A long noncoding RNA mediates both activation and repression of immune response genes

Abstract

An inducible program of inflammatory gene expression is central to antimicrobial defenses. This response is controlled by a collaboration involving signal-dependent activation of transcription factors, transcriptional co-regulators, and chromatin-modifying factors. We have identified a long noncoding RNA (lncRNA) that acts as a key regulator of this inflammatory response. Pattern recognition receptors such as the Toll-like receptors induce the expression of numerous lncRNAs. One of these, lincRNA-Cox2, mediates both the activation and repression of distinct classes of immune genes. Transcriptional repression of target genes is dependent on interactions of lincRNA-Cox2 with heterogeneous nuclear ribonucleoprotein A/B and A2/B1. Collectively, these studies unveil a central role of lincRNA-Cox2 as a broad-acting regulatory component of the circuit that controls the inflammatory response.

Figure 1. lincRNA-Cox2 expression is induced by TLR ligands in a MyD88 and NFκB dependent manner

A, The Circos plot shows genome-wide differential expression (RNA-seq) between untreated bone marrow derived macrophages (BMDM) and BMDMs stimulated with Pam₃CSK₄ (TLR1/2) (5 h). The inner track shows log₂ fold-change values for protein coding genes that are classified into immune genes (red, see methods) and other genes (blue). The outer track shows log₂ fold-change value for all lncRNAs. lincRNA-Cox2 is highlighted in red on Chromosome 1 (arrow). B, lincRNA-Cox2 encodes three splice variants. C–H, qRT-PCR was performed on bone marrow derived dendritic cells (BMDC) (C–F) or bone marrow derived macrophages (BMDM). Elevated levels of lincRNA-Cox2 and Ptgs2 were observed following LPS (TLR4) (C–D), Pam₃CSK₄ (TLR2) (E–H), R848 (TLR7/8) (G–H) but not with Poly I:C (TLR3) (E–H) stimulation. I–J. Induction of lincRNA-Cox2 and Ptgs2 were found to be dependent on MyD88 following qRT-PCR on BMDMs obtained from wild type (WT) or MyD88 KO mice. K–L, BMDMs treated for 30min with an NFκB inhibitor (1 μg/ml), followed by stimulation with LPS (100 ng/ml) resulted in reduced expression levels of lincRNA-Cox2 (K) and Ptgs2 (L) as examined by qRT-PCR. Data represents mean ±SD from three independent experiments.

Figure 2. lincRNA-Cox2 is a major regulator of immune genes

A, qRT-PCR was carried out on BMDMs stably expressing lentiviral shRNA specific to lincRNA-Cox2 (shRNA) or a control shRNA. Expression of lincRNA-Cox2 was measured. B, RNA-seq was performed on lincRNA-Cox2 knockdown or control (ctl shRNA) BMDMs that were either stimulated with Pam₃CSK₄ or unstimulated. The heat map shows mRNA levels for genes annotated in GO as immune genes. These genes are among the top 50 up-regulated immune genes in unstimulated cells when lincRNA-Cox2 was silenced or the top 50 down-regulated immune genes in stimulated cells when lincRNA-Cox2 was silenced. The 100 genes were ranked by absolute log₂ fold-change values and the top 80 differentially expressed genes displayed. C, Heatmap representation of differentially regulated genes from a custom designed gene codeset performed on RNA extracted from Ctl or lincRNA-Cox2 knockdown cells stimulated with Pam₃CSK₄ for 5 h. D–E, Cells were stimulated with Pam₃CSK₄ for 24 h, increased Ccl5 (Rantes) (D) and reduced IL6 (E) cytokine levels were identified in lincRNA-Cox2 knockdown cells by elisa (n.d means not detected). F–I, lincRNA-Cox2 was silenced in interferon α/β receptor (IFN α/β R) KO cells. Expression levels of lincRNA-Cox2 (F), Ccl5 (Rantes) (G), Irf7 (H) and Ifi204 (I) were measured using qRT-PCR to define direct targets of lincRNA-Cox2.

Figure 3. Differential gene expression following over expression of lincRNA-Cox2

A, BMDM stably over-expressing lincRNA-Cox2 or a Ctl vector were generated. qRT-PCR was carried out and over-expression of lincRNA-Cox2 was confirmed. B, Heatmap representation of differentially regulated genes from a custom designed gene codeset performed on RNA extracted from Ctl or lincRNA-Cox2 over expressing BMDMs. C, qRT-PCR was carried out on Ctl or lincRNA-Cox2 over expressing cells stimulated with Pam₃CSK₄ for 5 h, increased Il6 expression levels were identified using qRT-PCR. Data represents mean ±SD from three independent experiments.

Figure 4. lincRNA-Cox2 is localized to both the cytosolic and nuclear compartments, interacts with hnRNP-A/B and A2/B1 to regulate immune genes

A, BMDMs were labeled with a lincRNA-Cox2 probe using RNA FISH, and counterstained with DAPI (DNA). B–C, Biotinylated lincRNA-Cox2 or antisense RNA was incubated with nuclear extracts and interaction with endogenous hnRNP-A/B (B) or hnRNP-A2/B1 (C) assessed following IP/western (top panels). Expression levels of hnRNP- A/B (B) or hnRNP-A2/B1 (C) (lower panels) in input lysates were also examined. D, Cell lines with shRNA targeting hnRNP-A/B or hnRNP-A2/B1 were stimulated with Pam₃CSK₄, elevated Ccl5 (Rantes) production was identified in these cells by elisa. E, Heatmap representation of differentially regulated genes of Ctl, hnRNP-A/B or hnRNP-A2/B1 expressing BMDMs stimulated with Pam₃CSK₄ (100nM) for 5 h. F–G, Silencing of lincRNA-Cox2, hnRNP-A/B or hnRNP-A2/B1 promotes recruitment of RNA Pol II to the Ccl5 promoter as determined by CHIP analysis. H, hnRNP-A/B or hnRNP-A2/B1 was knocked down using lentiviral shRNA in lincRNA-Cox2 over-expressing BMDMs, Ccl5 expression levels were measured using qRT-PCR.