The Genome of the Chicken DT40 Bursal Lymphoma Cell Line

The chicken DT40 cell line is a widely used model system in the study of multiple cellular processes due to the efficiency of homologous gene targeting. The cell line was derived from a bursal lymphoma induced by avian leukosis virus infection. In this study we characterized the genome of the cell line using whole genome shotgun sequencing and single nucleotide polymorphism array hybridization. The results indicate that wild-type DT40 has a relatively normal karyotype, except for whole chromosome copy number gains, and no karyotype variability within stocks. In a comparison to two domestic chicken genomes and the Gallus gallus reference genome, we found no unique mutational processes shaping the DT40 genome except for a mild increase in insertion and deletion events, particularly deletions at tandem repeats. We mapped coding sequence mutations that are unique to the DT40 genome; mutations inactivating the PIK3R1 and ATRX genes likely contributed to the oncogenic transformation. In addition to a known avian leukosis virus integration in the MYC gene, we detected further integration sites that are likely to de-regulate gene expression. The new findings support the hypothesis that DT40 is a typical transformed cell line with a relatively intact genome; therefore, it is well-suited to the role of a model system for DNA repair and related processes. The sequence data generated by this study, including a searchable de novo genome assembly and annotated lists of mutated genes, will support future research using this cell line.

[1]  J. Buerstedde,et al.  Light chain gene conversion continues at high rate in an ALV‐induced cell line. , 1990, The EMBO journal.

[2]  M. Delany,et al.  Molecular and Cellular Evidence for the Alternative Lengthening of Telomeres (ALT) Mechanism in Chicken , 2011, Cytogenetic and Genome Research.

[3]  S. Ramaswamy,et al.  Systematic identification of genomic markers of drug sensitivity in cancer cells , 2012, Nature.

[4]  Bert Vogelstein,et al.  The structure of a human p110alpha/p85alpha complex elucidates the effects of oncogenic PI3Kalpha mutations. , 2007, Science.

[5]  Erin L. Doyle,et al.  Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting , 2011, Nucleic acids research.

[6]  H. Varmus,et al.  Proviral deletions and oncogene base-substitutions in insertionally mutagenized c-myc alleles may contribute to the progression of avian bursal tumors. , 1984, Proceedings of the National Academy of Sciences of the United States of America.

[7]  Steven J. M. Jones,et al.  ATR-X Syndrome Protein Targets Tandem Repeats and Influences Allele-Specific Expression in a Size-Dependent Manner , 2010, Cell.

[8]  Heng Li,et al.  A genetic variation map for chicken with 2.8 million single-nucleotide polymorphisms. , 2004, Nature.

[9]  W. S. Hayward,et al.  Activation of a cellular onc gene by promoter insertion in ALV-induced lymphoid leukosis , 1981, Nature.

[10]  Paul Theodor Pyl,et al.  The Genomic and Transcriptomic Landscape of a HeLa Cell Line , 2013, G3: Genes, Genomes, Genetics.

[11]  M. Watson,et al.  MicroRNA expression profiles in avian haemopoietic cells , 2013, Front. Genet..

[12]  François Laviolette,et al.  Ray: Simultaneous Assembly of Reads from a Mix of High-Throughput Sequencing Technologies , 2010, J. Comput. Biol..

[13]  Christopher A. Miller,et al.  VarScan 2: somatic mutation and copy number alteration discovery in cancer by exome sequencing. , 2012, Genome research.

[14]  B. Giroir,et al.  Cell lines derived from avian lymphomas exhibit two distinct phenotypes. , 1985, Virology.

[15]  David T. W. Jones,et al.  Signatures of mutational processes in human cancer , 2013, Nature.

[16]  Adam A. Margolin,et al.  The Cancer Cell Line Encyclopedia enables predictive modeling of anticancer drug sensitivity , 2012, Nature.

[17]  A. Berns,et al.  Highly efficient gene targeting in embryonic stem cells through homologous recombination with isogenic DNA constructs. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[18]  Akira Shinohara,et al.  Rad51‐deficient vertebrate cells accumulate chromosomal breaks prior to cell death , 1998, The EMBO journal.

[19]  J. Sale,et al.  Role for RAD 18 in Homologous Recombination in DT 40 Cells , 2006 .

[20]  J. Dodgson,et al.  An autosomal genetic linkage map of the chicken. , 1994, The Journal of heredity.

[21]  Emile Zuckerkandl,et al.  On the molecular evolutionary clock , 2005, Journal of Molecular Evolution.

[22]  Jay Shendure,et al.  The haplotype-resolved genome and epigenome of the aneuploid HeLa cancer cell line , 2013, Nature.

[23]  D. Higgs,et al.  The chromatin remodeller ATRX: a repeat offender in human disease. , 2013, Trends in biochemical sciences.

[24]  Aaron R. Quinlan,et al.  BIOINFORMATICS APPLICATIONS NOTE , 2022 .

[25]  J. Sale,et al.  Role for RAD18 in Homologous Recombination in DT40 Cells , 2006, Molecular and Cellular Biology.

[26]  N. Papalopulu,et al.  The oncogenic potential of the high mobility group box protein Sox3. , 2000, Cancer research.

[27]  Bert Vogelstein,et al.  The Structure of a Human p110α/p85α Complex Elucidates the Effects of Oncogenic PI3Kα Mutations , 2007, Science.

[28]  E. H. Humphries,et al.  Differential response to avian leukosis virus infection exhibited by two chicken lines. , 1984, Virology.

[29]  Benjamin J. Raphael,et al.  Mutational landscape and significance across 12 major cancer types , 2013, Nature.

[30]  H. Hakonarson,et al.  ANNOVAR: functional annotation of genetic variants from high-throughput sequencing data , 2010, Nucleic acids research.

[31]  D. Burt,et al.  Differences in gene density on chicken macrochromosomes and microchromosomes. , 2000, Animal genetics.

[32]  International Chicken Polymorphism Map Consortium.  Explorer A genetic variation map for chicken with 2.8 million single-nucleotide polymorphisms , 2012 .

[33]  David A. Scott,et al.  Genome engineering using the CRISPR-Cas9 system , 2013, Nature Protocols.

[34]  Z. Szallasi,et al.  Sequenza: allele-specific copy number and mutation profiles from tumor sequencing data , 2014, Annals of oncology : official journal of the European Society for Medical Oncology.

[35]  M. Delany,et al.  Dynamics of telomere erosion in transformed and non-transformed avian cells in vitro , 2004, Cytogenetic and Genome Research.

[36]  Paul Modrich,et al.  Isolation of MutSβ from Human Cells and Comparison of the Mismatch Repair Specificities of MutSβ and MutSα* , 1998, The Journal of Biological Chemistry.

[37]  M. Lynch.  Rate, molecular spectrum, and consequences of human mutation , 2010, Proceedings of the National Academy of Sciences.

[38]  S. Astrin,et al.  Chromosomal localization of ev-1, a frequently occurring endogenous retrovirus locus in white Leghorn chickens, by in situ hybridization , 1980, Journal of virology.

[39]  Carl-Fredrik Tiger,et al.  Identification of candidate cancer-causing genes in mouse brain tumors by retroviral tagging. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[40]  Colin N. Dewey,et al.  Sequence and comparative analysis of the chicken genome provide unique perspectives on vertebrate evolution , 2004, Nature.

[41]  Nansheng Chen,et al.  CooVar: Co-occurring variant analyzer , 2012, BMC Research Notes.

[42]  W. Hahn,et al.  Loss of ATRX, Genome Instability, and an Altered DNA Damage Response Are Hallmarks of the Alternative Lengthening of Telomeres Pathway , 2012, PLoS genetics.

[43]  Gonçalo R. Abecasis,et al.  The variant call format and VCFtools , 2011, Bioinform..

[44]  S. Astrin,et al.  Nucleotide sequence of acceptor site and termini of integrated avian endogenous provirus ev1: Integration creates a 6 bp repeat of host DNA , 1981, Cell.

[45]  Daniel R. Zerbino,et al.  Ensembl 2014 , 2013, Nucleic Acids Res..

[46]  Gonçalo R. Abecasis,et al.  The Sequence Alignment/Map format and SAMtools , 2009, Bioinform..

[47]  Richard Durbin,et al.  Sequence analysis Fast and accurate short read alignment with Burrows – Wheeler transform , 2009 .

[48]  Hans H. Cheng,et al.  The development and characterization of a 60K SNP chip for chicken , 2011, BMC Genomics.

[49]  Wen-Sui Lo,et al.  Genome-Wide Patterns of Genetic Variation in Two Domestic Chickens , 2013, Genome biology and evolution.

[50]  K. Lindblad-Toh,et al.  Whole-genome resequencing reveals loci under selection during chicken domestication , 2010, Nature.

[51]  Jean-Marie Buerstedde,et al.  Increased ratio of targeted to random integration after transfection of chicken B cell lines , 1991, Cell.

[52]  J. Sale,et al.  Rev1 is essential for DNA damage tolerance and non‐templated immunoglobulin gene mutation in a vertebrate cell line , 2003, The EMBO journal.

[53]  R. Grantham.  Amino Acid Difference Formula to Help Explain Protein Evolution , 1974, Science.

[54]  B. Clurman,et al.  Analysis of gene expression, copy number and palindrome formation with a Dt40 enriched cDNA microarray. , 2006, Sub-cellular biochemistry.

[55]  P. Mires.  Lines , 2006 .

[56]  M. Delany,et al.  Karyotype stability of the DT40 chicken B cell line: Macrochromosome variation and cytogenetic mosaicism , 2004, Chromosome Research.