Analysis of epigenetic features characteristic of L1 loci expressed in human cells

Abstract

Only a select few L1 loci in the human genome are expressed in any given cell line or organ, likely to minimize damage done to the genome. The epigenetic features and requirements of expressed L1 loci are currently unknown. Using human cells and comprehensive epigenetic analysis of individual expressed and unexpressed L1 loci, we determined that endogenous L1 transcription depends on a combination of epigenetic factors, including open chromatin, activating histone modifications, and hypomethylation at the L1 promoter. We demonstrate that the L1 promoter seems to require interaction with enhancer elements for optimal function. We utilize epigenetic context to predict the expression status of L1Hs loci that are poorly mappable with RNA-Seq. Our analysis identified a population of ‘transitional’ L1 loci that likely have greater potential to be activated during the epigenetic dysregulation seen in tumors and during aging because they are the most responsive to targeted CRISPR-mediated delivery of trans-activating domains. We demonstrate that an engineered increase in endogenous L1 mRNA expression increases Alu mobilization. Overall, our findings present the first global and comprehensive analysis of epigenetic status of individual L1 loci based on their expression status and demonstrate the importance of epigenetic context for L1 expression heterogeneity.

SCIFER: approach for analysis of LINE-1 mRNA expression in single cells at a single locus resolution

“…Similar analysis of actin (ACTB) demonstrated the expected enrichment of reads at the 3′ end of the gene locus in scRNA-Seq, with bulk RNA-Seq reads evenly distributed throughout gene exons (Additional file 1 B, top). Bulk RNA-Seq reads are equally distributed throughout the length of an L1 locus previously identified and authenticated as expressed (Additional file 1 B, bottom) [29]. Alignment of scRNA-Seq reads to the same L1 locus are enriched at the 3′ end (Additional file 1 B, bottom).…”

“…Following alignment, clustering of cells, and de-duplication of barcode-UMIs, the next step in SCIFER analysis is to parse expressed L1 loci from passively transcribed L1 sequences (also referred to as background) by cross referencing the list of L1 loci that were assigned samesense RNA-Seq alignments in the scRNA-Seq dataset with a list of full-length L1 loci validated to be expressed in MCF7 cells from a previous study (Additional file 17, Fig. 1, step 2) [29]. By summing the RPM of all L1 loci identified as expressed by this approach in each MCF7 cell, we determined the RPM levels per cell for each cluster (Fig.…”

“…Detection of full-length L1 mRNA is further complicated by chimeric transcripts generated via L1 splice sites and polyadenylation signals [23][24][25][26]. Because L1 transcripts produced as a part of other genes' transcripts are incapable of retrotransposition, they must be discarded to obtain an accurate quantification of fulllength L1 mRNA transcripts [10,12,13,18,20,[27][28][29].…”

“…We developed a method to rigorously analyze L1 mRNA expression at a single-locus resolution by filtering out truncated and passively transcribed L1 sequences [12,20,30]. This method includes cytoplasmic RNA extraction, selection of polyadenylated transcripts, stranded paired-end sequencing, unique alignment of transcripts to the reference genome, and visual validation of L1 transcript alignment to annotated, full-length L1 loci [12,20,29,30]. All existing methods developed for single-locus L1 expression [13,28], including our own [12,20,30], have limitations in their ability to reproducibly detect all L1 subfamilies in datasets with variable sequencing depth.…”

“…Despite this limitation it is a useful tool to discover expression patterns of unambiguously expressed L1 loci. Previous application of this approach demonstrated organ-, age, and sex-specific L1 loci expression as well as identified epigenetic features of expressed L1 loci [10,29]. The last frontier in L1 expression remains rigorous detection of individual L1 or other transposable elements expression in single cells.…”