High-definition mapping of retroviral integration sites identifies active regulatory elements in human multipotent hematopoietic progenitors

Abstract

IntroductionPioneering clinical studies have shown that transplantation of genetically modified hematopoietic stem cells may cure severe genetic diseases such as severe combined immunodeficiencies (SCID), 1,2 chronic granulomatous disease (CGD), 3 and lysosomal storage disorders. 4 Unfortunately, some of these studies showed also the limitations of retroviral gene transfer technology, which may cause severe and sometimes fatal adverse effects. In particular, insertional activation of proto-oncogenes by vectors derived from the Moloney murine leukemia virus (MLV) caused T-cell lymphoproliferative disorders in patients with X-linked SCID 5,6 and premalignant expansion of myeloid progenitors in patients with CGD. 3 Preclinical studies showed that HIV-derived lentiviral vectors are less likely to cause insertional gene activation, 7,8 although they can still interfere with normal gene expression at the posttranscriptional level, as observed in a clinical trial of gene therapy for ␤-thalassemia. 9 The molecular bases of vector-induced genotoxicity and the influence of vector design, transduction protocols, and the patient's genetic background in inducing severe adverse effects are still poorly understood. A better understanding of the interactions between retroviral vectors and the human genome may provide new cues to explain these phenomena and a rational basis for predicting genotoxic risks in gene therapy.A large number of studies have focused on the molecular mechanisms by which mammalian retroviruses choose their integration sites in the target cell genome. After entering a cell, retroviral RNA genomes are reverse transcribed into double-stranded DNA and assembled in preintegration complexes (PICs) containing viral and cellular proteins. PICs translocate to the nucleus and associate with the host cell chromatin, where the viral integrase mediates proviral insertion in genomic DNA. Integration is a nonrandom process, whereby PICs of different viruses recognize components or features of the host cell chromatin in a specific fashion. 10 For HIV and other lentiviruses, the LEDGF/p75 protein has been identified as the main factor tethering PICs to active chromatin, 11 whereas mechanisms underlying integration site selection of other retroviruses remain largely unknown. We recently showed that MLV-derived vectors integrate preferentially in hot spots near genes controlling growth and development of hematopoietic cells and flanked by defined subsets of transcription factor binding sites (TFBSs) and suggested that MLV PICs are tethered to active regulatory regions by basal components of the transcriptional machinery. 12,13 The MLV integrase and long terminal repeat enhancer are the main determinants of the selection of TFBS-rich regions of the genome. 13,14 We used ligation-mediated polymerase chain reaction (LM-PCR) and pyrosequencing to build a genomewide, high-definition map of Ͼ 32 000 MLV integration sites in the genome of human CD34 ϩ hematopoietic progenitor cells (HPCs) and used gene expression profiling, chroma...

Retroviral Infection of hES Cells Produces Random-like Integration Patterns

“…virus 1 (HIV-1) had a strong integration preference for the regions within genes in 293T cells as consistent with previously published results (Table 2A) (Cattoglio et al, 2010;Mitchell et al, 2004;Schroder et al, 2002). Compared with the events expected by random chance, lentiviral integrations occurred 1.9-fold more often (85.4% vs. 44.6%) in the regions within genes (significance of 1.3 × 10 -8…”

“…Interestingly, in 293T cells multiple genes that harbored the lentiviral integrants were involved in chromatin and chromosomal organizations. Similar results have previously been observed in HIV-infected hematopoietic progenitor cells (HPCs) (Cattoglio et al, 2010). In addition, lentiviral vectors integrated into genes that were involved in apoptosis or programmed cell death, thereby potentially affecting cell survival, rather than integrating near the genes as shown in retroviral infections.…”

“…It has been suggested that interactions among retroviral pre-integration complexes (PICs) and cellular proteins, such as transcription factors (TFs), determine viral integration sites via a mechanism in which cellular proteins tether PICs to certain genomic loci (Engelman and Cherepanov, 2008;Felice et al, 2009). However, only a fraction of host proteins associating with retroviral PICs have been identified, and other mechanisms that underlie such interactions are still poorly understood (Cattoglio et al, 2010). Moreover, cellular physiological states and cell-specific features, both of which determine the gene expression patterns of host cells as well as the epigenetic landscape of the host genome, may affect retroviral and lentiviral integrations, but the relevant studies have begun to be available only recently Biasco et al, 2011).…”

Measles virus envelope pseudotyped lentiviral vectors transduce quiescent human HSCs at an efficiency without precedent

“…The classical LV integration pattern, which is generally accepted to be safer than that of murine leukemia virus-based vectors in hCD34 1 cells, is conserved in H/F-LV transduction in the absence of cytokine stimulation (G 0 CD34 1 cells). 3,4,[51][52][53]76 A recent study determined the VSV-G-LV IS pattern in thymidinetreated CD34 1 cells inducing a blockage into the G 0 /G 1a phase of the cell cycle. 77 Of interest, they revealed ISs primarily in RefSeq genes, in agreement with our results.…”

Applications of DNA integrating elements: Facing the bias bully

“…[20][21][22] Recently, with the explosive growth of available epigenomics datasets, attention has shifted more and more to the epigenetic determinants of target site selection. In a comparison of 3700000 MuLV integration sites in K562 cells with the corresponding ENCODE data, the previously reported bias of MuLV 23 for regulatory elements such as enhancers and promoters was confirmed. 24 An especially broad study characterized integration bias of a wide range of retroviruses, MuLV, HIV, ASLV, Porcine Endogenous Retrovirus (PERV), Xenotropic Murine leukemia virus-related Virus (XMRV), Human T-lymphotropic Virus (HTLV), and Foamy Virus (FV) with respect to histone modifications and transcription factor binding as determined by ChIPseq.…”