Project Grant R35GM155432

DISSECTING MECHANISMS OF TRANSCRIPTIONAL REGULATION DURING STRESS - PROJECT SUMMARY MAMMALIAN CELLS ARE CONSTANTLY CHALLENGED BY A MULTITUDE OF ENVIRONMENTAL STRESSES. IN RESPONSE, CELLS TRIGGER CORRECTIVE MEASURES TO MAKE CRITICAL CELLULAR DECISIONS. DYSREGULATION OF THESE STRESS RESPONSE PROGRAMS ARE ASSOCIATED WITH AGEING, NEURODEGENERATION, AND CANCER. HOWEVER, THE MOLECULAR BASIS OF CELLULAR STRESS RESPONSE IS LARGELY UNKNOWN. WHILE STRESSED CELLS GLOBALLY SUPPRESS RNA AND PROTEIN SYNTHESIS, THEY SPECIFICALLY INDUCE STRESS RESPONSE GENES THAT DETERMINE CELL FATE. THE OVERARCHING GOAL OF MY RESEARCH IS TO UNDERSTAND HOW MAMMALIAN CELLS REGULATE GENE EXPRESSION DURING STRESS. IN THIS PROPOSED WORK, WE WILL INVESTIGATE THE MECHANISM OF TWO CONSERVED AND ILL-UNDERSTOOD GENE REGULATORY FEATURES OF STRESS RESPONSE - (1) ACCUMULATION OF GENE REGULATORY BIOMOLECULES WITHIN MEMBRANELESS COMPARTMENTS, CALLED CONDENSATES, AND (2) WIDESPREAD DISRUPTION OF TRANSCRIPTION TERMINATION. WE RECENTLY DISCOVERED THAT CELLS RAPIDLY ACCUMULATE THEIR MULTIVALENT PROTEOME, ESPECIALLY TRANSCRIPTION MODULATORS, WITHIN SUBCELLULAR CONDENSATES DURING STRESS. WHILE THE FUNCTIONS OF CONDENSATES REMAIN NEBULOUS, IT'S BEEN SHOWN THAT DYSREGULATION OR AGGREGATION OF CONDENSATE CONSTITUENTS ARE ASSOCIATED WITH NEURODEGENERATION. BUILDING ON OUR DISCOVERY, WE FOUND THAT HEAT SHOCK FACTORS (HSFS), MULTIVALENT TRANSCRIPTION FACTORS AND ESSENTIAL ARBITERS OF PROTEOSTASIS, FORM STRESS-INDUCED NUCLEAR CONDENSATES THAT LOCALIZE AT CELLULAR SITES OF ELEVATED TRANSCRIPTION. OUR DATA SUGGEST THAT STRESS-INDUCED HSF CONDENSATES INTENSELY ACTIVATE TRANSCRIPTION AT SPECIFIC GENOMIC LOCI AND ACT AS TRANSCRIPTION HUBS. WE WILL EMPLOY THIS EXEMPLARY SYSTEM TO GAIN A BROADER UNDERSTANDING OF HOW STRESS-INDUCED TRANSCRIPTIONAL CONDENSATES FUNCTION AND HOW SPATIAL ORGANIZATION DRIVES TRANSCRIPTIONAL CONTROL. ADDITIONALLY, WE FOUND THAT DISRUPTION OF TRANSCRIPTION INITIATION AND TERMINATION ARE CORRELATED WITH SEQUESTRATION OF RNA METABOLISM REGULATORS AND LINKED TO GLOBAL TRANSCRIPTIONAL DOWNREGULATION DURING STRESS. YET, DISRUPTED TERMINATION AT SPECIFIC GENOMIC LOCI LEADS TO EXTENSIVE TRANSCRIPTIONAL READTHROUGH VIA UNCLEAR MECHANISMS, CULMINATING IN THE SYNTHESIS OF NOVEL STRESS-SPECIFIC TRANSCRIPTS. THIS PHENOMENON IS ALSO A HALLMARK OF VIRAL INFECTIONS AND RENAL CANCERS. TOGETHER, THESE FINDINGS LEAD TO THE FOLLOWING QUESTIONS - (1) HOW DO STRESS-INDUCED HSF CONDENSATES FORM AND FUNCTION? WE WILL EMPLOY A COMBINATION OF ADVANCED IMAGING AND SPATIAL-OMICS TECHNOLOGIES THAT IDENTIFY MOLECULAR DRIVERS OF CONDENSATE FORMATION AND PROBE CONDENSATE'S FUNCTION IN TRANSCRIPTIONAL REGULATION WITHIN CELLULAR MODELS OF PROTEOTOXIC STRESS. (2) HOW DO CELLS MEDIATE STRESS-ASSOCIATED TRANSCRIPTION READTHROUGH? USING NASCENT RNA SEQUENCING AND EPIGENOMICS WE WILL IDENTIFY EPIGENETIC DRIVERS OF READTHROUGH IN VALIDATED CELLULAR MODELS OF OSMOTIC AND PROTEOTOXIC STRESS. WE WILL THEN USE SINGLE-MOLECULE IMAGING TO ELUCIDATE THE MOLECULAR DRIVERS OF TRANSCRIPTIONAL READTHROUGH AT INDIVIDUAL GENOMIC LOCI. OVERALL, THIS BODY OF WORK WILL ADVANCE THE UNDERSTANDING OF ORGANISMAL RESPONSE TO STRESS AND RESOLVE LONG-STANDING QUESTIONS ABOUT TRANSCRIPTIONAL DRIVERS OF STRESS RESPONSE. OUR WORK WILL HAVE WIDESPREAD IMPACT ON BOTH ORGANISMAL PHYSIOLOGY AND PATHOLOGY, UNCOVER NOVEL DISEASE MECHANISMS AND PAVE THE WAY FOR NEW TREATMENTS AGAINST MALADIES DRIVEN BY HARMFUL EXPOSURE TO STRESSORS AND ABERRANT STRESS RESPONSE PROGRAMS.