Abstract

BackgroundThe autonomous retroelement Long Interspersed Element-1 (LINE-1) mobilizes though a copy and paste mechanism using an RNA intermediate (retrotransposition). Throughout human evolution, around 500,000 LINE-1 sequences have accumulated in the genome. Most of these sequences belong to ancestral LINE-1 subfamilies, including L1PA2-L1PA7, and can no longer mobilize. Only a small fraction of LINE-1 sequences, approximately 80 to 100 copies belonging to the L1Hs subfamily, are complete and still capable of retrotransposition. While silenced in most cells, many questions remain regarding LINE-1 dysregulation in cancer cells.ResultsHere, we optimized CRISPR Cas9 gRNAs to specifically target the regulatory sequence of the L1Hs 5’UTR promoter. We identified three gRNAs that were more specific to L1Hs, with limited binding to older LINE-1 sequences (L1PA2-L1PA7). We also adapted the C-BERST method (dCas9-APEX2 Biotinylation at genomic Elements by Restricted Spatial Tagging) to identify LINE-1 transcriptional regulators in cancer cells. Our LINE-1 C-BERST screen revealed both known and novel LINE-1 transcriptional regulators, including CTCF, YY1 and DUSP1.ConclusionOur optimization and evaluation of gRNA specificity and application of the C-BERST method creates a tool for studying the regulatory mechanisms of LINE-1 in cancer. Further, we identified the dual specificity protein phosphatase, DUSP1, as a novel regulator of LINE-1 transcription.

Highlights

  • Long Interspersed Element-1 (LINE-1) is the only autonomous mobile element in the human genome

  • Targeting the LINE‐1 5’UTR promoter with deficient Cas9 (dCas9) C‐BERST To better understand LINE-1 transcriptional regulation in cancer cells, we utilized the dCas9 C-BERST method to map regulatory proteins bound to the LINE-1 promoter [54]

  • C Chromatin immunoprecipitation (ChIP) of dCas9 was performed in cells expressing each of the eight LINE-1 5’UTR guide RNAs (gRNA), as well as a non-targeting control (NS)

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Summary

Introduction

Long Interspersed Element-1 (LINE-1) is the only autonomous mobile element in the human genome. LINE-1 sequences have accumulated in our DNA through the process of retrotransposition, entailing the copy and paste of an RNA intermediate [1, 2]. An antisense promoter has been identified within the 5’UTR that has been shown to control expression of a third open reading frame on the antisense strand, ORF0, as well as alternative antisense. The autonomous retroelement Long Interspersed Element-1 (LINE-1) mobilizes though a copy and paste mechanism using an RNA intermediate (retrotransposition). Throughout human evolution, around 500,000 LINE-1 sequences have accumulated in the genome. Most of these sequences belong to ancestral LINE-1 subfamilies, including L1PA2-L1PA7, and can no longer mobilize. While silenced in most cells, many questions remain regarding LINE-1 dysregulation in cancer cells

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