Abstract
It is becoming increasingly appreciated that the non-coding genome may have a great impact on the regulation of chromatin structure and gene expression. The innate immune response can be mediated upon lipopolysaccharide stimulation of macrophages which leads to immediate transcriptional activation of early responsive genes including tumor necrosis factor alpha (Tnfα). The functional role of non-coding RNAs, such as lncRNAs and microRNAs, on the transcriptional activation of proinflammatory genes and the subsequent regulation of the innate immune response is still lacking mechanistic insights. In this study we wanted to unravel the functional role of the lncRNA SeT, which is encoded from the murine Tnfα gene locus, and miR-155 on the transcriptional regulation of the Tnfα gene. We utilized genetically modified mice harboring either a deletion of the SeT promoter elements or the mature miR-155 and studied the response of macrophages to lipopolysaccharide (LPS) stimulation. We found that decreased expression of the lncRNA SeT in murine primary macrophages resulted in increased mortality of mice challenged with LPS, which was corroborated by increased Tnfα steady state mRNA levels and a higher frequency of biallelically expressing macrophages. On the contrary, miR-155 deletion resulted in reduced Tnfα mRNA levels supported by a lower frequency of biallelically expressing macrophages upon stimulation with LPS. In both cases, in the absence of either lncRNA SeT or miR-155 we observed a deregulation of the Tnfα allele homologous pairing, previously shown to regulate the switch from mono- to bi-allelic gene expression. Although lncRNA SeT was not found to be a direct target of miR-155 its stability was increased upon miR-155 deletion. This study suggests a role of the non-coding genome in mediating Tnfα mRNA dosage control based on the regulation of homologous pairing of gene alleles and their subsequent biallelic expression.
Highlights
Innate immunity constitutes the first defensive line against pathogen invasion [1]
We questioned whether this reduction in the SeT mRNA levels would have an impact in the Tnfα mRNA levels expressed in macrophages upon their induction with LPS
Our analysis showed that the Tnfα mRNA levels expressed in bone marrow derived macrophages (BMDMs), upon their stimulation with LPS, from the knockout mice compared to their wild type counterparts were clearly increased (Fig 2A) in both the CX3CR1-Cre SeTfl/fl and the SeT-/- mice
Summary
Innate immunity constitutes the first defensive line against pathogen invasion [1]. Macrophages (Mφs), the main cellular component of innate immunity, can be activated by, among others, interleukins (IL-4, IL-10), interferon-γ (IFN-γ), or Gram-negative bacterial lipopolysaccharide (LPS), in order to exert their homeostatic role under normal, pathogenic and tumorigenic conditions [2,3,4,5,6]. The long non-coding RNAs (lncRNAs) are RNA transcripts exceeding 200 nucleotides (nt) in size that can be traced either in the nucleus or the cytoplasm in a polyadenylated or not form [12,13] Regarding their location relative to protein coding genes they can be further separated into intronic, long intergenic (lincRNAs), bidirectional, antisense and pseudogene lncRNAs [14]. Regarding their functional role, ncRNAs can interact with target RNA, DNA sequences or even proteins and are implicated in both gene silencing or activation or serve as a decoy mechanism competing with other regulatory proteins for binding in specific sites [15,16,17]. The ~2,6 kb NKILA lncRNA was found to downregulate cancer-related inflammation pathways by binding to the p65 component of the NF-κB complex and masking the phosphorylation sites responsible for IκB-mediated release of the NF-κB complex in the nucleus [23]
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