Transposable elements make up more than half of the human genome. These mobile DNAs leave evidence of their activity in the form of sequence insertions. We recognize these as interspersed repeats.
In humans, all active mobile DNAs are retrotransposons, also known colloquially as ‘copy-and-paste’ transposons. These propagate through a process known as retrotransposition, which involves an RNA intermediate that is reverse transcribed to make the new insertion sequence (Figure 1). Each new genomic insertion is initially highly homologous to the element that templated the RNA. This homology deteriorates over time such that very ancient sequences are difficult to recognize, and ancient families of elements are difficult to subclassify. In addition to accumulating nucleotide substitutions, interspersed repeat sequences can also be interrupted by insertions of other transposable elements.
Our modern genome contends with one autonomous retroelement, Long INterspersed Element-1 (LINE-1, L1) (Burns and Boeke, 2012; Hancks and Kazazian, 2012; Levin and Moran, 2011; Ostertag and Kazazian, 2001). An intact LINE–1 sequence measures approximately 6 kilobases in length and encodes two well-recognized proteins, open reading frame 1 protein (ORF1p) and open reading frame 2 protein (ORF2p). ORF1p trimerizes to form an RNA binding complex required for LINE-1 transposition (Martin et al., 2005; Martin et al., 2003; Hohjoh and Singer, 1996). ORF2p encodes two enzymatic activities also essential for retrotransposition, an endonuclease and a reverse transcriptase (Cost and Boeke, 1998; Feng et al,. 1996; Mathias et al., 1991). While there is evidence that ORF2p interacts preferentially with the RNA that encoded it to promote its efficient retrotransposition (Kulpa and Moran, 2006), ORF2p functions can be co-opted to transpose other RNA species. This happens commonly in the germline, such that Alu (Dewannieux et al., 2003) and SINE-VNTR-Alu (SVA) retroelements (Riaz et al., 2012; Hancks et al., 2011), as well as the occasional pseudogene (Wei et al., 2001) are copied into the genome.
Mechanisms of LINE-1 toxicity
Lead Investigator: Daniel Ardeljan
Half of human cancers overexpress a LINE-1 encoded protein. LINE-1 retrotransposition employs, but is also repressed by, host cell genes in every stage of its life cycle. Since LINE-1 expression is recognized as cytotoxic in cell culture , the question arises as to whether this retroelement is cytotoxic in human cancers. My work explores the interaction of LINE-1 machinery with cell growth and death, with a particular emphasis on perturbations in human cancer. The goal of this work is to identify the mechanisms that enable cancer cells to proliferate in the face of an increased LINE-1 burden, and in so doing reveal mechanisms of host cell adaptations to LINE-1.
LINE-1 mapping and somatic insertion discovery
The repetitive nature of transposable elements makes it very challenging to accurately locate them in the genome. To solve this issue we have developed a novel method to identify insertion sites: Transposon Insertion Profiling by Sequencing (TIPseq). TIPseq utilizes a targeted amplification called vectorette PCR (Arnold and Hodgson, 1991). This technique allows for the selective amplification of the unique genomic DNA flanking the 3’ end of LINE-1 elements. Next generation sequencing of these PCR amplicons produces reads which are mapped back to the human reference genome to determine the LINE-1 insertions sites.
We use TIPseq to study LINE-1 retrotransposition in a variety of samples, including cancers. We recently identified hundreds of new somatic LINE-1 insertions in pancreatic ductal adenocarcinoma (PDAC) tumors which are absent in matched normal samples (Rodić et al., 2015). These somatic insertions are typically 5’ truncated and clonal in primary and subsequent metastatic tumors (Figure 3), although this is not always the case. Our results show that LINE-1 retrotransposition contributes to the genetic evolution of gastrointestinal cancers. We are expanding our study to include other types of cancer.