The utility of transposon mutagenesis for cancer studies in the era of genome editing

DeNicola et al. Genome Biology (2015) 16:229 DOI 10.1186/s13059-015-0794-y REVIEW Open Access The utility of transposon mutagenesis for cancer studies in the era of genome editing Gina M. DeNicola1†, Florian A. Karreth1*†, David J. Adams2 and Chi C. Wong2,3* Abstract The use of transposons as insertional mutagens to identify cancer genes in mice has generated a wealth of information over the past decade. Here, we discuss recent major advances in transposon-mediated insertional mutagenesis screens and compare this technology with other screening strategies. Introduction Genome sequencing has revealed a plethora of mutations in cancer, with some tumors carrying tens of thousands of somatic mutations [1]. Importantly, the relevance of these mutations is not always intrinsically clear and as a result must be inferred from the types of mutations observed, their frequency across tumor types, and their predicted effects on protein function. Insertional mutagenesis screens provide a functional readout to complement these sequencing studies, as genes identified by insertional mutagens are likely to represent both functionally important and evolutionarily conserved cancer genes. Insertional mutagenesis studies can also highlight cancer genes or common pathways that are disrupted at low frequency or by processes not immediately obvious from the genome sequence alone. The first insertional mutagenesis efforts in mice were performed with the murine leukemia virus and the mouse mammary transforming virus to induce lymphoma and mammary tumors [2, 3], respectively, and led to the identification of numerous cancer pathways, including the WNT pathway [4]. However, these viruses were found to be of limited utility for mutagenesis in other tissue types owing to viral tropism and the fact * Correspondence: ; † Equal contributors 1 Meyer Cancer Center, Weill Cornell Medical College, New York, NY 10021, USA 2 Experimental Cancer Genetics, Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton CB10 1HH, UK Full list of author information is available at the end of the article that they only infect replicating cells [5]. Furthermore, as these retroviruses generate insertions that activate gene expression, they almost exclusively tag proto-oncogenes [5], restricting our ability to identify other types of cancer genes such as tumor suppressors. For these reasons, DNA transposons were developed as insertional mutagens [6]. Transposons are mobile elements that move through the genome by a cut-andpaste process (DNA transposons), or through an RNA intermediate in a copy-and-paste mechanism (retrotransposons) [7]. Endogenous transposons are ubiquitous in vertebrate genomes, comprising approximately 45 % of DNA sequence [8], but are largely silent as a result of inactivating mutations acquired through evolution. The introduction of exogenous DNA transposons allows insertional mutagenesis in a wider spectrum of tissues than the ones that are accessible with retroviruses, and thus the generation of new mouse tumor models [9, 10]. The most commonly used transposon systems are the Sleeping Beauty (SB) and piggyBac (PB) systems [11]. A typical transposon used for in vivo insertional mutagenesis contains splice acceptors (SAs) followed by polyadenylation signals (pA) in both orientations, and a unidirectional promoter upstream of a splice donor (SD). A transposon can either disrupt gene function when it integrates into the body of a gene, thereby intercepting and curtailing transcription through the SA–pA elements, or it can activate expression when inserted upstream of a gene as the promoter–SD module drives expression of downstream sequences (Fig. 1). The pattern and orientation of transposon integration sites therefore often provide a clue as to whether the affected gene encodes a tumor suppressor or an oncogene. Here, we discuss recent advances in cancer gene discovery using transposons and their role in the era of other mutagenesis tools such as clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9). © 2015 DeNicola et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. DeNicola et al. Genome Biology (2015) 16:229 Page 2 of 15 (a) SB SB SB SB TACWGTA TA PB PB TTAA TTAA SB PB PB PB Transposon reintegration Transposon mobilization (b) 1 2 3 4 5 6 7 Intragenic transposon insertion (I) (II) AAAAA 04 1 2 3 SA pA Transcriptional repression AAAAA promoter SD 4 5 6 7 Transcriptional activation Fig. 1 Transposons as insertional mutagens. a Sleeping Beauty (SB) and piggyBac (PB) (black rectangles) are mutagenic transposons that can be mobilized from donor loci (left panel) and reintegrated into other loci (right panel). Repeats in the transposon (arrowheads) are recognized by the Sleeping Beauty or piggyBac transposases (ovals), resulting in the transposon being excised from the genome. Reintegration of mobilized SB or PB transposons can occur at TA and TTAA sites, respectively, catalyzed by transposase activity. b Transposon insertion can promote or disrupt gene expression. In the example depicted in this panel, a transposon integrates between exons 3 and 4 (numbered gray boxes) of a gene. This can result in two possible outcomes: (I) the transposon disrupts gene function by hijacking transcription through the splice acceptor-polyadenylation signal (SA-pA) elements, leading to expression of a truncated transcript (exons 1–3); or (II) the transposon drives expression of the downstream gene sequences (exons 4–7) through the promoter-splice donor (SD) elements. Depending on the integration site, transposons can activate or abrogate expression of either the entire mRNA of a gene or only parts of it Transposon-mediated insertional mutagenesis In 2005, the groups of David Largaespada, Nancy Jenkins and Neal Copeland reported the use of the Sleeping Beauty transposon system as a tool for the identification of cancer-promoting genes in transgenic mice [12, 13]. Largaespada and colleagues performed whole-body transposon-mediated insertional mutagenesis (TMIM) with the first-generation T2/Onc transposon, accelerating tumorigenesis in mice null for the tumor suppressor p19Arf gene [12]. Using a more active transposon system (T2/Onc2), Dupuy and colleagues induced predominantly hematopoietic tumors following global mutagenesis in wild-type mice [13]. Following these landmark studies, a (...truncated)