Unbiased proteomic mapping of the LINE-1 promoter using CRISPR Cas9

Erica M Briggs; Paolo Mita; Xiaoji Sun; Susan Ha; Nikita Vasilyev; Zev R Leopold; Evgeny Nudler; Jef D Boeke; Susan K Logan

doi:10.1186/s13100-021-00249-9

Unbiased proteomic mapping of the LINE-1 promoter using CRISPR Cas9

Mob DNA. 2021 Aug 23;12(1):21. doi: 10.1186/s13100-021-00249-9.

Authors

Erica M Briggs^{1

2}, Paolo Mita^{1

2

3}, Xiaoji Sun^{3

4}, Susan Ha⁵, Nikita Vasilyev¹, Zev R Leopold⁵, Evgeny Nudler^{1

6}, Jef D Boeke^{1

3

7}, Susan K Logan^{8

9}

Affiliations

¹ Department of Biochemistry and Molecular Pharmacology, NYU Grossman School of Medicine, Alexandria Center for Life Sciences, 450 East 29th Street, Room 321, New York, NY, 10016, USA.
² Present Address: Opentrons Labworks, Queens, NY, USA.
³ Institute of Systems Genetics, NYU Grossman School of Medicine, New York, NY, 10016, USA.
⁴ Cellarity, Cambridge, MA, USA.
⁵ Department of Urology, NYU Grossman School of Medicine, Alexandria Center for Life Sciences, 450 East 29th Street, Room 321, New York, NY, 10016, USA.
⁶ Howard Hughes Medical Institute, NYU Grossman School of Medicine, New York, NY, USA.
⁷ Department of Biomedical Engineering, NYU Tandon School of Engineering, Brooklyn, NY, USA.
⁸ Department of Biochemistry and Molecular Pharmacology, NYU Grossman School of Medicine, Alexandria Center for Life Sciences, 450 East 29th Street, Room 321, New York, NY, 10016, USA. Susan.Logan@nyulangone.org.
⁹ Department of Urology, NYU Grossman School of Medicine, Alexandria Center for Life Sciences, 450 East 29th Street, Room 321, New York, NY, 10016, USA. Susan.Logan@nyulangone.org.

Abstract

Background: 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. 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.

Results: Here, 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.

Conclusion: Our 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.

Keywords: C-BERST; CRISPR Cas9 Restricted Spatial Tagging; Cancer; LINE-1; Transcriptional regulation.

Abstract

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