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Comparative Genome-Wide Profiling of Transposon and Retrovirus Integration Sites in Primary Human CD4+ T Cells

ABSTRACT: We mapped Sleeping Beauty (SB) and piggyBac (PB) insertions in primary human CD4+ T cells and compared their insertion profiles with those of the MLV retrovirus and the HIV lentivirus with respect to proximity to genes, TSSs, CpG islands, DNase I hypersensitive sites, repetitive elements, chromatin marks and transcriptional status of genes. Examination of two transposon systems in one cell type.

ORGANISM(S): Homo sapiens

SUBMITTER: Andreas Gogol-Döring

PROVIDER: E-GEOD-58744 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Comparative Genome-Wide Profiling of Transposon and Retrovirus Integration Sites in Primary Human CD4+ T Cells

Project description:We mapped Sleeping Beauty (SB) and piggyBac (PB) insertions in primary human CD4+ T cells and compared their insertion profiles with those of the MLV retrovirus and the HIV lentivirus with respect to proximity to genes, TSSs, CpG islands, DNase I hypersensitive sites, repetitive elements, chromatin marks and transcriptional status of genes.

2015-07-01 | GSE58744 | GEO

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Project description:DNA transposon-based gene delivery vectors represent a promising new branch of randomly integrating vector development for gene therapy. For the side-by-side evaluation of the piggyBac and Sleeping Beauty systems - the only DNA transposons currently employed in clinical trials - during therapeutic intervention, we treated the mouse model of Tyrosinemia type I. with liver-targeted gene delivery using both transposon vectors. For genome-wide mapping of transposon insertion sites we developed a new Next Generation Sequencing procedure called Streptavidin-Based Enrichment Sequencing, which allowed us to identify approximately 1 million integration sites for both systems. We revealed that a high proportion of piggyBac integrations are clustered in hot regions and found that they are frequently recurring at the same genomic positions among treated animals, indicating that the genome-wide distribution of Sleeping Beauty-generated integrations is closer to random. We also revealed that the piggyBac transposase protein exhibits prolonged activity, which predicts the risk of oncogenesis by generating chromosomal double-strand breaks. Safety concerns associated with prolonged transpositional activity draw attention to the importance of squeezing the active state of the transposase enzymes into a narrower time window.

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Project description:We generated an inducible DDdCas9VP192 activator cell line in H9 human embryonic stem cells using the Sleeping beauty transposition system. The guides targeting a LEUTX promoter and enhancers were introduced by PiggyBac transposition. Three sample types were generated: Promoter only, Promoter+Enhancer1, Promoter+Enhancer2. Cells were collected before and at 24 h, 48 h, and 72 h after doxycycline treatment, and subjected to STRT RNA-sequencing.

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Human somatic cell mutagenesis creates genetically tractable sarcomas

Project description:Creating spontaneous yet genetically tractable human tumors from normal cells presents a fundamental challenge. Retroviruses and transposons have been separately used as somatic cell insertional mutagens to identify cancer drivers in model organisms. Here we combined these mutagenic elements to enable cancer gene discovery starting with normal human cells. Lentivirus was used to seed gain- and loss-of-function gene disruption elements which were further deployed by Sleeping Beauty transposons throughout the genome of human bone explant mesenchymal cells. De novo tumors rapidly generated in this context were high-grade sarcomas corresponding to the spectrum of myxofibrosarcoma and undifferentiated pleomorphic sarcoma, aggressive neoplasms with a predilection for older adults. Tumor insertion sites were genome-wide and enriched in regions of recurrent somatic copy number alteration found in multiple cancers, with a bias towards those of sarcomas. Novel driver genes which sustain somatic alterations in cancer patients were pinpointed. We identify the gene HDLBP, which codes for the RNA binding protein vigilin, as a candidate tumor suppressor deleted at 2q37.3 in greater than one in ten tumors across multiple tissues of origin. Hybrid viral-transposon systems will accelerate the functional annotation of cancer genomes by enabling insertional mutagenesis screens in higher eukaryotes that are not amenable to germline transgenesis. Lentivirus was used to seed gain- and loss-of-function gene disruption elements which were further deployed by Sleeping Beauty (SB) transposons throughout the genome of human bone explant mesenchymal cells. Genomic locations of LV (lentiviral backbone) and SB insertion sites were mapped by a pooled strategy utilizing linear amplification mediated PCR (LAM-PCR), followed by Illumina next generation sequencing of the product pool.

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Cell of origin strongly influences genetic selection in a mouse model of T-ALL.

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2011-08-20 | E-GEOD-29849 | biostudies-arrayexpress

Homo sapiens

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Mus musculus

Project description:Insertion profiles of the piggyBac, Tol2 and Sleeping Beauty transposons and murine leukemia virus

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Cell of origin strongly influences genetic selection in a mouse model of T-ALL.

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