CRISPR/Cas-based screening of long non-coding RNAs (lncRNAs) in macrophages with an NF-κB reporter

Abstract

The innate immune system protects against infections by initiating an inducible inflammatory response. NF-κB is one of the critical transcription factors controlling this complex response, but some aspects of its regulation remain unclear. For example, although long non-coding RNAs (lncRNAs) have been shown to critically regulate gene expression, only a fraction of these have been functionally characterized, and the extent to which lncRNAs control NF-κB expression is unknown. Here, we describe the generation of a GFP-based NF-κB reporter system in immortalized murine bone marrow–derived macrophages (iBMDM). Activation of this reporter, using Toll-like receptor ligands, resulted in GFP expression, which could be monitored by flow cytometry. We also established a CRISPR/Cas9 gene deletion system in this NF-κB reporter line, enabling us to screen for genes that regulate NF-κB signaling. Our deletion-based approach identified two long intergenic non-coding(linc)RNAs, lincRNA-Cox2 and lincRNA-AK170409, that control NF-κB signaling. We demonstrate a potential novel role for lincRNA-Cox2 in promoting IκBα degradation in the cytoplasm. For lincRNA-AK170409, we provide evidence that this nuclearly-localized lincRNA regulates a number of inflammation-related genes. In conclusion, we have established an NF-κB–GFP iBMDM reporter cell line and a line that stably expresses Cas9. Our approach enabled the identification of lincRNA-Cox2 and lincRNA-AK170409 as NF-κB regulators, and this tool will be useful for identifying additional genes involved in regulating this transcription factor critical for immune function.

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

“…Consistent with prior work, we found that lincRNA-Cox2 --deficient macrophages had altered expression of numerous inflammatory genes (Carpenter et al, 2013; Covarrubias et al, 2017). In addition, we observed a profound cis function for lincRNA-Cox2 : lincRNA-Cox2-defi- cient mice had severely reduced expression of the neighboring gene Pfgs2, a gene that encodes for cyclooxygenase-2 (Cox2), a central enzyme of the prostaglandin biosynthesis pathway, across several tissues.…”

“…Although there are a number of studies, both by our lab and others, showing that lincRNA-Cox2 promotes and restrains different classes of innate immune gene expression, none have shown the function of this gene in vivo (Carpenter et al, 2013; Covarrubias et al, 2017; Hu et al, 2016; Tong et al, 2016; Xue et al, 2018). Here we expand our knowledge of the role of lincRNA-Cox2 by generating two murine models and multiple macrophage cell lines, which strengthens our earlier findings that lincRNA-Cox2 can inhibit expression of ISGs and enhance pro-inflammatory gene expression.…”

“…KO mice were born at expected Mende- lian frequencies with no obvious developmental abnormalities (Sauvageau et al, 2013). We and others have previously published that lincRNA-Cox2 acts to positively and negatively regulate the expression of distinct classes of innate immune genes (Carpenter et al, 2013; Covarrubias et al, 2017; Hu et al, 2016; Tong et al, 2016; Xue et al, 2018). In those studies, short hairpin RNA (shRNA)-based knockdown of lincRNA- Cox2 reduced the levels of pro-inflammatory cytokines like Interleukin-6 (Il6) in bone-marrow derived macrophages (BMDMs) activated with LPS, whereas a number of interferon- stimulated genes (ISGs) were expressed at higher levels.…”

“…These trans -acting lincRNAs, such as FOXF1 adjacent non-coding developmental regulatory RNA (FENDRR), long-intergenic non-coding-erythroid prosurvival (linc-EPS), and Nettoie Salmonella pasTheiler’s (NeST), can function to regulate chromatin states (Atianand et al, 2016; Gomez et al, 2013; Grote et al, 2013; Sauvageau et al, 2013), influence nuclear structure and organization (Rinn and Guttman, 2014), or interact with and regulate the behavior of proteins and/or other RNA molecules (Covarrubias et al, 2017; Kawasaki et al, 2018; Lee et al, 2016; Wang et al, 2017). …”

“…Previous work from our lab and others defined an immune-inducible lincRNA, lincRNA-Cox2 (synonym Ptgs2os2) with broad trans -regulatory activity on inflammatory responses (Carpenter et al, 2013; Covarrubias et al, 2017; Hu et al, 2016; Tong et al, 2016; Xue et al, 2018). In macrophages, where this lincRNA was highly induced upon inflammatory activation, lincRNA-Cox2 functioned to activate and repress distinct classes of innate immune genes (Carpenter et al, 2013).…”