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. 2023 Nov 30;14(1):20.
doi: 10.1186/s13100-023-00308-3.

An intronic LINE-1 regulates IFNAR1 expression in human immune cells

Carmen A Buttler  1 , Daniel Ramirez  1 , Robin D Dowell  1 , Edward B Chuong  2
Affiliations

An intronic LINE-1 regulates IFNAR1 expression in human immune cells

Carmen A Buttler et al. Mob DNA. .

Abstract

Background: Despite their origins as selfish parasitic sequences, some transposons in the human genome have been co-opted to serve as regulatory elements, contributing to the evolution of transcriptional networks. Most well-characterized examples of transposon-derived regulatory elements derive from endogenous retroviruses (ERVs), due to the intrinsic regulatory activity of proviral long terminal repeat regions. However, one subclass of transposable elements, the Long Interspersed Nuclear Elements (LINEs), have been largely overlooked in the search for functional regulatory transposons, and considered to be broadly epigenetically repressed.

Results: We examined the chromatin state of LINEs by analyzing epigenomic data from human immune cells. Many LINEs are marked by the repressive H3K9me3 modification, but a subset exhibits evidence of enhancer activity in human immune cells despite also showing evidence of epigenetic repression. We hypothesized that these competing forces of repressive and activating epigenetic marks might lead to inducible enhancer activity. We investigated a specific L1M2a element located within the first intron of Interferon Alpha/Beta Receptor 1 (IFNAR1). This element shows epigenetic signatures of B cell-specific enhancer activity, despite being repressed by the Human Silencing Hub (HUSH) complex. CRISPR deletion of the element in B lymphoblastoid cells revealed that the element acts as an enhancer that regulates both steady state and interferon-inducible expression of IFNAR1.

Conclusions: Our study experimentally demonstrates that an L1M2a element was co-opted to function as an interferon-inducible enhancer of IFNAR1, creating a feedback loop wherein IFNAR1 is transcriptionally upregulated by interferon signaling. This finding suggests that other LINEs may exhibit cryptic cell type-specific or context-dependent enhancer activity. LINEs have received less attention than ERVs in the effort to understand the contribution of transposons to the regulatory landscape of cellular genomes, but these are likely important, lineage-specific players in the rapid evolution of immune system regulatory networks and deserve further study.

Keywords: Enhancer; Interferon; LINE-1; Transposons.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
LINEs are predominantly epigenetically repressed, but a subset also exhibit enhancer-associated epigenetics. A A heatmap shows enrichment (red) and depletion (blue) of LINE families for the histone modifications H3K9me3 (top) and H3K27ac (bottom) in 16 publicly available human primary blood cell types obtained through the Blueprint Database [28]. Enrichment of ChIP signal at LINEs was quantified using Giggle [38]. Columns represent annotated LINE families with enrichment scores of more than 100 or depletion scores of less than -100 for either histone mark in any of the datasets shown. Unfiltered heatmaps of all LINE families, as well as SINE, ERV and DNA transposon families are available in (Additional file 1: Fig. S1). Most LINE families, especially younger L1M and L1P families are strongly enriched for repressive H3K9me3 and strongly depleted for activating H3K27ac, as expected. B Heatmaps and metaplots show ChIP signal of repressive H3K9me3 and enhancer associated H3K4me1 and H3K27ac at individual LINE-1s of lengths > 500 bp, in both primary naive B cells [28], and in the lymphoblastoid cell line GM12878 [29]. 10 kb windows were set around the 5’ end of the LINE-1 to cover the entire length of LINEs. Many LINE-1s are silenced by H3K9me3 as expected (navy), and some have enhancer associated epigenetic modifications (yellow). A subset, labeled as “bivalent” exhibit both repressive and enhancer associated signals (cyan)
Fig. 2
Fig. 2
IFNAR1.L1M2a was chosen as an example locus for experimental exploration. A IFNAR1.L1M2a (blue) contains a predicted distal enhancer element identified by the ENCODE consortium (orange) [43]. Within or nearby this predicted enhancer region are several immune-related transcription factor binding motifs (red, purple). We sought to knock out this potential enhancer region, dubbed IFNAR1.L1M2a.enh (black). B The IFNAR1 gene (navy) locus. Publicly available ChIP-seq data (red), obtained from the Blueprint Database [28] and ENCODE consortium [29], along with our CUT&Tag data, describing the epigenetic landscape at the IFNAR1.L1M2a locus. Naive B cells, class switched memory B cells and the B cell-like GM12878 cell line exhibit bivalent repressive and enhancer associated histone modifications across the L1M2a insertion (highlighted blue). PRO-seq (purple) and ATAC-seq (green) both support the presence of an enhancer at the predicted site in IFNAR1.L1M2a, potentially induced by type I IFN treatment
Fig. 3
Fig. 3
CRISPR knockout of IFNAR1.L1M2a.enh alters the local chromatin landscape. Wildtype GM12878 cells (light red), as well as a representative IFNAR1.L1M2a.enh knockout clone, L3.E4 (dark red) were characterized by CUT&Tag. The expected bivalent histone modifications and PolII occupancy are present in wildtype GM12878 cells, but both PolII and enhancer associated H3K27ac are lost, and upstream repressive H3K9me3 is increased in knockout clone L3.E4 cells
Fig. 4
Fig. 4
IFNAR1.L1M2a inducibly enhances transcription of IFNAR1 during type I IFN signaling. A IFNAR1 and three nearby genes, IFNAR2, IL10RB, and IFNGR2, were all considered to be possible targets of IFNAR1.L1M2a enhancer activity due to their proximity. B RNA sequencing of three wildtype clones (gray) and four IFNAR1.L1M2a.enh knockout clones (purple) for IFNAR1.L1M2a-proximal genes during a 24 h time course of IFNβ treatment. Pairwise differential expression analysis [48] at the untreated timepoint showed slightly but significantly lower baseline expression of IFNAR1 and IFNGR2 in knockout cells, and likelihood ratio tests [48] showed significant differences in response to IFNβ treatment of IFNAR1 and IL10RB. At the 24 h timepoint, IFNAR1 expression was slightly but significantly upregulated compared to the untreated timepoint in wildtype cells but not in knockout cells, and expression of IFNAR1 was significantly lower in knockout cells. C-D Immunofluorescence was used to confirm protein-level differences in IFNAR1 expression between wildtype cells (left, gray) and knockout cells (right, purple) in untreated (top, light) and IFNβ-treated (bottom, dark) conditions. In agreement with RNA-seq data, IFNAR1 expression was induced by IFNβ treatment in wildtype cells, but not in knockout cells. Scale bar indicates 25 microns
Fig. 5
Fig. 5
Deletion of IFNAR1.L1M2a.enh alters downstream transcriptional response to IFNβ. RNA sequencing of three wildtype (gray) and four IFNAR1.L1M2a.enh knockout clones (purple) during a 24 h time course of IFNβ treatment. A Likelihood Ratio Tests [48] of RNAsequencing data revealed many genes with significantly altered IFNβ responses in knockout cells compared to wildtype. Many of these genes were expected IFN stimulated genes (red) as identified by induction in wildtype cells at the 4 h timepoint [48] (Additional file 7: Table S6). Gene ontology [51] confirmed that significantly differentially responsive genes were enriched for immune-related biological processes (red). B RNA sequencing of representative expected ISGs showed a dampened initial induction in the knockout cells (purple) compared to wildtype cells (gray) and differential expression throughout the time course of IFNβ treatment according to likelihood ratio tests [48]
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