Novel principles of gamma-retroviral insertional transcription activation in murine leukemia virus-induced end-stage tumors

Abstract

BackgroundInsertional mutagenesis screens of retrovirus-induced mouse tumors have proven valuable in human cancer research and for understanding adverse effects of retroviral-based gene therapies. In previous studies, the assignment of mouse genes to individual retroviral integration sites has been based on close proximity and expression patterns of annotated genes at target positions in the genome. We here employed next-generation RNA sequencing to map retroviral-mouse chimeric junctions genome-wide, and to identify local patterns of transcription activation in T-lymphomas induced by the murine leukemia gamma-retrovirus SL3-3. Moreover, to determine epigenetic integration preferences underlying long-range gene activation by retroviruses, the colocalization propensity with common epigenetic enhancer markers (H3K4Me1 and H3K27Ac) of 6,117 integrations derived from end-stage tumors of more than 2,000 mice was examined.ResultsWe detected several novel mechanisms of retroviral insertional mutagenesis: bidirectional activation of mouse transcripts on opposite sides of a provirus including transcription of unannotated mouse sequence; sense/antisense-type activation of genes located on opposite DNA strands; tandem-type activation of distal genes that are positioned adjacently on the same DNA strand; activation of genes that are not the direct integration targets; combination-type insertional mutagenesis, in which enhancer activation, alternative chimeric splicing and retroviral promoter insertion are induced by a single retrovirus. We also show that irrespective of the distance to transcription start sites, the far majority of retroviruses in end-stage tumors colocalize with H3K4Me1 and H3K27Ac-enriched regions in murine lymphoid tissues.ConclusionsWe expose novel retrovirus-induced host transcription activation patterns that reach beyond a single and nearest annotated gene target. Awareness of this previously undescribed layer of complexity may prove important for elucidation of adverse effects in retroviral-based gene therapies. We also show that wild-type gamma-retroviruses are frequently positioned at enhancers, suggesting that integration into regulatory regions is specific and also subject to positive selection for sustaining long-range gene activation in end-stage tumors. Altogether, this study should prove useful for extrapolating adverse outcomes of retroviral vector therapies, and for understanding fundamental cellular regulatory principles and retroviral biology.

Avian Leukosis Virus Activation of an Antisense RNA Upstream of TERT in B-Cell Lymphomas

“…Other studies have reported integrations of hepatitis B virus and human papillomavirus DNAs in the TERT promoter region in human liver and cervical tumors, resulting in elevated TERT expression (44). While sense and antisense retroviral activation has been previously reported (1,41), to our knowledge, this is the first report of retroviral activation of a bidirectional promoter-associated lncRNA. In this work, we show that most of the proviral integrations in our tumors are in the antisense direction relative to TERT and drive the overexpression of a novel lncRNA transcribed from a bidirectional promoter.…”

“…Retroviruses can induce overexpression of host genes by multiple mechanisms (1,41). For instance, insertion of viral enhancer elements in the vicinity of host gene promoters can induce overexpression (1).…”

Infectivity and insertional mutagenesis of endogenous retrovirus in autoimmune NZB and B/W mice

“…In contrast to integrations in shortterm cell culture, where one expects little selection, tumours ex vivo have undergone strong selection for cell growth. In mouse end-stage tumours, irrespective of the distance to transcription start sites, the majority of integrations colocalized with common epigenetic enhancer markers in murine lymphoid tissues (Akagi et al, 2004;Sokol et al, 2014;Uren et al, 2008). Of 6132 tags we analysed previously in T lymphomas induced by exogenous MLV, 46.4 % were in genes.…”

“…In addition to the names of the genes, Table 2 lists the location of the insertions in or near the genes and the layout of the insertions. A separate column gives the number of additional insertions at a given locus in lymphomas recovered by us, or in lymphocytic/ myeloid tumours reported by others (Akagi et al, 2004;Sokol et al, 2014;Uren et al, 2008). Because the insertions were absent from the BALB/c-generated fusion Table 2.…”

Utility of next‐generationRNA‐sequencing in identifying chimeric transcription involving human endogenous retroviruses

“…The presence of retroviral sequence similar to gene fusions produces sequence signatures across the genome, and such signatures can be exploited to determine the orientation as well as the exact positions of endogenous and exogenous retroviruses if the chimeric junction, that is, the point of fusion with host flanking sequence, is covered in sequencing. Using paired‐end RNA‐seq in whole‐transcriptome analyses of mouse tumors we showed that integration of exogenous gamma‐retroviruses are exposed by at least one of 14 paired‐end sequence signatures that permit detection of multiple modes of insertional mutagenesis such as promoter insertion and antisense transcription (Fig. ) .…”