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

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 ances...

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Autores principales: Briggs, Erica M., Mita, Paolo, Sun, Xiaoji, Ha, Susan, Vasilyev, Nikita, Leopold, Zev R., Nudler, Evgeny, Boeke, Jef D., Logan, Susan K.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8381588/
https://www.ncbi.nlm.nih.gov/pubmed/34425899
http://dx.doi.org/10.1186/s13100-021-00249-9
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author Briggs, Erica M.
Mita, Paolo
Sun, Xiaoji
Ha, Susan
Vasilyev, Nikita
Leopold, Zev R.
Nudler, Evgeny
Boeke, Jef D.
Logan, Susan K.
author_facet Briggs, Erica M.
Mita, Paolo
Sun, Xiaoji
Ha, Susan
Vasilyev, Nikita
Leopold, Zev R.
Nudler, Evgeny
Boeke, Jef D.
Logan, Susan K.
author_sort Briggs, Erica M.
collection PubMed
description 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. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s13100-021-00249-9.
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spelling pubmed-83815882021-08-23 Unbiased proteomic mapping of the LINE-1 promoter using CRISPR Cas9 Briggs, Erica M. Mita, Paolo Sun, Xiaoji Ha, Susan Vasilyev, Nikita Leopold, Zev R. Nudler, Evgeny Boeke, Jef D. Logan, Susan K. Mob DNA Research 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. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s13100-021-00249-9. BioMed Central 2021-08-23 /pmc/articles/PMC8381588/ /pubmed/34425899 http://dx.doi.org/10.1186/s13100-021-00249-9 Text en © The Author(s) 2021 https://creativecommons.org/licenses/by/4.0/Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) . The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/ (https://creativecommons.org/publicdomain/zero/1.0/) ) applies to the data made available in this article, unless otherwise stated in a credit line to the data.
spellingShingle Research
Briggs, Erica M.
Mita, Paolo
Sun, Xiaoji
Ha, Susan
Vasilyev, Nikita
Leopold, Zev R.
Nudler, Evgeny
Boeke, Jef D.
Logan, Susan K.
Unbiased proteomic mapping of the LINE-1 promoter using CRISPR Cas9
title Unbiased proteomic mapping of the LINE-1 promoter using CRISPR Cas9
title_full Unbiased proteomic mapping of the LINE-1 promoter using CRISPR Cas9
title_fullStr Unbiased proteomic mapping of the LINE-1 promoter using CRISPR Cas9
title_full_unstemmed Unbiased proteomic mapping of the LINE-1 promoter using CRISPR Cas9
title_short Unbiased proteomic mapping of the LINE-1 promoter using CRISPR Cas9
title_sort unbiased proteomic mapping of the line-1 promoter using crispr cas9
topic Research
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8381588/
https://www.ncbi.nlm.nih.gov/pubmed/34425899
http://dx.doi.org/10.1186/s13100-021-00249-9
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