Endogenous Retrovirus-Derived Long Noncoding RNA Enhances Innate Immune Responses via Derepressing RELA Expression

Endogenous retroviruses are transposable genetic elements comprising 8% to 10% of the human and mouse genomes. Although most ERVs have been inactivated due to deleterious mutations, some are still transcribed. However, the biological functions of transcribed ERVs are largely unknown. Here, we identified a full-length ERV-derived lncRNA, designated lnc-EPAV, as a positive regulator of host innate immune responses. We found that silencing lnc-EPAV impaired virus-induced cytokine production, resulting in increased viral replication in cells. The lnc-EPAV-deficient mice exhibited enhanced susceptibility to viral challenge. We also found that lnc-EPAV regulated expression of RELA, an NF-κB subunit that plays a critical role in antiviral responses. ERV-derived lncRNA coordinated with a transcription repressor, SFPQ, to control Rela transcription. Our report provides new insights into the previously unrecognized immune gene regulatory mechanism of ERV-derived lncRNAs.

expression of inflammatory cytokines, interferons, and ISGs confirmed activation of the immune responses (see Fig. S1A in the supplemental material). By comparing the global expression patterns of ERVs, we found that the expression levels of ERVs were lower than those of coding genes (NM in RefSeq gene annotation) and known noncoding RNAs (NR in RefSeq gene annotation) in resting cells (Fig. 1A), consistent with the notion that most ERVs are genomic mutants or are silenced by the host due to evolutionary stress (25,26). Interestingly, expression levels of ERV were globally induced by poly(I·C) stimulation, while those of coding genes and known noncoding RNAs mainly remained unchanged (Fig. 1A), suggesting that ERVs are more sensitive to pathogenic stimuli.
Since FL-ERVs contain complete proviral sequences and likely serve as lncRNAs with comprehensive features when their coding regions are mutated, we next examined the dynamic expression changes of these FL-ERVs upon poly(I·C) treatment in mouse macrophages. We identified 5,322 FL-ERVs among a total of 896,922 ERV elements from the mouse genome by the use of LTR_FINDER (27). To further define the transcribed FL-ERV-derived noncoding RNAs with high confidence, the Coding Potential Assessment Tool (CPAT) algorithm (default coding probability cutoff value of Յ0.44 indicating noncoding sequence) (28) coupled with a strict threshold (uniquely aligned reads, Ն5; fragments per kilobase per million [FPKM] transcripts mapped, Ն0.1 per FL-ERV) was applied. Finally, we identified 1,278 FL-ERV-derived noncoding RNAs among 5,322 FL-ERVs. The corresponding heat map showed that most of the differentially FL-ERVderived noncoding RNAs (FPKM value of Ն1 and fold change value of Ն2) were rapidly upregulated after stimulation (Fig. 1B), consistent with the trend of global ERV expression shift (Fig. 1A). Among these, a lncRNA of the ERV1 family was found to be the most highly upregulated transcript (Fig. 1B). We named this lncRNA "lnc-EPAV" and characterized its potential functions in antiviral innate immunity. lnc-EPAV was transcribed from the positive strand of the intergenic region flanked by the coding genes Fibroblast growth factor receptor 4 (Fgfr4) and Nuclear receptor-binding SET-domain protein 1 (Nsd1) in the 13qB1 chromosome (Fig. 1C). We next verified whether lnc-EPAV was upregulated by RNA viruses. Northern blotting detected an ϳ4.7-kb lnc-EPAV transcript, in line with the full-length signal identified by RNA-seq ( Fig. 1C and D). Importantly, a stronger Northern blot band was observed upon poly(I·C), Sendai virus (SeV), and vesicular stomatitis virus (VSV) stimulation (Fig. 1D), confirming the increased expression of lnc-EPAV stimulated by both pathogenic mimics and viruses. Real-time fluorescence quantitative PCR (qPCR) independently confirmed such upregulation upon pathogenic stimulation (Fig. 1E). To determine whether expression of lnc-EPAV is conserved in different mouse strains, we assessed the transcription level of lnc-EPAV in other commonly used experimental mouse strains. The qPCR results showed that lnc-EPAV was also upregulated in BMDMs from BALB/c and 129/Sv mouse strains after VSV infection (Fig. S1B). All of these results demonstrate that lnc-EPAV expression can be upregulated by both pathogenic mimics and viruses.
lnc-EPAV is activated by NF-B subunit RELA. We hypothesized that the rapid upregulation of lnc-EPAV after pathogenic stimulation was mediated by immunerelated transcription factors (TFs). To identify such TFs, the TRANSFAC database was used to analyze the TF binding sites of promoter region (including the 5= long terminal repeat [5= LTR]) of lnc-EPAV. Gene Ontology (GO) annotation enrichment analysis was performed (Fig. S2A), and 10 putative immune system-related TFs in the lnc-EPAV promoter region were selected for further investigation. We assessed the effect of these TFs on lnc-EPAV promoter activation by a luciferase reporter assay. The results showed that overexpression of RELA significantly induced activation of the lnc-EPAV promoter ( Fig. 2A). RELA occupancy of lnc-EPAV promoter was also confirmed by chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) (Fig. 2B). Sequence analysis showed that there is a potential NF-B/RELA binding motif (at nucleotide [nt] ϩ256 to nt ϩ266 relative to transcription start sites [TSS]) at the lnc-EPAV 5= LTR region. To characterize the RELA binding motif, we generated a series of lnc-EPAV promoter truncation and mutation constructs for luciferase reporter assay (Fig. 2C, left). Overexpression of RELA induced the activation of lnc-EPAV promoter wild-type (WT), T1, and T2 constructs but failed to activate the T3 and mutant constructs that were devoid of NF-B/RELA binding motif (5=-TGTACTTTCCC-3=) (Fig. 2C, right). The results of these experiments suggest that the region spanning nt ϩ256 to nt ϩ266 of lnc-EPAV 5= LTR contains the binding site for RELA-mediated activation.
To further assess the functional role of RELA in VSV-induced lnc-EPAV expression, BMDMs were treated with NF-B-specific inhibitor pyrrolidine dithiocarbamate (PDTC), which prevented RELA from transferring to the nucleus and accumulating in the cytoplasm (Fig. S2B). This treatment reduced the level of expression of lnc-EPAV after VSV infection (Fig. 2D). Consistent with this result, the RNA levels of lnc-EPAV were significantly lower in Rela-silenced cells than in control cells upon VSV infection ( Fig. 2E; see also Fig. S2C). Taken together, these results demonstrate that NF-B subunit RELA is required for activated transcription of lnc-EPAV.
We next asked whether the RELA motif also existed in other ERV families. Four ERV family-derived lncRNAs were identified in this study, namely, ERV1, ERVL, ERVL-MaLR, and ERVK (Fig. S2D). By scanning the putative ERV-derived lncRNA promoter sequences (the 5= LTR sequence plus 500 nt before 5= LTR), we found that the RELA binding motif Impact of lnc-EPAV on Antiviral Immune Responses ® was globally located in the promoter region of four ERV families (Fig. S2E). However, the average number of RELA motifs in the members of the ERV1 and ERVL-MaLR families was higher than in the members of the ERVL and ERVK families. We speculated that the transcription of lnc-EPAV was controlled by several factors, including TFs and epigenetic modification. RELA is among the key factors that contribute to the upregulation of lnc-EPAV. lnc-EPAV enhances cellular antiviral responses. To investigate the role of lnc-EPAV in cellular antiviral responses, we designed short hairpin RNAs (shRNAs) targeting two different sites of lnc-EPAV and generated lnc-EPAV-silenced mouse J774A.1 macrophages. Endogenous lnc-EPAV was silenced efficiently as quantified by qPCR (Fig. S3A). We did not observe any off-target effects on shRNA putative target sequences ( Fig. S3B to I). Next, we measured the levels of replication of a recombinant VSV expressing green fluorescent protein (VSV-GFP) in lnc-EPAV-silenced cells. Silencing lnc-EPAV greatly enhanced VSV replication in terms of GFP-positive (GFP ϩ ) cell numbers (Fig. 3A). Consistent with this, both the viral RNA levels measured by qPCR and the virus titers determined by plaque assay showed that silencing lnc-EPAV significantly promoted viral replication in J774A.1 macrophages ( Fig. 3B and C). In addition, replication of VSV was dramatically attenuated by lnc-EPAV overexpression (Fig. S3J). These data suggest that lnc-EPAV is involved in cellular antiviral responses.
To explore the underlying mechanism by which lnc-EPAV modulates antiviral responses, we performed RNA-seq to analyze the global effects of lnc-EPAV in J774A.1 macrophages infected with VSV for 12 h. A total of 16 significant pathways were identified in lnc-EPAV-silenced cells through gene set enrichment analysis (GSEA) performed with KEGG gene sets (normalized enrichment scores [NES] greater than or equal to 1 or less than or equal to Ϫ1; false-discovery-rate [q] value, Յ0.05). Most enriched KEGG pathways were involved in pathogen infection and immune responses (Fig. 3D). GSEA was performed with the transcription factor target set (MSigDB C3-TFT) and identified NF-B/RELA as a master transcription factor associated with the immune responses in lnc-EPAV knockdown cells (Fig. 3E). Consistently, the expression levels of of NF-B/RELA target genes, including the beta interferon (IFN-␤), interleukin-6 (IL-6), and TNF-␣ genes, significantly decreased at both the mRNA level ( Fig. 3F to H) and the protein level ( Fig. 3I to K) in lnc-EPAV knockdown cells after VSV infection. These results implied the presence of cross talk between NF-B/RELA and lnc-EPAV.
We next evaluated the impact of lnc-EPAV knockdown on RELA expression. Depletion of endogenous lnc-EPAV significantly reduced Rela expression (Fig. 3L). Immunoblotting confirmed downregulation of RELA protein levels in lnc-EPAV-silenced cells (Fig. 3M). We hypothesized that lnc-EPAV might regulate antiviral responses through upregulation of RELA and, if so, that forced expression of RELA could reverse the effects of silencing lnc-EPAV on viral replication. To this end, exogenous RELA was overexpressed in the lnc-EPAV-silenced J774A.1 macrophages. Overexpression of RELA rescued the effects of silencing lnc-EPAV to inhibit VSV replication (Fig. 3N). These results suggest that lnc-EPAV may regulate the expression of RELA and its target genes during virus infection and may consequently inhibit viral replication.
SFPQ is a binding partner of lnc-EPAV in the nucleus. Although we have provided clues indicating that RELA was a key regulator in mediating lnc-EPAVdependent antiviral effects, the details of the molecular mechanism by which lnc-EPAV controls RELA expression are still lacking. qPCR of nuclear fractions and of cytoplasmic fractions revealed that lnc-EPAV was mostly located in the nucleus (Fig. 4A). These data hint that lnc-EPAV executed its function in the nucleus. To characterize the functional region of lnc-EPAV, we constructed a series of lnc-EPAV truncation constructs (Fig. 4B). Each lnc-EPAV truncation mutant was overexpressed in J774A.1 macrophages and was then assessed for its antiviral effects. Full-length lnc-EPAV and the E2 lnc-EPAV truncation mutant were found to affect the virus replication most significantly (Fig. 4C). These results suggest that RNA sequences (1,041 to 2,000 nt; E2) of lnc-EPAV are essential for its function. Impact of lnc-EPAV on Antiviral Immune Responses ® As nuclear lncRNAs usually interact with proteins to exert their functions, we applied RNA pulldown coupled with mass spectrometry (MS) to identify the interacting proteins of lnc-EPAV. Biotinylated full-length lnc-EPAV and lnc-EPAV E2 truncation mutants were incubated with nuclear extracts and pulled down with streptavidin magnetic beads. The associated proteins were analyzed by SDS-PAGE with silver staining (Fig. 4D; see also Fig. S4A) followed by mass spectrometry. By analyzing the full-length and E2 region sequences of lnc-EPAV interacting proteins, we identified two potential RNA binding proteins, namely, SFPQ and DDX21 (see Table S1 in the supplemental material; see also data available under ProteomeXchange identifier PXD011577). To confirm the binding of these proteins to lnc-EPAV, we first performed an independent RNA pulldown experiment. The results showed that the sense strand of lnc-EPAV bound both SFPQ and DDX21 but that the antisense strand failed to do so (Fig. 4E). Next, we asked if endogenous SFPQ and DDX21 were able to coimmunoprecipitate with lnc-EPAV. Only the anti-SFPQ antibody enriched lnc-EPAV and not the anti-DDX21 antibody ( Many lncRNAs are known to interact with nuclear proteins (e.g., TFs and RNA binding proteins) to regulate gene expression (29,30). SFPQ is a nuclear protein with DNA and RNA binding activity and exerts transcriptional inhibition of CYP17 (31) and IL-8 (32). We speculated that lnc-EPAV may cooperate with SFPQ to regulate downstream immune gene expression. To test this, we knocked down Sfpq by the use of shRNA, which led to reduced viral replication in terms of GFP ϩ cell numbers (Fig. 4H) and VSV titers (Fig. 4I). In line with a reduction in VSV loads, SFPQ knockdown resulted in increased levels of mRNA expression of immune genes, including Rela, Ifnb1, Il6, and Tnf ( Fig. 4J to M), and in increased levels of RELA protein (Fig. S4C). These results indicate that the binding of lnc-EPAV to SFPQ may derepress the transcription activity of immune genes and ultimately contribute to antiviral effects.
lnc-EPAV cooperates with SFPQ to regulate rela. We further explored the details of the mechanism by which lnc-EPAV interacts with SFPQ to regulate antiviral responses. To examine whether SFPQ directly bound to the promoter region of immune genes such as Rela, chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) was performed in BMDMs. Model-based ChIP-seq analysis (MACS) (33) was used to detect the statistically significant peaks of mapped reads. The distribution of putative SFPQ binding sites around the TSS gene was enriched (Fig. 5A). We then applied GO term enrichment analysis of the SFPQ putative target genes by ChIP assay and found that 172 were immune genes, including Rela (Table S2). To investigate whether SFPQ occupied the promoter region of Rela in resting macrophages and ceased to occupy the region after viral infection, we examined the SFPQ representative read coverage over the Rela promoter. Notably, a high level of binding signal of SFPQ was observed around the promoter region of Rela but the level was attenuated in macrophages after VSV stimulation (Fig. 5B). Such a change of occupancy upon VSV infection was confirmed by ChIP-qPCR and ChIP-PCR ( Fig. 5C and D). Meanwhile, the mRNA level of Rela was significantly increased after VSV infection (Fig. 5E). Immunoblotting confirmed the upregulation of RELA protein expression in VSV-infected cells  Impact of lnc-EPAV on Antiviral Immune Responses ® (Fig. 5F). We further assessed the effect of SFPQ on Rela promoter repression using luciferase reporters. Transient overexpression of SFPQ inhibited the transcriptional activity of Rela, while knockdown of SFPQ activated its transcriptional activity after VSV infection (Fig. 5G). Several studies showed that the VSV matrix (M) protein may shut down host cell translation (34,35). In order to examine whether SPFQ translation was shut down by VSV infection, the level of expression of SFPQ was quantified by immunoblot analysis. The level of protein expression of SFPQ was unchanged during the VSV infection within 24 h (Fig. 5H). These results indicate that the dissociation of SFPQ from Rela promoter may promote the transcriptional activation of Rela upon viral infection.
To explore whether lnc-EPAV functions through SFPQ, we performed ChIP-qPCR on lnc-EPAV-silenced macrophages. The results of ChIP-qPCR showed that the levels of avidity of SFPQ for Rela promoter DNA in the resting state wer esimilar in lnc-EPAVsilenced cells and control cells (Fig. 5I, left). After VSV infection, a significant decrease in SFPQ binding to the Rela promoter was observed in control cells, indicating activation of Rela transcription (Fig. 5I, Mock versus VSV-infected scrambled control cells). However, the level of SFPQ binding to the Rela promoter in lnc-EPAV-silenced cells before and after VSV infection remained the same (Fig. 5I, Mock versus VSV-infected lnc-EPAV-silenced cells). These results indicated that the absence of lnc-EPAV hindered the dissociation of SFPQ from Rela promoter under conditions of viral infection, leading to transcriptional repression. Next, we examined whether the positive effect of lnc-EPAV on Rela transcription is dependent on SFPQ. The results demonstrated that depletion of lnc-EPAV significantly reduced the Rela mRNA expression level but that the effect was absent from SFPQ knockdown cells (Fig. 5J). Consistently, lnc-EPAV overexpression promoted Rela expression, whereas it had no effect in SFPQ knockdown cells (Fig. 5K), indicating that lnc-EPAV acts upstream of SFPQ. Altogether, these results suggest that lnc-EPAV binds SFPQ and removes its occupancy in the Rela promoter, leading to transcription of Rela.
lnc-EPAV protects mice against viral infection. The aforementioned in vitro results provided a solid basis for in vivo studies. We thus created mice that lost lnc-EPAV by removing the full-length lnc-EPAV genomic locus using clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 genome-editing technology ( Fig. 6A and B). Homozygous female lnc-EPAV Ϫ/Ϫ mice exhibited growth deficiency due to unknown reasons, so we chose heterozygous mice and their littermates for experimentation. We challenged lnc-EPAV ϩ/ϩ and lnc-EPAV ϩ/Ϫ mice with VSV and found that the overall survival rate of the lnc-EPAV ϩ/Ϫ mice was much lower (Fig. 6C). VSV replication levels and titers were significantly higher in the liver and lung of lnc-EPAV ϩ/Ϫ mice than in those from lnc-EPAV ϩ/ϩ mice ( Fig. 6D and E), and there was more infiltration of inflammatory cells into the lungs of lnc-EPAV ϩ/Ϫ mice following infection (Fig. 6F). In addition, the infected lnc-EPAV ϩ/Ϫ mice developed more-severe neurological symptoms as well as decreased movement and limb paralysis in comparison to the lnc-EPAV ϩ/ϩ mice on day 3 or 4 postinfection. The levels of Ifnb1 mRNA expression in liver, lung, and spleen of lnc-EPAV ϩ/Ϫ mice were decreased after infection (Fig. 6G). In agreement with this, the level of IFN secretion induced by VSV infection was much lower in serum of lnc-EPAV ϩ/Ϫ mice than in that of lnc-EPAV ϩ/ϩ mice (Fig. 6H). Collectively, these data indicate that lnc-EPAV is an important positive regulator of antiviral immune responses in vivo.

DISCUSSION
Sequences derived from ERVs constitute a substantial fraction of human and mouse genomes. However, the biological roles of ERVs are still poorly understood. In particular,  Impact of lnc-EPAV on Antiviral Immune Responses involvement of any full-length ERV-derived lncRNAs in host immune responses has not yet been reported. In this study, we demonstrated that an ERV-derived lncRNA (named lnc-EPAV) functioned as a positive regulator of virus-induced host antiviral immune responses. lnc-EPAV expression was rapidly upregulated by viral RNA mimics or RNA viruses. Transcriptome analysis of lnc-EPAV-silenced macrophages showed that lnc-EPAV was critical for induction of NF-B/RELA target genes during viral infection.  lnc-EPAV deficiency led to reduced interferon production, resulting in enhanced susceptibility to VSV infection in mice. Mechanically, the expression of lnc-EPAV competitively displaced SFPQ from the Rela promoter to release its inhibitory effect, resulting in upregulation of RELA, which in turn promoted the expression of lnc-EPAV in a positive-feedback manner. This work revealed, for the first time, that ERV-derived lncRNA could enhance innate immune responses through derepressing a key immune gene, Rela.
Normally, due to evolutionary pressure, ERVs have been inactivated by accumulation of point mutations, insertions, or deletions to avoid deleterious impacts in host genome. The shutdown of ERV activity can also be achieved by epigenetic repression, including that resulting from DNA methylation and histone modifications. To counteract these silencing effects, ERVs hijack host transcription factors to their LTR regions. The LTR region plays a vital role as it contains all the transcriptional elements, including the TATA box, enhancers, and transcription factor binding sites, which are required for initiation of transcription of ERVs (36). In this study, an NF-B/RELA binding site was identified in the LTR of lnc-EPAV. Some studies estimated that nearly 15% of coding regions simultaneously work as both exon sequence and TF recognition sites (37). Overexpression of RELA significantly induced the activation of lnc-EPAV, whereas silencing of RELA had the opposite effect on virus-induced lnc-EPAV expression. Recruiting RELA to the ERV LTR region may help lnc-EPAV utilize host immune signaling and facilitate its transcription. In addition, we analyzed the key antiviral innate immune response transcription factor binding sites in the LTR region of the 32 upregulated FL-ERV-derived lncRNAs shown in the heat map in Fig. 1B. We found that three representative lncRNAs from different ERV families contained RELA, IRFs, and E74-like ETS transcription factor 4 (ELF4) binding sites (38). These results indicated that other factors might be involved in the regulation of ERV expression. We speculated that the transcription of lnc-EPAV was controlled by several factors, including trans-acting factors (e.g., TFs, epigenetic modification) and cis-regulatory elements in promoter or LTR regions (39). For example, ERV activation upon loss of histone methylation occurring in a lineage-specific manner depends on specific sets of transcription factors available to LTR regions (40). Therefore, we hypothesized that TFs and epigenetic modifications may work together to regulate the expression of lnc-EPAV.
lncRNAs cooperate with other molecules, usually proteins, to exert their regulatory functions. For example, lnc-DC, NRON, and lncRNA-ACOD1 interact with STAT3, NFAT, and GOT2, respectively (41)(42)(43). In this study, SFPQ was identified as a lnc-EPAVinteracting protein involved in antiviral innate immune responses. We investigated whether the SFPQ binding motif (44) in lnc-EPAV was also present in other ERV families. Interestingly, the SFPQ binding motif was specifically present in members of the ERV1 family rather than in those of other ERV families (e.g., EFVK, ERVL, and ERVL-MaLR).
SFPQ is a multifunctional protein that is involved in various biological processes, including paraspeckle function, RNA splicing, intron retention, miRNA synthesis, virus replication, and transcription regulation (45)(46)(47). Here we showed that SFPQ acted as a transcriptional repressor of key immune gene Rela. In agreement with our findings, it has been reported that SFPQ can also repress the transcription of immune genes such as IL-8 (32). SFPQ protein belongs to a conserved family of Drosophila behavior human splicing (DBHS) proteins (48). DBHS proteins encompass two RNA recognition motif domains (RRM1 and RRM2) to interact with lncRNA. SFPQ can also bind to DNA through its DNA binding domain (DBD) (48,49). These properties provide a molecular basis for the use of SFPQ by lnc-EPAV to regulate Rela expression in the nucleus.
The human and mouse SFPQ proteins share 95.25% identity, which implies conserved function. By analyzing public human SFPQ ChIP-seq data (GSE58444) (50), we observed an enriched distribution of reads around the TSS. Interestingly, we found a strong SFPQ-bound peak at the RELA promoter (see Fig. S5A in the supplemental material). By ChIP-qPCR and ChIP-PCR, we experimentally confirmed that SFPQ bound to the promoter region of RELA and that the occupancy of SFPQ at the RELA promoter was reduced upon VSV infection in human HEK293T cells (Fig. S5B). Consistent with the phenotype of SFPQ knockdown mouse cells, the expression levels of RELA were increased in SFPQ knockdown human cells (Fig. S5C). We hypothesize that human ERV (HERV)-derived lncRNAs may cooperate with human SFPQ to exert function although ERV-derived lncRNAs are not conserved in different species (51). We used RNA immunoprecipitation coupled with deep sequencing (RIP-seq) to examine whether SFPQ bound human ERV-derived transcripts in nuclei. By scanning 506,566 human ERV loci (ERV length, Ն200 nt) from the RepeatMasker database with strict cutoff values (fold change, Ն 3; FPKM, Ն1), we identified 1,025 putative SFPQ-bound human ERV-derived transcripts in the nucleus (see Table S3 in the supplemental material). The protein-RNA binding between SFPQ and three representative transcribed HERVs (MER9a2, LTR5A, and MLT2A1) was validated with independent RIP followed by reverse transcription-PCR (RT-PCR) ( Fig. S5D and E). These findings indicated the biological importance and evolutionary prevalence of such a regulatory mechanism. So our current understanding is that although lnc-EPAV is not evolutionary conserved, interactions of SPFQ with ERV-derived lncRNAs is conserved between mouse and human.
The NF-B transcription factor has vital roles in cellular processes involved in immune responses, inflammation, and oncogenesis (52)(53)(54). Although results of many studies investigating regulation of NF-B/RELA activity through several posttranslational modifications, including acetylation, phosphorylation, and ubiquitination, have been reported previously (55)(56)(57), regulation at the transcriptional level is still poorly understood. Here we report that an ERV-derived lncRNA coordinated with a transcription repressor SFPQ to control Rela transcription. In turn, RELA promoted the transcription of lnc-EPAV to form a positive-feedback loop (Fig. 7). Our findings regarding lnc-EPAV offer an insight into the previously unrecognized immune regulatory mechanism of ERV-derived lncRNAs. proteins on the beads were separated by SDS-PAGE followed by silver staining and mass spectrometry (MS) identification. Mouse anti-SFPQ antibody, mouse anti-DDX21 antibody, or IgG control was added to the BMDM nuclear extracts and incubated at 4°C for 4 h followed by incubation with SureBeads protein G magnetic beads (Bio-Rad, USA) at 4°C for 2 h. RNA/protein complexes were immunoprecipitated, and RNA was extracted and quantified with RT-PCR.
ChIP-qPCR, ChIP-seq, and data analysis. BMDMs were fixed with 1% formaldehyde and quenched with glycine. Purified chromatin was sonicated to a level of 300 to 500 bp using ultrasonic processing (Scientz-IID, China) and incubated with mouse anti-RELA antibody (CST, USA), mouse anti-SFPQ antibody (Sigma, USA), or mouse IgG control (Abmart, China). DNA-protein complexes were immunoprecipitated by the use of salmon sperm DNA-blocked SureBeads protein G magnetic beads (Bio-Rad, USA), followed by reverse cross-linking processes. The DNA was then purified and quantified by qPCR and PCR.
For ChIP-seq library construction, purified DNA was processed for end repair, followed by a 3=-end dA-adding reaction and Y-shape adaptor ligation. The final sequencing library was obtained by PCR amplification, and sequencing was then performed via the use of an Illumina HiSeq X Ten platform. For data analysis, model-based analysis of ChIP-seq (MACS) was used to identify enrichment regions (33). The ChIPseeker R package was used for ChIP peaks annotation and visualization (65).
Statistical analysis. GraphPad Prism software was used for statistical analyses. The analyses of results were performed with a two-tailed unpaired Student=s t test. Survival curves were analyzed using a log rank (Mantel-Cox) test. Data are presented as means Ϯ standard errors of the means (SEM). Data are representative of results from at least three independent experiments. P values of Ͻ0.05 were considered statistically significant.
Data availability. All sequencing data have been deposited in the NCBI SRA database under accession number PRJNA503657. Data from the proteomics studies are available via ProteomeXchange with identifier PXD011577.