Project Grant F31DK145216

ESTABLISHING THE FUNCTION OF POU2F3 CO-ACTIVATOR OCA-T2 IN TUFT CELL DEVELOPMENT - PROJECT SUMMARY TUFT CELLS ARE CHEMOSENSORY CELLS, RESPONSIBLE FOR MODULATING TYPE 2 INFLAMMATORY RESPONSES WITHIN THE MUCOSAL LININGS OF VERTEBRATES. THESE CELLS ARE CRITICAL FOR THE MAINTENANCE OF A HEALTHY GUT MICROBIOME, AND THEIR FUNCTIONS ARE IMPLICATED IN CHRONIC INFLAMMATORY CONDITIONS, SUCH AS ULCERATIVE COLITIS. THE FUNCTION OF DEVELOPMENT OF TUFT CELL SUBTYPES IS A MAJOR QUESTION IN THE FIELD. TUFT CELL IDENTITY IS INVARIABLY DEPENDENT UPON EXPRESSION OF THE LINEAGE MASTER REGULATOR POU2F3. MY LAB RECENTLY PUBLISHED TWO NOVEL, SPECIFIC CO-ACTIVATORS OF POU2F3, NAMED OCA-T1 AND OCA-T2. OCA-T1-/- MICE LACK SMALL INTESTINAL TUFT CELLS, DEMONSTRATE A QUANTIFIABLE REDUCTION IN COLONIC TUFT CELLS, AND MAINTAIN TUFT CELLS WITHIN THE STOMACH. THIS PHENOTYPE INCOMPLETELY PHENO-COPIES POU2F3 DEFICIENCY; THUS, MY PROJECT SEEKS TO CLOSE THIS GAP BY INVESTIGATING THE ROLE OF OCA-T2 IN TUFT CELL DEVELOPMENT IN VIVO. SPECIFICALLY, I HYPOTHESIZE THAT EXPRESSION OF OCA-T2 IS REQUIRED FOR THE EARLY DEVELOPMENT OF TUFT CELLS, DUE TO ITS UNIQUE ABILITY TO ESTABLISH A TUFT CELL-SPECIFIC EPIGENOME, WHICH IS MAINTAINED BY OCA-T1 OR OCA-T2 IN A CONTEXT-SPECIFIC MANNER. WE HAVE GENERATED TWO DISTINCT OCA-T2-/- MOUSE MODELS, WHICH HARBOR GERMLINE KNOCKOUT OF EITHER EXON 1 OR EXON 2 OF THE GENE ENCODING OCA-T2. MUCH LIKE IN OCA-T1 KNOCKOUT, TUFT CELL STAINING OF OCA-T2-/- MICE SHOWS A SURPRISING LOSS OF SMALL INTESTINAL AND COLONIC TUFT CELLS. THESE FINDINGS SUGGEST DISTINCT, ESSENTIAL FUNCTIONS OF BOTH OCA-T1 AND OCA-T2 IN THE DIFFERENTIATION OF INTESTINAL TUFT CELLS. INTERESTINGLY, RNA-FISH STAINING REVEALED CRYPT-BIASED EXPRESSION WITHIN THE MURINE SMALL INTESTINE. I WILL CHARACTERIZE THE PHYSIOLOGY OF INTESTINAL AND COLONIC INFLAMMATION IN THESE MICE (AIM 1). THE GOAL OF THIS AIM IS TO ESTABLISH THE DISTINCT FUNCTION OF OCA-T2 IN THE GASTROINTESTINAL EPITHELIUM. TUFT CELL-ENRICHED SINGLE CELL RNA-SEQ (SCRNASEQ) OF THE MURINE SMALL INTESTINAL EPITHELIUM HAS IDENTIFIED ENRICHED OCA-T2 EXPRESSION WITHIN A TUFT CELL PROGENITOR POPULATION. IN A POU2F3-NAIVE CELL LINE CONTEXT, LENTIVIRAL OVEREXPRESSION OF OCA-T2 DEMONSTRATES A REMARKABLE ABILITY TO DRIVE TRANSCRIPTION OF A GREATER NUMBER OF GENES, AND TO A GREATER EXTENT, THAN OCA-T1. OF NOTE, OCA-T2 UPREGULATES BOTH POU2F3 AND OCA-T1, WHEREAS OCA-T1 FAILS TO UPREGULATE OCA-T2. TO INVESTIGATE THIS REGULATORY LOGIC FURTHER, I WILL EMPLOY BOTH EX VIVO AND IN VITRO TRANSCRIPTOMICS AND EPIGENOMICS (AIM 2). THE GOAL OF THIS AIM IS TO DETERMINE THE FUNCTIONAL SPECIFICITY OF THE OCA-TS AND EVALUATE CHANGES IN GENE EXPRESSION AND CHROMATIN STATE ESTABLISHED AND MAINTAINED BY EACH CO-ACTIVATOR. THIS PROJECT WILL DEFINE THE DISTINCT ROLE OF THE NOVEL POU2F3 CO-ACTIVATOR OCA-T2 IN THE TRANSCRIPTIONAL REGULATION OF TUFT CELL DEVELOPMENT, CLOSING THE GAP BETWEEN OCA-T1-/- PHENOTYPES AND POU2F3-/- PHENOTYPES. THIS UNDERSTANDING WILL OPEN NOVEL AVENUES FOR THERAPEUTIC INTERVENTION IN CHRONIC INTESTINAL INFLAMMATION.