Project Summary The mammalian genome is pervasively transcribed to generate a complex non-coding transcriptome. A novel class of long non-coding RNA (lncRNA) called DoGs for “Downstream-of-Gene”-containing transcripts was recently discovered. In response to various stressors such as viral infections, DoG RNAs are expressed by readthrough transcription that continues past the ends of some protein-coding genes for at least 5kb. Remarkably, this previously unknown RNA species as a group accounts for up to 30% of all intergenic transcripts, yet their function remains completely unknown. I discovered that innate immune sensing of viral nucleic acids and activation of the type I interferon (IFN) antiviral response leads to expression of DoG RNAs from thousands of genes, most notably from the IFN gene itself. I therefore hypothesize that a DoG RNA may function to regulate its upstream gene of origin, and that this is an essential regulatory process in innate antiviral responses. Mechanistically I propose that a DoG RNA remodels the chromatin and/or transcriptional landscape of its upstream gene to achieve a poised state. This would be beneficial for organisms because rapid responses to viral infections can be achieved. In Aim 1, I will evaluate the role of the DoG RNA from the Ifnb1 gene (Ifnb1-DoG RNA) in regulating IFN-β expression using genetic, transcriptional, and post- transcriptional approaches to manipulate DoG RNA levels. In Aim 2, I will investigate the molecular basis of Ifnb1-DoG RNA function by characterizing the chromatin and transcriptional landscape of the Ifnb1 gene locus. I will test whether DoG RNA expression affects post-transcriptional processing, epigenetic marks, and chromatin architecture. In Aim 3, I will determine the physiological importance of DoG RNA in vivo. I will generate Ifnb1- DoG RNA deficient mice by first generating guide RNA knock-in mice that targets downstream of Ifnb1 and then by crossing these with dCas9 transgenic mice. I will study the effect of the Ifnb1-DoG RNA on innate immunity at homeostasis and during viral infections with these mice. The type I IFN response is a critical component of our antiviral defense mechanism, but can lead to human disease such as type I interferonopathies and systemic lupus erythematosus when dysregulated. This study aims to advance our understanding of type I IFN regulation by functionally characterizing a novel class of lncRNAs. Insights gained from this study can potentially be leveraged to develop improved therapeutics for infectious and IFN-mediated autoimmune diseases.