MLV integration site selection is driven by strong enhancers and active promoters

ABSTRACTRetroviruses integrate into the host genome inpatterns specific to each virus. Understanding thecauses of these patterns can provide insight intoviral integration mechanisms, pathology andgenome evolution, and is critical to the developmentof safe gene therapy vectors. We generated murineleukemia virus integrations in human HepG2 andK562 cells and subjected them to second-gener-ation sequencing, using a DNA barcoding techniquethat allowed us to quantify independent integrationevents. We characterized >3700000 unique integra-tion events in two ENCODE-characterized cell lines.We find that integrations were most highly enrichedin a subset of strong enhancers and active pro-moters. In both cell types, approximately half theintegrations were found in <2% of the genome,demonstrating genomic influences even narrowerthan previously believed. The integration pattern ofmurine leukemia virus appears to be largely drivenby regions that have high enrichment for multiplemarks of active chromatin; the combination ofhistone marks present was sufficient to explainwhy some strong enhancers were more prone tointegration than others. The approach we used isapplicable to analyzing the integration pattern ofany exogenous element and could be a valuable pre-clinical screen to evaluate the safety of gene therapyvectors.INTRODUCTIONRetroviruses have played important roles in pathology (1)and genome evolution (2–4). In practice, the introductionof exogenous DNA into host cell chromosomes has bothexperimental and medical utility: it can be used to disruptendogenous genes, as in insertional mutagenesis screens(5), as well as to introduce functional alleles in thecontext of gene therapy (6). In both cases, an understand-ing of the integration pattern of the vector of choice isimportant for proper experimental design. Our under-standing of the integration preferences for differentinsertional elements is rapidly changing. Initially,because of a lack of data to prove otherwise, it wasassumed that retroviral integration occurred randomly(7,8). With the completion of the human genome andmore efﬁcient sequencing technologies, it becamepossible to map many hundreds of integration events,and data quickly emerged that demonstrated that viralintegrations were non-random and speciﬁc to the viralsubtypes (9–12). These original ﬁndings were stillseverely limited because functional genome annotationbeyond transcribed genes was essentially non-existent.Since that time, more chromatin features have beenidentiﬁed, along with tens of thousands of integrationsites (13). This has led to the inference that murineleukemia virus (MLV) integrates near regulatoryelements like promoters and enhancers (14). Consistentwith this, previous studies have detected associationsbetween MLV integration and individual chromatinmarks (15,16). In addition, MLV integration data havebeen correlated with areas of active chromatin representedby DNAse hypersensitive sites (DHS) (17,18).Annotations from the ENCODE pilot regions wereused in conjunction with pyrosequencing to analyzehuman immunodeﬁciency virus integration site selection(19). Now, the ENCODE consortium has carried outdetailed studies that deeply characterize and annotatenot just the pilot regions, but the entire genome of aselect number of cell lines (20,21). These data potentiallygive researchers a comprehensive set of annotations thatallows for high-resolution examination of the speciﬁcgenomic features that drive viral integration site selection,