Neurofibromin 2 (NF2) Is Necessary for Efficient Silencing of LINE-1 Retrotransposition Events in Human Embryonic Carcinoma Cells
Frisbie, Trenton
2022
Abstract
Transposable elements are DNA sequences that can mobilize to new genomic locations and have had a profound impact on human genome evolution. Although ~45% of the human genome consists of transposable element-derived sequences, Long INterspersed Element-1 (LINE-1 or L1) is the only active transposable element in the human genome. L1s can mobilize (i.e., retrotranspose) in the germline, during early development, and in select somatic cells and the resultant retrotransposition events can alter gene expression, generate structural variation, and create pathogenic mutations. Given the mutagenic potential of L1 retrotransposition, it is no surprise that a variety of cellular mechanisms have evolved to restrict unabated L1 retrotransposition. Previous studies revealed that reporter genes integrated into the genome of human embryonic carcinoma cells (hECs) by L1 retrotransposition are efficiently silenced by a process that we have termed L1-REPEL (L1-delivered REPorter gEne siLencing). L1-REPEL is mitotically stable, reversible, and correlates with changes in chromatin status at the L1 integration site, suggesting an epigenetic mechanism that requires both initiation and maintenance phases. L1-REPEL is specific to the mechanism of L1 genomic integration (target-primed reverse transcription or TPRT), which utilizes both endonuclease and reverse transcriptase enzymatic activities to facilitate retrotransposition. Thus, we hypothesize that cellular factors recognize TPRT intermediates leading to the establishment of an epigenetic mark required for L1-REPEL in hECs. Here, we designed and implemented a forward genetic screen using a genome-wide CRISPR/Cas9-based system to elucidate cellular factors that mediate L1-REPEL in PA-1 hECs. We identified 20 highly enriched candidate L1-REPEL factors, including our top candidate gene, neurofibromin 2 (NF2) – a tumor suppressor gene that encodes the NF2/merlin protein. Comprehensive validation experiments revealed that NF2/merlin expression was necessary for efficient L1-REPEL in PA-1 cells. Additionally, we determined that the expression of NF2/merlin isoform 1 efficiently re-established L1-REPEL in NF2 knockout cells. We further demonstrated that NF2 knockout was insufficient to reactivate L1-REPEL in cells containing a previously silenced insertion, suggesting that NF2/merlin is required, either directly or indirectly, to initiate L1-REPEL. Finally, we found that culturing cells in differentiation media further attenuated L1-REPEL, suggesting that NF2 knockout and cellular differentiation may act independently or, perhaps, synergistically to attenuate L1-REPEL. Thus, our data indicate that NF2/merlin, a tumor suppressor gene implicated in human disease, may also play a role in silencing L1 retrotransposition events during early human development.Deep Blue DOI
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Transposable Elements Long Interspersed Element-1 Epigenetics NF2
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