Data Availability StatementRC-seq FASTQ files are available from the European Nucleotide

Data Availability StatementRC-seq FASTQ files are available from the European Nucleotide Archive (ENA) under the identifier PRJEB1785. Despite sequencing L1 integration sites at up to 250 depth by RC-seq, we found no tumour-specific, endonuclease-dependent L1 insertions. Whole genome sequencing analysis of the tumours carrying the MeCP2 and EGFR L1 mutations also revealed no endonuclease-dependent L1 insertions. In a complementary in vitro assay, wild-type and endonuclease mutant L1 reporter constructs each mobilised very inefficiently in four cultured GBM cell lines. Conclusions These experiments altogether highlight the consistent absence of canonical L1 retrotransposition in GBM tumours and cultured cell lines, purchase Doramapimod as well as atypical L1-associated sequence rearrangements following DNA damage in vivo. Electronic supplementary material The online version of this article (doi:10.1186/s13100-016-0076-6) contains supplementary material, which is available to authorized users. Background Glioblastoma multiforme (GBM) is the most common and aggressive brain tumour in adults [1]. Ninety-five percent of diagnosed GBM tumours originate (primary GBM), while the remainder progress from a lower grade glioma (secondary GBM) [2]. Primary and secondary GBM tumours are histologically indistinguishable [3]. To date, genomic analyses have elucidated somatic mutations and intra-tumoural heterogeneity governing GBM progression and resistance to therapy [4C6]. Defects in several DNA repair mechanisms, especially in the repair of DNA double strand breaks (DSBs), are known to enable genomic aberrations, such as deletions and amplifications, in GBM [7, 8]. Despite the extensive genomic analyses performed thus far, the GBM genome may yet harbour additional etiological clues that could improve treatment and patient outcomes. L1 retrotransposons are endogenous mutagens known to cause sporadic disease, including cancer [9]. A full-length human L1 is ~6?kb long [10, 11] and contains a 5 untranslated region (UTR), two non-overlapping open reading frames that encode purchase Doramapimod respectively for a 40KDa RNA binding protein (ORF1p) [12, 13] and a 150KDa protein with both endonuclease (EN) and reverse transcriptase (RT) activities (ORF2p) [14, 15], and a 3UTR. The L1 5UTR bears an internal promoter with sense and antisense activity [16, 17] and a recently described antisense open reading frame (ORF0) [18]. Canonical L1 mobilisation depends on the transcription and translation of L1 and the formation of a ribonucleoprotein particle purchase Doramapimod (RNP) consisting of ORF1p and ORF2p, and their encoding mRNA. Once the RNP enters the nucleus, the L1-encoded EN can cleave genomic DNA [15] and, typically, generate a new L1 insertion via target-primed reverse transcription (TPRT) [19]. Hallmarks of L1 integration by TPRT include use of an L1 purchase Doramapimod EN recognition motif (5-TT/AAAA), target site duplications (TSDs), and an L1 poly-A tail [20]. Endonuclease-independent (ENi) L1 mobilisation can also occur into pre-existing DNA double strand breaks, producing insertions that lack TPRT hallmarks [21C24]. Notably, L1 can mobilise other polyadenylated RNAs, such as retrotransposons, in [25C27]. L1 and elements can also participate in DNA rearrangements driven by recombination [28, 29]. Although TPRT-mediated L1 mobilisation occurs in many cancers [30C38] and neural cells [39C42], several recent studies employing high-throughput sequencing have reported a surprising absence of somatic L1 insertions in brain tumours [6, 30C32, 35]. We hypothesised that L1-associated DNA rearrangements in GBM might occur via recombination or an atypical retrotransposition mechanism and therefore may lack the TPRT hallmarks required for L1 insertion recognition by previous genomic analyses. Alternatively, we considered that L1 insertions in GBM could be restricted to sub-clonal and highly heterogeneous events. We therefore applied deep retrotransposon capture sequencing (RC-seq) [34, MMP9 42] to 14 brain tumour patients (9 GBM and 5 lower grade glioma) and detected tumour-specific L1-associated mutations lacking TPRT hallmarks in 4 GBM tumour samples, and also found no examples of TPRT-driven L1 mobilisation. Complementary assays using an engineered L1 reporter assay [43] revealed negligible in vitro L1 activity in all tested GBM cell lines. These experiments confirm that L1 mobilisation is absent or very rare in GBM tumours and cell lines. Unusual endonuclease-independent L1 retrotransposition or L1-associated recombination events can however occasionally occur, and may impact the expression of genes relevant to GBM aetiology and neural cell morphology. Methods Patient samples Tissues were obtained from 14 patients undergoing surgical removal of a brain tumour at the Department of Clinical Neurosciences, Western General Hospital, Edinburgh, UK. All patients gave informed consent for tumour and peri-tumoural.