RESUMEN
Our respiratory system is vital to protect us from the surrounding nonsterile environment; therefore, it is critical for a state of homeostasis to be maintained through a balance of inflammatory cues. Recent studies have shown that actively transcribed noncoding regions of the genome are emerging as key regulators of biological processes, including inflammation. lincRNA-Cox2 is one such example of an inflammatory inducible long intergenic noncoding RNA functioning to fine-tune immune gene expression. Using bulk and single-cell RNA sequencing, in addition to FACS, we find that lincRNA-Cox2 is most highly expressed in the lung and is most upregulated after LPS-induced lung injury (acute lung injury [ALI]) within alveolar macrophages, where it functions to regulate inflammation. We previously reported that lincRNA-Cox2 functions to regulate its neighboring protein Ptgs2 in cis, and in this study, we use genetic mouse models to confirm its role in regulating gene expression more broadly in trans during ALI. Il6, Ccl3, and Ccl5 are dysregulated in the lincRNA-Cox2-deficient mice and can be rescued to wild type levels by crossing the deficient mice with our newly generated lincRNA-Cox2 transgenic mice, confirming that this gene functions in trans. Many genes are specifically regulated by lincRNA-Cox2 within alveolar macrophages originating from the bone marrow because the phenotype can be reversed by transplantation of wild type bone marrow into the lincRNA-Cox2-deficient mice. In conclusion, we show that lincRNA-Cox2 is a trans-acting long noncoding RNA that functions to regulate immune responses and maintain homeostasis within the lung at baseline and on LPS-induced ALI.
Asunto(s)
Lesión Pulmonar Aguda , Ciclooxigenasa 2 , Inflamación , Macrófagos Alveolares , ARN Largo no Codificante , Lesión Pulmonar Aguda/inducido químicamente , Lesión Pulmonar Aguda/genética , Lesión Pulmonar Aguda/metabolismo , Animales , Ciclooxigenasa 2/genética , Ciclooxigenasa 2/metabolismo , Modelos Animales de Enfermedad , Inflamación/genética , Inflamación/metabolismo , Lipopolisacáridos/farmacología , Macrófagos Alveolares/metabolismo , Ratones , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismoRESUMEN
Determining the layers of gene regulation within the innate immune response is critical to our understanding of the cellular responses to infection and dysregulation in disease. We identified a conserved mechanism of gene regulation in human and mouse via changes in alternative first exon (AFE) usage following inflammation, resulting in changes to the isoforms produced. Of these AFE events, we identified 95 unannotated transcription start sites in mice using a de novo transcriptome generated by long-read native RNA-sequencing, one of which is in the cytosolic receptor for dsDNA and known inflammatory inducible gene, Aim2. We show that this unannotated AFE isoform of Aim2 is the predominant isoform expressed during inflammation and contains an iron-responsive element in its 5'UTR enabling mRNA translation to be regulated by iron levels. This work highlights the importance of examining alternative isoform changes and translational regulation in the innate immune response and uncovers novel regulatory mechanisms of Aim2.
Asunto(s)
Empalme Alternativo , Proteínas de Unión al ADN/genética , Exones , Inmunidad Innata/genética , Inflamación/genética , Macrófagos/metabolismo , Regiones no Traducidas 5' , Animales , Células Cultivadas , Proteínas de Unión al ADN/metabolismo , Perfilación de la Expresión Génica , Humanos , Inflamación/inmunología , Inflamación/metabolismo , Macrófagos/inmunología , Ratones , Regiones Promotoras Genéticas , TranscriptomaRESUMEN
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. Transcriptomic 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.
Asunto(s)
Ciclooxigenasa 2/metabolismo , Inmunidad/genética , Modelos Genéticos , ARN Largo no Codificante/metabolismo , Animales , Elementos de Facilitación Genéticos/genética , Eliminación de Gen , Regulación de la Expresión Génica , Células HEK293 , Humanos , Lipopolisacáridos/farmacología , Pulmón/metabolismo , Macrófagos/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Mutación/genética , ARN/metabolismo , Empalme del ARN/genética , ARN Largo no Codificante/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Bazo/metabolismo , Transcripción GenéticaRESUMEN
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.