PROJECT SUMMARY/ABSTRACT
Viral respiratory infections are the most common causes of exacerbations in children and adults with allergic
asthma. Because pathogenic viruses initially replicate in the mucosa, the epithelium plays a key role in mounting
innate inflammatory responses. Upon detection of replicating virus, the interferon regulatory factor-1 (IRF1) is
highly induced, producing type III interferon (IFNL), a key mediator of mucosal innate immunity. We have
discovered that the IRF1 gene is regulated by a previously unknown upstream enhancer-like promoter
(Epromoter) whose activity is induced by viral infection and silenced by cell-state changes produced by allergic
asthma. In preliminary studies, we find that the potential IRF1 Epromoter displays enhancer activity of virus-
inducible IRF1 transcription in primary human small airway epithelial cells (hSAECs). The IRF1 Epromoter
transcribes an unannotated IRF1 antisense upstream (AU) long noncoding RNA, IRF1-AU. Expression of the
5.8 kb IRF1-AU is inducible by viral infection and changed by mesenchymal transition. In this R21 application,
we will investigate the hypothesis that IRF1 Epromoter is a cell-state regulated epigenetic regulator of
mucosal IRF1-IFNL response. We will dissect the relative contributions of IRF1 Epromoter as the enhancer of
IRF1 and the promoter of IRF1-AU on viral inducible IRF1-IFNL response in two hypotheses: 1. The IRF1-
Epromoter maintains the IRF1-IFNL pathway in a highly inducible state by direct (looping) interactions
with the proximal IRF1 promoter. We will test the presence of direct looping interactions by chromatin
conformation capture (3C/4C) studies using KRAB-dCas9 silencing or CRISPR/Cas9 excision of IRF1-
Epromoter. We will measure effects on the accessibility of IRF1 proximal promoter by quantifying binding of
IRF3 and NF¿B transcription factors, recruitment of transcriptional elongation machinery and accumulation of
euchromatin marks by chromatin immunoprecipitation (XChIP) assays. Studies are also designed to examine
the effects of cell-state transitions. 2. The inducible IRF1-AU is a ribonucleo-protein complex that facilitates
transcriptional elongation of IRF1. We will modulate IRF1-AU using gain- and loss-of-function approaches
and challenge wild type and mesenchymal transitioned hSAECs to rhinovirus. Changes in chromatin looping,
recruitment of IRF3/NF¿B and transcriptional elongation will be measured. Using established affinity purification
(AP)-LC-MS, we will identify the composition of the IRF1-AU ribonucleoprotein complex. We are uniquely
qualified to conduct these studies based on our track record of CRISPR/Cas9 genome targeting, XChIP and
discovery proteomics. Upon completion of these studies, we will have elucidated a novel mechanism for
epigenetic control of mucosal IRF1-IFNL production that will position us for a R01 level project. This project
would establish the role of the IRF1 Epromoter and IRF1-AU in innate anti-viral immunity in vivo, and to identify
how cell-state changes produced by allergic asthma modify its composition and function.
