Project Grant R21AI180682

SILENCER-ENHANCER TRANSITION DYSREGULATES INTERLEUKIN-6 IN MUCOSAL EPITHELIAL CELL PLASTICITY - PROJECT SUMMARY/ABSTRACT RESPIRATORY VIRUSES, SUCH AS INFLUENZA A VIRUS, RHINOVIRUS (RV), RESPIRATORY SYNCYTIAL VIRUS (RSV) AND CORONAVIRUS, TRIGGER INNATE INFLAMMATION AND LUNG TISSUE DAMAGE IN PATIENTS OF ALL AGES, PARTICULARLY PREMATURE INFANTS AND ELDERLY ADULTS. DURING RESPIRATORY VIRAL INFECTION, INTERLEUKIN-6 (IL-6) PLAYS A PIVOTAL ROLE IN PROMPTLY AND TRANSIENTLY ACTIVATING AND REGULATING THE PROPER MUCOSAL ANTIVIRAL IMMUNITY TO FIGHT AND CLEAR THE PATHOGEN. HOWEVER, EXCESSIVE AND PERSISTENT IL-6 PRODUCTION MAY PROMOTE VIRUS SURVIVAL AND EXACERBATION OF CLINICAL SYMPTOMS. THEREFORE, THE REGULATORY MECHANISMS THAT SHAPE AND FINE-TUNE THE PRECISE IL-6 RESPONSE TO RESPIRATORY VIRUSES NEEDS TO BE WELL UNDERSTOOD. IN ORDER TO GAIN INSIGHT INTO THE GENOME-WIDE CONTROL OF GENE TRANSCRIPTION IN MUCOSAL EPITHELIAL ANTIVIRAL RESPONSE, WE APPLIED PRECISION NUCLEAR RUN-ON NEXT GENERATION SEQUENCING (PRO-SEQ) TO HUMAN SMALL AIRWAY EPITHELIAL CELLS (HSAECS) INFECTED WITH RV. IN THE IL-6 GENE LOCUS, WE HAVE IDENTIFIED AN UPSTREAM CIS-REGULATORY ELEMENT (URE) THAT RESIDES WITHIN AN OPEN CHROMATIN DOMAIN AND ENCODES NONCODING RNAS BIDIRECTIONALLY. OUR PRELIMINARY DATA SHOWED THAT THIS UNANNOTATED URE IS A DUAL-FUNCTION REGULATORY ELEMENT OF IL-6 EXPRESSION, ACTING AS A SILENCER IN NORMAL AIRWAY EPITHELIAL CELLS BUT AS AN ENHANCER IN EPITHELIAL CELLS THAT UNDERGO CELLULAR PLASTICITY TO DEVELOP MESENCHYMAL CHARACTERISTICS AS SEEN IN CHRONIC AIRWAY REMODELING. IN THIS R21 APPLICATION, WE WILL INVESTIGATE THE HYPOTHESIS THAT THE IL-6 URE IS AN EPIGENETIC SILENCER OF IL-6 EXPRESSION IN MUCOSAL EPITHELIAL CELLS, AND THAT THE SILENCER-ENHANCER TRANSITION DYSREGULATES IL-6 EXPRESSION IN RESPONSE TO CELLULAR PLASTICITY. OUR PROJECT SEEKS TO UNDERSTAND HOW THE FUNCTIONAL SWITCH OF THE IL-6 URE FROM SILENCER TO ENHANCER ACTIVITY ASSOCIATES WITH IL-6 EXPRESSION, EITHER THROUGH CHROMATIN INTERACTIONS WITH IL-6 PROMOTER (CHROMATIN LOOPING) OR VIA FUNCTIONS OF ITS BIDIRECTIONAL NON- CODING RNAS. WE HAVE ASSEMBLED A HIGHLY SKILLED INTERDISCIPLINARY TEAM TO DISSECT THE CONTRIBUTIONS OF OUR IDENTIFIED IL-6 URE IN TWO SPECIFIC AIMS: AIM 1, THE CHROMATIN INTERACTION BETWEEN IL-6 URE AND PROMOTER DETERMINES ITS FUNCTION AS A SILENCER OR ENHANCER IN DIFFERENT CELLULAR CONTEXTS. AIM 2, THE BIDIRECTIONAL NON- CODING RNAS TRANSCRIBED AT IL-6 URE SERVES AS DOCKING SITES TO RECRUIT EPIGENETIC REGULATORS THAT AFFECTS THE TRANSCRIPTIONAL ELONGATION OF IL-6. WE ARE THE FIRST TO IDENTIFY IL-6 URE AND ITS DERIVED NONCODING RNAS THAT WILL ADVANCE US TO UNDERSTAND HOW DYNAMIC INTERACTIONS BETWEEN CIS-REGULATORY ELEMENTS FACILITATE PRECISE GENE REGULATORY RESPONSE TO EPITHELIAL CELL PLASTICITY. OUR FINAL GOAL IS TO OFFER NOVEL TRANSLATIONAL TARGETS THAT SELECTIVELY ALTER IL-6 EXPRESSION IN CERTAIN CELL SETTINGS WHILE NOT IN OTHERS, THEREFORE SIGNIFICANTLY REDUCING SIDE EFFECTS FOR THERAPEUTICS OF IL-6 ASSOCIATED CLINICAL DISORDERS.