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Cattoglio, C, Pellin, D, Rizzi, E, Maruggi, G, Corti, G, Miselli, F et al.. High-definition mapping of retroviral integration sites identifies active regulatory elements in human multipotent hematopoietic progenitors. Blood 116: 5507-5517

Istituto Scientifico H. San Raffaele, Milan, Italy.
Blood (Impact Factor: 10.43). 12/2010; 116(25):5507-17. DOI: 10.1182/blood-2010-05-283523
Source: PubMed

ABSTRACT Integration of retroviral vectors in the human genome follows nonrandom patterns that favor insertional deregulation of gene expression and increase the risk of their use in clinical gene therapy. The molecular basis of retroviral target site selection is still poorly understood. We used deep sequencing technology to build genomewide, high-definition maps of > 60 000 integration sites of Moloney murine leukemia virus (MLV)- and HIV-based retroviral vectors in the genome of human CD34(+) multipotent hematopoietic progenitor cells (HPCs) and used gene expression profiling, chromatin immunoprecipitation, and bioinformatics to associate integration to genetic and epigenetic features of the HPC genome. Clusters of recurrent MLV integrations identify regulatory elements (alternative promoters, enhancers, evolutionarily conserved noncoding regions) within or around protein-coding genes and microRNAs with crucial functions in HPC growth and differentiation, bearing epigenetic marks of active or poised transcription (H3K4me1, H3K4me2, H3K4me3, H3K9Ac, Pol II) and specialized chromatin configurations (H2A.Z). Overall, we mapped 3500 high-frequency integration clusters, which represent a new resource for the identification of transcriptionally active regulatory elements. High-definition MLV integration maps provide a rational basis for predicting genotoxic risks in gene therapy and a new tool for genomewide identification of promoters and regulatory elements controlling hematopoietic stem and progenitor cell functions.

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Available from: Giorgio Corti, Aug 19, 2015
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    • "This lowers, but does not completely alleviate, the risk of genotoxicity according to studies addressing the oncogenic potential of these vectors either in vitro or in vivo (Modlich et al, 2009; Montini et al, 2006, 2009). The common consensus drawn by these studies is that viral vector integration is an active process catalysed by the tethering of the viral preintegration complex to open chromatin regions in the host cell genome as characterized by DNaseI hypersensitive sites and epigenetics marks (Cattoglio et al, 2010; Deichmann et al, 2011; Felice et al, 2009). For example, the host‐cell encoded LEDGF/p75 binds to the lentiviral integrase to direct integration to active transcription units. "
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    • "rocessed through an automated bioinformatic pipeline that eliminated small and redundant sequences and were mapped to the UCSC hg19 release of the human genome ( Cattoglio et al . , 2010 ) . Sequences with 90% or greater identity to the human genome were considered genuine integration sites . As control dataset , we used 40 , 000 random sites from Cattoglio et al . ( 2010 ) . Genomic features ( CpG islands and DNaseI hypersensitive sites ) were annotated when their genomic coordinates over - lapped for at least 1 nucleotide with a 50 - kb interval surrounding each integration site . We used UCSC tracks for both cytosine – phosphate – guanosine ( CpG ) islands and Jurkat DNaseI hypersensitive sites ."
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    ABSTRACT: The early steps of HIV-1 replication involve the entry of HIV-1 into the nucleus, which is characterized by viral interactions with nuclear pore components. HIV-1 developed an evolutionary strategy to usurp the nuclear pore machinery and chromatin in order to integrate and efficiently express viral genes. In the current work, we studied the role of nucleoporins 153 and 98 (Nup153 and Nup98) in infection of human Jurkat lymphocytes by HIV-1. We showed that Nup153-depleted cells exhibited a defect in nuclear import, while depletion of Nup 98 caused a slight defect in HIV integration. To explore the biochemical viral determinants for the requirement of Nup153 and Nup98 during HIV-1 infection, we tested the ability of these nucleoporins to interact with HIV-1 cores. Our findings showed that both nucleoporins bind HIV-1 cores suggesting that this interaction is important for HIV-1 nuclear import and/or integration. Distribution analysis of integration sites in Nup153-depleted cells revealed a reduced tendency of HIV-1 to integrate in intragenic sites, which in part could account for the large infectivity defect observed in Nup153-depleted cells. Our work strongly supports a role for Nup153 in HIV-1 nuclear import and integration.
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    • "The table indicates the percentage of HIV integrations occurring in intragenic , intergenic , transcriptional start site ( TSS ) - proximal regions , and in close proximity to CpG islands and in DNaseI hypersensitive sites . Computer - generated random control sequences were obtained from Cattoglio et al . ( 2010 )"
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    ABSTRACT: a b s t r a c t The early steps of HIV-1 replication involve the entry of HIV-1 into the nucleus, which is characterized by viral interactions with nuclear pore components. HIV-1 developed an evolutionary strategy to usurp the nuclear pore machinery and chromatin in order to integrate and efficiently express viral genes. In the current work, we studied the role of nucleoporins 153 and 98 (Nup153 and Nup98) in infection of human Jurkat lymphocytes by HIV-1. We showed that Nup153-depleted cells exhibited a defect in nuclear import, while depletion of Nup 98 caused a slight defect in HIV integration. To explore the biochemical viral determinants for the requirement of Nup153 and Nup98 during HIV-1 infection, we tested the ability of these nucleoporins to interact with HIV-1 cores. Our findings showed that both nucleoporins bind HIV-1 cores suggesting that this interaction is important for HIV-1 nuclear import and/or integration. Distribution analysis of integration sites in Nup153-depleted cells revealed a reduced tendency of HIV-1 to integrate in intragenic sites, which in part could account for the large infectivity defect observed in Nup153-depleted cells. Our work strongly supports a role for Nup153 in HIV-1 nuclear import and integration.
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