Our research focuses on human mobile genetic elements, or "jumping genes," named long interspersed element-1 (LINE-1 or L1) retrotransposons. LINE-1 elements comprise roughly 17 percent of human DNA. It is estimated that an average human genome contains ~80–100 LINE-1 elements that can "jump" (i.e., retrotranspose) to new locations. De novo LINE-1 retrotransposition events in either the germline or during early development have resulted in sporadic cases of human diseases, including hemophilia, muscular dystrophy, X-linked retinitis pigmentosa, and choroideremia. LINE-1 retrotransposition events in somatic cells also may lead to intra-individual genetic variation.
Retrotransposition-competent LINE-1 elements encode two proteins (ORF1p and ORF2p) that are required for their mobility. ORF1p has RNA-binding and nucleic acid chaperone activities. ORF2p has endonuclease and reverse transcriptase activities. The LINE-1 proteins also can promote the mobilization of short interspersed elements (e.g., Alu elements), noncoding RNAs (e.g., U6 snRNA (small nuclear RNA), and some messenger RNAs, leading to processed pseudogene formation. These sequences comprise at least 11 percent of human genomic DNA. Thus, LINE-1–mediated retrotransposition events have been instrumental in shaping our genome, accounting for nearly 1 billion bases of human DNA. Despite their abundance, we are only beginning to understand the mechanism of LINE-1 retrotransposition, the impact of LINE-1 retrotransposition in our genome, and how cellular factors influence LINE-1 mobility.
The Mechanism of LINE-1 Retrotransposition
As a postdoctoral fellow in Haig Kazazian's laboratory at the University of Pennsylvania, I developed an assay to monitor LINE-1 retrotransposition in cultured human cells (Figure 1). This assay, combined with other technologies, such as epitope tagging, has allowed us to distinguish the proteins encoded by our engineered LINE-1 constructs from those encoded by endogenous elements. The use of these assays has been instrumental in developing a working model for how LINE-1 elements mobilize to new genomic locations (Figure 2). We are conducting experiments to gain a detailed mechanistic understanding of LINE-1 retrotransposition.
The Impact of LINE-1 Retrotransposition on the Genome
We are only beginning to realize how LINE-1 elements impact the human genome. Our previous work revealed that LINE-1 is not simply an insertional mutagen, but that its retrotransposition can lead to the generation of structural variation (i.e., intrachromosomal genomic deletions and duplications) in human DNA. However, since these studies were conducted in a transformed human cervical cancer cell line (HeLa), the results may not truly reflect the consequences of LINE-1 retrotransposition in "normal" cells.
To overcome these limitations, we have begun to study LINE-1 retrotransposition in human embryonic stem (hES) cells. We have demonstrated that a number of federally approved human embryonic stem cell lines can accommodate LINE-1 retrotransposition in vitro, that the resultant retrotransposition events can occur within genes, and that LINE-1 retrotransposition events can lead to the generation of small intrachromosomal deletions (Figure 3). In addition, Frans Cremers (Radboud University, Netherlands) and his colleagues have demonstrated that LINE-1 insertions can occur early in human embryogenesis. Thus, hES cells provide a model to study LINE-1 retrotransposition in a developmentally relevant cell type. We are generating a library of LINE-1 retrotransposition events in hES cells to examine the spectrum of mutations associated with LINE-1 retrotransposition, the frequency at which LINE-1 retrotransposes into genes, and the consequences of new LINE-1 retrotransposition events on gene expression. These studies will provide a comprehensive understanding of how LINE-1 retrotransposition impacts the genome.
Cellular Factors That Influence LINE-1 Retrotransposition
Little is known about cellular factors that affect LINE-1 retrotransposition. We recently uncovered an alternative pathway of LINE-1 retrotransposition in Chinese hamster ovary cells that contain mutations in proteins involved in the nonhomologous end-joining pathway of DNA repair. This alternative pathway allows endonuclease-deficient LINE-1 elements to integrate at sites of DNA damage or dysfunctional telomeres. We speculate that endonuclease-independent LINE-1 retrotransposition is an ancestral mechanism of RNA-mediated DNA repair associated with non-LTR (long terminal repeat) retrotransposons. Moreover, our data have revealed similarities between the mechanism of endonuclease-independent LINE-1 retrotransposition and the action of telomerase, which supports the hypothesis that the reverse transcriptase activities encoded by non-LTR retrotransposons and telomerase were derived from a common ancestor. We are examining how mutations in other DNA repair genes affect endonuclease-independent LINE-1 retrotransposition. In addition, we are using candidate gene approaches, as well as genetic and biochemical screens, to identify host factors that either interact with the LINE-1 retrotransposition apparatus or regulate aspects of LINE-1 and/or Alu element retrotransposition. We hypothesize that the identification of such genes will open new frontiers in the biology of host-retrotransposon interactions.
LINE-1 Elements and Genetic Variation
Finding actively mobile LINE-1 elements in individual genome sequences is akin to finding a needle in a haystack. Although an average human is estimated to contain ~80–100 active LINE-1 elements, inter-individual genetic variation in LINE-1 content likely plays an important role in determining the absolute number of active elements in the extant population. Genomic technologies developed in collaboration with the laboratories of Richard Badge (University of Leicester, United Kingdom) and Evan Eichler (HHMI, University of Washington) have allowed us to selectively identify LINE-1 elements that have amplified in the human lineage. We have used this platform to identify retrotransposition-competent LINE-1 elements in the genomes of individuals representative of diverse geographic populations. The available data suggest that active LINE-1 elements are underrepresented in the human genome reference sequence and are far more prevalent in the human population than previously appreciated. We are beginning to investigate how these active LINE-1 elements impact human genetic variation.
In collaboration with Fred Gage (Salk Institute), we have found that human LINE-1 elements can retrotranspose in rat and human neural progenitor cells and in the brain of transgenic mice in vivo. These data overturned a long-held dogma that human LINE-1 retrotransposition is restricted to germ cells and suggest that somatic retrotransposition may contribute to intra-individual differences and somatic mosaicism in select cell types. We are continuing this collaboration to determine the developmental consequences of LINE-1 retrotransposition in somatic cells.