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

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.

Subfamily-specific differential contribution of individual monomers and the tether sequence to mouse L1 promoter activity

“…Thus, additional efforts should be devoted to cellular niches that are known to support high levels of L1 activity, such as during early embryogenesis, gametogenesis, neurogenesis, and tumorigenesis (reviewed in [ 57 ]). Lastly, while there has been significant progress in mapping transcriptional regulators of human L1 expression [ 58 , 59 ], the field has been lagging in understanding transcription regulation of mouse L1 promoters. Future efforts should also focus on characterizing both cis and trans regulatory elements for mouse L1 expression, including those both common and unique to specific mouse L1 subfamilies.…”

Factors Regulating the Activity of LINE1 Retrotransposons

“…However, this has only been shown for some types of cancer cells [385]. Activity of Dual Specificity Phosphatase 1 (DUSP1) downregulate L1 in cancers cells [386]. DUSP1 is also known as mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1), which is involved in the negative regulation of cellular proliferation and the suppression of inflammation [386].…”

An eQTL-based Approach Reveals Candidate Regulators of LINE-1 RNA Levels in Lymphoblastoid Cells

“…Much of the work on L1 regulation relies on overexpressing full-length L1 elements in cell lines that can tolerate these manipulations (Liu et al 2018; Mita et al 2020). However, there are some approaches aimed at characterizing transcription factors that bind endogenous L1 promoters (Sun et al 2018; Briggs et al 2021), in addition to those that implement gene network approaches to find potential regulators that may act through alternative mechanisms (Chung et al 2019). These, in our view, complement plasmid-based approaches through a more physiological study of L1 regulators.…”