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. 2013 Aug 16;341(6147):789-92.
doi: 10.1126/science.1240925. Epub 2013 Aug 1.

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

Susan Carpenter  1 , Daniel Aiello, Maninjay K Atianand, Emiliano P Ricci, Pallavi Gandhi, Lisa L Hall, Meg Byron, Brian Monks, Meabh Henry-Bezy, Jeanne B Lawrence, Luke A J O'Neill, Melissa J Moore, Daniel R Caffrey, Katherine A Fitzgerald
Affiliations

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

Susan Carpenter et al. Science. .

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.

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Figures

Figure 1
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
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
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
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.
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References

    1. Kawai T, Akira S. Toll-like receptor and RIG-I-like receptor signaling. Ann N Y Acad Sci. 2008 Nov;1143:1. - PubMed
    1. Medzhitov R, Horng T. Transcriptional control of the inflammatory response. Nature reviews Immunology. 2009 Oct;9:692. - PubMed
    1. Hargreaves DC, Horng T, Medzhitov R. Control of inducible gene expression by signal-dependent transcriptional elongation. Cell. 2009 Jul 10;138:129. - PMC - PubMed
    1. Foster SL, Medzhitov R. Gene-specific control of the TLR-induced inflammatory response. Clinical immunology. 2009 Jan;130:7. - PMC - PubMed
    1. Murray PJ, Smale ST. Restraint of inflammatory signaling by interdependent strata of negative regulatory pathways. Nature immunology. 2012 Sep 18;13:916. - PMC - PubMed

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