LINE-1 Retrotransposons: A Source of Replication Stress in Cancer
Embargo until
2023-05-01
Date
2018-12-17
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Johns Hopkins University
Abstract
Long Interspersed Element-1 (LINE-1, L1) is the only protein-coding, functional retrotransposon in the human genome. Retrotransposition-competent LINE-1 generates new genomic copies of itself through a copy-and-paste mechanism. This activity has generated much of the genome’s repetitive content over evolutionary time. The genome defends against this mutagenesis both transcriptionally, by methylating the LINE-1 promoter, and post-transcriptionally, using a myriad of host-cell proteins to interfere with LINE-1 RNA processing and preventing LINE-1 insertions into DNA.
LINE silencing breaks down during oncogenesis, resulting in aberrant RNA and protein expression in a multitude of carcinomas. The extent to which malignancies express LINE-1 proteins and support retrotransposition is puzzling when considering the degree of cytotoxicity conferred by LINE-1. Ectopic expression of LINE-1 in human cancer cells in culture slows their growth and damages their DNA. Loss of LINE-1 repression in mouse germline, including both ovary and testis, causes toxicity that results in infertility. Consequently, the fact that cancers tolerate LINE-1 expression and continue to proliferate highlights a gap in knowledge regarding the role of LINE-1 retrotransposons in cell survival.
The work in this dissertation begins to address this knowledge gap. First, I review the literature surrounding the use of LINE-1 expression as a cancer biomarker. Next, I describe the development and validation of monoclonal antibodies that detect the LINE-1 open reading frame 2 protein (ORF2p), an endonuclease and reverse transcriptase that nicks genomic DNA and reverse transcribes LINE-1 cDNA during insertion. Detection of this protein has been historically difficult due to low antigen abundance, but through a series of biochemical assays to validate antibody sensitivity and specificity, I am able to detect the protein in cancers that are known to be experiencing de novo retrotransposition. Finally, the third chapter of this dissertation focuses on identifying genetic determinants of fitness in cells expressing LINE-1. Through a series of CRISPR knockout screens and RNA-sequencing studies, I uncover a mechanism of LINE-1-dependent cytotoxicity suggesting that de novo insertions conflict with DNA replication. I propose a model in which retrotransposition intermediates collide with replication forks and induce replication fork stalling, which creates unique molecular vulnerabilities in LINE-1-expressing cells.
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Keywords
LINE-1, Replication Stress