Publications by authors named "Ruby Banerjee"

26 Publications

  • Page 1 of 1

Werner Helicase Is a Synthetic-Lethal Vulnerability in Mismatch Repair-Deficient Colorectal Cancer Refractory to Targeted Therapies, Chemotherapy, and Immunotherapy.

Cancer Discov 2021 Aug 9;11(8):1923-1937. Epub 2021 Apr 9.

Wellcome Sanger Institute, Cambridge, United Kingdom.

Targeted therapies, chemotherapy, and immunotherapy are used to treat patients with mismatch repair-deficient (dMMR)/microsatellite instability-high (MSI-H) colorectal cancer. The clinical effectiveness of targeted therapy and chemotherapy is limited by resistance and drug toxicities, and about half of patients receiving immunotherapy have disease that is refractory to immune checkpoint inhibitors. Loss of Werner syndrome ATP-dependent helicase (WRN) is a synthetic lethality in dMMR/MSI-H cells. To inform the development of WRN as a therapeutic target, we performed WRN knockout or knockdown in 60 heterogeneous dMMR colorectal cancer preclinical models, demonstrating that WRN dependency is an almost universal feature and a robust marker for patient selection. Furthermore, models of resistance to clinically relevant targeted therapy, chemotherapy, and immunotherapy retain WRN dependency. These data show the potential of therapeutically targeting WRN in patients with dMMR/MSI-H colorectal cancer and support WRN as a therapeutic option for patients with dMMR/MSI-H cancers refractory to current treatment strategies. SIGNIFICANCE: We found that a large, diverse set of dMMR/MSI-H colorectal cancer preclinical models, including models of treatment-refractory disease, are WRN-dependent. Our results support WRN as a promising synthetic-lethal target in dMMR/MSI-H colorectal cancer tumors as a monotherapy or in combination with targeted agents, chemotherapy, or immunotherapy..
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http://dx.doi.org/10.1158/2159-8290.CD-20-1508DOI Listing
August 2021

PiggyBac mutagenesis and exome sequencing identify genetic driver landscapes and potential therapeutic targets of EGFR-mutant gliomas.

Genome Biol 2020 07 30;21(1):181. Epub 2020 Jul 30.

The Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridgeshire, CB10 1SA, UK.

Background: Glioma is the most common intrinsic brain tumor and also occurs in the spinal cord. Activating EGFR mutations are common in IDH1 wild-type gliomas. However, the cooperative partners of EGFR driving gliomagenesis remain poorly understood.

Results: We explore EGFR-mutant glioma evolution in conditional mutant mice by whole-exome sequencing, transposon mutagenesis forward genetic screening, and transcriptomics. We show mutant EGFR is sufficient to initiate gliomagenesis in vivo, both in the brain and spinal cord. We identify significantly recurrent somatic alterations in these gliomas including mutant EGFR amplifications and Sub1, Trp53, and Tead2 loss-of-function mutations. Comprehensive functional characterization of 96 gliomas by genome-wide piggyBac insertional mutagenesis in vivo identifies 281 known and novel EGFR-cooperating driver genes, including Cdkn2a, Nf1, Spred1, and Nav3. Transcriptomics confirms transposon-mediated effects on expression of these genes. We validate the clinical relevance of new putative tumor suppressors by showing these are frequently altered in patients' gliomas, with prognostic implications. We discover shared and distinct driver mutations in brain and spinal gliomas and confirm in vivo differential tumor suppressive effects of Pten between these tumors. Functional validation with CRISPR-Cas9-induced mutations in novel genes Tead2, Spred1, and Nav3 demonstrates heightened EGFRvIII-glioma cell proliferation. Chemogenomic analysis of mutated glioma genes reveals potential drug targets, with several investigational drugs showing efficacy in vitro.

Conclusion: Our work elucidates functional driver landscapes of EGFR-mutant gliomas, uncovering potential therapeutic strategies, and provides new tools for functional interrogation of gliomagenesis.
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http://dx.doi.org/10.1186/s13059-020-02092-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7392733PMC
July 2020

Analysis pipelines for cancer genome sequencing in mice.

Nat Protoc 2020 02 6;15(2):266-315. Epub 2020 Jan 6.

Institute of Molecular Oncology and Functional Genomics, School of Medicine, Technische Universität München, Munich, Germany.

Mouse models of human cancer have transformed our ability to link genetics, molecular mechanisms and phenotypes. Both reverse and forward genetics in mice are currently gaining momentum through advances in next-generation sequencing (NGS). Methodologies to analyze sequencing data were, however, developed for humans and hence do not account for species-specific differences in genome structures and experimental setups. Here, we describe standardized computational pipelines specifically tailored to the analysis of mouse genomic data. We present novel tools and workflows for the detection of different alteration types, including single-nucleotide variants (SNVs), small insertions and deletions (indels), copy-number variations (CNVs), loss of heterozygosity (LOH) and complex rearrangements, such as in chromothripsis. Workflows have been extensively validated and cross-compared using multiple methodologies. We also give step-by-step guidance on the execution of individual analysis types, provide advice on data interpretation and make the complete code available online. The protocol takes 2-7 d, depending on the desired analyses.
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http://dx.doi.org/10.1038/s41596-019-0234-7DOI Listing
February 2020

Functional linkage of gene fusions to cancer cell fitness assessed by pharmacological and CRISPR-Cas9 screening.

Nat Commun 2019 05 16;10(1):2198. Epub 2019 May 16.

Wellcome Sanger Institute, Wellcome Genome Campus, Cambridge, CB10 1SA, UK.

Many gene fusions are reported in tumours and for most their role remains unknown. As fusions are used for diagnostic and prognostic purposes, and are targets for treatment, it is crucial to assess their function in cancer. To systematically investigate the role of fusions in tumour cell fitness, we utilized RNA-sequencing data from 1011 human cancer cell lines to functionally link 8354 fusion events with genomic data, sensitivity to >350 anti-cancer drugs and CRISPR-Cas9 loss-of-fitness effects. Established clinically-relevant fusions were identified. Overall, detection of functional fusions was rare, including those involving cancer driver genes, suggesting that many fusions are dispensable for tumour fitness. Therapeutically actionable fusions involving RAF1, BRD4 and ROS1 were verified in new histologies. In addition, recurrent YAP1-MAML2 fusions were identified as activators of Hippo-pathway signaling in multiple cancer types. Our approach discriminates functional fusions, identifying new drivers of carcinogenesis and fusions that could have clinical implications.
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http://dx.doi.org/10.1038/s41467-019-09940-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6522557PMC
May 2019

PiggyBac transposon tools for recessive screening identify B-cell lymphoma drivers in mice.

Nat Commun 2019 03 29;10(1):1415. Epub 2019 Mar 29.

The Wellcome Trust Sanger Institute, Genome Campus, Hinxton, Cambridge, CB10 1SA, UK.

B-cell lymphoma (BCL) is the most common hematologic malignancy. While sequencing studies gave insights into BCL genetics, identification of non-mutated cancer genes remains challenging. Here, we describe PiggyBac transposon tools and mouse models for recessive screening and show their application to study clonal B-cell lymphomagenesis. In a genome-wide screen, we discover BCL genes related to diverse molecular processes, including signaling, transcriptional regulation, chromatin regulation, or RNA metabolism. Cross-species analyses show the efficiency of the screen to pinpoint human cancer drivers altered by non-genetic mechanisms, including clinically relevant genes dysregulated epigenetically, transcriptionally, or post-transcriptionally in human BCL. We also describe a CRISPR/Cas9-based in vivo platform for BCL functional genomics, and validate discovered genes, such as Rfx7, a transcription factor, and Phip, a chromatin regulator, which suppress lymphomagenesis in mice. Our study gives comprehensive insights into the molecular landscapes of BCL and underlines the power of genome-scale screening to inform biology.
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http://dx.doi.org/10.1038/s41467-019-09180-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6440946PMC
March 2019

Evolutionary routes and KRAS dosage define pancreatic cancer phenotypes.

Nature 2018 02 24;554(7690):62-68. Epub 2018 Jan 24.

Anthropology & Human Genomics, Department of Biology II, Ludwig-Maximilians Universität, 82152 Martinsried, Germany.

The poor correlation of mutational landscapes with phenotypes limits our understanding of the pathogenesis and metastasis of pancreatic ductal adenocarcinoma (PDAC). Here we show that oncogenic dosage-variation has a critical role in PDAC biology and phenotypic diversification. We find an increase in gene dosage of mutant KRAS in human PDAC precursors, which drives both early tumorigenesis and metastasis and thus rationalizes early PDAC dissemination. To overcome the limitations posed to gene dosage studies by the stromal richness of PDAC, we have developed large cell culture resources of metastatic mouse PDAC. Integration of cell culture genomes, transcriptomes and tumour phenotypes with functional studies and human data reveals additional widespread effects of oncogenic dosage variation on cell morphology and plasticity, histopathology and clinical outcome, with the highest Kras levels underlying aggressive undifferentiated phenotypes. We also identify alternative oncogenic gains (Myc, Yap1 or Nfkb2), which collaborate with heterozygous Kras in driving tumorigenesis, but have lower metastatic potential. Mechanistically, different oncogenic gains and dosages evolve along distinct evolutionary routes, licensed by defined allelic states and/or combinations of hallmark tumour suppressor alterations (Cdkn2a, Trp53, Tgfβ-pathway). Thus, evolutionary constraints and contingencies direct oncogenic dosage gain and variation along defined routes to drive the early progression of PDAC and shape its downstream biology. Our study uncovers universal principles of Ras-driven oncogenesis that have potential relevance beyond pancreatic cancer.
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http://dx.doi.org/10.1038/nature25459DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6097607PMC
February 2018

Copy number variation arising from gene conversion on the human Y chromosome.

Hum Genet 2018 Jan 5;137(1):73-83. Epub 2017 Dec 5.

Wellcome Trust Sanger Institute, Wellcome Genome Campus, Hinxton, Cambridge, CB10 1SA, UK.

We describe the variation in copy number of a ~ 10 kb region overlapping the long intergenic noncoding RNA (lincRNA) gene, TTTY22, within the IR3 inverted repeat on the short arm of the human Y chromosome, leading to individuals with 0-3 copies of this region in the general population. Variation of this CNV is common, with 266 individuals having 0 copies, 943 (including the reference sequence) having 1, 23 having 2 copies, and two having 3 copies, and was validated by breakpoint PCR, fibre-FISH, and 10× Genomics Chromium linked-read sequencing in subsets of 1234 individuals from the 1000 Genomes Project. Mapping the changes in copy number to the phylogeny of these Y chromosomes previously established by the Project identified at least 20 mutational events, and investigation of flanking paralogous sequence variants showed that the mutations involved flanking sequences in 18 of these, and could extend over > 30 kb of DNA. While either gene conversion or double crossover between misaligned sister chromatids could formally explain the 0-2 copy events, gene conversion is the more likely mechanism, and these events include the longest non-allelic gene conversion reported thus far. Chromosomes with three copies of this CNV have arisen just once in our data set via another mechanism: duplication of 420 kb that places the third copy 230 kb proximal to the existing proximal copy. Our results establish gene conversion as a previously under-appreciated mechanism of generating copy number changes in humans and reveal the exceptionally large size of the conversion events that can occur.
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http://dx.doi.org/10.1007/s00439-017-1857-9DOI Listing
January 2018

Punctuated bursts in human male demography inferred from 1,244 worldwide Y-chromosome sequences.

Nat Genet 2016 06 25;48(6):593-9. Epub 2016 Apr 25.

Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA.

We report the sequences of 1,244 human Y chromosomes randomly ascertained from 26 worldwide populations by the 1000 Genomes Project. We discovered more than 65,000 variants, including single-nucleotide variants, multiple-nucleotide variants, insertions and deletions, short tandem repeats, and copy number variants. Of these, copy number variants contribute the greatest predicted functional impact. We constructed a calibrated phylogenetic tree on the basis of binary single-nucleotide variants and projected the more complex variants onto it, estimating the number of mutations for each class. Our phylogeny shows bursts of extreme expansion in male numbers that have occurred independently among each of the five continental superpopulations examined, at times of known migrations and technological innovations.
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http://dx.doi.org/10.1038/ng.3559DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4884158PMC
June 2016

Formin Is Associated with Left-Right Asymmetry in the Pond Snail and the Frog.

Curr Biol 2016 Mar 25;26(5):654-60. Epub 2016 Feb 25.

Institute of Evolutionary Biology, University of Edinburgh, Edinburgh EH9 3JT, UK; Edinburgh Genomics, School of Biological Sciences, University of Edinburgh, Edinburgh EH9 3JT, UK.

While components of the pathway that establishes left-right asymmetry have been identified in diverse animals, from vertebrates to flies, it is striking that the genes involved in the first symmetry-breaking step remain wholly unknown in the most obviously chiral animals, the gastropod snails. Previously, research on snails was used to show that left-right signaling of Nodal, downstream of symmetry breaking, may be an ancestral feature of the Bilateria [1 and 2]. Here, we report that a disabling mutation in one copy of a tandemly duplicated, diaphanous-related formin is perfectly associated with symmetry breaking in the pond snail. This is supported by the observation that an anti-formin drug treatment converts dextral snail embryos to a sinistral phenocopy, and in frogs, drug inhibition or overexpression by microinjection of formin has a chirality-randomizing effect in early (pre-cilia) embryos. Contrary to expectations based on existing models [3, 4 and 5], we discovered asymmetric gene expression in 2- and 4-cell snail embryos, preceding morphological asymmetry. As the formin-actin filament has been shown to be part of an asymmetry-breaking switch in vitro [6 and 7], together these results are consistent with the view that animals with diverse body plans may derive their asymmetries from the same intracellular chiral elements [8].
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http://dx.doi.org/10.1016/j.cub.2015.12.071DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4791482PMC
March 2016

Multiplexed pancreatic genome engineering and cancer induction by transfection-based CRISPR/Cas9 delivery in mice.

Nat Commun 2016 Feb 26;7:10770. Epub 2016 Feb 26.

Department of Medicine II, Klinikum rechts der Isar, Technische Universität München, 81675 Munich, Germany.

Mouse transgenesis has provided fundamental insights into pancreatic cancer, but is limited by the long duration of allele/model generation. Here we show transfection-based multiplexed delivery of CRISPR/Cas9 to the pancreas of adult mice, allowing simultaneous editing of multiple gene sets in individual cells. We use the method to induce pancreatic cancer and exploit CRISPR/Cas9 mutational signatures for phylogenetic tracking of metastatic disease. Our results demonstrate that CRISPR/Cas9-multiplexing enables key applications, such as combinatorial gene-network analysis, in vivo synthetic lethality screening and chromosome engineering. Negative-selection screening in the pancreas using multiplexed-CRISPR/Cas9 confirms the vulnerability of pancreatic cells to Brca2-inactivation in a Kras-mutant context. We also demonstrate modelling of chromosomal deletions and targeted somatic engineering of inter-chromosomal translocations, offering multifaceted opportunities to study complex structural variation, a hallmark of pancreatic cancer. The low-frequency mosaic pattern of transfection-based CRISPR/Cas9 delivery faithfully recapitulates the stochastic nature of human tumorigenesis, supporting wide applicability for biological/preclinical research.
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http://dx.doi.org/10.1038/ncomms10770DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4773438PMC
February 2016

Chromosome engineering in zygotes with CRISPR/Cas9.

Genesis 2016 Feb 25;54(2):78-85. Epub 2016 Jan 25.

Wellcome Trust Sanger Institute, Wellcome Genome Campus, Hinxton, CB10 1SA, Cambridge, United Kingdom.

Deletions, duplications, and inversions of large genomic regions covering several genes are an important class of disease causing variants in humans. Modeling these structural variants in mice requires multistep processes in ES cells, which has limited their availability. Mutant mice containing small insertions, deletions, and single nucleotide polymorphisms can be reliably generated using CRISPR/Cas9 directly in mouse zygotes. Large structural variants can be generated using CRISPR/Cas9 in ES cells, but it has not been possible to generate these directly in zygotes. We now demonstrate the direct generation of deletions, duplications and inversions of up to one million base pairs by zygote injection.
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http://dx.doi.org/10.1002/dvg.22915DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4819711PMC
February 2016

G&T-seq: parallel sequencing of single-cell genomes and transcriptomes.

Nat Methods 2015 Jun 27;12(6):519-22. Epub 2015 Apr 27.

1] Sanger Institute-EBI Single-Cell Genomics Centre, Wellcome Trust Sanger Institute, Hinxton, UK. [2] Department of Human Genetics, University of Leuven, Leuven, Belgium.

The simultaneous sequencing of a single cell's genome and transcriptome offers a powerful means to dissect genetic variation and its effect on gene expression. Here we describe G&T-seq, a method for separating and sequencing genomic DNA and full-length mRNA from single cells. By applying G&T-seq to over 220 single cells from mice and humans, we discovered cellular properties that could not be inferred from DNA or RNA sequencing alone.
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http://dx.doi.org/10.1038/nmeth.3370DOI Listing
June 2015

A next-generation dual-recombinase system for time- and host-specific targeting of pancreatic cancer.

Nat Med 2014 Nov 19;20(11):1340-1347. Epub 2014 Oct 19.

Department of Internal Medicine II, Klinikum rechts der Isar, Technische Universität München, München, Germany.

Genetically engineered mouse models (GEMMs) have dramatically improved our understanding of tumor evolution and therapeutic resistance. However, sequential genetic manipulation of gene expression and targeting of the host is almost impossible using conventional Cre-loxP-based models. We have developed an inducible dual-recombinase system by combining flippase-FRT (Flp-FRT) and Cre-loxP recombination technologies to improve GEMMs of pancreatic cancer. This enables investigation of multistep carcinogenesis, genetic manipulation of tumor subpopulations (such as cancer stem cells), selective targeting of the tumor microenvironment and genetic validation of therapeutic targets in autochthonous tumors on a genome-wide scale. As a proof of concept, we performed tumor cell-autonomous and nonautonomous targeting, recapitulated hallmarks of human multistep carcinogenesis, validated genetic therapy by 3-phosphoinositide-dependent protein kinase inactivation as well as cancer cell depletion and show that mast cells in the tumor microenvironment, which had been thought to be key oncogenic players, are dispensable for tumor formation.
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http://dx.doi.org/10.1038/nm.3646DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4270133PMC
November 2014

Fine mapping of the pond snail left-right asymmetry (chirality) locus using RAD-Seq and fibre-FISH.

PLoS One 2013 12;8(8):e71067. Epub 2013 Aug 12.

School of Biology, University of Nottingham, University Park, Nottingham, UK.

The left-right asymmetry of snails, including the direction of shell coiling, is determined by the delayed effect of a maternal gene on the chiral twist that takes place during early embryonic cell divisions. Yet, despite being a well-established classical problem, the identity of the gene and the means by which left-right asymmetry is established in snails remain unknown. We here demonstrate the power of new genomic approaches for identification of the chirality gene, "D". First, heterozygous (Dd) pond snails Lymnaea stagnalis were self-fertilised or backcrossed, and the genotype of more than six thousand offspring inferred, either dextral (DD/Dd) or sinistral (dd). Then, twenty of the offspring were used for Restriction-site-Associated DNA Sequencing (RAD-Seq) to identify anonymous molecular markers that are linked to the chirality locus. A local genetic map was constructed by genotyping three flanking markers in over three thousand snails. The three markers lie either side of the chirality locus, with one very tightly linked (<0.1 cM). Finally, bacterial artificial chromosomes (BACs) were isolated that contained the three loci. Fluorescent in situ hybridization (FISH) of pachytene cells showed that the three BACs tightly cluster on the same bivalent chromosome. Fibre-FISH identified a region of greater that ∼0.4 Mb between two BAC clone markers that must contain D. This work therefore establishes the resources for molecular identification of the chirality gene and the variation that underpins sinistral and dextral coiling. More generally, the results also show that combining genomic technologies, such as RAD-Seq and high resolution FISH, is a robust approach for mapping key loci in non-model systems.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0071067PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3741322PMC
March 2014

Massively parallel sequencing reveals the complex structure of an irradiated human chromosome on a mouse background in the Tc1 model of Down syndrome.

PLoS One 2013 15;8(4):e60482. Epub 2013 Apr 15.

Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, United Kingdom.

Down syndrome (DS) is caused by trisomy of chromosome 21 (Hsa21) and presents a complex phenotype that arises from abnormal dosage of genes on this chromosome. However, the individual dosage-sensitive genes underlying each phenotype remain largely unknown. To help dissect genotype--phenotype correlations in this complex syndrome, the first fully transchromosomic mouse model, the Tc1 mouse, which carries a copy of human chromosome 21 was produced in 2005. The Tc1 strain is trisomic for the majority of genes that cause phenotypes associated with DS, and this freely available mouse strain has become used widely to study DS, the effects of gene dosage abnormalities, and the effect on the basic biology of cells when a mouse carries a freely segregating human chromosome. Tc1 mice were created by a process that included irradiation microcell-mediated chromosome transfer of Hsa21 into recipient mouse embryonic stem cells. Here, the combination of next generation sequencing, array-CGH and fluorescence in situ hybridization technologies has enabled us to identify unsuspected rearrangements of Hsa21 in this mouse model; revealing one deletion, six duplications and more than 25 de novo structural rearrangements. Our study is not only essential for informing functional studies of the Tc1 mouse but also (1) presents for the first time a detailed sequence analysis of the effects of gamma radiation on an entire human chromosome, which gives some mechanistic insight into the effects of radiation damage on DNA, and (2) overcomes specific technical difficulties of assaying a human chromosome on a mouse background where highly conserved sequences may confound the analysis. Sequence data generated in this study is deposited in the ENA database, Study Accession number: ERP000439.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0060482PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3626651PMC
October 2013

The zebrafish reference genome sequence and its relationship to the human genome.

Nature 2013 Apr 17;496(7446):498-503. Epub 2013 Apr 17.

Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, UK.

Zebrafish have become a popular organism for the study of vertebrate gene function. The virtually transparent embryos of this species, and the ability to accelerate genetic studies by gene knockdown or overexpression, have led to the widespread use of zebrafish in the detailed investigation of vertebrate gene function and increasingly, the study of human genetic disease. However, for effective modelling of human genetic disease it is important to understand the extent to which zebrafish genes and gene structures are related to orthologous human genes. To examine this, we generated a high-quality sequence assembly of the zebrafish genome, made up of an overlapping set of completely sequenced large-insert clones that were ordered and oriented using a high-resolution high-density meiotic map. Detailed automatic and manual annotation provides evidence of more than 26,000 protein-coding genes, the largest gene set of any vertebrate so far sequenced. Comparison to the human reference genome shows that approximately 70% of human genes have at least one obvious zebrafish orthologue. In addition, the high quality of this genome assembly provides a clearer understanding of key genomic features such as a unique repeat content, a scarcity of pseudogenes, an enrichment of zebrafish-specific genes on chromosome 4 and chromosomal regions that influence sex determination.
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http://dx.doi.org/10.1038/nature12111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3703927PMC
April 2013

Mutant nucleophosmin and cooperating pathways drive leukemia initiation and progression in mice.

Nat Genet 2011 May 27;43(5):470-5. Epub 2011 Mar 27.

Mouse Genomics Team, The Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, UK.

Acute myeloid leukemia (AML) is a molecularly diverse malignancy with a poor prognosis whose largest subgroup is characterized by somatic mutations in NPM1, which encodes nucleophosmin. These mutations, termed NPM1c, result in cytoplasmic dislocation of nucleophosmin and are associated with distinctive transcriptional signatures, yet their role in leukemogenesis remains obscure. Here we report that activation of a humanized Npm1c knock-in allele in mouse hemopoietic stem cells causes Hox gene overexpression, enhanced self renewal and expanded myelopoiesis. One third of mice developed delayed-onset AML, suggesting a requirement for cooperating mutations. We identified such mutations using a Sleeping Beauty transposon, which caused rapid-onset AML in 80% of mice with Npm1c, associated with mutually exclusive integrations in Csf2, Flt3 or Rasgrp1 in 55 of 70 leukemias. We also identified recurrent integrations in known and newly discovered leukemia genes including Nf1, Bach2, Dleu2 and Nup98. Our results provide new pathogenetic insights and identify possible therapeutic targets in NPM1c+ AML.
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http://dx.doi.org/10.1038/ng.796DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3084174PMC
May 2011

PiggyBac transposon mutagenesis: a tool for cancer gene discovery in mice.

Science 2010 Nov 14;330(6007):1104-7. Epub 2010 Oct 14.

Wellcome Trust Sanger Institute, Genome Campus, Hinxton-Cambridge CB10 1SA, UK.

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.
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http://dx.doi.org/10.1126/science.1193004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3719098PMC
November 2010

Loss of Rb proteins causes genomic instability in the absence of mitogenic signaling.

Genes Dev 2010 Jul 15;24(13):1377-88. Epub 2010 Jun 15.

Division of Molecular Biology, The Netherlands Cancer Institute, Amsterdam, The Netherlands.

Loss of G1/S control is a hallmark of cancer, and is often caused by inactivation of the retinoblastoma pathway. However, mouse embryonic fibroblasts lacking the retinoblastoma genes RB1, p107, and p130 (TKO MEFs) are still subject to cell cycle control: Upon mitogen deprivation, they enter and complete S phase, but then firmly arrest in G2. We now show that G2-arrested TKO MEFs have accumulated DNA damage. Upon mitogen readdition, cells resume proliferation, although only part of the damage is repaired. As a result, mitotic cells show chromatid breaks and chromatid cohesion defects. These aberrations lead to aneuploidy in the descendent cell population. Thus, our results demonstrate that unfavorable growth conditions can cause genomic instability in cells lacking G1/S control. This mechanism may allow premalignant tumor cells to acquire additional genetic alterations that promote tumorigenesis.
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http://dx.doi.org/10.1101/gad.580710DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2895197PMC
July 2010

Abnormal behavior in a chromosome-engineered mouse model for human 15q11-13 duplication seen in autism.

Cell 2009 Jun;137(7):1235-46

Osaka Bioscience Institute, Suita, Osaka 565-0874, Japan.

Substantial evidence suggests that chromosomal abnormalities contribute to the risk of autism. The duplication of human chromosome 15q11-13 is known to be the most frequent cytogenetic abnormality in autism. We have modeled this genetic change in mice by using chromosome engineering to generate a 6.3 Mb duplication of the conserved linkage group on mouse chromosome 7. Mice with a paternal duplication display poor social interaction, behavioral inflexibility, abnormal ultrasonic vocalizations, and correlates of anxiety. An increased MBII52 snoRNA within the duplicated region, affecting the serotonin 2c receptor (5-HT2cR), correlates with altered intracellular Ca(2+) responses elicited by a 5-HT2cR agonist in neurons of mice with a paternal duplication. This chromosome-engineered mouse model for autism seems to replicate various aspects of human autistic phenotypes and validates the relevance of the human chromosome abnormality. This model will facilitate forward genetics of developmental brain disorders and serve as an invaluable tool for therapeutic development.
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http://dx.doi.org/10.1016/j.cell.2009.04.024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3710970PMC
June 2009

Definition of the zebrafish genome using flow cytometry and cytogenetic mapping.

BMC Genomics 2007 Jun 27;8:195. Epub 2007 Jun 27.

Department of Pathology, Brigham and Women's Hospital, Boston, Massachusetts 02115, USA.

Background: The zebrafish (Danio rerio) is an important vertebrate model organism system for biomedical research. The syntenic conservation between the zebrafish and human genome allows one to investigate the function of human genes using the zebrafish model. To facilitate analysis of the zebrafish genome, genetic maps have been constructed and sequence annotation of a reference zebrafish genome is ongoing. However, the duplicative nature of teleost genomes, including the zebrafish, complicates accurate assembly and annotation of a representative genome sequence. Cytogenetic approaches provide "anchors" that can be integrated with accumulating genomic data.

Results: Here, we cytogenetically define the zebrafish genome by first estimating the size of each linkage group (LG) chromosome using flow cytometry, followed by the cytogenetic mapping of 575 bacterial artificial chromosome (BAC) clones onto metaphase chromosomes. Of the 575 BAC clones, 544 clones localized to apparently unique chromosomal locations. 93.8% of these clones were assigned to a specific LG chromosome location using fluorescence in situ hybridization (FISH) and compared to the LG chromosome assignment reported in the zebrafish genome databases. Thirty-one BAC clones localized to multiple chromosomal locations in several different hybridization patterns. From these data, a refined second generation probe panel for each LG chromosome was also constructed.

Conclusion: The chromosomal mapping of the 575 large-insert DNA clones allows for these clones to be integrated into existing zebrafish mapping data. An accurately annotated zebrafish reference genome serves as a valuable resource for investigating the molecular basis of human diseases using zebrafish mutant models.
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http://dx.doi.org/10.1186/1471-2164-8-195DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1925092PMC
June 2007

Isolation and characterization of murine Cds (CDP-diacylglycerol synthase) 1 and 2.

Gene 2005 Aug;356:19-31

Institute of Ophthalmology, University College London, 11-43 Bath Street, London, EC1V 9EL, UK.

Phototransduction in Drosophila is a phosphoinositide-mediated signalling pathway. Phosphatidylinositol 4,5-bisphosphate (PIP2) plays a central role in this process, and its levels are tightly regulated. A photoreceptor-specific form of the enzyme CDP-diacylglycerol synthase (CDS), which catalyzes the formation of CDP-diacylglycerol from phosphatidic acid, is a key regulator of the amount of PIP2 available for signalling. cds mutants develop light-induced retinal degeneration. We report here the isolation and characterization of two murine genes encoding this enzyme, Cds1 and Cds2. The genes encode proteins that are 73% identical and 92% similar but exhibit very different expression patterns. Cds1 shows a very restricted expression pattern but is expressed in the inner segments of the photoreceptors whilst Cds2 shows a ubiquitous pattern of expression. Using fluorescent in situ hybridization we have mapped Cds1 and Cds2 to chromosomes 5E3 and 2G1 respectively. These are regions of synteny with the corresponding human gene localization (4q21 and 20p13). Transient transfection experiments with epitope tagged proteins have also demonstrated that both are associated with the endoplasmic reticulum.
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http://dx.doi.org/10.1016/j.gene.2005.04.037DOI Listing
August 2005

Complex haplotypes, copy number polymorphisms and coding variation in two recently divergent mouse strains.

Nat Genet 2005 May 24;37(5):532-6. Epub 2005 Apr 24.

The Wellcome Trust Sanger Institute, Hinxton, Cambs, CB10 1SA, UK.

Inbred mouse strains provide the foundation for mouse genetics. By selecting for phenotypic features of interest, inbreeding drives genomic evolution and eliminates individual variation, while fixing certain sets of alleles that are responsible for the trait characteristics of the strain. Mouse strains 129Sv (129S5) and C57BL/6J, two of the most widely used inbred lines, diverged from common ancestors within the last century, yet very little is known about the genomic differences between them. By comparative genomic hybridization and sequence analysis of 129S5 short insert libraries, we identified substantial structural variation, a complex fine-scale haplotype pattern with a continuous distribution of diversity blocks, and extensive nucleotide variation, including nonsynonymous coding SNPs and stop codons. Collectively, these genomic changes denote the level and direction of allele fixation that has occurred during inbreeding and provide a basis for defining what makes these mouse strains unique.
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http://dx.doi.org/10.1038/ng1551DOI Listing
May 2005

The DNA sequence of the human X chromosome.

Nature 2005 Mar;434(7031):325-37

The Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, UK.

The human X chromosome has a unique biology that was shaped by its evolution as the sex chromosome shared by males and females. We have determined 99.3% of the euchromatic sequence of the X chromosome. Our analysis illustrates the autosomal origin of the mammalian sex chromosomes, the stepwise process that led to the progressive loss of recombination between X and Y, and the extent of subsequent degradation of the Y chromosome. LINE1 repeat elements cover one-third of the X chromosome, with a distribution that is consistent with their proposed role as way stations in the process of X-chromosome inactivation. We found 1,098 genes in the sequence, of which 99 encode proteins expressed in testis and in various tumour types. A disproportionately high number of mendelian diseases are documented for the X chromosome. Of this number, 168 have been explained by mutations in 113 X-linked genes, which in many cases were characterized with the aid of the DNA sequence.
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http://dx.doi.org/10.1038/nature03440DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2665286PMC
March 2005

Candidate testis-determining gene, Maestro (Mro), encodes a novel HEAT repeat protein.

Dev Dyn 2003 Aug;227(4):600-7

MRC Mammalian Genetics Unit, Harwell, Didcot, United Kingdom.

Mammalian sex determination depends on the presence or absence of SRY transcripts in the embryonic gonad. Expression of SRY initiates a pathway of gene expression resulting in testis development. Here, we describe a novel gene potentially functioning in this pathway using a cDNA microarray screen for genes exhibiting sexually dimorphic expression during murine gonad development. Maestro (Mro) transcripts are first detected in the developing male gonad before overt testis differentiation. By 12.5 days postcoitus (dpc), Mro transcription is restricted to the developing testis cords and its expression is not germ cell-dependent. No expression is observed in female gonads between 10.5 and 14.5 dpc. Maestro encodes a protein containing HEAT-like repeats that localizes to the nucleolus in cell transfection assays. Maestro maps to a region of mouse chromosome 18 containing a genetic modifier of XX sex reversal. We discuss the possible function of Maestro in light of these data.
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http://dx.doi.org/10.1002/dvdy.10342DOI Listing
August 2003
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