Genetic Models Reveal cis and trans Immune-Regulatory Activities for lincRNA-Cox2

Abstract

SUMMARY An inducible gene expression program is a hallmark of the host inflammatory response. Recently, long intergenic non-coding RNAs (lincRNAs) have been shown to regulate the magnitude, duration, and resolution of these responses. Among these is lincRNA- Cox2, a dynamically regulated gene that broadly controls immune gene expression. To evaluate the in vivo functions of this lincRNA, we characterized multiple models of lincRNA-Cox2-deficient mice. LincRNA- Cox2-deficient macrophages and murine tissues had altered expression of inflammatory genes. Tran- scriptomic studies from various tissues revealed that deletion of the lincRNA-Cox2 locus also strongly impaired the basal and inducible expression of the neighboring gene prostaglandin-endoperoxide synthase (Ptgs2), encoding cyclooxygenase-2, a key enzyme in the prostaglandin biosynthesis pathway. By utilizing different genetic manipulations in vitro and in vivo, we found that lincRNA-Cox2 functions through an enhancer RNA mechanism to regulate Ptgs2. More importantly, lincRNA-Cox2 also functions in trans, independently of Ptgs2, to regulate critical innate immune genes in vivo.

The how and why of lncRNA function: An innate immune perspective

“…There are 10 different TLR genes in the human genome and 13 TLR genes in mice [38][39][40], each binding a different PAMP [41]. Using this extensively studied biological system, we identified the first example of a TLR-stimulated lncRNA, lincRNA-Cox2, which was capable of positively and negatively regulating distinct types of innate immune genes [42][43][44][45][46]. Knockdown of lincRNA-Cox2 resulted in impaired production of proinflammatory genes (i.e., IL-6), while IFNrelated genes were hyperactivated in the absence of lincRNA-Cox2 [42][43][44][45][46].…”

“…Using this extensively studied biological system, we identified the first example of a TLR-stimulated lncRNA, lincRNA-Cox2, which was capable of positively and negatively regulating distinct types of innate immune genes [42][43][44][45][46]. Knockdown of lincRNA-Cox2 resulted in impaired production of proinflammatory genes (i.e., IL-6), while IFNrelated genes were hyperactivated in the absence of lincRNA-Cox2 [42][43][44][45][46]. Numerous other studies have made use of the TLR-signaling biological system, uncovering and characterizing dozens of novel lncRNAs that act in a wide range of mechanisms to either positively or negatively regulate this pathway as reviewed in Carpenter et al and Hadjicharalambous et al [47,48].…”

“…There are many examples of lncRNAs that regulate their neighboring coding genes, for example: lnc-MARCKS, lnc-TNFAIP3, AS-IL1α, lnc-IL7R, and IL-1β-eRNA [52][53][54][55][56]. Recently we used genetic mouse models to show that lincRNA-Cox2 can function as an enhancer RNA in cis to regulate its neighboring gene Ptgs2 [46]. For these reasons, one aspect to consider when selecting a candidate, no matter the class, is to investigate the effect of the transcriptional expression on neighboring coding genes.…”

“…Multiple studies have demonstrated that lncRNA expression is more cell type specific compared to protein-coding genes [94][95][96]. Such specific expression patterns can often provide important clues into the specific biology that the gene could be involved in [46] (Tables 1 and 2). A variety of consortiums exist for both human and mouse and can be utilized to determine cell type specificity of a lncRNA candidate further.…”

“…We recently used multiple genetic mouse models to dissect both cis and trans functions of lincRNA-Cox2 in vivo [46]. Working with a complete lincRNA-Cox2 knockout [185], in which the gene is replaced with a LacZ cassette, we observed a strong cis defect on the neighboring protein-coding gene Ptgs2.…”