Transcription Factor Binding Sites Are Genetic Determinant of Retroviral Integration in the Human Genome

Gamma-retroviruses and lentiviruses integrate non-randomly in mammalian genomes, with specific preferences for active chromatin, promoters and regulatory regions. Gamma-retroviral gene transfer vectors show a high frequency of insertional gene activation, and may induce neoplastic or pre-neoplastic clonal expansions in patients treated with genetically modified cells. An analysis of >10,000 integration sites of a Moloney leukemia virus (MLV)-derived vector in human CD34+ hematopoietic progenitors showed that genes involved the control of growth, differentiation and development of the hematopoietic and immune system are targeted at high frequency and enriched in integration hot spots, suggesting that the cell gene expression program is instrumental in directing MLV integration. To investigate the role of transcriptional regulatory networks in retroviral target site selection, we analyzed the distribution of transcription factor binding sites (TFBSs) flanking >4,000 MLV- and HIV-derived proviruses in human hematopoietic and non-hematopoietic cells. We show that MLV vectors integrate in genomic regions enriched in cell-type specific subsets of TFBSs, independently from their relative position with respect to genes and transcription start sites. Conversely, HIV vectors appear to avoid TFBS-rich genomic regions. Hierarchical clustering and a principal component analysis of TFBSs flanking retroviral integration sites in CD34+ and HeLa cells showed that TFBS subsets are vector- and cell type-specific. Analysis of sequences flanking the integration sites of vectors carrying mutations in their long terminal repeats (LTRs), and of HIV vectors packaged with a MLV integrase, indicates that the MLV integrase and LTR enhancer are the viral determinants of the selection of TFBS-rich regions in the genome. Chromatin immunoprecipitation analysis shows that transcription factors bind the LTR enhancers in the cell nucleus before integration, and may therefore synergize with the integrase in tethering retroviral pre-integration complexes to transcriptionally active regulatory regions. This study identifies TFBSs as differential genomic determinants of retroviral target site selection in the human genome, and indicates that gamma-retroviruses and lentiviruses have evolved dramatically different strategies to interact with the host cell chromatin. These differences predict a higher risk in using gamma-retroviral vs. lentiviral vectors for human gene therapy applications, independently from the design of the vector and the transgene expression cassette.