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Identification of Virulence Determinants During Host-Pathogen Interaction Using Tn-Seq Technology.
Peek CT, Ibberson CB, Cassat JE
(2020) Methods Mol Biol 2069: 155-175
MeSH Terms: Animals, DNA Transposable Elements, Gene Library, High-Throughput Nucleotide Sequencing, Mice, Staphylococcal Infections, Staphylococcus aureus, Virulence Factors
Show Abstract · Added March 25, 2020
Transposon sequencing (Tn-seq) is a powerful genetic tool that enables the detection of essential genes within a given environment. The application of Tn-seq to Staphylococcus aureus has generated transposon libraries in numerous strains with inactivation of virtually every nonessential gene in the genome. This exciting technology coupled with increasingly available computational tools has been deployed in animal models of infection to identify essential S. aureus genes within specific host environments. In this chapter, we describe the application of Tn-seq to a murine model of osteomyelitis as a paradigm for using this powerful technology to elucidate mechanisms of bacterial pathogenesis.
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Signatures of Recent Positive Selection in Enhancers Across 41 Human Tissues.
Moon JM, Capra JA, Abbot P, Rokas A
(2019) G3 (Bethesda) 9: 2761-2774
MeSH Terms: DNA Transposable Elements, Databases, Genetic, Enhancer Elements, Genetic, Evolution, Molecular, Genome-Wide Association Study, Genomics, Humans, Immunity, Organ Specificity, Quantitative Trait, Heritable, Selection, Genetic
Show Abstract · Added March 3, 2020
Evolutionary changes in enhancers are widely associated with variation in human traits and diseases. However, studies comprehensively quantifying levels of selection on enhancers at multiple evolutionary periods during recent human evolution and how enhancer evolution varies across human tissues are lacking. To address these questions, we integrated a dataset of 41,561 transcribed enhancers active in 41 different human tissues (FANTOM Consortium) with whole genome sequences of 1,668 individuals from the African, Asian, and European populations (1000 Genomes Project). Our analyses based on four different metrics (Tajima's , , H12, ) showed that ∼5.90% of enhancers showed evidence of recent positive selection and that genes associated with enhancers under very recent positive selection are enriched for diverse immune-related functions. The distributions of these metrics for brain and testis enhancers were often statistically significantly different and in the direction suggestive of less positive selection compared to those of other tissues; the same was true for brain and testis enhancers that are tissue-specific compared to those that are tissue-broad and for testis enhancers associated with tissue-enriched and non-tissue-enriched genes. These differences varied considerably across metrics and tissues and were generally in the form of changes in distributions' shapes rather than shifts in their values. Collectively, these results suggest that many human enhancers experienced recent positive selection throughout multiple time periods in human evolutionary history, that this selection occurred in a tissue-dependent and immune-related functional context, and that much like the evolution of their protein-coding gene counterparts, the evolution of brain and testis enhancers has been markedly different from that of enhancers in other tissues.
Copyright © 2019 Moon et al.
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Direct reprogramming to human nephron progenitor-like cells using inducible piggyBac transposon expression of SNAI2-EYA1-SIX1.
Vanslambrouck JM, Woodard LE, Suhaimi N, Williams FM, Howden SE, Wilson SB, Lonsdale A, Er PX, Li J, Maksimovic J, Oshlack A, Wilson MH, Little MH
(2019) Kidney Int 95: 1153-1166
MeSH Terms: Cells, Cultured, Cellular Reprogramming, DNA Transposable Elements, Gene Transfer Techniques, Genetic Engineering, Homeodomain Proteins, Humans, Intracellular Signaling Peptides and Proteins, Nephrons, Nuclear Proteins, Primary Cell Culture, Protein Tyrosine Phosphatases, Regeneration, Snail Family Transcription Factors
Show Abstract · Added March 28, 2019
All nephrons in the mammalian kidney arise from a transient nephron progenitor population that is lost close to the time of birth. The generation of new nephron progenitors and their maintenance in culture are central to the success of kidney regenerative strategies. Using a lentiviral screening approach, we previously generated a human induced nephron progenitor-like state in vitro using a pool of six transcription factors. Here, we sought to develop a more efficient approach for direct reprogramming of human cells that could be applied in vivo. PiggyBac transposons are a non-viral integrating gene delivery system that is suitable for in vivo use and allows for simultaneous delivery of multiple genes. Using an inducible piggyBac transposon system, we optimized a protocol for the direct reprogramming of HK2 cells to induced nephron progenitor-like cells with expression of only 3 transcription factors (SNAI2, EYA1, and SIX1). Culture in conditions supportive of the nephron progenitor state further increased the expression of nephron progenitor genes. The refined protocol was then applied to primary human renal epithelial cells, which integrated into developing nephron structures in vitro and in vivo. Such inducible reprogramming to nephron progenitor-like cells could facilitate direct cellular reprogramming for kidney regeneration.
Copyright © 2019 International Society of Nephrology. All rights reserved.
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14 MeSH Terms
Integration Mapping of piggyBac-Mediated CD19 Chimeric Antigen Receptor T Cells Analyzed by Novel Tagmentation-Assisted PCR.
Hamada M, Nishio N, Okuno Y, Suzuki S, Kawashima N, Muramatsu H, Tsubota S, Wilson MH, Morita D, Kataoka S, Ichikawa D, Murakami N, Taniguchi R, Suzuki K, Kojima D, Sekiya Y, Nishikawa E, Narita A, Hama A, Kojima S, Nakazawa Y, Takahashi Y
(2018) EBioMedicine 34: 18-26
MeSH Terms: DNA Transposable Elements, High-Throughput Nucleotide Sequencing, Humans, Lentivirus, Polymerase Chain Reaction, Receptors, Antigen, T-Cell, Retroviridae, T-Lymphocytes
Show Abstract · Added December 13, 2018
Insertional mutagenesis is an important risk with all genetically modified cell therapies, including chimeric antigen receptor (CAR)-T cell therapy used for hematological malignancies. Here we describe a new tagmentation-assisted PCR (tag-PCR) system that can determine the integration sites of transgenes without using restriction enzyme digestion (which can potentially bias the detection) and allows library preparation in fewer steps than with other methods. Using this system, we compared the integration sites of CD19-specific CAR genes in final T cell products generated by retrovirus-based and lentivirus-based gene transfer and by the piggyBac transposon system. The piggyBac system demonstrated lower preference than the retroviral system for integration near transcriptional start sites and CpG islands and higher preference than the lentiviral system for integration into genomic safe harbors. Integration into or near proto-oncogenes was similar in all three systems. Tag-PCR mapping is a useful technique for assessing the risk of insertional mutagenesis.
Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.
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amplification is a mechanism of acquired resistance to EGFR inhibitors identified by transposon mutagenesis and clinical genomics.
Fan PD, Narzisi G, Jayaprakash AD, Venturini E, Robine N, Smibert P, Germer S, Yu HA, Jordan EJ, Paik PK, Janjigian YY, Chaft JE, Wang L, Jungbluth AA, Middha S, Spraggon L, Qiao H, Lovly CM, Kris MG, Riely GJ, Politi K, Varmus H, Ladanyi M
(2018) Proc Natl Acad Sci U S A 115: E6030-E6038
MeSH Terms: Cell Line, Tumor, DNA Transposable Elements, Drug Resistance, Neoplasm, Enzyme Inhibitors, ErbB Receptors, Gene Amplification, Gene Expression Regulation, Neoplastic, Humans, Lung Neoplasms, Proto-Oncogene Proteins c-fyn, Proto-Oncogene Proteins c-yes, Proto-Oncogene Proteins pp60(c-src)
Show Abstract · Added September 10, 2020
In ∼30% of patients with -mutant lung adenocarcinomas whose disease progresses on EGFR inhibitors, the basis for acquired resistance remains unclear. We have integrated transposon mutagenesis screening in an -mutant cell line and clinical genomic sequencing in cases of acquired resistance to identify mechanisms of resistance to EGFR inhibitors. The most prominent candidate genes identified by insertions in or near the genes during the screen were , a gene whose amplification is known to mediate resistance to EGFR inhibitors, and the gene encoding the Src family kinase YES1. Cell clones with transposon insertions that activated expression of exhibited resistance to all three generations of EGFR inhibitors and sensitivity to pharmacologic and siRNA-mediated inhibition of Analysis of clinical genomic sequencing data from cases of acquired resistance to EGFR inhibitors revealed amplification of in five cases, four of which lacked any other known mechanisms of resistance. Preinhibitor samples, available for two of the five patients, lacked amplification. None of 136 postinhibitor samples had detectable amplification of other Src family kinases ( and ). amplification was also found in 2 of 17 samples from fusion-positive lung cancer patients who had progressed on ALK TKIs. Taken together, our findings identify acquired amplification of as a recurrent and targetable mechanism of resistance to EGFR inhibition in -mutant lung cancers and demonstrate the utility of transposon mutagenesis in discovering clinically relevant mechanisms of drug resistance.
Copyright © 2018 the Author(s). Published by PNAS.
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Transposon-modified antigen-specific T lymphocytes for sustained therapeutic protein delivery in vivo.
O'Neil RT, Saha S, Veach RA, Welch RC, Woodard LE, Rooney CM, Wilson MH
(2018) Nat Commun 9: 1325
MeSH Terms: Adoptive Transfer, Animals, Cell Engineering, Cell- and Tissue-Based Therapy, DNA Transposable Elements, Erythropoietin, Gene Expression, Genetic Vectors, Hematopoiesis, Herpesvirus 4, Human, Humans, Mice, Ovalbumin, Receptors, Antigen, T-Cell, T-Lymphocytes, Transgenes, Vaccination
Show Abstract · Added September 24, 2018
A cell therapy platform permitting long-term delivery of peptide hormones in vivo would be a significant advance for patients with hormonal deficiencies. Here we report the utility of antigen-specific T lymphocytes as a regulatable peptide delivery platform for in vivo therapy. piggyBac transposon modification of murine cells with luciferase allows us to visualize T cells after adoptive transfer. Vaccination stimulates long-term T-cell engraftment, persistence, and transgene expression enabling detection of modified cells up to 300 days after adoptive transfer. We demonstrate adoptive transfer of antigen-specific T cells expressing erythropoietin (EPO) elevating the hematocrit in mice for more than 20 weeks. We extend our observations to human T cells demonstrating inducible EPO production from Epstein-Barr virus (EBV) antigen-specific T lymphocytes. Our results reveal antigen-specific T lymphocytes to be an effective delivery platform for therapeutic molecules such as EPO in vivo, with important implications for other diseases that require peptide therapy.
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Transposable Element Exaptation into Regulatory Regions Is Rare, Influenced by Evolutionary Age, and Subject to Pleiotropic Constraints.
Simonti CN, Pavlicev M, Capra JA
(2017) Mol Biol Evol 34: 2856-2869
MeSH Terms: Animals, Biological Evolution, DNA Transposable Elements, Gene Expression Regulation, Genetic Pleiotropy, Humans, Mice, Promoter Regions, Genetic, Regulatory Sequences, Nucleic Acid
Show Abstract · Added March 14, 2018
Transposable element (TE)-derived sequences make up approximately half of most mammalian genomes, and many TEs have been co-opted into gene regulatory elements. However, we lack a comprehensive tissue- and genome-wide understanding of how and when TEs gain regulatory activity in their hosts. We evaluated the prevalence of TE-derived DNA in enhancers and promoters across hundreds of human and mouse cell lines and primary tissues. Promoters are significantly depleted of TEs in all tissues compared with their overall prevalence in the genome (P < 0.001); enhancers are also depleted of TEs, though not as strongly as promoters. The degree of enhancer depletion also varies across contexts (1.5-3×), with reproductive and immune cells showing the highest levels of TE regulatory activity in humans. Overall, in spite of the regulatory potential of many TE sequences, they are significantly less active in gene regulation than expected from their prevalence. TE age is predictive of the likelihood of enhancer activity; TEs originating before the divergence of amniotes are 9.2 times more likely to have enhancer activity than TEs that integrated in great apes. Context-specific enhancers are more likely to be TE-derived than enhancers active in multiple tissues, and young TEs are more likely to overlap context-specific enhancers than old TEs (86% vs. 47%). Once TEs obtain enhancer activity in the host, they have similar functional dynamics to one another and non-TE-derived enhancers, likely driven by pleiotropic constraints. However, a few TE families, most notably endogenous retroviruses, have greater regulatory potential. Our observations suggest a model of regulatory co-option in which TE-derived sequences are initially repressed, after which a small fraction obtains context-specific enhancer activity, with further gains subject to pleiotropic constraints.
© The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.
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Comparative analysis of chimeric ZFP-, TALE- and Cas9-piggyBac transposases for integration into a single locus in human cells.
Luo W, Galvan DL, Woodard LE, Dorset D, Levy S, Wilson MH
(2017) Nucleic Acids Res 45: 8411-8422
MeSH Terms: Bacterial Proteins, CRISPR-Associated Protein 9, CRISPR-Cas Systems, Cell Line, Tumor, DNA Transposable Elements, Endonucleases, Gene Knockout Techniques, Gene Targeting, Gene Transfer Techniques, Humans, Hypoxanthine Phosphoribosyltransferase, Mutagenesis, Insertional, Recombinant Fusion Proteins, Reproducibility of Results, Transcription Activator-Like Effector Nucleases, Transcription Activator-Like Effectors, Transposases, Zinc Fingers
Show Abstract · Added September 11, 2017
Integrating DNA delivery systems hold promise for many applications including treatment of diseases; however, targeted integration is needed for improved safety. The piggyBac (PB) transposon system is a highly active non-viral gene delivery system capable of integrating defined DNA segments into host chromosomes without requiring homologous recombination. We systematically compared four different engineered zinc finger proteins (ZFP), four transcription activator-like effector proteins (TALE), CRISPR associated protein 9 (SpCas9) and the catalytically inactive dSpCas9 protein fused to the amino-terminus of the transposase enzyme designed to target the hypoxanthine phosphoribosyltransferase (HPRT) gene located on human chromosome X. Chimeric transposases were evaluated for expression, transposition activity, chromatin immunoprecipitation at the target loci, and targeted knockout of the HPRT gene in human cells. One ZFP-PB and one TALE-PB chimera demonstrated notable HPRT gene targeting. In contrast, Cas9/dCas9-PB chimeras did not result in gene targeting. Instead, the HPRT locus appeared to be protected from transposon integration. Supplied separately, PB permitted highly efficient isolation of Cas9-mediated knockout of HPRT, with zero transposon integrations in HPRT by deep sequencing. In summary, these tools may allow isolation of 'targeted-only' cells, be utilized to protect a genomic locus from transposon integration, and enrich for Cas9-mutated cells.
Published by Oxford University Press on behalf of Nucleic Acids Research 2017.
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18 MeSH Terms
De novo RNA sequence assembly during in vivo inflammatory stress reveals hundreds of unannotated lincRNAs in human blood CD14 monocytes and in adipose tissue.
Xue C, Zhang X, Zhang H, Ferguson JF, Wang Y, Hinkle CC, Li M, Reilly MP
(2017) Physiol Genomics 49: 287-305
MeSH Terms: Adipose Tissue, Adolescent, Adult, DNA Transposable Elements, Endotoxemia, Gene Expression Profiling, Genome-Wide Association Study, Humans, Inflammation, Lipopolysaccharide Receptors, Male, Middle Aged, Monocytes, Polymorphism, Single Nucleotide, RNA, Long Noncoding, Young Adult
Show Abstract · Added June 6, 2017
Long intergenic noncoding RNAs (lincRNAs) have emerged as key regulators of cellular functions and physiology. Yet functional lincRNAs often have low, context-specific and tissue-specific expression. We hypothesized that many human monocyte and adipose lincRNAs would be absent in current public annotations due to lincRNA tissue specificity, modest sequencing depth in public data, limitations of transcriptome assembly algorithms, and lack of dynamic physiological contexts. Deep RNA sequencing (RNA-Seq) was performed in peripheral blood CD14 monocytes (monocytes; average ~247 million reads per sample) and adipose tissue (average ~378 million reads per sample) collected before and after human experimental endotoxemia, an in vivo inflammatory stress, to identify tissue-specific and clinically relevant lincRNAs. Using a stringent filtering pipeline, we identified 109 unannotated lincRNAs in monocytes and 270 unannotated lincRNAs in adipose. Most unannotated lincRNAs are not conserved in rodents and are tissue specific, while many have features of regulated expression and are enriched in transposable elements. Specific subsets have enhancer RNA characteristics or are expressed only during inflammatory stress. A subset of unannotated lincRNAs was validated and replicated for their presence and inflammatory induction in independent human samples and for their monocyte and adipocyte origins. Through interrogation of public genome-wide association data, we also found evidence of specific disease association for selective unannotated lincRNAs. Our findings highlight the critical need to perform deep RNA-Seq in a cell-, tissue-, and context-specific manner to annotate the full repertoire of human lincRNAs for a complete understanding of lincRNA roles in dynamic cell functions and in human disease.
Copyright © 2017 the American Physiological Society.
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16 MeSH Terms
Kidney-specific transposon-mediated gene transfer in vivo.
Woodard LE, Cheng J, Welch RC, Williams FM, Luo W, Gewin LS, Wilson MH
(2017) Sci Rep 7: 44904
MeSH Terms: Acute Kidney Injury, Animals, DNA Transposable Elements, Erythropoietin, Gene Expression, Gene Expression Regulation, Gene Transfer Techniques, Genes, Reporter, Genetic Vectors, Hydrodynamics, Immunosuppressive Agents, Kidney, Male, Mice, Organ Specificity, Promoter Regions, Genetic, Transfection
Show Abstract · Added September 11, 2017
Methods enabling kidney-specific gene transfer in adult mice are needed to develop new therapies for kidney disease. We attempted kidney-specific gene transfer following hydrodynamic tail vein injection using the kidney-specific podocin and gamma-glutamyl transferase promoters, but found expression primarily in the liver. In order to achieve kidney-specific transgene expression, we tested direct hydrodynamic injection of a DNA solution into the renal pelvis and found that luciferase expression was strong in the kidney and absent from extra-renal tissues. We observed heterogeneous, low-level transfection of the collecting duct, proximal tubule, distal tubule, interstitial cells, and rarely glomerular cells following injection. To assess renal injury, we performed the renal pelvis injections on uninephrectomised mice and found that their blood urea nitrogen was elevated at two days post-transfer but resolved within two weeks. Although luciferase expression quickly decreased following renal pelvis injection, the use of the piggyBac transposon system improved long-term expression. Immunosuppression with cyclophosphamide stabilised luciferase expression, suggesting immune clearance of the transfected cells occurs in immunocompetent animals. Injection of a transposon expressing erythropoietin raised the haematocrit, indicating that the developed injection technique can elicit a biologic effect in vivo. Hydrodynamic renal pelvis injection enables transposon mediated-kidney specific gene transfer in adult mice.
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17 MeSH Terms