Cancer Gene Discovery Using the Sleeping Beauty Transposon

Epidemiological and molecular data support the hypothesis that cancer results from a series of acquired somatic mutations. Discovering the initial mutations required for oncogenesis has long been a goal of cancer research. To date, the majority of causative mutations have been identified based on their ability to act in a dominant fashion and/or because they are activated by chromosomal translocations. Forward genetic screens are necessary for unbiased discovery of the remaining unknown oncogenic mutations. Two recent projects have demonstrated the feasibility of using the Sleeping Beauty transposon as an insertional mutagen for cancer gene discovery. In this article we discuss the history of cancer gene discovery and propose novel forward genetic screens using Sleeping Beauty transposon aimed at specific tissues and accelerating the discovery of recessive tumor suppressor genes.     

Tandem gene arrays: a challenge for functional genomics

In sequenced plant genomes, 15% or more of the identified genes are members of tandem-arrayed gene families. Because mutating only one gene in a duplicated pair often produces no measurable phenotype, this poses a particular challenge for functional analysis. To generate phenotypic knockouts, it is necessary to create deletions that affect multiple genes, select for rare meiotic recombination between tightly linked loci, or perform sequential mutant screens in the same plant line. Successfully implemented strategies include PCR-based screening for fast neutron-induced deletions, selection for recombination between herbicide resistance markers, and localized transposon mutagenesis. Here, we review the relative merits of current genetic approaches and discuss the prospect of site-directed mutagenesis for generating elusive knockouts of tandem-arrayed gene families.     

A Genetic Screen Using the PiggyBac Transposon in Haploid Cells Identifies Parp1 as a Mediator of Olaparib Toxicity

Genetic perturbation screens have the potential to dissect a wide range of cellular phenotypes. Such screens have historically been difficult in diploid mammalian cells. The recent derivation of haploid embryonic stem cells provides an opportunity to cause loss of function mutants with a random mutagen in a mammalian cell with a normal genetic background. We describe an approach to genetic screens that exploits the highly active piggyBac transposon in haploid mammalian cells. As an example of haploid transposon (HTP) screening, we apply this approach to identifying determinants of cancer drug toxicity and resistance. In a screen for 6-thioguanine resistance we recovered components of the DNA mismatch repair pathway, a known requirement for toxicity. In a further screen for resistance to the clinical poly(ADP-ribose) polymerase (PARP) inhibitor olaparib we recovered multiple Parp1 mutants. Our results show that olaparib toxicity to normal cells is mediated predominantly via Parp1, and suggest that the clinical side effects of olaparib may be on target. The transposon mutant libraries are stable and can be readily reused to screen other drugs. The screening protocol described has several advantages over other methods such as RNA interference: it is rapid and low cost, and mutations can be easily reverted to establish causality.     

A PATO-compliant zebrafish screening database (MODB): management of morpholino knockdown screen information

Background  

The zebrafish is a powerful model vertebrate amenable to high throughput in vivo genetic analyses. Examples include reverse genetic screens using morpholino knockdown, expression-based screening using enhancer trapping and forward genetic screening using transposon insertional mutagenesis. We have created a database to facilitate web-based distribution of data from such genetic studies.     

Manipulating the <Emphasis Type="Italic">Xenopus</Emphasis>genome with transposable elements

The study of amphibian embryogenesis has provided important insight into the mechanisms of vertebrate development. The frog Xenopus laevis has been an important model of vertebrate cell biology and development for many decades. Genetic studies in this organism are not practical because of the tetraploid nature of the genome and the long generation time of this species. Recently, a closely related frog, namely Xenopus tropicalis, has been proposed as an alternative system; it shares all of the physical characteristics that make X. laevis a useful model but has the advantage of a diploid genome and short generation time. The rapid accumulation of genetic resources for this animal and the success of pilot mutagenesis screens have helped propel this model system forward. Transposable elements will provide invaluable tools for manipulating the frog genome. These integration systems are ideally suited to transgenesis and insertional mutagenesis strategies in the frog. The high fecundity of the frog combined with the ability to remobilize transposon transgenes integrated into frog genome will allow large-scale insertional mutagenesis screens to be performed in laboratories with modest husbandry capacities.     

Characterization of Mos1-mediated mutagenesis in Caenorhabditis elegans: a method for the rapid identification of mutated genes

Insertional mutagenesis with a heterologous transposon provides a method to rapidly determine the molecular identity of mutated genes. The Drosophila transposon Mos1 can be mobilized to cause mutations in Caenorhabditis elegans (Bessereau et al. 2001); however, the mutagenic rate was initially too low for use in most forward genetic screens. To increase the effectiveness of Mos1-mediated mutagenesis we examined the conditions influencing Mos1 transposition. First, optimal transposition occurs 24 hr after expression of the transposase and is unlikely to occur in differentiated sperm or oocytes. Second, transposition is limited to germ-cell nuclei that contain donor elements, but the transposase enzyme can diffuse throughout the gonad syncytium. Third, silencing of transposition is caused by changes in the donor array that occur over time. Finally, multiple transposition events occur in individual germ cells. By using screening techniques based on these results, Mos1 mutagenicity was increased to within an order of magnitude of chemical mutagens.     

Toward forward genetic screens in malaria-causing parasites using the piggyBac transposon

The ability to analyze gene function in malaria-causing Plasmodium parasites has received a boost with a recent paper in BMC Genomics that describes a genome-wide mutagenesis system in the rodent malaria species Plasmodium berghei using the transposon piggyBac. This advance holds promise for identifying and validating new targets for intervention against malaria. But further improvements are still needed for the full power of genome-wide molecular genetic screens to be utilized in this organism.     

A piggyBac transposon-based mutagenesis system for the fission yeast Schizosaccharomyces pombe

The TTAA-specific transposon piggyBac (PB), originally isolated from the cabbage looper moth, Trichoplusia ni, has been utilized as an insertional mutagenesis tool in various eukaryotic organisms. Here, we show that PB transposes in the fission yeast Schizosaccharomyces pombe and leaves almost no footprints. We developed a PB-based mutagenesis system for S. pombe by constructing a strain with a selectable transposon excision marker and an integrated transposase gene. PB transposition in this strain has low chromosomal distribution bias as shown by deep sequencing-based insertion site mapping. Using this system, we obtained loss-of-function alleles of klp5 and klp6, and a gain-of-function allele of dam1 from a screen for mutants resistant to the microtubule-destabilizing drug thiabendazole. From another screen for cdc25-22 suppressors, we obtained multiple alleles of wee1 as expected. The success of these two screens demonstrated the usefulness of this PB-mediated mutagenesis tool for fission yeast.     

Silent mischief: bacteriophage Mu insertions contaminate products of Escherichia coli random mutagenesis performed using suicidal transposon delivery plasmids mobilized by broad-host-range RP4 conjugative machinery

Random transposon mutagenesis is the strategy of choice for associating a phenotype with its unknown genetic determinants. It is generally performed by mobilization of a conditionally replicating vector delivering transposons to recipient cells using broad-host-range RP4 conjugative machinery carried by the donor strain. In the present study, we demonstrate that bacteriophage Mu, which was deliberately introduced during the original construction of the widely used donor strains SM10 λpir and S17-1 λpir, is silently transferred to Escherichia coli recipient cells at high frequency, both by hfr and by release of Mu particles by the donor strain. Our findings suggest that bacteriophage Mu could have contaminated many random-mutagenesis experiments performed on Mu-sensitive species with these popular donor strains, leading to potential misinterpretation of the transposon mutant phenotype and therefore perturbing analysis of mutant screens. To circumvent this problem, we precisely mapped Mu insertions in SM10 λpir and S17-1 λpir and constructed a new Mu-free donor strain, MFDpir, harboring stable hfr-deficient RP4 conjugative functions and sustaining replication of Π-dependent suicide vectors. This strain can therefore be used with most of the available transposon-delivering plasmids and should enable more efficient and easy-to-analyze mutant hunts in E. coli and other Mu-sensitive RP4 host bacteria.     

Forward and reverse genetics through derivation of haploid mouse embryonic stem cells

All somatic mammalian cells carry two copies of chromosomes (diploidy), whereas organisms with a single copy of their genome, such as yeast, provide a basis for recessive genetics. Here we report the generation of haploid mouse ESC lines from parthenogenetic embryos. These cells carry 20 chromosomes, express stem cell markers, and develop into all germ layers in vitro and in vivo. We also developed a reversible mutagenesis protocol that allows saturated genetic recessive screens and results in homozygous alleles. This system allowed us to generate a knockout cell line for the microRNA processing enzyme Drosha. In a forward genetic screen, we identified Gpr107 as a molecule essential for killing by ricin, a toxin being used as a bioweapon. Our results open the possibility of combining the power of a haploid genome with pluripotency of embryonic stem cells to uncover fundamental biological processes in defined cell types at a genomic scale.     

Spontaneous mutagenesis is enhanced in Apex heterozygous mice

Germ line DNA directs the development of the next generation and, as such, is profoundly different from somatic cell DNA. Spermatogenic cells obtained from young adult lacI transgenic mice display a lower spontaneous mutant frequency and greater in vitro base excision repair activity than somatic cells and tissues obtained from the same mice. However, spermatogenic cells from old lacI mice display a 10-fold higher mutant frequency. This increased spontaneous mutant frequency occurs coincidentally with decreased in vitro base excision repair activity for germ cell and testicular extracts that in turn corresponds to a decreased abundance of AP endonuclease. To directly test whether a genetic diminution of AP endonuclease results in increased spontaneous mutant frequencies in spermatogenic cell types, AP endonuclease heterozygous (Apex(+/-)) knockout mice were crossed with lacI transgenic mice. Spontaneous mutant frequencies were significantly elevated (approximately twofold) for liver and spleen obtained from 3-month-old Apex(+/-) lacI(+) mice compared to frequencies from Apex(+/+) lacI(+) littermates and were additionally elevated for somatic tissues from 9-month-old mice. Spermatogenic cells from 9-month-old Apex(+/-) lacI(+) mice were significantly elevated twofold compared to levels for 9-month-old Apex(+/+) lacI(+) control mice. These data indicate that diminution of AP endonuclease has a significant effect on spontaneous mutagenesis in somatic and germ line cells.     

Clonal Expansion Analysis of Transposon Insertions by High-Throughput Sequencing Identifies Candidate Cancer Genes in a PiggyBac Mutagenesis Screen

Somatic transposon mutagenesis in mice is an efficient strategy to investigate the genetic mechanisms of tumorigenesis. The identification of tumor driving transposon insertions traditionally requires the generation of large tumor cohorts to obtain information about common insertion sites. Tumor driving insertions are also characterized by their clonal expansion in tumor tissue, a phenomenon that is facilitated by the slow and evolving transformation process of transposon mutagenesis. We describe here an improved approach for the detection of tumor driving insertions that assesses the clonal expansion of insertions by quantifying the relative proportion of sequence reads obtained in individual tumors. To this end, we have developed a protocol for insertion site sequencing that utilizes acoustic shearing of tumor DNA and Illumina sequencing. We analyzed various solid tumors generated by PiggyBac mutagenesis and for each tumor >10⁶ reads corresponding to >10⁴ insertion sites were obtained. In each tumor, 9 to 25 insertions stood out by their enriched sequence read frequencies when compared to frequencies obtained from tail DNA controls. These enriched insertions are potential clonally expanded tumor driving insertions, and thus identify candidate cancer genes. The candidate cancer genes of our study comprised many established cancer genes, but also novel candidate genes such as Mastermind-like1 (Mamld1) and Diacylglycerolkinase delta (Dgkd). We show that clonal expansion analysis by high-throughput sequencing is a robust approach for the identification of candidate cancer genes in insertional mutagenesis screens on the level of individual tumors.     

Identification of mutations that delay somatic or reproductive aging of Caenorhabditis elegans

Aging is an important feature of animal biology characterized by progressive, degenerative changes in somatic and reproductive tissues. The rate of age-related degeneration is genetically controlled, since genes that influence lifespan have been identified. However, little is known about genes that affect reproductive aging or aging of specific somatic tissues. To identify genes that are important for controlling these degenerative changes, we used chemical mutagenesis to perform forward genetic screens in Caenorhabditis elegans. By conducting a screen focused on somatic aging, we identified mutant hermaphrodites that displayed extended periods of pharyngeal pumping, body movement, or survival. One of these mutations is a novel allele of the age-1 gene. age-1 encodes a phosphatidylinositol-3-kinase (PI3K) that functions in the insulin/insulin-like growth factor-1 (IGF-1) signaling pathway. age-1(am88) creates a missense change in the conserved PIK domain and causes dramatic extensions of the pharyngeal pumping and body movement spans, as well as a twofold extension of the lifespan. By conducting screens focused on reproductive aging in mated hermaphrodites, we identified mutants that displayed increased progeny production late in life. To characterize these mutations, we developed quantitative measurements of age-related morphological changes in the gonad. The am117 mutation delayed age-related declines in progeny production and morphological changes in the gonad. These studies provide new insights into the genetic regulation of age-related degenerative changes in somatic and reproductive tissues.     

Mutagenesis is elevated in male germ cells obtained from DNA polymerase-beta heterozygous mice

Gametes carry the DNA that will direct the development of the next generation. By compromising genetic integrity, DNA damage and mutagenesis threaten the ability of gametes to fulfill their biological function. DNA repair pathways function in germ cells and serve to ameliorate much DNA damage and prevent mutagenesis. High base excision repair (BER) activity is documented for spermatogenic cells. DNA polymerase-beta (POLB) is required for the short-patch BER pathway. Because mice homozygous null for the Polb gene die soon after birth, mice heterozygous for Polb were used to examine the extent to which POLB contributes to maintaining spermatogenic genomic integrity in vivo. POLB protein levels were reduced only in mixed spermatogenic cells. In vitro short-patch BER activity assays revealed that spermatogenic cell nuclear extracts obtained from Polb heterozygous mice had one third the BER activity of age-matched control mice. Polb heterozygosity had no effect on the BER activities of somatic tissues tested. The Polb heterozygous mouse line was crossed with the lacI transgenic Big Blue mouse line to assess mutant frequency. The spontaneous mutant frequency for mixed spermatogenic cells prepared from Polb heterozygous mice was 2-fold greater than that of wild-type controls, but no significant effect was found among the somatic tissues tested. These results demonstrate that normal POLB abundance is necessary for normal BER activity, which is critical in maintaining a low germline mutant frequency. Notably, spermatogenic cells respond differently than somatic cells to Polb haploinsufficiency.     

Computational identification of insertional mutagenesis targets for cancer gene discovery

Insertional mutagenesis is a potent forward genetic screening technique used to identify candidate cancer genes in mouse model systems. An important, yet unresolved issue in the analysis of these screens, is the identification of the genes affected by the insertions. To address this, we developed Kernel Convolved Rule Based Mapping (KC-RBM). KC-RBM exploits distance, orientation and insertion density across tumors to automatically map integration sites to target genes. We perform the first genome-wide evaluation of the association of insertion occurrences with aberrant gene expression of the predicted targets in both retroviral and transposon data sets. We demonstrate the efficiency of KC-RBM by showing its superior performance over existing approaches in recovering true positives from a list of independently, manually curated cancer genes. The results of this work will significantly enhance the accuracy and speed of cancer gene discovery in forward genetic screens. KC-RBM is available as R-package.     

Transposon-mediated mutagenesis of somatic cells in the mouse for cancer gene identification

To successfully treat cancer we will likely need a much more detailed understanding of the genes and pathways meaningfully altered in individual cancer cases. One method for achieving this goal is to derive cancers in model organisms using unbiased forward genetic screens that allow cancer gene candidate discovery. We have developed a method using a “cut-and-paste” DNA transposon system called Sleeping Beauty (SB) to perform forward genetic screens for cancer genes in mice. Although the approach is conceptually similar to the use of replication competent retroviruses for cancer gene identification, the SB system promises to allow such screens in tissues previously not amenable to forward genetic screens such as the gastrointestinal tract, brain, and liver. This article describes the strains useful for SB-based screens for cancer genes in mice and how they are deployed in an experiment.     

A meta-analysis of kidney microarray datasets: investigation of cytokine gene detection and correlation with rt-PCR and detection thresholds

Background

Large-scale mutagenesis screens in the zebrafish employing the mutagen ENU have isolated several hundred mutant loci that represent putative developmental control genes. In order to realize the potential of such screens, systematic genetic mapping of the mutations is necessary. Here we report on a large-scale effort to map the mutations generated in mutagenesis screening at the Max Planck Institute for Developmental Biology by genome scanning with microsatellite markers.

Results

We have selected a set of microsatellite markers and developed methods and scoring criteria suitable for efficient, high-throughput genome scanning. We have used these methods to successfully obtain a rough map position for 319 mutant loci from the Tübingen I mutagenesis screen and subsequent screening of the mutant collection. For 277 of these the corresponding gene is not yet identified. Mapping was successful for 80 % of the tested loci. By comparing 21 mutation and gene positions of cloned mutations we have validated the correctness of our linkage group assignments and estimated the standard error of our map positions to be approximately 6 cM.

Conclusion

By obtaining rough map positions for over 300 zebrafish loci with developmental phenotypes, we have generated a dataset that will be useful not only for cloning of the affected genes, but also to suggest allelism of mutations with similar phenotypes that will be identified in future screens. Furthermore this work validates the usefulness of our methodology for rapid, systematic and inexpensive microsatellite mapping of zebrafish mutations.