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

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

Erica M Briggs  1   2 , Paolo Mita  1   2   3 , Xiaoji Sun  3   4 , Susan Ha  5 , Nikita Vasilyev  1 , Zev R Leopold  5 , Evgeny Nudler  1   6 , Jef D Boeke  1   3   7 , Susan K Logan  8   9
Affiliations

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

Erica M Briggs et al. Mob DNA. .

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.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Utilizing the C-BERST method to _target LINE-1 5’UTR. A An APEX2 tagged dCas9 is recruited to the LINE-1 5’UTR promoter using L1Hs specific gRNAs. Cells are incubated with biotin-phenol for 30 min and treated with hydrogen peroxide for 1 min, triggering the biotinylation of proteins located at the LINE-1 5’UTR promoter. Biotinylated proteins are enriched through streptavidin immunoprecipitation and identified through mass spectrometry. Figure and method based on Gao et al. [54]. B Alignment of L1PA2-L1PA7 to L1Hs. Blue lines designate correct alignment to L1Hs, cream lines designate mismatch alignment to L1Hs, and arrowheads mark additional misaligned sequence missing from L1Hs. Eight guide RNAs (gRNA) were designed to _target the 5’UTR of LINE-1. Preference went to guides that aligned well with L1Hs, but lacked alignment to older LINE-1 sequences (L1PA2-L1PA7). C Chromatin immunoprecipitation (ChIP) of dCas9 was performed in cells expressing each of the eight LINE-1 5’UTR gRNAs, as well as a non-_targeting control (NS). ChIP reads were aligned to LINE-1 L1Hs, as well as older L1PA2-L1PA7, using MapRRCon analysis software [32]. Red line denotes the _target location of each gRNA. Representative data shown for gRNA 3, 4, 7 and NS. Complete data set for all gRNAs can be found in Supplemental Figure 1
Fig. 2
Fig. 2
C-BERST identification of 5’UTR biotinylated transcription factors. A Western blot of LINE-1 ORF1p expression in whole cell lysates of E006AA-hT and LNCaP cells. (Tubulin loading control). B Relative LINE-1 mRNA levels as assessed by qPCR. C Western blot of C-BERST streptavidin immunoprecipitation. Cells expressing gRNA-4, gRNA-7, or gRNA-NS were harvested, and biotinylated proteins were collected by streptavidin immunoprecipitation. A no hydrogen peroxide control (No H2O2) was included in the harvest to eliminate endogenously biotinylated proteins. Input, flow through, and immunoprecipitation (IP) were blotted with streptavidin HRP. D Proteins identified with C-BERST that were enriched at least 1.5 × in both LINE-1 _targeting gRNAs (gRNA-4, gRNA-7) when compared to the non-_targeting control (gRNA NS). Transcription factors have been highlighted in red. E Complete list of transcription factors enriched at least 1.5 × in both LINE-1 _targeting gRNAs (gRNA-4, gRNA-7) when compared to non-_targeting control (gRNA NS). Proteins previously identified to regulate LINE-1 are highlighted in green. Blue squares denote ENCODE ChIP data availability. Red squares denote peak on specified LINE-1 location by MapRRCon analysis
Fig. 3
Fig. 3
MapRRCon ChIP plots of C-BERST identified proteins. MapRRCon analysis of C-BERST enriched proteins in A E006AA-hT and B LNCaP cells. Each plot is an analysis of available ENCODE ChIP data, in multiple cell lines (listed on the right side of each plot). Position of observed peaks is noted by the full length L1Hs diagram along the bottom of the figure. Additional MapRRCon plots can be found in Supplemental Figure 2
Fig. 4
Fig. 4
DUSP1 modulates LINE-1 mRNA expression. A qPCR of LINE-1 mRNA levels in E006AA-hT cells expressing shRNA to DUSP1 or non-_targeting control (NS). B qPCR of relative LINE-1 levels in E006AA-hT cells that were treated with vehicle (DMSO) or DUSP1 inhibitor BCI. C qPCR of relative LINE-1 mRNA levels in E006AA-hT or LNCaP cells overexpressing vector only (VO) or DUSP1. D qPCR of DUSP1 mRNA levels in panel of prostate cancer cell lines (including E006AA-hT renal cell carcinoma line). E qPCR of relative LINE-1 mRNA levels (left) and western blots of LINE-1 ORF1p, DUSP1, and hsp90 (loading control) protein levels (right) in PC3 cells that were treated with siScramble or siDUSP1 siRNA. ORF1 protein levels relative to hsp90 were quantified, siScramble was set to 1
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