario, Canada.

Health care workers (HCWs) are at higher risk for SARS-CoV-2 infection and may play a role in transmitting the infection to vulnerable patients and members of the community. This is particularly worrisome in the context of asymptomatic infection. We performed a cross-sectional study looking at asymptomatic SARS-CoV-2 infection in HCWs. We screened asymptomatic HCWs for SARS-CoV-2 via PCR. Complementary viral genome sequencing was performed on positive swab specimens. A seroprevalence analysis was also performed using multiple assays. Asymptomatic health care worker cohorts had a combined swab positivity rate of 29/5776 (0.50%, 95%CI 0.32-0.75) relative to a comparative cohort of symptomatic HCWs, where 54/1597 (3.4%) tested positive for SARS-CoV-2 (ratio of symptomatic to asymptomatic 6.8:1). SARS-CoV-2 seroprevalence among 996 asymptomatic HCWs with no prior known exposure to SARS-CoV-2 was 1.4-3.4%, depending on assay. A novel in-house Coronavirus protein microarray showed differing SARS-CoV-2 protein reactivities and helped define likely true positives vs. suspected false positives. Our study demonstrates the utility of routine screening of asymptomatic HCWs, which may help to identify a significant proportion of infections.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0247258PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7886177PMC
February 2021

Landscape of somatic single nucleotide variants and indels in colorectal cancer and impact on survival.

Nat Commun 2020 07 20;11(1):3644. Epub 2020 Jul 20.

Department of Preventive Medicine, USC Norris Comprehensive Cancer Center, Keck School of Medicine, University of Southern California, 1441 Eastlake Avenue, Los Angeles, CA, 90089, USA.

Colorectal cancer (CRC) is a biologically heterogeneous disease. To characterize its mutational profile, we conduct targeted sequencing of 205 genes for 2,105 CRC cases with survival data. Our data shows several findings in addition to enhancing the existing knowledge of CRC. We identify PRKCI, SPZ1, MUTYH, MAP2K4, FETUB, and TGFBR2 as additional genes significantly mutated in CRC. We find that among hypermutated tumors, an increased mutation burden is associated with improved CRC-specific survival (HR = 0.42, 95% CI: 0.21-0.82). Mutations in TP53 are associated with poorer CRC-specific survival, which is most pronounced in cases carrying TP53 mutations with predicted 0% transcriptional activity (HR = 1.53, 95% CI: 1.21-1.94). Furthermore, we observe differences in mutational frequency of several genes and pathways by tumor location, stage, and sex. Overall, this large study provides deep insights into somatic mutations in CRC, and their potential relationships with survival and tumor features.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-020-17386-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7371703PMC
July 2020

Genome-wide germline correlates of the epigenetic landscape of prostate cancer.

Nat Med 2019 10 7;25(10):1615-1626. Epub 2019 Oct 7.

Ontario Institute for Cancer Research, Toronto, Ontario, Canada.

Oncogenesis is driven by germline, environmental and stochastic factors. It is unknown how these interact to produce the molecular phenotypes of tumors. We therefore quantified the influence of germline polymorphisms on the somatic epigenome of 589 localized prostate tumors. Predisposition risk loci influence a tumor's epigenome, uncovering a mechanism for cancer susceptibility. We identified and validated 1,178 loci associated with altered methylation in tumoral but not nonmalignant tissue. These tumor methylation quantitative trait loci influence chromatin structure, as well as RNA and protein abundance. One prominent tumor methylation quantitative trait locus is associated with AKT1 expression and is predictive of relapse after definitive local therapy in both discovery and validation cohorts. These data reveal intricate crosstalk between the germ line and the epigenome of primary tumors, which may help identify germline biomarkers of aggressive disease to aid patient triage and optimize the use of more invasive or expensive diagnostic assays.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41591-019-0579-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7418214PMC
October 2019

The Paf1 Complex Broadly Impacts the Transcriptome of .

Genetics 2019 07 15;212(3):711-728. Epub 2019 May 15.

Department of Biological Sciences, University of Pittsburgh, Pennsylvania 15260

The Polymerase Associated Factor 1 complex (Paf1C) is a multifunctional regulator of eukaryotic gene expression important for the coordination of transcription with chromatin modification and post-transcriptional processes. In this study, we investigated the extent to which the functions of Paf1C combine to regulate the transcriptome. While previous studies focused on the roles of Paf1C in controlling mRNA levels, here, we took advantage of a genetic background that enriches for unstable transcripts, and demonstrate that deletion of affects all classes of Pol II transcripts including multiple classes of noncoding RNAs (ncRNAs). By conducting a differential expression analysis independent of gene annotations, we found that Paf1 positively and negatively regulates antisense transcription at multiple loci. Comparisons with nascent transcript data revealed that many, but not all, changes in RNA levels detected by our analysis are due to changes in transcription instead of post-transcriptional events. To investigate the mechanisms by which Paf1 regulates protein-coding genes, we focused on genes involved in iron and phosphate homeostasis, which were differentially affected by deletion. Our results indicate that Paf1 stimulates phosphate gene expression through a mechanism that is independent of any individual Paf1C-dependent histone modification. In contrast, the inhibition of iron gene expression by Paf1 correlates with a defect in H3 K36 trimethylation. Finally, we showed that one iron regulon gene, , is coordinately controlled by Paf1 and transcription of upstream noncoding DNA. Together, these data identify roles for Paf1C in controlling both coding and noncoding regions of the yeast genome.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1534/genetics.119.302262DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6614894PMC
July 2019

The Proteogenomic Landscape of Curable Prostate Cancer.

Cancer Cell 2019 03;35(3):414-427.e6

Department of Medical Biophysics, University of Toronto, Toronto, ON M5G 1L7, Canada; Ontario Institute for Cancer Research, Toronto, ON M5G 0A3, Canada; Department of Pharmacology and Toxicology, University of Toronto, Toronto, ON M5S 1A8, Canada; Department of Human Genetics, University of California, 12-109 CHS, 10833 Le Conte Avenue, Los Angeles, CA 90095, USA; Department of Urology, University of California, Los Angeles, CA 90024, USA; Jonsson Comprehensive Cancer Centre, University of California, Los Angeles, CA 90024, USA; Institute for Precision Health, University of California, Los Angeles, CA 90024, USA. Electronic address:

DNA sequencing has identified recurrent mutations that drive the aggressiveness of prostate cancers. Surprisingly, the influence of genomic, epigenomic, and transcriptomic dysregulation on the tumor proteome remains poorly understood. We profiled the genomes, epigenomes, transcriptomes, and proteomes of 76 localized, intermediate-risk prostate cancers. We discovered that the genomic subtypes of prostate cancer converge on five proteomic subtypes, with distinct clinical trajectories. ETS fusions, the most common alteration in prostate tumors, affect different genes and pathways in the proteome and transcriptome. Globally, mRNA abundance changes explain only ∼10% of protein abundance variability. As a result, prognostic biomarkers combining genomic or epigenomic features with proteomic ones significantly outperform biomarkers comprised of a single data type.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ccell.2019.02.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6511374PMC
March 2019

Widespread and Functional RNA Circularization in Localized Prostate Cancer.

Cell 2019 02;176(4):831-843.e22

Research Centre of CHU de Québec-Université Laval, Québec City, Québec, Canada.

The cancer transcriptome is remarkably complex, including low-abundance transcripts, many not polyadenylated. To fully characterize the transcriptome of localized prostate cancer, we performed ultra-deep total RNA-seq on 144 tumors with rich clinical annotation. This revealed a linear transcriptomic subtype associated with the aggressive intraductal carcinoma sub-histology and a fusion profile that differentiates localized from metastatic disease. Analysis of back-splicing events showed widespread RNA circularization, with the average tumor expressing 7,232 circular RNAs (circRNAs). The degree of circRNA production was correlated to disease progression in multiple patient cohorts. Loss-of-function screening identified 11.3% of highly abundant circRNAs as essential for cell proliferation; for ∼90% of these, their parental linear transcripts were not essential. Individual circRNAs can have distinct functions, with circCSNK1G3 promoting cell growth by interacting with miR-181. These data advocate for adoption of ultra-deep RNA-seq without poly-A selection to interrogate both linear and circular transcriptomes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cell.2019.01.025DOI Listing
February 2019

Molecular landmarks of tumor hypoxia across cancer types.

Nat Genet 2019 02 14;51(2):308-318. Epub 2019 Jan 14.

Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada.

Many primary-tumor subregions have low levels of molecular oxygen, termed hypoxia. Hypoxic tumors are at elevated risk for local failure and distant metastasis, but the molecular hallmarks of tumor hypoxia remain poorly defined. To fill this gap, we quantified hypoxia in 8,006 tumors across 19 tumor types. In ten tumor types, hypoxia was associated with elevated genomic instability. In all 19 tumor types, hypoxic tumors exhibited characteristic driver-mutation signatures. We observed widespread hypoxia-associated dysregulation of microRNAs (miRNAs) across cancers and functionally validated miR-133a-3p as a hypoxia-modulated miRNA. In localized prostate cancer, hypoxia was associated with elevated rates of chromothripsis, allelic loss of PTEN and shorter telomeres. These associations are particularly enriched in polyclonal tumors, representing a constellation of features resembling tumor nimbosus, an aggressive cellular phenotype. Overall, this work establishes that tumor hypoxia may drive aggressive molecular features across cancers and shape the clinical trajectory of individual tumors.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41588-018-0318-2DOI Listing
February 2019

The Evolutionary Landscape of Localized Prostate Cancers Drives Clinical Aggression.

Cell 2018 05 19;173(4):1003-1013.e15. Epub 2018 Apr 19.

Informatics and Biocomputing Program, Ontario Institute for Cancer Research, Toronto, ON M5G 1L7, Canada; Department of Medical Biophysics, University of Toronto, Toronto, ON M5G 1L7, Canada; Department of Pharmacology and Toxicology, University of Toronto, Toronto, ON M5S 1A8, Canada. Electronic address:

The majority of newly diagnosed prostate cancers are slow growing, with a long natural life history. Yet a subset can metastasize with lethal consequences. We reconstructed the phylogenies of 293 localized prostate tumors linked to clinical outcome data. Multiple subclones were detected in 59% of patients, and specific subclonal architectures associate with adverse clinicopathological features. Early tumor development is characterized by point mutations and deletions followed by later subclonal amplifications and changes in trinucleotide mutational signatures. Specific genes are selectively mutated prior to or following subclonal diversification, including MTOR, NKX3-1, and RB1. Patients with low-risk monoclonal tumors rarely relapse after primary therapy (7%), while those with high-risk polyclonal tumors frequently do (61%). The presence of multiple subclones in an index biopsy may be necessary, but not sufficient, for relapse of localized prostate cancer, suggesting that evolution-aware biomarkers should be studied in prospective studies of low-risk tumors suitable for active surveillance.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cell.2018.03.029DOI Listing
May 2018

Mitochondrial mutations drive prostate cancer aggression.

Nat Commun 2017 09 22;8(1):656. Epub 2017 Sep 22.

Informatics and Biocomputing Program, Ontario Institute for Cancer Research, Toronto, ON, Canada, M5G 0A3.

Nuclear mutations are well known to drive tumor incidence, aggression and response to therapy. By contrast, the frequency and roles of mutations in the maternally inherited mitochondrial genome are poorly understood. Here we sequence the mitochondrial genomes of 384 localized prostate cancer patients, and identify a median of one mitochondrial single-nucleotide variant (mtSNV) per patient. Some of these mtSNVs occur in recurrent mutational hotspots and associate with aggressive disease. Younger patients have fewer mtSNVs than those who diagnosed at an older age. We demonstrate strong links between mitochondrial and nuclear mutational profiles, with co-occurrence between specific mutations. For example, certain control region mtSNVs co-occur with gain of the MYC oncogene, and these mutations are jointly associated with patient survival. These data demonstrate frequent mitochondrial mutation in prostate cancer, and suggest interplay between nuclear and mitochondrial mutational profiles in prostate cancer.In prostate cancer, the role of mutations in the maternally-inherited mitochondrial genome are not well known. Here, the authors demonstrate frequent, age-dependent mitochondrial mutation in prostate cancer. Strong links between mitochondrial and nuclear mutational profiles are associated with clinical aggressivity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-017-00377-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5610241PMC
September 2017

Genomic hallmarks of localized, non-indolent prostate cancer.

Nature 2017 01 9;541(7637):359-364. Epub 2017 Jan 9.

Genome Technologies Program, Ontario Institute for Cancer Research, Toronto, Canada.

Prostate tumours are highly variable in their response to therapies, but clinically available prognostic factors can explain only a fraction of this heterogeneity. Here we analysed 200 whole-genome sequences and 277 additional whole-exome sequences from localized, non-indolent prostate tumours with similar clinical risk profiles, and carried out RNA and methylation analyses in a subset. These tumours had a paucity of clinically actionable single nucleotide variants, unlike those seen in metastatic disease. Rather, a significant proportion of tumours harboured recurrent non-coding aberrations, large-scale genomic rearrangements, and alterations in which an inversion repressed transcription within its boundaries. Local hypermutation events were frequent, and correlated with specific genomic profiles. Numerous molecular aberrations were prognostic for disease recurrence, including several DNA methylation events, and a signature comprised of these aberrations outperformed well-described prognostic biomarkers. We suggest that intensified treatment of genomically aggressive localized prostate cancer may improve cure rates.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nature20788DOI Listing
January 2017

Germline BRCA2 mutations drive prostate cancers with distinct evolutionary trajectories.

Nat Commun 2017 01 9;8:13671. Epub 2017 Jan 9.

Princess Margaret Cancer Centre, University Health Network, Toronto, Ontario, Canada M5G 1L7.

Germline mutations in the BRCA2 tumour suppressor are associated with both an increased lifetime risk of developing prostate cancer (PCa) and increased risk of aggressive disease. To understand this aggression, here we profile the genomes and methylomes of localized PCa from 14 carriers of deleterious germline BRCA2 mutations (BRCA2-mutant PCa). We show that BRCA2-mutant PCa harbour increased genomic instability and a mutational profile that more closely resembles metastastic than localized disease. BRCA2-mutant PCa shows genomic and epigenomic dysregulation of the MED12L/MED12 axis, which is frequently dysregulated in metastatic castration-resistant prostate cancer (mCRPC). This dysregulation is enriched in BRCA2-mutant PCa harbouring intraductal carcinoma (IDC). Microdissection and sequencing of IDC and juxtaposed adjacent non-IDC invasive carcinoma in 10 patients demonstrates a common ancestor to both histopathologies. Overall we show that localized castration-sensitive BRCA2-mutant tumours are uniquely aggressive, due to de novo aberration in genes usually associated with metastatic disease, justifying aggressive initial treatment.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/ncomms13671DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5227331PMC
January 2017

A comprehensive assessment of somatic mutation detection in cancer using whole-genome sequencing.

Nat Commun 2015 Dec 9;6:10001. Epub 2015 Dec 9.

Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1SA, UK.

As whole-genome sequencing for cancer genome analysis becomes a clinical tool, a full understanding of the variables affecting sequencing analysis output is required. Here using tumour-normal sample pairs from two different types of cancer, chronic lymphocytic leukaemia and medulloblastoma, we conduct a benchmarking exercise within the context of the International Cancer Genome Consortium. We compare sequencing methods, analysis pipelines and validation methods. We show that using PCR-free methods and increasing sequencing depth to ∼ 100 × shows benefits, as long as the tumour:control coverage ratio remains balanced. We observe widely varying mutation call rates and low concordance among analysis pipelines, reflecting the artefact-prone nature of the raw data and lack of standards for dealing with the artefacts. However, we show that, using the benchmark mutation set we have created, many issues are in fact easy to remedy and have an immediate positive impact on mutation detection accuracy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/ncomms10001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4682041PMC
December 2015

A genome scale overexpression screen to reveal drug activity in human cells.

Genome Med 2014 29;6(4):32. Epub 2014 Apr 29.

Department of Molecular Genetics, University of Toronto, Toronto, M5S 3E1, Canada ; Banting and Best Department of Medical Research, University of Toronto, Toronto, M5S 3E1, Canada ; Terrence Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, 160 College Street, Toronto, Ontario M5S 3E1, Canada ; Donnelly Sequencing Center, University of Toronto, 160 College Street, Toronto, Ontario M5S 3E1, Canada ; Department of Pharmaceutical Sciences, University of British Columbia, 6619-2405 Wesbrook Mall, Vancouver, BC V6T 1Z3, Canada.

Target identification is a critical step in the lengthy and expensive process of drug development. Here, we describe a genome-wide screening platform that uses systematic overexpression of pooled human ORFs to understand drug mode-of-action and resistance mechanisms. We first calibrated our screen with the well-characterized drug methotrexate. We then identified new genes involved in the bioactivity of diverse drugs including antineoplastic agents and biologically active molecules. Finally, we focused on the transcription factor RHOXF2 whose overexpression conferred resistance to DNA damaging agents. This approach represents an orthogonal method for functional screening and, to our knowledge, has never been reported before.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/gm549DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4062067PMC
June 2014

Mapping the cellular response to small molecules using chemogenomic fitness signatures.

Science 2014 Apr;344(6180):208-11

The Donnelly Centre, University of Toronto, Toronto, Ontario M5S 3E1, Canada.

Genome-wide characterization of the in vivo cellular response to perturbation is fundamental to understanding how cells survive stress. Identifying the proteins and pathways perturbed by small molecules affects biology and medicine by revealing the mechanisms of drug action. We used a yeast chemogenomics platform that quantifies the requirement for each gene for resistance to a compound in vivo to profile 3250 small molecules in a systematic and unbiased manner. We identified 317 compounds that specifically perturb the function of 121 genes and characterized the mechanism of specific compounds. Global analysis revealed that the cellular response to small molecules is limited and described by a network of 45 major chemogenomic signatures. Our results provide a resource for the discovery of functional interactions among genes, chemicals, and biological processes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1126/science.1250217DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4254748PMC
April 2014

CHIP-MYTH: a novel interactive proteomics method for the assessment of agonist-dependent interactions of the human β₂-adrenergic receptor.

Biochem Biophys Res Commun 2014 Mar 19;445(4):746-56. Epub 2014 Feb 19.

Donnelly Centre, University of Toronto, Ontario, Canada; Department of Molecular Genetics, University of Toronto, Ontario, Canada; Department of Biochemistry, University of Toronto, Ontario, Canada. Electronic address:

G-protein coupled receptors (GPCRs) are involved in a variety of disease processes and comprise major drug targets. However, the complexity of integral membrane proteins such as GPCRs makes the identification of their interacting partners and subsequent drug development challenging. A comprehensive understanding of GPCR protein interaction networks is needed to design effective therapeutic strategies to inhibit these drug targets. Here, we developed a novel split-ubiquitin membrane yeast two-hybrid (MYTH) technology called CHIP-MYTH, which allows the unbiased characterization of interaction partners of full-length GPCRs in a drug-dependent manner. This was achieved by coupling DNA microarray technology to the MYTH approach, which allows a quantitative evaluation of interacting partners of a given integral membrane protein in the presence or absence of drug. As a proof of principle, we applied the CHIP-MYTH approach to the human β2-adrenergic receptor (β2AR), a target of interest in the treatment of asthma, chronic obstructive pulmonary disease (COPD), neurological disease, cardiovascular disease, and obesity. A CHIP-MYTH screen was performed in the presence or absence of salmeterol, a long-acting β2AR-agonist. Our results suggest that β2AR activation with salmeterol can induce the dissociation of heterotrimeric G-proteins, Gαβγ, into Gα and Gβγ subunits, which in turn activates downstream signaling cascades. Using CHIP-MYTH, we confirmed previously known and identified novel β2AR interactors involved in GPCR-mediated signaling cascades. Several of these interactions were confirmed in mammalian cells using LUminescence-based Mammalian IntERactome (LUMIER) and co-immunoprecipitation assays. In summary, the CHIP-MYTH approach is ideal for conducting comprehensive protein-protein interactions (PPI) screenings of full-length GPCRs in the presence or absence of drugs, thus providing a valuable tool to further our understanding of GPCR-mediated signaling.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bbrc.2014.02.033DOI Listing
March 2014

Miniature short hairpin RNA screens to characterize antiproliferative drugs.

G3 (Bethesda) 2013 Aug 7;3(8):1375-87. Epub 2013 Aug 7.

Department of Molecular Genetics, University of Toronto, Toronto, Ontario M5S 3E1, Canada.

The application of new proteomics and genomics technologies support a view in which few drugs act solely by inhibiting a single cellular target. Indeed, drug activity is modulated by complex, often incompletely understood cellular mechanisms. Therefore, efforts to decipher mode of action through genetic perturbation such as RNAi typically yields "hits" that fall into several categories. Of particular interest to the present study, we aimed to characterize secondary activities of drugs on cells. Inhibiting a known target can result in clinically relevant synthetic phenotypes. In one scenario, drug perturbation could, for example, improperly activate a protein that normally inhibits a particular kinase. In other cases, additional, lower affinity targets can be inhibited as in the example of inhibition of c-Kit observed in Bcr-Abl-positive cells treated with Gleevec. Drug transport and metabolism also play an important role in the way any chemicals act within the cells. Finally, RNAi per se can also affect cell fitness by more general off-target effects, e.g., via the modulation of apoptosis or DNA damage repair. Regardless of the root cause of these unwanted effects, understanding the scope of a drug's activity and polypharmacology is essential for better understanding its mechanism(s) of action, and such information can guide development of improved therapies. We describe a rapid, cost-effective approach to characterize primary and secondary effects of small-molecules by using small-scale libraries of virally integrated short hairpin RNAs. We demonstrate this principle using a "minipool" composed of shRNAs that target the genes encoding the reported protein targets of approved drugs. Among the 28 known reported drug-target pairs, we successfully identify 40% of the targets described in the literature and uncover several unanticipated drug-target interactions based on drug-induced synthetic lethality. We provide a detailed protocol for performing such screens and for analyzing the data. This cost-effective approach to mammalian knockdown screens, combined with the increasing maturation of RNAi technology will expand the accessibility of similar approaches in academic settings.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1534/g3.113.006437DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3737177PMC
August 2013

Effects of the Paf1 complex and histone modifications on snoRNA 3'-end formation reveal broad and locus-specific regulation.

Mol Cell Biol 2013 Jan 29;33(1):170-82. Epub 2012 Oct 29.

Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania, USA.

Across diverse eukaryotes, the Paf1 complex (Paf1C) plays critical roles in RNA polymerase II transcription elongation and regulation of histone modifications. Beyond these roles, the human and Saccharomyces cerevisiae Paf1 complexes also interact with RNA 3'-end processing components to affect transcript 3'-end formation. Specifically, the Saccharomyces cerevisiae Paf1C functions with the RNA binding proteins Nrd1 and Nab3 to regulate the termination of at least two small nucleolar RNAs (snoRNAs). To determine how Paf1C-dependent functions regulate snoRNA formation, we used high-density tiling arrays to analyze transcripts in paf1Δ cells and uncover new snoRNA targets of Paf1. Detailed examination of Paf1-regulated snoRNA genes revealed locus-specific requirements for Paf1-dependent posttranslational histone modifications. We also discovered roles for the transcriptional regulators Bur1-Bur2, Rad6, and Set2 in snoRNA 3'-end formation. Surprisingly, at some snoRNAs, this function of Rad6 appears to be primarily independent of its role in histone H2B monoubiquitylation. Cumulatively, our work reveals a broad requirement for the Paf1C in snoRNA 3'-end formation in S. cerevisiae, implicates the participation of transcriptional proteins and histone modifications in this process, and suggests that the Paf1C contributes to the fine tuning of nuanced levels of regulation that exist at individual loci.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/MCB.01233-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3536304PMC
January 2013

Functional analysis with a barcoder yeast gene overexpression system.

G3 (Bethesda) 2012 Oct 1;2(10):1279-89. Epub 2012 Oct 1.

Department of Molecular Genetics, University of Toronto, Ontario M5S 1A8, Canada.

Systematic analysis of gene overexpression phenotypes provides an insight into gene function, enzyme targets, and biological pathways. Here, we describe a novel functional genomics platform that enables a highly parallel and systematic assessment of overexpression phenotypes in pooled cultures. First, we constructed a genome-level collection of ~5100 yeast barcoder strains, each of which carries a unique barcode, enabling pooled fitness assays with a barcode microarray or sequencing readout. Second, we constructed a yeast open reading frame (ORF) galactose-induced overexpression array by generating a genome-wide set of yeast transformants, each of which carries an individual plasmid-born and sequence-verified ORF derived from the Saccharomyces cerevisiae full-length EXpression-ready (FLEX) collection. We combined these collections genetically using synthetic genetic array methodology, generating ~5100 strains, each of which is barcoded and overexpresses a specific ORF, a set we termed "barFLEX." Additional synthetic genetic array allows the barFLEX collection to be moved into different genetic backgrounds. As a proof-of-principle, we describe the properties of the barFLEX overexpression collection and its application in synthetic dosage lethality studies under different environmental conditions.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1534/g3.112.003400DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3464120PMC
October 2012

Comparative chemogenomics to examine the mechanism of action of dna-targeted platinum-acridine anticancer agents.

ACS Chem Biol 2012 Nov 5;7(11):1892-901. Epub 2012 Sep 5.

Department of Molecular Genetics and the Donnelly Centre, University of Toronto, Toronto, ON, M5S3E1 Canada.

Platinum-based drugs have been used to successfully treat diverse cancers for several decades. Cisplatin, the original compound of this class, cross-links DNA, resulting in cell cycle arrest and cell death via apoptosis. Cisplatin is effective against several tumor types, yet it exhibits toxic side effects and tumors often develop resistance. To mitigate these liabilities while maintaining potency, we generated a library of non-classical platinum-acridine hybrid agents and assessed their mechanisms of action using a validated genome-wide screening approach in Saccharomyces cerevisiae and in the distantly related yeast Schizosaccharomyces pombe. Chemogenomic profiles from both S. cerevisiae and S. pombe demonstrate that several of the platinum-acridines damage DNA differently than cisplatin based on their requirement for distinct modules of DNA repair.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/cb300320dDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3500413PMC
November 2012

Identification of yeast genes that confer resistance to chitosan oligosaccharide (COS) using chemogenomics.

BMC Genomics 2012 Jun 22;13:267. Epub 2012 Jun 22.

Department of Cell and Systems Biology, University of Toronto, Mississauga, Ontario, Canada.

Background: Chitosan oligosaccharide (COS), a deacetylated derivative of chitin, is an abundant, and renewable natural polymer. COS has higher antimicrobial properties than chitosan and is presumed to act by disrupting/permeabilizing the cell membranes of bacteria, yeast and fungi. COS is relatively non-toxic to mammals. By identifying the molecular and genetic targets of COS, we hope to gain a better understanding of the antifungal mode of action of COS.

Results: Three different chemogenomic fitness assays, haploinsufficiency (HIP), homozygous deletion (HOP), and multicopy suppression (MSP) profiling were combined with a transcriptomic analysis to gain insight in to the mode of action and mechanisms of resistance to chitosan oligosaccharides. The fitness assays identified 39 yeast deletion strains sensitive to COS and 21 suppressors of COS sensitivity. The genes identified are involved in processes such as RNA biology (transcription, translation and regulatory mechanisms), membrane functions (e.g. signalling, transport and targeting), membrane structural components, cell division, and proteasome processes. The transcriptomes of control wild type and 5 suppressor strains overexpressing ARL1, BCK2, ERG24, MSG5, or RBA50, were analyzed in the presence and absence of COS. Some of the up-regulated transcripts in the suppressor overexpressing strains exposed to COS included genes involved in transcription, cell cycle, stress response and the Ras signal transduction pathway. Down-regulated transcripts included those encoding protein folding components and respiratory chain proteins. The COS-induced transcriptional response is distinct from previously described environmental stress responses (i.e. thermal, salt, osmotic and oxidative stress) and pre-treatment with these well characterized environmental stressors provided little or any resistance to COS.

Conclusions: Overexpression of the ARL1 gene, a member of the Ras superfamily that regulates membrane trafficking, provides protection against COS-induced cell membrane permeability and damage. We found that the ARL1 COS-resistant over-expression strain was as sensitive to Amphotericin B, Fluconazole and Terbinafine as the wild type cells and that when COS and Fluconazole are used in combination they act in a synergistic fashion. The gene targets of COS identified in this study indicate that COS's mechanism of action is different from other commonly studied fungicides that target membranes, suggesting that COS may be an effective fungicide for drug-resistant fungal pathogens.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/1471-2164-13-267DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3505485PMC
June 2012

Multiple means to the same end: the genetic basis of acquired stress resistance in yeast.

PLoS Genet 2011 Nov 10;7(11):e1002353. Epub 2011 Nov 10.

Laboratory of Genetics, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.

In nature, stressful environments often occur in combination or close succession, and thus the ability to prepare for impending stress likely provides a significant fitness advantage. Organisms exposed to a mild dose of stress can become tolerant to what would otherwise be a lethal dose of subsequent stress; however, the mechanism of this acquired stress tolerance is poorly understood. To explore this, we exposed the yeast gene-deletion libraries, which interrogate all essential and non-essential genes, to successive stress treatments and identified genes necessary for acquiring subsequent stress resistance. Cells were exposed to one of three different mild stress pretreatments (salt, DTT, or heat shock) and then challenged with a severe dose of hydrogen peroxide (H(2)O(2)). Surprisingly, there was little overlap in the genes required for acquisition of H(2)O(2) tolerance after different mild-stress pretreatments, revealing distinct mechanisms of surviving H(2)O(2) in each case. Integrative network analysis of these results with respect to protein-protein interactions, synthetic-genetic interactions, and functional annotations identified many processes not previously linked to H(2)O(2) tolerance. We tested and present several models that explain the lack of overlap in genes required for H(2)O(2) tolerance after each of the three pretreatments. Together, this work shows that acquired tolerance to the same severe stress occurs by different mechanisms depending on prior cellular experiences, underscoring the context-dependent nature of stress tolerance.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.pgen.1002353DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3213159PMC
November 2011

Compound prioritization methods increase rates of chemical probe discovery in model organisms.

Chem Biol 2011 Oct;18(10):1273-83

Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, Ontario M5S 3E1, Canada.

Preselection of compounds that are more likely to induce a phenotype can increase the efficiency and reduce the costs for model organism screening. To identify such molecules, we screened ~81,000 compounds in Saccharomyces cerevisiae and identified ~7500 that inhibit cell growth. Screening these growth-inhibitory molecules across a diverse panel of model organisms resulted in an increased phenotypic hit-rate. These data were used to build a model to predict compounds that inhibit yeast growth. Empirical and in silico application of the model enriched the discovery of bioactive compounds in diverse model organisms. To demonstrate the potential of these molecules as lead chemical probes, we used chemogenomic profiling in yeast and identified specific inhibitors of lanosterol synthase and of stearoyl-CoA 9-desaturase. As community resources, the ~7500 growth-inhibitory molecules have been made commercially available and the computational model and filter used are provided.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.chembiol.2011.07.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4193902PMC
October 2011

Competitive genomic screens of barcoded yeast libraries.

J Vis Exp 2011 Aug 11(54). Epub 2011 Aug 11.

Banting and Best Department of Medical Research and Department of Molecular Genetics, University of Toronto, Canada.

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily. The tempo of these advances is accelerating, promising greater depth and breadth. In light of these extraordinary advances, the need for fast, parallel methods to define gene function becomes ever more important. Collections of genome-wide deletion mutants in yeasts and E. coli have served as workhorses for functional characterization of gene function, but this approach is not scalable, current gene-deletion approaches require each of the thousands of genes that comprise a genome to be deleted and verified. Only after this work is complete can we pursue high-throughput phenotyping. Over the past decade, our laboratory has refined a portfolio of competitive, miniaturized, high-throughput genome-wide assays that can be performed in parallel. This parallelization is possible because of the inclusion of DNA 'tags', or 'barcodes,' into each mutant, with the barcode serving as a proxy for the mutation and one can measure the barcode abundance to assess mutant fitness. In this study, we seek to fill the gap between DNA sequence and barcoded mutant collections. To accomplish this we introduce a combined transposon disruption-barcoding approach that opens up parallel barcode assays to newly sequenced, but poorly characterized microbes. To illustrate this approach we present a new Candida albicans barcoded disruption collection and describe how both microarray-based and next generation sequencing-based platforms can be used to collect 10,000-1,000,000 gene-gene and drug-gene interactions in a single experiment.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3791/2864DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3211125PMC
August 2011

Dosage suppression genetic interaction networks enhance functional wiring diagrams of the cell.

Nat Biotechnol 2011 May 15;29(6):505-11. Epub 2011 May 15.

Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada.

Dosage suppression is a genetic interaction in which overproduction of one gene rescues a mutant phenotype of another gene. Although dosage suppression is known to map functional connections among genes, the extent to which it might illuminate global cellular functions is unclear. Here we analyze a network of interactions linking dosage suppressors to 437 essential genes in yeast. For 424 genes, we curated interactions from the literature. Analyses revealed that many dosage suppression interactions occur between functionally related genes and that the majority do not overlap with other types of genetic or physical interactions. To confirm the generality of these network properties, we experimentally identified dosage suppressors for 29 genes from pooled populations of temperature-sensitive mutant cells transformed with a high-copy molecular-barcoded open reading frame library, MoBY-ORF 2.0. We classified 87% of the 1,640 total interactions into four general types of suppression mechanisms, which provided insight into their relative frequencies. This work suggests that integrating the results of dosage suppression studies with other interaction networks could generate insights into the functional wiring diagram of a cell.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nbt.1855DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7386433PMC
May 2011

A comprehensive platform for highly multiplexed mammalian functional genetic screens.

BMC Genomics 2011 May 6;12:213. Epub 2011 May 6.

Donnelly Centre and Banting & Best Department of Medical Research, University of Toronto, Toronto, Canada.

Background: Genome-wide screening in human and mouse cells using RNA interference and open reading frame over-expression libraries is rapidly becoming a viable experimental approach for many research labs. There are a variety of gene expression modulation libraries commercially available, however, detailed and validated protocols as well as the reagents necessary for deconvolving genome-scale gene screens using these libraries are lacking. As a solution, we designed a comprehensive platform for highly multiplexed functional genetic screens in human, mouse and yeast cells using popular, commercially available gene modulation libraries. The Gene Modulation Array Platform (GMAP) is a single microarray-based detection solution for deconvolution of loss and gain-of-function pooled screens.

Results: Experiments with specially constructed lentiviral-based plasmid pools containing ~78,000 shRNAs demonstrated that the GMAP is capable of deconvolving genome-wide shRNA "dropout" screens. Further experiments with a larger, ~90,000 shRNA pool demonstrate that equivalent results are obtained from plasmid pools and from genomic DNA derived from lentivirus infected cells. Parallel testing of large shRNA pools using GMAP and next-generation sequencing methods revealed that the two methods provide valid and complementary approaches to deconvolution of genome-wide shRNA screens. Additional experiments demonstrated that GMAP is equivalent to similar microarray-based products when used for deconvolution of open reading frame over-expression screens.

Conclusion: Herein, we demonstrate four major applications for the GMAP resource, including deconvolution of pooled RNAi screens in cells with at least 90,000 distinct shRNAs. We also provide detailed methodologies for pooled shRNA screen readout using GMAP and compare next-generation sequencing to GMAP (i.e. microarray) based deconvolution methods.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/1471-2164-12-213DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3115879PMC
May 2011

Highly-multiplexed barcode sequencing: an efficient method for parallel analysis of pooled samples.

Nucleic Acids Res 2010 Jul 11;38(13):e142. Epub 2010 May 11.

Department of Molecular Genetics, University of Toronto, 1 King's College Circle, Toronto, Ontario M5S 1A8, Canada.

Next-generation sequencing has proven an extremely effective technology for molecular counting applications where the number of sequence reads provides a digital readout for RNA-seq, ChIP-seq, Tn-seq and other applications. The extremely large number of sequence reads that can be obtained per run permits the analysis of increasingly complex samples. For lower complexity samples, however, a point of diminishing returns is reached when the number of counts per sequence results in oversampling with no increase in data quality. A solution to making next-generation sequencing as efficient and affordable as possible involves assaying multiple samples in a single run. Here, we report the successful 96-plexing of complex pools of DNA barcoded yeast mutants and show that such 'Bar-seq' assessment of these samples is comparable with data provided by barcode microarrays, the current benchmark for this application. The cost reduction and increased throughput permitted by highly multiplexed sequencing will greatly expand the scope of chemogenomics assays and, equally importantly, the approach is suitable for other sequence counting applications that could benefit from massive parallelization.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/nar/gkq368DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2910071PMC
July 2010

Genotype to phenotype: a complex problem.

Science 2010 Apr;328(5977):469

Computer Science and Artificial Intelligence Laboratory, Department of Electrical Engineering and Computer Science, Massachusetts Institute of Technology (MIT), Cambridge, MA 02139, USA.

We generated a high-resolution whole-genome sequence and individually deleted 5100 genes in Sigma1278b, a Saccharomyces cerevisiae strain closely related to reference strain S288c. Similar to the variation between human individuals, Sigma1278b and S288c average 3.2 single-nucleotide polymorphisms per kilobase. A genome-wide comparison of deletion mutant phenotypes identified a subset of genes that were conditionally essential by strain, including 44 essential genes unique to Sigma1278b and 13 unique to S288c. Genetic analysis indicates the conditional phenotype was most often governed by complex genetic interactions, depending on multiple background-specific modifiers. Our comprehensive analysis suggests that the presence of a complex set of modifiers will often underlie the phenotypic differences between individuals.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1126/science.1189015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4412269PMC
April 2010

A comparative analysis of DNA barcode microarray feature size.

BMC Genomics 2009 Oct 13;10:471. Epub 2009 Oct 13.

Department of Molecular Genetics, University of Toronto, 1 King's College Circle, Toronto, Ontario M5S 1A8, Canada.

Background: Microarrays are an invaluable tool in many modern genomic studies. It is generally perceived that decreasing the size of microarray features leads to arrays with higher resolution (due to greater feature density), but this increase in resolution can compromise sensitivity.

Results: We demonstrate that barcode microarrays with smaller features are equally capable of detecting variation in DNA barcode intensity when compared to larger feature sizes within a specific microarray platform. The barcodes used in this study are the well-characterized set derived from the Yeast KnockOut (YKO) collection used for screens of pooled yeast (Saccharomyces cerevisiae) deletion mutants. We treated these pools with the glycosylation inhibitor tunicamycin as a test compound. Three generations of barcode microarrays at 30, 8 and 5 microm features sizes independently identified the primary target of tunicamycin to be ALG7.

Conclusion: We show that the data obtained with 5 microm feature size is of comparable quality to the 30 microm size and propose that further shrinking of features could yield barcode microarrays with equal or greater resolving power and, more importantly, higher density.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/1471-2164-10-471DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2765990PMC
October 2009

Quantitative phenotyping via deep barcode sequencing.

Genome Res 2009 Oct 21;19(10):1836-42. Epub 2009 Jul 21.

Department of Molecular Genetics, University of Toronto, Ontario, Canada.

Next-generation DNA sequencing technologies have revolutionized diverse genomics applications, including de novo genome sequencing, SNP detection, chromatin immunoprecipitation, and transcriptome analysis. Here we apply deep sequencing to genome-scale fitness profiling to evaluate yeast strain collections in parallel. This method, Barcode analysis by Sequencing, or "Bar-seq," outperforms the current benchmark barcode microarray assay in terms of both dynamic range and throughput. When applied to a complex chemogenomic assay, Bar-seq quantitatively identifies drug targets, with performance superior to the benchmark microarray assay. We also show that Bar-seq is well-suited for a multiplex format. We completely re-sequenced and re-annotated the yeast deletion collection using deep sequencing, found that approximately 20% of the barcodes and common priming sequences varied from expectation, and used this revised list of barcode sequences to improve data quality. Together, this new assay and analysis routine provide a deep-sequencing-based toolkit for identifying gene-environment interactions on a genome-wide scale.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1101/gr.093955.109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2765281PMC
October 2009

Chemical-genetic profiling of imidazo[1,2-a]pyridines and -pyrimidines reveals target pathways conserved between yeast and human cells.

PLoS Genet 2008 Nov 28;4(11):e1000284. Epub 2008 Nov 28.

Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada.

Small molecules have been shown to be potent and selective probes to understand cell physiology. Here, we show that imidazo[1,2-a]pyridines and imidazo[1,2-a]pyrimidines compose a class of compounds that target essential, conserved cellular processes. Using validated chemogenomic assays in Saccharomyces cerevisiae, we discovered that two closely related compounds, an imidazo[1,2-a]pyridine and -pyrimidine that differ by a single atom, have distinctly different mechanisms of action in vivo. 2-phenyl-3-nitroso-imidazo[1,2-a]pyridine was toxic to yeast strains with defects in electron transport and mitochondrial functions and caused mitochondrial fragmentation, suggesting that compound 13 acts by disrupting mitochondria. By contrast, 2-phenyl-3-nitroso-imidazo[1,2-a]pyrimidine acted as a DNA poison, causing damage to the nuclear DNA and inducing mutagenesis. We compared compound 15 to known chemotherapeutics and found resistance required intact DNA repair pathways. Thus, subtle changes in the structure of imidazo-pyridines and -pyrimidines dramatically alter both the intracellular targeting of these compounds and their effects in vivo. Of particular interest, these different modes of action were evident in experiments on human cells, suggesting that chemical-genetic profiles obtained in yeast are recapitulated in cultured cells, indicating that our observations in yeast can: (1) be leveraged to determine mechanism of action in mammalian cells and (2) suggest novel structure-activity relationships.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.pgen.1000284DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2583946PMC
November 2008
-->