Striking heterogeneity of somatic L1 retrotransposition INAUGURAL ARTICLE in single normal and cancerous gastrointestinal cells Katsumi Yamaguchia,1, Alisha O. Soaresa, Loyal A. Goffa, Anjali Talasilaa, Jungbin A. Choia, Daria Ivenitskya, Sadik Karmaa, Benjamin Brophya, Scott E. Devineb, Stephen J. Meltzerc, and Haig H. Kazazian Jr.a,1 aDepartment of Genetic Medicine, The Johns Hopkins University School of Medicine, Baltimore, MD 21205; bInstitute for Genome Sciences, University of Maryland School of Medicine, Baltimore, MD 21201; and cDepartment of Medicine, The Johns Hopkins University School of Medicine, Baltimore, MD 21205 This contribution is part of the special series of Inaugural Articles by members of the National Academy of Sciences elected in 2018. Contributed by Haig H. Kazazian Jr., November 1, 2020 (sent for review September 16, 2020; reviewed by Mark A. Batzer and John V. Moran) Somatic LINE-1 (L1) retrotransposition has been detected in early et al. (14) showed that L1s are mobile in certain regions of the embryos, adult brains, and the gastrointestinal (GI) tract, and brain, particularly the hippocampus of mice. Other studies of many cancers, including epithelial GI tumors. We previously found human brain, including of single cells, solidified the consensus numerous somatic L1 insertions in paired normal and GI cancerous that somatic retrotransposition of L1 occurs in the brain, but at tissues. Here, using a modified method of single-cell analysis for low levels (∼0.2 insertions per neuron) (14–19). Somatic L1 in- somatic L1 insertions, we studied adenocarcinomas of colon, pan- sertion has also been found at very low levels in human adult creas, and stomach, and found a variable number of somatic L1 colon, stomach, esophagus, and liver (20, 21), but such data are insertions in tumors of the same type from patient to patient. We lacking for other organs. detected no somatic L1 insertions in single cells of 5 of 10 tumors In contrast, the level of L1 retrotransposition varies greatly studied. In three tumors, aneuploid cells were detected by FACS. In among different types of epithelial tumors (21–31). Retro- one pancreatic tumor, there were many more L1 insertions in an- transposition also varies from patient to patient within the same euploid than in euploid tumor cells. In one gastric cancer, both tumor type: Some tumors possess many somatic L1 insertions, aneuploid and euploid cells contained large numbers of likely while others have none (24, 25). In addition, investigators have clonal insertions. However, in a second gastric cancer with aneu- “ ” ploid cells, no somatic L1 insertions were found. We suggest that reported an artifactual detection of somatic L1 insertions from GENETICS when the cellular environment is favorable to retrotransposition, both whole tissue and single-cell analysis. Chimera artifacts arise aneuploidy predisposes tumor cells to L1 insertions, and retro- either during multiple displacement amplification (MDA) or the transposition may occur at the transition from euploidy to aneu- ligation and PCR steps of library preparation (32–35). Chimeras ploidy. Seventeen percent of insertions were also present in are sequences extending from a reference or polymorphic non- normal cells, similar to findings in genomic DNA from normal tis- reference L1Hs to a downstream flanking sequence less than 5 to sues of GI tumor patients. We provide evidence that: 1) The num- 10 kb away (33). ber of L1 insertions in tumors of the same type is highly variable, Here, we used a modified method to analyze single cells from 2) most somatic L1 insertions in GI cancer tissues are absent from adenocarcinomas of the colon, pancreas, and stomach for so- normal tissues, and 3) under certain conditions, somatic L1 retro- matic L1 insertions. We found that many presumptive new in- transposition exhibits a propensity for occurring in aneuploid cells. sertions close to reference human L1s were, in fact, artifacts: They could not be validated in any cells. They were absent from somatic retrotransposition | gastrointestinal cancer | single cells genomic DNA (gDNA) of tissues and their 5′ ends could not be recovered by PCR of the 5′ flank. As others have reported, we hile protein-coding DNA sequences make up only 1.5% of found significant patient-to-patient variability in the number of Wthe human genome, remnants of transposable elements somatic L1 insertions in tumors of the same type. Among in- comprise nearly 50% (1). The 500,000 known LINE-1s (L1s), sertions detected in tumors, about 17% (10 of 59) were also one group of retrotransposons, comprise 17% of total human DNA, but nearly all are inactive, the result of ancient truncated Significance insertions and full-length insertions that previously mutated during human evolution. We humans have some 100 to 150 ac- Using a modified method, we analyzed single cells from ade- tive L1s in our genomes (2), but only a handful of these are very nocarcinomas of the colon, pancreas, and stomach and their “ ” active or hot (2, 3). They become mobile only when their DNA paired normal tissues for somatic LINE-1 retrotransposition is transcribed into RNA, and this RNA is reverse-transcribed events. We found that somatic retrotransposition varied sig- back into DNA and inserted into the genome. This process of nificantly from patient to patient with cancers of the same retrotransposition has been modeled in cell culture (4). The L1- type, as well as in different cells within a particular cancer. encoded proteins, endonuclease and reverse transcriptase, tend to act predominantly in cis on the L1 transcript that encoded Author contributions: K.Y., L.A.G., and H.H.K. designed research; K.Y., A.O.S., A.T., J.A.C., them (5–7). Paradoxically, these autonomous elements also drive D.I., S.K., B.B., and S.E.D. performed research; S.J.M. contributed new reagents/analytic tools; S.J.M. provided samples; K.Y., A.O.S., and S.E.D. analyzed data; and K.Y. and H.H.K. the insertion of nonautonomous Alus (8), SVAs (SINE-R, VNTR, wrote the paper. Alu – and )(911), processed pseudogenes (6, 7), and other small Reviewers: M.A.B., Louisiana State University; and J.V.M., University of Michigan, RNAs (12). This process of in trans retrotransposition depends Ann Arbor. upon the L1 endonuclease and reverse transcriptase encoded by the The authors declare no competing interest. L1 ORF2 protein. Published under the PNAS license. For many years, investigators believed that L1 retrotransposition 1To whom correspondence may be addressed. Email: [email protected] or hkazazi1@ occurred exclusively in germ cells and the very early human embryo, jhmi.edu. > because so many L1s and Alus (together, 1.5 million) are fixed This article contains supporting information online at https://www.pnas.org/lookup/suppl/ in the genome. In addition, it was difficult to find L1 RNA doi:10.1073/pnas.2019450117/-/DCSupplemental. outside of the testis and ovary (13). However, in 2005, Muotri www.pnas.org/cgi/doi/10.1073/pnas.2019450117 PNAS Latest Articles | 1of8 Downloaded by guest on September 24, 2021 present in normal cells adjacent to the tumor, similar to previous single-copy sites on different chromosomes. To use the MDA DNA for library findings in gDNA from normal tissues (21, 29). production, we required a positive PCR product from all 16 sites, except for the colon cancer samples, where we worked with cells that had 14 to 16 Materials and Methods positive sites. Even with the highest-quality MDA kit, this stringent criterion was fulfilled in only 0 to 50% of single-cell DNA tested per single FACS- To detect somatic L1 insertions in single cells, we modified previous methods sorting experiment. (36) at both the wet bench and computational phases. These changes led to After MDA and its quality-control procedure, we carried out asymmetric, more efficient detection of somatic L1 insertions and improved accuracy in one-sided PCR off the 3′ ends of human-specific L1s using the specific ACA reducing chimeras. Briefly, we isolated single tumor and nontumor cell nu- sequence 94 bp upstream of the polyA tract (38). To obtain a very high clei by FACS sorting. When possible, we separated aneuploid nuclei on the fraction of 3′ flanking sequences of these L1s, we then circularized the basis of DNA content [nuclei having a DNA content peak greater than a product (Fig. 2) and carried out inverse PCR around the circle in both di- euploid peak: A peak between G1 and G2 (Fig. 1B) or a peak greater than G2 rections using two oligonucleotides, both of which were located on the (Fig. 1D)]. Distance along the abscissa is proportional to DNA content. In L1Hs-specific sequence (SI Appendix, Supplemental Methods). Additional Fig. 1B, aneuploid cells (∼240 to 260) have less DNA than euploid cells (∼260 adapters were then added to the resulting linearized products, increasing to 280), while in Fig. 1D, aneuploid cells (∼260 to 280) have more DNA than the specificity for human-specific L1s by anchoring the 3′ end of the L1 euploid cells (∼180 to 200). primer to the specific G residue in L1Hs 9 bp upstream of the polyA tract. The We then amplified single-cell DNA (∼12 pg) by MDA to 0.5 to 5 μg (37). resulting library was quality-checked for flanking sequences 3′ to human- MDA DNA was then quality-checked by PCR amplification of 16 different specific L1s by topoisomerase cloning and Sanger sequencing of 5 to 10 Fig. 1. FACS analysis of nuclei from cases 352 and 6041. (A) The 352 normal pancreas nuclei; G2 phase (R11) nuclei were sorted. (B) The 352 pancreatic cancer nuclei; G2 phase (R11) and sub G2 (R10) nuclei are sorted. (C) The 6041 normal gastric nuclei; G2 phase (R11) nuclei are sorted.