Nathalie Conte

PiggyBac Transposon Mutagenesis: A Tool for Cancer Gene Discovery in Mice

Piggybacking on Cancer Genes Transposons are mobile segments of DNA that can insert in or near important genes to cause mutations that disrupt gene function. Rad et al. (p. 1104, published online 14 October) adapted a mutagenic transposon called Piggybac, originally derived from a moth, into a tool for discovery of cancer-causing genes in mice. Mobilization of Piggybac in mice was associated with the development of leukemias and solid tumors. In many instances the causative mutations, which were identified by mapping the Piggybac integration sites, were within genes not previously implicated in cancer. Mutations induced by a transposable element in mice can be used to identify cancer-causing genes. Transposons are mobile DNA segments that can disrupt gene function by inserting in or near genes. Here, we show that insertional mutagenesis by the PiggyBac transposon can be used for cancer gene discovery in mice. PiggyBac transposition in genetically engineered transposon-transposase mice induced cancers whose type (hematopoietic versus solid) and latency were dependent on the regulatory elements introduced into transposons. Analysis of 63 hematopoietic tumors revealed that PiggyBac is capable of genome-wide mutagenesis. The PiggyBac screen uncovered many cancer genes not identified in previous retroviral or Sleeping Beauty transposon screens, including Spic, which encodes a PU.1-related transcription factor, and Hdac7, a histone deacetylase gene. PiggyBac and Sleeping Beauty have different integration preferences. To maximize the utility of the tool, we engineered 21 mouse lines to be compatible with both transposon systems in constitutive, tissue- or temporal-specific mutagenesis. Mice with different transposon types, copy numbers, and chromosomal locations support wide applicability.

Extensive genomic copy number variation in embryonic stem cells

Recent analysis of the human and mouse genomes has revealed that highly identical duplicated elements account for >5% of the sequence content. These elements vary in copy number between individuals. Copy number variations (CNVs) contribute significantly to genetic differences among individuals and are increasingly recognized as a causal factor in human diseases with different etiologies. In inbred mouse strains, CNVs have been fixed by inbreeding, but they are highly variable among strains. Within strains, de novo germ-line CNVs can occur, leading to interindividual variation. By analyzing the genome of clonal isolates of mouse ES cells derived from common parental lines, we have uncovered extensive and recurrent CNVs. This variation arises during mitosis and can be cotransmitted into the mouse germ line along with engineered alleles, contributing to genetic variability. The frequency and extent of these genomic changes in ES cells suggests that all somatic tissues in individuals will be mosaics composed of variants of the zygotic genome. Human ES (hES) cells and derived somatic lineages may be similarly affected, challenging the concept of a stable somatic genome.

Histone deacetylase 1 and 2 are essential for normal T-cell development and genomic stability in mice

Histone deacetylase 1 and 2 (HDAC1/2) regulate chromatin structure as the catalytic core of the Sin3A, NuRD and CoREST co-repressor complexes. To better understand the key pathways regulated by HDAC1/2 in the adaptive immune system and inform their exploitation as drug targets, we have generated mice with a T-cell specific deletion. Loss of either HDAC1 or HDAC2 alone has little effect, while dual inactivation results in a 5-fold reduction in thymocyte cellularity, accompanied by developmental arrest at the double-negative to double-positive transition. Transcriptome analysis revealed 892 misregulated genes in Hdac1/2 knock-out thymocytes, including down-regulation of LAT, Themis and Itk, key components of the T-cell receptor (TCR) signaling pathway. Down-regulation of these genes suggests a model in which HDAC1/2 deficiency results in defective propagation of TCR signaling, thus blocking development. Furthermore, mice with reduced HDAC1/2 activity (Hdac1 deleted and a single Hdac2 allele) develop a lethal pathology by 3-months of age, caused by neoplastic transformation of immature T cells in the thymus. Tumor cells become aneuploid, express increased levels of c-Myc and show elevated levels of the DNA damage marker, γH2AX. These data demonstrate a crucial role for HDAC1/2 in T-cell development and the maintenance of genomic stability.

The piggyBac Transposon Displays Local and Distant Reintegration Preferences and Can Cause Mutations at Noncanonical Integration Sites

ABSTRACT The DNA transposon piggyBac is widely used as a tool in mammalian experimental systems for transgenesis, mutagenesis, and genome engineering. We have characterized genome-wide insertion site preferences of piggyBac by sequencing a large set of integration sites arising from transposition from two separate genomic loci and a plasmid donor in mouse embryonic stem cells. We found that piggyBac preferentially integrates locally to the excision site when mobilized from a chromosomal location and identified other nonlocal regions of the genome with elevated insertion frequencies. piggyBac insertions were associated with expressed genes and markers of open chromatin structure and were excluded from heterochromatin. At the nucleotide level, piggyBac prefers to insert into TA-rich regions within a broader GC-rich context. We also found that piggyBac can insert into sites other than its known TTAA insertion site at a low frequency (2%). Such insertions introduce mismatches that are repaired with signatures of host cell repair pathways. Transposons could be mobilized from plasmids with the observed noncanonical flanking regions, indicating that piggyBac could generate point mutations in the genome.

Disease model discovery from 3,328 gene knockouts by The International Mouse Phenotyping Consortium.

Although next-generation sequencing has revolutionized the ability to associate variants with human diseases, diagnostic rates and development of new therapies are still limited by a lack of knowledge of the functions and pathobiological mechanisms of most genes. To address this challenge, the International Mouse Phenotyping Consortium is creating a genome- and phenome-wide catalog of gene function by characterizing new knockout-mouse strains across diverse biological systems through a broad set of standardized phenotyping tests. All mice will be readily available to the biomedical community. Analyzing the first 3,328 genes identified models for 360 diseases, including the first models, to our knowledge, for type C Bernard–Soulier, Bardet–Biedl-5 and Gordon Holmes syndromes. 90% of our phenotype annotations were novel, providing functional evidence for 1,092 genes and candidates in genetically uncharacterized diseases including arrhythmogenic right ventricular dysplasia 3. Finally, we describe our role in variant functional validation with The 100,000 Genomes Project and others.

Chromosome instability induced by Mps1 and p53 mutation generates aggressive lymphomas exhibiting aneuploidy-induced stress

Significance Normal cells rarely missegregate chromosomes, but the majority of cancer cells have a chromosomal instability (CIN) phenotype that makes errors more common and results in abnormal chromosomal content (aneuploidy). Although aneuploidy promotes transformation via gain of oncogenes and loss of tumor suppressors, it also slows cell proliferation and disrupts metabolic homeostasis. Aneuploidy therefore represents a liability as well as a source of selective advantage for cancer cells. We provoked CIN in murine T cells by weakening the spindle-assembly checkpoint and then studied the consequences. We found that CIN dramatically accelerates cancer in a genetically predisposed background and that the resulting aneuploid cancers are metabolically deranged, a vulnerability that may open new avenues to treating aneuploid cancers. Aneuploidy is a hallmark of human solid cancers that arises from errors in mitosis and results in gain and loss of oncogenes and tumor suppressors. Aneuploidy poses a growth disadvantage for cells grown in vitro, suggesting that cancer cells adapt to this burden. To understand better the consequences of aneuploidy in a rapidly proliferating adult tissue, we engineered a mouse in which chromosome instability was selectively induced in T cells. A flanked by Lox mutation was introduced into the monopolar spindle 1 (Mps1) spindle-assembly checkpoint gene so that Cre-mediated recombination would create a truncated protein (Mps1DK) that retained the kinase domain but lacked the kinetochore-binding domain and thereby weakened the checkpoint. In a sensitized p53+/− background we observed that Mps1DK/DK mice suffered from rapid-onset acute lymphoblastic lymphoma. The tumors were highly aneuploid and exhibited a metabolic burden similar to that previously characterized in aneuploid yeast and cultured cells. The tumors nonetheless grew rapidly and were lethal within 3–4 mo after birth.

Loss of Rassf1a cooperates with Apc(Min) to accelerate intestinal tumourigenesis

Promoter methylation of the RAS-association domain family 1, isoform A gene (RASSF1A) is one of the most frequent events found in human tumours. In this study we set out to test the hypothesis that loss of Rassf1a can cooperate with inactivation of the adenomatous polyposis coli (Apc) gene to accelerate intestinal tumourigenesis using the Apc-Min (ApcMin/+) mouse model, as mutational or deletional inactivation of APC is a frequent early event in the genesis of intestinal cancer. Further, loss of RASSF1A has also been reported to occur in premalignant adenomas of the bowel. RASSF1A has been implicated in an array of pivotal cellular processes, including regulation of the cell cycle, apoptosis, microtubule stability and most recently in the β-catenin signalling pathway. By interbreeding isoform specific Rassf1a knockout mice with Apc+/Min mice, we showed that loss of Rassf1a results in a significant increase in adenomas of the small intestine and accelerated intestinal tumourigenesis leading to the earlier death of adenocarcinoma-bearing mice and decreased overall survival. Comparative genomic hybridization of adenomas from Rassf1a−/−; Apc+/Min mice revealed no evidence of aneuploidy or gross chromosomal instability (no difference to adenomas from Rassf1a+/+; Apc+/Min mice). Immunohistochemical analysis of adenomas revealed increased nuclear β-catenin accumulation in adenomas from Rassf1a−/−; Apc+/Min mice, compared to those from Rassf1a+/+; Apc+/Min mice, but no differences in proliferation marker (Ki67) staining patterns. Collectively these data demonstrate cooperation between inactivation of Rassf1a and Apc resulting in accelerated intestinal tumourigenesis, with adenomas showing increased nuclear accumulation of β-catenin, supporting a mechanistic link via loss of the known interaction of Rassf1 with β-TrCP that usually mediates degradation of β-catenin.

Detailed molecular characterisation of acute myeloid leukaemia with a normal karyotype using targeted DNA capture.

Advances in sequencing technologies are giving unprecedented insights into the spectrum of somatic mutations underlying acute myeloid leukaemia with a normal karyotype (AML–NK). It is clear that the prognosis of individual patients is strongly influenced by the combination of mutations in their leukaemia and that many leukaemias are composed of multiple subclones, with differential susceptibilities to treatment. Here, we describe a method, employing targeted capture coupled with next-generation sequencing and tailored bioinformatic analysis, for the simultaneous study of 24 genes recurrently mutated in AML–NK. Mutational analysis was performed using open source software and an in-house script (Mutation Identification and Analysis Software), which identified dominant clone mutations with 100% specificity. In each of seven cases of AML–NK studied, we identified and verified mutations in 2–4 genes in the main leukaemic clone. Additionally, high sequencing depth enabled us to identify putative subclonal mutations and detect leukaemia-specific mutations in DNA from remission marrow. Finally, we used normalised read depths to detect copy number changes and identified and subsequently verified a tandem duplication of exons 2–9 of MLL and at least one deletion involving PTEN. This methodology reliably detects sequence and copy number mutations, and can thus greatly facilitate the classification, clinical research, diagnosis and management of AML–NK.

Mutational History of a Human Cell Lineage from Somatic to Induced Pluripotent Stem Cells.

The accuracy of replicating the genetic code is fundamental. DNA repair mechanisms protect the fidelity of the genome ensuring a low error rate between generations. This sustains the similarity of individuals whilst providing a repertoire of variants for evolution. The mutation rate in the human genome has recently been measured to be 50–70 de novo single nucleotide variants (SNVs) between generations. During development mutations accumulate in somatic cells so that an organism is a mosaic. However, variation within a tissue and between tissues has not been analysed. By reprogramming somatic cells into induced pluripotent stem cells (iPSCs), their genomes and the associated mutational history are captured. By sequencing the genomes of polyclonal and monoclonal somatic cells and derived iPSCs we have determined the mutation rates and show how the patterns change from a somatic lineage in vivo through to iPSCs. Somatic cells have a mutation rate of 14 SNVs per cell per generation while iPSCs exhibited a ten-fold lower rate. Analyses of mutational signatures suggested that deamination of methylated cytosine may be the major mutagenic source in vivo, whilst oxidative DNA damage becomes dominant in vitro. Our results provide insights for better understanding of mutational processes and lineage relationships between human somatic cells. Furthermore it provides a foundation for interpretation of elevated mutation rates and patterns in cancer.

Identifiers for the 21st century: How to design, provision, and reuse persistent identifiers to maximize utility and impact of life science data

In many disciplines, data are highly decentralized across thousands of online databases (repositories, registries, and knowledgebases). Wringing value from such databases depends on the discipline of data science and on the humble bricks and mortar that make integration possible; identifiers are a core component of this integration infrastructure. Drawing on our experience and on work by other groups, we outline 10 lessons we have learned about the identifier qualities and best practices that facilitate large-scale data integration. Specifically, we propose actions that identifier practitioners (database providers) should take in the design, provision and reuse of identifiers. We also outline the important considerations for those referencing identifiers in various circumstances, including by authors and data generators. While the importance and relevance of each lesson will vary by context, there is a need for increased awareness about how to avoid and manage common identifier problems, especially those related to persistence and web-accessibility/resolvability. We focus strongly on web-based identifiers in the life sciences; however, the principles are broadly relevant to other disciplines.