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Borowsky, Mark L

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Borowsky

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Mark L

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Borowsky, Mark L
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    Small RNA pathway genes identified by patterns of phylogenetic conservation and divergence
    (2013) Tabach, Yuval; Billi, Allison C.; Hayes, Gabriel D.; Newman, Martin; Zuk, Or; Gabel, Harrison; Kamath, Ravi; Yacoby, Keren; Chapman, Brad; Garcia, Susana M. D. A.; Borowsky, Mark L; Kim, John K.; Ruvkun, Gary
    Genetic and biochemical analyses of RNA interference (RNAi) and microRNA (miRNA) pathways have revealed proteins such as Argonaute/PIWI and Dicer that process and present small RNAs to their targets. Well validated small RNA pathway cofactors, such as the Argonaute/PIWI proteins show distinctive patterns of conservation or divergence in particular animal, plant, fungal, and protist species. We compared 86 divergent eukaryotic genome sequences to discern sets of proteins that show similar phylogenetic profiles with known small RNA cofactors. A large set of additional candidate small RNA cofactors have emerged from functional genomic screens for defects in miRNA- or siRNA-mediated repression in C. elegans and D. melanogaster1,2 and from proteomic analyses of proteins co-purifying with validated small RNA pathway proteins3,4. The phylogenetic profiles of many of these candidate small RNA pathway proteins are similar to those of known small RNA cofactor proteins. We used a Bayesian approach to integrate the phylogenetic profile analysis with predictions from diverse transcriptional coregulation and proteome interaction datasets to assign a probability for each protein for a role in a small RNA pathway. Testing high-confidence candidates from this analysis for defects in RNAi silencing, we found that about half of the predicted small RNA cofactors are required for RNAi silencing. Many of the newly identified small RNA pathway proteins are orthologues of proteins implicated in RNA splicing. In support of a deep connection between the mechanism of RNA splicing and small RNA-mediated gene silencing, the presence of the Argonaute proteins and other small RNA components in the many species analysed strongly correlates with the number of introns in that species.
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    Genomic Analysis Identifies Targets of Convergent Positive Selection in Drug Resistant Mycobacterium tuberculosis
    (2013) Farhat, Maha; Shapiro, B Jesse; Kieser, Karen; Sultana, Razvan; Jacobson, Karen R; Victor, Thomas C; Warren, Robin M; Streicher, Elizabeth M; Calver, Alistair; Sloutsky, Alex; Kaur, Devinder; Posey, Jamie E; Plikaytis, Bonnie; Oggioni, Marco R; Gardy, Jennifer L; Johnston, James C; Rodrigues, Mabel; Tang, Patrick K C; Kato-Maeda, Midori; Borowsky, Mark L; Muddukrishna, Bhavana; Kreiswirth, Barry N; Kurepina, Natalia; Galagan, James; Gagneux, Sebastien; Birren, Bruce; Rubin, Eric; Lander, Eric S; Sabeti, Pardis; Murray, Megan
    Mycobacterium tuberculosis is successfully evolving antibiotic resistance, threatening attempts at tuberculosis epidemic control. Mechanisms of resistance, including the genetic changes favored by selection in resistant isolates, are incompletely understood. Using 116 newly and 7 previously sequenced M. tuberculosis genomes, we identified genomewide signatures of positive selection specific to the 47 resistant genomes. By searching for convergent evolution, the independent fixation of mutations at the same nucleotide site or gene, we recovered 100% of a set of known resistance markers. We also found evidence of positive selection in an additional 39 genomic regions in resistant isolates. These regions encode pathways of cell wall biosynthesis, transcriptional regulation and DNA repair. Mutations in these regions could directly confer resistance or compensate for fitness costs associated with resistance. Functional genetic analysis of mutations in one gene, ponA1, demonstrated an in vitro growth advantage in the presence of the drug rifampicin.
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    Multiplexed Illumina sequencing libraries from picogram quantities of DNA
    (BioMed Central, 2013) Bowman, Sarah K.; Simon, Matthew D; Deaton, Aimee; Tolstorukov, Michael; Borowsky, Mark L; Kingston, Robert
    Background: High throughput sequencing is frequently used to discover the location of regulatory interactions on chromatin. However, techniques that enrich DNA where regulatory activity takes place, such as chromatin immunoprecipitation (ChIP), often yield less DNA than optimal for sequencing library preparation. Existing protocols for picogram-scale libraries require concomitant fragmentation of DNA, pre-amplification, or long overnight steps. Results: We report a simple and fast library construction method that produces libraries from sub-nanogram quantities of DNA. This protocol yields conventional libraries with barcodes suitable for multiplexed sample analysis on the Illumina platform. We demonstrate the utility of this method by constructing a ChIP-seq library from 100 pg of ChIP DNA that demonstrates equivalent genomic coverage of target regions to a library produced from a larger scale experiment. Conclusions: Application of this method allows whole genome studies from samples where material or yields are limiting.
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    The Microglial Sensome Revealed by Direct RNA Sequencing
    (2013) Hickman, Suzanne E.; Kingery, Nathan D.; Ohsumi, Toshiro; Borowsky, Mark L; Wang, Li-chong; Means, Terry K.; Khoury, Joseph
    Microglia, the principal neuroimmune sentinels of the brain, continuously sense changes in their environment and respond to invading pathogens, toxins and cellular debris. Microglia exhibit plasticity and can assume neurotoxic or neuroprotective priming states that determine their responses to danger. We used direct RNA sequencing, without amplification or cDNA synthesis, to determine the quantitative transcriptomes of microglia of healthy adult and aged mice. We validated our findings by fluorescent dual in-situ hybridization, unbiased proteomic analysis and quantitative PCR. We report here that microglia have a distinct transcriptomic signature and express a unique cluster of transcripts encoding proteins for sensing endogenous ligands and microbes that we term the “sensome”. With aging, sensome transcripts for endogenous ligand recognition are downregulated, whereas those involved in microbe recognition and host defense are upregulated. In addition, aging is associated with an overall increase in expression of microglial genes involved in neuroprotection.
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    Transposon activation mutagenesis as a screening tool for identifying resistance to cancer therapeutics
    (BioMed Central, 2013) Chen, Li; Stuart, Lynda; Ohsumi, Toshiro K; Burgess, Shawn; Varshney, Gaurav K; Dastur, Anahita; Borowsky, Mark L; Benes, Cyril; Lacy-Hulbert, Adam; Schmidt, Emmett V
    Background: The development of resistance to chemotherapies represents a significant barrier to successful cancer treatment. Resistance mechanisms are complex, can involve diverse and often unexpected cellular processes, and can vary with both the underlying genetic lesion and the origin or type of tumor. For these reasons developing experimental strategies that could be used to understand, identify and predict mechanisms of resistance in different malignant cells would be a major advance. Methods: Here we describe a gain-of-function forward genetic approach for identifying mechanisms of resistance. This approach uses a modified piggyBac transposon to generate libraries of mutagenized cells, each containing transposon insertions that randomly activate nearby gene expression. Genes of interest are identified using next-gen high-throughput sequencing and barcode multiplexing is used to reduce experimental cost. Results: Using this approach we successfully identify genes involved in paclitaxel resistance in a variety of cancer cell lines, including the multidrug transporter ABCB1, a previously identified major paclitaxel resistance gene. Analysis of co-occurring transposons integration sites in single cell clone allows for the identification of genes that might act cooperatively to produce drug resistance a level of information not accessible using RNAi or ORF expression screening approaches. Conclusion: We have developed a powerful pipeline to systematically discover drug resistance in mammalian cells in vitro. This cost-effective approach can be readily applied to different cell lines, to identify canonical or context specific resistance mechanisms. Its ability to probe complex genetic context and non-coding genomic elements as well as cooperative resistance events makes it a good complement to RNAi or ORF expression based screens.
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    MolBioLib: A C++11 Framework for Rapid Development and Deployment of Bioinformatics Tasks
    (Oxford University Press, 2012) Ohsumi, Toshiro K.; Borowsky, Mark L
    Summary: We developed MolBioLib to address the need for adaptable next-generation sequencing analysis tools. The result is a compact, portable and extensively tested C++11 software framework and set of applications tailored to the demands of next-generation sequencing data and applicable to many other applications. MolBioLib is designed to work with common file formats and data types used both in genomic analysis and general data analysis. A central relational-database-like Table class is a flexible and powerful object to intuitively represent and work with a wide variety of tabular datasets, ranging from alignment data to annotations. MolBioLib has been used to identify causative single-nucleotide polymorphisms in whole genome sequencing, detect balanced chromosomal rearrangements and compute enrichment of messenger RNAs (mRNAs) on microtubules, typically requiring applications of under 200 lines of code. MolBioLib includes programs to perform a wide variety of analysis tasks, such as computing read coverage, annotating genomic intervals and novel peak calling with a wavelet algorithm. Although MolBioLib was designed primarily for bioinformatics purposes, much of its functionality is applicable to a wide range of problems. Complete documentation and an extensive automated test suite are provided.
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    Complex Reorganization and Predominant Non-Homologous Repair Following Chromosomal Breakage in Karyotypically Balanced Germline Rearrangements and Transgenic Integration
    (Nature Publishing Group, 2012) Chiang, Colby; Jacobsen, Jessie C.; Ernst, Carl; Hanscom, Carrie; Heilbut, Adrian; Blumenthal, Ian; Mills, Ryan E.; Kirby, Andrew; Rudiger, Skye R.; McLaughlan, Clive J.; Bawden, C. Simon; Reid, Suzanne J.; Faull, Richard L. M.; Snell, Russell G.; Hall, Ira M.; Ohsumi, Toshiro K.; Shen, Yiping; Borowsky, Mark L; Daly, Mark; Lee, Charles; Morton, Cynthia; MacDonald, Marcy; Gusella, James; Talkowski, Michael; Lindgren, Amelia M.
    We defined the genetic landscape of balanced chromosomal rearrangements at nucleotide resolution by sequencing 141 breakpoints from cytogenetically-interpreted translocations and inversions. We confirm that the recently described phenomenon of “chromothripsis” (massive chromosomal shattering and reorganization) is not unique to cancer cells but also occurs in the germline where it can resolve to a karyotypically balanced state with frequent inversions. We detected a high incidence of complex rearrangements (19.2%) and substantially less reliance on microhomology (31%) than previously observed in benign CNVs. We compared these results to experimentally-generated DNA breakage-repair by sequencing seven transgenic animals, and revealed extensive rearrangement of the transgene and host genome with similar complexity to human germline alterations. Inversion is the most common rearrangement, suggesting that a combined mechanism involving template switching and non-homologous repair mediates the formation of balanced complex rearrangements that are viable, stably replicated and transmitted unaltered to subsequent generations.
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    Atypical Case Of Wolfram Syndrome Revealed Through Targeted Exome Sequencing In A Patient With Suspected Mitochondrial Disease
    (BioMed Central, 2012) Lieber, Daniel Solomon; Vafai, Scott B.; Horton, Laura C; Slate, Nancy G; Liu, Shangtao; Borowsky, Mark L; Calvo, Sarah; Schmahmann, Jeremy; Mootha, Vamsi
    Background: Mitochondrial diseases comprise a diverse set of clinical disorders that affect multiple organ systems with varying severity and age of onset. Due to their clinical and genetic heterogeneity, these diseases are difficult to diagnose. We have developed a targeted exome sequencing approach to improve our ability to properly diagnose mitochondrial diseases and apply it here to an individual patient. Our method targets mitochondrial DNA (mtDNA) and the exons of 1,600 nuclear genes involved in mitochondrial biology or Mendelian disorders with multi-system phenotypes, thereby allowing for simultaneous evaluation of multiple disease loci. Case Presentation: Targeted exome sequencing was performed on a patient initially suspected to have a mitochondrial disorder. The patient presented with diabetes mellitus, diffuse brain atrophy, autonomic neuropathy, optic nerve atrophy, and a severe amnestic syndrome. Further work-up revealed multiple heteroplasmic mtDNA deletions as well as profound thiamine deficiency without a clear nutritional cause. Targeted exome sequencing revealed a homozygous c.1672C > T (p.R558C) missense mutation in exon 8 of WFS1 that has previously been reported in a patient with Wolfram syndrome. Conclusion: This case demonstrates how clinical application of next-generation sequencing technology can enhance the diagnosis of patients suspected to have rare genetic disorders. Furthermore, the finding of unexplained thiamine deficiency in a patient with Wolfram syndrome suggests a potential link between WFS1 biology and thiamine metabolism that has implications for the clinical management of Wolfram syndrome patients.
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    Identifying candidate ncRNAs that direct changes in chromatin structure
    (BioMed Central, 2013) Ray, Mridula Kumari; Wang, Yanqun; Borowsky, Mark L; Sadreyev, Ruslan; Kingston, Robert
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    Short-Term Genome Evolution of Listeria Monocytogenes in a Non-Controlled Environment
    (BioMed Central, 2008) Orsi, Renato H; Lauer, Peter; Nusbaum, Chad; Birren, Bruce W; Ivy, Reid A; Graves, Lewis M; Swaminathan, Bala; Wiedmann, Martin; Borowsky, Mark L; Young, Sarah K.; Galagan, James E.; Sun, Qi
    Background: While increasing data on bacterial evolution in controlled environments are available, our understanding of bacterial genome evolution in natural environments is limited. We thus performed full genome analyses on four Listeria monocytogenes, including human and food isolates from both a 1988 case of sporadic listeriosis and a 2000 listeriosis outbreak, which had been linked to contaminated food from a single processing facility. All four isolates had been shown to have identical subtypes, suggesting that a specific L. monocytogenes strain persisted in this processing plant over at least 12 years. While a genome sequence for the 1988 food isolate has been reported, we sequenced the genomes of the 1988 human isolate as well as a human and a food isolate from the 2000 outbreak to allow for comparative genome analyses. Results: The two L. monocytogenes isolates from 1988 and the two isolates from 2000 had highly similar genome backbone sequences with very few single nucleotide (nt) polymorphisms (1 – 8 SNPs/isolate; confirmed by re-sequencing). While no genome rearrangements were identified in the backbone genome of the four isolates, a 42 kb prophage inserted in the chromosomal comK gene showed evidence for major genome rearrangements. The human-food isolate pair from each 1988 and 2000 had identical prophage sequence; however, there were significant differences in the prophage sequences between the 1988 and 2000 isolates. Diversification of this prophage appears to have been caused by multiple homologous recombination events or possibly prophage replacement. In addition, only the 2000 human isolate contained a plasmid, suggesting plasmid loss or acquisition events. Surprisingly, besides the polymorphisms found in the comK prophage, a single SNP in the tRNA Thr-4 prophage represents the only SNP that differentiates the 1988 isolates from the 2000 isolates. Conclusion: Our data support the hypothesis that the 2000 human listeriosis outbreak was caused by a L. monocytogenes strain that persisted in a food processing facility over 12 years and show that genome sequencing is a valuable and feasible tool for retrospective epidemiological analyses. Short-term evolution of L. monocytogenes in non-controlled environments appears to involve limited diversification beyond plasmid gain or loss and prophage diversification, highlighting the importance of phages in bacterial evolution.