PMC

(A) Top 10 lncRNAs downregulated in TLR2-activated BMDMs. Expression levels (log₂-FC) are from the RNA-seq dataset from BMDMs stimulated with Pam3CSK4 for 5 hr ().
(B and C) RT-qPCR analysis of lincRNA-EPS expression in BMDMs (B) and DCs (C) stimulated with TLR ligands.
(D) RT-qPCR analysis of lincRNA-EPS expression in WT, Trif-KO or MyD88/Trif-DKO BMDMs stimulated with TLR ligands for 6 hr.
(E) WT BMDMs were pretreated with BAY11-7082 (10 μM) or DMSO, followed by LPS stimulation for 6 hr. Expression of lincRNA-EPS (left) and IL-1α (right) was quantified by RT-qPCR. Data are shown relative to DMSO treated cells. ^*, p < 0.05; ^**, p < 0.01.
(F) lincRNA-EPS expression in WT and IKKβ-deficient BMDMs that were treated or not with LPS for 6 hr. ^*, p < 0.05; ^**, p < 0.01.
(G and H) lincRNA-EPS expression in BMDMs exposed to microbial infections (G) or proinflammatory cytokines (H) for indicated times.
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(A) RT-qPCR analysis of RNAs purified from nuclear (red) and cytosolic (black) compartments in BMDMs.
(B) Single molecule RNA FISH detecting endogenous lincRNA-EPS molecules (red) in resting BMDMs. DNA (blue) was stained with DAPI, and autofluorescence detected by staining with probes against GFP (green). A representative image (100× magnification) is shown. DIC, differential interference contrast.
(C) Quantification of lincRNA-EPS foci detected by RNA FISH in resting BMDMs. Results are shown as % of cells that showed either a nuclear or cytosolic lincRNA-EPS foci in >500 randomly selected cells. ^**p < 0.01.
(D) Quantification of cells with lincRNA-EPS foci (nuclear or cytosolic) detected by RNA FISH in BMDMs in response to LPS stimulation for 6 hr. ^**p < 0.01.
(E) Schematic for the isolation of chromatin-associated RNAs following cross-linking (formaldehyde and glutaryldehyde).
(F) Western blotting of histone H3 (upper panel) in nuclear fractions isolated by sucrose-gradient fractionation. Purified RNA was reverse-transcribed using oligo-dT primer, and analyzed by qPCR (lower panel).
(G) Histone H3 RIP followed by RT-qPCR analysis of co-purified RNAs in formaldehyde cross-linked BMDMs.

(A) RT-qPCR analysis of lincRNA-EPS levels in iBMDMs expressing ectopic lincRNA-EPS or empty vector control (EV Ctl).
(B) Heatmap of gene expression in iBMDMs expressing ectopic lincRNA-EPS or EV Ctl that were treated or not with LPS for 6 hr, and analyzed by NanoString. Data are shown for 23 genes (out of 94 genes) that were suppressed by at least 2-fold in cells expressing ectopic lincRNA-EPS.
(C and D) Rescue of lincRNA-EPS function in lincRNA-EPS^−/− macrophages through ectopic expression. lincRNA-EPS levels in WT iBMDMs, or lincRNA-EPS^−/− iBMDMs expressing ectopic lincRNA-EPS or EV Ctl analyzed by RT-qPCR (C). These cells were stimulated with LPS for 6 hr and analyzed by RT-qPCR to measure lincRNA-EPS regulated IRGs (D). ^*, p < 0.05; ^**, p < 0.01.
(E and F) iBMDMs expressing ectopic lincRNA-EPS or EV Ctl were stimulated as indicated, or infected with E. coli (10 MOI; multiplicity of infection). Levels of secreted IL-1β was quantified by ELISA (E), and cell lysates analyzed by western blot (F).
(G and H) Addback of ASC expression in iBMDMs expressing ectopic lincRNA-EPS was confirmed by immunoblotting ASC (G), and IL-1β levels measured by ELISA (H). n.s, non-specific.
See also .

(A–D) Cytokine levels in serum (A), peritoneal fluid (B), liver (C) and spleen (D) of WT and lincRNA-EPS^−/− (KO) mice challenged i.p. with E. coli LPS (5 mg/kg/mice) for 5 hr. Data are shown as mean ± SEM (n = 4–6 mice per group). ^*, p < 0.05; ^**, p < 0.01, ^***, p < 0.001; ns, not significant.
(E) Heatmap of gene expression in spleens isolated from WT and lincRNA-EPS^−/− mice challenged i.p. with LPS for 5 hr, and analyzed by NanoString. Image represents fold change relative to untreated WT mice for all genes that were differentially expressed at least 2-fold in response to LPS (n = 4–6 mice per group).
(F) Basal gene expression profiles of IRGs in vivo in spleens isolated from WT and lincRNA-EPS^−/− mice, and analyzed by RT-qPCR. ^*, p < 0.05; ^**, p < 0.01; ns, not significant.
(G) Survival data of WT and lincRNA-EPS^−/− mice in response to LPS challenge. The numbers of mice that survived in each condition is provided. The statistical test of differences was calculated using Log-rank (Mantel-Cox) test with p < 0.05 considered as significant.
(H) Integrated model depicting lincRNA-EPS as a transcriptional brake that controls basal and inducible expression of IRGs in macrophages. TF, transcription factor.
See also .

(A) SDS-PAGE analysis of proteins purified from in vitro binding assay using biotinylated lincRNA-EPS or antisense control RNA, and macrophage nuclear extracts. The highlighted protein bands were subjected to Mass Spectrometry analysis.
(B) Western blot confirms lincRNA-EPS and hnRNPL interaction in vitro.
(C and D) hnRNPL RIP followed by RT-qPCR analysis of co-purified RNAs in non-cross-linked BMDMs (C) and formaldehyde cross-linked BMDMs (D). Immunoprecipitation of hnRNPL was assessed by western blot (inset, panel C).
(E) Western blot assessing the knockdown of hnRNPL in iBMDMs stably expressing shRNAs targeting non-overlapping regions of hnRNPL, and the control shRNA against GFP.
(F and G) Control iBMDMs or those expressing hnRNPL specific shRNAs were stimulated with LPS, and subjected to RT-qPCR analysis of Cxcl10 mRNA (F), and ELISA against Cxcl10 protein levels (G).
(H) RT-qPCR analysis of unspliced and spliced forms of lincRNA-EPS in iBMDMs expressing control or hnRNPL specific shRNAs. ^*, p < 0.05.
(I) Schematic of lincRNA-EPS deletion mutants used in RNA-protein binding assays.
(J) hnRNPL binds 3′-region of lincRNA-EPS. In vitro RNA: protein binding assay was performed using biotinylated full-length or deletion mutants of lincRNA-EPS and the nuclear extracts isolated from BMDMs, captured using streptavidin beads, and subjected to western blot against hnRNPL.
(K) The 3′-region (2000 – 2531 nt) of lincRNA-EPS is necessary and sufficient to suppress Cxcl10 expression. lincRNA-EPS^−/− iBMDMs expressing full-length or deletion mutants of lincRNA-EPS were stimulated with LPS for 6 hr, and mRNA levels analyzed by RT-qPCR. ^*, p < 0.05; ^**, p < 0.01.
(L) Schematic of CANACA motifs in the 3′-region (2000 – 2531 nt) of lincRNA-EPS. The nucleotide mutations in CANACA tracts are highlighted.
(M) lincRNA-EPS binds hnRNPL through a CANACA motif (2386 – 2391 nt) embedded in its 3′-region. In vitro RNA: protein binding assay was performed using biotinylated WT or CANACA tract mutant versions of the full-length lincRNA-EPS, captured using streptavidin beads, and subjected to western blot against hnRNPL.
(N) Functional role of CANACA motif (2386 – 2391 nt) of lincRNA-EPS. lincRNA-EPS^−/− BMDMs expressing WT or the point mutants of the full-length lincRNA-EPS (defective in hnRNPL binding) were stimulated with LPS for 6 hr, and mRNA levels analyzed by RT-qPCR. ^**, p < 0.01.
See also .

(A) Numbers of differentially expressed genes (≥ 2-FC over WT; Q-value < 0.05) in lincRNA-EPS^−/− BMDMs relative to WT cells in resting (0 hr) and LPS treated cells (2 and 6 hr). Data are from RNA-seq performed in biological duplicates.
(B) Gene ontology (GO) analysis of differentially expressed genes (≥ 2-FC over WT; Q-value < 0.05 in RNA-seq) between lincRNA-EPS^−/− BMDMs and WT BMDMs following LPS treatment for 6 hr. The top 5 most significantly enriched GO terms (−log₁₀ P-values for biological processes) in differentially expressed genes relative to all other genes in the genome (background model) are shown.
(C) Venn diagram represents the number of immune genes (defined in extended experimental methods) that were differentially expressed (≥ 2-FC over WT; Q-value < 0.05 in RNA-seq) in lincRNA-EPS^−/− BMDMs.
(D) Heatmap of top 50 upregulated genes in lincRNA-EPS^−/− BMDMs relative to WT cells at 6 hr post-LPS treatment. log2-FC values were calculated from RNA-seq (FPKM+1), and are equivalent to lincRNA-EPS KO/WT. A subset of common IRGs are highlighted in red.
(E) Circos plot of differentially expressed genes (≥ 2-FC over WT in RNA-seq) for untreated cells (inner track) and LPS-stimulated cells (middle track 2 hr, outer track 6 hr). Immune genes (defined in extended experimental methods) are highlighted in red and all other genes are in blue.
(F) RT-qPCR analysis of lincRNA-EPS regulated IRGs in untreated, and LPS-treated WT and lincRNA-EPS^−/− BMDMs.
(G) mRNA levels of cytokine genes in WT and lincRNA-EPS^−/− BMDMs stimulated with LPS for 6 hr. ^**, p < 0.01.
(H) Protein levels of indicated cytokines in WT and lincRNA-EPS^−/− BMDMs stimulated with LPS for 12 hr, and analyzed by ELISA. ^**, p < 0.01; nd, non-detectable.
See also .

(A) ChIP-qPCR analysis of RNA pol II at lincRNA-EPS regulated IRGs in macrophages expressing ectopic lincRNA-EPS or EV Ctl that were treated or not with LPS for 5 hr. ChIP purified DNA was analyzed by qPCR targeting the TSSs of indicated genes.
(B) ChIP-qPCR analysis of RNA pol II at lincRNA-EPS regulated IRGs in WT and lincRNA-EPS^−/− (KO) BMDMs that were treated or not with LPS for 5 hr.
(C and D) ChIP-qPCR analysis of H3K4me3 (C) and RNA pol II Ser2P levels (D) in WT and lincRNA-EPS^−/− BMDMs that were treated or not with LPS for 5 hr.
(E and F) RNA antisense purification (RAP) of endogenous lincRNA-EPS in BMDMs, followed by RT-qPCR analysis of retrieved RNA (E), and qPCR analysis of co-purified DNA (F). qPCR analysis of Cxcl10, Ccl5 and ASC genomic regions was performed using oligos directed around their TSS. #, not detected.
(G) ATAC-seq results showing fold change of ATAC-seq signals (KO/WT) versus ATAC-seq signals at WT promoters (+/− 1 kb of TSS) for lincRNA-EPS regulated genes identified in RNA-seq (red: upregulated; blue: downregulated) (; ), and all other genes (gray dots). lincRNA-EPS target genes showing more open chromatin are highlighted. Dash lines represent 1.2 fold change.
(H) NucleoATAC analysis showing nucleosome signals in WT (blue) and lincRNA-EPS^−/− BMDMs (KO; red) at basal state. Aggregate nucleosome signals are shown within the promoters (+/− 1 kb of TSS) of genes that are regulated by lincRNA-EPS (above panel), or within the promoters of all genes on a genome-wide level (bottom panel). Repositioning of −1 nucleosomes away from TSS in lincRNA-EPS^−/− BMDMs is highlighted by an arrow.
(I and J) Genome tracks of Cxcl10 (I) and Gpr84 (J) showing chromatin accessibility (normalized ATAC-seq signal), and nucleosome positioning (NucleoATAC signals) centered around their transcription start sites in WT and lincRNA-EPS^−/− BMDMs at basal conditions.
See also .