Project Grant R01HL174794

TRANSCRIPTIONAL REGULATION OF MYELOPOIESIS - ABSTRACT MYELOID CELLS ARE AMONGST THE FIRST RESPONDERS TO PATHOGENIC INSULTS, BUT THERE REMAINS A SUBSTANTIAL KNOWLEDGE GAP CONCERNING MECHANISMS THAT CONTROL THEIR DEVELOPMENT IN THE BONE MARROW (BM). WE HAVE FOUND THAT THE TRANSCRIPTION FACTOR THPOK (T-HELPER-INDUCING POZ-KRUPPEL FACTOR) PLAYS A CRITICAL UNEXPECTED ROLE IN MYELOID LINEAGE DIFFERENTIATION, AS THPOK NULL MICE SHOW EXPANSION OF NEUTROPHIL PROGENITORS AND PRECURSORS IN THE BM, LEADING TO NEUTROPHILIA AND PROTECTION AGAINST SEPSIS. LOSS OF THPOK EXPRESSION CAUSES WIDESPREAD TRANSCRIPTIONAL REPROGRAMMING THROUGHOUT MYELOPOIESIS. HERE, WE PROPOSE TO TEST THE HYPOTHESIS THAT THPOK SERVES AS A TRANSCRIPTIONAL CHECKPOINT FOR NEUTROPHIL COMMITMENT AND GRANULOCYTE LINEAGE OUTPUT IN 3 SPECIFIC AIMS. AIM 1 WILL DEFINE HOW GROWTH FACTOR (GF)/CYTOKINE RECEPTOR SIGNALING REGULATES THPOK EXPRESSION IN MYELOID PROGENITORS. THE UPSTREAM FACTORS THAT CONTROL THPOK INDUCTION DURING MYELOPOIESIS ARE NOT KNOWN BUT ARE LIKELY TO INCLUDE BM-DERIVED GF / CYTOKINE(S), WHICH ARE KNOWN TO DIRECT MYELOID LINEAGE CHOICE. HERE WE WILL DEFINE THE SIGNALING CASCADE/S FROM GF/CYTOKINE RECEPTOR TO THPOK TRANSCRIPTIONAL CIS ELEMENT THAT CONTROL THPOK INDUCTION AT EACH SPECIFICATION STEP. WE WILL USE IN VITRO HEMATOPOIETIC PROGENITOR CULTURES, AN EXISTING PANEL OF KNOCK-IN MOUSE MODELS HARBORING MUTATIONS IN THE THPOK CIS ELEMENTS TO DELINEATE THE UPSTREAM PATHWAYS THAT DRIVE THPOK EXPRESSION DURING MYELOPOIESIS. AIM 2 WILL ELUCIDATE THE MODUS OPERANDI BY WHICH THPOK REGULATES GENE EXPRESSION DURING MYELOPOIESIS. THE MECHANISM BY WHICH THPOK REGULATES MYELOPOIESIS REMAIN LARGELY UNCLEAR. WE PROPOSE THAT THPOK ACTS IN PART BY RECRUITING CO- FACTORS TO MODULATE CHROMATIN ACCESSIBILITY, INCLUDING THE NURD COMPLEX. WE HAVE GENERATED 3 DIFFERENT THPOK AND 2 GFI1 MUTANT MOUSE MODELS THAT ALTER THEIR ABILITY TO INTERACT WITH COFACTORS OR WITH DNA BINDING SITES. USING MYELOID AND GRANULOCYTE PROGENITORS FROM THESE MUTANT MICE, CHROMATIN ACCESSIBILITY ASSAY, CHIP SEQ, AND RNASEQ WE WILL DETERMINE THE FUNCTIONAL OUTCOMES OF THPOK AND GFI1 INTERACTION ON MYELOPOIESIS. AIM 3 WILL TEST THE HYPOTHESIS THAT THPOK IS A CRITICAL REGULATOR OF INFECTION-INDUCED EMERGENCY MYELOPOIESIS. EMERGENCY MYELOPOIESIS IS CRITICAL FOR ENHANCED RELEASE OF NEUTROPHILS AND MONOCYTES FROM THE BM DURING INFECTION, BUT THE UNDERLYING REGULATION REMAINS POORLY UNDERSTOOD. OUR PRELIMINARY DATA SUGGEST A ROLE FOR THPOK. USING OUR ESTABLISHED THPOK LOSS- AND GAIN-OF-FUNCTION MOUSE MODELS, WE WILL EXAMINE THE FUNCTIONAL CONSEQUENCES OF THPOK-MEDIATED GENE REGULATION ON LPS AND GCSF-MEDIATED EMERGENCY MYELOPOIESIS. THIS PROPOSAL REPRESENTS THE FIRST STUDY TO DISSECT THE ROLE OF THPOK IN MYELOID DEVELOPMENT AND FUNCTION, AND IF SUCCESSFUL WILL REPRESENT AN IMPORTANT ADVANCE IN THE FIELD. THE PROPOSED STUDIES WILL ULTIMATELY ALLOW DEVELOPMENT OF NOVEL TREATMENT STRATEGIES TO REPROGRAM MYELOPOIESIS UNDER DIFFERENT PATHOLOGICAL CONDITIONS SUCH AS NEUTROPENIA, SEPSIS, AND MYELOPROLIFERATIVE DISORDER THROUGH TARGETING OF THPOK AND ITS ASSOCIATED PATHWAYS.